Characterization of Polysaccharide and Volatile Compounds Produced by Kefir Grains Grown in Soymilk

ABSTRACT: The characteristics of polysaccharides isolated from milk and soymilk kefir grains, and the composition of flavor and volatile compounds from soymilk kefir were investigated. Soymilk kefir grains revealed lower polysaccharide content than milk kefir grains, with a polysaccharide profile consisting primarily of glucose and galactose, with the former predominating. The apparent molecular weight of the polysaccharide was estimated at 1.7 × 10⁶ Da. In comparison to nonfermented soymilk, the concentrations of the key volatile compounds for soymilk kefir (such as acetaldehyde, acetone, diacetyl, and ethanol) increased, while n-hexanal concentration decreased. The acetaldehyde level for soymilk kefir was slightly higher, but levels for the other volatile compounds were lower than for milk kefir.     

Chemical composition and sensory analysis of cheese whey‐based beverages using kefir grains as starter culture

The aim of the present work was to evaluate the use of the kefir grains as a starter culture for tradicional milk kefir beverage and for cheese whey‐based beverages production. Fermentation was performed by inoculating kefir grains in milk (ML), cheese whey (CW) and deproteinised cheese whey (DCW). Erlenmeyers containing kefir grains and different substrates were statically incubated for 72 h at 25 °C. Lactose, ethanol, lactic acid, acetic acid, acetaldehyde, ethyl acetate, isoamyl alcohol, isobutanol, 1‐propanol, isopentyl alcohol and 1‐hexanol were identified and quantified by high‐performance liquid chromatography and GC‐FID. The results showed that kefir grains were able to utilise lactose in 60 h from ML and 72 h from CW and DCW and produce similar amounts of ethanol (～12 g L^?1), lactic acid (～6 g L^?1) and acetic acid (～1.5 g L^?1) to those obtained during milk fermentation. Based on the chemical characteristics and acceptance in the sensory analysis, the kefir grains showed potential to be used for developing cheese whey‐based beverages.     

Kefir grains as a starter for whey fermentation at different temperatures: chemical and microbiological characterisation

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.     

一种新型开菲尔风味复合发酵剂的研制

为研制一款新型的风味突出、理化性质稳定的开菲尔风味复合发酵剂,该研究从源自中国新疆的10种开菲粒中分离获得51株乳酸菌、22株酵母菌。51株乳酸菌中,3株乳酸菌的产香能力和蛋白分解能力突出,单菌发酵乳的全质构特性最稳定。其中,开菲尔乳杆菌MLK5的乙醛质量浓度为15.82 mg/L、双乙酰质量浓度为3.99 mg/L、氨基氮质量浓度为597.09 mg/L,干酪乳杆菌SLC1的乙醛质量浓度为20.02 mg/L、双乙酰质量浓度为4.69 mg/L、氨基氮质量浓度为684.92 mg/L,肠膜明串珠菌NLM2的乙醛质量浓度为18.01 mg/L、双乙酰质量浓度为4.44 mg/L、氨基氮质量浓度为600.58 mg/L。22株酵母菌中,马克思克鲁维酵母菌菌株FY1的乙醇产量适宜,遗传稳定性好,乳糖利用率最高为56.56%。最终确定,发酵剂中最佳比例为乳酸菌:酵母菌=5:1,3株乳酸菌间最优例为肠膜明串珠菌:开菲尔乳杆菌:干酪乳杆菌为1:2:1。复合发酵乳的凝乳时间为6.0 h、酸度为84.65 ºT、持水力为62.31%、乙醇体积分数为0.53%、乳酸菌活菌数为3.89×109 CFU/mL、酵母菌活菌数为4.61×106 CFU/mL、感官评分为89.21,与开菲尔粒发酵乳的各项指标类似。而复合发酵乳中的挥发性风味物质间构成和谐,更易于消费者接受。上述研究结果,有助于稳定和简化开菲尔的工业生产,为开发不同风味酸乳产品提供参考。     

Comparative study of the biochemical changes and volatile compound formations during the production of novel whey-based kefir beverages and traditional milk kefir

Cheese whey (CW) and deproteinised cheese whey (DCW) were investigated for their suitability as novel substrates for the production of kefir-like beverages. Lactose consumption, ethanol production, as well as organic acids and volatile compounds formation, were determined during CW and DCW fermentation by kefir grains and compared with values obtained during the production of traditional milk kefir. The results showed that kefir grains were able to utilise lactose from CW and DCW and produce similar amounts of ethanol (7.8–8.3 g/l), lactic acid (5.0 g/l) and acetic acid (0.7 g/l) to those obtained during milk fermentation. In addition, the concentration of higher alcohols (2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol, 2-methyl-1-propanol, and 1-propanol), ester (ethyl acetate) and aldehyde (acetaldehyde) in cheese whey-based kefir and milk kefir beverages were also produced in similar amounts. Cheese whey and deproteinised cheese whey may therefore serve as substrates for the production of kefir-like beverages similar to milk kefir.     

Microbiological, physicochemical, and sensory characteristics of kefir during storage

Changes in certain microbiological, physicochemical, and sensory parameters of kefir were studied during refrigerated storage. Kefir batches were prepared using 1% and 5% added kefir grains, and samples for analysis were taken 24 h after inoculation and then after 2, 7, 14, 21, and 28 days of storage at 5 ± 1 °C. After fermentation for 24 h after inoculation, lactobacilli and lactococci were present at levels of 10⁸ cfu/ml, and yeasts and acetic acid bacteria were present at levels of 10⁵ and 10⁶ cfu/ml, respectively. The lactic acid flora decreased by about 1.5 log units between days 7 and 14 and then stabilized at that level. Yeast and acetic acid bacterial counts, lactose, and pH all remained constant over the storage period, while the total fat content and dry matter decreased. The percentage inoculate did exert an influence, and the sample batches made using 1% added kefir grains had higher lactic acid bacterial counts, lactose, and pH, while the sample batches made using 5% added kefir grains had higher yeast and acetic acid bacterial counts and viscosity. The total fat and dry matter contents were similar in both sample batches. Sensory analysis of the kefir samples revealed maximum acceptability levels in the first 2 days of storage.     

Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir starter made from them

The characteristics of cell growth, lactic acid production, amino acid release and consumption by single-strain cultures of lactic acid bacteria (isolated from kefir grains), and by a multiple-strain kefir starter prepared from them, were studied. The change in the levels of free amino acids was followed throughout the kefir process: single-strain kefir bacteria and the kefir starter (Lactococcus lactis C15-1%+Lactobacillus helveticus MP12-3%+(Streptococcus thermophilus T15+Lactobacillus bulgaricus HP1 = 1:1)-3%) were cultivated in pasteurized (92 degrees C for 20 min) cow's milk (3% fat content) at 28 degrees C for 5 h (the kefir starter reached pH 4.7) and subsequently grown at 20 degrees C for 16 h; storage was at 4 degrees C for 168 h. The strain L. helveticus MP12 was unrivaled with respect to free amino acid production (53.38 mg (100 g)(-1)) and cell growth (17.8 x 10(8) CFU ml(-1)); however, it manifested the lowest acidification activity. L. bulgaricus HP1 released approximately 3.7 times less amino acids, nearly 5 times lower cell growth, and produced about 1.2 times more lactic acid. S. thermophilus T15 demonstrated dramatically complex amino acid necessities for growth and metabolism. With L. lactis C15, the highest levels of growth and lactic acid synthesis were recorded (18.3 x 10(8) CFU ml(-1) and 7.8 g l(-1) lactic acid at the 21st hour), and as for free amino acid production, it approximated L. bulgaricus HP1 (17.03 mg (100 g)(-1) maximum concentration). In the L. lactis C15 culture, the amino acids were used more actively throughout the first exponential growth phase (by the 10th hour) than during the second growth phase. The unique properties of the L. helveticus MP12 strain to produce amino acids were employed to create a symbiotic bioconsortium kefir culture, which, under conditions of kefir formation, enhanced lactic acid production and shortened the time required to reach pH 4.7; intensified cell growth activity, resulting in a respective 90- and 60-fold increase in the concentration of lactobacilli and cocci in the mixed culture compared to individual cultures; and accumulated free amino acids in the final kefir with higher total concentrations (56.88 mg (100 g)(-1)) and an individual concentration of essential amino acids (1.5 times) greater than that of yogurt.     

Growth and activity of some lactic streptococci cultivated on soymilk medium infected with bacteriophage

Pure cultures of S. lactis, S. cremoris, S. diacetilactis and S. thermophilus were cultivated on soymilk medium containing 3% lactose and skim milk medium which were infected or not infected with bacteriophage. The cultures were incubated for 24 h at the optimum temperature of each organism. The growth, lactic acid production and activity of each culture were periodically examined. The growth and the acidity development of the cultivated cultures on soymilk medium infected with bacteriophage increased gradually during the incubation period, whereas the cultures grown on skim milk infected with bacteriophage showed less growth and acid production. Moreover, the growth and the rate of lactic acid production of the cultures cultivated on soymilk medium were higher than those of the cultures grown on skim milk medium (control). Therefore, soymilk medium containing 3% lactose could be recommended as a protective medium against bacteriophage infection for lactic acid starter.     

Effect of different growth conditions on biomass increase in kefir grains

Kefir is a functional dairy product and the effects of kefir consumption on health have been well documented. Kefir grains have naturally high numbers of lactic acid bacteria and yeasts and are used in manufacturing kefir. The biomass of kefir grains slowly increases after successive fermentations. The effects of adding whey protein isolate, modified whey protein (MWP, fat replacer; Carbery Inc., Cork, Ireland), or inulin to milk and different atmospheric conditions (ambient or 6% CO₂) during fermentation on the increase in biomass of kefir grains were investigated. Reconstituted milks (10% milk powder) enriched with whey protein isolate (2%), MWP (2%), and inulin (2%) were inoculated with kefir grains and fermented in ambient and 6% CO₂ incubators at 25°C until a final pH of 4.6 was reached. Biomass increments of kefir grains were determined weekly over 30 d. Lactic acid bacteria and yeast contents of kefir grains were also determined. The highest biomass increase (392%) was found in kefir grains grown in milk supplemented with whey protein isolate under ambient atmospheric conditions. Application of CO₂ did not provide a significant supporting effect on the biomass of kefir grains. Addition of MWP significantly accelerated the formation of kefir grain biomass (223%). The use of whey protein isolate, MWP, or inulin in milk did not cause any adverse effects on the microbial flora of kefir grains.     

Review: functional properties of kefir

Kefir is a unique cultured dairy product due to combined lactic acid and alcoholic fermentation of lactose in milk. Kefir is produced by microbial activity of "kefir grains" which have a relatively stable and specific balance of lactic acid bacteria and yeast. Due to the claimed health benefits of kefir which include reduction of lactose intolerance symptoms, stimulation of the immune system, lowering cholesterol, and antimutagenic and anticarcinogenic properties, kefir has become an important functional dairy food and consequently, research on kefir has increased in the past decade. In the following review, recent studies on the functional properties of kefir are reviewed.     

Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.     

Effect of fat replacers on kefir quality

The purpose of the study was to determine the effects of fat replacers on the quality of non‐fat kefir. Skim milk fortified with Dairy Lo^® (DL) and inulin (INU) was fermented with kefir grains to manufacture kefir. The results of compositional, microbiological, rheological and sensorial analyses were compared with whole kefir (WK) and non‐fat kefir (NFK) controls. Results for dry matter, pH and lactic acid ranged between 82.4 and 109.1 g kg^?1, 4.26 and 4.40, and 7.0 and 9.2 g L^?1, respectively. Acetaldehyde and ethanol contents of samples were between 2.89 and 7.28 mg L^?1, and 151.46 and 323.89 mg L^?1, respectively. In all samples, Lactobacillus spp., Streptococcus spp. and yeast counts were between 9.1 and 9.9, 9.3 and 9.9, and 5.2 and 5.6 log cfu mL^?1, respectively. Kefir samples had non‐Newtonian behaviour and pseudoplastic fluid with thixotropy. At the first day, DL had the highest apparent viscosity (3.119 Pa s) while NFK had the lowest value (1.830 Pa s). In the sensory evaluation, odour and taste scores of samples were not different. Dairy Lo^® and inulin could be used without any adverse effect for the production of non‐fat kefir. Copyright © 2009 Society of Chemical Industry     

Preparation of a Yogurt-like Product Containing Egg White

The fermentation of liquid egg white by lactic acid bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus , to develop a new yogurt-type product has been studied. Egg white was prepared by pasteurization, blending, and neutralization to minimize antimicrobial activity, and by combining additives to make the media more suitable for growth of lactic acid bacteria. Skim milk, gums (guar gum, CMC, xanthan gum), soymilk, and glucose were studied. Optimum results were obtained when 47.4% egg white was combined with 28.4% alkali treated soymilk. 19.0% skim milk. 1.9% glucose, 2.8% sucrose, 0.5% xanthan gum, and 0.01% vanilla extract. The composition per 100 gram of the final product included 7.52% protein, 0.57% fat, and 62 calories. Microbiological and sensory tests showed the product to be free of pathogens and to have an extended shelf life at refrigeration temperature.     

Inhibitory power of kefir: the role of organic acids

Milk and MRS broth fermented with kefir grains from different households were examined for inhibitory activity toward gram-negative and gram-positive strains. Fermented milk obtained with 10 g per 100 ml of inoculum (final pH 3.32 to 4.25) and MRS broth fermented with 1 and 10 g per 100 ml of inocula (final pH 4.18 to 5.25) had inhibitory power demonstrated by spot test and agar well diffusion assay. This inhibitory effect could be assigned to the undissociated form of lactic and acetic acid produced during the fermentation process. Kefir supernatants inhibited the growth of Escherichia coli 3 in nutrient broth at 37 degrees C for 24 h. However, supernatants of yogurt or milk artificially acidified with lactic and acetic acids allowed the growth of E. coli 3 in the same conditions. A bacteriostatic effect of milk fermented with kefir grains over E. coli 3 was also demonstrated.     

开菲尔粒中主要组成菌的分离鉴定及微囊化纯培养混合发酵指标分析

开菲尔粒是一个复杂的微生物共生体系,包含很多有益生作用的微生物。本文研究了一种开菲尔粒的主要组成菌并制成发酵剂。通过形态学特征初步分离纯化得出:该开菲尔粒样品主要由两株酵母菌、三株乳酸菌以及两株醋酸菌构成。经16S r DNA序列分析进一步确定其种属,得出其分别为Kluyveromyces marxianus和Pichia kudriavzevii、Lactobacillus pontis和Lactobacillus kefiri、Acetobacter lovaniensis和Acetobacter cibinongensis。采用分离出的菌株纯培养微囊化之后进行混合发酵,得到具有优良稳定发酵性能的混合发酵剂,测定结果显示发酵乳的营养成分、挥发性成分和抑菌性与原粒对比十分接近。      

The anti‐allergenic properties of milk kefir and soymilk kefir and their beneficial effects on the intestinal microflora

Food allergy is now recognized as a worldwide problem, and like other atopic disorders its incidence appears to be increasing. Kefir is reported to possess the ability to reduce intestinal permeation of food antigens; however, no experimental study has clearly evaluated the relationships between kefir consumption, allergen‐specific IgE response, and intestinal microflora. The aim of this study was to evaluate the effect of oral consumption of milk kefir and soymilk kefir on in vivo IgE and IgG1 production induced by ovalbumin (OVA) in mice. The effects of kefir administration on the murine intestinal microflora were also examined. Oral administration of milk kefir and soymilk kefir for 28 days significantly increased the fecal populations of bifidobacteria and lactobacilli, while it significantly decreased those of Clostridium perfringens. Milk kefir and soymilk kefir also significantly decreased the serum OVA‐specific IgE and IgG1 levels for both groups, but not those of the IgG2a analogues. Consumption of milk kefir and soymilk kefir suppressed the IgE and IgG1 responses and altered the intestinal microflora in our supplemented group, suggesting that milk kefir and soymilk kefir may be considered among the more promising food components in terms of preventing food allergy and enhancement of mucosal resistance to gastrointestinal pathogen infection. Copyright © 2006 Society of Chemical Industry     

Isolation and characterization of the microbial population of different South African kefir grains

Kefir, a slightly acidic fermented milk, is produced by adding lactic acid bacteria and yeasts, in the form of grains, to milk. The bacteria and yeasts present in the kefir grains are known to vary widely. Selective growth media and morphological and biochemical characteristics were used for the isolation and identification of the microbes present in the grains from eight different sources in South Africa. The kefir grains were activated in milk for only 24 h to prevent any changes in the microbial population of the grains. The microbial numbers varied between 6.4 × 10⁴ and 8.5 × 10 ⁸  cfu/g on the media selective for the bacterial species and between 1.5 × 10 ⁵ and 3.7 × 10 ⁸   cfu/g on the media selective for the yeast species. The bacterial genera that were identified included Lactobacillus , Leuconostoc and Lactococcus and the yeast genera included Zygosaccharomyces , Candida and Saccharomyces . The distribution frequencies of the microbes in the different grains were determined and most of the grains were dominated by two microbial species. No pediococci, acetic acid bacteria or propionibacteria were detected.     

Chemical and microbiological characterisation of kefir grains

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.     

应用序贯设计优化混合乳开菲尔的发酵条件

采用序贯设计对以牛乳和豆乳为原料研制混合乳开菲尔的发酵条件进行了优化。首先应用部分析因设计对影响开菲尔发酵的牛乳与豆乳比例、酵母接种量、乳酸菌接种量、蔗糖添加量、发酵温度、发酵时间6个因子进行了分析,筛选出蔗糖添加量和发酵时间为显著因子,然后通过最陡爬坡设计逼近最大响应区域,应用中心复合设计法确定蔗糖添加量(x4)和发酵时间(x6)的最佳组合,最终得到混合乳开菲尔发酵的最优发酵条件为牛乳与豆乳比例为7∶3,酵母接种量为1.00×105 CFU/mL,乳酸菌接种量为1.00×107 CFU/mL,蔗糖添加量为1.6%,发酵温度为22℃,发酵时间为22 h。     

Turkish kefir and kefir grains: microbial enumeration and electron microscobic observation†

Microbial populations in kefir and kefir grains were enumerated by plating. Total lactic acid bacteria, lactoccocci, lactobacilli and yeast populations increased during fermentation and increased slightly during cold storage. Kefir grains had a lactic acid bacteria : yeast ratio of 10 ⁹  : 10 ⁶ . In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods, which indicated yeast colonization on the surface and middle part of the kefir grain. Three types of lactobacilli (short, long and curved) were noted throughout the grain. Lactococci were not observed under SEM; preparation of kefir grains for SEM may have caused removal of lactococci from the grains.