Production of Kefir from Soymilk with Or Without Added Glucose, Lactose, Or Sucrose JE-RUEI LIU and CHIN-WEN LIN

JFS: Food Microbiology and Safety

Production of Kefir from Soymilk With or Without Added Glucose, Lactose, or Sucrose JE-RUEI LIU AND CHIN-WEN LIN

ABSTRACT: The effects of added glucose, lactose, and sucrose on microbial growth, acid, and ethanol production, and galactosidase activity in soymilk fermented with kefir grains were studied. Immediately after the addition of kefir grains to soymilk, the lactic-acid bacterial counts were higher, but the yeast counts were lower than in milk kefir. After fermentation for 32 h, the concentrations of yeast, lactic acid, and ethanol in soymilk were significantly lower than those in milk kefir. Addition of 1% glucose to soymilk stimulated growth of lactic-acid bacteria and yeast, the production of lactic acid and ethanol, and the -galactosidase activity. Nevertheless - galactosidase activity was suppressed by 1% glucose. Key Words: kefir, soymilk, carbohydrates, growth characteristic, galactosidase activity

Introduction Because both kefir and soymilk are beneficial to health, in EFIR IS AN ACIDIC AND MILDLY ALCOHOLIC FERMENTED MILK this study we attempted to produce soymilk kefir and also inves- Kalleged to have originated in the Caucasus mountains. This tigated the effects of different added carbohydrates on the beverage differs from other milk products in that it is not the re- growth characteristics of kefir grains in soymilk. sult of the metabolic activity of a single species, but of fermentation with a mixed microflora confined to a matrix of discrete kefir Results and Discussion grains that are recovered after fermentation (Marshall and Cole IGURE 1 SHOWS THE CHANGES IN LACTIC-ACID BACTERIAL 1985). Kefir grains are small, gelatinous, yellowish, and irregular- Fcounts during fermentation of soymilk and milk (control) ly shaped masses resembling individual miniature florets of a with kefir grains. Immediately after the addition of kefir grains to head of cauliflower. Kefir grains contain a complex flora of lactic- the milk and soymilk, the concentrations of lactic-acid bacteria acid bacteria, yeasts, and sometimes acetic-acid bacteria. The were 6 log CFU/mL and 7 log CFU/mL, respectively. This indi- flora is held together in a slimy polysaccharide matrix named ke- cates that part of the microflora contained in the kefir grain is firan, which is the result of the microbial metabolism of milk lac- transferred to milk and soymilk. Although the initial counts of tose (La Riviere and others 1967; Marshall and others 1984). Yeast lactic-acid bacteria in soymilk were higher than those in milk, the and lactic-acid bacteria coexist in a symbiotic association and are microorganisms grew more slowly in soymilk, even with the addi- responsible for an acid-alcohol fermentation. Various lactic-acid tion of carbohydrates. After incubation for 32 h, the lactic-acid bacteria and yeasts have been identified in kefir grains, includ- bacterial counts increased by 2.3 log cycles from 5.9 0.1 to 8.2 ing Lactobacillus brevis, L. helveticus, L. kefir, Leuconostoc me- 0.2 log CFU/mL in milk, but by only 1.6 log cycles from 7.4 senteroides, Kluyveromyces lactis, K. marxianus, and Pichia fer- 0.2 to 9.0 0.1 log CFU/mL in soymilk without added carbohy- mentans (Angulo and others 1993; Lin and others 1999). The mi-

Food Microbiology and Safety croorganisms constituting the kefir grains produce lactic acid, an- tibiotics, and bactericide, which inhibit the development of de- grading and pathogenic microorganisms in kefir milk (Angulo and others 1993). Furthermore, kefiran is reported to possess antitumor activity (Shiomi and others 1982). Soybeans contain a number of anticarcinogens (Messina and others 1994), and a recent National Cancer Institute workshop rec- ommended that the role of soy foods in cancer prevention be investigated. There is much evidence from studies on experimental animal and human subjects that substituting soy protein for animal protein in the diet reduces the concentration of total and low- density lipoprotein cholesterol in plasma and serum (Carroll and Kurowska 1995). Soymilk, obtained by aqueous extraction of whole soybeans, is well-known and growing in popularity in many areas of the world (Haumann 1984). Soymilk may afford nutrition and health benefits, since it contains no cholesterol or lactose and only small quantities of saturated fatty acids. However, many peo- ple find the taste of soymilk undesirable. Soymilk is commonly characterized as having a beany, grassy, or soy flavor, which report- edly can be improved by lactic acid fermentation (Murti and others 1992; Granata and Morr 1996). Kikuchi and others (1998) showed that both soymilk and Bifidobacterium-fermented soymilk Fig. 1—Growth curves of lactic acid bacteria in milk and soymilk have lowering plasma cholesterol effects. fermented with kefir grains

716 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 4, 2000 © 2000 Institute of Food Technologists drate. The major carbohydrates present in soymilk are sucrose, Figure 4 shows the production of ethanol during fermentation raffinose, and stachyose, whereas in milk it is lactose (Pinthong of milk or soymilk containing 1% glucose, lactose, sucrose, or and others 1980). The fact that lactic-acid bacteria from kefir without added carbohydrate. At the end of fermentation, grains grew well in soymilk, even when no extra carbohydrate soymilk containing 1% glucose or lactose had ethanol concentra- was added, means that these organisms can utilize soymilk car- tions similar to that of milk kefir (0.25% to 0.26%). Soymilk with- bohydrates for growth. out carbohydrate addition showed the lowest ethanol produc- The initial counts of yeasts in milk and soymilk without added tion (0.11 0.01%). carbohydrate were 5.8 0.1 log CFU/mL and 4.7 0.2 log CFU/ Ethanol, together with carbon dioxide, gives kefir its stimulat- mL, respectively (Fig. 2). In milk, yeast reached the stationary ing and effervescent characteristics. The ethanol content in kefir growth phase at 20 h of fermentation, with a concentration of 6.6 ranges widely in the literature. For example, Marshall and Cole 0.1 log CFU/mL. Soymilk kefir with 1% glucose had the highest (1985) reported that 0.17 % ethanol makes a good kefir, and yeast density (6.4 0.1 log CFU/mL) of the soymilks at the end Duitschaever and others (1987) reported that 0.12% to 0.18 % of fermentation, while soymilk without added carbohydrate had ethanol was produced in sequential fermentation of kefir. Korol- the lowest. The yeast count did not differ significantly between eva (1988) indicated that many factors affect the percentage of milk and 1%-glucose soymilk kefir at the end of fermentation. ethanol in kefir, such as time and temperature of fermentation, Yeast is important in kefir fermentation because of the produc- starter culture, and containers. Our study showed that the addition of ethanol and carbon dioxide, which give the kefir drink its tion of 1% glucose or lactose to soymilk enhances the production unique taste. Kefir grains usually contain lactose-fermenting yeasts such as Kluyveromyces lactis, Kluyveromyces marxianus, Torula kefir, as well as nonlactose-fermenting yeasts such as Sac- charomyces cerevisiae and Pichia fermentans (Angulo and others 1993; Lin and others 1999). Because the type of fermentable carbohydrates present in soymilk and milk are different, the growth characteristics of microorganisms in kefir grains may change, resulting in dissimilarity of the microflora present in kefir grains. From the above results, we conclude that the addition of 1% glucose greatly enhances growth of both lactic-acid bacteria and yeast in soymilk. Lactic acid is extremely important for producing high-quality fermented milk, and appropriate concentrations are needed to ensure proper flavor with minimum syneresis during storage (Karleskind and others 1991; Granata and Morr 1996). Previous studies indicated that yogurt culture can not produce adequate lactic acid in soymilk (Lee and others 1990; Karleskind and others 1991). We found that at the end of fermentation, the concentration of lactic acid in milk kefir (1.6 0.3%) was significantly higher than in soymilk kefir without added carbohydrate (0.9 0.1%) (Fig. 3). However, adding 1% glucose or lactose to soymilk result- ed in lactic-acid concentrations similar to those of milk kefir, showing that the addition of these carbohydrates improves the ability of microorganisms in kefir grains to produce lactic acid in Fig. 3—Lactic acid production in milk and soymilk fermented with soymilk. kefir grains Food Microbiology and Safety

Fig. 2—Growth curves of yeasts in milk and soymilk fermented with Fig. 4—Ethanol production in milk and soymilk fermented with kefir kefir grains grains

Vol. 65, No. 4, 2000—JOURNAL OF FOOD SCIENCE 717 c e a d b 28 OF 30 39 46 73

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different carbohydrates on the growth characteristics of different carbohydrates on the growth E To measure the concentrations of lactic acid, 1 g of each the concentrations measure To The concentrations of ethanol in samples were determined Lactic-acid bacteria were enumerated on spread plates of kefir grains in soymilk. Future research may include investiga- kefir grains in soymilk. Future research its effects on health. tions of the kefiran from soy kefir grains and Determination of lactic-acid production Determination of ethanol Fig. 5—Viscosity of milk and soymilk fermented with kefir grains Fig. 5—Viscosity of milk and soymilk fermented W milk and inoculated with 5% kefir grains. All inoculated soymilk samples were incubated at 20 Enumeration of culture microorganisms Table 1—Mean 1—Mean Table kefir grains with fermented and soymilk Soymilk glucoseSoymilk + 1% Soymilk + 1% lactose 1694 Soymilk + 1% sucrose 4 h for cell counting fermentation, samples were taken every and analysis of other properties. sample was mixed with 9 mL deionized distilled water, and 0.1 distilled water, sample was mixed with 9 mL deionized N NaOH was used as the titrant. Phenolphthalein was used as lactic acidity was expressed as percent Titratable the indicator. acid in the kefir. MRS agar (Merck, Darmstadt, Germany) containing 200 mg/L cycloheximide after anaerobic incubation at 30 De- enumerated on potato dextrose agar (Difco, were Yeasts troit, Mich., U.S.A.) containing 100 mg/L chlortetracycline after aerobic incubation at 25 logarithmic colony forming units (log CFU)/mL kefir. Milk C C for 20 -galactosidase C for 24 h. The Lactobacillus fer- C, and stored at 4 C for short- and long- -galactosidase activity. -galactosidase and may -galactosidase 80 —Vol. 65, No. 4, 2000 65, No. —Vol. -galactosidase activity was -galactosidase activity C or -Galactosidase is needed to digest -Galactosidase -galactosidase from Materials and Methods - galactosidase activity was higher in soymilk without - galactosidase activity , but that exogenously supplied glucose inhibits the , but that exogenously supplied glucose JOURNAL OF FOOD SCIENCE Sterilized reconstituted skim milk (10% w/v), soymilk, and Soymilk was prepared by a modification of the procedure Kefir grains were collected from households in northern Some lactic-acid bacteria possess bacteria possess Some lactic-acid The fermentable carbohydrates in soybeans and soybean soybeans in carbohydrates The fermentable After 32 h of fermentation, the After 32 h of fermentation, The viscosity of soymilk kefir was lower than that of milk ke- The viscosity of soymilk kefir was lower until used. Kefir manufacture Preparation of soymilk Kefir grains soymilk containing 1% glucose, lactose, or sucrose were each of Mital and others (1974). One kg dry, mature, whole soy- mature, and others (1974). One kg dry, of Mital beans was soaked in 3 L distilled water at 25 soak water was decanted, and the beans were washed and ground in 6 L boiling distilled water in a blender (Waring Divi- sion, Dynamics Corporation, New Hartford, Conn., U.S.A.) The resulting suspension was filtered through 3 layers of cheesecloth, autoclaved 15 min at 121 Taiwan. In the laboratory, they were propagated at 20 they were the laboratory, In Taiwan. h with twice- or thrice-weekly transfers in sterilized cow milk or soymilk (50 g/L) and kept at 4 term storage, respectively. term storage, 718 significantly higher in milk than in any of the soymilk kefirs, significantly higher while the carbohydrate added than in milk kefir (Table 1). The addition of 1). added than in milk kefir (Table carbohydrate the 1% glucose or lactose to soymilk increased products are low-molecular-weight oligosaccharides such as su- oligosaccharides low-molecular-weight products are 1975). (Mital and Steinkraus and stachyose crose, raffinose, sugars: fruc- up of 3 simple and stachyose are made Raffinose and galactose. tose, glucose, series. galactosido-sucrose sugars of the Characteristics of Soymilk Kefir . . . . Kefir Soymilk of Characteristics of ethanol. use galacto-oligosaccharides for growth and acid production (Mi- use galacto-oligosaccharides Measuring sugar content of a fermented tal and others 1973). an accurate index of galactosidase activity dairy product is not and then galactose are rapidly metabolized because first glucose bacteria (Kilara and Shahani 1976). to lactic acid by culture activity in kefir must be measured Therefore, the galactosidase the activity of microorganisms in kefir directly to determine grains. but decreased activity of kefir grains by Garro and others This result is consistent with that reported raffinose stimulate the (1996), who showed that stachyose and synthesis and activity of mentum in soymilk were fir (Fig. 5), and the kefir grains subcultured in milk (results not much smaller than those subcultured the kefir grain consists shown). At least 24% of the dry matter of by the lactobacilli in of kefiran, which is thought to be produced temperature, medium the grain. Growth conditions (such as carbon source have a composition, and incubation time) and exopolysaccha- considerable influence on lactic-acid bacterial others 1992). Thus, ride yield and composition (Cerning and in this study may the smaller size of the kefir grains in soymilk less kefiran when suggest that lactic-acid bacteria produce grown in soymilk. synthesis of the enzyme.

Food Microbiology and Safety by enzymatic methods described by Bernt and Gutmann phosphate buffer (pH 7.0) was added to the assay mixture, (1974). and the mixture was vortexed for 10 s and incubated at 37 C for 10 min. The reaction was terminated by adding 1 mL 0.5 M Lactase assays Na2CO3. The sample was then centrifuged at 16,000 g for 15 -Galactosidase activity was measured by determining the min to remove cell debris. The release of o-nitrophenol was de- rate of hydrolysis of o-nitrophenol--galactopyranoside (Sig- termined at 420 nm. A standard curve was used to determine ma Chemical Co., St. Louis, Mo., U.S.A.), and -galactosidase the amount of o-nitrophenol that was liberated. One unit of activity was measured by determining the rate of hydrolysis of enzyme activity released 1 M of o-nitrophenol/ min. o-nitrophenol--galactopyranoside (Sigma Chemical Co., St. Louis, Mo., U.S.A.) Hydrolysis of these substrates results in the Determination of viscosity release of o-nitrophenol, a highly chromogenic compound that Viscosity was determined with a Brookfield Viscometer can be measured spectrophotometrically (Citti and others (Stoughton, Mass., U.S.A.) equipped with a helipath stand 1965). A 1-mL sample was added to 50 mL 0.1 M phosphate and a LV-3 spindle. buffer (pH 7.0) containing 0.001 M MgSO4 and 0.005 M -mer- captoethanol. Then 1 mL of the diluted sample was with- Statistical analysis drawn, and 2 drops of chloroform and 1 drop of 0.1 % sodium Data were analyzed using the general linear model proce- dodecyl sulfate were added to it. The mixture was vortexed for dure of the SAS software package (SAS Institute 1996). Duncan 10 s and incubated at 37 C for 5 min. multiple range test (Montgomery 1991) was used to detect dif- Next, 1 mL of 15 mM o-nitrophenol--galactopyranoside or ferences between treatment means. All experiments were rep- 10 mM o- nitrophenol--galactopyranoside solution in 0.02 M licated three times.

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