Cultural condition affecting the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter

In the present study, the growth and production of beta-galactosidase by Bifidobacterium longum CCRC 15708 in a 5-L jar fermenter as influenced by cultivation temperature (27-42 degrees C), medium pH (4.5-7.5) and agitation speed (5-200 rpm) were evaluated. In general, it was found that a cultivation temperature of 37 degrees C proved optimal for both growth and beta-galactosidase production by the test organism. Although the growth of the test organism was the highest in the culture with pH controlled at 4.5-6.5, the culture with pH controlled at 6.5 resulted in the highest production of beta-galactosidase. Further, agitation at 100 rpm or more was found to enhance both the growth and production of beta-galactosidase. Fermentation conducted in a jar fermenter having the pH of the culture medium, the cultivation temperature, and the agitation speed controlled at 6.5, 37 degrees C, and 100 rpm, respectively, a maximum beta-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml was achieved in 10 h of fermentation. There are ca 2.0 and 12.3 fold greater than the reported maximum beta-galactosidase and transgalactosylation activity, respectively, produced by B. longum CCRC 15708 in a flask culture system.     

Survival of bifidobacteria after spray-drying

To investigate the survival of bifidobacteria after spray-drying, Bifidobacterium infantis CCRC 14633, B. infantis CCRC 14661, B. longum ATCC 15708, B. longum CCRC 14634 and B. longum B6 were first spray-dried with different carrier media including 10% (w/w) gelatin, gum arabic and soluble starch. B. infantis CCRC 14633 and B. longum were also determined in skim milk. It was found that survival of bifidobacteria after spray-drying varied with strains and is highly dependent on the carriers used. Among the test organisms, B. longum B6 exhibited the least sensitivity to spray-drying and showed the highest survival of ca. 82.6% after drying with skim milk. Comparisons of the effect of carrier concentrations revealed that spray-drying at 10% (w/w) gelatin, gum arabic or soluble starch resulted in the highest survival of bifidobacteria. In addition, among the various outlet-air temperatures tested, bifidobacteria showed the highest survival after drying at 50 degrees C. Elevation of outlet-air temperature caused increased inactivation of bifidobacteria. However, the inactivation caused by increased outlet-air temperature varied with the carrier used, with the greatest reduction observed using soluble starch and the least with skim milk.     

Growth of bifidobacteria in soymilk and their survival in the fermented soymilk drink during storage

Growth of Bifidobacterium infantis CCRC 14633 and B. longum B6, in soymilk was investigated in the present study. It was found that soymilk could support the growth of both organisms tested. B. infantis grew better than B. longum in soymilk. Supplementation of bifitose (isomaltooligosccharie), glucose, lactose or galactose to soymilk increased the growth of B. infantis and B. longum as determined after 48 h of fermentation. On the other hand, addition of yeast extract, peptone, tryptone, casitone or N-Z-Case plus to soymilk enabled B. infantis to reach its maximum population in a shorter cultivation time of 24 h. Acid production by B. longum and B. infantis in soymilk was mainly non-growth associated, while in the yeast extract-supplemented soymilk, acid produced by B. infantis was found to be growth-associated. Populations of B. longum reduced more than did B. infantis in the prepared fermented soymilk drink during storage period. Viable population of both test organisms reduced less in the fermented drink held at 5 degrees C than at 25 degrees C. After a 10-day storage at 5 degrees C, viable B. infantis and B. longum reduced by 0.44 and 3.18 log CFU/ml, respectively, in the fermented drink. Addition of sucrose to the fermented drink resulted in an increased reduction of viable bifidobacteria during the storage period. This phenomenon was most prominent with B. infantis in the fermented drink held at 25 degrees C.     

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.     

Production of oligosaccharides in yogurt containing bifidobacteria and yogurt cultures

Yogurts were prepared by using yogurt cultures combined to mixed cultures of bifidobacteria (Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) and by adding a preincubation step (1.5 h at 50 degrees C) with bifidobacteria to the conventional method of manufacture in order to produce oligosaccharides. The survival of bifidobacteria was drastically affected during storage of yogurts, except for products containing B. animalis, in which viable counts remained at >10(6) cfu/g after 28 d of storage at 4 degrees C. Oligosaccharides with a degree of polymerization of 3 were produced during the preincubation step (0.31 to 0.68%), and the amount in the final products varied according to the species of bifidobacteria inoculated during the preincubation step or the concentration of bifidobacteria used as second inoculum during the fermentation process. In fact, the higher concentration of oligosaccharides measured at the end of the fermentation process (0.72%) and the 28 d-storage period (0.67%) was obtained for yogurts containing B. infantis. However, yogurts containing B. breve showed higher beta-galactosidase activities and had lower lactose concentrations after the fermentation process and the storage period than the other yogurts. The use of a mixed cultures of bifidobacteria (B. animalis, B. infantis, or B. breve) thus allows the production of yogurts in which bifidobacteria can survive in relatively high cell numbers and contain appreciable amount of oligosaccharides.     

Growth-enhancing supplements for various species of the genus Bifidobacterium

Various biological materials were tested for their growth-promoting activity of several bifidobacterial species in a synthetic medium containing ample sources of inorganic salts, vitamins, nitrogen, and carbon. It was found that only Bifidobacterium adolescentis and B. longum (ATCC 15708) grew optimally or near optimally in the synthetic medium. All the other bifidobacteria tested grew optimally only in the synthetic medium supplemented with a growth promoter. The best growth promoters for all bacteria were bovine casein digest and yeast extract rather than human milk whey. Other growth promoters, including human and bovine milk wheys, hog gastric mucin, and bovine serum albumin digest were effective with some bacterial species but not with others. Bifidobacteria also grew well when the bovine casein digest (20 mg/ml) was used as the nitrogen source. Only the yeast extract was able to improve growth under these circumstances. The nature of these growth factors has not yet been determined.     

Antioxidative activities of soymilk fermented with lactic acid bacteria and bifidobacteria

To further the goal of developing a probiotic dietary adjunct using soymilk, soymilk is fermented with lactic acid bacteria (Lactobacillus acidophilus CCRC 14079 or Streptococcus thermophilus CCRC 14085) and bifidobacteria (Bifidobacterium infantis CCRC 14633 or Bifidobacterium longum B6) individually, and in conjunction. We investigate several antioxidative activities including the inhibition of ascorbate autoxidation, the scavenging effect of superoxide anion radicals and hydrogen peroxide, and the reducing activity exerted by different varieties of fermented soymilks. In addition, the effect of spray-drying and freeze-drying on changes in antioxidative activity is examined. We find that in fermented soymilk both the inhibition of ascorbate autoxidation, and the reducing activity and scavenging effect of superoxide anion radicals varied with the starters used, but nevertheless are significantly higher than those found in unfermented soymilk. In general, antioxidative activity in soymilk fermented with lactic acid bacteria and bifidobacteria simultaneously is significantly higher (P < 0.05) than that fermented with either individually. Moreover, antioxidative activity increases as the fermentation period is extended. However, unfermented soymilk shows an H2O2-scavenging effect, while there is no scavenging effect except for the accumulation of H2O2 in fermented soymilk. Finally, we find that freeze-drying causes a significantly lesser (P < 0.05) reduction in the antioxidative activity of soymilk than does spray-drying. Irrespective of the drying method and the starters used for fermentation. The antioxidative activity of fermented soymilk reduces after drying yet remains higher than that of dried unfermented soymilk.     

Viability of microencapsulated bifidobacteria in simulated gastric juice and bile solution

Microencapsulated cells of Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 were prepared by spray drying the cell suspension containing the test organism and 10% (w/w) of the carrier material of either gelatin, soluble starch, skim milk or gum arabic. Survival of these microencapsulated and free cells of bifidobacteria in simulated gastric juice (pH 2.0 and 3.0) and bile solution (0.5% and 2.0%) was then examined. B. infantis CCRC 14633 was more susceptible than B. longum B6 to the simulated gastric environment and bile solution tested. Microencapsulated bifidobacteria exhibited a lower population reduction than free cells during exposure to simulated gastric environment and bile solution. This phenomenon was most pronounced when the test organism was exposed to gastric juice at pH 2.0 or 2.0% bile solution. Moreover, it was also observed that the protective effect exerted by encapsulation with spray drying varied with the carriers used and the strains of bifidobacteria.     

响应面法优化长双歧杆菌增殖培养基

为了提高长双歧杆菌发酵液中的活菌数,对其增殖培养基进行响应面优化。通过单因素试验筛选出长双歧杆菌的最佳碳源为乳糖,并发现低聚木糖、菊糖、低聚异麦芽糖、低聚果糖、苯丙氨酸、蛋氨酸、脯氨酸、谷氨酸及赖氨酸均能显著促进长双歧杆菌的生长。利用Design Expert 8.06软件设计Plackett-Burman 试验筛选出影响长双歧杆菌生长的3个最重要因子,通过Box-Behnken试验及响应面分析确定3个因子的最佳添加量为：低聚木糖1.7g/L、菊糖3.6g/L、脯氨酸0.4g/L,用优化后的增殖培养基培养长双歧杆菌,18h后其活菌数达(1.75±0.02)×109CFU/mL,比优化前提高了95.64%。     

Production and molar yield of 2-phenylethanol by Pichia fermentans L-5 as affected by some medium components

In this study, the effects of carbon sources and various levels of sucrose (0-30%) yeast extract (0-1.25%) and phenylalanine (0-0.2%) on the production of 2-phenylethanol by Pichia fermentans L-5 were investigated. Results revealed that among the 11 carbon sources tested, glycerol and sucrose were suitable carbon sources for P. fermentans L-5 resulting in production of higher amounts of 2-phenylethanol. In the medium that contained 18% sucrose, P. fermentans L-5 produced the highest amount of 2-phenylethanol, 283.4 mg/l with a molar yield of 0.77, was noted among the various levels of sucrose tested. Increasing the amount of phenylalanine added to the culture medium to 1.0% resulted in increased production of phenylethanol. Enhanced production of 2-phenylethanol, 453.1 mg/l by P. fermentans L-5 with a molar yield of 0.71 for phenylethanol was obtained in the medium containing 18% sucrose, 0.25% yeast extract and 0.1% phenylalanine after 16 h of fermentation.     

动物双歧杆菌乳亚种HCS04-002发酵条件优化

为实现动物双歧杆菌乳亚种（Bifidobacterium animalis subsp. lactis）HCS04-002的高活菌数培养,获得其生长的最适发酵条件,对发酵工艺和发酵培养基分别进行优化。以菌泥收率为考察指标,通过单因素及正交试验对培养温度、接种量和初始pH值等发酵工艺参数进行了优化;以发酵液活菌数为响应值,通过单因素试验和Box-Behnken试验优化发酵培养基。结果表明,动物双歧杆菌乳亚种HCS04-002最佳发酵条件为：培养温度39 ℃、接种量3%、初始pH值为7.2;最佳发酵培养基组分为：酵母蛋白胨24 g/L、酵母浸出物30 g/L、葡萄糖19 g/L、乳糖11 g/L。在此优化条件下,菌株HCS04-002菌悬液活菌数达2.73×109 CFU/mL。     

产乳糖酶酵母Kluyveromyces lactis培养产酶发酵条件的研究

通过正交试验和单因素试验确定了本实验室乳酸克鲁维酵母菌株产乳糖酶的发酵培养基组分(W/V,%)为乳糖8、葡萄糖0.5、酵母膏0.7、尿素0.15、KH2PO41.5、MgSO40.1、MnCl20.01,并考察了其发酵工艺条件,优化得到的最高产酶量平均达到1.930ONPGU/ml。     

蒺藜提取液对长双歧杆菌及青春双歧杆菌生长的影响

以MRS培养基为对照,OD600及pH值为响应,研究了蒺藜提取液浓度对2株双歧杆菌——长双歧杆菌及青春双歧杆菌生长的影响.结果表明;蒺藜提取液能促进长双歧杆菌和青春双歧杆菌的生长,但最适浓度因菌而已,长双歧杆菌和青春双歧杆菌在MRS培养基中的最佳蒺藜提取液的加量分别为2.0％和1.5％,37℃培养18h后,培养液的OD600分别达到1.388和1.564.     

Production of bacteriocin‐like inhibitory substances (BLIS) by Bifidobacterium lactis using whey as a substrate

The objective of this work was to evaluate the production of bacteriocin‐like inhibitory substances (BLIS) by Bifidobacterium animalis subsp. lactis in whey supplemented with yeast extract, inulin, Tween‐80 or l ‐cysteine. Cell growth, acidification, glucose and lactose consumption as well as BLIS production were measured during fermentations carried out in shake flasks. The best additive for both cell growth and BLIS production was shown to be yeast extract, which gave the highest concentrations of biomass (9.9 log cfu/mL) and BLIS (800 AU/mL). In a bench‐scale fermentor, B. lactis growth and BLIS production were between 6% and 25% higher than in flasks depending on the conditions assayed.     

耐有机溶剂脂肪酶产生菌的筛选及其粗酶酶学性质

以体积分数2%苯为筛选压力,利用罗丹明B平板显色法和摇瓶发酵法,从采集的花生地土壤样品中分离筛选得到1株中度耐热、耐碱脂肪酶产生菌,编号为H2。通过形态观察、生理生化特性实验及其16S rDNA基因序列对菌种进行鉴定。结果表明,H2菌株与短小芽孢杆菌(Bacillus pumilus)的亲缘关系最紧密。通过研究得到该菌株的摇瓶发酵条件:产酶培养基为:蛋白胨3%、酵母膏1%、NaCl 0.5%、橄榄油1%,pH7.0,摇瓶发酵温度为28℃,摇床转速为180r/min,发酵周期为48～60h。所产脂肪酶在40℃、pH9.0时酶活性最高,对pH值和温度的适应范围较宽,pH6.0～10.0比较稳定,35～50℃具有较高酶活性。     

Growth-promoting factors for Bifidobacterium longum

The ability of various biological materials to promote growth of Bifidobacterium Iongum (ATCC 15708) was tested using Bacto B₁₂ assay medium (Difco Corp.). Supplements included yeast extract, beef extract, malt extract, α-lactalbumin, β-lactoglobulin, trypticase soy broth, phytone-peptone and unknown factors from Escherichia coli spent broth. Growth of B. longum 15708 was monitored by measuring turbidity and pH. Yeast extract, α-lactalbumin and β-lactoglobulin were the best growth promoters. Growth in the presence of E. coli spent broth was maximal; however, fresh enzymatically hydrolyzed E. coli broth was as effective. Beef extract and trypticase soy broth were effective to some extent. Malt extract and phytone-peptone did not significantly enhance growth. All materials lost growth promoting activity when their disulfide bonds were reduced-alkylated.     

海洋细菌NBRC10260产琼胶酶发酵条件优化

通过正交试验法对产琼胶酶海洋菌株NBRC102603发酵条件进行优化,优化培养基为:蛋白胨4.0g/L、酵母粉1.25g/L、琼胶粉5.0g/L;在装液量150mL(500mL三角瓶)、28℃、150r/min、接种量为1%条件下发酵48h酶活力达到最高,为56.89U/mL,比未优化前提高了2.01倍.     

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.     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria

This study was conducted to investigate the catabolism and fermentation of human milk oligosaccharides (HMO) by individual strains of bifidobacteria. Oligosaccharides were isolated from a pooled sample of human milk using solid-phase extraction, and then added to a growth medium as the sole source of fermentable carbohydrate. Of five strains of bifidobacteria tested (Bifidobacterium longum biovar infantis, Bifidobacterium bifidum, Bifidobacterium longum biovar longum, Bifidobacterium breve, and Bifidobacterium adolescentis), B. longum bv. infantis grew better, achieving triple the cell density then the other strains. B. bifidum did not reach a high cell density, yet generated free sialic acid, fucose and N-acetylglucosamine in the media, suggesting some capacity for HMO degradation. Thin layer chromatography profiles of spent fermentation broth suggests substantial degradation of oligosaccharides by B. longum bv. infantis, moderate degradation by B. bifidum and little degradation by other strains. While all strains were able to individually ferment two monosaccharide constituents of HMO, glucose and galactose, only B. longum bv. infantis and B. breve were able to ferment glucosamine, fucose and sialic acid. These results suggest that as a potential prebiotic, HMO may selectively promote the growth of certain bifidobacteria strains, and their catabolism may result in free monosaccharides in the colonic lumen.