Proteolytic activities of combined fermentation with Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 reduce antigenic response to cow milk proteins

Cow milk protein is one of the leading food allergens. This study aimed to develop an effective method for reducing milk sensitization by evaluating antigenicity of fermented skim milk protein using Lactobacillus helveticus KLDS 1.8701, Lactobacillus plantarum KLDS 1.0386, and a combination of both strains. The proteolytic systems of strains in terms of genotype and phenotype are characterized by complete genome sequence, and evaluation the antigenicity of skim milk proteins was determined by ELISA and liquid chromatography with tandem mass spectrometry. Our results showed that the genomes encoded a variety of peptidase genes. For fermented skim milk, the degree of hydrolysis of the combined strains was higher than that of individual strain. Electrophoresis showed that the band color density of α-casein (α-CN) by fermentation of the combined strains was reduced when compared with control group. The fermentation process of the combined strains inhibited α-CN, β-lactoglobulin, and α-lactalbumin antigenicity by 69.13, 36.10, and 20.92, respectively. Major allergic epitopes of α-CN and β-lactoglobulin were cleaved by abundant proteases of combined strains. In all, this study showed that the fermentation process involving both L. helveticus and L. plantarum strains could reduce cow milk protein allergenicity through the combination of cell-envelope proteinase and peptidase on α-CN.     

嗜酸乳杆菌和双歧杆菌发酵乳的流变特性研究

以两株嗜酸乳杆菌(KLDS AD1、KLDS AD2)和3株双歧杆菌(长双歧杆菌KLDS 2.0001、婴儿双歧杆菌KLDS 2.0002和KLDS 2.0604)分别发酵的酸乳为研究对象,测定其pH值、滴定酸度、质构及流变学特性。pH值和滴定酸度测定结果表明嗜酸乳杆菌产酸能力强于双歧杆菌。质构测定结果表明嗜酸乳杆菌发酵的酸奶质地较为结实。5种酸乳的剪切力升速和降速曲线都能形成触变环,触变环的面积大小为KLDS 2.0604＞KLDS 2.0001＞KLDS AD2＞KLDS 2.0002＞KLDS AD1,即婴儿双歧杆菌KLDS 2.0604在剪切力破坏下其组织状态的恢复力最差,很难恢复到起始状态。表观黏度曲线在下降时只有婴儿双歧杆菌KLDS 2.0002没有出现突增现象,其弹性最差。综合得出嗜酸乳杆菌KLDS AD2发酵乳的组织状态、黏弹性最好。     

植物乳杆菌KLDS1.0391在酸奶体系中的细菌素产生特点

植物乳杆菌KLDS1.0391能够合成细菌素,也是益生菌,在食品中既可作为益生菌使用,也可作为辅助发酵剂用于生物防控,该研究主要考察了KLDS1.0391菌株在酸奶体系中细菌素的产生特点。研究结果表明,在发酵的6 h期间,抑菌活性随发酵时间延长而增强,发酵结束时,添加植物乳杆菌KLDS1.0391酸奶组的抑菌活性显著高于仅使用酸奶发酵剂的对照组(P<0.01)。与对照组相比,加入辅助发酵剂的实验组的感官品质未发生明显的变化。植物乳杆菌KLDS1.0391具备开发益生酸奶的潜力。     

Lactobacillus casei LcY decreases milk protein immunoreactivity of fermented buttermilk but also contains IgE-reactive proteins

This study evaluates Lactobacillus casei LcY protein immunactive properties and assesses its potential to decrease milk protein immunoreactivity in fermented buttermilk before and after simulated digestion. Competitive ELISA analysis of α-lactalbumin, β-lactoglobulin, α-casein, β-casein, κ-casein, bovine serum albumin and lactoferrin determined significant (P < 0.001) immunoreactivity reduction after fermentation, and even greater decrease after three-stage simulated digestion. Immunoblotting of fermented buttermilk with human allergic sera revealed that α-casein remained the most allergenic milk protein. The LcY cell fractionation and further separation by SDS-PAGE and 2DE, immunoblotting and MALDI-TOF MS/MS identification all highlighted that cyclopropane-fatty-acyl-phospholipid synthase and carboxylate-amine ligase in the cell wall/membrane protein fraction reacted with human IgE antibodies. In silico analysis of these proteins' allergenic potential indicated that these are new allergens rather than already known cross-reacting allergens.To our best knowledge, this is the first study providing evidence of human IgE reactive protein presence in lactic acid bacteria.     

植物乳杆菌KLDS 1.0318对小鼠免疫调节作用初步研究

目的:探讨植物乳杆菌KLDS 1.0318对小鼠免疫调节功能的影响。方法:将健康的BALB/c小鼠随机分为4组,即正常对照组、乳酸菌低剂量组、乳酸菌中剂量组、乳酸菌高剂量组。灌胃不同剂量植物乳杆菌KLDS 1.0318菌悬液后,分别测定各组脾脏指数和胸腺指数,脾淋巴细胞增殖、NK细胞活性、巨噬细胞能量代谢水平,巨噬细胞吞噬能力。结果:与正常对照组相比,植物乳杆菌KLDS 1.0318中、高剂量组能够显著提高小鼠的脾脏和胸腺指数,同时能够促进脾淋巴细胞增殖,增强小鼠NK细胞活性,提高小鼠巨噬细胞吞噬中性红能力及其能量代谢水平。结论:植物乳杆菌KLDS 1.0318可以通过促进机体的特异性免疫和非特异性免疫功能来增强机体的免疫作用。     

Comparison of the immunogenicity of yak milk and cow milk

This is a comparison of potential allergenicity in a mouse model. Balb/C mice were sensitized by intraperitoneal injections (administered in three doses) of skimmed milk, casein, and whey protein from cow or yak milk. After 4 weeks, the mice were challenged and killed, sera were collected, and the spleens removed for analysis. An enzyme-linked immunosorbent assay was used to determine the specific antibodies (IgG and IgE) and cytokine. The number of mice with diarrhea was higher in the cow milk protein-sensitized group than in the yak milk protein-sensitized group. Serum (IgG, IgE) antibodies and histamine levels were also higher in cow milk protein-sensitized mice. Cytokine production by spleen-derived T cells showed high levels of interleukin-4 and interleukin-5 production and low levels of interferon-γ in cow milk protein and yak milk protein-sensitized mice. Lymphocyte proliferation ratio induced by yak milk protein was lower than cow milk protein. All results indicated that Maiwa yak milk protein may less allergenic than cow milk protein in mice.     

Occurrence and persistence of diacetyl in unfermented and fermented milks

The occurrence and persistence of diacetyl in samples of unfermented raw and processed milk have been investigated; in addition, samples of milk fermented by different lactic acid bacteria have also been analysed. Diacetyl was determined by using a simplified gas chromatographic method in which acetone was the extracting agent. Diacetyl in unfermented raw milk of cow, buffalo, goat and sheep was found to be 45.1, 65.2, 42.8 and 41.2 mg kg^?1, respectively. The pre-fermentative diacetyl in milk was highly stable, and its initial value irrelevantly changed after the milk had been stored at 20 °C for 24 h or freeze-dried for 8 h and finally, having been thermically treated at 80 °C for 30 min. Significant amounts of diacetyl were detected also in all the processed milks. At 26 °C growth temperature, the tested lactic acid bacteria produced diacetyl in milk in the following order: Streptococcus thermophilus < Lactobacillus paracasei < Lactobacillus rhamnosus. Diacetyl increased during fermentation of 24 h, while a spontaneous and significant loss was registered when fermentation was prolonged to 48 h.     

植物乳杆菌P-8发酵乳的代谢物图谱

发酵乳是由上百种生物分子组成的一个复杂的食品体系,目前没有单一的分析平台能够完整地测定其所有的代谢物。本试验通过监测植物乳杆菌P-8单菌发酵乳和植物乳杆菌P-8与商业发酵剂YC-X11复配发酵乳的发酵特性,采用超高效液相色谱和四极杆飞行时间串联质谱技术结合统计学方法分析发酵乳的代谢物图谱。试验结果表明,植物乳杆菌P-8与商业发酵剂YC-X11复配发酵所需时间为5 h,远远少于植物乳杆菌P-8发酵所需时间(13 h);复配发酵乳活菌数、滴定酸度均显著增加(P<0.05)。通过代谢组学的分析方法,共鉴定出62种代谢物,主要差异代谢物为有机酸(7种)、多肽(4种)、游离氨基酸(4种)、核酸代谢物(4种)和芳香类物质(3种)等。利用代谢组学方法能够对发酵乳代谢物进行鉴定和分析,并作为其潜在的生物标记物。     

非热加工技术在低或无致敏婴幼儿配方粉中应用的研究进展

牛乳营养丰富，对婴幼儿来说，是母乳较好的替代品，绝大多数婴幼儿配方粉都是基于牛乳制备的，非热加工技术在降低牛乳过敏原致敏性，减少营养成分损失等方面表现出较多优势。本文介绍了牛乳中主要过敏原的结构和B细胞表位，总结了低或无致敏婴儿配方粉的研究进展，重点阐述了非热加工技术包括高压、微波、发酵及其与酶解联合处理对乳蛋白致敏性的影响，以及在婴幼儿配方粉中的应用，并对其未来的发展做出了展望，以期能够为营养充足、适口、低或无致敏性新型婴幼儿配方粉的开发提供理论依据。     

The role of plNC8HK-plnD genes in bacteriocin production in Lactobacillus plantarum KLDS1.0391

The function of plNC8HK gene and plnD gene on the bacteriocin production of Lactobacillus plantarum KLDS1.0391 was studied. The results of bioinformatics analysis showed that PlNC8HK protein was a histidine protein kinase and a cytoplasmic membrane protein with six transmembrane helices. PlnD protein was a response regulator of the LytR/AlgR family. Quantitative real-time PCR revealed that plNC8HK and plnD genes of L. plantarum KLDS1.0391 were up-regulated significantly (P < 0.01) when L. plantarum KLDS1.0391 co-cultured with Lactobacillus helveticus KLDS1.9207. The plNC8HK-plnD mutant was also constructed. The change of antibacterial activity, AI-2 activity and cell number of plNC8HK-plnD mutant in co-culture and mono-culture demonstrated that plNC8HK and plnD genes were essential for bacteriocin production in L. plantarum KLDS1.0391, and the increase of bacteriocin production correlated closely with plNC8HK and plnD genes in co-culture. These results are beneficial to understand the role of two components system in the bacteriocin production of L. plantarum.     

鼠李糖乳杆菌GG发酵驼乳与牛乳的发酵特性和降糖活性比较

本试验以耐酸、耐氧、定植力强及功能性丰富的鼠李糖乳杆菌GG (Lactobacillus rhamnosus GG,L-GG)单菌发酵的驼乳和牛乳为研究对象,通过鼠李糖乳杆菌GG的生长曲线确定发酵时间,评价不同发酵时间和不同储藏时间下,发酵驼乳与牛乳的发酵特性(pH、滴定酸度和活菌数)、蛋白质水解活性和降糖活性(α-淀粉酶和α-葡萄糖苷酶抑制活性)。结果表明,pH、滴定酸度、活菌数和蛋白质水解活性存在一定的内在联系。随着发酵时间的增加,两种发酵乳的pH下降,滴定酸度上升,活菌数和蛋白质水解活性也在不断增加。在储藏期间,pH、滴定酸度、活菌数和蛋白质水解活性逐渐趋于稳定。发酵驼乳较发酵牛乳的pH低,滴定酸度、活菌数和蛋白质水解活性高,在维持发酵品质和延长货架期方面优于发酵牛乳。经过发酵后的原料乳降糖活性提高,在发酵和储藏期间发酵驼乳和牛乳都表现出很高的α-淀粉酶抑制活性,在储藏期间表现出较高的α-葡萄糖苷酶抑制活性,整体上看发酵驼乳的降糖活性优于发酵牛乳。     

海鲈鱼鱼肉发酵过程中小清蛋白IgE结合能力的变化

为探究微生物发酵对鱼肉过敏原小清蛋白Ig E结合能力的影响,选用木糖葡萄球菌(Staphylococcus xylosus,Sx)作为发酵剂,以海鲈鱼(Lateolabrax japonicus)为研究对象,利用兔抗鲈鱼多克隆抗体及鱼过敏患者血清,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDSPAGE)、免疫印迹及间接酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)对发酵过程中鱼肉过敏原蛋白进行鉴定,通过模拟胃液(simulated gastric fluid,SGF)消化实验分析发酵后的鱼肉蛋白的消化稳定性。SDSPAGE图谱显示,在发酵过程中大量蛋白质被降解,但分子质量为10 k D左右的小清蛋白(parvalbumin,PV)依然存在。免疫印迹及间接ELISA结果显示,发酵60 h后PV的Ig G结合能力下降26.9%,Ig E结合能力下降22.3%。在体外SGF消化实验中,未发酵的鱼肉蛋白20 min后PV被胃蛋白酶分解,而发酵60 h后的鱼肉中PV被酶解的时间提前至5 min。研究结果表明,经Sx发酵后鱼肉蛋白的Ig E结合能力降低,并且发酵后的PV更易被胃蛋白酶分解。     

牛羊乳促嗜酸乳杆菌增菌发酵差异性及冷藏存活率比较研究

益生菌的活菌数量是衡量益生菌产品及制剂的营养价值和保健功能的主要指标之一。本研究以嗜酸乳杆菌为发酵剂分析比较牛乳发酵奶和羊乳发酵奶中嗜酸乳杆菌增菌发酵的活力差异,及在冷藏期活菌数量的变化趋势,探讨了牛羊乳在促进嗜酸乳杆菌发酵方面的差异性及在冷藏期存活率变化规律。结果表明:嗜酸乳杆菌在羊乳基质中表现为较好的增菌发酵效能,总活菌数为1.66×1010CFU/mL,大于牛乳基质中的总活菌数3.02×109CFU/mL;羊乳发酵奶和牛乳发酵奶在21d贮藏期内嗜酸乳杆菌的活菌数均维持在较高活力水平,而在21～26d的贮藏期内,其存活性显著降低(p<0.05),存活率分别为84.15%和84.39%。由此说明羊乳基质促嗜酸乳杆菌增菌发酵效能更佳,牛、羊乳发酵奶在贮藏期活菌数变化规律基本一致。     

嗜酸乳杆菌KLDS1.0901表层蛋白对菌株黏附特性的影响

采用体外实验探究了嗜酸乳杆菌KLDS1.0901的浓度、生长阶段、表层蛋白对其黏附Caco-2细胞能力的影响。将未处理、去除表层蛋白和热致死的KLDS1.0901采用荧光标记灌胃给小鼠,取不同部位的肠黏膜,进行流式细胞检测。通过透射电镜和SDS-PAGE对表层蛋白粗品进行分析鉴定。结果表明,随着菌体浓度的增加,KLDS1.0901黏附率增大直至浓度为108 CFU/mL黏附率达到饱和。菌体处于对数生长末期时,黏附特性更优。Caco-2单细胞层与KLDS1.0901表层蛋白共同孵育1 h黏附量显著下降(p<0.05),且加入的表层蛋白浓度越高,对其黏附抑制作用越强。未处理的KLDS1.0901细胞壁外围包裹着表层结构,但除去表层蛋白后表层结构即消失。表层蛋白粗品中表层蛋白含量约为15.6%,分子量在43 kDa左右。综上,嗜酸乳杆菌KLDS1.0901含有丰富的表层蛋白,且能较好的黏附于小鼠肠道,具有益生菌的潜力。     

干酪乳杆菌纯种发酵燕麦乳工艺及其产品质量分析

针对目前益生菌发酵乳制品多为混合菌种发酵动物源乳制品的研究现状,本研究以燕麦为主要原料,通过制备纯燕麦乳,酶解,添加20%牛乳和7%蔗糖以及适量乳化剂与稳定剂,组成发酵培养基,以发酵乳制品中选育出的生长繁殖力强,发酵活力高的干酪乳杆菌05-20为试验菌株,研究接种量、发酵温度、发酵时间等单因素对干酪乳杆菌纯种发酵燕麦乳产品质量的影响。采用响应面试验优化干酪乳杆菌纯种发酵燕麦乳产品的工艺条件,分析干酪乳杆菌纯种发酵燕麦乳的产品质量,包括:感官、理化与营养成分、益生菌活菌含量与功能性成分、食品安全性、贮藏稳定性。结果表明:干酪乳杆菌纯种发酵燕麦乳的最适工艺条件为:接种量5.0%,发酵温度37℃,发酵时间5.3 h。该发酵乳呈微黄色,质地均匀细腻,酸甜可口,具有浓郁的燕麦香气和发酵香气,发酵乳活菌数达8.8×108 CFU/mL,滴定酸度49.05°T,蛋白质含量1.76 g/100 g,脂肪含量0.75 g/100 g,必需氨基酸含量占总氨基酸含量的55.92%,不饱和脂肪酸含量约占总脂肪酸含量的79.20%,燕麦β-葡聚糖的含量(92.00±1.29)μg/mL,总酚含量(13.00±0.38)μg/mL,抗消化物质质量分数(19.61±0.02)%,未检测出致病微生物和毒素,保质期为24 d。研究结果为工业化生产以植物蛋白燕麦为主的干酪乳杆菌纯种发酵乳制品提供了理论依据,也为新型益生菌发酵其它植物蛋白乳制品提供了新途径。     

弱后酸化保加利亚乳杆菌KLDS1.1011的筛选及其全基因组注释研究

为了研究影响保加利亚乳杆菌后酸化的关键基因,为酸奶发酵剂的开发提供分子水平的理论基础。本实验以实验室现有的8株保加利亚乳杆菌作为出发菌株,通过对其生长性能以及对酸敏感性筛选出KLDS1.0207、KLDS1.0205、KLDS1.1001、KLDS1.1011产酸差异性明显的4株保加利亚乳杆菌,通过对4株保加利亚乳杆菌单菌株发酵乳的凝乳时间、滴定酸度、乳糖消耗进行检测,分析得到菌株KLDS1.1011后酸化能力最弱。通过Illumina HiSeq与Illumina MiSeq测序平台对菌株菌株KLDS1.1011进行基因组测序,得到KLDS1.1011基因组全长1887491 bp,平均G+C含量为39.83%,基因组中共预测出2098个CDS,其总长度为1622760 bp,编码区域总长度占全基因组比例85.97%,编码基因的平均长度为773 bp;利用同源聚类分析方式比较保加利亚乳杆菌KLDS1.1011和KLDS1.0207基因组,发现两者有1631个共有基因,KLDS1.1011有353个特有基因,KLDS1.0207有320个特有基因,进而通过对特有基因进行KEGG通路的注释以及后酸化相关通路的分析得到6个与后酸化相关的基因,1.1011_GM000805、1.1011_GM002068、1.1011_GM000803、1.1011_GM000804四个基因是关于生物膜的形成,菌株的物质转运、1.1011_GM000260基因属于丙酮酸代谢通路,是乳酸生成过程的重要通路、1.1011_GM000194基因是蛋白水解的关键酶基因。在基因水平上为菌株KLDS1.1011较弱的后酸化特性提供了重要的理论依据。     

与特定乳酸菌共培养对植物乳杆菌KLDS1.0391细菌素合成的诱导作用

植物乳杆菌KLDS1.0391与76株乳酸菌分别共培养后,测定培养物的抑菌活性和活菌数,判断与乳酸菌共培养对植物乳杆菌细菌素合成的影响及细菌素合成与菌体密度的关系。结果表明：植物乳杆菌KLDS1.0706、KLDS4.0315、KLDS4.0351、罗伊氏乳杆菌KLDS1.0736这4株菌与植物乳杆菌KLDS1.0391共培养后,抑菌活性显著增加(P＜0.01)。与罗伊氏乳杆菌共培养过程中细菌素的抑菌活性与植物乳杆菌KLDS1.0391的细胞密度呈现明显的正相关性,并且发现只有活的诱导菌与植物乳杆菌KLDS1.0391共培养才能够诱导细菌素的合成。     

The nutrient requirements of Lactobacillus acidophilus LA-5 and their application to fermented milk

Lactobacillus acidophilus LA-5 is a suitable probiotic for food application, but because of its slow growth in milk, an increase in its efficiency is desired. To shorten the time required for fermentation, the nutrient requirements of L. acidophilus LA-5 were analyzed, including the patterns of consumption of amino acids, purines, pyrimidines, vitamins, and metal ions. The nutrients required by L. acidophilus LA-5 were Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn²⁺, and when they were added to milk, the fermentation time of fermented milk prepared by L. acidophilus LA-5 alone was shortened by 9 h, with high viable cell counts that were maintained during storage of nutrient-supplemented fermented milk compared with the control. For fermented milk prepared by fermentation with Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and L. acidophilus LA-5, viable cell counts of L. acidophilus LA-5 increased 1.3-fold and were maintained during storage of nutrient-supplemented fermented milk compared with the control. Adding nutrients had no negative effect on the quality of the fermented milk. The results indicated that suitable nutrients enhanced the growth of L. acidophilus LA-5 and increased its viable cell counts in fermented milk prepared by L. acidophilus LA-5 alone and mixed starter culture, respectively.     

优选保加利亚乳杆菌发酵特性的测定

对实验室研究较为成熟的4株保加利亚乳杆菌进行发酵产酸、后酸化测定,同时对单菌株发酵、与嗜热链球菌组合发酵制备的酸奶进行质构测定和感官评价,旨为菌株开发成直投式酸奶发酵提供基础。