Transcriptomic analysis of molecular mechanisms underlying the biodegradation of organophosphorus pesticide chlorpyrifos by Lactobacillus delbrueckii ssp.bulgaricus in skimmed milk

Bioremediation of organophosphorus pesticides in contaminated foodstuffs using probiotics has been increasingly under the spotlight in recent years,though the biodegradation mechanism and derived intermediate products remain unclear.This study aimed to help fill this knowledge gap and examined the degradation mechanism of organophosphorus pesticide,chlorpyrifos,in milk by Lactobacillus delbrueckii ssp.bulgaricus using gas chromatography-tandem mass spectrometry(GC-MS/MS)combined with transcriptome analysis.After the strain was cultured for 20 h in the presence of chlorpyrifos,differential expressions of 383 genes were detected,including genes probably implicated during chlorpyrifos degradation such as those related to hydrolase,phosphoesterase,diphosphatase,oxidoreductase,dehydratase,as well as membrane transporters.GC-MS/MS analysis revealed the changes of secondary metabolites in L.bulgaricus during milk fermentation due to chlorpyrifos stress.6-Methylhexahydro-2H-azepin-2-one,2,6-dihydroxypyridine and methyl 2-aminooxy-4-methylpentanoate as intermediates,along with the proposed pathways,might be involved in chlorpyrifos biodegradation by L.bulgaricus.

Lactobacillus delbrueckii subsp.bulgaricus KSFY07对D-半乳糖诱导的氧化衰老小鼠运动能力的提升作用

该研究通过D-半乳糖建立小鼠衰老模型,观察了一株新发现的乳酸菌(Lactobacillus delbrueckii subsp.bulgaricus KSFY07,LDSBKSFY07)对衰老小鼠运动能力提升的作用并阐述了其作用机制。研究通过观察衰老小鼠跑步能力、游泳耐力、血液生化指标、组织病理学变化和mRNA表达变化、小鼠肠道内容物微生物变化来检测LDSBKSFY07提升小鼠运动能力的作用及机制。动物实验结果显示LDSBKSFY07(1.5×10^(9) CFU/kg BW灌胃)能够延长衰老小鼠的跑步时间和力竭游泳时间,降低衰老小鼠血清中的血尿素氮、血乳酸、丙二醛的水平,增加肝糖原、肌糖原的水平及超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶的酶活力。组织病理学观察提示LDSBKSFY07减轻了小鼠肝脏和肾脏肾组织的衰老损伤。qPCR结果显示LDSBKSFY07具备下调衰老小鼠肝脏、肾脏和骨骼肌组织诱导型一氧化氮合酶、肿瘤坏死因子α的mRNA表达和上调神经型一氧化氮合酶、Cu/Zn-SOD、Mn-SOD、过氧化氢酶表达的能力。另外,LDSBKSFY07还可以下调衰老小鼠骨骼肌组织的syncytin-1 mRNA表达。同时,LDSBKSFY07还增加了衰老小鼠肠道中厚壁菌门、乳酸杆菌、双歧杆菌的丰度和降低了拟杆菌门的丰度。而且,LDSBKSFY07对组织、血清和肠道菌群状态的调节作用优于维生素C(150 mg/kg BW灌胃)具有减缓小鼠衰老和提升其运动能力的效果。

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。

不同处理对Lactobacillus delbrueckii subsp. Bulgaricus LB细胞膜脂肪酸的影响

研究了pH值为6．0（温度为32，37，42和42℃），pH值为5．0和5．5五种培养条件、两个不同生长阶段对Lb. ddbrueckii subsp. Bulgaricus LB细胞膜脂肪酸组成的影响，以及冷冻干燥过程中细胞膜脂肪酸组成的变化。实验菌株Lb.LB中主要检测到8种脂肪酸。不同处理条件下Lb．LB细胞膜脂肪酸种类一样，但质量分数存在差异。C12：0和C19cyc9质量分数差异最大，变化范围分别为0．86％－2，16％@4．03％～7．51％。冷冻干燥过程中助．LB细胞膜中各脂肪酸质量分数变化甚微。

Response of Osmotic Adjustment of Lactobacillus bulgaricus to NaCl Stress

Growth and osmotic response of Lactobacillus bulgaricus ATCC 11842 under hyperosmotic constraint were investigated in a chemically defined medium （CDM） and MRS medium. NaCl could inhibit the growth of L. bulgaricus which decreased with increasing NaCl concentration. In the MRS, NaCl of 1.0 mol·L-1 was the biggest salt stress concentration; in the CDM, 0.8 mol·L-1 was the biggest inhibition concentration. In contrast to what was observed in other lactic acid bacteria, proline, glycine betaine and related molecules were unable to relieve inhibition of growth of L. bulgaricus under osmotic constraint. This was correlated to the absence of sequences homologous to the genes coding for glycine-betaine and/or proline transporters described in Lactococcus lactis and Bacillus subtilis. The amino acid aspartate and alanine were proved to be osmoprotective under NaCl stress. Addition of peptone （0.25% w/v） in the presence of salt led to a stimulation of the growth, as the decrease of the lag time and generation time, and the final biomass increased from 0.31 to 0.64.

Lactobacillus bulgaricus inhibits colitis-associated cancer via a negative regulation of intestinal inflammation in azoxymethane/dextran sodium sulfate model

BACKGROUND Colitis-associated cancer(CAC)accounts for 2%-3%of colorectal cancer(CRC)cases preceded by inflammatory bowel diseases(IBD)such as Crohn's disease and ulcerative colitis.Intestinal microbiota has been reported to play a central role in the pathogenesis of IBD and CAC.Recently,numerous prebiotics and probiotics have being investigated as antitumor agents due to their capacity to modulate inflammatory responses.Previous studies have indicated that lactic acid bacteria could be successfully used in managing sporadic CRC,however little is known about their role in CAC.AIM To investigate the effect of the probiotic Lactobacillus bulgaricus(L.bulgaricus)during the development of an experimental model of colitis associated colon cancer(CAC).METHODS C57BL/6 mice received an intraperitoneal injection of azoxymethane(10 mg/kg),followed by three cycles of sodium dextran sulphate diluted in water(5%w/v).Probiotic group received daily L.bulgaricus.Intestinal inflammation was determined by scoring clinical signs.Cytokines levels were determined from colon and/or tumor samples by ELISA BD OptEIATM kits.The level of significance was set at P<0.05.Graphs were generated and statistical analysis performed using the software GraphPad Prism 6.0.RESULTS L.bulgaricus treatment inhibited of total tumor volume and mean size of tumors.In addition,the probiotic also attenuated the clinical signs of intestinal inflammation inducing a decrease in intestinal and tumor levels of IL-6,TNF-α,IL-17,IL-23 and IL-1β.CONCLUSION Our results suggest a potential chemopreventive effect of probiotic on CAC.L.bulgaricus regulates the inflammatory response and preventing CAC.

脱脂乳对L.bulgaricus和S.thermophilus生长的影响

将保加利亚乳杆菌(L.bulgaricus,LB)和嗜热乳链球菌(S.thermophilus,ST)分别接种于不同浓度的脱脂乳中培养,检测两菌株在生长过程中酸度、pH值、活菌数的变化.结果表明:20%脱脂乳培养基对两菌株的生长都有一定的促进作用,二者在20%和14%脱脂乳中生长时,最终的活菌数分别为LB(2.0×109cfu./mL,6.0×108cfu./mL)和ST(2.2×109cfu./mL,7.2×108cfu./mL).

Production of High Quality Functional Labneh Cheese Using Exopolysaccharides Producing Encapsulated Lactobacillus Bulgaricus

Production of polysaccharides by lactic acid bacteria is vital and its technology is very important, too in dairy study. The selection ofexpolysaccharides producing encapsulated Lactobacillus bulgaricus in alginate gel and the factors affecting the production of polysaccharides such as time, temperature, pH and the growth with Streptococcus thermophilus were studied, pH 5.5 and temperature 35℃/18hr were found to be optimal for exopolysaccharides production by encapsulated L. bulgaricus and free culture yielding 600 mg/L and 380 mg/L respectively. Co-culturing L. bulgaricus with S. thermophilus increased exopolysaccharides production at 35 ℃/18hr to 660 mg/L by encapsulated cells and 400 mg/L by free culture. Results of this study were applied in the manufacture of Labneh cheese vitally containing probiotic species ofL. acidophilus, L. gasseri, L. johnsonii and protection of these bacteria by polysaeeharide produced encapsulated L. bulgaricus. The use of exopolysaccharides producing encapsulated L. bulgaricus and probiotic bacteria can provide acceptable quality to functional Labneh. This product can be used as therapeutic and diabetic milk product with highly acceptable qualities. It is noted that all of the results are the means of triplicate experiments.

Application of Pectin Crude Extract from Krung Kha Mao Leaves （Cissampelos pareira L.） for Immobilization of Lactobacillus casei subsp, rhamnosus TISTR 108 and Lactobacillus delbrueckii subsp, bulgaricus TISTR 1339 on Lactic Acid Production

The research was first to perform the utilization of novel and cheap pectin crude extract from Krung Kha Mao leaves （Cissampelos pareira L.） to immobilize cells of Lactobacillus casei subsp, rhamnosus TISTR 108 and Lactobacillus delbrueckii subsp, bulgaricus TISTR 1339 for lactic acid production, the optimum condition of immobilized cells was produced significantly at 5% of probability, that the highest lactic acid has been 38.50 and 33.66 g/L in steady state of whey medium for 96 and 108 h, respectively. Both strains were immobilized by 4% （w/w） of pectin crude extract from Krung Kha Mao （KKM pectin） leaves, 1.52 mm inner diameters of silicone tube and 5% （v/v） inoculum for immobilization. Efficiency of lactic acid production was compared by immobilized cells ofL. casei subsp, rhamnosus TISTR 108 when KKM pectin, commercial citrus pectin, commercial apple pectin and sodium alginate were used as supporting materials to produce lactic acid 38.50, 38.76, 30.43 and 34.56 g/L, respectively, the productivity of lactic acid has been 0.40, 0.40, 0.36 and 0.36 g/L h, respectively.

Exopolysaccharides Produced from <i>Lactobacillus delbrueckii</i>subsp. <i>bulgaricus</i>

Lactobacillus delbrueckii subsp. bulgaricus, which has been widely used as a fermented milk starter, is a type of probiotic, and certain strains are able to produce exopolysaccharide (EPS). EPS produced from L. bulgaricus contributes to the physical and biological function of dairy products by regulating immune response, and this tendency seems to place EPS with acidic groups. To date, six types of chemical structure have been determined and are basically composed from glucose (Glc), galactose (Gal), and rhamnose (Rha). Eps clusters on chromosome DNA control the EPS synthesis and are transcribed as one mRNA 14 genes with 18kb on L. bulgaricus Lfi5. Furthermore, L. bulgaricus is able to utilize lactose (Lac) as carbohydrate source, repeating units of EPS are synthesized from Glc 6-phosphate, generated by an Embden-Meyerhof (EM) pathway in cellular carbohydrate assimilation. This review discusses EPS produced from L. bulgaricus.

Influence of Ethanol on Probiotic and Culture Bacteria <i>Lactobacillus bulgaricus</i>and <i>Streptococcus thermophilus</i>within a Therapeutic Product

Probiotic bacteria in plain yogurt namely of Lactobacillus ssp. have been reported to treat thrush, diarrhea, athlete’s foot, jock itch and vaginal yeast infections. Lactobacillus delbrueckii ssp. bulgarius (LB-12) and Streptococcus thermophilus (ST-M5) are lactic acid bacteria widely used in the manufacture of yogurt. Alcohol is used in manufacture of some medications such as cough syrups and some products such as eggnog and rum-raisin ice cream. The objectives were to study the effect of food grade ethanol on the growth of yogurt culture bacteria and the physico-chemical characteristics of therapeutic yogurt. The treatments were 0% (control), 2.5%, 5%, and 7.5% v/v ethanol in plain yogurt. The ethanol was incorporated by stirring it into one day old plain yogurt. Product characteristics were studied weekly for a month of refrigerated (4?C) storage. Data were analyzed using Proc Mixed model of Statistical Analysis System. The ethanol amount × storage period interaction effect was significant for Lactobacillus bulgaricus counts while the ethanol amount × storage period effect was not significant for Streptococcus thermophilus counts, viscosity, pH and titratable acidity (TA). Therapeutic yogurts with ethanol, at these concentrations, can successfully be manufactured without adversely influencing counts of its probiotic bacteria over product shelf life.

Effect of Mild Sonication Conditions on the Attributes of <i>Lactobacillus delbrueckii</i>ssp. <i>bulgaricus</i>LB-12

Lactobacillus delbrueckii ssp. bulgaricus is a widely used bacterium for the production of some fermented dairy products. Mild sonication intensity condition is a non-destructive technique that uses sound waves to cause cavitation in aqueous solutions and may improve the permeability of membranes, speed up the transfer of substrates and promote cellular growth and propagation. The objective was to determine the effect of mild sonication intensities at different temperatures on growth, bile tolerance and protease activity of Lactobacillus delbrueckii ssp. bulgaricus LB-12. The treatments were four sonication intensities (8.07, 14.68, 19.83 and 23.55 W/cm2) randomized at three different temperatures (4℃, 22℃ and 40℃). The energy input (1500 J) was kept constant in all treatments. Control samples did not receive any sonication treatment. Growth and bile tolerance were determined every 2 h for 12 h of incubation. Protease activity was determined at 0, 12 and 24 h. Mild sonication conditions included 1) mild sonication intensities, 2) temperatures and 3) times, all three of which played a role in influencing the desirable attributes of Lactobacillus delbrueckii ssp. bulgaricus LB-12. Of all the mild sonication intensities studied, 14.68 W/cm2 had the best overall influence at certain time points forimproving the bile tolerance and growth at 4℃ and protease activity at 40℃. Mild sonication intensity of 23.55 W/cm2 had the best overall influence at certain time points for protease activity of at 22℃. Some mild sonication conditions could be recommended for improvement of some characteristics of Lactobacillus delbrueckii ssp. bulgaricus LB-12.

Effect of Flaxseed on Bile Tolerances of <i>Lactobacillus acidophilus, Lactobacillus bulgaricus</i>, and <i>Streptococcus thermophilus</i>

Consumption of flaxseed provides health benefits. Bile tolerance allows survival of probiotics in the intestinal tract. The objective was to determine whether or not flaxseed enhances bile tolerance of Lactobacillus acidophilus (L. acidophilus) LA-K, Lactobacillus delbruekii ssp. bulgaricus (L. bulgaricus) LB-12, and Streptococcus salivarius ssp. thermophilus (S. thermophilus) ST-M5. Control and experimental (62 g flaxseed/L) broths containing 0.3% oxgall were prepared for each culture, sterilized, cooled, inoculated, and plated for 8 h. Growth of each microorganism in both the control and experimental broths was evaluated by the slope of the regression line of its log count versus time after inoculation. Flaxseed significantly enhanced growth of L. acidophilus but not L. bulgaricus and S. thermophilus over 8 h compared to its corresponding control. Therefore, flaxseed improved the bile tolerance of L. acidophilus but not of S. thermophilus and L. bulgaricus.

Amino Acid Biosynthesis and Proteolysis in <i>Lactobacillus Bulgaricus</i>Revisited: A Genomic Comparison

The amino acid biosynthesis and proteolytic system of Lactobacillus bulgaricus (L.Bulgaricus ) is important for its growth in niche-specific environments, as well as for flavour formation in the food industry. Comparative analyses of 4 completed sequences of the L.Bulgaricus strain genome on a genomic scale revealed that genes involved in amino acids synthesis were undergoing reductive evolution. However, the selected industrial strains, namely, L.Bulgaricus 2038 and L.Bulgaricus ND02, retained more complete genes in the amino acid synthesis and proteolytic system category than the laboratory strains, and have some unique genes and pathways for obtaining amino acids that enable these bacteria to adapt to their various environmental niches.

Influence of Different Sterilization Conditions on the Growth and Exopolysaccharide of <i>Streptococcus thermophilus</i>and Co-Cultivation with <i>Lactobacillus delbrueckii</i>subsp. <i>bulgaricus</i>OLL1073R-1

The sterilization conditions between experiment and dairy industrial level are different and concern about influence on product’s qualities. In this study, the fermentation properties of Streptococcus thermophilus, alone and in combination with Lactobacillus delbrueckii subsp. bulgaricus, were evaluated in skim milk that had been subjected to distinct sterilization conditions. Growth, organic acid generation, and EPS production were determined using pasteurized or autoclaved milk. When S. thermophilus was cultivated in pasteurized skim milk, the growth was strain-dependent. On the other hand, growth of S. thermophilus was accelerated in autoclaved milk. Exocellular polysaccharide (EPS) production by L. bulgaricus was not affected by the combination of S. thermophilus strains. Thus, we observed that yogurt fermented by L. bulgaricus was minimally affected by the combination of S. thermophilus strains;growth of L. bulgaricus was maintained under the constant environment. These results should facilitate the development of fermented milk produced from L. bulgaricus in the dairy industry.

保加利亚乳杆菌发酵转化人参皂苷工艺研究

目的以人参为原料,通过保加利亚乳杆菌发酵提高人参皂苷含量。方法利用单因素试验和响应面法优化发酵工艺,并对发酵过程中原型人参皂苷生物转化可能途径进行分析。结果在发酵培养基为MRS液体培养基的前提下,最适发酵条件为发酵温度40℃,发酵时间3d,接种量3%,转化稀有人参皂苷含量在150μg/mL。经对比发现,原参中检测出Re、Rg1、Rb1、Rc、Rb2、Rd、Rh17种皂苷,经过发酵后的人参中检测出Re、Rg1、Rb1、Rc、Rb2、Rh1、Rd、R-rg3、CK 9种皂苷。同时原参中的常规皂苷含量经发酵后有所下降,稀有皂苷含量有所增加,且总酚、黄酮含量增加,总糖含量减少,发酵过程中人参皂苷生物转化的可能途径与人参皂苷含量变化趋势一致。结论保加利亚乳杆菌发酵人参能够有效将原型皂苷转化成稀有人参皂苷,为人参的深加工奠定基础,为人参发酵产品的开发和利用提供参考。

基于pH下降速率的反馈补料策略高密度培养保加利亚乳杆菌NQ2508

保加利亚乳杆菌是酸奶发酵剂和益生菌制剂的重要菌种,其高密度培养对于提高菌种在应用产品生产中的效率具有重要的现实意义。葡萄糖浓度和发酵pH是影响保加利亚乳杆菌生长的关键因子,底物葡萄糖被转化为细胞菌体和主要代谢产物乳酸,探究葡萄糖消耗速率与表征乳酸生成的发酵pH下降速率的关系,是研究保加利亚乳杆菌代谢流方向的重要方法。该实验通过培养基组成和培养条件对保加利亚乳杆菌生长的影响进行研究,同时对葡萄糖消耗速率-pH降低速率相关模型研究,并基于底物质量守恒定律建立了一条基于pH下降速率的保加利亚乳杆菌高密度培养的反馈补料发酵策略。通过化学中和法控制发酵pH为6.0~5.0,基于pH下降速率进行反馈补料将葡萄糖质量浓度控制在8~2 g/L,发酵液的活菌数达到3.6×10^(9)CFU/mL,是批次分批培养的2.4倍。该策略的应用结果为提高保加利亚乳杆菌的工业化生产效率提供参考。

保加利亚乳杆菌预处理制备液态黑蒜技术研究

文章研究了保加利亚乳杆菌预处理制备液态发酵黑蒜技术。以新鲜大蒜为原料,在180℃高温下分别对大蒜瓣进行9,10 min蒜氨酸酶灭活,将灭活后的蒜瓣处理成粒径为4 mm的颗粒,加水至液料比为4∶1,添加保加利亚乳杆菌比例为4%、5%、6%,室温下共存16,24,32 h,于恒温80℃发酵制备液态黑蒜,以未添加保加利亚乳杆菌的大蒜制备的液态黑蒜为参照(简称未处理),考察共存时间、保加利亚乳杆菌比例和灭酶时间对液态黑蒜品质的影响。液态黑蒜制备的最优条件为高温180℃、蒜氨酸酶灭活10 min、保加利亚乳杆菌比例6%、共存时间32 h、发酵时间18 d,此时总酸含量为48.48 g/kg,氨基酸态氮含量为7.79 g/100 g,还原糖含量为44.61 g/100 g,褐变值为62.49,总酚含量为8.779 mg/g,DPPH·、·OH清除率分别为66.92%、45.97%,相比于未处理的液态黑蒜,总酸含量是其3.88倍,氨基酸态氮含量是其1.41倍,还原糖含量是其2.23倍,褐变度是其1.31倍,总酚含量是其1.48倍,DPPH·、·OH清除率分别是其1.40倍和1.15倍。保加利亚乳杆菌预处理制备液态黑蒜技术可以提升黑蒜的品质,缩短黑蒜的发酵时间,具有较广阔的应用前景。

添加富含乳脂肪球膜乳清蛋白粉对发酵乳代谢组学的影响

目的:研究添加富含乳脂肪球膜(MFGM)乳清蛋白粉(M-WPI)前、后的保加利亚乳杆菌发酵乳中保加利亚乳杆菌存活率,发酵乳风味及抗氧化能力。采用代谢组学分析添加M-WPI前、后保加利亚乳杆菌发酵乳的显著差异代谢标志物及其作用通路。方法:通过样本主成分分析、偏最小二乘判别分析、热图分析和KEGG富集分析等方法确定加入M-WPI前、后发酵乳代谢过程中的差异性代谢产物,并以此分析M-WPI对发酵乳产生的影响。结果表明:在脱脂乳中加入M-WPI,可以提高保加利亚乳杆菌存活率,增加发酵乳的风味及抗氧化活性。采用主成分分析(PCA)和偏最小二乘判别分析(PLS-DA)共确定77种显著差异代谢物,涉及的主要代谢通路为19条。加入M-WPI后葡萄糖、半乳糖等,代谢物上调,提高了保加利亚乳杆菌存活率;癸酸、苹果酸等有机酸上调,能提高发酵乳的口感及改善发酵乳风味。保加利亚乳杆菌发酵乳中上调的代谢物以氨基酸类较多,其中丙氨酸、天冬氨酸和泛酸等参与β-丙氨酸代谢,与发酵乳抗氧化活性提高有关。本研究为加入M-WPI发酵乳营养评价以及相关加工技术和产品开发提供参考。

银耳多糖对椰浆酸奶感官品质及益生菌生长的影响

银耳多糖是银耳中重要的天然多功能活性成分,保加利亚乳杆菌和嗜热链球菌则是用于酸奶发酵最常见的益生菌。为了探究银耳多糖对保加利亚乳杆菌和嗜热链球菌生长的影响,本研究测定添加不同浓度银耳多糖的MRS液体培养基中保加利亚乳杆菌和嗜热链球菌的OD_(600),绘制生长曲线,并对MRS固体培养基上两种菌的活菌数进行计数。为扩大银耳多糖应用范围,使用保加利亚乳杆菌和嗜热链球菌对含有不同浓度银耳多糖的椰浆进行发酵,对发酵所得的椰浆制品进行感官评价并测定活菌数。研究结果显示,银耳多糖浓度越高,保加利亚乳杆菌和嗜热链球菌的OD_(600)越大,活菌数量也逐渐增多。在对数生长期内,培养基中银耳多糖的浓度越高,保加利亚乳杆菌和嗜热链球菌的生长速度越快。以上结果说明银耳多糖可以有效促进保加利亚乳杆菌和嗜热链球菌的增殖。当添加的银耳多糖浓度为8 g/100 mL时,发酵椰浆制品中的活菌数为1.28×10^(7)CFU·mL^(-1),产品此时的感官评分最高,品质最好。