Cholesterol-lowering And In Vitro Probiotic Characteristics Of Lactic Acid Bacteria Isolated From Traditionally Fermented Yak Milk In The Qinghai-Tibetan Plateau

The history of making and drinking traditional fermented yak milk on the Tibetan plateau dates back over a thousand years;the yogurt is rich and has a particular biological function of the lactic acid bacteria,which is under extreme environmental conditions of global precious and unique lactic acid bacteria germplasm resources.Cholesterol is an essential substance in the human body,having important physiological functions.Cholesterol oxidation leads to the formation of arterial plaques,with the likelihood of cardiovascular disease when there is an over-intake of cholesterol.Therefore,an increasing number of studies have given attention to the lactic acid bacteria with high cholesterol-lowering effects.In this study,202 yak yoghurt samples were collected from six regions of the Tibetan Plateau(Nagchu,Tianzhu,Maqu,Yushu,Hongyuan and Ruoergai),and 720 strains of lactic acid bacteria were isolated and purified by special culture medium.The cholesterol-lowering ability of these 720 lactic acid bacteria was determined by lecithin-cholesterol micelles,and the assimilated water-soluble cholesterol of selected 72 lactic acid bacteria was studied.Furthermore,this cholesterol-lowering mechanism of these 72 lactic acid bacteria was explored.16 S rRNA gene sequencing was used to analyze the 72 strains of lactic acid bacteria.At last,through a series of in vitro simulation tests,screening probiotic strains with probiotic properties were observed.The results are as follows:1.The 720 lactic acid bacteria had the ability to assimilate cholesterol of lecithin cholesterol micelles.There were 177 strains(24.54%)that possessed 50% of the cholesterol-lowering rate.Moreover,for 12 strains of lactic acid bacteria had a degradation rate higher than 80%,accounting for 1.7% of all strains.Among them,the the lactic acid bacteria BX39 had the best ability of cholesterol-lowering with a rate of 90.05%.2.From the results of the screening,it was determined that 72 lactic acid bacteria were able to assimilate water-soluble cholesterol.There were 27 strains(37.5%)with a cholesterol-lowering rate between 90-95%.The highest ability of cholesterol lowering was lactic acid bacteria BT18 with 90.05%.Cholesterol-lowering rates between 80-90% were achieved for 30 strains,accounting for about 41.6% of all strains.By comparing the two kinds of determination of cholesterol degradation rate of the strains,the test of water soluble cholesterol had better stability between all the repeated degradations,having more reliable results.3.The results showed that the assimilation of cholesterol was related to the activities of bile salt hydrolase,though it was not positively correlated with the activity of lactic acid bacteria.Interestingly,the strain of BX56 cholesterol removal was as high as 93.85%,though the strain did not produce a calcium ring.This indicates that this strain did not secrete bile salt hydrolase,denoting that the strain of lactic acid bacteria with high cholesterol removal was not the cause of bile salt hydrolase.4.The results revealed that there were 64 strains of Lactobacillus reuteri,4 strains of Lactobacillus lactis,3 strains of Lactobacillus helveticus,and one strain of Lactobacillus plantarum were isolated from 72 strains of lactic acid bacteria.5.A series of in vitro probiotic properties of 72 lactic acid bacteria were studied.At the acid resistance test,11 strains of 72 lactic acid bacteria could survive at a pH of 2.0;a strain of Lactobacillus helveticus BX95 demonstrated the best acid resistance,with the survival rate reaching 87.75%,and the number of bacteria reaching 8.59 lg cfu/mL.The surface hydrophobicity rate of Lactobacillus helveticus BX95 was more than 20%.In the simulated pH 3.0 artificial gastric juice test,the survival rate of BX95 was up to 110.46%.When the artificial gastric juice was simulated at pH 2.5,the survival rate of the strain was 90.77%.Compared to the other strains,the L.helveticus BX95 was considered better in artificial intestinal fluid.In the resistant 0.3% bile salt test,the shortest lag time was 6.63 h of L.helveticus BX95 among the 12 strains.It also exhibited the ability to inhibit four pathogens.Compared with the existing research results,the results of the above studies showed that the lactic acid bacteria in traditional fermented yak yogurt had obvious advantages in lowering cholesterol,and the removal of water-soluble cholesterol was up to 96%.Therefore,the traditional fermented yak yoghurt on the Qinghai-Tibet Plateau is an ideal medium for screening LAB strains with high cholesterol-lowering properties.Moreover,it may be related to the harsh environment characterized by high altitude,cold temperature,and strong ultraviolet radiation,and the unique environment of LAB which is characterized by high concentrations of nutrients.At the same time,the results of this study also provide an important scientific basis and theoretical support for the development and utilization of lactic acid bacteria germplasm resources in traditional fermented yak yoghurt.