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.     

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.     

Enhancing the Biotransformation of Isoflavones in Soymilk Supplemented with Lactose Using Probiotic Bacteria during Extended Fermentation

ABSTRACT: Soymilk (SM) lacks lactose; hence supplementation of SM with lactose is likely to enhance the growth of probiotic bacteria and biotransformation of isoflavone glycosides to isoflavone aglycones. In this study, 11 strains of probiotic bacteria including Lactobacillus rhamnosus, L. salivarius, L. plantarum, L. acidophilus, L. paracasei, HOWARU L. rhamnosus, L. delbrueckii subsp. bulgaricus, Bifidobacterium lactis type Bi-07, B. longum, HOWARU B. bifidum, and B lactis type Bi-04 were inoculated individually or as mixed cultures into SM and soymilk supplemented with lactose (SML). A total of 2% of lactose was added to 1 L of SM with the aim of improving the growth of probiotic organisms and promoting the biotransformation of isoflavone isomers to bioactive isoflavone aglycomes. Samples of SM were incubated at 37 °C and 10 mL aliquots of SM were taken at 0, 24, 48, and 72 h to monitor the growth of probiotic bacteria and changes in isoflavone contents using high-performance liquid chromatography (HPLC). Results indicated that SML fermented with probiotics had higher viable counts by >2.4 log CFU/mL than that in SM at the end of the 72 h fermentation period. Mixed cultures grew at different rates and in general Lactobacilius spp. had >1.02 log CFU/mL more cells than Bifidobacterium spp. at the end of the fermentation period. The total aglycone content in SM at 72 h of fermentation was 0.924 mg/100 mL, whereas that in SML was 1.623 mg/100 mL. Addition of lactose not only improved the growth of probiotic bacteria in SM but also enhanced the biotransformation of isoflavone glucosides to the more bioactive isoflavone aglycones. Mixed cultures did not improve the biotransformation of bioactive isoflavones when compared to single cultures.     

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.     

Production of beta-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk

ABSTRACT:  The study determined β-glucosidase activity of commercial probiotic organisms for hydrolysis of isoflavone to aglycones in fermenting soymilk. Soymilk made with soy protein isolate (SPI) was fermented with Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and Lactobacillus casei LAFTI^® L26 at 37 °C for 48 h and the fermented soymilk was stored for 28 d at 4 °C. β-Glucosidase activity of organisms was determined using ρ-nitrophenyl β-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94, and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4 °C for 28 d was 20%, 15%, and 11% greater ( P < 0.05) than initial cell counts, respectively. All the bacteria produced β-glucosidase, which hydrolyzed isoflavone β-glycosides to isoflavone aglycones. The decrease in the concentration of β-glycosides and the increase in the concentration of aglycones were significant ( P < 0.05) in the fermented soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.     

乳酸菌在豆乳中的生长特性及其与酵母菌联合发酵作用

本研究通过分析植物乳杆菌45、植物乳杆菌571、干酪乳杆菌、嗜酸乳杆菌、鼠李糖乳杆菌以及联合增香酵母PL09发酵豆乳过程中的酸度、活菌数、电泳图、胞外多糖含量、黏度等的变化,研究不同乳酸菌在豆乳中的生长特性及乳酸菌和酵母菌联合发酵豆乳时的特性。结果表明:植物乳杆菌45和植物乳杆菌571单独发酵豆乳8 h后pH分别达到4.43和4.42,活菌数分别达到3.11×108和2.78×108 CFU/mL,并且总糖含量降低较快;鼠李糖乳杆菌和干酪乳杆菌能够在豆乳中发酵分别产生胞外多糖180.82和174.45 μg/mL;乳酸菌和增香酵母PL09联合用于发酵豆乳时,增香酵母PL09能够促进乳酸菌在豆乳中的产酸能力,并且提高活菌数及产品的黏度,为开发乳酸菌和酵母菌的联合发酵豆乳提供指导。     

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。     

Fermentation of reconstituted skim milk supplemented with soy protein isolate by probiotic organisms

ABSTRACT:  Utilization of lactose and production of organic acid were determined in reconstituted skim milk (RSM) and RSM supplemented with soy protein isolate (SPI) (RSMS) by 6 probiotic organisms, including L. acidophilus 4461, L. acidophilus 4962 , L. casei 290, L. casei 2607, B. animalis subsp. lactis bb12, and B. longum 20099. The viable counts of probiotic organisms of RSM and RSMS were enumerated and pH measured during fermentation. Our results showed that 3% to 10% more lactose was utilized by all the 6 probiotic microorganisms from RSMS than RSM. All 6 probiotic organisms produced significantly more acetic acid in RSMS than RSM. However, the viable microbial populations in RSMS were lower than those in RSM due to lower pH of the former. It appears that addition of SPI enhanced lactose utilization and acetic acid production but slightly reduced the lactic acid production and the growth of probiotic microorganisms.     

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.     

Proteolytic profiles and angiotensin-I converting enzyme and alpha-glucosidase inhibitory activities of selected lactic acid bacteria

ABSTRACT:  This study was conducted to examine the growth, proteolytic profiles as well as angiotensin-I converting enzyme (ACE) and α-glucosidase (α-glu) inhibitory potentials of selected strains of lactic acid bacteria (LAB). Two strains each of yogurt bacteria ( Streptococcus thermophilus —1275 and 285, and Lactobacillus delbrueckii ssp. bulgaricus —1092 and 1368), and probiotics ( L. acidophilus —4461 and 33200, and L. casei —2607 and 15286, and 1 strain of Bifidobacterium longum 5022), were cultivated in reconstituted skim milk (RSM) at 37 °C and their proteolytic profiles and ACE as well as α-glu inhibitory activities were determined. Among all the strains of lactic acid bacteria studied, yogurt bacteria grew very well, with the exception of L. delbrueckii ssp. bulgaricus 1368 which showed a slower growth during the initial 3 h of incubation. The growth pattern corresponded well with the decrease in pH for the organisms. All the organisms showed an increase in proteolysis with time. The variations in proteolytic capabilities translated into corresponding variations in ACE inhibitory potential of these organisms. Bifidobacterium longum 5022 showed the highest ACE inhibitory potential followed by L. delbrueckii ssp. bulgaricus 1368, L. casei 15286, S. thermophilus 1275, and L. acidophilus 4461. Organisms with high intracellular enzymatic activities grew well. Also, aminopeptidases of strains of L. acidophilus 4461 and S. thermophilus 1275 that could better utilize proline containing substrates showed enhanced ACE inhibitory potential. Lactic acid bacteria possessed the ability to inhibit α-glu activity, which endowed them an antidiabetic property as well.     

Reduction of Levels of Volatile Components Associated with the "Beany" Flavor in Soymilk by Lactobacilli and Streptococci

ABSTRACT: Methanol, acetaldehyde, ethanol, and hexanal were the 4 major volatiles detected in unfermented soymilk. Eight of the cultures of lactobacilli or streptococci completely eliminated hexanal in the soymilk during fermentation. However, there were considerable variations in the effects of the cultures on the other 3 compounds. All 8 caused significant reduction in levels of methanol. Streptococcus thermophilus OSU-2 was the only culture that significantly lowered the concentration of ethanol in the soymilk. All except Lactobacillus acidophilus C19 and Lactobacillus casei E5 significantly lowered the level of acetaldehyde. Of the cultures tested, L. acidophilus L1 offered the best potential for producing fermented soymilk with an improved volatile profile.     

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.     

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).     

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.     

大豆益生元发酵豆乳的制备及其对益生菌数量的影响

该文采用干酪乳杆菌与鼠李糖乳杆菌混种发酵大豆低聚糖,优化大豆益生元发酵豆乳制备工艺。通过单因素及正交试验,得到最佳工艺为:干酪乳杆菌与鼠李糖乳杆菌比例1∶1、果胶的添加量0.3%、豆粉与水质量体积比1∶12(g/mL)、脱脂乳粉添加量24%、大豆低聚糖添加量25%、接种量5%、发酵温度37℃、发酵时间7 h、后熟时间18 h^24 h。食用150 mL/d此条件下制备的大豆益生元发酵豆乳不仅感官品质最佳,并且对益生菌的生长具有显著的增殖作用。     

嗜酸乳杆菌、双歧杆菌和干酪乳杆菌的选择性计数

介绍了干酪乳杆菌、嗜酸乳杆菌和双歧杆菌的选择性计数方法。LC培养基只能计数干酪乳杆菌,MRS-水杨素（或山梨醇）培养基可以计数嗜酸乳杆菌和干酪乳杆菌;而MRS培养基可以计数这3种益生菌,通过减法原则从嗜酸乳杆菌、干酪乳杆菌和双歧杆菌混合物中单独计数。另外,MRS-NNLP培养基也可用于选择性计数双歧杆菌。     

Changes in antioxidant capacity,isoflavone profile,phenolic and vitamin contents in soymilk during extended fermentation

In this study, the influence of lactic acid bacteria fermentation on the antioxidant capacity, phenolic compounds and vitamins profile of soymilk with different final fermentation pH values (pH 4.55, 4.15 and 3.85) was examined. Fermented soymilk (FSM) with a final pH of 3.85 exhibited relatively higher antioxidant capacity than samples terminated at pH 4.55 or 4.15, as shown from results of Folin–Ciocalteu assay (57.3–63.8 mg GAE/100 mL FSM), DPPH-radical scavenging efficiency (30.0–36.5%) and ferricyanide reducing power (1.13–1.58 mg AAE/100 mL FSM). Among all products, Lactobacillus rhamnosus WQ2-fermented soymilk showed the highest antioxidant level among the four Lactobacillus species, while Lactobacillus acidophilus CSCC 2400 possessed the best ability to deglycosylate isoflavone glucoside (IG), reducing IG concentration by 60.8%. Four vitamins, three phenolic acids, one flavanol, and six isoflavones were detected in fermented and non-fermented soymilk by HPLC-DAD. The contents of antioxidant compounds in FSM were closely related to reducing power and anti-radical ability. In addition, antioxidant capacity was highly correlated with proteolytic activity. This study also demonstrated the potential of extended fermentation to enhance the overall health-promoting property of the products.     

乳酸菌发酵大豆糖蜜生产乳酸及糖代谢变化

选用德氏乳杆菌保加利亚亚种KLDS1.8501、嗜酸乳杆菌KLDS1.0327、嗜酸乳杆菌ATCC11975、植物乳杆菌植物亚种CICC23168、干酪乳杆菌ATCC393、植物乳杆菌NAU322分别接种于大豆糖蜜,用高效液相色谱法测定乳酸的产生以及碳水化合物的利用情况,分析不同乳酸菌发酵大豆糖蜜生产乳酸能力及糖代谢能力。结果表明,在15 °Brix大豆糖蜜中,37?℃、pH?6.0条件下发酵24?h,植物乳杆菌植物亚种CICC23168的活细胞数达到6.66×109?CFU/mL,乳酸产生量为12.18?g/L,总糖消耗量为22.48?g/L,与其他菌株相比有明显优势。因此,植物乳杆菌植物亚种CICC23168是能利用大豆糖蜜发酵产乳酸的潜力菌株。     

Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage.

A total of 26 Lactobacillus strains were isolated from various mild yoghurts and novel-type probiotic dairy products and from a starter culture preparation and were identified by using DNA-DNA hybridization technique. The species present in those products were found to be Lactobacillus acidophilus, L. johnsonii, L. crispatus, L. casei, L. paracasei and L. rhamnosus. DNA homology analysis revealed that some strains had been misclassified by their investigators. Three strains designated as L. acidophilus (L. acidophilus LA-1, L. acidophilus ATCC 43121 and the Lactobacillus strain from Biogarde culture) were found to belong to L. johnsonii and L. acidophilus L1 to be L. crispatus. Strains designated as L. casei were found to be members of three separate species: L. casei, L. paracasei and L. rhamnosus. Viable numbers of lactobacilli in mild and probiotic yoghurts varied greatly including some products with very low Lactobacillus counts. The majority of the probiotic yoghurts, however, contained viable counts above 10(5) per g even at the end of the best before use period.     

Metabolic activities of Lactobacillus spp. strains isolated from kefir

A total of 21 strains of Lactobacillus species were isolated from Turkish kefir samples, in order to select the most suitable strains according to their metabolic activities including probiotic properties. As a result of the identification tests, 21 Lactobacillus isolates were identified as L. acidophilus (4%), L. helveticus (9%), L. brevis (9%), L. bulgaricus (14%), L. plantarum (14%), L. casei (19%) and L. lactis (28%). The amount of produced lactic acid, hydrogen peroxide, proteolytic activity, and acetaldehyde productions of Lactobacillus spp. were determined. Different amounts of lactic acid were produced by strains studies; however, lactic acid levels were 1.7-11.4 mg/mL. All strains produced hydrogen peroxide. L. bulgaricus Z14L strain showed no proteolytic activity, L. casei Z6L strain produced the maximum amount (0.16 mg/mL) of proteolytic activity. Acetaldehyde concentration produced in Lactobacillus strains ranged between 0.88-3.52 microg/mL.