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2020-12-10T09:04:17.050Z

1970-01-01T00:00:00.000Z

237234171

s2

Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds. A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds. Several methods have been described for rapid detection and quantitation of griseofulvin in body fluids and in fermentation media. Most of them are based on spectrophotometry (1, 3, 5-7, 10, 18, 22), spectrofluorometry (2,4,8,13,16), and colorimetry (19,23). Some spectrophotometric methods minimized error due to irrelevant materials by measuring absorbancies of the extracts at several equally spaced wavelengths and calculating the concentration of griseofulvin mathematically (1, 3, 5, 6). Holbrook et al. (11) converted griseofulvin to isogriseofulvin with methanesulfonic acid in methanol and determined its amount by measuring the resulting shift in ultraviolet (UV) absorption. Fischer and Riegelman (9) quantitated griseofulvin and griseofulvin-4'alcohol by measurement of fluorescence directly on thin-layer chromatograms. MacMillan (17) described a sensitive color test to detect dechlorogriseofulvin in the presence of griseofulvin. He reported that dechlorogriseofulvin gave an intense blue-violet color with nitric acid, whereas griseofulvin gave a pale yellow color. A method for griseofulvin determination in fermented broths of Penicillium griseofulvum and P. nigricans has been described in which the antibiotic was extracted from the fermentation broth with chloroform, and iodine was added in stochiometric ratios (24). Compounds which are structurally related to griseofulvin, such as dechlorogriseofulvin, can interfere with the analysis. Rezabek (20) and Kleine-Natrop et al. (14) assayed for griseofulvin on the basis of colony growth of Trichophyton persicolor and Trichophyton rubrum, respectively. Other biological methods for griseofulvin determination were re-ported by Knoll et al. (15) and Stepanisshcheva and Ziserman (21). The present study describes a rapid and accurate method for determination of griseofulvin and dechlorogriseofulvin by thin-layer chromatography (TLC) and gas-liquid chromatography (GLC). MATERIALS AND METHODS A griseofulvin-producing isolate of Penicilliwn urticae was used in this study. The fungus was cultured in 500-ml Erlenmeyer flasks containing 25 g of shredded wheat that was moistened with 50 ml of Mycological Broth (Difco) supplemented with 0.5% each of yeast and malt extract. After 10 to 14 days of growth at 28 C, fungal cultures from each flask were transferred into a Waring Blendor and extracted with 100 ml of chloroform. The chloroform extracts were filtered through anhydrous sodium sulfate and a 10 1uliter sample of the extract was spotted onto thinlayer chromatographic plates (0.25 mm) (MN-Kieselgel G-HR, Brinkman Instruments, Westbury, N.Y.), along with authentic griseofulvin. The plates were developed in chloroform-acetone (93:7, v/v) to a height of 10 cm. They were examined for the presence of griseofulvin or dechlorogriseofulvin, or both, first under long-wave UV light and then in normal light after being sprayed with 50% sulfuric acid and heated at 110 C for 30 min. GLC analyses were made with a Barber Colman series 5000 gas chromatograph equipped with a hydrogen-flame ionization detector and disc integrator. The liquid phases used were 1% QF-1 and 1 to 2% SE-30 coated onto Anakrom ABS 80 mesh (Analab Corp., Hamden, Conn.) by the method of Horning et al. (12). The GLC supports were packed into silanized glass columns. Precautions similar to those taken for steroids (12) were rigorously observed when preparing the GLC columns, column supports, and associated equipment to prevent "active sites" which would have caused decomposition of the antibiotics. Griseofulvin and dechlorogriseofulvin were isolated and purified from chloroform extracts of P. urticae by precipitation from chloroform solution with nhexane, followed by silica gel column chromatography (0.05to 0.20-mm mesh) (Brinkman Instruments, Westbury, New York) of the precipitate with chloroform as the eluting solvent. Fractions (25 ml) were collected automatically and subsequently monitored by GLC for the presence of griseofulvin and dechlorogriseofulvin. The fractions containing griseofulvin and dechlorogriseofulvin were combined, evaporated to dryness, and recrystallized from n-hexane-chloroform solution. Analytical confirmation of the structures of the purified griseofulvin and dechlorogriseofulvin from P. urticae was based on melting points, TLC, GLC, and UV, infrared (IR), nuclear molecular resonance (NMR), and mass spectral analyses. Melting points were taken on a Fischer-Johns melting point apparatus; UV spectra were determined in methanol solution with a model DB-G spectrometer (Beckman Instruments, Inc., Fullerton, Calif.). Infrared spectra were measured with a Perkin-Elmer model 257 spectrometer as a thin film coated onto a KBr window. NMR spectra were performed with a Varian A-60A spectrometer in deuterated chloroform. Samples for mass spectra were introduced into the mass spectrometer by the direct-probe method. RESULTS AND DISCUSSION Griseofulvin and dechlorogriseofulvin were determined accurately in crude extracts from P. urticae by TLC and GLC analysis. These two compounds appeared together as a bright blue fluorescent spot at RF 0.65 on our TLC system. The difference in polarity between these two compounds was insufficient for their complete separation. The limit of detection of griseofulvin by TLC was 0.05 ,ug. Thus, this chromatographic method provided a rapid and sensitive technique for tentative identification of both griseofulvin and dechlorogriseofulvin. Two GLC systems were then used to separate these compounds (Fig. 1, Table 1). This permitted accurate quantitation of the individual compounds without extensive purification. Spectrophotometric and spectrofluorometric methods would not make this distinction on a sample containing a mixture of these compounds. The colorimetric method described by MacMillan (17) would detect both substances in a purified sample mixture, but interfering substances in a crude extract might affect accuracy. No metabolites in the extracts from P. urticae interfered with either the GLC or TLC analyses. The use of TLC is valuable for preliminary screening, since several extracts can be evaluated on one chromatographic plate in minimal time. Extracts that appear to contain griseofulvin or dechlorogriseofulvin, or both, can then be simultaneously analyzed qualitatively and quantitatively by GLC. In addition to being an excellent qualitative and quantitative method for analysis of these compounds, GLC also served as a monitor during purification. Use of GLC showed that griseofulvin and dechlorogriseofulvin were not resolved from each other by silica gel column chromatography; however, they were separated by fractional recrystallization. This was consistent with previously reported data (17). The melting point and UV, IR, NMR, and mass spectra of the metabolite identified as griseofulvin from P. urticae and an authentic griseofulvin standard were compared to confirm that the compound observed on TLC and GLC was griseo-fulvin. No authentic standard for dechlorogriseofulvin was available; however, purified dechlorogriseofulvin from P. urticae was identified by comparison of its UV, IR, NMR, and mass spectra with those of griseofulvin. The melting point (220 C), UV spectra [wavelength (X) maxima in methyl alcohol at 291, 236, and 325 nm], and IR spectra of griseofulvin from P. urticae and authentic griseofulvin were identical. The molecular weights of these compounds, as determined by mass spectroscopy [molecular extinction coefficient (m/e) 352], and of the major fragments (m/e 214 and m/e 138) were also identical. In addition, the mass spectra were identical in their overall fragmentation patterns. The mass spectrum of dechlorogriseofulvin showed parent peak at m/e 318 with major fragmentation at m/e 180 and m/e 138. This is consistent with the spectrum of griseofulvin less chlorine. The UV spectrum of dechlorogriseofulvin was identical with griseofulvin and the IR spectrum had only minor differences. The NMR spectra for griseofulvin from P. urticae and authentic griseofulvin were identical. The NMR spectrum of dechlorogriseofulvin showed the presence of an additional proton at 6.08 parts per million (ppm) which was coupled with a proton at 6.26 ppm (J = 2 Hz). This is consistent with the expected coupling of protons that are meta to each other such as the protons at carbon 5 coupling with the proton replacing the chlorine group at carbon 7 in dechlorogriseofulvin. The absorptions of the two aromatic methoxy groups in griseofulvin (4.00 ppm and 4.05 ppm) were shifted in dechlorogriseofulvin so as to be superimposed at 3.92 ppm. The above analytical data prove that the two compounds, from crude extracts of P. urticae which were analyzed via TLC and GLC, were griseofulvin and dechlorogriseofulvin.

v3-fos

2020-12-10T09:04:12.317Z

1970-05-01T00:00:00.000Z

237231559

s2

Effect of Chloramphenicol on Host-Bacteriophage Relationships in the Lactic Streptococci Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml3. Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 μg/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml3 phage in strain ML3-CMrI, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one. ogous attacking phages but not to phage mls. Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 ,g/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml3 phage in strain ML3-CMrI, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phageresistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one. Studies of bacteriophage resistance in the lactic streptococci are of considerable interest since phage can destroy these bacteria when they are used as starter cultures in the manufacture of cheese (2,6). The use of phage-resistant mutants to circumvent phage attack has been examined in the past, with attention directed towards the employment of mutants that either do not allow phage penetration, or are lysogenic, or exist as phage carriers (5,8,10,11,13,14). Indirect means of obtaining phage-resistant mutants have been described for other bacterial genera, including the method of screening drug-resistant mutants for phage resistance. A recent report (9) indicated that choramphenicol (CM)-resistant mutants of group A streptococci were also resistant to lysis by virulent phage. The biochemical events which result from exposure of growing bacteria to CM, which acts primarily to inhibit protein synthesis, have been the subject of intensive investigation (3). This communication describes the effect of CM treatment on phage-host relationships in lactic streptococci. I (16). Bacteriophages. In phage infection experiments with S. lactis ML3 and its homologous phage, a whey suspension of phage ml s containing 2.4 X 1010 plaqueforming units per ml was used. In testing mutants of ML3 for their spectrum of resistance to heterologous phage types, the collection of S. lactis and S. cremoris phages held by the New Zealand Dairy Research Institute was used. Phage assays were carried out by the agar overlay method (1) on LYP agar (16) plates. To test the reaction of a bacterial strain to a particular phage, a sample of the bacteria was layered in semisolid agar on an LYP agar plate, and the phage suspension was spotted on. Isolation of phage-resistant mutants. Mutant colonies of S. lactis ML3 resistant to phage ml3 were isolated from plates layered with semisolid agar containing about 5 X 108 cells and phage at a multiplicity of 5. These were streaked on plates to purify, 707 they were grown in broth, and the cultures were checked for phage resistance. Isolation of CM-resistant mutants. Mutants of S. lactis ML3 resistant to CM were obtained during the process of continuous transfer of ML3 in LYP broth containing increasing amounts of CM. Other CMresistant mutants of ML3 were isolated from LYP agar plates containing CM which had been spread with about 108 colony-forming units from an ML3 culture previously subjected to mutagenic treatment with either NG (15) or ultraviolet (UV) irradiation. Treatment with NG was carried out as follows. A logphase LYP broth culture received 1,000 ,ug of NG/ml before incubation at 30 C for 20 min. The cells were centrifuged, resuspended in LYP broth, and incubated at 30 C for 3 hr before they were spread on CMcontaining plates. For UV treatment, the cells of a log-phase culture were centrifuged, washed in Ringer solution, and finally resuspended in this medium. A sample (3 ml) of the suspension was placed at a distance of 29 cm from a Hanovia UV-lamp and irradiated for 30 sec. Samples were then withdrawn, incubated at 30 C for 3 hr, and spread on CM-containing plates. Bacterial growth curves. The growth or lysis of logphase LYP broth cultures of S. lactis ML3 and derivative CM-and phage-resistant mutants, in the presence or absence of CM and phage, was determined by viable cell counts and turbidity measurements in a Spectronic 20 colorimeter/spectrophotometer set at 560 nm. One-step growth experiments were carried out as described by Adams (1). Premature lysis of cells was achieved by diluting infected cells into 1.0-ml samples of Ringer solution, held at 4 C, containing two drops of chloroform and 25% (v/v) Chance no. 12 Ballotini beads, followed by shaking on a Mickel disintegrator for 2 hr at 4 C. To determine whether phage penetration of bacterial cells occurred after phage adsorption in the presence of CM, cultures of CM-treated and untreated cells were infected with phage and, after allowing time for adsorption, the bacteria were separated from unadsorbed phage by low-speed centrifugation (4,000 X g for 5 min). The cells were then suspended in a solution containing (per liter): 1 mM MgSO4, 0.1 mM CaCl2, and 0.1 g of gelatin (12). This suspension was agitated in a Waring Blendor for 2 min at 10,000 rev/min before cooling in ice water. Samples were removed for assay of infective centers or for further centrifugation to collect released phage. RESULTS Mutagenesis studies with S. lactis ML3. The killing effects of NG and UV irradiation were examined initially to determine suitable mutagenic doses for treatment of S. lactis ML3. The effect of NG treatment on ML3 is presented as a function of dose ( Fig. 1) and as a function of time (Fig. 2). ML3 was found to be very resistant in comparison with gram-negative bacteria (15). The cells of an NG-treated culture, examined The results of a UV survival experiment ( Fig. 3), carried out with a suspension of ML3 cells in Ringer solution, indicated that irradiation at a distance of 29 cm for 30 sec gave about 90% killing of cells, and this was subsequently chosen as the mutagenic dose. CM resistance and phage resistance in S. lactis ML3. Continuous transfer of ML3 in LYP broth containing increasing concentrations of CM produced, after 73 transfers, a culture able to grow slowly in the presence of 280 jig of CM/ml. At levels of CM above this value, no bacterial growth could be obtained. The natural level of resistance is 2 ,g/ml. A culture of ML3_CMrI, isolated after 66 transfers, grew normally in the presence of 210 ,ug of CM/ml and was chosen for examination in subsequent growth curve studies in the presence or absence of CM and phage ml 3. ML3-CMrI did not revert to CM susceptibility when subcultured in the absence of the antibiotic. During the period of training, after addition of each CM increment, the growing culture was tested for its susceptibility to 25 stock S. lactis and S. cremoris phages, seven of which, under normal conditions, are able to form plaques when spotted on an ML3 culture plated in semi-solid agar on LYP agar. The phage relationships of ML3 and the culture of ML3 enriched for CMr mutants are shown in Table 1. In the course of transfer in CM-containing broth, the ML3 culture lost its resistance to hp phage for a period of 29 transfers; it acquired resistance to z8 phage after 14 transfers and to sk2 phage after 72 transfers; and it became susceptible to pl phage after 50 transfers. Apart from phage-relationship changes, other significant observations made during the course of training were as follows. At a level of 16 jAg of CM/ml, there was a distinct change from slow growth to normal growth in the presence of increasing CM concentrations. At 42 ,ug of CM/ ml and thereafter, there was a marked increase in the number of phage-resistant colonies appearing in the phage ml,3 lytic areas. At levels between 175 and 190 ,g of CM/ml, cultures required 48 hr of incubation at 30 C to yield cell numbers previously obtained in 24 hr. Above 190 MAg of CM/ml, it became necessary to alternate subculture in LYP broth plus the appropriate CM increment, with subculture in normal LYP broth to achieve satisfactory growth in the presence of the increased CM concentration. Single CM-resistant colonies were isolated from plates, containing a range of CM concentrations, that had been spread with NG-treated and UV-irradiated S. lactis ML3 cultures. Each colony was suspended in LYP broth, grown to log phase, and then tested for resistance to both the homologous phage and to CM. In this way, nine mutants resistant to CM concentrations ranging up to 200 ,ug/ml were isolated from the NG-treated culture. All of the mutants tested were susceptible to phage ml3. Sixteen mutants, resistant to similar CM concentrations and sensitive to ml,3, were isolated from the UV-irradiated culture and, in addition, four mutants were isolated at a level of 500 ,g of CM/ml after examination of 200 plates. These four mutants were resistant to phage ml,3. In the course of daily subculture in LYP broth in the absence of CM, they lost their resistance to CM after about 4 days, but retained their resistance to phage ml,3 for about 6 weeks before reversion to phage sensitivity was observed in two of the cultures. An identical number of plates spread with the NGtreated culture and containing the same high concentration of CM failed to yield any CM-resistant mutants. A total of 100 phage-resistant forms of S. lactis ML3 were examined for resistance to CM by subculturing to tubes of LYP broth containing a range of CM concentrations; none of the strains tested had acquired CM resistance in addition to their phage resistance. Effect of CM on the multiplication of S. lactis phage m13. The mutant S. lactis strain, ML3-CMrI, trained to grow in the presence of 210 ,ug of CM/ml, was compared with normal ML3 in a number of growth curve experiments to determine the effect of CM on phage multiplication. Each strain was grown to early log phase, and its subsequent growth was examined both when untreated and after receiving additions of (i) an m13 phage suspension, (ii) CM solution at two different concentration levels, and (iii) ml 3 phage suspension plus CM solution at the two different concentration levels ( Fig. 4 and 5). At a concentration which did not inhibit bacterial growth, CM clearly had no effect on phage multiplication in ML3, and the same concentration did not inhibit phage lysis in ML3_CMrI. On the other hand, at a concentration of 50 ,ug of CM/ml, growth of ML3 was halted, whereas ML3_CMrI continued to grow normally. As would be expected under these conditions, ML3 was not lysed by the phage. However, ML3_CMrI was also not lysed by phage ml 3 in the presence of 50 ,ug of CM/ml, despite the fact that normal cell growth occurred at this concentration. As a control experiment, the effect of CM on phage per se was tested, and no decrease in viability was observed. Adsorption experiments. To determine whether phage was adsorbed to ML3_CMrI in the presence of CM, the titer of free phage in an adsorption mixture of ML3_CMrI plus 50 jug of CM/ml was compared with that in ML3-CMrI during the course of an incubation period of 19 min. There was a rapid adsorption by both strains of approximately 45 % of the plaque-forming units within 2 min. Adsorption continued at a decreased rate until about 90% of the particles were adsorbed by 19 min. Phage penetration. An experiment was carried out to determine whether addition of phage ml3 to ML3-CMrI in the presence of CM resulted in phage adsorption but no penetration. Adsorption mixtures of ML3_CMrI plus phage and ML3-CMrI plus 50 ,ug of CM/ml plus phage were examined, after a 10-min adsorption period, for phage penetration. A phage multiplicity of 0.1 was used. The mixtures were assayed for infective centers at the end of the 10-min adsorption period FIG. 6. One-step growth and premature lysis curves for S. lactis ML3 and ML3-CMrl. The experiments were performed in L YP broth at 30 C, with an input ratio of phage to bacteria of 1:10. The number of infected bacteria was determined by neutralization of unadsorbed phage with antiphage serum, followed by plating at dilutions suitable for plaque counts. and then subjected to blendor treatment before determining survival of infective centers by plaque assay. At the same time, the infected bacteria in both mixtures were centrifuged at 5,000 rev/min, and the supernatants were assayed for any free phage that may have been separated from the bacteria by the blendor treatment. Approximately 94%O of the infective centers in the two adsorption mixtures survived the blendor treatment. The free phage titers in the two supernatants, after the final low-speed centrifugation, were: ML3_CMrI plus phage, 1.9 X 102/ml; ML3_CMrI plus CM plus phage, 1.4 X 102/nl. That is, similar small amounts of phage, probably representing unadsorbed phage, were found in the two mixtures, indicating that adsorption of phage leads to penetration in both cases. Premature lysis and one-step growth curves. The results of one of four such experiments for each of the strains ML3 and ML3-CMrI are depicted in Fig. 6. Strain ML3_CMrI was tested both with and without 50 ,ug of CM/ml added to the culture before addition of phage. In the onestep growth curves for ML3, and ML3_CMrI without CM added, phage m13 showed a latent period of about 25 min, followed by a rapid rise in titer which reached its maximum in 6 min. The average burst size in each case was about 43 particles per cell. The other three experiments revealed similar latent and burst periods, although the actual increase in phage titer was variable. In the presence of CM, however, no increase in phage titer was observed for ML3_CMrI during the course of the one-step growth curve. The premature lysis curves for ML3 and ML3-CMrI revealed similar eclipse periods followed by the usual phage proliferation. No mature phage particles were detected in the ML3_CMrI culture containing added CM during the entire incubation period. As a further check on the fate ofphagepenetrating the host cells in an adsorption mixture of ML3_CMrI plus 50 ,ug of CM/ml plus phage ml3, a test was made for the appearance of lysogenic forms. The mixture was incubated for 4 hr after infection, and samples were then withdrawn and spread on LYP agar plates. Single colonies were selected and tested for resistance to 3 phage. One hundred colonies were examined in this way, and none was phage resistant, indicating that no lysogenic association resulted from infection of ML3_CMrI with m13 phage in the presence of CM. DISCUSSION At least three types of mutation have been shown to affect resistance to CM in S. lactis ML3. The stepwise mutation associated with training host cells to grow in the presence of increasing increments of CM produced forms resistant to CM up to a maximum concentration level of 280 jig/ml. In association with this stepwise mutation, there was a change in the response of the strain to infection with heterologous phage types, generally in the direction of increased resistance to these phages. However, it remained susceptible to the homologous phage. After treatment with NG or UV irradiation, both potent mutagenic agents for many bacterial genera, at levels sufficient to give 90% inactivation of the cells, mutants showing resistance to concentrations of CM up to 200 ,ug/ml could be selected. These one-step mutants remained susceptible to attack by the homologous phage. The third type of mutant was selected at low frequency, and again involved a one-step mutation event. In this case, ML3 yielded, after UV irradiation, mutants resistant to 500 Ag of CM/ml; these mutants were also resistant to phage ml 3. During the course of subsequent daily subculture, some of these mutants reverted to CM sensitivity after four transfers and to phage sensitivity after 43 transfers. Mutants selected for phage resistance were not CM resistant. VOL. 19, 1970 711 Ios It seems likely that there are at least two different loci for CM resistance on the ML3 chromosome: the one for high-level resistance (500 Mg! ml), although not identical with the phage-resistance locus, is associated with it, whereas the one or more concerned in the stepwise mutation are independent of it. The locus affected by the singlestep mutations conferring resistance to CM levels up to 200 ,g/ml may not be the same as that involved in the stepwise mutational events. It has been shown that resistance to CM in Escherichia coli may be due to one of three possible mechanisms (17): (i) resistance of the protein-synthesizing machinery; (ii) decreased permeation to the site of antibiotic action; (iii) inactivation of the drug. The demonstration that CM intereferes with the multiplication of phage in S. lactis ML3_CMrI indicates that the second of these mechanisms does not operate in this case. If ability to inactivate the drug resulted from the mutation to CM-resistance, the mutation would be one in which the necessary enzyme was formed in greater amount or had a higher affinity for the CM substrate. In either case, after addition of CM to the growing culture, the growth rate would be decreased until inactivation was completed. No reduction in growth rate was observed when normally bacteriostatic concentrations of CM were added to strain ML3CMrI. It seemslikely, therefore, that in this strain the mutation to CM resistance confers resistance on the proteinsynthesizing ability of the host cells. At or below a concentration of 2 ug/ml, CM had no effect on the growth of ML3 in LYP broth, and lysis occurred after the usual period of incubation in the presence of phage ml 3. The same was true for strain ML3_CMrI which had been trained to grow in the presence of 280 MAg of CM/ml. When the concentration of CM in each adsorption mixture was raised to 50 Mg/ml, growth of ML3 was inhibited and no phage lysis occurred; phage lysis of ML3_CMrI was also inhibited by this concentration of CM, but cell growth remained unaffected. In analogous experiments (R. J. Richards, Ph.D. thesis, Ohio State University, 1960), it was shown that subinhibitory concentrations of penicillin decreased the time required for lysis initiation by a S. lactis host-phage system in broth, whereas oxytetracycline caused a delay in the onset of lysis, and streptomycin had no effect. The magnitude of the penicillin and oxytetracycline effects increased as the antibiotic concentration increased. Since it could be demonstrated that CM had no effect on phage ml 3 per se, the possibility existed that the CM affected the phage-host interaction in one or more of the following ways. (i) Phage adsorption was inhibited; (ii) phage penetration was inhibited; (iii) penetrating phage established a lysogenic relationship with the host. When cells are infected with temperate phages, either a lytic response or a lysogenic response can occur. In some bacteria, CM brings about an increase in the lysogenic response, apparently by inhibiting the protein synthesis that is required for the lytic response (4). This effect is not specific but is brought about also by other conditions that retard biosynthetic operations. The adsorption and penetration experiments carried out with ML3_CMrI provided evidence that CM at a concentration of 50 ,g/ml did not affect adsorption to and penetration of the host cells by phage ml 3. In addition, it was shown that lysogenic forms did not arise as a result of the phage infection, since surviving cells were not resistant to the homologous phage. The one-step growth and premature lysis experiments confirmed that no intracellular multiplication of phage took place even though phage adsorption and penetration occurred. Thus, any infection that occurred may be termed abortive. It may be concluded that, in an adsorption mixture of ML3_CMrI plus phage m13 plus 50 ,ug of CM/ml, the CM interferes with the synthesis of mature phage particles without affecting the synthesis of host cell material, or that phage is synthesized but is nonviable (defective) when synthesized in the presence of CM. When abortive infection occurs, phage development is halted before the completion of mature phage particles. With a variety of chemical manipulations, it is possible to allow phage development to proceed to completion with the formation of structurally mature phage which, however, are non-infective. This results from the incorporation of an unnatural amino acid or pyrimidine into the protein or nucleic acid of the phage. No defective phage of phage components were detected with an electron microscope after artificial disruption of the phage-infected ML3_CMrI. Phage production is dependent on the host cell metabolism for energy and for the synthesis of raw materials. Therefore, any interference with the energy metabolism or the synthetic enzyme system of the host cell may be expected to have an effect on phage production. However, it is possible to interfere with bacterial multiplication without affecting phage growth, and vice versa, showing that the nutritional requirements for the two processes are not identical. CM inhibition of deoxyribonucleic acid (DNA) synthesis has been observed in phage-infected bacteria (7). Several new enzymes, which are necessary for synthesis of phage DNA, are produced in the first few minutes after phage infection. It is possible that CM inhibits phage DNA synthesis indirectly by blocking the formation of these enzymes. It is concluded that the block in phage development in ML3-CMrI probably involves inhibition of phage component synthesis so that, although phage penetrates the cell, either the genome is not replicated or phage coat proteins are not synthesized. Thus, no mature phage particles are formed. ACKNOWLEDGMENT It is a pleasure to acknowledge the capable technical assistance of Diana Mellor.

v3-fos

2022-07-07T15:02:43.334Z

1970-01-01T00:00:00.000Z

250318563

s2

Characteristics and Productivity of Some Pigeon Breeds in Bangladesh (Aves: Columbidae) This is our hurried responsibility to introduce those pigeon breeds that have economic value. In this case, very common and productive breeds but moderately unknown in history or confusing on their scientific identity need to be focused elaborately. A survey in the country, as well as experiences on these pigeons, inspired to write this valuable fundamental composition. Pet shops, markets, and lofts have sulli (moos-sulli), chuva chandan (chua chandan), bombai (sotobanca/Italian crested mondain), kokah (laugher), and parvin (Australian red) pigeons are carrying a remarkable productivity in the country. Prices of adult and squabs are reasonable to all sorts of people. Introduction Bangladesh is very rich with many varieties of pigeons from the past. Many people kept pigeons with maintaining proper care and breeding. In past, people reared pigeons just for meat purposes (local pigeon) and few were tumblers and highflyer pigeons for flying amusement. (Levi, 1941) described moos-sulli/Syrian coop tumbler with their characteristics in his book 'The Pigeon'. In Bangladesh, this breed is called 'ghia sulli' for its only self-yellow colour. (Kabir, 2014), (Kabir, 2014a), (Kabir, 2015) just compiled some tumbler/roller/highflyer pigeons of Bangladesh where this moos-sulli (so-called ghia sulli) was noticeable. In most of the areas of Bangladesh, this breed is available with its perfect markings. It has no leg feathers and cannot tumble when fly and is considered a beautiful cage bird (Kabir, 2021). Italian sotobanca or crested mondain had been noted by Levi (1941), in his book too. In Bangladesh, one type of pigeon is available which is called bombai pigeon. According to this name, this is an Indian breed (Bombay of India) but it has no concrete history of origin in any book of pigeons. A keen observation about sotobanca, this bombai pigeon shows many similarities with them. So this bombai can be a cross product of sotobanca. (Kabir, 2018) mentioned bombai pigeon in his paper as a successful pigeon breed for starting a firsttime pigeon farm. In 1985, Moragacha village of Kumarkhali upazila of Bangladesh, there was a male black bombai (Italian crested mondain), its pair flew from the launch from getting back from Sirajganj of Bangladesh. This Moragacha village was renowned for tumblers and lotan pigeons at a time, and this village carries a nostalgic event with the author of this paper (Kabir, 2020). Kokah pigeon is a special type of vocalized pigeon, available throughout Bangladesh but in the Rangpur division, this is many (Rahman, 1999). Emperor Akbar had around 20,000 pigeons and among them, 500 were specially sorted out. Kabir (2014), published an article on the Mughal pigeons with the availability of kokah pigeons. In other books, there is no trace about this pigeon but their voice is similar to Thai laugher but phenotypically this breed is different than others. There is no single evidence about "chuva chandan" and "Australian red" breeds in books except only one pdf on the internet (Self, 2022). Rahman (1999), only mentioned just the names of bombai, parvin, and chua chandan pigeons in his book without description. The objective of this study is to enhance for keeping these pigeon breeds for sufficient profit with the fulfilling their hobby. Materials and Methods Sulli, chuva chandan, parvin, bombai, and kokah pigeons were available in pet shops of Kushtia, Saidpur, Dinajpur, and Dhaka Katabon Animal Market Kabir (2013), and pigeon markets of Kushtia, Saidpur, and Mirpur Dhaka, Bangladesh (Kabir, 2014c). Direct observation and survey method provided immense thought about their breeding characteristics, comparison with other breeds, and their acceptance value. Weekly visit in these shops and markets enriched the parameters of this article. Pigeon keepers helped to contribute their year-wise data on the above parameters. This study was completed in the year 2021. Results and Discussion Moos-sulli. Wendell Mitchell Levi (1941), described moos-sulli breed in his book 'The Pigeon' (page 217) in tumbler varieties. In Bangladesh, this pigeon is called 'ghia sulli'. Its colour is very nice like ghee and a popular breed (Table 1; Figure 1). This is not a flying bird; rearers keep in the cage as a show bird. In most of the pet shops, this pigeon is available and the price is reasonable at all. Moreover, its breeding performance, as well as care to the young, is mentionable. In Bangladesh, this is considered a non-tumbling tumbler (Kabir, Hawkeswood, & Makhan, 2020). Its body shape, eye colour, beak, and head size are somewhat a true tumbler. Levi (1941), said this breed into coop-tumbler because it tumbles in a very short space. Among the tumbler group, in Bangladesh, this sulli pigeon is very common, especially in Dhaka city. Till now, there is no mixing record of this breed but sometimes white feathers can be expressed with its yellow plumages (Table 1; Figure 1). Levi (1941), described this breed with muff but in Bangladesh, this bird is totally clean-legged. Based on the shape of the head mostly this breed is plainheaded but crested variety also common. In fact, this is a breed (domestic) (Columba livia domestica) not a species. Figure 1. Ghia sulli (Moos-sulli) (Columba livia domestica) Chuva chandan. This is an Indian breed, same with the moos-sulli. Pigeon belongs to the tumbler family but this is a show breed. Remarkable coloured with yellowish head-neck and the body is shades of grey (Table 1; Figure 2). Commonly found in Bangladesh at a reasonable price and has an enchanting face at a glance. Its eye is always black and clean-legged. Mostly plain-headed but crested variety is also available throughout Bangladesh. Moreover, it cannot tumble. The beak is short and is clean-legged. Breeders do not mix this pigeon with others so that this is a purebreed till now; a very docile and calm pigeon. Its crest is always displayed with peak crest. The breeding biology of this pigeon is mentionable and possible to sell its baby anytime. Based on the nice and exceptional plumage colour, this pigeon could snatch anybody's eyesight. Plain-headed variety is more abundant than the crested ones (Table 1; Figure 2). Acceptance, availability, price, overall reproductive performances are distinguished on their commercial value. Figure 2. Chua chandan (chuva chandan) (Columba livia domestica) Bombai pigeon. When I read in class four, saw a big large-sized black male pigeon (so-called bombai) in my village Moragacha. Its pair flew from the launch when my cousin is returning from Sirajganj of Bangladesh. Then in our village, this male got a pair with other local (indigenous) black female and this pair produced some black and intermediate-sized offspring. In my childhood place, Moragacha village under Kumarkhali upazila of Bangladesh had many nostalgic events with pigeons (Kabir, 2020). My father kept tumblers and loton pigeons in this village. Finally, all of those black bombai pigeons were caught by domestic cats. In many books, I did not find the actual international name of this bombai pigeon. From the name bombai, it is easily understandable that this is an Indian breed. On the internet, after keen searching Italian crested mondain is similar to this bombai pigeon (Beaumont, 1962). It has four self colours-black, red, yellow, and white but black is rare. All are very nice with their broad crest (ear to ear); robust body and very powerful birds at all. Carrying a sharp beak and completely clean-legged (Table 1; Figure 3). Breeding is mentionable but sometimes its huge bodyweight makes hazards in incubation. Alternative use of the pair could ensure proper hatching. It is commonly found in any pet shops and markets of Bangladesh. Legs are comparatively short. Pigeon keeper collects this pigeon without any doubt and does not cross with others so that this is purebred as a whole. Due to heavyweight, they cannot fly more. Figure 3. Red bombai (sotobanca) (Columba livia domestica) Kokah Pigeon. The history of the laugher pigeon is known but on kokah pigeon, this is completely unknown in any books. Rahman (1999), mentioned this pigeon as a kokah breed in his book 'Kingdom of Pigeons' (in Bangla). Its voice is completely similar to Thai laugher pigeon but its physical appearance is the same as with the local pigeon. The plumage colour is brick-red. The beak is very sharp; loose plumages; peak crested and always clean legged (Table 1; Figure 4). Till now, this is purebred at all and found abundant. Price is moderately high (depending on its voice quality) and available in Bangladesh especially Rajshahi, Natore, and Rangpur. After hatching, this pair shows more aggressiveness to each other so alternative use of birds ensures the stability of the young (Rahman, 1999). Its continuous narrow cooing is a long-lasting phenomenon. Early in the morning, it makes a huge sound. Normally small pigeons and body stamina are not very strong. Till now it has no alternative colours except brick-red. In addition, it has no plain-headed and muffed variety. With the brick-red plumages, sometimes black feathers can be seen. Slim bodied with lightweight. The eye colour is orange or red (Table 1; Figure 4). Figure 4. Kokah (laugher) (Columba livia domestica) Parvin. In Bangladesh, this breed is called 'parvin' but on the internet supplement, this is somewhat Australian red/Darpan/Barpan (Self, 2022). Always red or yellow and very few are whites. A normal looked pigeon having a broad crest and muff. Plain-headed variety is not available (Table 1; Figure 5). Medium elongated body and medium to large-sized. It has an affectionate face with reasonable market value; not very common in the pet shop or pigeon markets. Breeding performance is acceptable like other pigeons. Cool tempered pigeon and possible to rear with other pigeons. Yellow coloured eye with small and flattened head. It is show bird and cannot fly well due to muff (Table 1; Figure 5). More or less broad crest and muffed pigeon; it has no other extra-ordinary qualities to distinguish from other breeds. All are self-coloured; this is purebred in Bangladesh. Pigeon breeders normally do not mix this exceptional breed with others for earning a good profit. Conclusion The mentionable five breeds are more pronounced perspective Bangladesh with their economic advantages. If any pigeon keepers rear these pigeons, be economically benefited after fulfilling their hobby. In this regard, this article is carrying remarkable merit. For a long time staying in Bangladesh and for some/many differences we can recognize them in the separate breed and could deliver their name internationally. These pigeons are available in the country, outstanding breeding performance, and the market value of adults and squabs are desirable.

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Inactivation of Vesicular Stomatitis Virus by Disinfectants Twenty-four chemical disinfectants considered to be viricidal were tested. Ten disinfectants were not viricidal for vesicular stomatitis virus within 10 min at 20 C when an LD50 titer of 108.5 virus units per 0.1 ml were to be inactivated. Quantitative inactivation experiments were done with acid, alkaline, and a substituted phenolic disinfectant to determine the kinetics of the virus inactivation. Substituted phenolic disinfectants, halogens, and cresylic and hydrochloric acids were viricidal. Basic compounds such as lye and sodium metasilicate were not viricidal. MATERIALS AND METHODS The techniques used to obtain these data were the same as described previously (8) with the following exceptions. Disinfectant tests. Virus-disinfectant contact times were 5 and 10 min. The 30-min interval was eliminated since 10 min has been established as a maximal practical exposure time for effective viricides in animal disease control work. Speed of inactivation. Experiments were completed with seven time intervals. These were 0.5, 1, 2, 3, 4, 7.5, and 10 min. Additional time intervals of 30 and 120 min and 24 hr were used for NaOH. The disinfectant was diluted at the end of each time interval by the addition of 9 ml of phosphate-buffered saline (PBS) to the disinfectant-virus mixture, and, where acid or base was used, sterile 0.1 N NaOH or 0.1 N HCI was added to neutralize the pH of the PBS. Quantitative determinations of virus titer were measured by using serial 10-fold dilutions of virusdisinfectant mixture in embryonating chicken eggs for each time interval. The titer of virus was estimated by the LD5o method of Reed and Muench (7). Disinfectants. The 24 disinfectants are listed in Table 1. RESULTS Of 24 disinfectants, 10 were not viricidal with the previously described procedure. Alkaline chemicals tested were not viricidal for VSV, e.g., VSV survived for 24 hr in 10% NaOH (pH 12.2). Virulent virus was demonstrated after treatment with 10% KOH (pH 13.3), 10% Na2CO3 (pH 11.1), and 5% Na2SiO3 (pH 12.1) for 10 min. The quatemary ammonium compound, disinfectant G, was not viricidal at concentrations of 0.1 to 5.0%. In addition, sulfuric acid, acetic acid, isopropanol, ethyl alcohol, and Formalin were not viricidal at concentrations tested. The results are listed in Table 2 with the range of concentrations. Identical results were obtained for at least four replications of each disinfectant. The 14 other disinfectants were viricidal, but in some cases at concentrations greater than suggested by the manufacturer. The minimum viricidal concentration and the range of concentrations tested are listed in Table 3. Consistent results were not obtained with disinfectant E. The manufacturer's lot 1 was viricidal, whereas a second lot was not viricidal at the same concentration. The speed of inactivation was determined with NaOH, acetic acid, and disinfectant L. The death curve with NaOH and acetic acid was not linear, being initially rapid, followed by a decrease in rate. Virulent virus was not detectable after 30 min with 5% acetic acid, whereas, with 10% NaOH, it was still virulent after 24 hr. Disinfectant L at 2% concentration was rapidly viricidal and virulent virus was not detectable after 2 min. The virus survived 0.5% Table 1. Table 1. b Results variable depending on lot tested. propanol were not viricidal at a rate rapid enough to be useful against VSV. Alkaline chemicals have been employed as viricides in vesicular disease outbreaks in the United States because of their application for the inactivation of foot-and-mouth disease (FMD) virus. FMD virus was much more susceptible to change in pH as a means of virus destruction than VSV (4). VSV was resistant to destruction by alkaline chemicals in the present study. None of the alkaline chemicals, NaOH, KOH, Na2CO3, or Na2SiO3, was effective against VSV. However, in the United States, when a vesicular condition is found, it must be assumed to be FMD and chemicals used must be recognized as effective against FMD virus until the agent is proved to be VSV. Whereas acids have also been used to control VSV, it was determined that acetic acid was of only marginal value, since variable results were obtained with a 5 % concentration and lower concentrations were not viricidal. The results obtained with mineral acids depended on the particular acid, possibly owing to the different degrees of ionization (pK). The pK values of the mineral acids were hydrochloric acid, 0.784; sulfuric acid, 0.510; and acetic acid, 0.004. The most consistent viricides for VSV were the phenolic types when a sufficient concentration was used, but the effective concentration was higher than the manufacturer's recommendations in some cases. Organic iodine (U) and sodium hypochlorite were both active viricides. The virus was inactivated in 10 min even with the presence of the chorioallantoic membrane and allantoic fluid in the virus mixture. However, halogens are more susceptible to inactivation by organic material than other disinfectants. The rate of inactivation was determined for three different types of disinfectants. Disinfectant L (2%) was rapidly viricidal by an apparent first-order reaction, but, when it was diluted to 0.5%, a diphasic inactivation curve was evident. The diphasic curve was also evident for acetic acid and NaOH, even though the pH of the NaOH remained stable at pH 12.2 over the time of exposure. It appeared that the survivor curve was diphasic when the concentration of disinfectant was below that which was rapidly viricidal. Disinfectants at viricidal concentrations produced logarithmic virus survival curves or curves with a slight change in slope. The survival of virulent virus particles may have been due to the size of the aggregate and degree of aggregation. When a sufficiently high concentration of phenolic and halogen type of disinfectants was present, the additional time required to inactivate the virus was minimal. Based on laboratory evidence, substituted phenolics, halogens, or cresylic acids are recommended for use when vesicular stomatitis virus is to be destroyed on an infected premise. These chemicals are in the proper concentration under clean conditions and can be used with greater personal safety than acids and bases.

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1970-03-01T00:00:00.000Z

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Thermal Inactivation of Newcastle Disease Virus The rate of destruction of hemagglutinins and infectivity of Newcastle disease virus was determined over a temperature range of 37.8 to 60 C. From the calculated values of δH and δS, it was concluded that inactivation of the hemagglutinating activity and viral infectivity was due to protein denaturation. The significance of virus-contaminated foods in human diets is not yet established. However, food, including milk, has been implicated epidemiologically with several outbreaks of viral diseases (1,6,7,8,12,17). Poliovirus and echovirus have been isolated from the soil of fields irrigated with sewage, and a few vegetables grown in these fields have been shown to contain cytopathic agents (4). Although it is known that viruses do not replicate in foods, echovirus, coxsackievirus, and poliovirus have been shown to survive on vegetables stored under household conditions for as long as 2 months (5). These findings seem to indicate that viruses pathogenic to man can gain entrance to foods during their production, processing, or preparation. Food processes designed for the elimination of harmful organisms from food cannot be assumed to eliminate foodborne viruses (13). Thus, it becomes a matter of practical importance to determine the thermal resistance of viruses. It is also of interest to study the kinetics of the thermal inactivation, since it may give some insight to the mechanism of inactivation. The present study reports experiments measuring energy of activation and entropy of activation of the thermal inactivation of hemagglutinin and infectivity properties of Newcastle disease virus (NDV). MATERIAL for 20 min, and stored at -40 C in 15-ml portions until used. Hemagglutinin titration. The virus in 0.5-ml amounts was serially diluted by factors of two in test tubes containing immunological buffer (NaH2PO4-H20, 27.6 g; NaCl, 29.25 g; NaOH, 6.05 g/liter). To each tube of virus, a 0.5-ml quantity of a 0.25% suspension of chicken red blood cells in immunological buffer was added. The virus titer was read after 1 hr at room temperature. The titer was determined from the highest dilution of virus which provided a visible pattern of agglutination. Agglutination patterns were read as ++++, +++, ++, +, 0 and were expressed as hemagglutinating units per ml. Virus infectivity titration. Tenfold dilutions of virus samples were made in phosphate-buffered saline containing 1% rabbit serum. One-tenth milliliter amounts of each dilution were injected into the allantoic cavity of six 10-day-old fertile hens' eggs and incubated for 1 to 5 days. Each day the eggs were candled, and the dilutions at which embryos died were recorded. The end point was taken as the highest dilution which resulted in 50% mortality of embryos. The 50% lethal end point (LDt,) was determined by plotting the logarithm of the per cent mortality of the dilution just above 50% and just below 50% mortality versus the dilution of virus. The curve was drawn through these two points, and the point where the curve intersected the 50% survival line was noted. The reciprocal of this value was regarded as the LD o per 0.1 ml. Thermal inactivation studies. A series of glass tubes (8 mm in diameter by 150 mm in length) containing 3-ml amounts of diluted infective allantoic fluid (pH 7.7) were heated for various times in a thermostatically controlled water bath which was constant to 0.1 C and were then immediately cooled in ice water and assayed for hemagglutinins and infectivity by the methods described previously. RESULTS Inactivation of hemagglutinin. The inactivation curves obtained for NDV hemagglutinin when allantoic fluids containing virus were heated at four different temperatures, ranging from 43.3 to 60 C, are shown in Fig. 1. The thermal inac-tivation curves are considered linear over the temperature region studied, indicating first-order 8 -kinetics. There was no reduction in hemagglutinin 6 _ activity after 75 min at 43. 3 C, but at the higher 4temperatures there was a loss which was a function of temperature. 3 The velocity constant (k) for the inactivation 2 at a given temperature was calculated from the equation k = (2.303 log V/Vo)/It, where Vo is the initial activity and V is the activity after heating Arrhenius plot, is presented in Fig. 2 An Arrhenius plot of the velocity constants is also presented in Fig. 2 Table 1. The calculated value of activation energy for inactivation of NDV infectivity falls within the range cited for denaturation of various proteins (18). DISCUSSION NDV is a ribonucleic acid (RNA) virus containing an inner coiled helix of ribonucleoprotein surrounded by an outer protein envelope. In addition, the virus contains some lipid material. The hemagglutination properties of the virus are associated principally with the protein of the outer coat. Denaturation of this outer protein should then result in a loss of hemagglutinin activity. The large values of AH (70.5 kcal/mole) and AS (143.4 cal per mole per degree) obtained in this study for inactivation of hemagglutinin activity are compatible with the requirement for protein denaturation (18). The two-component curve which characterized the thermal inactivation of NDV infectivity has been reported for other viruses (3,15,16,19). The shape of these curves has been attributed by some to an inherent heterogeneity of the virus, such that a small proportion of the thermally resistant particles account for the decreased slope of the latter segment of these curves. However, in the present study, it would appear that the fraction of resistant virus, which can be estimated by extrapolating the secondary portion of the inactivation curve to the ordinate, is not some fixed value but is dependent on the heating temperature and disappears at low temperatures. It is difficult to reconcile this result with the theory of a heterogeneous resistance. Others have postulated that the change in inactivation rate at high temperature is due to factors operating during the reaction, such as the formation of aggregates, adsorption to the walls of the vessel, or the presence of virus particles in aerosol droplets above the surface of the liquid (14), all of which may protect against inactivation. Polioviruses being treated with formaldehyde become progressively more resistant to inactivation by this compound, and it has been suggested that this is due to a hardening of the protein coat as the reaction proceeds (10). Nevertheless, it appears that the two-component inactivation curves for viruses will have to be accepted until the question is resolved as to whether this phenomenon is due to an artifact or to a particular inactivation mechanism. Inactivation of NDV infectivity can be caused by destruction of the RNA, denaturation of the nucleoprotein, or denaturation of the protein in the outer coat resulting in an inability of the virus to attach to host cells. Denaturation of protein is associated with a large value of AH and AS due to the rupture of a large number of hydrogen bonds which results in a collapse or unfolding of the secondary structure of the molecule. Smaller values of AH and AS are required for heat inactivation of viral RNA. Values of about 20 kcal/mole for AH and -19 cal per mole per degree for AS for thermal inactivation of tobacco mosaic virus RNA and 31 kcal/mole for AH and 4 cal per mole per degree for AS for thermal inactivation of a poliovirus RNA have been reported (9,11). Ginoza (11) postulated that inactivation of RNA is due to a rupture of the chain. The rather large values of AH and AS obtained in the present study for inactivation of NDV infectivity would indicate that loss of infectivity in the high temperature range was due to protein denaturation rather than destruction of RNA. In support of this conclusion is the fact that a poliovirus, a rhinovirus, and foot-and-mouth disease virus heated at high temperature (50 to 65 C) show a marked reduction in viral infectivity but only a slight loss in infectivity of the extracted RNA (2,9). It should be pointed out that it has been found with some viruses, at least, that at low heating temperatures (about 43 C or less) loss of infectivity is due to inactivation of RNA (9). It appears that this phenomenon may also apply to NDV. Note that there was no damage to protein (hemagglutinin) after heating for 75 min at 43.3 C (Fig. 1), whereas this same timetemperature treatment caused over 90% loss in infectivity (Fig. 2).

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2020-12-10T09:03:04.017Z

1970-02-01T00:00:00.000Z

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Spoilage Bacteria in Canned Foods Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established. MATERIALS AND METHODS Isolation of the organisms. Cans of mushrooms taken from a batch which showed heavy sulfide spoilage were incubated at 55 C, half the cans being sampled after 7 days and the remainder after a total of 14 days. In all cases, a sample of brine was withdrawn under aseptic conditions, and a 2-ml amount was inoculated into each of four tubes of sulfite agar (12). The tubes were incubated for 72 hr at 55 C, and the black colonies were transferred into "vanilla" tubes containing the same medium for purification and isolation (9). The isolated organisms were maintained in ATCC medium code No. 42 (1), and the biological characteristics were examined by the methods described in the Manual of Microbiological Methods (14) and Laboratory Methods in Microbiology (6). Organisms. Two strains of C. nigrificans were used for the comparative study of the media. The strain ATCC 7946 was obtained from the American Type Culture Collection and strain FPI 68102 was isolated from canned mushrooms opened at the Food Processing Institute. Stock cultures were maintained in ATCC medium code No. 42 (1). Spores of these two strains were prepared as follows. tubes of liver broth (12), kept at 55 C for 72 hr, and then transferred into 1 liter of the same medium in long-necked flasks for cultivation. The culture tubes and flasks were stratified with liquid paraffin. After incubation for 10 days at 55 C, the cultures were filtered through a sterile Toyo no. 5 filter paper to remove liquid paraffin and sediment. After heating at 100 C for 20 min to destroy vegetative cells, the spores were separated from the medium in a centrifuge (4,000 rev/min for 15 min), suspended in sterile normal 0.85% saline, shaken with a test tube mixer to facilitate washing, and centrifuged again (4,000 rev/min for 15 min). This procedure was repeated three times. Finally, the spores were suspended in sterile normal saline to give the stock suspension. Media. A versatile medium for determination and enumeration of C. nigrificans was a modification of a basal type (BETI medium). Seven liquid and 10 solid media were compared for outgrowth of C. nigrificans (Table 1). Decimal dilutions of both spore suspensions of strains ATCC 7946 and FPI 68102 [stcck spore concentrations: ATCC 7946, 49,000 spores/ml; FPI 68102, 24,000 spores/ml by most probable number (MPN) determination with liver broth] were prepared with sterile normal saline, and 1 ml of each dilution was inoculated into duplicate screw-capped test tubes (18 by 170 mm, outer diameter) which contained 15 ml of the test media. The liquid media were subsequently stratified with liquid paraffin, and a strip of lead acetate paper (6) was inserted with its lower end above the medium. All tubes were incubated at 55 C, and growth was observed daily for 7 days. Plate count of C. nigrificans. Each spore dilution (1 ml) was transferred into each of five tubes of BETI broth and a similar amount into each of two petri dishes (bottom: 90-mm inner diameter by 20-mm depth) and subsequently mixed with 15 ml of BETI agar. After setting, the agar was stratified with approximately 70 ml of hard BETI agar. All tubes and plates were kept at 55 C for 7 days in an ordinary incubator. The MPN values were read from Sharf's table (13), depending on the number of positive tubes APPL. MICROBIOL. in each dilution, and the total spore counts were also determined by counting colonies on the plates. Ten different spore concentrations were studied. These were randomly selected. (9) a L, liquid media; S, solid media. b BETI broth and BETI agar: basal type of BETI medium contained beef extract, 3 g; tryptone, 10 g; yeast extract, 1 g; soluble starch, 1 g; dipotassium phosphate, 1.25 g; Fe(NH4)2(SO4)2, 0.1 g; dextrose, 5 g; in 1 liter of distilled water as a liquid medium (BETI broth). The pH of the medium was adjusted to 7.0 before sterilization at 121 C for 20 min. When required in the form of a solid medium, two concentrations of agar were used: solid medium (15 g) and hard agar (20 g) in 1 liter of the above basal medium. BETI agar was used for the solid culture in tubes and on plates, and hard BETI agar was used as the agar for stratification on plate cultures. c Drained from commercial canned mushrooms with a pH of 6.3. d NORDISK, the committee for the methods of food examination, Norway. The agar contained tryptone (15 g), yeast extract (10 g), and agar (15 g) in 1 liter of distilled water. The pH was adjusted to 7.0, and the medium was sterilized for 15 min at 121 C. To 100 ml of this base medium, 1 ml of 5% ferric citrate solution, 1 ml of 5% aqueous solution of anhydrous sodium sulfite, and 1 ml of aqueous solution of potassium permanganate were added immediately before use. RESULTS Five strains were isolated from canned mushrooms, four from cans opened on the 7th day and one from a can opened on the 14th day of incubation. The isolates were rod-shaped, gramnegative, 0.4 to 0.5 by 3.0 to 6.0 Am in size, and moderately motile. They formed eliptical subterminal spores. The deep colonies in sulfite agar were surrounded by a blackened area of the medium. The color changed to black as a result of the fine black particles in BETI broth culture. Gelatin was not liquefied and no indole was produced. Hydrogen sulfide was produced from cystine, but acid was not produced from glucose, fructose, galactose, mannose, xylose, arabinose, rhamnose, sucrose, maltose, lactose, raffinose, starch, inulin, glycerol, mannitol, or salicin. Nitrate was not reduced to nitrite. The optimal temperature for growth was between 50 to 55 C, and the strains were obligate anaerobes. The comparative outgrowth of C. nigrificans in 7 liquid and 10 solid media is shown in Tables 2 and 3. The MPN values and the plate counts of 10 different spore concentrations of C. nigrificans were enumerated, and the results were shown in Table 4. 10-' a Incubated at 55 C; the same spore suspension was used in the comparative study of both liquid and solid media. There was no apparent change in the control tube. (3) except that they were gram-negative as reported by Campbell and Postgate for Desulfotomaculum nigrificans (5). The fact that four strains were isolated from cans incubated 7 days but only one from cans incubated 14 days might be the result of the inhibiting action of hydrogen sulfide (10) which accumulated in the contents of the can. The odor of sulfide was much more pronounced after 14 days than 7 days. The experimental results (Tables 2 and 3) showed that the modified media, BETI broth (media no. L6), and BETI agar (media no. S8) showed a slight improvement on the recovery and on the quicker growth of C. nigrificans over 7 liquid and 10 solid media which were commonly used. The recovery of spores from the broth was probably 102 times higher than that of the agar tube culture. The growth of C. nigrificans in liquid media could be detected by darkening of the lead acetate strips as a result of hydrogen sulfide production, and it was detectable only by this test when glucose liver broth (media no. L2) and canned mushroom brine (media no. L7) were used. It should be noted that this test can not be used as a method for detecting hydrogen sulfide production Baars' Medium (media no. L3) and Postgate's Medium B (media no. L4), since these media when incubated at 55 would show blackening of all strips, including the control, by chemically produced hydrogen sulfide. Bufton (4) examined several media and showed that none was quantitatively satisfactory for C. nigrificans. Postgate (11) published a procedure permitting colony counts on impure cultures and natural samples for which only MPN determinations were hitherto reliable. The experimental results, however, showed the mentioned method to be a proper procedure of simple plate culture for viable count of C. nigrificans. The points of the procedure are the following. (i) BETI agar should be used as the plate culture medium since it improves recovery (Table 3). (ii) Hard BETI agar is a suitable medium as a cover for the culture medium. Two per cent agar may be adequate for the deep colonies, but it cannot detect hydrogen sulfide which is produced by surface colonies on the plates. (iii) The depth of the covering medium must be more than 10 mm to make the conditions sufficiently anaerobic for the growth of C. nigrificans. VOL. 19, 1970 285

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Mechanical characterization of buckwheat noodles mixed with seaweed (fu-nori) The present study was conducted to clarify the effect of incorporation of seaweed, i.e. funori (Gloiopeltis tenax (Turner) J. Agardh) into buckwheat noodles on their mechanical characteristics. Mechanical analysis of buckwheat noodles with funori showed that incorporation of funori into buckwheat noodles enhanced breaking stress and energy. On the other hand, incorporation of funori into buckwheat noodles enhanced decreased solubility of the albumin plus globulin fraction. The present study findings suggest that the endogenous protein may be an important factor responsible for the mechanical characteristics of buckwheat noodles with seaweed. Assami et al., (2019): Mechanical characterization of buckwheat noodles with seaweed 6 (A) (B) (A) (B) called “hegi-soba”. These buckwheat noodles are prepared by incorporating into buckwheat dough a kind of seaweed, i.e. funori, (Gloiopeltis tenax (Turner) J. Agardh) as a dough-binder (Zen-men-kyo, 2014). Before ingestion, the noodles prepared with funori are usually put on a unique wooden-tray which is called “hegi”; so these buckwheat noodles are called “hegi-soba”. Although this buckwheat dish is traditionally utilized only in Niigata region, many Japanese people currently often enjoy these local buckwheat noodles. Hegi-soba noodles have a unique masticatory sense with refreshing sense on ingestion. Mechanical characterization of “hegi-soba” noodles is an interesting subject in view of buckwheat research. In this background, the present study was conducted to characterize noodles made from buckwheat flour with seaweed. MATERIALS AND METHODS Materials Buckwheat flour (Fagopyrum esculentum Moench, var. Kitawase-soba), which was harvested in Hokkaido (in 2017), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80oC until use. Ground seaweed, i.e. fu-nori in Japanese, Gloiopeltis tenax J. Agardh) used in this study was a commercial product (Oowaki-manzou-shoten Co., Fukui, Japan). Fig. 1. Buckwheat noodles. (A), non added seaweed; and (B) added seaweed (1.7% addition). INTRODUCTION Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003; Ikeda, 2002). Buckwheat flour contains various beneficial components for human health such as protein, polyphenolics, rutin and minerals at high levels (Ikeda 2002; Ikeda and Yamashita 1994). Thus, buckwheat can contribute as an important dietary source of such beneficial components. There is a large variety of buckwheat products produced on a global basis (Ikeda, 2002). Attention has been currently paid to the palatability and acceptability of buckwheat products from the perspective of their cooking and processing. However, there are still unanswered questions on the palatability and acceptability of buckwheat products. As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japanese yam flour, are often added in preparing buckwheat noodles (ZMCS, 2004). A variety of buckwheat noodles with various dough-binders has been traditionally available in Japan. We reported mechanical effects by addition of various dough-binders to common and Tartary buckwheat noodles in view of two analysis, i.e., tensile analysis and breaking analysis (Ikeda, et al., 2005). However, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buckwheat products. In Niigata district, located in the middle region of Japan, there is a famous buckwheat dish. This dish is Fagopyrum 36(1):5-9 (2019) 7 called "hegi-soba". These buckwheat noodles are prepared by incorporating into buckwheat dough a kind of seaweed, i.e. funori, (Gloiopeltis tenax (Turner) J. Agardh) as a dough-binder (Zen-men-kyo, 2014). Before ingestion, the noodles prepared with funori are usually put on a unique wooden-tray which is called "hegi"; so these buckwheat noodles are called "hegi-soba". Although this buckwheat dish is traditionally utilized only in Niigata region, many Japanese people currently often enjoy these local buckwheat noodles. Hegi-soba noodles have a unique masticatory sense with refreshing sense on ingestion. Mechanical characterization of "hegi-soba" noodles is an interesting subject in view of buckwheat research. In this background, the present study was conducted to characterize noodles made from buckwheat flour with seaweed. Materials Buckwheat flour (Fagopyrum esculentum Moench, var. Kitawase-soba), which was harvested in Hokkaido (in 2017), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80 o C until use. Ground seaweed, i.e. fu-nori in Japanese, Gloiopeltis tenax J. Agardh) used in this study was a commercial product (Oowaki-manzou-shoten Co., Fukui, Japan). Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003;Ikeda, 2002). Buckwheat flour contains various beneficial components for human health such as protein, polyphenolics, rutin and minerals at high levels (Ikeda 2002;Ikeda and Yamashita 1994). Thus, buckwheat can contribute as an important dietary source of such beneficial components. INTRODUCTION There is a large variety of buckwheat products produced on a global basis (Ikeda, 2002). Attention has been currently paid to the palatability and acceptability of buckwheat products from the perspective of their cooking and processing. However, there are still unanswered questions on the palatability and acceptability of buckwheat products. As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japanese yam flour, are often added in preparing buckwheat noodles (ZMCS, 2004). A variety of buckwheat noodles with various dough-binders has been traditionally available in Japan. We reported mechanical effects by addition of various dough-binders to common and Tartary buckwheat noodles in view of two analysis, i.e., tensile analysis and breaking analysis (Ikeda, et al., 2005). However, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buckwheat products. In Niigata district, located in the middle region of Japan, there is a famous buckwheat dish. This dish is Mechanical measurements For the study of the effects of the seaweed on the mechanical characteristics of buckwheat noodles, buckwheat noodles were prepared by hand. The mechanical characteristics of buckwheat noodles were evaluated by breaking analysis. Prior to the mechanical analysis, the buckwheat flour which had been stored at -80 o C was placed in a desiccator at room temperature until the flour exhibited a constant moisture content. The moisture of the flour was measured with a moisture analyzer (ML-50, A&D Co. Ltd., Japan). Seaweed was boiled, and sticky seaweed was added to buckwheat flour. The buckwheat dough was prepared just prior to mechanical analysis to have a moisture content of 42% by adding the appropriate amount of distilled water. Then the buckwheat noodles were made from the buckwheat dough using a handmade pasta machine (SP-150, Imperta Co., Torino, Italy). Figure 1 shows buckwheat noodles prepared in this study. The buckwheat noodles obtained were subjected to mechanical analysis. Before the mechanical analysis, buck-wheat noodles prepared were heated in boiling water for 150 seconds and subsequently were cooled for 150 seconds at 4 o C. Immediately after cooling, mechanical measurements of the noodles were performed. The breaking analysis of the buckwheat noodles was performed with Rheoner RE2-3305C (Yamaden Co. Ltd., Japan). Measurements of breaking analysis were performed with a load cell of 200N and measurement speed of 0.50 mm/ sec. A wedge-style plunger (No.49: W 13mm, D 30mm, H 25mm) was used in measurements with the Rheoner RE2-3305C. Mechanical measurements were replicated twenty times for each sample. Protein determination For chemical analysis of the combined fractions of buckwheat albumin plus globulin (AG) in the heated noodle samples which had been subjected to the mechanical measurements, the noodle samples were lyophilized and then ground into flour. The flours obtained were extracted with a ten-fold (v/w) volume of 0.2M NaCl for 1hr at 4oC. After extraction, the suspensions were centrifuged at 17,000 Xg for 20 min. Protein concentration was determined using the Bradford method with bovine serum albumin as a standard protein. Statistical analysis Statistical analysis was conducted using a personal computer with the program Excel (Microsoft Co., USA), Ekuseru-Toukei 2015 (Social Survey Research Information Co., Japan) and SPSS Ver.23.0 (IBM, USA). Figure 2 shows the breaking characteristics of hegi-soba buckwheat noodles prepared with funori-seaweed. The breaking stress and energy of the hegi-soba noodles gradually increased as the added concentration of funori seaweed increased (Fig. 2 (A and B)). A significant high breaking stress (Fig. 2 (A)) was found with hegi-soba buckwheat noodles with a concentration of funori seaweed with 1.4% or over as compared the buckwheat without funori seaweed (P<0.05). Similarly, a significant high breaking energy (Fig. 2 (B)) was found with buckwheat noodles with a concentration of funori seaweed with 1.7% as compared the buckwheat without funori seaweed (P<0.05). These findings characterize showed the unique mastication characteristics of hegi-soba noodles. Figure 3 shows the NaCl-soluble protein content of buckwheat noodles made with seaweed. The NaCl-soluble protein exhibits the combined fraction of the major buckwheat proteins, i.e., albumin plus globulin (Ikeda, 2002), designated as the AG fraction below. Changes by the addition of the seaweed in solubility of the AG fraction were found (Fig. 3). Incorporation of seaweed into buckwheat dough was found to reduce the solubility of the AG fraction in buckwheat dough as the funori seaweed added increased (Fig. 3). The seaweed contains dietary fiber at high levels (Ooishi, 1993). Judged from our previous findings (Ikeda and Kusano 1983), this phenomenon may be due to in-solubilization of proteins arisen by dietary fiber in seaweed. Interest in the nutritional function of dietary fiber for humans is currently increasing. Dietary fiber has many beneficial effects on human such as blood glucose increase suppression and antihypertensive (Mori and Tsuji, 1997). Considering in view of current nutritional science concerning the beneficial effects of dietary fiber, the intake of buckwheat noodles with seaweed with high level of dietary fiber, should be recommended as a key source of dietary fiber. Protein compositions of buckwheat noodles made with seaweed Relationships of the observed breaking characteristics (Fig. 2) to the protein components ( Fig. 3) was analyzed. The AG fraction content (Fig. 3) negatively correlated to their observed breaking stress (Fig. 2 (A)) with r = -0.934 (P<0.01), breaking energy ( Fig. 2 (B)) with r = -0.942 (P<0.01). These findings suggest that proteins in the AG fraction may be an important factor involved in the observed changes in mechanical characteristics arisen by the addition of funori seaweed. Finally, the present study shows changes in mechanical characteristics of buckwheat noodles made with seaweed. The present study suggests that changes in the protein of AG fraction in buckwheat noodles with seaweed may be an important factor affecting the mechanical characteristics of buckwheat noodles, although the exact mechanism remains uncertain. The present findings provide a scientific basis in the understanding of palatability and acceptability of buckwheat noodles.

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Changes of physicochemical properties and correlation analysis of common buckwheat starch during germination In order to clarify the physicochemical properties of starch during germination of common buckwheat, Xinong9976 was selected as the experimental material to study the main nutrients, particle structure, particle size distribution, transparency, aging value, pasting properties and the correlation between pasting properties and starch composition and main nutrients. The results showed that main nutrients were significantly different. The diameter of starch granules ranged from 2.36 to 8.89μm, and the shapes of starch granules were irregular with obvious holes and cracks on the surface. There were significant differences in starch transparency, aging value and pasting properties at different germination stages. Peak viscosity, through viscosity and final viscosity of germinated common buckwheat was significantly positive correlated with amylopectin content (P < 0.05) and breakdown, final viscosity and setback were significantly negatively correlated with amylose content (P < 0.05). The correlation analysis of starch pasting properties and main nutrients showed that breakdown, setback and crude fat content were significantly negatively correlated (P < 0.01), peak viscosity, through viscosity and final viscosity were significantly negatively correlated with crude fat content (P < 0.05), while the starch pasting properties had no significant correlation with other nutrients. Gao et al. (2019): Common buckwheat starch during germination 44 INTRODUCTION Common buckwheat (Fagopyrum esculentum) belongs to Polygonaceae, it is an important minor grain crop in China (Nam et al. 2018). Common buckwheat is rich in nutrients, containing 10.6% -15.5% of protein, 1.2% 2.8% of fat, 63% -71.2% of starch, as well as flavonoids, mineral elements and cysteine (Gimenez-Bastida and Zielinski 2015). Recently, with the improvement of people’s living standard, it has become fashionable to pursue a balanced diet with scientific based nutrition. Therefore, common buckwheat products with the reputation of “the same origin of food and medicine” will be widely welcomed by people. Besides, the development and research of common buckwheat healthy food have broad market prospects, high economic value and social value (Yoshimoto et al. 2004). There have been many studies on the cultivation methods, yield and protein content of common buckwheat in China and abroad (Salehi et al. 2018, Fang et al. 2018, Khan et al. 2012) and studies on the starch properties have also been reported. However, changes of physicochemical properties of germinated common buckwheat starch is not reported. Starch is the main nutritional component of common buckwheat. Its physicochemical properties affect the nutritional properties of related products and are also related to the development of new uses of common buckwheat starch (Stibilj et al. 2004). Germination is a dynamic process where plants come from resting state to the state with many physiological activities. Germination treatments can enhance the respiration of plants and significantly increase the species and number of enzymes (Mamilla and Mishra 2017). After germination, the starch content of mung bean decreased (Liu et al. 2014), while the amylase activity of kidney bean increased, leading to the changes in starch structure and composition (Yanli et al. 2018). Studies have showed that buckwheat has a high peak viscosity, hot paste stability and cold paste stability (Hara et al. 2007). Tartary buckwheat flavone content significantly increased after germination (Xiao-Peng et al. 2015). In addition, germination treatment can improve the edible and health value of buckwheat, enhance the resistance of starch paste and improve the stability of starch paste (Hara et al. 2007). Therefore, the study on the characteristics of germinated common buckwheat starch is of great significance to the development and utilization. In this experiment, the main nutrients, particle structure, physicochemical properties and the correlation between gelatinization characteristic value and main nutrients of germinated common buckwheat starch were studied by using cv. Xinong9976 as material to provide basis for the development and utilization of common buckwheat sprouts and bean flour and the deep processing of starch. MATERIALS AND METHODS 1. Experimental materials Cv. Xinong9976, a common buckwheat variety, provided by small grain laboratory of Northwest A&F University, was used for the experiment. Common buckwheat seeds with full grain and no disease were selected and were sterilized with 0.1% H2O2 for 10 min and soaked in the distilled water for 24 h. Then seeds were placed in a petri dish with two layers of filter paper, the cultivation of the sample under 25 ̊C in dark for germination, respectively taking sample after 2, 4, 6 d, removed shell, dried at 40 ̊C. The dried sample was grinded and passed through a 0.100 mm mesh. Added 80% ethanol, 50 ̊C, with the ultrasonic treatment (500 w) 30 min to remove flavonoids, then added the volume of distilled water, heated under the condition of 30 ̊C for 24 h. Centrifuged (4000 rpm, 10 min) three times, scraped off the grayish-brown soft layer. Finally, dried at 40 ̊C for 48 h and sieved with a 0.150 mm mesh. 2. Measurement of physicochemical properties The morphology of starch granules was observed by scanning electron microscope (JSM-6390, Jeol Ltd, Tokyo Japan) at 2000 x magnification, the particle size distribution was determined by a laser diffraction particle size analyzer and the pasting properties were measured using a rapid visco analyzer (RVA) (Newport Scientific, Pty Ltd, Warriewood, Australia). Starch transparency was determined using a modified version of (Zhou et al. 2014). 0.5g of the starch sample was blended with 50 mL distilled water and heated in a boiling water bath for 30 min. After the starch was completely gelatinized by stirring, the starch was removed and cooled to room temperature. Distilled water was used as a blank for zero adjustment and the transparency was measured at the wavelength of 620 nm with a visible-light spectrophotometer. The starch aging value was determined as follows: 0.5g of the starch sample was blended with 50 mL distilled waFagopyrum 36(2):43-50 (2019) 45 Germination time/d Water/% Ash/% Crude fat/% Crude protein/% Total starch/% Amylose/% Amylopectin/% 0 12.26 ± 0.09 a 1.12 ± 0.03 c 1.37 ± 0.01 a 9.22 ± 0.06 c 68.41 ± 0.11 a 24.11±0.28 a 44.30±0.39 a 2 11.21 ± 0.03 b 1.36 ± 0.02 b 1.04 ± 0.01 b 9.42 ± 0.10 c 58.77 ± 0.81 b 22.89±0.17 b 35.88±0.97 b 4 9.78 ± 0.01 c 1.44 ± 0.01 b 0.92 ± 0.01 c 10.83 ± 0.17 b 52.80 ± 0.09 c 21.85±0.25 c 30.95±0.32 c 6 7.73 ± 0.01 d 1.69 ± 0.03 a 0.84 ± 0.01 d 13.79 ± 0.20 a 47.66 ± 0.11 d 19.77±0.20 d 27.89±0.31 d ter and heated in a boiling water bath for 30 min and added distilled water to keep the total volume constant. Removed and cooled to room temperature, refrigerated for 24h, and defrosted at room temperature, and then centrifuged at 4000 rpm for 10 min, finally weighed the sediment quality. The starch aging value index was determined as follows: starch aging value = (weight of starch paste before centrifugation – weight of sediment quality) X 100. 3. Data analysis Three parallel tests were conducted in the experiment. SPSS 17.0 was used for statistical analysis, Origin 9.0 was used for drawing, and LSD minimum significant difference test was used for the determination of significance of differences. RESULTS AND DISCUSSION Changes of the concentration of main nutrients Main nutrients of common buckwheat were shown in table 1. The results showed that after germination, the contents of water, crude fat, total starch, amylose and amylopectin decreased significantly (P < 0.05), while the contents of ash and crude protein increased significantly (P < 0.05). Among them, the crude fat mass fraction decreased the most, from 1.37% to 0.84%, a decrease of 38.69% while the ash quality score increased the most, from 1.12% to 1.69%, increasing by 51%. In addition, the relative standard deviations of main nutrients in water, ash, crude fat, crude protein, total starch, amylose and amylopectin at different stages were 19.14%, 16.76%, 22.38%, 19.50%, 15.66%, 8.30% and 20.61%, respectively, indicating that the nutritional composition of the main nutrients in different stages of the germinated common buckwheat was significantly different. After germination, the crude protein mass fraction increased exponentially, which may be caused by the decreased protease activity in the seeds of common buckwheat during the germination process, which effectively weakened the hydrolysis of related proteins and thus promoted the protein accumulation (Ikeda et al. 1984). The decrease of total starch mass fraction may be due to the activation of α-amylase and β-amylase in the sprouting of common buckwheat, which could promote the degradation of starch and provide part of sugars needed for the germination (Mohan et al. 2010). The decrease of fat mass fraction might be due to the action of lipase, which could decompose part of the fat into the energy required for the germination and growth of common buckwheat seeds. Starch grain structure As can be seen on Fig. 1a, the starch particles of mature common buckwheat seeds were complete with clear gaps, mostly spherical and oval in shape, with smooth surface and no holes or cracks. After the germination of 2 d, most starch granules were irregular in shape, while a few were spherical in shape. In addition, some of the crystalline structures of starch were destroyed, and a few starch granules showed cracks on the surface (Fig. 1b). In 4 d, the starch granules were disordered. A small number of starch granules were spherical in shape, while some starch granules were deformed and condensed together with the surrounding granules (Fig. 1c). And in 6 d, the starch granules were polygonal in shape with few of them being spherical. The crystalline structures of most starch granules were destroyed, and obvious cracks and holes appeared on the surface of most granules (Fig. 1d). The table 2 showed that in the process of germination, starch granule size distribution was more dispersed, which indicated that the size of starch granules had obTable 1 Changes of main nutrients of common buckwheat before and after germination Note: different letters in the same column mean significant difference of P<0.05, the same as below. Gao et al. (2019): Common buckwheat starch during germination 46 Transparency can reflect the mutual solubility of starch and water, and the transparency of buckwheat starch is positively proportional to the absorbance value, the higher the absorbance value is, the higher the transparency of the starch is (Li et al. 1997). As can be seen from Fig. 2, the transparency of common buckwheat starch first increased and then decreased with the extension of time in the germination process. And the transparency in different stages was significantly different (P < 0.05), the most transparent stage was 2 d and the absorbance value was 1.75, indicating that starch particles were completely expanded at this time, and there was no mutual association among the starch molecules after gelatinization. While the lowest transparency period was 6 d and the absorbance value was 1.00, which decreased by 36.30% compared with that of mature seeds. The average absorbance of starch in different germination stages was 1.41. After germination of 2d, starch transparency decreased significantly (P < 0.05), which may be due to the starch retrogradation, rearrangement of starch molecules and Note: D10, D50 and D90 represented the critical particle size values when the minimum particle size was added up to 10%, 50% and 90% of the sample. Germination time/d D10 D50 D90 Average sphericity Average aspect ratio 0 4.97 7.86 8.89 0.84 1.45 2 4.68 6.64 7.98 0.76 1.43 4 3.29 5.17 6.97 0.68 1.40 6 2.36 4.05 5.84 0.57 1.37 Table 2 Starch particle size distribution during the germination of common buckwheat d. Starch granules in 6d (×2000). Fig.1. Scanning electron microscopy of starch granules of common buckwheat at different germination stages a. Starch granules in 0d (×2000); b. Starch granules in 2d (×2000); c. Starch granules in 4d (×2000); vious differences. Chang found that the corn starch size ranged from 5.76 to 8.64 μm (Chang et al. 2004). Common buckwheat starch particles between 4.00 to 9.00 microns in diameter, which was smaller than the corn starch granules. After germination, the diameter, average sphericity and average aspect ratio of starch granules decreased significantly with the increase of germination time. In 6 d, the average diameter of starch granules decreased from 7.24 μm to 4.08 μm, a decrease of 43.65%. The average sphericity and the average aspect ratio decreased by 32.14% and 5.52%, respectively. Starch transparency Transparency is one of the important external characteristics of starch, which is directly related to the appearance and use of starch products (Wang et al. 2017). Fig.2. Changes of the transparency of common buckwheat starch at different germination stages Fagopyrum 36(2):43-50 (2019) 47 scattering of light, thus reducing light transmission and starch transparency (Zhou et al. 2017). Starch aging value The essence of starch aging is that gelatinized starch molecules re-form hydrogen bonds during the cooling process(Jiamjariyatam et al. 2014). The aging process of starch can be regarded as the reverse process of gelatinization, but the degree of starch crystallization decreases after aging (Verma et al. 2018). It could be seen from Fig. 3 that there were significant differences in the aging value of common buckwheat starch in different germination stages (P < 0.05). The maximum aging value was 71.20% in mature grains. Subsequently, the aging value decreased gradually with the extension of germination time. Both 4d and 6d, the aging value decreased by 28.84%, 39.35%, respectively, which might be related to the weakened ability of buckwheat starch molecules to form hydrogen bonds again after germination (Liu et al. 2006). Starch aging not only makes food taste worse, but also reduces the digestibility (Verma et al. 2018). However, the aging value of common buckwheat gradually decreased during germination, indicating that common buckwheat sprouts were good in taste, easy to digest and had broad market development value. Pasting viscosity Starch granules rapidly absorb water in aqueous solution due to thermal expansion, resulting in the fracture of intramolecular and intermolecular hydrogen bonds, and the process of gradual diffusion of starch granules is called starch paste (Jane et al. 1992). The pasting temperature was different in different germination stages due to the size of starch granules. The starch pasting properties of common buckwheat in the process of germination were shown in table 3, the results showed that starch pasting viscosities gradually decreased and significantly different (P < 0.05) in different period. After germination, peak viscosity, through viscosity, breakdown, final viscosity, setback, peak time and pasting temperature were lower than those of the mature grain starch. Peak viscosity refers to the increase in the viscosity of starch paste caused by the friction between starch particles after full water absorption and expansion, which can reflect the expansion capacity of starch (Xiao-Li et al. 2008). As shown in table 3, the peak viscosity was 1394 2982 cp, the average peak viscosity was 2242 cp, and the difference was great. Through viscosity is caused by the sharp decrease in the viscosity of starch paste due to the fact that starch particles no longer friction with each other after they have expanded to the limit (Xiao-Li et al. 2008). The through viscosity was between 1335 and 2819 cp, and the average was 2131 cp. The breakdown is the difference between peak viscosity and through viscosity, which can reflect the thermal stability of starch paste. The smaller the breakdown is, the better the thermal stability is (Karim et al. 2000). The average breakdown was 111 cp, and the breakdown in mature grains was 2.76 times higher than that after germination, indicating that the starch Fig.3. Changes of the aging value of common buckwheat starch at different germination stages Germination time/d peak viscosity /cp Through viscosity /cp breakdown/cp final viscosity /cp setback/cp peak time /min pasting temperature /°C INTRODUCTION Common buckwheat (Fagopyrum esculentum) belongs to Polygonaceae, it is an important minor grain crop in China (Nam et al. 2018). Common buckwheat is rich in nutrients, containing 10.6% -15.5% of protein, 1.2% -2.8% of fat, 63% -71.2% of starch, as well as flavonoids, mineral elements and cysteine (Gimenez-Bastida and Zielinski 2015). Recently, with the improvement of people's living standard, it has become fashionable to pursue a balanced diet with scientific based nutrition. Therefore, common buckwheat products with the reputation of "the same origin of food and medicine" will be widely welcomed by people. Besides, the development and research of common buckwheat healthy food have broad market prospects, high economic value and social value (Yoshimoto et al. 2004). There have been many studies on the cultivation methods, yield and protein content of common buckwheat in China and abroad (Salehi et al. 2018, Fang et al. 2018, Khan et al. 2012) and studies on the starch properties have also been reported. However, changes of physicochemical properties of germinated common buckwheat starch is not reported. Starch is the main nutritional component of common buckwheat. Its physicochemical properties affect the nutritional properties of related products and are also related to the development of new uses of common buckwheat starch (Stibilj et al. 2004). Germination is a dynamic process where plants come from resting state to the state with many physiological activities. Germination treatments can enhance the respiration of plants and significantly increase the species and number of enzymes (Mamilla and Mishra 2017). After germination, the starch content of mung bean decreased (Liu et al. 2014), while the amylase activity of kidney bean increased, leading to the changes in starch structure and composition (Yanli et al. 2018). Studies have showed that buckwheat has a high peak viscosity, hot paste stability and cold paste stability (Hara et al. 2007). Tartary buckwheat flavone content significantly increased after germination (Xiao-Peng et al. 2015). In addition, germination treatment can improve the edible and health value of buckwheat, enhance the resistance of starch paste and improve the stability of starch paste (Hara et al. 2007). Therefore, the study on the characteristics of germinated common buckwheat starch is of great significance to the development and utilization. In this experiment, the main nutrients, particle structure, physicochemical properties and the correlation between gelatinization characteristic value and main nutrients of germinated common buckwheat starch were studied by using cv. Xinong9976 as material to provide basis for the development and utilization of common buckwheat sprouts and bean flour and the deep processing of starch. Experimental materials Cv. Xinong9976, a common buckwheat variety, provided by small grain laboratory of Northwest A&F University, was used for the experiment. Common buckwheat seeds with full grain and no disease were selected and were sterilized with 0.1% H 2 O 2 for 10 min and soaked in the distilled water for 24 h. Then seeds were placed in a petri dish with two layers of filter paper, the cultivation of the sample under 25˚C in dark for germination, respectively taking sample after 2, 4, 6 d, removed shell, dried at 40˚C. The dried sample was grinded and passed through a 0.100 mm mesh. Added 80% ethanol, 50˚C, with the ultrasonic treatment (500 w) 30 min to remove flavonoids, then added the volume of distilled water, heated under the condition of 30˚C for 24 h. Centrifuged (4000 rpm, 10 min) three times, scraped off the grayish-brown soft layer. Finally, dried at 40˚C for 48 h and sieved with a 0.150 mm mesh. Measurement of physicochemical properties The morphology of starch granules was observed by scanning electron microscope (JSM-6390, Jeol Ltd, Tokyo Japan) at 2000 x magnification, the particle size distribution was determined by a laser diffraction particle size analyzer and the pasting properties were measured using a rapid visco analyzer (RVA) (Newport Scientific, Pty Ltd, Warriewood, Australia). Starch transparency was determined using a modified version of (Zhou et al. 2014). 0.5g of the starch sample was blended with 50 mL distilled water and heated in a boiling water bath for 30 min. After the starch was completely gelatinized by stirring, the starch was removed and cooled to room temperature. Distilled water was used as a blank for zero adjustment and the transparency was measured at the wavelength of 620 nm with a visible-light spectrophotometer. The starch aging value was determined as follows: 0.5g of the starch sample was blended with 50 mL distilled wa- ter and heated in a boiling water bath for 30 min and added distilled water to keep the total volume constant. Removed and cooled to room temperature, refrigerated for 24h, and defrosted at room temperature, and then centrifuged at 4000 rpm for 10 min, finally weighed the sediment quality. The starch aging value index was determined as follows: starch aging value = (weight of starch paste before centrifugation -weight of sediment quality) X 100. Data analysis Three parallel tests were conducted in the experiment. SPSS 17.0 was used for statistical analysis, Origin 9.0 was used for drawing, and LSD minimum significant difference test was used for the determination of significance of differences. Changes of the concentration of main nutrients Main nutrients of common buckwheat were shown in table 1. The results showed that after germination, the contents of water, crude fat, total starch, amylose and amylopectin decreased significantly (P < 0.05), while the contents of ash and crude protein increased significantly (P < 0.05). Among them, the crude fat mass fraction decreased the most, from 1.37% to 0.84%, a decrease of 38.69% while the ash quality score increased the most, from 1.12% to 1.69%, increasing by 51%. In addition, the relative standard deviations of main nutrients in water, ash, crude fat, crude protein, total starch, amylose and amylopectin at different stages were 19.14%, 16.76%, 22.38%, 19.50%, 15.66%, 8.30% and 20.61%, respectively, indicating that the nutritional composition of the main nutrients in different stages of the germinated common buckwheat was significantly different. After germination, the crude protein mass fraction increased exponentially, which may be caused by the decreased protease activity in the seeds of common buckwheat during the germination process, which effectively weakened the hydrolysis of related proteins and thus promoted the protein accumulation (Ikeda et al. 1984). The decrease of total starch mass fraction may be due to the activation of α-amylase and β-amylase in the sprouting of common buckwheat, which could promote the degradation of starch and provide part of sugars needed for the germination (Mohan et al. 2010). The decrease of fat mass fraction might be due to the action of lipase, which could decompose part of the fat into the energy required for the germination and growth of common buckwheat seeds. Starch grain structure As can be seen on Fig. 1a, the starch particles of mature common buckwheat seeds were complete with clear gaps, mostly spherical and oval in shape, with smooth surface and no holes or cracks. After the germination of 2 d, most starch granules were irregular in shape, while a few were spherical in shape. In addition, some of the crystalline structures of starch were destroyed, and a few starch granules showed cracks on the surface (Fig. 1b). In 4 d, the starch granules were disordered. A small number of starch granules were spherical in shape, while some starch granules were deformed and condensed together with the surrounding granules (Fig. 1c). And in 6 d, the starch granules were polygonal in shape with few of them being spherical. The crystalline structures of most starch granules were destroyed, and obvious cracks and holes appeared on the surface of most granules (Fig. 1d). The table 2 showed that in the process of germination, starch granule size distribution was more dispersed, which indicated that the size of starch granules had ob- Transparency can reflect the mutual solubility of starch and water, and the transparency of buckwheat starch is positively proportional to the absorbance value, the higher the absorbance value is, the higher the transparency of the starch is (Li et al. 1997). As can be seen from Fig. 2, the transparency of common buckwheat starch first increased and then decreased with the extension of time in the germination process. And the transparency in different stages was significantly different (P < 0.05), the most transparent stage was 2 d and the absorbance value was 1.75, indicating that starch particles were completely expanded at this time, and there was no mutual association among the starch molecules after gelatinization. While the lowest transparency period was 6 d and the absorbance value was 1.00, which decreased by 36.30% compared with that of mature seeds. The average absorbance of starch in different germination stages was 1.41. After germination of 2d, starch transparency decreased significantly (P < 0.05), which may be due to the starch retrogradation, rearrangement of starch molecules and Note: D 10 , D 50 and D 90 represented the critical particle size values when the minimum particle size was added up to 10%, 50% and 90% of the sample. vious differences. Chang found that the corn starch size ranged from 5.76 to 8.64 μm (Chang et al. 2004). Common buckwheat starch particles between 4.00 to 9.00 microns in diameter, which was smaller than the corn starch granules. After germination, the diameter, average sphericity and average aspect ratio of starch granules decreased significantly with the increase of germination time. In 6 d, the average diameter of starch granules decreased from 7.24 μm to 4.08 μm, a decrease of 43.65%. The average sphericity and the average aspect ratio decreased by 32.14% and 5.52%, respectively. Starch transparency Transparency is one of the important external characteristics of starch, which is directly related to the appearance and use of starch products ). Fig.2. Changes of the transparency of common buckwheat starch at different germination stages scattering of light, thus reducing light transmission and starch transparency ). Starch aging value The essence of starch aging is that gelatinized starch molecules re-form hydrogen bonds during the cooling process (Jiamjariyatam et al. 2014). The aging process of starch can be regarded as the reverse process of gelatinization, but the degree of starch crystallization decreases after aging (Verma et al. 2018). It could be seen from Fig. 3 that there were significant differences in the aging value of common buckwheat starch in different germination stages (P < 0.05). The maximum aging value was 71.20% in mature grains. Subsequently, the aging value decreased gradually with the extension of germination time. Both 4d and 6d, the aging value decreased by 28.84%, 39.35%, respectively, which might be related to the weakened ability of buckwheat starch molecules to form hydrogen bonds again after germination (Liu et al. 2006). Starch aging not only makes food taste worse, but also reduces the digestibility (Verma et al. 2018). However, the aging value of common buckwheat gradually decreased during germination, indicating that common buckwheat sprouts were good in taste, easy to digest and had broad market development value. Pasting viscosity Starch granules rapidly absorb water in aqueous solution due to thermal expansion, resulting in the fracture of intramolecular and intermolecular hydrogen bonds, and the process of gradual diffusion of starch granules is called starch paste (Jane et al. 1992). The pasting temperature was different in different germination stages due to the size of starch granules. The starch pasting properties of common buckwheat in the process of germination were shown in table 3, the results showed that starch pasting viscosities gradually decreased and significantly different (P < 0.05) in different period. After germina-tion, peak viscosity, through viscosity, breakdown, final viscosity, setback, peak time and pasting temperature were lower than those of the mature grain starch. Peak viscosity refers to the increase in the viscosity of starch paste caused by the friction between starch particles after full water absorption and expansion, which can reflect the expansion capacity of starch (Xiao- Li et al. 2008). As shown in table 3, the peak viscosity was 1394 -2982 cp, the average peak viscosity was 2242 cp, and the difference was great. Through viscosity is caused by the sharp decrease in the viscosity of starch paste due to the fact that starch particles no longer friction with each other after they have expanded to the limit (Xiao- Li et al. 2008). The through viscosity was between 1335 and 2819 cp, and the average was 2131 cp. The breakdown is the difference between peak viscosity and through viscosity, which can reflect the thermal stability of starch paste. The smaller the breakdown is, the better the thermal stability is (Karim et al. 2000). The average breakdown was 111 cp, and the breakdown in mature grains was 2.76 times higher than that after germination, indicating that the starch Table 3 Characteristic values of gelatinization of common buckwheat starch during germination had good thermal stability after germination and was suitable for the development of noodles and thickening agents. The final viscosity can reflect the retrogradation property of starch (Xiao-Li et al. 2008). After germination, the final viscosity decreased significantly, reaching 58.28% in 6d. The setback is the difference between final viscosity and through viscosity, which can reflect the stability of cold paste of starch. It can be seen from table 3 that the starch was not easy to age after germination and was suitable for making food such as common buckwheat instant noodles. Correlation analysis of pasting properties and starch composition Starch is the main component of common buckwheat grain, accounting for 60 -75% of the grain. The contents, composition and properties of buckwheat starch directly affect the processing technology of buckwheat food (Xin-Hua et al. 2009). The table 4 showed that peak viscosity, through viscosity and final viscosity of germinated common buckwheat was significantly positive correlated with amylopectin content (P < 0.05), which was the same as the relationship between the pasting viscosity and starch composition of the rice starch or germinated brown rice starch reported in previous studies, that was, the higher the content of amylopectin was, the higher the peak viscosity, through viscosity and final viscosity were. Break-down, final viscosity and setback were significantly negatively correlated with amylose content (P < 0.05). Studies have shown that the short-term retrogradation of starch is mainly caused by the gelation order and dehydration crystallization of amylose molecules. However, setback was significantly negatively correlated with amylose content (P < 0.05), indicating that germinated common buckwheat with low amylose content was easy to retrogradate. Correlation analysis of pasting properties and other nutrients The correlation analysis of starch pasting properties and other nutrients was shown in table 5. In 6d, pasting properties was positively correlated and negatively correlated with the main nutrients. Breakdown and setback were significantly negatively correlated with crude fat content (P < 0.01), indicating that the thermal stability and cold paste stability gradually increased after germination with the decrease of crude fat content. Peak viscosity, through viscosity and final viscosity were negatively correlated with crude fat content (P < 0.05). There was no significant relationship between pasting viscosity and water content, which may be due to the fast growth rate and the need to consume more water for growth, resulting in low water content. Similarly, pasting viscosity had no significant relationship with ash content and crude protein content. Table 4 Correlation coefficient between pasting properties and starch composition (r/p) Note: Linear correlation coefficient between r. **: Significant correlation at the level of 0.01, *: Significant correlation at the level of 0.05, the same below. CONCLUSIONS The results showed that in comparison to non-germinated grain, after germination, the concentration of main nutrients of common buckwheat were significantly different, where the content of crude fat, total starch, amylose and amylopectin decreased significantly while the content of ash and crude protein increased significantly. Starch granules were arranged in a disorderly manner, most of which were irregular in shape, few of which were spherical in shape. Moreover, the crystal structure of most starch granules was destroyed, and obvious cracks and voids appeared on the surface. In addition, starch size of mature common buckwheat was 4-9 μm. Pasting properties were closely related to the starch composition, and peak viscosity, through viscosity, final viscosity and setback were sig-nificantly positively correlated with amylopectin content, while breakdown, final viscosity and setback were significantly negatively correlated with amylose content. In addition, breakdown, setback and fat content were significantly negatively correlated, and peak viscosity, through viscosity and final viscosity were significantly negatively correlated with fat content, while pasting properties were not significantly correlated with other nutrients.

v3-fos

2019-04-27T13:09:06.200Z

1970-01-01T00:00:00.000Z

135167625

s2

CASSAVA FORTIFICATION AND QUALITY EVALUATION The broad objective of this work is to improve the nutrient content of cassava flour by inclusion of cowpeas seed flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. Cassava tuber flour was fortified with cowpeas flour and from cassava leaves at 20% and 30% of dry weight. Standard methods were used for the determination of parameter such as protein and carbohydrates. All samples were analysed for potassium, phosphorus, calcium, magnesium, iron, and cyanide. Unfortified cassava had significantly lower (P<0.05) values (protein: 0.942%, P: 0.093%, K: 0.749 Mg: 0.052%, Fe: 5.008 ppm) than fortification with both cowpeas flour and cassava leaf flour. Fortification with cowpeas flour did not significantly (P>0.05) change the Ca content however they were significant (P<0.05) increases cassava leaf flour. Cyanide content increased significantly for Treatment LF 20 and LF 30 but remained unchanged for Treatment CW 20 and CW 30 . Both cowpeas and cassava leaves had significantly (P<0.05) lower carbohydrate content than cassava tuber flour. Both cowpeas and cassava leaves are excellent for fortification but cassava leaves have to be used with additional pre-treatments to reduce the cyanide content in them. Organoleptic qualities analysed indicate high acceptability of fortification of cassava tuber flour with cowpeas flour. cowpeas leaves. The broad objective of this to improve the nutrient content of cassava flour by inclusion of cowpeas flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. It is envisaged that this will enhance the protein content of cassava, decrease the incidence of protein malnutrition among the less privileged cassava in sub-tropical Africa and other countries. It is significant that the can be easily adopted domestically and at cottage level. of cowpeas and cassava leaves as cassava fortificant will improve its production and accessibility and also diversify the use of INTRODUCTION Cassava (Manihot esculenta Crantz) is one among the important cultivated woody shrub in the family Euphorbiaceae and is used for food and feed purpose. Throughout the world, in tropical and subtropical areas, this crop is cultivated as an annual crop. Cassava roots are rich in carbohydrates [1]. Dependence on cassava diets therefore may lead to serious protein deficiency problems. Such malnutrition problem has been reported among consumers that rely primarily on cassava flour and other cassava products as major food source with little or no high protein food sources as complements. Cassava usually not eaten alone most times as a full meal but is rather taken with vegetable stew/soup/sauce that can provide other nutrients like protein. The animal origin diet like meat, fish and egg are expensive items for people in low-income families in African region. The current exorbitant cost of animal protein especially for low income earners deters the inclusion of such animal protein source in the stew that cassava is eaten with. Improving the protein content of cassava may be an alternative and affordable option. The enriching of the cassava meal with high nutritional way can solve the problems of mal nutrition [2]. This makes the need to improve the protein quality of cassava imperative [3,4] and to search for cheaper but good quality protein sources that are readily available [5]. In SADC region, the research on cassava crop seems scanty when compared to rice, maize and wheat. Fortification of cassava flour with plant protein is a viable affordable alternative to tackle specifically the problem of protein energy malnutrition in those areas affected by malnutrition. The plant protein can be sourced from unexploited indigenous legumes with high protein content (18.1 to 25.8 %) like cowpeas and cassava leaves, soybeans, and Bambara nuts among others. Cowpeas and cassava leaves unlike other mentioned plant protein sources have not found used in many food formulations as soybean [6]. This study addresses the problem of protein deficiency in cassava, a major staple food, using food-to-food fortification approach with the use of cowpeas and cassava leaves. The broad objective of this work is to improve the nutrient content of cassava flour by inclusion of cowpeas seed flour and cassava leaf powder to assess the effects of the cowpeas flour and cassava leaf powder inclusion on the nutrient quality and acceptability of the flour. It is envisaged that this will enhance the protein content of cassava, decrease the incidence of protein malnutrition among the less privileged cassava consumers in sub-tropical Africa and other developing countries. It is significant that the technology involved can be easily adopted domestically and at cottage level. Utilization of cowpeas and cassava leaves as cassava fortificant will improve its production and accessibility and also diversify the use of cassava. Experimental details Cassava root (Manihot esculenta Crantz) (Variety M7) was procured from Chiredzi research station, Chiredzi, Zimbabwe. Cowpea seed (variety CBC 2) was procured from Chiredzi research station, Chiredzi Zimbabwe and cassava leaves will be procured from Africa University Research Block, Mutare Zimbabwe. Preparation of Cowpeas into flour (CW100): The cowpea seed is winnowed to remove any trash and milled using a hammer mill. The first 5 kg of the milled flour is discarded to make sure there is no contamination of the flour. The flour is put in a polythene bag until used for the study. Preparation of cassava flour (Control): Harvested cassava roots are washed to remove all the dirty, the washed roots are peeled care should be taken that all peeled roots are kept under water to avoid discolouration. After peeling the roots are cut into small chips and fermented in water for 60 h. After 60 h the fermented cassava is drained to remove the water and sun dried. When completely dry the cassava is milled using a hammer mill. First 10 kg of the milled cassava is discarded to avoid contamination. The flour is put in polythene bags until used for the study. Preparation of cassava leaves flour (LF100): Cassava shoots approximately 20 cm in length are harvested, the hard petioles are removed and the leaves are sun dried. When completely dry the leaves are milled using an electric miller. The flour is put in a polythene bag until used for the study. Preparation of the fortified cassava flour 20% cowpea concentration (CW20): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 300g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 30% cowpea concentration (CW30): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 450g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 20% cassava leaf concentration (LF20): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 300g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. 30% cassava leaf concentration (LF30): Cassava flour was randomly sampled by scoping the flour to make up a sample of 1500g. Cowpeas flour was also randomly sampled to make up 450g. The cassava flour and cowpea flour were thoroughly mixed together and the mixed flour was divided to make three equal samples. Chemical analysis Protein and Carbohydrate were determined according to AOAC [7]. The micronutrients including magnesium, potassium, cyanide, calcium and iron were evaluated using an Atomic Absorption Spectrophotometer (Buck 210VGP) Germany according to AOAC [7]. Sensory analysis The organoleptic evaluation of the biscuits samples was carried out for consumer acceptance and preference. Samples of the biscuits prepared from the cassava tuber flour and the different cowpeas and cassava leaf composite flour. Consumers evaluated the treatments on overall appreciation, taste, color and odour of the flour. Scores were given against the choice and preferences of the respondents. Statistical analysis All data collected to be statistically analyzed using the GenSTAT Analysis of Variance (ANOVA) software. Differences between means were determined using the Least Significant Difference (LSD) test at 0.05 level. Nitrogen (N) Data regarding the fortification of cassava tuber flour (CTF) with cowpeas flour (CWF) and cassava leaf flour (CLF) showed significant (P<0.05) differences for the nitrogen content. Results show that CTF (Control) has the least amount of nitrogen (0.151%) in comparison to CLF (Treatment LF100) and when fortified with either CLF or CWF. Results show that CLF (Treatment LF100) have has higher nitrogen (4.780%) content than CWF (Treatment CW100) (3.445%). After fortifying the CTF, Treatment LF30 produced the highest amount of nitrogen (1.220%) while treatment CW20 had the least (0.727%). Treatment CW30 and Treatment LF20 produced result which were not significantly (P>0.05) different from each other (table 1). Treatment LF30 produced the highest P content (0.161%) and it was not significantly different from Treatment CW20 and CW30 which produced 0.136% and 0.143% respectively. Potassium (K) The Control treatment had the least (0.749%)) potassium (K) content with respect to CW100 and LF100 which had 1.304% and 1.873% respectively (table 1). Results from this investigation show that treatments from fortification of CTF with CLF produced significantly (P<0.05) higher K content than treatments from fortification with CWF. Treatment LF30 produced the highest K content (0.990%) followed by Treatment LF20 (0.943%) while Treatment CW20 and CW30 had 0.847 and 0.867% respectively. Magnesium (Mg) The Mg content from the Control treatment, Treatment CW100 and Treatment LF100 was significantly (P<0.05) differently from each other with treatment LF100 having the highest Mg content (0.302%). Similarly, fortification of CTF with CLF produced significantly more Mg content as observed in Treatment LF20 (0.090%) and LF30 (0.107%) compared to Treatment CW20 and CW30 with Mg content; 0.067% and 0.070% respectively (table 1). Iron (Fe) Results pertaining to Fe content show that the Control treatment has significantly the lowest (5.008 ppm) Fe content in comparison to CW100 and LF100 with 60.034 ppm and 80.517 ppm respectively ( fig. 1). Fortification of CTF with CWF from level treatment CW20 to level treatment CW30 did not significantly (P>0.05) increase the Fe content. However, Fe content increased significantly (P<0.05) from level treatment LF20 to level Treatment LF30. Calcium (Ca) Results from the investigation reveal that the calcium (Ca) content in the Control treatment and CW100 was not significantly (P>0.05) different from each other ( fig. 2). Similarly, fortification of CTF with CWF was not significant (P>0.05) at all levels. However, Ca in CLF was 8 times more than that in CTF such that the Treatments LF20 and LF30 produced Ca content which was significantly (P<0.05) more than Treatment CW20 and CW30. The more the proportion of CLF used in the fortification of the CTF the more the Ca content that was obtained and this is true for Treatment LF20 and LF30. Cyanide (CN) Data pertaining to the CN content in the Control treatment, CW100, LF100 and fortified CTF at different levels is shown in fig. 3. The CN content in the Control treatment and CW100 was not significantly (P>0.05) differently from each other. Similarly, the fortified CTF with both levels of CWF (CW20 and CW30) did not significantly influence any change to the CN content. However, fortification of CTF with CLF increased the CN content significantly (P<0.05) as observed from Treatment LF20 and LF30 ( fig. 3). Fig. 3: Shows the influence of different fortifying agents on CN content Carbohydrate Results from the investigation reveal that LF100 has significantly (P<0.05) lower carbohydrates compared to CW100 (36.044%) and CTF (73.225%). Similarly, fortification of CTF with CWF and CLF at different levels significantly (P<0.05) reduced the carbohydrate content ( fig. 4). Overall appreciation Results for the overall appreciation of the unfortified cassava and fortified cassava with either cowpeas or cassava leaves are shown in table 2. The fig. show that 36 out of the 50 respondents scored very good to the overall appreciation of the Treatment CW20 and 18 respondents also scored very good to Treatment CW30. Treatment CW30, LF20 and LF30 had each 2 respondents who scored excellent on the overall appreciation of the levels of cassava fortification. Treatment LF20 and LF30 had 4 respondents each who scored poor on the overall appreciation. Fig. 4: Shows the influence of different fortifying agents on carbohydrate content Overall taste Data pertaining to the overall taste preferences recorded from the survey is shown in table 3. Out of the 50 respondents 6respondnents scored excellent for the overall taste of cassava. Twenty-eight respondents scored very good for the overall taste of the Control treatment while only 2 respondents did not like the taste of cassava. After fortification of cassava, Treatment CW20 and Treatment CW30 recorded 12 and 4 respondents respectively who scored excellent for the overall taste. The preference for Treatment LF20 and LF30 was somewhat lower as there were more respondents who scored poor and fair compared to the fortification of cassava with cowpeas. Appearance of the flour Data regarding the opinions of the respondents towards the appearance of the treatments is shown in table 4. For the Control treatment, CW20 and CW30 all the respondents scored good, very good and excellent. However, for Treatment LF20 and LF30 the preferences of the respondents were showing that the fortification of cassava with different levels of cassava leaves was unpopular as the color of the flour became more colored than being white. There were fewer respondents who score very good and excellent than those who scored fair to poor preferences of appearance. Fortification with cowpeas were the most preferred treatments. Odour of the flour Data from the respondents regarding their preferences towards the odour of the flours under study is shown in table 5. The Control treatment, Treatment CW20 and Treatment CW30 reveal that they have a got moderate to no odour which is most preferred by the respondents. However, fortification of cassava tuber flour with flour from cassava leaves, as in Treatment LF20 and LF30 was relatively unpopular since the treatments somewhat produced a more moderate to strong odour. DISCUSSION Traditionally, the roots of cassava are harvested and processed by many methods and produce different food products for diverse purposes. Cassava which is an important staple in the Sub-Tropics is low in protein and deficient in essential amino acids. However, the protein content of all composite flours with different levels of both CWF and CLF increased as a result of the significantly more nitrogen that is in cowpeas and cassava leaves than cassava tubers. Therefore, the more the proportion of cowpea or cassava leaf flour is added the more the protein content is obtained in the composite flour. It has been reported that fortification of cassava with soybean or cowpea extract increased the protein content of cassava [6,8]. In a similar study using soya bean by Collins and Falasinnu, they observed that legumes are generally high in their protein content and proposed that they make an ideal source for protein supplementation [9]. This observation agrees with previous findings of several researchers [10][11][12]. The results of the P content of CTF, CWF and CLF are shown in table 1. Significantly (P<0.05) unfortified cassava tuber flour has got less phosphorus than CWF and CLF. The increase in the P content of the CTF was as a result of adding either CWF or CLF. These results agree with the work by Anuonye et al. [13] on fortifying cassava with yams. There was also a corresponding increase in most of the other elements with increase in either CWF or CLF. The K content increased significantly (P<0.05) with fortification of the cassava tuber flour, similarly Mg and Fe content from CWF and CLF is significantly (P<0.05) superior to CTF. The Ca content of cassava tuber flour and cowpea flour does not differ significantly (P>0.05) from each. As a result, fortification of CTF will not benefit in increased Ca content of the product. Similar results were observed in the work of Anuonye et al. [14]. However, the Ca content in cassava leaves is significantly more than that in the tubers [15]. Therefore, as expected, to improve the Ca content in CTF fortification with the flour from its leaves will benefit as expressively observed for Treatment LF20 and LF30. This is attributed to the replacement of the cassava tuber flour with calcium-rich flour from cassava leaves. Fig. 1 shows that cassava tubers have significantly much less Fe content than cowpeas and cassava leaves. The increase in the Fe content of the fortified CTF is as a result of adding the flour of cowpeas and/or cassava leaves. Treatment LF20 produced the same effect with Treatment CW30 revealing that cassava leaves are very rich in Fe content. Results obtained in this study show that CTF has significantly (P<0.05) more CHO content that the products from the fortification with cowpea flour or flour from cassava leaves. Correspondingly, there was a significant (P<0.05) decrease in the CHO content of the CTF with addition of flour from cowpeas and cassava leaves. This is attributed to the fact that there is poor CHO content in both cowpeas like any other legume and cassava leaves. As a result, there was no significant difference in the CHO content by increasing the level of either CWF or CLF. These results are similar to those obtained in the work of Obadina et al. [16] with soya beans. The total CN content of the simple cassava tuber-flour is very low and statistical not significantly different with the CN content in cowpeas flour ( fig. 3). Fortification of CTF with CWF at all levels did not result in any change of the CN content. However, fortification of CTF with CLF increased the CN content in the composite flour significantly (P<0.05). This is attributed to the very high CN content in CLF and the replacement of the low CN content flour with high CN content flour in the composite flour. The organoleptic Test investigated the overall consumers' appreciation as well as taste preferences, preferences in terms of color appearance and odour of the flour. Unlike those countries in central Africa were cassava is a staple and there are many dishes that have been developed from cassava, in Zimbabwe our staple is maize. An appreciable number of Zimbabweans do eat cassava and the numbers are increasing as more and more people are being to eat a wide range food. So, it was expected that the results from this investigation was likewise influenced by the fact that cassava is not a very popular dish in the region. For the overall appreciation however, the product from fortifying with cowpeas flour was rated better from good to excellent. Similarly, with regards to taste preferences and appearance fortified cassava tuber flour with cowpeas flour scored more from good to excellent as well. With regards to the odour of the composite flours, more respondents said that the composite flour with cowpeas had no odour to questionable odour. However, for the composite flour with cassava leaves more respondents said that it had a moderate to strong odour. These results are attributed to the fact that cowpeas are already an acceptable food unlike cassava leaves. CONCLUSION The results of this study have shown that substitution of both cowpeas flour and cassava leaves in cassava tuber flour is possible. Both cowpeas flour-fortified cassava tuber flour and cassava leaf flour-fortified cassava tuber flour could be used to fight macronutrient and micronutrient deficiencies. The mineral composition of the cassava tuber flour was enhanced as a result of the flour substitution. The composite flour could help in reducing protein energy and micronutrient deficiency prevalent in developing countries such as Zimbabwe. Cassava tuber flour can be fortified by adding flour from cassava leaves however, these results shows that the fortification with cassava leaf flour leads to increase in cyanide content and organoleptic challenges. It is concluded that protein, phosphorus, potassium, magnesium and iron content in cassava tuber flour could be enriched with up to 30% cowpeas flour without organoleptic challenges.

v3-fos

2020-12-10T09:01:31.857Z

1970-06-01T00:00:00.000Z

237230264

s2

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min. Although the degree of freshness or spoilage of meats is often evaluated by plate counts, it is known that spoilage is not the result of bacterial numbers per se but is caused by biochemical changes brought about by the growing flora. Investigations on the mechanism of spoilage of fresh refrigerated meats over the past several years have led to the proposal of a number of techniques for assessing its presence and extent. Among these are techniques based upon the phenomena of extract-release volume (ERV), water-holding capacity (WHC), meat swelling, and viscosity (1,2,5,6). All four of these are based primarily on changes in hydration capacity of meat proteins, which is lowest for fresh meat but gradually increases as spoilage occurs. Although the pH of fresh beef is around 5.6 to 5.8 and gradually increases to as much as 8.5 when beef becomes putrid, the increase from freshness to incipient spoilage generally does not exceed 0.3 to 0.5 of a pH unit. This, along with the fact that the change is usually not uniformly distributed in a meat sample, makes direct pH measurements unsuitable for the purpose of detecting incipient spoilage. Also, beef is often judged as spoiled without any noticeable pH changes The present report describes a more direct 1 technique for detecting incipient beef spoilage by accurate titration of the basic (alkaline) substances or functions produced in beef by the spoilage flora. The technique employs the measurement of the quantity of dilute acid required to bring beef homogenates to pH 5.00. The volumes of acid have been correlated with log bacterial numbers, ERV, and pH on beef from different sources and in different stages of spoilage. MATERIAL3 AND METHODS Titrations were carried out by blending 10-g samples of beef in 100 ml of deionized water for 2 min and filtering through cheesecloth to eliminate connective tissue. Duplicate samples of the homogenate containing 2 g of meat each were titrated with 0.02 N HCI by using an autotitrator (model iT-T and ABU1, Radiometer, Copenhagen). The amount of acid required to bring the homogenates to pH 5.00 was recorded. The initial pH of homogenates was read simultaneously on the titrator. The relationships between titration and spoilage, aging, and fat content were studied employing semitendinous (ST) muscle. To study the effect of spoilage on titration values, 15-g samples of ground muscle were stored at 5 C in small beakers covered with aluminum foil. Log bacterial numbers, pH, and titration values were determined on the stored meat at 2-day intervals. For the study of aging, 15-g samples of meat were stored at 5 C in gas-impermeable plastic bags as previously described (3). The effect of fat on titration values was determined by adding known quantities of beef fat to fat-free ST ground muscle followed by immediate titration. ERV values, log bacterial numbers, and percentage of fat were determined as previously described (1). RESULTS AND DISCUSSION The relationship between log bacterial numbers, pH, and titration values in fresh ground ST muscle undergoing spoilage at 5 C is presented in Fig. 1. During spoilage, log bacterial numbers, titration values, and pH showed a marked increase from the 2nd day of storage to the 10th; spoilage was detected on the 5th day, at which point the log bacterial count was 9.1 /g, the titra- FiG. 1. Relationship between bacterial nwnbers, pH, and titration values on ground semitendinosus muscle held at 5 C for 10 days. Beef stored in beakers and covered with aluminum foil underwent spoilage in the usual manner, whereas spoilage was delayed in beef stored in gas-impermeable plastic bags. 10 ui 9 z 0 0 8 7 tion volume was 2.9 ml, and the pH was 6.3. Beef stored in plastic bags, however, showed only a slight increase in both pH and titration values after 10 days of storage when the log bacterial count did not exceed 8.90/g. This meat was judged acceptable, even at the end of this 10day holding period of 5 C. With respect to titration volume of ground beef, the method of sampling may affect results. Since ground beef spoilage at refrigerator temperatures is largely due to surface growth, surface samples would yield higher titration volumes than those taken from the interior. To minimize this difference, the entire batch of ground beef [1 to 1.5 lb (453 to 679 g) portions] was thoroughly mixed by use of spatula, followed by the removal of test samples in a random manner. Replicate samples from ground beef treated in this manner gave results with low degrees of variation. When beef cuts are to be tested, both surface and interior portions should be mixed as for ground beef. The effect of mixing surface samples with interior samples, where there are generally fewer bacteria, is to dilute the generally higher level of titration substances present in surface samples and to neutralize any microbially produced organic acids that may be present in subsurface portions. A pattern similar to that presented in Fig. 1 is presented in Fig. 2, employing retail-store stew beef which was ground in the laboratory. Statistically, the correlation coefficients (r) between titration values and pH and between titration values and log bacterial numbers were significantly above the 1% level; however, between titration values and ERV, r was significantly above the 2% level. The effect of fat content on titration is presented in Table 1, from which it can be seen that mean values for four replicates decreased from 1.73 ml to 0.91 ml as the percentage of fat increased to 50. It was concluded from these findings that fat alone does not contribute to the acid-titratable groups in fresh beef, although there is some evidence that fat may affect titratable functions as beef undergoes spoilage. In an effort to determine the performance of titration on market meats, hamburger meat was obtained from 23 retail chain stores, and titration values, log bacterial numbers, ERV, pH, and fat content are presented in Table 2. Titration values, log bacterial numbers, ERV, and pH are given at freshness (day of purchase) and at onset of spoilage. The titration volumes of fresh hamburger meats ranged from 0.53 to 2.15 with a mean value of 1.32 i 0.36, whereas values at the onset of spoilage ranged from 1.34 to 3.41 with a mean of 2.58 i 0.56. The average time for the onset of spoilage was 5 days, with a range of 2 to 7 days at 5 C. With respect to its degree of sensitivity to the changes that occur when fresh beef undergoes spoilage, the titration volume increased by 95.4% from freshness to spoilage, based on mean values. The per cent increase with respect to bacterial numbers was 24.8, with the mean log number for fresh beef being 7.41 ± 0.61 and 9.25 i 0.56 at the first signs of detectable spoilage. With respect to ERV, the per cent increase was 44.1; the mean at freshness was 34, whereas the mean value at spoilage decreased 7.0 7.0 6.3 6.5 6.7 6.5 6.6 6.3 6.4 6.3 6.7 6.8 6.5 6.5 6.9 6.8 6.9 to 19. The r between values for titration, log bacterial numbers, and ERV at the two different times is very highly significant (P < 0.001). In an effort to determine the titration value of ground beef at the onset of spoilage, titration volumes were determined on the 23 samples of retail-store hamburger and related to the previously established ERV of 25 and log bacterial numbers of 8.50/g (1). These values are presented in Table 3. Employing an ERV of 25, the corresponding titration values ranged from 1.33 to 2.68 with a mean of 2.01 i 0.37, whereas the mean was 2.10 + 0.35 when a log bacterial number of 8.50/g was employed as reference. On the basis of these findings, ground beef that requires in excess of 2 ml of 0.02 N HC1 for titration of 1 g to pH 5.00 may be expected to be in some state of microbial spoilage. Of the 23 retailstore samples, only two required more than 2 ml of acid for titration at freshness (no. 13 and 21, Table 2). It may be noted further from Table 2 that sample 13 had a titration value of 2.15, a log bacterial count per gram of 7.91, but an ERV of 25 at freshness; whereas sample 21 had a titration value of 2.05, a log bacterial count of 8.32, and an ERV of 29 at freshness. Both of these samples of meat were apparently undergoing incipient spoilage at the time of purchase. A simplified technique for the rapid detection of spoilage in ground beef can be achieved by adding 2 ml of 0.02 N HCl/g of meat to the blended and filtered homogenate and checking the final pH of the homogenate. Using this method, when the pH is >5.0, the meat may be presumed to be in some state of incipient spoilage. Although the identification of the basic substances that are titrated by this technique is not known at this time, all available evidence suggests that they are microbially produced and their appearance is time-dependent. When fresh beef was inoculated with meat spoilage flora to log 8.5 to 9.0/g and tested immediately, titration values remained low and no signs of spoilage resulted. The homogenizing step, along with the constant stirring that accompanied titration, suggests that the substances in question are mainly nonvolatile. In a previous report from this laboratory (4), amino sugar complexes were shown to increase along with bacterial numbers and hydration capacity. The possibility exists that these compounds are at least partly responsible for the increased amounts of acid necessary to lower the pH of spoiling beef. Further research towards identification of the basic functions and their role in meat spoilage is in progress.

v3-fos

2020-12-10T09:04:12.914Z

1970-04-01T00:00:00.000Z

237233015

s2

Chemical States of Bacterial Spores: Heat Resistance and Its Kinetics at Intermediate Water Activity Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is prominently nonlogarithmic for both chemical forms of the spore. Log survival varies approximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, Ea, and Q10 cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed. Traditionally, bacterial spores have been considered to have two kinds of heat resistance, wet and dry. In general, wet has referred to test environments in which liquid-phase water was present, dry to measurements made in the absence of liquid water. Murrell and Scott (10) showed that in the absence of liquid water the water vapor activity (a,) or relative humidity of the test environment had a very large effect on spore heat resistance. They showed that heat resistance was maximal at environmental a, values in the 0.2 to 0.4 range. The heat resistance effects they obtained by variation of the relative humidity of the test environment were large, amounting to many-fold. Thus, those categories of spore heat resistance based on the water status of the test environment could no longer be confined to two, wet and dry, but must be increased to at least three general classes: wet, meaning in the presence of liquid water at a, values near 1; dry, meaning the total absence of water activity in the test environment; and third, resistance in the absence of liquid water but at an intermediate water vapor activity. In the last category, environmental water activity must be specified precisely because of the large variation of resistance with relative humidity. As a practical probability, most heat challenges of bacterial spores in the absence of liquid water would fall into the third category since quite rigorous precautions are required to insure the total absence of water activity (3). This inadequacy of the traditional term "dry heat" as a sufficient specification of a nonaqueous environment for spore heat resistance has also been pointed out by Angelotti et al. (5), Pflug and Schmidt (11), and others, but the term seems to persist for a description of environments with water activities from zero to near one so long as obvious liquid water is absent. Since Murrell and Scott's work (10), several recent studies of Angelotti (5), Fox and Pflug (7), Mullican and Hoffman (9), Hoffman, Gambill, and Buchanan (8), and Bruch and Smith (6) on so-called dry-heat resistance have included comments on the pertinence of these water activity effects to the results. Unfortunately, in none of these studies could the level of water activity at the lethal temperature be specified quantitatively. For ordinary aqueous heat resistance, we have shown that mature bacterial spores can exist in sensitive and resistant states (1,3,4). These different resistance states are prepared and interconverted by in vitro chemical pretreatments of the spores. The changes of aqueous heat resistance between the states can amount to about a thousand-fold. These changes or differences in heat resistance reside within the spores rather than being a response to environmental conditions during the heat resistance test since the changed resistance properties persist when the reagents used to effect the change of state are removed and the spore is transferred to a new environment for measurement of its heat resistance capacity. Thus, to the extent of three orders of magnitude, aqueous spore heat resistance for Bacillus stearothermophilus is an inducible property, and meaningful measurements of heat resistance potential are not possible without knowing the chemical state of a spore sample. Chemical events accompanying these changes of heat resistance state have been described (1,4). Later, we showed (3) that these same chemical states of the spore also have different resistances to strictly dry heat and we presented a method for dry-heat resistance measurements designed to avoid artifacts due to interference by unknown spore chemical state, water activity effects, and uncertain mechanical recovery of the dry-heat-challenged spores. Here we show that the sensitive and resistant states of spores also exist for heat resistance at intermediate environmental water activity. Characteristics of the nonlogarithmic survivor curves and their temperature dependence are given for each of the chemical states at an optimal environmental water activity. A method is presented for testing heat resistance at intermediate water activity. The method is designed to avoid the interfering factors mentioned above and also to furnish known and easily controlled environmental water activity at the test temperature. Some consequences of the existence of a maximum in the survivor versus a. relation are given for the temperature dependence of survival rates of spores encapsulated with a definite weight of water. MATERIALS AND METHODS Preparation of spore crop and the spore chemical states. Spores of B. stearothermophilus NCA 1518 were grown and cleaned as previously described (3). The preparation of the sensitive state, hydrogen form, spores, and the three preparations of the resistant state spores were the same as before (3). Method for measurement of heat resistance at controlled water activity. The general approach to water activity control was direct rather than through the use of humidity-controlling solutions. It was arranged to have a known weight of water in an otherwise evacuated sealed glass tube of known volume at a known temperature. The amounts of water required to give the desired a, at the lethal test temperature were taken from handbook tables of the properties of saturated steam. The ratio of space volume to spore weight should be chosen such that these amounts of water are large by comparison with any expected emission or uptake of water by the spores themselves. Borosilicate glass thermal death time (TDT) tubes (9 by 150 mm) were preconstricted to facilitate later flame sealing. The volume of the tubes up to the middle of the constriction (about 4 ml) was then determined by filling with water. The tubes were segregated into lots whose members had a volume variation range of less than 0.1 ml. The tubes were then filled and covered with distilled deionized water and autoclaved for 1 hr to leach out soluble alkali near the surface of the glass (2). The leaching process was repeated and the tubes were allowed to dry. Very small piles (200 Mg) of the "dry" (freeze dried) spores were weighed into the tubes. The amount (steam tables) of water required to give the desired water activity at the test temperature was then injected by a Hamilton microliter syringe as a drop on the inner wall of the tube. The area of the tube around the water drop was pressed against a piece of dry ice until the drop froze firmly. The open top of the tube was then quickly connected to a piece of gum-rubber tubing connected to, but sealed off by a spring clamp from, an oil pump vacuum. The TDT tube with the drop still frozen was then inserted up to the constriction into powdered dry ice in a Dewar flask. After about 1.5 min, the spring clamp to the preexisting oil pump vacuum was removed and vacuum was pulled for about 20 sec. The TDT tube still attached to the oil pump vacuum was then quickly sealed in a gas-oxygen flame and the seal was annealed in a smoky flame. The sealed TDT tube was then canted against the sharp edge of a slab of dry ice until needed for the lethal heating. The water drop can be moved quickly from place to place within the sealed tube by chilling a small area against dry ice. This serves as a vacuum test and was applied to each tube both before and after the lethal heating. Also, it was confirmed that no significant loss of water occurred during the above described vacuum sealing procedure. This was done by weighing, before and after drying, severed portions of previously prepared TDT tubes into which the water had been collected into one end by such spot chilling with dry ice. For the lethal heating step, the tubes were enclosed singly in flat wire cages (-1.5 by 10 by 12 cm) fabricated from 0.64-cm mesh wire (hardware wire cloth). Prior to insertion of the tubes into the cages, the chilled spot on the tube was thawed with fingers. The tubes were heated for measured times in an oil bath controlled to better than 0.01 C. After the elapsed heating time, the wire cages were quickly plunged into cool water for a few seconds. The cages were then set upright to a depth of about 2.54 cm in warm (-40 C) water for a few seconds to drive the condensed water away from the spores. The tubes were then removed from the cages, quickly wiped free of oil, and again slanted against the sharp edge of a slab of dry ice to collect all the condensed water into one spot away from the spores. If plating for survivor count was to be delayed to another day, the tubes at this point were stored in crushed dry ice. The tubes were then snapped open after scoring thoroughly to facilitate a clean, even break. The spores were washed out with four 1-ml rinses of Tryptone broth into a Teflon homogenizer cup (A. H. Thomas Co., Catalog #4288, size A) and homogenized thoroughly enough to disperse them singly, as judged by direct microscopic count on oil-cleared membrane filters (12). The homogenized spore suspension was then further diluted as required with Tryptone broth for plating (Tryptone, 1%-glucose 0.5%-soluble starch, 0.1%). Incubation was for 2 days at 53 C. Direct microscopic counts were made on the Tryptone broth dilutions by the method of Snell (12) for each tube. The number of spores in the original little pile of spores was based on this direct count. This avoided needing to know and control the moisture content of the original spore sample and the problems of accurate weighing in the microgram range as well as that of correcting for possible loss in opening the evacuated tubes. After rinsing the spores out of the opened tubes, the volume of the two halves of each of the tubes was measured by filling (level meniscus) with water. With some practice in flame sealing and the preselection for volume uniformity mentioned above, the standard deviation of the measured volumes of the opened tubes within a lot was about 0.4% of the mean. However, the volume of both halves of each tube was always checked because of the obvious possibility of serious volume variation from the flame sealing operation. In actual practice, such variations did not occur. RESULTS Variation of survivors with a,. In Fig. 1 are plots for a given heat treatment of log survivors versus water activity for each chemical state of the spores. Log survivors fall off very steeply on each side of the moderately broad maximum. Fortunately, this maximum is at about the same position for each chemical state. The optimal water activity (0.28) chosen for this study was taken from the central portion of these maxima. The position of the maximum was not significantly changed when the heating time for the resistant state (preparation 3) was heated for 50 min at 150 C rather than 30 min, although the height of the maximum was reduced by about 2.5 log units. The water activity at the maximum agrees closely with that reported by Murrell and Scott (10) for their bithermal method. Survivor curve characteristics for the sensitive and resistant states. It is obvious from Fig. 2 and 3 that the log survivors versus heating time curves are indeed curves without the possibility of reasonable approximation by straight lines (log death). This curvilinearity holds for both chemical states but is most prominent in the log survivor curves for the resistant-state spores. Since the log survivor versus time plots are not linear, the D value concept is not applicable to heat inactiva- tion of spores under these conditions of intermediate water activity. For describing heat inactivation at a, = 0.28 under these conditions, an empirical expression, [(log initial count/g) -(log survivors/g)]a = ktmin + C, was chosen which gave a reasonable fit to the log survivor curves. The exponent a for VOL. 19,1970 each chemical state was chosen from the least squares slope of plots of log [(log initial count/g) -(log survivors/g)] versus log tmin. For the sensitive-state spores, a value of the exponent a of 0.58 was selected from such slopes. For the resistant state, a was taken as 0.33. The log initial count/g was 11.47 for the original unheated resistant-state preparation and 11.85 for the sensitive-state preparation. Thus, the expressions used to describe heat inactivation at a, 0.28 under these experimental conditions were (11.47 -log 5)0 33 = k'tmin + C for resistant state (1) (11.85 -log S)o -18 = k'tmin + C for sensitive state (2) where S is the number of survivors per gram of spores at heating time t in minutes, and k' and C are constants. As shown in Fig. 4, plotting (log Soriginallog S)0 33 for the resistant state and (log Soriginallog S)0 58 for the sensitive state versus time gave reasonably straight lines for several test temperatures. The least squaresdetermined slopes of these lines yielded coefficients of t in equations 1 and 2 for various temperatures. These coefficients of t were used as reaction velocity constants (k') in determining the temperature dependence of heat inactivation ( Table 1). For convenience, the Arrhenius plots for both states have been included in Fig. 5, but it should be noted that the rate expressions (equations 1 and 2) for the two states are different and so equal values of the rate constants (k') between states do not mean equal numbers of survivors will result from equal reaction times. 5, It is apparent that the temperature dependence of spore heat survival for each of the chemical states at a, 0.28 is similar to that for strictly dry heat resistance (3), even though the survivor curve shapes and the general level of heat sensitivity are quite different. The activation energies for both chemical states at both environmental humidity conditions of heat challenge (strictly dry and a, 0.28) are all about 40,000 cal. However, the log survivor versus time relations for strictly dry heat resistance are straight, that is, follow "logarithmic death," whereas these same relations for heat challenge at intermediate water activity of 0.28 are prominently curved. The general level of heat sensitivity of the chemical forms under strictly dry conditions is about like that for ordinary aqueous heat resistance. That for the same chemical forms at the intermediate water activity is much higher. DISCUSSION It is evident from Fig. 2 and 3 that mature bacterial spores can be manipulated chemically into different states for the property of resistance to heat at intermediate water activity. Since, like heat resistance at both strictly dry and strictly wet environmental conditions, heat resistance at intermediate water activity is a spore property manipulatable by in vitro pretreatments, the same implications for experimental strategy which we have stressed previously (2, 3) for the a, = 0 and a, 1 conditions also hold for this intermediate humidity situation. The most important of these is that meaningful estimates of heat resistance potential cannot be made without knowing the chemical state of the spores. This knowledge of chemical state can be gained by converting deliberately a portion of the spore sample into each chemical state before measuring heat resistance at controlled water activity. As pointed out previously (2), if artifacts are to be avoided in the measurement of the aqueous heat resistance of the different spore chemical states, special care must be taken to use a testing medium inert with respect to its capacity to induce change of chemical state during the test. In fact, aqueous conditions can be arranged deliberately for the adaptation of spores to heat (4) by using a noninert suspending medium for heating. Nonaqueous heat resistance of spores, both dry and at intermediate water activity, is generally not subject to these changes of chemical state effects during the lethal heat challenge itself. However, we have pointed out how inadvertent changes of spore resistance state could occur by interaction of spores and their supporting surface in the preparatory phases of nonaqueous heat resistance measurement (3). Even in the case of aqueous heat resistance, it has been found possible to sensitize spores to heat in the presence of complex biological mixtures at their ordinary pH (2). It is not necessary to have acid conditions present during lethal heating of spores in such wet, complex biological mixtures, probably because free calcium ion is absent by virtue of the complexing action of the organic materials. Such sensitization to heat for facilitating spore killing in a practical situation should be straightforward in the case of nonaqueous heat resistance, providing the spores are or can be made physically available to the sensitizing reagent. Merely washing the sensitizing acid away before lethal heating would restore the original nonacid condition of an inert substrate on which the spores were supported. Conceivably, in some situations, the spores could be chemically sensitized before encapsulation in solid matrices. From the survivor versus a, plots of Fig. 1, some inferences may be drawn about the apparent temperature dependence of heat resistance of spores encapsulated within a given volume with a given weight (as contrasted with activity) of water which considerably exceeds the water-holding capacity of the enclosed spores, that is, where the encapsulating volume exceeds spore volume by several hundred-fold. In the absence of liquid water, specification of the water activity within an enclosed space requires knowledge of three terms: volume of space, weight of water, and temperature. Of course, if pure liquid water is known to be present at all temperatures of interest, a, is 1 by definition. For spores encapsulated in solid matrices such as plastics, crystals, tightly joined surfaces, etc., neither the volume of the encapsulating space nor the weight of water is likely to be known, leaving temperature as the only measurable variable, and water activity unknown. In Fig. 1 it may be seen that the depend-ence of survivors on water activity is quite sharp on either side of the maximum. This could make for an apparent unusual temperature dependence of heat resistance for spores encapsulated with a given weight of water since, when volume and weight of water (short of saturation) are fixed, a, varies inversely with temperature. If, for example, the particular tube used for the last point on Fig. 1 (Resistant State) had been heated at a temperature 100 higher, the spores would tend to be protected against the higher temperature. Instead of 0.50, the water activity at the 100 higher temperature would be only 0.39 for these encapsulating conditions. Thus, on the right side of the maximum, the lethal effect of an increase in heating temperature would tend to be compensated by the temperature-induced lowering of water activity. On the left side of the maximum in the a, versus survivor curve, the killing effect of raising the temperature would be reinforced by an a, change toward a less favorable (for survival) value. If, on the other hand, the tubes mentioned were heated at a 100 lower temperature the situation would be reversed with survival for tubes on the left side of the maximum being reinforced, whereas survival on the right side would be opposed by such a lowering of heating temperature. It is thus apparent that for such casually (with a given weight of water) encapsulated spores, it is not possible to determine temperature dependence of heat lethality in the usual sense. The temperature dependence of spore heat survival rate is commonly expressed as a z value, the temperature change required for a 10-fold change in survival rate. Such z values are considered to be a property of the spore in a given, presumably constant, environment. For example, in both aqueous and strictly dry heat environments water activity is essentially independent of temperature. However, as discussed above, when spores are encapsulated with a given weight of water, the kind of environment is also temperature dependent; water activity itself is a function of temperature. On the left side of the a, versus survivor maximum, apparent z values would be low, on the right side, high. Even in cases where the encapsulating fit around a spore is close with the resulting expected buffering of environmental water activity by the spore itself, the required lowering of equilibrium water content with increasing temperature would be expected to give some environmental disturbance. Only when volume and water content of the encapsulating space are known and manipulatable is it possible directly to determine a z value in the usual sense of its being a spore property. It appears that such considerations of environ-mental water activity temperature dependence can explain the anomalous z values reported by Angelotti et al. (5) for their paper system and possibly also for their highly torqued stainlesssteel-surface system. The cellulose moisture sorption isotherm and the geometry of the system used for drying the paper would indicate an appreciable final water content in the paper, probably at least 2%. Such a moisture content of 2%, when released by high temperature, would result in an a, of about 0.06 at 125 C, about 0.04 at 140 C. Such values lie on the steep, left side of the curves of Fig. 1 at which point lethality of heat is reinforced by temperature-induced lowering of aw, in other words, where apparent z values would be low. Doubling or halving, or more, the assumed value of 2% moisture content for the paper would not affect this qualitative argument for low z values since the a, would still be on the steep, left side of the a, versus survivor plot of Fig. 1. As we have reported (3), extremely low water activities still can elevate spore heat resistance over that at a strictly dry condition. Thus, the abnormally low z value operationally observed for the paper system appears to have been due to lack of environmental constancy during its measurement rather than being an expression of a temperature characteristic which could be interpreted as a "wet kill mechanism." Although sufficient information on environmental water activity is not available for the other unusual z value in the highly torqued stainless-steel system, its direction (high) would be expected for wetter-than-optimal conditions on the right side of the curves of Fig. 1. The cavities provided by the roughness of the no. 4 stainless finish of the washers should not have been large enough to accommodate spore size particles. One possible source for sufficient water in the environment would be water released from mechanical disintegration of most of the spores. Another recent report of an anomalously high (139 C) z value is that of Bruch and Smith (6) for spores on Teflon film. Here, the Kapton film interlayer was known to have an appreciable water-holding capacity and is a reasonable source for sufficient water to furnish a wetter-thanoptimal environment with the tendency to apparent high z values. Some inferences can be drawn on the question of efficient heating conditions for killing spores encapsulated under conditions in which environmental water activity is highly temperature dependent. For random encapsulating conditions in which the water activities of the cavities are distributed over the whole of a curve like that of Fig. 1, temperature cycling should bq useful in successively moving spores off the optimum (for survival) of the curve down to the steep sides where heat sensitivity is much greater. Murrell and Scott's comprehensive report (10) on the effect of water activity on spore heat resistance clearly established the large effect of environmental water activity on the heat resistance of several species of bacterial spores in the absence of liquid water. They showed that the optimal relative humidity for heat survival was in the range 0.2 to 0.4 a, and that in this optimum environmental a, region the great interspecies differences in heat resistance largely disappeared. There are several differences between our work here and that of Murrell and Scott (10). These differences fall in three principal categories: (i) experimental methods of water activity control, (ii) heat survivor curve shape, and (iii) knowledge and control of the chemical resistance state of the spore samples used. For the bulk of their data, Murrell and Scott used several conventional humidity-controlling solutions packaged with, but separated from, the spores by various two-container arrangements. In all these arrangements, the whole system was evacuated prior to final sealing so that equilibration of water activity would be speeded during lethal heating. They also made less extensive use of a bithermal method. Each of these variations of the methods was more than adequate to show the striking maximum in the curve relating a, and D value. However, the a, at the maximum was somewhat variable, and at particular a, values off the maximum, D value variations among the methods amounted to many-fold. It was recognized that the behavior of the controlling solutions at high temperature was somewhat uncertain and the water activity control was described as approximate. We have chosen a simpler, direct method of controlling water activity which insures that the gaseous water concentration in the spore-encapsulating environment is known unequivocally once equilibration to the lethal temperature has been achieved. The usable range is not limited to low temperatures. So long as the spore weight is kept sufficiently low that any water uptake or emission by the spores is negligible by comparison with the total water content of the whole encapsulating environment, there appears to be no reason to use humidity-controlling solutions. Both with our proposed method and that of Murrell and Scott, temperature lags will occur during the come-up and come-down periods of the lethal heating operation. Such temperature lags should be less with our method because of its smaller mass and the fact of two containers in the system of Murrell and Scott. We have made some measurements of the time required for disappearance of the water drop in our method and these times are measured in seconds. For example, in a 4-ml volume, 2.55 mg of water took 15 sec to disappear at 1480 from one spot. When the 2.55 mg of water was divided into three roughly equal spots on the wall of the evacuated tube, disappearance took about 5 sec. Water spread over the upper 1 to 2 cm of the tube was more difficult to observe precisely but disappeared very quickly. During the temperature come-up period with our method and also with arrangements 1 and 5 of Murrell and Scott (10), the spores temporarily would see a water activity lower than that intended. We did obtain increases of survival by spreading the water over the upper part of the tube as compared to letting it vaporize from a single spot, but the differences were not large. The second major difference between our work and that of Murrell and Scott lies in the radically different log survivor curve shapes. They reported a linear log survivor versus time relation and expressed survival in the usual D-value terms. Our log survivor curves, on the other hand, are, as pointed out above, definitely and prominently curvilinear and not susceptible to even rough approximation by the single D value concept. The reasons for this difference in the survival versus time relation are not definitely known. The experimental physical arrangements and means of water activity control are quite different for the two methods. Another possibility for the origin of the curve shape discrepancy may lie in the fact that Murrell and Scott determined resistance at temperatures which are, at least for the resistant form of spores like B. stearothermophilus, quite low. Under the conditions of the resultant long survival times such as curve # 2 of their Fig. 2, it would be difficult to distinguish between a curvilinear log survivor relation like our equation 1 and classic logarithmic death. Both expressions would have about the same appearance of linearity with low slope over the early portion of the log survivor drop. For example, in our Fig. 3, if only the data in the first three quarters of the time period were available, an interpretation of log death could easily be made. At such low temperatures, only in tests observing survival over several log units would the curvilinearity following the long lag period become evident. The third main category of difference between this work and that of Murrell and Scott lies in the control of the chemical state of the spore sample. Here, we converted the spores to a known heat-resistance state before measuring heat resistance. Murrell and Scott used naturally occur-ring spore crops which can be composed of various mixtures of the heat-resistance forms.

v3-fos

2020-04-16T09:09:14.417Z

2020-01-01T00:00:00.000Z

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Qualitative analysis of some bioactive components of methanolic leaf extract of M. citrifolia (Noni) Medicinal plants offer endless opportunities for new drugs discovery due to their supremacy for the possession of phytochemical compounds known for diverse antimicrobial activities. The world ever increasing demand for therapeutic drugs from natural products with particular interest in edible plants for safety purposes is now catching researchers’ attention. This study therefore aimed at determining the presence of some bioactive phytochemical components of methanolic leaf extract of M. citrifolia L. Qualitative screening of leaf extract has confirmed the existence of Tannins, steroids, saponins, flavonoids and alkaloids in the mixture. And these bioactive compounds correspond to phytochemicals with antimicrobial, nematicide, pesticidal, antioxidant, ant-inflammatory, cytotoxic, anti-allergy, and anti-carcinogenic properties (bioactive compounds) earlier documented by previous researchers. INTRODUCTION Voucher number SK 3255/17) (Appendix A). Thereafter leaves were brought to the Biocontrol Laboratory, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia (UPM), washed under running tap water to get rid of dust and debris and rinsed in sterile distilled water. Leaves were first allowed to stay for 6 hrs under the laminar air flow to dry up the wet leaves surface. And finally dried at 40-45 o C in a mechanical convection oven (Memmert, Germany). An electrical grinder Retsch SK100 standard Guβeisen, 2002 was used to gried the dried leaves into the powdered form for use [5]. Test for Tannins To determine the presence of Tannins, Braemer's test was performed. Following the method described by [6], where 2 g of the powdered M. citrifolia leaf was dissolved in 10 ml of methanol, then macerated and filtered by means of cotton wool and funnel. Thereafter, 2 ml of the filtrate was added to 2 ml of 10 % alcoholic ferric chloride (1:1). Formation of greenish grey coloration of the mixture indicates the presence of tannins. Test for Steroids To test for the presence of steroids, Lieberman Burchardt test was used. For the test, 2 g of the leaf powder was added to 20 ml of methanol in a 150 ml conical flask and covered for 30 min, mixture was filtered using cotton wool and funnel. Filtrate was poured into a 50 ml beaker and placed on a water bath until filtrate was completely evaporated. 6 ml of chloroform was added to the evaporated extract and mixed thoroughly. Then 2 ml of the chloroform mixture was transferred into a test tube where few drops of acetic anhydrite was added, followed by addition of few drops of H 2 SO 4 , which was added slowly to the wall of the test tube. Formation of dark green colour designated the presence of steroids [9]. Plants steroids are derivatives of cyclization of the triterpene squalene [10] and [11]. Test for Saponins To determine the presence of saponin in the phytochemical components of M. citrifolia leaf. 70 ml of sterile distilled water was placed in a beaker containing 3 g of plant powdered leaf, mixed then boiled for 2 min. Mixture was filtered into a new test tube using cotton wool and funnel to produce an aqueous extract. 2 ml of the aqueous extract was discharged into a graduated test tube, and vigorously agitated. The formation of 1cm form that persists for 3 minutes designated the presence of saponins [12]. Test for Flavonoid Ammonium test was employed to test for the presence of Flavonoid in leaf extract of M. citrifolia, following the method as described by [13] and [14]. To achieve this, 0.2 g of M. citrifolia leaf powder was added to 10 ml of ethyl acetate in a 100 ml conical flask. Mixture was then heated for 5 min in a water bath, allowed to cool and filtered. 4 ml of the filtrate was discharged into a test tube where 1 ml of diluted ammonia solution was added to the mixture, agitated and kept at room temperature for a few seconds then observed. Appearance of layer of yellow coloration at the bottom of the test tube indicates the presence of flavonoid Test for Alkaloids Dragendroff reagent test: For this test, 0.2 g of M. citrifolia leaf powder was added to 20 ml of diluted H 2 SO 4 in methanol in a conical flask. Mixture was boiled for 5 minutes in a water bath, cool and filtered. Three drops of dragendroff reagent were added to the filtrate. Formation of creamy, orange solution indicates the presence of alkaloids [15]. RESULTS AND DISCUSSION The qualitative analysis of phytochemical components of M. citrifolia leaf extract using the conventional phytochemical screening assay (chemical tests) detected the presence of tannins, steroids, saponins, flavonoids, and alkaloids (Table 1 and Figure 1) which is in agreement with the findings of [16] and [17]. During the Braemer's test, the M. citrifolia leaf extract turned greenish grey which was an indication for the presence of Tannins in the solution according to the reports of [18] and [8]. Tannins belong to the class polyphenol compounds with an astringent property, soluble in acetone, alcohol, and water. Similarly, Lieberman test (Burchardt test) of the leaf extract solution turned dark green in colour ( Figure 2) which was an indication for the presence of steroids. This finding is in line with the report of [8]. The Frothing test also showed the formation of I cm form height above the mixture which persisted for more than 3 min. According to [6], the appearance of up to 1 cm form height above mixture that lasts for up to 2-3 minutes is an indication for the presence of saponins (Figure 3). Saponins are naturally produce by many plants for defense against pest and pathogens, they are easily converted to drugs, cosmetics and taste modifiers and are therefore considered economically viable compounds [19] and [20]. Result for the Ammonium test (test for flavonoids) showed layer of yellow coloration at the bottom of the test tub, which is an indication for the presence of flavonoid in the leaf extract, according to [21] and [14] ( Figure 4). Flavonoids is another member of the compounds class polyphenols which are known for their Pesticidal, antimicrobial, antioxidant, chemotherapy activities, and their mechanism of action against microorganisms includes; complex activities with the cell wall, cell lysis, membrane disruption, inactivation of enzymes and death [22]. Findings of the Dragendroff test (test for Alkaloids) shows formation of layer of creamy-orange solution ( Figure 5). This result is in agreement with the report of [15], that by this test, the formation of creamy-orange colouration of the test solutions suggests the presence of alkaloids in the test solution. Alkaloids have a toxic effect on microbials, in human medication and or as biopesticides [22].

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Aflatoxigenic Isolates of Aspergillus flavus from Pecans Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production. While investigating a bakery mold problem involving pecans, it was noted that a number of pecan halves (meats) being cultured for internal fungi yielded colonies of Aspergillus flavus Link ex Fries. Inasmuch as the presence of aflatoxin in foods may constitute a health hazard, a study was made to determine the frequency and toxigenic potential of these strains as well as the suitability of pecans as a substrate for aflatoxin production. MATERIALS AND METHODS The presence of A. flavus in pecans was determined by plating the seed on either 6% malt-salt agar (1) or rose bengal-streptomycin agar (RBM-2) after surface sterilization by immersion for 2 min in a solution of sodium hypochlorite (10 ml of bleach, 10 ml of 95% ethyl alcohol, and 80 ml of water). The RBM-2 medium was prepared as described by Tsao (9), except that the streptomycin sulfate was added at the rate of 0.06 g/liter. The plates were incubated at room temperature and examined every other day for 3 weeks. Colonies of A. flavus were isolated onto malt extract agar slants (7) for identification and toxin analysis. A total of 2,061 pecan pieces were plated out during this study; the source and nature of each sample are shown in Table 1. Samples 1-10 were taken from pecan lots used in the production of commercial bakery products; samples 11-16 were packaged pecans purchased in local supermarkets. Screening for aflatoxin. For screening of mold isolates, 50 ml of 2% yeast extract plus 20% sucrose [YES medium (3)] in a 250-ml Erlenmeyer flask was inoculated with 106 spores, incubated at room temperature for 7 days, and extracted with two 100-ml portions of chloroform (CHCl,) on a gyratory shaker. 1 The pooled extracts were filtered and evaporated to dryness on a flash evaporator; the residue was cooled and resuspended in 5 ml of CHC13. Visual estimates of aflatoxin content were made by comparing thinlayer chromatograms of appropriate dilutions of the extracts with aflatoxin standard obtained from the Southern Utilization Research and Development Laboratory, USDA, New Orleans, La. Thin-layer chromatograms (20 by 20 cm, 0.25 mm thickness of silica gel G-HR) were developed in acetone-chloroform (1:9, v/v) in an unequilibrated tank. Chloroform extracts were purified by column chromatography and precipitation with hexane in the first part of this study. However, thin-layer chromatography (TLC) results indicated that visual estimates could be made easily and accurately without further purification. Consequently, column chromatography and hexane precipitation were discontinued in the latter part of this study. For screening batches of pecans, pecans were extracted directly by the method of Pons et al. (6) and extracts were examined by TLC to determine the aflatoxin content. For screening individual nuts, moldy pecans were extracted by the method of Cucullu et al. (2) for determining aflatoxins in individual peanuts and peanut sections. Presumptive TLC results were confirmed by spectrophotometric analysis and chick embryo bioassay. Spectrophotometric determinations of aflatoxin were made by the method of Nabney and Nesbitt (5) on a Shimadzu model MPS-50L recording spectrophotometer. The method of Verrett et al. (10) was used for chick embryo bioassays. Further confirmation of aflatoxins was obtained by administering extracts per os to 1-day-old Peking ducklings and examining for bile duct cell proliferation (8). Extracts for duckling bioassays and spectrophotometric analyses were purified further by preparative TLC. RESULTS AND DISCUSSION The level of A. flavus in the bakery pecans (lots 1-10) ranged from 2 to 21 % (average 9%), whereas in market pecans the level ranged from 0 to 85% (Table 1). Of the 120 colonies isolated and identified, 105 (87.5%) were A. flavus and 15 (12.5%) were A. parasiticus Speare. Table 2 shows the results of screening mold isolates for aflatoxin production in YES medium. Presumptive TLC results showed that 29.1% of the isolates were negative; 13.4% produced aflatoxins B1, B2, G1, and G2, and 57.5 % produced only aflatoxins B1 and B2. Diener and Davis (4) reported that 90% of their toxigenic isolates of A. flavus from agricultural commodities produced primarily aflatoxin B. In this study, 80% of the toxigenic isolates produced only aflatoxins B1 and B2. Almost all the negative isolates and those producing only the B toxins were A. flavus; most of the isolates producing all four aflatoxins were A. parasiticus. Isolates were considered to be negative when aflatoxin was not detected in 3 ,uliters of undiluted chloroform extract. In all but one instance, when four toxins were produced in YES medium there was more aflatoxin G produced than B. The one exception was A. parasiticus ( # PC101) which produced more aflatoxin B than G. Table 3 gives the number of isolates producing aflatoxin within several arbitrarily chosen ranges. All isolates of A. parasiticus produced aflatoxin in amounts of 10 ;g/ml or more, whereas the majority of A. flavus isolates produced aflatoxin in lower ranges (less than 10 lg/ml). Though A. parasiticus isolates seem to produce more total aflatoxin (B plus G) than do A. flavus isolates, this does not necessarily indicate a greater potential hazard. As has already been pointed out in Table 2, most of the A. parasiticus isolates produce more aflatoxin G than B and, to date, aflatoxin B1 is regarded as the most carcinogenic of the aflatoxins. Spectrophotometric determinations on chloroform extracts of 20 randomly selected samples confirmed preliminary positive TLC results. Maximum absorption typical of aflatoxins was obtained at 363 nm from samples that were positive on TLC but not from those that were negative. Toxigenicity of randomly selected strains of A. flavus and A. parasiticus was further confirmed by bioassay. Culture extracts from eight strains of A. flavus that were negative by TLC were also negative by the chick embryo bioassay. Two of these cultuies were shown to be negative by duckling bioassay. Extracts of six cultures that were suspected to produce four aflatoxins by TLC screening were shown to be toxic to chick embryos. Toxigenicity of three of these cultures was further confirmed by duckling bioassay in which administration per os resulted in bile duct cell proliferation typical of aflatoxin. Extracts of 18 randomly selected cultures that produced only aflatoxins B, and B2 were also toxic to chick embryos. Confirmation of the toxigenicity of 5 of these 18 isolates was obtained by duckling bioassay. LILLARD, HANL Eighty-five isolates were found to be aflatoxigenic when grown on YES medium. Since many of these isolates were obtained from pecans used commercially in bakery products, the ability of randomly selected isolates to produce aflatoxin on cooked pecans was also determined. Nine isolates of A. flavus and four of A. parasiticus were. grown on 25 g of crushed, autoclaved pecans. The five isolates that were negative in YES medium were also negative on autoclaved pecans. The eight isolates showing positive TLC results after growth on YES medium also gave positive TCL results after growth on autoclaved pecans. A. flavus and A. parasiticus were isolated from moldy, raw pecans obtained at retail commercial outlets. Results of direct extraction for aflatoxin of 22 of these pecan samples (50 to 100 g each) were inconclusive. Strong presumptive evidence of aflatoxin in commercial pecans was obtained by extracting individual moldy raw pecans by the method of Cucullur et al. (2). Fluorescent compounds with RF values identical to standard aflatoxin were extracted from several individual sections of moldy pecans. However, due to the small amount extracted, confirmation by bioassay or ultraviolet spectroscopy was not possible. In view of the known carcinogenic properties of aflatoxin, the detection of these compounds in market pecans poses a potential health hazard to the consumer. Consideration should be given to the conditions under which pecans are stored and processed. Additional research is needed to determine the conditions which would minimize ,Ip N, AND LILLARD APPL. MICROBIOL. mold growth and the threat of aflatoxin contamination of pecans.

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