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.     

不同醇沉竹荪多糖组分对青春双歧杆菌体外增殖作用的影响

本文以竹荪为原料提取多糖成分并以乙醇分级制备竹荪多糖(DPs)不同组分,通过青春双歧杆菌体外增殖试验初步评价竹荪多糖及其分级醇沉组分的益生元功效。结果显示在α-淀粉酶溶液中水解4 h后,20%、40%、50%、60%竹荪多糖醇沉组分水解度分别为9.10%、8.02%、8.88%、11.67%,与对照组菊粉(水解度13.15%)相比差异显著(P<0.05);80%竹荪多糖醇沉组分、未分级醇沉组分与菊粉对照组相比差异不显著(P>0.05)。在模拟胃液中水解6 h后,20%、40%、50%竹荪多糖醇沉组分水解度分别为17.30%、14.52%、9.61%,与对照组菊粉(水解度5.72%)相比差异显著(P<0.05);60%竹荪多糖醇沉组分、80%竹荪多糖醇沉组分、未分级醇沉组分水解度与对照组相比无显著差异(P>0.05)。随着竹荪多糖添加量的增加,青春双歧杆菌的菌体浓度呈现先增大而后不断减小的趋势。分级醇沉浓度达到80%的竹荪多糖组分和未分级的多糖组分,青春双歧杆菌体外增殖作用与pH下降显著且效果优于菊粉(P<0.05),并在20 h的发酵时间内两者表现出相似的效果。不同竹荪多糖醇沉组分对青春双歧杆菌均有增殖效果,且不同竹荪多糖醇沉组分的增殖效果和抗水解能力随乙醇浓度的增加而增强,未分级的多糖组分同样可以加强青春双歧杆菌增殖能力,具有益生元潜力。     

The effect of hydrolysed tragacanth gum and inulin on the probiotic viability and quality characteristics of low‐fat yoghurt

This study investigates the effect of supplementation of tragacanth gum (GT, 0.05% w/v), low molecular weight gum tragacanth (LMWGT, 0.5% w/v) and inulin (0.5% w/v) on the viability of Bifidobacterium bifidum and the quality parameters of low‐fat yoghurt during a three‐week storage period. The count of probiotics was found to be 7.8 log cfu/g in inulin, and LMWGT enriched yoghurts at the end of the storage period. The minimum water holding capacity and the maximum syneresis values were also obtained in the low‐fat yoghurt enriched with GT throughout the storage time. The samples containing inulin and LMWGT revealed sensory attributes that were judged superior compared to those in GT.     

一株Bifidobacterium longum CCFM760的生理特性、安全性评价和基因分析

长双歧杆菌(Bifidobacterium longum CCFM760)是一株从江苏省无锡市滨湖区两岁健康幼女粪便分离筛选得到的具有良好缓解便秘功效的益生菌。作者对其生长产酸能力、人工模拟肠胃液耐受性、抑菌能力、低聚糖利用能力、抗生素耐受能力和总超氧化物歧化酶(SOD)活性进行了检测。该菌具有良好的生长产酸能力和人工模拟肠胃液耐受性,对大肠杆菌和金黄色葡萄球菌有明显的抑制作用,能显著利用7种低聚糖,对14种抗生素敏感,培养液中总SOD活性达到(99.14±9.91)U/mL。此外,对该菌进行基因组草图测序,并从基因组角度解析了其低聚糖代谢能力、抗生素敏感性以及抗氧化能力。以上结果表明,B.longum CCFM760是一株安全的、具有实际应用潜力的益生菌。     

鲟鱼肝酶解条件优化及酶解液对双歧杆菌增殖效果的研究

为提高鲟鱼肝脏的加工附加值，使用碱性蛋白酶，以水解度为评价指标，在单因素试验的基础上，通过Plackett-Burman试验、响应面分析法优化鲟鱼肝蛋白酶解条件。在此条件下，进一步探究了酶解液对双歧杆菌的促生长效果。结果表明，最优酶解条件为碱性蛋白酶添加量10 540 U/g鱼肝蛋白、体系初始pH值10.0、酶解时间8.6 h、酶解温度45 ℃、料液比1∶10（g∶mL），此条件下水解度（81.7%）是优化前（40.4%）的1.02倍。鲟鱼肝蛋白酶解液对青春双歧杆菌、两歧双歧杆菌、动物双歧杆菌、长双歧杆菌、短双歧杆菌和婴儿双歧杆菌6种均具有显著的促生长效果。研究结果为鲟鱼加工副产物利用提供了新途径，同时也为双歧杆菌促生长因子的研发提供新思路。     

Effect of different levels of fat and inulin on the microbial growth and metabolites in probiotic yogurt containing nonviable bacteria

Effects of different levels of fat and inulin on bacterial cell counts, degree of proteolysis and concentrations of organic acids in the yogurt containing inactivated cells of probiotic strains Bifidobacterium animalis and Lactobacillus acidophilus were investigated. Results showed that both L. acidophilus and B. animalis grew well in the yogurt samples reaching cell counts higher than 10⁶ CFU mL^?1 at the final pH of 4.5. Inulin at the concentration of 1% had no significant effects on the production of organic acids and cell counts of L. acidophilus, but promoted the growth of B. animalis with a reduction in the degree of proteolysis. Generally, different fat levels showed significant effects on the production of organic acids and nonsignificant effects on the cell counts of probiotic bacteria and degree of proteolysis. In case of lactic acid, the ratio of L‐ (+)to D‐ (?) isomer ranged from 50/50 to 80/20 in yogurt samples.     

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。     

Lactobacillus delbrueckii subsp. bulgaricus和Streptococcus thermophilus接种比例对Bifidobacterium lactis益生菌发酵乳品质的影响

将Lactobacillus delbrueckii subsp.bulgaricus ND02(LB-ND02)和Streptococcus thermophilus ND03(ST-ND03)按1∶1、1∶10、1∶100、1∶1000接种于脱脂乳中,同时接入益生菌Bifidobacterium lactis V9(B.lactis V9,接种量为2.0×107g-1),于42℃进行发酵。通过对发酵及贮藏过程中发酵乳指标的测定,评价LB-ND02和ST-ND03的接种比例对发酵乳品质的影响。结果表明,随着LB-ND02接种比例减小,凝乳时间显著延长,B.lactis V9活菌数显著提高。4℃贮藏28 d后,随LB-ND02接种比例减小,B.lactis V9存活率差异显著,后酸化也显著减弱。研究发现,LB-ND02和ST-ND03的接种比例,显著影响发酵乳的发酵时间、B.lactis V9活菌数、后酸化及黏度。