4083 Lactobacillus acidophilus Utilization ofSugars and Production of a Fermented Soybean Product..
N. J. Stern~. C. W. Hesseltine, H. L. Wang and F. Konishi' Nonhem Regional Research Laboratorv. Ae:ricultural Research Service. U.S. Oepanment of Agriculture. Peoria. Illinois 61604
Data are from pontOlU ofa lhesis submitted 10 the Depanmenl of Food ana Nutrluon. Soulhern Bacto-Yeast extract, 7.5 g; Bacto-tryptone, 12.5 g: Bacto· illinoIS University. Carbonaale. by N. J. Slem. III llama! fulfillment of the requirement for the oJetzree of Master of SCIence. dextrose, 10.0 g; sodium citrate, 5.0 g; thiamine hydro· , pre.ent aadress: Food SCIence Technology DellanmenL Virgtnia. PolYlechnlc Insulute. BIac:Ju. bul'2. Vil'lttnta 24061. chloride, 0.001 g; sodium chloride, 5.0 g: dipotassiuIT Dell-anmeill of Food and Nutnllon. Southern illinoIS University. Carbonaale. illinoIS 62901. phosphate, 5.0 g; manganese chloride. 0.14 g; magnesium sulfate, 0.8 g; ferrous sulfate. 0.04 g; sorbitan monooleate Abstract complex, 0.2 g) (Baltimore Biological. Cockeysville. Mary Eight strains of Lactobacillus acidophilus. obtained from the ARS land); and 0.1% Tween 80. Stock culture transfers were Culture Collection. were tested for their abilitY to use monosaccharides made on a monthlv basis to maintain viabilitY. [fructose. galactose. glucose and 0 (+) mannosej. disaccharides (lactose. " , maltose and sucrose) and two oligosaccharides (raffinose and stachyose). Inoculum Inoculum was grown and standardized in a As judged by titratable acidity and pH changes during growth. stnin vari broth of 4.5% A.PT. media~ and 0.1% Tween 80. The ation existed in the ability of the microoreanisms to utilize the sue:ars. Strain NRRL 8-1910 was asuperior utilizerof raffinose and stachyose as A.P.T.-Tween 80 broth was sterilized in 12 x 125 mm glass measured by medium pH changes. Therefore. 8-1910 was selected as tubes by autoclaving at l2loC for 15 min. After cooling inoculum for the successful production of a yoghun-like soybean prod the sterile medium, specific strains were aseptically added uct, which has potential as a good protein food source. and grown stationary for 24 hr at 37°C. Cells were then harvested in a Safety-Head centrifuge (Clay-Adams, Inc.) Resume at about 3500 rpm for 10 min. Supernatant was discarded: On a fait l'etude de huit lignees de Lactobacillus acidophi/us, en prove nance de la Collection de cultures ARS pour leur aptitude a utiliser les the cells were suspended in sterile 0.1% peptone solution monosaccharides (fructose. galactose. glucose et D (+) mannose}, les di and centrifuged two additional times. After the final wash. saccharides (lactose. maltose et sucrose), et deux oli20saccharides (raffi cells were resuspended in peptone, and optical density was nose et stachyose). O'apres les changements de l'acidItl~ titrable et du pH adjusted to 10-12% light transmission at 500 nm in a avec la croissance. on a observe que I' aptitude aI'utilisation des sucres va riait avec la Iignee. Les changements du pH medium ont indique que la Bausch and Lomb Spectronic 20. lignee NRRL 8-1910 etait superieure aux autres pour I'utilisation du raf Carbohydrate utilization Test medium (50 ml) for car finose et du stachyose. En consequence. on a choisi cene Iignee comme bohydrate utilization was sterilized in 250-rnl Erlenmeyer inoculum pour la production reussie d'un simili-yoghoun a base de soja. lequel a un potentiel comme une bonne source de proteines alimentaires. flasks. The medium consisted of peptone, 1.0%; yeast ex tract, 1.0%; Tween 80. 0.1%; and the specific sugar se Introduction lected, 0.5%. The sugar solutions were sterilized by Milli Lactobacillus acidophi/us ferments glucose by homo pore filtration and, subsequently, added to the test fermentation into lactic acid (Breed et ai., 1957). Mital and medium. After specific sugar addition, standardized inocu Steinkraus (1974) have shown that L acidophi/us strain lum (2.0%) of each strain of the bacterium was added sepa ATCC 4356 uses glucose and sucrose but not melibiose. rately to the flasks and incubated (stationary) at 37°C. raffinose and stachyose. Aside from generalizations about At successive 12-hr incubation intervals (for a total of L. acidophilus, the literature contains little about various 48 hr), 10 ml of uniformly suspended L acidophilus cul strains of the species using other specific sugars. One pur tures were examined for growth and acid production. A pose ofour study was to determine how eight strains of the Beckman Model G pH meter was used to measure pH microorganism used fructose, galactose, glucose. lactose, changes. Reading;s were standardized with sterile test me maltose. D( +) mannose, raffinose. stachyose and sucrose. dium~ as controls: Culture samples (5.0 ml at each 12 hr) A second purpose was to produce a yoghurt-like product were titrated with 0.025 N NaOH with phenolphthalein as from soybean using "preferred" strains of the lactobacilli. the indicator. Titratable aciditv was determined bv sub The L. acidophilus strain that utilized best the three pri tracting the amount ofacid preS'ent in an uninoculated me mary soluble sugars in soybeans (sucrose 5.0%. stachyose dium sample from that found in the test culture. 3.8% and raffinose 1.1%) (Smith and Circle, 1972) was cho Fermentation ofsoybean milk A schematic outline of sen to ferment soybean milk. Also included were organ the fermentation process is shown in Figure 1. oleptic evaluations of the fermented product. Soyb~an milk was prepared along with subsequent dena turatlon as follows: Beeson soybeans were thoroughly Materials and Methods washed and dehulled after soaking; for 24 hr in distilled Cultures Eight lyophilized strains of L. acidophilus water at room temperature. The soaked beans were placed were supplied by the ARS Culture Collection, maintained in distilled water and blended into a thick slurry in a War at the Northern Regional Research Laboratory. The ing Blender at maximum rpm (high speed). The ratio of lyophilized strains were grown in a medium consisting of water to dry beans was 9: 1. The slurry was filtered throuE:h liver extract. 10%; yeast extract. 0.5%; tryptone. 1.0%; several layers of cheesecloth into a'lOOO-ml Erlenmeyer K~HPOH 0.2%: glucose. 0.5%; and unweighed particles of flask. Portions of50 rnl raw soybean milk was then poured liver chips. The cultures were maintained on ag;ar slants into 100-ml beakers. covered with cotton-filled gauze lids. containing 2.0% agar: 4.5% A.P.T." media (conSisting of: and denatured by autoclaving at 121°C for 5 nUn: imme-
J. InSI. Can. SCI. Techno!. .... liment. Vo!. 10. No.3. July 1917 197 Soak soybeans (24 hr] Table I. The utilization of various sugars by eight strains of LActobacillus acidophilus ~valuated by determining titratable acidity' after 48 Discard excess water hr of incubation. Add water to a NRRL strains water:soybean ratio of 9:1 Sugar 629 1833 1858 1910 1911 1912 2092 2178
Blend 3 minutes at high Fructose 6.1 4.7 7.0 6.6 9.7 10.3 5.9 9.8 speed Galactose 1.6 1.5 7.8 3.4 7.1 9.6 3.1 4.4 Glucose 7.6 7.4 13.1 5.9 9.2 12.3 8.0 6.6 Filter through cheesecloth Lactose 5.9 4.7 3.4 5.7 13.3 8.2 3.2 6.3 Maltose 4.3 3.3 3.2 5.5 10.0 12.3 5.1 7.6 Autoclave 5 minutes o (+ ) mannose 3.4 6.0 6.2 5.3 11.6 13.5 3.3 9.5 Raffinose 0.9 1.3 1.9 2.4 5.4 2.1 2.2 1.8 While hot add 0.5% gelatin Stachyose 1.2 1.4 1.7 3.5 1.9 2.7 2.0 1.9 and COVeT Sucrose 3.0 4.4 13.4 8.5 3.6 5.2 8.9 10.5 Cool to 37°C and inoculate 'Titratable acidity - ml O.025N NaOH required to neutralize 5.O-ml sample: average of duplicate experiments. Least significant difference is with 5% inoculum l.ll for all strains; significance level is 0.05 for fructose and 0.0 I for all other sugars. Cover and incubate at 37°C for twenty - four hours B-1911,"B-1912 and B-2178 to be superior (p < 0.05) in the utilization of the fructose, whereas B-1912 was sig nificantly higher (p < 0.01) in the titratable acidity pro Soybean Yoghurt duced in galactose medium compared to other strains. Al though all strains utilized glucose well, NRRL B-1858 and Fig. l. Schematic outline ofsoybean yoghun production. B-1912 were the best at the 1.0% confidence level. Fre quently, monosaccharides are convened from one isomer diately, previously sterilized gelatin was added in a con to another by enzymes, such as isomerase. The differences centration of 0.5% gelatin. The milk was cooled to below in strain utilization ofspecific sugars are attributable to the 37°C, inoculated with various concentrations of previously varying levels of enzymes that conven sugars into glucose. standardized inoculum, and incubated at various tempera For both lactose and maltose, NRRL B-1911 and B tures for various times. Inocula tested were 2.0%, 5.0% and 1912 were superior in acidity produced at the 1.0% con 10.0%; incubation temperatures, 34°, 37° and 43°C; incu fidence level than any of the other strains (Table 1). These bation times, lO, 14. 18,20. 24 and 48 hr. Also determined two apparently contained significant levels of lactase and was dry soybean concentration in the milk, which varied maltase to break down the disaccharides into their con from 6: 1 to 8: 1 to lO: l-water:dry soybeans. Observations stituent monosaccharides of glucose and galactose. How were made on the effects of adding 0.1% to 3.0% concen ever, data indicate sig:nificant strain variation in rate of trations of xanthan gum.. corn starch or gelatin as stabiliz lactose utilization. Two strains. B-1858 and B-2l78, were ing: ag:ents. outstanding: in their use of sucrose (p < 0.01) Presumably. ~ Definition of optimum product development was the difference in strain utilization ofsucrose is due to levels based on pH of the end products and their viscosity which of invertase present: the enzyme bre~ down sucrose to is determined by the radius of a circle formed by dropping !Zlucose and fructose for further catabohsm. a 20-ml sample from a height of 10 cm. The syneresis was ~ A comparison of titratable acidity (Table l) and the measured by decanting the whey portion of a 50-ml pH change after 48-hr growth (Figures 2 and 3 ) best dem sample into a graduate cylinder. Measuring texture was onstrates the utilization of raffinose and stachyose by the based on a subjective opinion of coarseness. eiaht L. acidophiius strains. The pH measures the free pro A panel of 14 experienced tasters described and rated to~ in solution. whereas the titratable acidity measures the intensity of odors and flavors of all samples at room the total amount of acid developed by the microorganisms. temperature. Flavor intensity value (FlY) of individual de Lower pH and greater titratable aci~iry ~dicate better scriptions were based on 1 for weak: 2, moderate: and 3. growth that, in turn, suggests better utIlizatIon of a sugar strong. Usually two samples were presented in random or by the strain being tested. As indicated by pH change (Fig. der at each tasting to prevent sample interaction. and sam 2), NRRL B-19lO and B-1911 utilized raffinose sig ples with the least odor were tasted first. Statistical differ nificantly better than all the other strains. These results co ences between samples were determined by analysis of incide in part with the findings of Mital et al. (1974) but variance (Snedecor and Cochran. 1967) based on overall differ from those of Wheater ( 1955), who found 62% of the standard deviations within trials or on variations in the 29 L acidophiius tested utilized raffinose. The two strains mean score for the same material in different trials. in our study that utilized raffinose significantly probably had sufficient levels of invertase and an alpha-galactosi Results and Discussion dase to enable a hydrolysis of raffinose (Mital and Carbohydrate utilization As determined by titratable Steinkraus. 1975). " " aciditv of the g:rowth medium after 48-hr incubation. !Zlu The two strains utilizin!Z raffinose also utilized stach cose utilization was uniformlv !Zood for all ei!Zht strains of yose (Figure 3) with NRRL B-19lO being outstanding. L. acidophilus (Table I). Titratable acidity showed NRRL NRRL B-191O had not completed growth at the end of 48
198 Can. Insl. Food Sci. Technol. J. Vol. 10. No.3. July 1977 6.6 &.&
6.0 6.0
5.0 5.0
=A =A
NRRl B· and NRRl 8· and straills IIsti strain lISId -+- 629 -+- 629 4.0 -,,-1833 4.0 -,,-1833 -0-1858 -0-1858 -6-1910 -6-1910 -0- 1911 -0- 1911 -.- 1912 -.- 1912 -.-2092 -.-2092 -e";" 2178 -e- 2178
12 24 3& 48 12 24 3& 48 Time Ihours I Time Ihoursl Fig. 2. The pH change of raffinose medium by eight strains of Lactoba· Fig. J. The pH change of stachyose medium by eight strains of Lactoba· cillus acidophilus. cillus addophilus. hr. whereas 8-1911 reached a growth plateau after 12-hr vors associated 'with soy products (Wang et al.. 1974). The incubation. Again. the presence of an alpha-galactosidase NRRL taste panel determined that the grassy/beany fia allowed 8-1910 to utilize stachyose fully as it did. vor of soybeans was reduced considerablY in the soYbean Soybean yoghurt production Strain 8-19i O. which best uti milk fermented yoghurt (Table 2). The grassy/ beany fla- lized raffinose and stachyose, was selected for the soYbean yoghurt production. Other experiments indicated that Table 2. Flavor intensity values' assigned by a 14-member taste panel. 5.0% inoculum. an incubation time of 24 hr at 37°C and Soybean Yoghurt addition of0.5% 2elatin to the medium would 2ive the best with product. Several temperatures (85°. 100° and·121 ° C) and Descnptions Soybean milk Soybean yoghurt 3'C sucrose times (15 and 5 min and 3 sec) were used in denaturin2 min~ Grassvi beanv :U 0.1 0.7 soybean milk before deciding upon 121 °C for 5 Rancid . 0.6 1.4 0.5 Denaturing soy proteins allows for a greater water-hOlding Bitter 0.9 0.6 capacity by the yoghurt and therefore less syneresis. When soYbean milk was not denatured. much syneresis occurred. 'Flavor intensitv values based on: I = weak: 1 = moderate: 3 .. stron!!. A 0.4 difference indicates signirlcance in description between productS. - High temperature also destroys the lipo'xygenase systems that partially contribute to rancidity. Incubation at 37°C vor of soybean products is a common complaint of West minimized syneresis and resulted in a smooth curd. erners. The soYbean voe:hurt with 3.0% sucrose added was A distinct advantalZe in choosinlZ the strain that fer judged more ctesirabie than soybean milk or soybean yog ments both stachyose and raffinose lies in the fact that the hurt. The taste panel's only complaint was that a rancid human gastrointestinal tract does not possess alpha-galac "off-flavor" existed in the voe:hurt itself and if this flavor tosidase (Gitzelman and Auricchio. 1965). When a food could be eliminated. the yoghurt would be acceptable as a containing these sugars is ingested. they pass undigested to substitute for traditional foods. For example. dairy yog the lower g:ut where various bacteria evolve undesirable hurt has been a lon2 standine: tradition in Iranian diets. methane. carbon dioxide and hydrolZen g:asses (GalL 1968: and soybean yoghurt would be an inexpensive. substitute Richards et al.. 1968: and Steggerdil et·al.. 1966). protein source for that country. The American Soybean Alone: with a reduction of undesirable flatulence fac Association has served as liaison between USDA and the tors. fermenting; soybean milk reduces objectionable fla- Iranian Government in the development of an acceptable
J. lnst. C~n. Sc,. Tochnol. .">limen!. Vol. 10. No. J. July 1977 199 Gitzelman. R. ~nd Auncchlo. F. S. 1965. The handlinll oi sova alphaRalilCtosldes bv a normal .. product. Iranian taste preferences for an extremely tart a Ralaetosemlc chlhl. Pedta"":> JIll!I: 23 I. ... - . flavored yoghurt was taken into consideration during the \ittal. B. K.. SteInkraus. K. H.. Jnd :-;avlor. H. 8. 1974. Growth of lactIC aCId bilet."a ,n so' mdks. J. Food Set. 39: 1018. . . development of our fermented soybean product. Mita!. 8. K. and Stelllkraus. K. H. 1975. UlIhzallon oiohgosacchandes by IaClIC ileld bacte,", dut· InR fermentallon oi SOy milk. J. Food SeL dO: 114. RacklS. j, 1.. Sessa. D. 1.. Steggerda. F. R.. ShimIZU. T.. Anderson. 1.. anll Pearl. S. L. 1970. Sov Acknowledgements bean lactO" relallng to gas produCllon by Intesllnal bactena. J. Food SCL 35: bJ4. Richards. E. A.. Steggerda. F. R.. and Murata. A. 1%8. Relallonshlp of bean substrat.. and cer· We thank Ms. K. A. Warner for conductim~ the taste tilln tnt..lInai bact."a to !!"5 produellon 10 the dog. Ga..roen
200 Can. Inst. Food SCI. Technol. J. Vol. 10. No.3. July 1977