Stability of β-glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage

ABSTRACT: Microorganisms possess endogenous enzymes, however the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glucosides to their bioactive aglycone forms. Fifteen probiotic microorganisms including bifidobacterium, Lactobacillus acidophilus , and Lactobacillus casei were screened for β-glucosidase activity using p-nitrophenyl-β-d-glucopyranoside as a substrate. Six strains were selected on the basis of β-glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and storage for 8 wk at frozen (-80°C), refrigerated (4°C), room (24.8°C), and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, whereas bifidobacterium strains showedleast activity. However, p-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher ( P < 0.05) β-glucosidase activity and better stability than that at higher temperatures (24.8°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage of isoflavone β-glucosides to bioactive isoflavone aglycone forms with potential health benefits.     

Fermentation of calcium-fortified soymilk with Lactobacillus: effects on calcium solubility, isoflavone conversion, and production of organic acids

ABSTRACT:  The objective of this study was to enhance calcium solubility and bioavailability from calcium-fortified soymilk by fermentation with 7 strains of Lactobacillus , namely, L. acidophilus ATCC 4962, ATCC33200, ATCC 4356 , ATCC 4461 , L. casei ASCC 290, L. plantarum ASCC 276, and L. fermentum VRI-003. The parameters that were used are viability, pH, calcium solubility, organic acid, and biologically active isoflavone aglycone content. Calcium-fortified soymilk made from soy protein isolate was inoculated with these probiotic strains, incubated for 24 h at 37 °C, then stored for 14 d at 4 °C. Soluble calcium was measured using atomic absorption spectrophotometry (AA). Organic acids and bioactive isoflavone aglycones, including diadzein, genistein, and glycetein, were measured using HPLC. Viability of the strains in the fermented calcium-fortified soymilk was > 8.5 log₁₀ CFU/g after 24 h fermentation and this was maintained for 14-d storage at 4 °C. After 24 h, there was a significant increase ( P < 0.05) in soluble calcium. L. acidophilus ATCC 4962 and L. casei ASCC 290 demonstrated the highest increase with 89.3% and 87.0% soluble calcium after 24 h, respectively. The increase in calcium solubility observed was related to lowered pH associated with production of lactic and acetic acids. Fermentation significantly increased ( P < 0.05) the level of conversion of isoflavones into biologically active aglycones, including diadzein, genistein, and glycetein. Our results show that fermenting calcium-fortified soymilk with the selected probiotics can potentially enhance the calcium bioavailability of calcium-fortified soymilk due to increased calcium solubility and bioactive isoflavone aglycone enrichment.     

Probiotic Cheddar Cheese: Influence of Ripening Temperatures on Proteolysis and Sensory Characteristics of Cheddar Cheeses

ABSTRACT:  Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI L26, Lb. acidophilus 4962, or Lb. acidophilus LAFTI L10 were used as an adjunct in the production of Cheddar cheeses, which were ripened at 4 and 8 °C for 24 wk. Effects of ripening temperatures and probiotic adjuncts on proteolysis and sensory evaluation of the cheeses were examined. Higher ripening temperature increased the level of proteolysis in the cheeses. Product of proteolysis and organic acids released during ripening were shown to be important for the flavor of Cheddar cheeses. There were positive and significant correlations between the levels of soluble nitrogen, lactic, acetic, and butyric acids, percentage hydrolysis of α_s1-CN and β-CN to the scores of cheddary flavor ( P < 0.05). Scores for sour-acid and vinegary flavors were higher in cheeses with the addition of Bifidobacterium sp. or Lb. casei 279 ripened at 8 °C. The scores were positively and significantly correlated to the level of lactic, acetic, and free amino acids in the cheeses ( P < 0.05). The results show that both 4 and 8 °C have potential for use in the ripening of probiotic Cheddar cheeses.     

HYDROLYSIS OF ISOFLAVONE PHYTOESTROGENS IN SOYMILK FERMENTED BY LACTOBACILLUS AND BIFIDOBACTERIUM COCULTURES

This study evaluated the hydrolysis of isoflavones in soymilk fermented at 37C for 48 h by four different Lactobacillus and Bifidobacterium cocultures. The hydrolysis of isoflavone β-glucosides significantly increased ( P <  0.05) the bioactive aglycones from 36 to over 90% of total isoflavones in soymilk fermented with any of the four Lactobacillus and Bifidobacterium cocultures as compared with unfermented soymilk. Compared with three other cocultures of Lactobacillus and Bifidobacterium, fermentation of soymilk with the Lactobacillus paracasei/Bifidobacterium longum cocultures yielded better isoflavone hydrolytic potential (Otieno-Shah index) and the highest β-glucosidase activity after 12 h of incubation.

PRACTICAL APPLICATIONS

Isoflavones are known as phytoestrogens because they are present in soy products and have estrogen-like activity. During fermentation, the majority of glucoside isoflavones in soymilk are converted to bioactive aglycones via microorganism-derived β-glucosidase. In human intestines, aglycone isoflavones are absorbed faster and in greater amounts than their glucosides. Using probiotic Lactobacillus and Bifidobacterium cocultures to ferment soymilk efficiently increases the bioactive aglycone concentrations. Hence, fermenting soymilk with this coculture could enhance the nutritional value of the product.     

Oat-based Symbiotic Beverage Fermented by Lactobacillus plantarum, Lactobacillus paracasei ssp. casei, and Lactobacillus acidophilus

ABSTRACT: Oats and probiotics have long been recognized for their health benefits. The objectives of this study were (1) to study the ability of Lactobacillus plantarum (B-28), Lactobacillus paracasei ssp. casei (B-29) isolated from a traditional Bulgarian cereal-based fermented beverage, and Lactobacillus acidophilus from Chr. Hansen, Milwaukee, Wis., U.S.A., to remove cholesterol from the media and to adhere to the Caco-2 cell line, (2) to optimize the fermentation conditions to develop a beverage using these probiotics and oats with acceptable sensory and nutritional qualities, and (3) to assess the quality and shelf-life of this beverage and survivability of probiotics in the beverage. Lactobacillus acidophilus , B-28, and B-29 were found to remove 70.67%± 2.35%, 20.26%± 2.63%, and 16.75%± 3.83% of cholesterol from media and the percentage of adhesion was 4.69%± 0.78%, 1.92%± 0.78%, and 8.36%± 0.78%, respectively. The blend of oat flour, sugar, inulin, and whey protein concentrate was cooked in water and fermented for 12 h at 37°C by 2 ± 10⁶ colony-forming units (CFU) /mL each of B-28 and B-29 and 2 ± 10⁸ CFU/mL of L. acidophilus . The beverage had 0.87%± 0.03% of dietary fiber and had better sensory qualities compared with the commercially available similar product. The probiotics survived for 10 wk of storage at 4°C, except for L. acidophilus , which survived for about 4 wk. The population of B-28 was 1.77 ± 10⁶ to 1.29 ± 10⁷ CFU/mL and that of B-29 was 7.39 ± 10⁷ to 4.49 ± 10⁸ CFU/mL throughout the storage period. The oat-based symbiotic beverage is a functional drink providing both probiotics and prebiotics at the same time.     

Using of Lactobacillus and Bifidobacterium to product the isoflavone aglycones in fermented soymilk

The study determined isoflavone aglycone contents in soymilk and sugar-soymilk fermented with a single culture of two strains of Lactobacillus paracasei, two strains of Lactobacillus acidophilus, and one strain of Bifidobacterium longum respectively at 37 degrees C for 24 h. Isoflavone concentration was analyzed by HPLC. The viable count of bacteria in all of the fermented soymilk ranged from 7 to 9 log CFU/ml. The isoflavone aglycone had a significant increase of 62%-96% of isoflavone in all of the fermented soymilk compared to 17% in non-fermented soymilk (P<0.05). The five strains of microorganisms produced lesser amount of isoflavone aglycones in fermented sucrose-soymilk than in other fermented soymilk.     

Evaluation of enzymic potential for biotransformation of isoflavone phytoestrogen in soymilk by Bifidobacterium animalis,Lactobacillus acidophilus and Lactobacillus casei

Three strains of Lactobacillus acidophilus, two of Lactobacillus casei and one of Bifidobacterium were screened for β-glucosidase activity using ρ-nitrophenyl-β-d-glucopyranoside as a substrate and their potential for the breakdown of isoflavone glucosides to the biologically active aglycones in soymilk. Isoflavones quantification with HPLC and β-glucosidase activity were performed after 0, 12, 24, 36, and 48 h of incubation in soymilk at 37 °C. All six micro-organisms produced β-glucosidase, which hydrolysed the predominant isoflavone β-glucosides. There was a significant increase and decrease (P < 0.05) in the concentration of isoflavone aglycones and glucosides, respectively, in fermented soymilk. Based on the concentration of isoflavones during peak β-glucosidase activity, the hydrolytic potential was calculated. L. acidophilus 4461 had the highest aglycone concentration of 76.9% after 24 h of incubation, up from 8% in unfermented soymilk (at 0 h). It also had the best isoflavone hydrolytic index of 2.01, signifying its importance in altering the biological activity of soymilk.     

Effect of lactulose on biotransformation of isoflavone glycosides to aglycones in soymilk by lactobacilli

ABSTRACT:  Lactobacillus acidophilus 4461, L. acidophilus 4962, L. casei 290, and L. casei 2607 were used to hydrolyze isoflavone glycosides (IG) to biologically active forms—isoflavone aglycones (IA)—in soymilk (SM) prepared from soy protein isolate (SPI) and soymilk supplemented with 0.5% (w/v) of lactulose (SML). L. acidophilus 4461 utilized the highest level of lactulose (3.01 mg/mL) and L. acidophilus 4962 utilized the least (0.86 mg/mL) at 24 h of incubation. The pH values decreased to 4.00 to 5.00 in SML, while they remained relatively high (6.15 to 6.36) in SM. Supplementation with lactulose significantly ( P < 0.05) enhanced the viable counts of all the 4 Lactobacillus strains. At the end of incubation, the viable counts of Lactobacillus ranged from 8.08 to 8.25 log CFU/mL in SML compared to 6.99 to 7.11 log CFU/mL in SM. Supplementation with lactulose increased the biotransformation of IG to IA after 6 h of incubation. The presence of lactulose in the medium enhanced the biotransformation level of IG to IA by Lactobacillus up to 21.9%. The hydrolysis level of malonyl genistin and acetyl genistin in SML was much higher than in SM by all the 4 probiotic organisms. The biotransformation of IG to IA occurred rapidly during the 1st 12 h of incubation in both SML and SM. Among the 4 Lactobacillus strains, L. acidophilus 4461 biotransformed the highest level (88.8%) of IG to IA in SML compared to 68.2% in SM after 24 h of incubation.     

Skim milk powder supplementation affects lactose utilization, microbial survival and biotransformation of isoflavone glycosides to isoflavone aglycones in soymilk by Lactobacillus

Four probiotic bacteria, Lactobacillus acidophilus 4461, L. acidophilus 4962, Lactobacillus casei 290 and L. casei 2607, were used for fermentation of soymilk (SM) prepared from soy protein isolate (SPI) supplemented with skim milk powder (SMP) (SSM). Soymilk and reconstituted skim milk (RSM) were used as controls. Lactose utilization in SSM by these probiotic organisms ranged from 14.97 to 18.15mg/ml, compared to 14.12-16.06mg/ml for RSM. The pH in SSM dropped to 4.07-4.29 compared to 6.15-6.36 for SM and 4.10-4.96 for RSM. The microbial viable counts were also significantly enhanced by up to 0.98logCFU/ml by the supplementation of SMP to SM. The biotransformation level of isoflavone glycosides (IG) to isoflavone aglycones (IA) in SSM ranged from 81.4% to 85.1%, which was 13.9-19.0% higher than that for SM, after 24h of incubation. Most of IG in SSM was completely converted to IA, except malonyl glycitin and malonyl genistin. At the end of the incubation, IA comprised up to 76.8% of total isoflavones in SSM.     

Enzymic Transformation of Isoflavone Phytoestrogens in Soymilk by β-Glucosidase-Producing Bifidobacteria

ABSTRACT: Five strains of bifidobacteria were screened for β-glucosidase activity using p-nitrophenyl-β-D-glucopyranoside as the substrate, and selected strains were used to ferment soymilk. Enumeration of viable bifidobacteria and quantification of isoflavones using HPLC were performed at 0, 12, 24, 36, and 48 h of incubation. Four strains produced β-glucosidase. B. pseudolongum and B. longum -a displayed the best growth in soymilk, with an increase of 1.3 log₁₀ CFU/mL after 12 h. B. animalis, B. longum -a, and B. pseudolongum caused hydrolysis of isoflavone malonyl-, acetyl- and β-glucosides to form aglycones, and transformed daidzein to equol in soymilk. Fermentation of soymilk with Bifidobacterium sp. resulted in a significant increase (p < 0.05) in the concentration of aglycones.     

Conversion of isoflavone glucosides to aglycones in soymilk by fermentation with lactic acid bacteria

ABSTRACT:  Four lactic acid bacteria (LAB), Lactobacillus paraplantarum KM, Enterococcus durans KH, Streptococcus salivarius HM and Weissella confusa JY, were isolated from humans and tested for their capabilities of converting isoflavone glucosides to aglycones in soymilk. Changes in growth, pH, and titratable acidity (TA) were investigated during fermentation at 37 °C for 12 h. After 6 to 9 h of fermentation, each population of 4 LAB reached 10⁸ to 10⁹ CFU/mL. The initial pH of 6.3 ± 0.1 decreased while the TA of 0.13%± 0.01% increased as fermentation proceeded, resulting in the final range between 4.1 ± 0.2 and 4.6 ± 0.1 for pH and between 0.51%± 0.02% and 0.67%± 0.06% for TA after the 12 h of fermentation. The glucoside concentrations were significantly decreased in soymilks fermented with either L. paraplantarum KM, S. salivarius HM, or W. confusa JY with fermentation time ( P < 0.05). L. paraplantarum KM was the best in percent conversion of glucosides to corresponding aglycones, resulting in 100%, 90%, and 61% hydrolysis of genistin, daidzin, and glycitin, respectively, in 6 h. Consequently, the aglycone concentrations in soymilk fermented with L. paraplantarum KM were 6 and 7-fold higher than the initial levels of daidzein and genistein, respectively, after 6 h of fermentation. Changes in the daidzin and genistin levels were not significant in soymilk fermented with E. durans KH. The rates of hydrolysis of glucosides varied depending on the species of LAB. Especially, L. paraplantarum KM seems to be a promising starter for bioactive-fermented soymilk based on its growth, acid production, and isoflavone conversion within a short time.     

Effect of Lactic Acid Fermentation on Enrichment of Antioxidant Properties and Bioactive Isoflavones in Soybean

ABSTRACT: The antioxidant properties of 80% ethanolic extracts from soybean obtained with lactic acid fermentation using ABTS•⁺ [2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt] free radical decolorization assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay were measured, and the relationship between the observed antioxidant properties and the compositional changes in the isoflavone isomers was evaluated. Fermentation of soybean with 4 bacterial strains producing β-glucosidase for 48 h at 37°C— Lactobacillus plantarum KFRI 00144, Lactobacillus delbrueckii subsp. latisKFRI01181, Bifidobacteriathermophilum KFRI00748, and Bifidobacteria breve K-101—resulted in a significant increas (P < 0.05) in the antioxidant capacity expressed as Trolox equivalent antioxidant capacity (TEAC) (mM) and % scavenging activity. The significant bioconversion (P < 0.001) of the isoflavone glucosides (daidzin + genistin) into their responding bioactive aglycones (daidzein + genistein) during soy fermentation was observed. There was a good linear correlation between the concentration of isoflavone aglycones and the scavenging activity of ABTS ( R = 0.9045) assay, and DPPH ( R = 0.8299) assay in each extract. Among each extract, B. thermophilum KFRI 00748 showed a particularly high antioxidant activity (19.8 mM TEAC) and increased by 4.1 times compared with that of the control (4.8 mM TEAC), which fermented without strains. These results indicated that fermented soybean could be regarded as a potent antioxidant and radical scavenging dietary source due to their remarkable content of bioactive isoflavones.     

Effect of Versagel on the growth and metabolic activities of selected lactic acid bacteria

ABSTRACT:  In this study, the influence of a protein-based fat replacer, Versagel ^® added at 1% and 2% (w/v) to reconstituted skim milk (RSM), on the growth and metabolic activities of selected strains of yogurt starters ( Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus ) as well as probiotic organisms ( Lactobacillus casei , Lactobacillus acidophilus , and Bifidobacterium longum ) was examined. Addition of Versagel resulted in significantly improved growth of S. thermophilus and B. longum but inhibited that of L. casei , L. acidophilus , and L. delbrueckii ssp. bulgaricus . This is also reflected in the extent of reduction of pH in RSM with added Versagel by these organisms. Among the biochemical activities, proteolytic activity of all the organisms except B. longum was adversely affected by the presence of Versagel, although the angiotensin-I converting enzyme (ACE) inhibitory and α-glucosidase (α-glu) inhibitory activities were improved. Versagel at 1% level influenced the growth, while ACE inhibitory and α-glu inhibitory activities of the organisms were better at 2% level.     

Contribution of Lactococcus lactis subsp lactis IFPL 359 and Lactobacillus casei subsp casei IFPL 731 to the Proteolysis of Caprine Curd Slurries

ABSTRACT: Proteolysis was studied in low-fat caprine curd slurries pre-incubated (24 h at 30 °C) with an extra dose of rennet, 10.0 or 22.0 μg g^-1 (LR and HR slurries, respectively), and treated at 70 °C for 15 min prior to incubation with sonicated cells of Lactococcus lactis subsp lactis IFPL 359 alone or combined with Lactobacillus casei subsp casei IFPL 731. The addition of lactobacilli to slurries containing lactococci increased the conversion of water-soluble nitrogen to nonprotein nitrogen and amine nitrogen. Major differences were found for some free amino acids, such as glutamic acid and proline, and hydrolysis of most hydrophobic peptides produced by lactococci, which are associated with bitterness in cheese.     

6 株乳酸菌在豆乳中的发酵特性

以干酪乳杆菌（Lactobacillus casei）、短乳杆菌（L. brevis）、嗜酸乳杆菌（L. acidophilus）、植物乳杆菌（L. plantarum）、鼠李糖乳杆菌（L. rhamnosus）和保加利亚乳杆菌（L. bulgaricus）分别发酵豆乳,测定发酵期间pH值、滴定酸度、游离氨基氮,发酵结束后的活菌数和质构参数,并且对所得产品进行感官评价。结果表明：发酵过程中前5 株菌发酵豆乳的pH值显著下降,而L. bulgaricus下降缓慢,发酵24 h pH值仅为5.2。这6 株菌发酵产品的活菌数均达到1.0×108 CFU/mL以上。结果表明L. casei、L. brevis、L. acidophilus和L. plantarum发酵得到的产品的坚实度、稠度、黏度、黏附性指数均较高,感官评定结果表明这4 株菌发酵豆乳产品得分均较高,容易被消费者接受,适合用于生产发酵豆乳产品。     

Viability of probiotic micro-organisms (Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12) in ice cream

The purpose of this research was to determine the survival of two probiotic micro-organisms in ice creams (4% fat). The micro-organisms were Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp . lactis Bb-12 . To meet this objective, an ice cream mixture was formulated and subjected to three treatments. Treatment 1 was inoculated with L. acidophilus , treatment 2 with B. lactis and the third treatment was inoculated with a mixture of both bacteria inoculated in 1 : 1 proportions. The inoculation was with 4% culture for each treatment. The final products were stored at −25°C for 60 days. The ice cream inoculated with L. acidophilus had a final concentration of 2 × 10 ⁶ cfu/g and the survival rate was 87%. The treatment inoculated with B. lactis had a final concentration of 9 × 10 ⁶ cfu/g, with a logarithmic decrease of 10%. When both micro-organisms were inoculated together, the survival rate was 86%.     

植物乳杆菌58发酵豆乳产大豆异黄酮苷元条件的优化

本研究通过测定植物乳杆菌58在不同碳源中的生长和产β-葡萄糖苷酶情况,筛选菌株的最适碳源,并确定接种至豆乳的最佳时间.在接种量、糖含量、发酵时间和发酵温度等单因素实验基础上,根据Box-Behnken中心组合原理进行响应面实验设计,以大豆异黄酮苷元含量为指标,进一步优化菌株58发酵豆乳产大豆异黄酮苷元条件.结果显示,植...     

Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria

In the present study, soymilk is fermented with lactic acid bacteria (Streptococcus thermophilus BCRC 14085, Lactobacillus acidophilus BCRC 14079) and bifidobacteria (Bifidobacterium infantis BCRC 14633, B. longum B6) individually, and in combination. The change in the content of various isoflavones (aglycones, glucoside, acetyl- and malonyl-glucosides) and the beta-glucosidase activity in soymilk during fermentation is investigated. It is observed that fermented soymilk contains a lower total isoflavone content (81.94-86.61 microg/ml) than soymilk without fermentation (87.61 microg/ml). Regardless of starter organism employed, fermentation causes a major reduction in the contents of glucoside, malonylglucoside and acetylglucoside isoflavones along with a significant increase of aglycone isoflavones content. The level of change in the content of various isoflavones and beta-glucosidase activity after fermentation varies with the starter organism. Among all the fermented soymilks tested, soymilk fermented with S. thermophilus showed the highest beta-glucosidase activity and the greatest increase in the contents of aglycones. The percentage of daidzein, genistein and glycitein to total isoflavone content in S. thermophilus-fermented soymilk increases from an initial 14.24%, 6.89% and 2.45%, respectively, to 36.20%, 28.80% and 12.44% after 24h of fermentation. Finally, the increase of aglycones and decrease of glucoside isoflavones during fermentation coincides with the increase of beta-glucosidase activity observed in fermented soymilk.     

Viability of probiotic micro-organisms (Lactobacillus casei Shirota and Bifidobacterium animalis subspp. lactis) in a milk-based dessert with cranberry sauce

The objective of this study was to evaluate the survival of two probiotic micro-organisms, Lactobacillus casei Shirota and Bifidobacterium animalis subspp . lactis , in a milk-based dessert (2.7% fat) with cranberry sauce added. B. lactis had a final concentration of 1.99 × 10⁶ cfu/g after 21 days of study, with a logarithmic decrease of 8.87%. On other hand, L. casei Shirota had a final concentration of 2.05 × 10⁷ cfu/g at the end of the same period, a logarithmic decrease of 8.41%. Statistical analysis showed that significant differences existed between both micro-organisms and over various storage times, the more viable micro-organism being L. casei Shirota , which decreased less than one logarithmic cycle after 21 days.     

Biotransformation of Isoflavones by Bifidobacteria in Fermented Soymilk Supplemented with D-Glucose and L-Cysteine

ABSTRACT: Soymilk prepared using soy-protein isolate supplemented with D-glucose and L-cysteine was fermented with 4 strains of Bifidobacterium. Enumeration of bifidobacteria and quantification of isoflavones using HPLC were performed at 0, 12, 24, 36, and 48 h of incubation. Supplementation did not significantly enhance ( p > 0.05) the growth of bifidobacteria between 0 and 12 h, but did after 12 h. The increase in concentration of isoflavone aglycones and equol was significantly lower ( p < 0.05) in supplemented soymilk after 24 h when compared to plain soymilk. Supplementation increased the concentration of aglycones by 0.796 mg/100 mL in soymilk fermented with B. animalis between 12 and 24 h, and the population by 1.27 log₁₀ CFU/mL ( p < 0.05).