Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Effect of enzymatic hydrolysis of proteins on growth of Bifidobacterium bifidus in milk

The effect of enzymatic hydrolysis of proteins in milk using neutrase on the growth of the probiotic strain Bifidobacterium bifidus was evaluated by estimation of microbial growth, acidity, viscosity and flavour production. A significant increase in the growth of B bifidus was observed in neutrase‐hydrolysed milk. The setting time of bifidus‐cultured milk was advanced by about 12 h at 5% degree of hydrolysis. Enzymatic hydrolysis of proteins prior to cultivation also significantly increased the viscosity of the product. An approximately 60% increase in viscosity of the product was observed in neutrase‐hydrolysed milk. Production of steam‐volatile monocarbonyls as an indication of development of flavour was also higher in neutrase‐hydrolysed milk. The concentration of steam‐volatile monocarbonyls was 2.47 µmol per 100 ml in neutrase‐hydrolysed milk but only 1.84 µmol per 100 ml in control milk at the setting point of the curd. © 2002 Society of Chemical Industry     

Early-Life Development of the Bifidobacterial Community in the Infant Gut

The establishment of the gut microbiota poses implications for short and long-term health. Bifidobacterium is an important taxon in early life, being one of the most abundant genera in the infant intestinal microbiota and carrying out key functions for maintaining host-homeostasis. Recent metagenomic studies have shown that different factors, such as gestational age, delivery mode, or feeding habits, affect the gut microbiota establishment at high phylogenetic levels. However, their impact on the specific bifidobacterial populations is not yet well understood. Here we studied the impact of these factors on the different Bifidobacterium species and subspecies at both the quantitative and qualitative levels. Fecal samples were taken from 85 neonates at 2, 10, 30, 90 days of life, and the relative proportions of the different bifidobacterial populations were assessed by 16S rRNA–23S rRNA internal transcribed spacer (ITS) region sequencing. Absolute levels of the main species were determined by q-PCR. Our results showed that the bifidobacterial population establishment is affected by gestational age, delivery mode, and infant feeding, as it is evidenced by qualitative and quantitative changes. These data underline the need for understanding the impact of perinatal factors on the gut microbiota also at low taxonomic levels, especially in the case of relevant microbial populations such as Bifidobacterium. The data obtained provide indications for the selection of the species best suited for the development of bifidobacteria-based products for different groups of neonates and will help to develop rational strategies for favoring a healthy early microbiota development when this process is challenged.     

Microencapsulated Bifidobacterium bifidum and Lactobacillus gasseri in Combination with Quercetin Inhibit Colorectal Cancer Development in ApcMin/+ Mice

Recent studies have suggested that flavonoids such as quercetin and probiotics such as Bifidobacterium bifidum (Bf) and Lactobacillus gasseri (Lg) could play a relevant role in inhibiting colon cancer cell growth. Our study investigated the role of dietary supplementation with microencapsulated probiotics (Bf and Lg) along with quercetin in the development of mouse colorectal cancer (CRC). Methods: Adenomatous polyposis coli/multiple intestinal neoplasia (Apc^Min/+) mice were fed a standard diet or the same diet supplemented with microencapsulated probiotics (Bf and Lg strains, 10⁷ CFU/100 g food) or both probiotics strains plus microencapsulated quercetin (15 mg/100 g food) for 73 days. Changes in body and organ weights, energy metabolism, intestinal microbiota, and colon tissue were determined. The expression of genes related to the Wnt pathway was also analyzed in colon samples. Results: Dietary supplementation with microencapsulated probiotics or microencapsulated probiotics plus quercetin reduced body weight loss and intestinal bleeding in Apc^Min/+ mice. An improvement in energy expenditure was observed after 8 weeks but not after 10 weeks of treatment. A supplemented diet with microencapsulated Bf and Lg reduced the number of aberrant crypt foci (ACF) and adenomas by 45% and 60%, respectively, whereas the supplementation with Bf, Lg and quercetin decreased the number of ACF and adenomas by 57% and 80%, respectively. Microencapsulated Bf and Lg in combination with quercetin could exert inhibition of the canonical Wnt/β-catenin signaling pathway in the colon of Apc^Min/+ mice Conclusions: The administration of microencapsulated Bf and Lg, individually or in combination with quercetin, inhibits the CRC development in Apc^Min/+ mice.     

乳化法制备两歧双歧杆菌微胶囊

以两歧双歧杆菌BB01和BB28为试验菌株,活茵数及包埋产率为指标,研究了海藻酸钠浓度、CaCI：浓度、乳化时间及固定化时间对乳化法制备两歧双歧杆菌微胶囊的影响．结果表明：当两歧双歧杆菌BB01微胶囊制备的条件分别为海藻酸钠浓度2％、CaCl2浓度1％、乳化时间10min、固定化时间15min时,对应的活菌数分别为4．9×10^9cfu／mL、3．06×10^9cfu／mL、2．47×10^9cfu／mL和4．37×10^9cfu／mL,包埋产率分别为80％、56．7％、40．9％和54．1％;当两歧双歧杆菌BB28微胶囊制备的条件分别为海藻酸钠浓度2．5oA、CaCl。浓度1％、乳化时间15min、固定化时间15min时,对应的活菌数分别为4．0×10^9cfu／mL、41×10^9cfu／mL、1．59×10^9cfu／mL和5．55×10^9cfu／mL,包埋产率分别为80％、92％、87％和76．1％．     

双歧杆菌生理功能研究进展

双歧杆菌是存在于人和动物肠道内重要的益生菌,它与机体的许多生理、病理现象密切相关,因此成为人体健康的重要指标之一。它在调节肠道菌群、降低人体胆固醇、抗肿瘤以及在延缓人体衰老等方面的生理功能,更成为各领域专家研究的热点。本文综述了双歧杆菌热点生理功能在国内外的研究进展及应用发展前景。      

海藻酸钠二次包衣对益生菌微胶囊包埋效果的影响研究

为了考察海藻酸钠二次包衣对两歧双歧杆菌F-35包埋效果的影响,本研究分别制备得到了海藻酸钠微胶囊（AL-M）、蛋白质微胶囊（WP-M）和海藻酸钠二次包衣的微胶囊（AL-CM）,并从三个方面（壁材降解速度、益生菌释放速度和益生菌存活情况）对这三种微胶囊在连续模拟胃肠液环境下的降解进行了比较。结果表明,海藻酸钠二次包衣能够显著降低胃蛋白酶对乳清蛋白的降解作用。相比于AL-M和WP-M,AL-CM会延缓两歧双歧杆菌F-35在模拟肠液中的释放速率,并且在连续的模拟胃肠道中经过二次包以后的微胶囊中对两歧双歧杆菌F-35保护效果最好,两歧双歧杆菌F-35最终的存活率高达51.0%。      

Production of Lacto-N-biose I Using Crude Extracts of Bifidobacterial Cells

Lacto-N-biose I (LNB) is supposed to represent the bifidus factor in human milk oligosaccharides, and can be practically produced from sucrose and GlcNAc using four bifidobacterial enzymes, 1,3-β-galactosyl-N-acetylhexosamine phosphorylase, sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase, recombinantly produced by Escherichia coli. Here the production of LNB by the same enzymatic method without using genetically modified enzymes to consider the use of LNB for a food ingredient was reported. All four enzymes were produced as the intracellular enzymes of Bifidobacterium strains. The mixture of the crude extracts contained all four enzymes, with other enzymes interfering with the LNB production, namely, phosphoglucomutase, fructose 6-phosphate phosphoketolase, and glycogen phosphorylase. The first two interfering enzymes were selectively inactivated by heat treatment at 47 °C for 1 h in the presence of pancreatin, and glycogen phosphorylase was disabled by hydrolyzing its possible acceptor molecules using glucoamylase. Finally, 91 % of GlcNAc was converted into LNB in the 100-mL reaction mixture containing 300 mM GlcNAc.     

含金属硫蛋白鸡蛋水解物对两种乳酸菌体外生长的影响

以乳酸菌生长过程中的pH、OD值和菌落总数为评价指标,研究了普通鸡蛋、含金属硫蛋白鸡蛋、普通鸡蛋水解物以及含金属硫蛋白的鸡蛋水解物对动物双歧杆菌、嗜酸乳杆菌以及两种混合菌的影响。结果表明,鸡蛋水解物作为氮源能够促进乳酸菌的增殖,经比较,最佳氮源为含金属硫蛋白鸡蛋水解物,其动物双歧杆菌、嗜酸乳杆菌和混合菌发酵液OD值为普通鸡蛋水解物的1.05倍、1.08倍和1.09倍。含金属硫蛋白的鸡蛋水解物在动物双歧杆菌、嗜酸乳杆菌以及两种混合菌发酵液的最佳添加量分别为2.0%、1.5%和2.0%,活菌数分别可达7.5×109、3.21×109和2.33×1010 CFU/mL。本研究为开发乳酸菌发酵金属硫蛋白鸡蛋肽产品提供指导。     

活性乳酸菌小麦苗汁酸奶冰淇淋的研制

研究了活性乳酸菌小麦苗汁酸奶冰淇淋的生产工艺。结果表明,选用嗜菌、保菌和双歧杆菌混和发酵,加入8%～10%的麦苗汁,选用包括亚麻仔胶在内的复合稳定剂能使得成品风味口感俱佳,膨胀率达90.42%～91.25%,融化率为2.99%～3.08%,活菌数在3个月中保持较高水平。