Microbial Coagulation of Alfalfa Green Juice

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p /87/9226-6$2./ Copyright 1987, Americn Society for Microbiology Vol. 53, No. 9 Microbil Cogultion of Alflf Green Juice N. GODESSART, R. PARES, AND A. JUAREZ* Deprtmento de Microbiologi, Fcultd de Biologi, Universidd de Brcelon, 871 Brcelon, Spin Received 28 April 1987/Accepted 1 June 1987 Of the different bcteril strins isolted from lflf rw mteril, nine were ble to cogulte the protein frction of lflf green juice. The two strins showing the highest efficiency were further used for cogultion experiments. They were clssified s Erwini crotovor nd Escherichi coli. Juice smples inoculted (1:1 to 1:1) with sttionry-phse cultures were efficiently cogulted. The mount of protein recovered ws equivlent to tht obtined when the juice ws het treted. A miniml incubtion temperture of 3 C ws required. The protein cogulum ppered fter 8 to 1 h of incubtion. During this period no bcteril growth ws pprent, but glucose ws ctively fermented. For both strins no extrcellulr enzymtic ctivity could be shown in the culture superntnts. The fermenttive metbolism during the incubtion period seems to be responsible for protein cogultion. Green, lefy plnts re very importnt protein source. Nevertheless, the proteins from this source re lrgely unvilble to monogstric nimls, including humns, becuse nonruminnts lck the mechnism necessry for rupturing plnt cell wlls nd thus relesing the protein. The urgent need to develop lterntive protein sources to relieve the world's protein mlnutritionl problem hs incresed the interest in developing methods to frctionte lefy plnts (9, 14). The lef protein concentrtes thus obtined hve been extensively studied over the pst 3 yers (15) to evlute their importnce s protein source, initilly for feeding cttle nd poultry nd with the perspective of their incorportion in the humn diet. Alflf (Medicgo stiv) is n excellent source of protein nd xnthophyll nd hs been one of the most frequently used plnts in the frctiontion processes previously developed (11). In ll of these processes the lflf is first mechniclly pressed. This step yields fiber-free green juice (3, 6-8, 11, 13), nd the bulk of the lflf cn be recovered s fibrous residue with nutritionl qulity equivlent to dehydrted lflf mel. The protein frction present in the green juice is recovered by heting the juice to 6 to 8 C. Protein cogultes in this temperture rnge (4, 6). The use of chemicl flocculnts hs lso been reported (1). After cogultion, the cogulum cn be recovered by low-speed centrifugtion, further frctionted, nd desiccted. In the industril process both cogultion methods, especilly the former, re expensive nd my represent n importnt increse in lef protein concentrte production costs. Therml cogultion increses by 22% the overll energy input required in the production of dry protein concentrte (17). An lterntive to such cogultion techniques ws reported by Sthmnn (17). If plnt green juices re incubted nerobiclly spontneous fermenttion occurs, cusing protein cogultion. As the fermenttion goes on, one of the resons for the cogultion my be decrese in the ph of the juice. Finl ph vlues rnge from 3.8 to 4.6. The nutritive qulity of the lef protein concentrtes obtined by this method is better thn tht of hettreted lef protein concentrtes. Additionlly, importnt * Corresponding uthor. energy svings cn be obtined by using this method. Our objectives were to isolte bcteril strins with cogulting ctivity nd to study lterntive processes for developing microbiologicl cogultion nmethod. MATERIALS AND METHODS Alflf juice preprtion. Chopped lflf ws pressed in helicoidl screw press, s described by Edwrds et l. (6). Fiber-free juice ws obtined by filtrtion through 4-mesh screen. Het tretment. Juice smples were het treted s described by De Fremery et l. (4). Aliquots were distributed in 1-ml flsks to depth of 1 mm. The flsks were then plced in wter bth t the desired temperture, shken until the smple reched the bth temperture, held for selected times, nd then cooled in wter bth. Smple preprtion nd cogultion test. The finl step of cogultion ws tested visully s the ppernce of green cogulum nd cler yellow-brown superntnt. For quntittive evlution, smples were centrifuged t 1, x g for 2 s (miniml centrifugl force necessry for cogulum seprtion). The superntnt ws recovered nd centrifuged t 15, x g for 2 s to eliminte suspended prticles. Either pigment or protein concentrtion ws evluted in the liquid phse. The cogulum ws desiccted nd weighed, nd its protein content ws determined. Bcteril strins, isoltion, nd growth. All the bcteri tested were isolted from lflf rw mteril. Rw smples obtined in n lflf-processing fctory (Aprolf, S. A., Suchs, Lerid, Spin) were suspended in tryptic soy broth (TSB) (Difco Lbortories, Detroit, Mich.), mixed, nd incubted overnight t 3 C. Seril dilutions were plted on tryptic soy gr (Difco). Colonies were picked nd purified. When needed, bcteril strins were clssified by routine biochemicl tests. Escherichi coli AFS-34 nd Erwini crotovor AFS-35 were clssified by using n API-5E dignostic kit (Anlytb products, Plinview, N.Y.). Cells were grown in 5-ml flsks, ech one contining 1 ml of the following medi: TSB (Difco), nutrient broth (Difco), Todd-Hewitt broth (Difco), nd M-9 miniml medium (12). After inocultion with n overnight culture, bcteri were incubted in n orbitl shker (2 rpm) t 226

2 VOL. 53, 1987 BACTERIAL PROTEIN COAGULATION 227 A *.9 6 o.7 *.5 ox 4 Q Cogultion Temperture (C) FIG. 1. Effect of the cogultion temperture on the protein (A) nd pigment (A) concentrtion in the juice superntnt obtined upon centrifugtion. A43 vlues were obtined fter diluting the fresh juice nd the treted smples 1:3 nd 1:4, respectively. A43 vlues between.4 nd.5 indicte n efficient cogultion, s obtined with het-treted smples under stndrd conditions. 3 C. At different growth phses, portions were mixed with lflf green juice. Streptomycin-resistnt (Smr) mutnts from Erwini crotovor AFS-35 were obtined by plting n overnight culture on LB (12) pltes contining 25,ug of the ntibiotic per ml. Resistnt clones were picked, purified twice, nd tested for cogultion ctivity. E. coli AFS-34 ws n Smr isolte. Anlyticl methods. The reltive mounts of pigments present in the superntnt fter cogultion were determined spectophotometriclly t 43 nm, s described by De Fremery et l. (4). For soluble-protein determintion, nd to void interference with phenolic compounds, the protein ws precipitted with cold 1% trichlorcetic cid nd suspended with.1 N NOH, s described by Agusti nd Beltrn (1). Furthermore, protein ws determined by the Brdford method (2) with the Protein Assy Kit (Bio-Rd Lbortories, Richmond, Clif.) with bovine serum lbumin s stndrd. After TCA precipittion, deproteinized superntnts were used for glucose determintion with o-toluidine s described by Dubowsky (5). The protein content in the dried cogulum ws determined fter its C nd N content ws evluted with n elementl nlyzer (no. NA-15; Crlo Erb Strumentzione, Miln, Itly). The protein concentrtion is ssumed to be %N x Assy for extrcellulr proteses. E. coli AFS-34 nd Erwini crotovor AFS-35 were tested for extrcellulr proteolytic ctivity to determine their bility to hydrolyze csein nd geltin, s described by Smibert nd Krieg (16). RESULTS Determintion of prmeters tht define the cogultion of the protein frction in green juice. Protein is recovered from green juice s cogulum tht includes other cellulr components (e.g., pigments). To ssess the effectivity of technique for recovering lefy plnt protein, the protein content in the dried cogulum is usully determined. We hve lso tried to dpt lterntive esy methods to evlute cogultion simultneously in lrge number of smples nd to serch for strins with high cogultion ctivity W 2 Of the overll mount of protein present in the juice, significtive percentge is pigment ssocited (chloroplstic protein frction). Protein cogultion therefore implies decrese in the pigment-ssocited protein level in the soluble frction. The opticl density t 43 nm is routinely used for mesuring the overll protein content of vegetble pigments (1). For this reson we tried to use the opticl density t 43 nm s n indictor of the degree of cogultion. To test this, juice smples were het treted t vrious tempertures. The different mounts of protein thus cogulted were seprted by centrifugtion, nd the opticl density of the superntnts t 43 nm ws mesured. The results obtined (Fig. 1) showed good correltion between the A43 nd the predicted cogultion efficiency of the het tretment. For cogulted smples, independently of the method used, n A43 of.5 to.6 (smples diluted 1:4) in the superntnt indictes n optiml degree of cogultion. These bsorbnce vlues were obtined when the smples were heted t 55 to 6 C, high enough temperture for mximl recovery of the green protein frction (4). We hve routinely used this pigment determintion s mesure of the degree of cogultion. As n lterntive method we tried to correlte the degree of cogultion with the percentge of soluble protein tht remined in the superntnt. Using het-treted smples s mentioned bove, we evluted the soluble protein level once the cogulum ws seprted by centrifugtion. A good correltion could be observed between the cogultion efficiency nd the soluble protein in the superntnt (Fig. 1). Thus we routinely used both methods when ssessing the cogulting ctivity of the bcteril strins. Isoltion of bcteril strins with high cogultion bility. Previously published work on microbil green juices cogultion hd studied only the role of the ntive bcteril popultion (17). We tried insted to isolte bcteril strins with specific cogulting ctivity. A totl of 6 strins from n lflf-processing fctory, isolted s previously described, were tested for their bility to cogulte the protein present in the lflf juice. Juice smples were inoculted 1:1 with erly-sttionry-phse cultures nd incubted t 3 C. Cogultion ws first evluted by visul inspection nd by mesuring the pigment concentrtion in the superntnt. Of the 6 strins tested, only 9 showed cogulting ctivity. They were txonomiclly clssified s Proteus spp. (two of them), Pseudomons sp., n Escherichi sp., n Erwini sp., Streptococcus sp., n Actinomyces sp., nd two enterobcteri not further clssified. Of the nine strins isolted, two, in n verge of 1 independent experiments, gve the best results for cogultion. These two strins (AFS-34 nd AFS-35) were further chrcterized nd were used for studying nd improving the microbiologicl cogultion of the lflf juice. Strins AFS-34 nd AFS-35 were clssified (API 5E) s E. coli nd Erwini crotovor, respectively. Chrcteriztion of the cogultion process cused by strins AFS-34 nd AFS-35. To stndrdize microbil cogultion system, we used strins AFS-34 nd AFS-35 s cogulting gents nd performed more detiled study of the process. The lflf juice ws inoculted (1:1) with 24-h culture, nd smples were tken t 2-h intervls during the first 1 h of incubtion nd fter 24 h. The following prmeters were mesured: cogultion (determining the protein concentrtion in the superntnt s previously described), bcteril growth, glucose concentrtion in the superntnt, nd ph. Additionlly, the cogultion ws evluted in noninoculted smples to compre the effect of

3 228 GODESSART ET AL t 6 -o 4 4 S * 3 2 \ Time (h) FIG. 2. Soluble protein present in noninoculted smples (A) nd smples inoculted with AFS-34 () or AFS-35 (A), collected t 2-h intervls nd t 24 h fter inocultion. The rrow denotes ppernce of the cogulum. the ntive bcteril popultion. In inoculted smples, the cogulum ppered between 8 nd 1 h fter inocultion. Results shown in Fig. 2 to 4 re the verge of four independent experiments. In ll of them, the optiml cogultion time ws 1 h. Both strins cused cogultion with equivlent kinetics. No bcteril growth ws observed during the incubtion period, either for the cogulting strins or for the utoctonous microflor (Fig. 3). Strins AFS-34 nd AFS-35 were enumerted on medium supplemented with streptomycin, which inhibited growth of the utoctonous microbil popultion. After 1 h of incubtion only 15 to 2% of the soluble protein remined in the superntnt (Fig. 2). On the other hnd, for the sme incubtion period, 8% of the protein in noninoculted smples ws soluble. To test whether the 2% protein cogultion ws due to the microbil ctivity of the utoctonous flor or to hydrolysis cused by the endogenous plnt enzymes, we mesured cogultion under conditions of no utoctonous microbil ctivity. Since the juice cnnot be sterilized without cusing protein precipittion, we decided to inhibit the metbolic ctivity of the utoctonous microflor by dding streptomycin to the green juice (1 jig- ml-'). Under these conditions, no survivors could be isolted from noninoculted smples, nd no differences in protein cogultion were observed. Nevertheless, when juice smples contining streptomycin were inoculted with either AFS-34 ( Smr isolte) or Smr derivtive of AFS-35, the mount of protein cogulted remined unltered. The protein content of the cogulum ws lso nlyzed to determine the cogulting efficiency of both strins compred with tht of het-treted nd noninoculted smples. For equl volume smples, the dry weight of the cogulum ws equivlent in het-treted nd inoculted smples, nd differences in their protein content were miniml (Tble 1). Thus under the conditions used, bcteril floccultion of the lflf juice seems to be equivlent to the therml process. To obtin informtion bout the nture of the process, we studied the physiologicl ctivity of both strins in the lflf juice. First, we mesured growth by using Smr derivtives to void interference with the ntive flor of the juice (Fig. 3). During the incubtion period no bcteril growth ws detected, lthough glucose ws ctively fermented in inoculted smples nd lrge mounts of gs were generted. This glucose fermenttion ws not pprent in noninoculted smples (Fig. 4). Sthmnn reported ph decrese from ph 6. to ph 3.5 to 4. (17). Our results showed moderte decrese in the ph vlues to 5.2 to 5.3 once cogultion hd occurred in inoculted smples. Nevertheless, noninoculted smples tht showed no cogultion for the sme incubtion time underwent similr drop in ph. The superntnts of strins AFS-34 nd AFS-35 did not hve flocculting ctivity. We lso tested both strins for the production of extrcellulr proteses, with negtive results. Effect of incubtion temperture, inoculum size, nd physiologicl stte of the culture on cogultion. We first estblished incubtion conditions by using s inoculum sttionry-phse cultures which were diluted 1:1 with the lflf juice nd incubted t 3 C to detect cogulting ctivity in the different strins tested. Once we hd selected strins AFS-34 nd AFS-35 nd chrcterized cogultion process under the conditions mentioned bove, we decided to chnge some of the conditions to study how they ffect cogultion nd whether the cogultion level cn be improved. First we tested whether the culture ge ffected cogultion. Smples grown in TSB were collected during the erly nd mid-log phses, nd erly nd lte sttionry phses. They were used in 1:1 rtio s inoculum for cogultion nd incubted under stndrd conditions. Cogultion ws mesured 8 h fter inocultion. The results obtined showed tht cells collected during the sttionry phse gve better efficiency in protein cogultion thn cells collected during the log phse (dt not shown). We lso tested the effect of the culture medium. Cultures grown in TSB, nutrient broth, Todd-Hewitt broth, nd M-9 miniml medium were used. After 24 h of incubtion, smples were studied for cogultion. No significtive differences between bcteril cells grown in the different medi ws found. A very importnt point ws to determine the miniml inoculum size tht produced n optiml cogultion efficiency. Cultures grown in TSB for 24 h were used s inoculum nd diluted 1:1, 1:1, 1:1,, or 1:1,, nd IX ';i- Dj n 1A Time APPL. ENVIRON. MICROBIOL. FIG. 3. Vible cell counts for the utoctonous bcteril popultion (A) nd strins AFS-34 () nd AFS-35 (-) during incubtion of the green juice. The rrow denotes ppernce of the cogulum. (h) 9 24

4 VOL. 53, 1987 BACTERIAL PROTEIN COAGULATION z I- u C, Time (h) FIG. 4. Vrition of the glucose concentrtion nd ph during the incubtion of noninoculted smples (A, A) nd smples inoculted with strins AFS-34 (@, ) or AFS-35 (U, O). The rrow denotes ppernce of the cogulum. cogultion ws evluted. A good cogultion efficiency (Fig. 5) ws obtined with miniml popultion of 17 cells ml-', corresponding to 1:1 dilution of the initil culture. We lso studied the effect of lower incubtion temperture. After inocultion (1:1), juice smples were incubted simultneously t 23 nd 3 C. After 8 h of incubtion, the cogulum ws removed nd the soluble protein in the superntnt ws mesured (Tble 2). The results clerly showed tht t 23 C none of the strins hd ny ctivity. Higher tempertures were lso tested (37 C). No significnt increse in cogultion efficiency ws observed t 37 C, lthough the cogultion time ws slightly reduced. Definition of continuous cogultion process. Strins AFS-34 nd AFS-35 did not growth on the lflf juice during the cogultion period. Furthermore, miniml cell popultion of bout 17 cells ml-' ws necessry for cogultion. For this reson, we tried to nlyze whether the frction of vible cells tht remined in the superntnt could be used s inoculum for new cogultion cycle. In series of five 1-dy experiments, the efficiency of the resulting superntnts ws tested (Tble 3). Probbly becuse of the decrese in the vible cell counts, good results were obtined only with the first superntnt. After second cogultion cycle, the resulting superntnt contined very low number of cells from either AFS-34 or AFS-35 nd did not show flocculting ctivity. TABLE 1. Protein content of cogul from het-treted nd inoculted smples % Concn in cogulum Smple C N Protein Het treted Inoculted with AFS Inoculted with AFS Noninoculted DISCUSSION In this report microbiologicl cogultion method for lflf green juice is described. Cogultion could be esily evluted s decrese in either soluble protein or pigment concentrtion. Two bcteril strins, E. coli AFS-34 nd Erwini crotovor AFS-35, showed the highest cogultion efficiency of the 6 strins we isolted from lflf rw mteril. Both strins cused protein cogultion under well defined conditions. The mount of protein recovered ws equivlent to the protein obtined when, in industril processes, the green juice is het treted. With respect to the spontneous fermenttion reported by Sthmnn (17), some dvntges in our process were pprent. When the lflf juice is inoculted with ny of our strins, cogultion tkes plce fter short period. Additionlly, the cogultion efficiency obtined is firly constnt if smples re inoculted nd incubted during such period. Protein cogultion in the green juice is probbly not cused by n extrcellulr enzyme secreted by ny of the strins. This conclusion is bsed on the observtions tht cell-free superntnts from e A4c 1 ol-e osi.7 t ND 1 lo 1i imoevlum (Cll.mgi-1) 1' 1" FIG. 5. Cogultion efficiency of different inoculum sizes of strins AFS-34 (-) nd AFS-35 (), evluted s the concentrtion of soluble pigments in the superntnt (A43).

5 221 GODESSART ET AL. TABLE 2. Reltionship between cogultion efficiency nd incubtion temperture of inoculted juice' Inoculum % Soluble protein t following tempb 23 C 3C AFS AFS None Smples were centrifuged fter 8 h of incubtion. bcogultion is expressed s the percentge of soluble protein in the superntnt. cultures of either strin AFS-34 or strin AFS-35 did not retin cogulting ctivity nd tht no extrcellulr proteolytic ctivity could be shown for ny of the strins. Neither strin ws ble to grow during the incubtion period. Bcteril growth ws lso not pprent for the utoctonous microbil popultion during the sme period. The high protein concentrtion in the juice my be the fctor tht inhibits bcteril growth. The fct tht in inoculted nd noninoculted smples bcteril growth is pprent only fter frction of the juice protein is cogulted supports this hypothesis. Although no growth ppered in inoculted smples, glucose ws ctively fermented. The fct tht mutnt derivtives of strins AFS-34 nd AFS-35 tht were unble to ferment glucose did not cogulte (mnuscript in preprtion) supports the hypothesis tht cogultion seems to be due to sugr-fermenttive metbolism crried out by strins AFS-34 nd AFS-35 under nonproliferting conditions. An ctive role of the utoctonous microflor on protein cogultion in inoculted smples cn be ruled out. Smples tht re inoculted incorporte bcteril popultion 1 times higher thn the utoctonous one. Additionlly, if the metbolic ctivity of the utoctonous microflor is inhibited with streptomycin, both strin AFS-34 ( Smr isolte) nd Smr derivtive of strin AFS-35 cogulte the green juice protein with identicl efficiency s in streptomycin-free medium. Under the conditions we used, the decrese of the ph in the superntnts ws not s importnt s in the results reported by Sthmnn (17). A decrese of only.5 to.7 ph units does not pper to be responsible for cogultion. TABLE 3. Cogulting cpcity of juice superntnt No. of vible Expt Strin cells in smple A43 1 AFS-34 2 x AFS-35 2 x AFS-34 7 x AFS x AFS x AFS-35 7 x AFS x AFS-35 2 x AFS-34 2 x AFS-35 2 x Juice smples were inoculted with either strin AFS-34 or strin AFS h cultures (finl cell concentrtion, 2 x 18 ml-'). Upon cogultion, the superntnt ws used s cogulting inoculum for the next experiment. Five experiments were connected; the cogulting inoculum for ech ws the superntnt obtined in the previous one. Probbly multifctoril system including ph, fermenttion products, nd secondry metbolites resulting from sugr fermenttion with no cell prolifertion, etc., cused protein cogultion. The incubtion temperture is n importnt fctor in the process. Tempertures lower thn 3 C seem to be indequte. Higher temnpertures did not increse the cogultion efficiency. Sttionry-phse cultures seem to be the most efficient in protein cogultion. The nture of the culture medi showed no effect t ll. The fcts tht the mximl effective dilution of strins AFS-34 nd AFS-35 is 1:1 with respect to the finl volume nd tht no growth ws observed during incubtion led us to conclude tht the obtention of the bcteril cell mss is one of the most importnt limiting fctors of the process. The reutiliztion of the cogulting bcteri from the superntnt upon cogultion is now under investigtion. The results described here suggest tht the use of microorgnisms my be n lterntive method for protein recovery from lflf nd other lefy-plnt juices. ACKNOWLEDGMENTS We thnk Isidre Csls for technicl ssistnce. This work ws supported by grnt from the Generlitt de Ctluny Comissi6 Interdeprtmentl de Recerc i Innovci6 Tecnol6gic. LITERATURE CITED APPL. ENVIRON. MICROBIOL. 1. Agusti J. A., nd J. P. Beltrn Quntittive determintion of the protein citrus lef extrcts: comprtive study. Anl. Biochem. 127: Brdford, M. M A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizting the principle of protein-dye binding. Anl. Biochem. 72: De Fremery, D., E. M. Bickoff, nd G.. Kohler Pro-Xn process: stbility of proteins nd crotenoid pigments in freshly expressed lflf juice. J. Agr. Food Chem. 2: De Fremery, D., R. E. Miller, R. H. Edwrds, B. E. Knuckles, E. M. Bickoff, nd G.. Kohler Centrifugl seprtion of white nd green protein frctions from lflf juice following controlled heting. J. Agr. Food Chem. 28: Dubowsky, K. M An o-toluidine method for body fluid glucose determintion. Clin. Chem. 8: Edwrds, R. H., R. E. Miller, D. De Fremery, B. E. Knuckles, E. M. Bickoff, nd G.. Kohler Pilot plnt production of n edible white frction of lef protein concentrte from lflf. J. Agric. Food Chem. 23: Hollo, J Le proteine non convenzili per l'limentzione umn: le proteine estrtte d foglie. Riv. Itl. Sostnze Grsse 52: Hollo, J., nd L. Koch Commercil production in Hungry, p In N. W. Pirie (ed.), Lef protein: its gronomy, preprtion, qulity nd use. IBP Hndbook 2. Blckwell Scientific Publictions, Oxford. 9. Kinsell, J. E Evlution of plnt lef s source of food protein. Chem. Ind. 17: Knuckles, B. E., R. H. Edwrds, G.. Kohler, nd L. F. Whitney Flocculnts in the seprtion of green nd soluble white protein frctions from lflf. J. Agric. Food Chem. 28: Knuckles, B. E., R. R. Spencer, M. E. Lzr, E. M. Bickoff, nd G.. Kohler Pro-Xn process: incorportion nd evlution of sugr cne rolls in wet frctiontion of lflf. J. Agr. Food Chem. 18: Miller, J. H Experiments in moleculr genetics. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, N.Y. 13. Miller, R. E., D. De Fremery, E. M. Bickoff, nd G.. Kohler Soluble protein concentrte from lflf by low temperture cid precipittion. J. Agric. Food Chem. 23: Pirie, N. W The present position of reserch on the use of

6 VOL. 53, 1987 BACTERIAL PROTEIN COAGULATION 2211 lef protein s humn food. Plnt Foods Hum. Nutr. 1: Pirie, N. W Lef protein: its gronomy, preprtion, qulity nd use. IBP Hndbook 2. Blckwell Scientific Publictions, Oxford. 16. Smibert, R. M., nd N. R. Krieg Generl chrcteriztion, p In P. Gerhrdt, R. G. E. Murry, R. N. Coshlow, E. W. Nester, W. A. Wood, N. R. Krieg, nd G. B. Phillips (ed.), Mnul of methods for generl bcteriology. Americn Society for Microbiology, Wshington, D.C. 17. Sthmnn, M. A Anerobic fermenttion for cogultion of plnt juice protein nd preservtion of protein nd fibrous residues, p In Proc. 2nd Int. Green Crop Drying Congress. University of Ssktchewn, Ssktoon, Ssktchewn, Cnd.