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Rheological Characteristics and Microstructure of Probiotic Soy Yogurt Prepared from Germinated Soybeans

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Rheological Characteristics and Microstructure of Probiotic Soy Yogurt Prepared from Germinated Soybeans M. YANG et al.: Rheology of Sogurt from Germinated Soybeans, Food Technol. Biotechnol. 50 (1) 73–80 (2012) 73 ISSN 1330-9862 original scientific paper (FTB-2743) Rheological Characteristics and Microstructure of Probiotic Soy Yogurt Prepared from Germinated Soybeans Mei Yang1,2, Jing Fu1,3 and Li Li1* 1Research and Development Center of Food Proteins, College of Light Industry and Food Science, South China University of Technology, Guangzhou 510641, PR China 2Research Group for Bioactive Products, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China 3Bio-Resources Key Laboratory of Shaanxi Province, Shaanxi University of Technology, Hanzhong 723001, PR China Received: February 24, 2011 Accepted: June 28, 2011 Summary Soy yogurts (sogurts) were prepared from germinated soybeans [Glycine max (L.) Mer- rill] with different hypocotyl lengths. This study is mainly focused on the effects of soy- bean germination on the rheological characteristics and the microstructure of sogurt. Results show that, after soybean germination, sogurt had lower storage module (G’) and loss mod- ule (G’’) in the linear viscoelastic region than the sogurt from ungerminated soybean. Shear sweep test demonstrated that the flow behaviour index (n) of sogurt obviously rose from 0.225 to 0.241 (p<0.05), while the yield stress (t0) and consistency coefficient (k)de- creased remarkably from 3.25 to 1.98 Pa and from 12.61 to 8.37 Pa·sn, respectively. Further- more, the three-dimensional network of sogurt became more ordered and open, with the diameter of interspaces increased from 1–2 to 5–6 mm. Protein hydrolase system assays indicated that endopeptidase, aminopeptidase, dipeptidyl aminopeptidase and carboxy- peptidase in sprouted seeds showed different activities towards synthetic substrates. It can be concluded that, after the limited proteolysis of soybean storage proteins by endopepti- dases and exopeptidases in germinated soybeans, the rheological properties and micro- structure of sogurts can be improved. Key words: soybean germination, soy yogurt (sogurt), probiotics, rheological properties, mi- crostructure, protein hydrolase g Introduction enhancement with -aminobutyric acid (3) and active isoflavone aglycone (4). Nevertheless, due to the un- Soybean is one of the widely consumed oil crops pleasant beany flavour, insufficient acidity, rigid and worldwide, with health benefits because it is rich in un- brittle gel structure (5), sogurt has not been generally saturated fatty acids and in low cholesterol. Fermented accepted yet. soybean foods are especially popular in the Asian coun- After sprouting, soybean is reported to be more nu- tries such as China, Japan and Indonesia. Soy yogurt is tritional with increased levels of polypeptides, free amino prepared from the fermented soymilk by lactic acid bac- acids, isoflavones, dietary fibres, vitamins and minerals teria (LAB). Recently, there has been increased interest (6). At the same time, the anti-nutritional factors such as in the improvement of sogurt, e.g. by the addition of trypsin inhibitor, haemagglutinin and phytic acid are combined probiotics (1), prebiotic supplementation (2), significantly reduced (7,8). Degradation of soybean pro- *Corresponding author; Phone: ++86 20 8711 4262; Fax: ++86 20 8711 4263; E-mail: [email protected] 74 M. YANG et al.: Rheology of Sogurt from Germinated Soybeans, Food Technol. Biotechnol. 50 (1) 73–80 (2012) teins is recognized as one of the most complicated and Lactobacillus bulgaricus AS1.1482 and Streptococcus important physiological and biochemical changes during thermophilus IFFI 6038 were provided by Microbiological seedling growth. Globular storage proteins b-conglyci- Culture Collection Center (Guangdong, PR China). Lac- nin (7S) and glycinin (11S) constitute approx. 70 % of the tobacillus helveticus B02 was obtained from Chr. Hansen total soybean proteins, the former is a trimeric glycopro- Food Ingredients Co., Ltd. (Tianjin, PR China). De Man, tein consisting of a’, a and b subunits (9), the latter is a Rogosa and Sharpe (MRS) broth was used for the incu- hexamer composed of six kinds of subunits, each one bation of stock culture, while the reconstituted skimmed containing acidic and alkaline chains linked by a disul- milk (RSM) (w=12 %) was used for subculture. After ster- phide bond (10). It is generally thought that soy storage ilization, the media were inoculated and then incubated proteins are first partly cleaved by endopeptidases at in an ordinary incubator at 37 °C for 12–15 h, followed specific bonds, and then further degraded by unrestric- by refrigeration at 4 °C until further assays. tive proteases (11). According to literature reports, there are extensive Sogurt preparation peptidases that are capable of degrading soy storage pro- Sogurt was prepared as described before (20). Brief- teins during seed sprouting. Two endopeptidases were ly, after surface-sterilization, soybeans were incubated at assayed by Bond and Bowles (12), one of which is a car- 25 °C with 80 % relative humidity in the dark until the boxyl endopeptidase, exhibiting optimum activity at acid- hypocotyl length reached 3 or 6 cm. After peeling, the ic pH, while another is a neutral metalloendopeptidase, seeds were homogenized with double distilled water at showing degrading activity towards the a’ and a sub- 85 °C and a ratio of 1:10 (m/V). The resulting slurries units of 7S and the acidic chains of 11S. Proteases G1 were filtered to yield soymilk, and then mixed with re- and G2 are also responsible for the hydrolysis of the constituted skimmed milk (w=12 %) at a ratio of 7:3 (by acidic subunits of glycinin. The former is a legumain- volume). Next, the mixed media were supplemented with -like cysteine protease and it shows high specificity for 8%(m/V) sucrose and then sterilized at 100 °C for 20 cleavage sites, the latter is presumed to be a collection of min. After cooling to 42 °C, they were inoculated with enzymes (8). Moreover, protease C1 is a serine protease the combined strain starters Lactobacillus bulgaricus AS1. reported to be responsible for the cleavage of a’, a sub- 1482, Streptococcus thermophilus IFFI 6038 and Lactobacil- units of 7S globular protein in the N-terminal region lus helveticus B02 (y=5 %, at a volume ratio of 1.5:1.5:2), (13). Protease C2, a papain-like cysteine protease that followedby4hofincubation at 42 °C. Sogurt was sub- shows low specificity towards particular split site or sequently removed and refrigerated at 4 °C for further sequence, can degrade all subunits of b-conglycinin and assay. In this work, soy yogurt samples prepared from acidic polypeptides of glycinin (14). After enzymolysis, the soaked beans without germination were labelled S0 the generated polypeptides and oligopeptides can be sogurt, while samples prepared from soybean seeds with further cleaved by exopeptidases, and the free amino 3- and 6-cm hypocotyls were labelled S3 and S6 sogurt, acids are then released from the carboxylic or amino respectively. As a reference, reconstituted skimmed milk terminal domain. There are also some literature data on yogurt (RSMY) was prepared as well, by fermenting RSM the activities of carboxypeptidases (8,15) and aminopep- using the same procedure. tidases (12,16,17) in soybean seedlings. Latest research has demonstrated that different soy- Measurement of rheological properties bean subunits within b-conglycinin (18) and glycinin (19) The rheological properties of specimens were ana- have different effects on gel properties of heat-induced lyzed with a rheometer (AR550, TA Instruments-Waters and glucono-d-lactone (GDL)-induced soy protein aggre- LLC, New Castle, DE, USA), using the method of Don- gations. Soy storage proteins are partly hydrolyzed after kor et al. (21) with minor modifications. A 40-mm stain- seed sprouting, thus, the network structure of lactic acid less steel parallel plate with a gap of 1 mm was used to bacteria-coagulated soy protein gel may be affected as measure the samples at (25.0±0.5) °C with two repeti- well. However, there is still little research related to this tions. Strain sweep (0–50 %) was first operated at 1 Hz topic. Our previous research had indicated the improve- to determine the linear viscoelastic range. Specimens were ment of the physicochemical, textural and sensory char- subjected to frequency sweep ranging from 0.1 to 10 Hz acteristics of sogurt made from soybean seedlings (20). at 0.5 % constant strain, then followed by a shear sweep The object of this work is to further study the effects of with increasing shear rate from 0 to 500 per s in 180 s. germination on the rheological properties and micro- Herschel-Bulkley model was used to obtain the flow be- structure of sogurt. haviour parameters. The flow model is expressed by the following equation: n Materials and Methods t=t0+k·g /1/ Materials and chemicals where t is the shear stress, t0 is the yield stress, k is the consistency coefficient, g is the shear rate and n is the [ ] Soybean Glycine max (L.) Merrill was purchased flow behaviour index. from a local supermarket (Guangzhou, PR China). Skim- med milk powder, with protein content of w=32.7 %, was purchased from Fonterra Co., Ltd (Tauranga, New Zea- Microstructure observation land). Substrates for the assay of peptidase activities were The microstructures of the specimens were observed purchased from Sigma-Aldrich, Inc. (Schnelldorf, Ger- by scanning electron microscope (SEM) following the many) and Chem-Impex International, Inc. (Wood Dale, method of Supavititpatana et al. (22). After critical point IL, USA). Other reagents were analytically pure. drying by liquid carbon dioxide, specimens were sub- M. YANG et al.: Rheology of Sogurt from Germinated Soybeans, Food Technol. Biotechnol. 50 (1) 73–80 (2012) 75 sequently fractured at room temperature, placed on alu- then the released p-nitroaniline was monitored at 410 nm minium stubs, sputter-coated with platinum for 150 s to measure the absorbance.
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