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.

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.

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.

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.

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。

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

This study proposed a quenching protocol for metabolite analysis of Lactobacillus delbrueckii subsp.bulgaricus.Microbial cells were quenched with 60% methanol/water,80% methanol/glycerol,or 80% methanol/water.The effect of the quenching process was assessed by the optical density（OD）-based method,flow cytometry,and gas chromatography-mass spectrometry（GC-MS）.The principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were employed for metabolite identification.The results indicated that quenching with 80% methanol/water solution led to less damage to the L.bulgaricus cells,characterized by the lower relative fraction of prodium iodide（PI）-labeled cells and the higher OD recovery ratio.Through GC-MS analysis,higher levels of intracellular metabolites（including focal glutamic acid,aspartic acid,alanine,and AMP） and a lower leakage rate were detected in the sample quenched with 80% methanol/water compared with the others.In conclusion,we suggested a higher concentration of cold methanol quenching for L.bulgaricus metabolomics due to its decreasing metabolite leakage.

Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions （different phases, initial pH values, temperatures, and nitrogen sources） using real-time polymerase chain reaction （RT-PCR）. The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains （P〈0.05）. All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level.