In Vitro Modulating Effects Of Flaxseed Oil And Ala On Gut Microbiota

Flaxseed oil?FSO?,which has many excellent physiological functions is rich in?-linolenic acid?ALA?and is among the three essential fatty acids that the human body cannot synthesize.In addition,the intestinal environment is the largest micro-ecological system of the human body,and the intestinal flora is closely related to human health.There is no relevant report on the modulation effect of intestinal flora by FSO and ALA currently.In this paper,the effects of FSO and its functional component ALA on intestinal flora structure and intestinal probiotics were studied by using in vitro animal fecal anaerobic culture system,providing theoretical and experimental basis for clarifying the role and mechanism of FSO and ALA in improving intestinal micro-ecology as functional factors.Major findings are as follows:The process of supercritical CO₂ extraction of FSO was optimized by single factor and response surface method and it is found that the optimal extraction conditions,including extraction time,temperature and pressure are 4.58h,51°C and31MPa respectively,under which,the FSO extraction rate is 23.95%.The chemical composition of FSO was analyzed by GC-MS and the results indicate that the relative content of ALA in the FSO by supercritical extraction is 75.22%,and the ratio of unsaturated fatty acid to saturated fatty acid is 9.33,which are much higher than the two oils produced by cold press process and solvent leaching process in the market.Using the in vitro animal fecal anaerobic culture system,the effects of adding three different doses of FSO and ALA on the intestinal flora of normal diet rats and high-fat diet rats were studied.The results show that FSO and ALA could promote the growth of intestinal probiotics,inhibit the proliferation of harmful bacteria in the intestine,and increase the content of short-chain fatty acids in the fermentation broth of intestinal flora.Using Illumina-MiSeq high-throughput sequencing technology analysis methods,the results of PCA,PCoA,NMDS and cluster analysis illustrate that the addition of FSO or ALA has significant effects on intestinal flora structure,especially for high-fat diet rats'intestinal flora structure.Adding FSO can increase the abundance of intestinal probiotics Lactobacillus and Phascolarctobacterium,and reduce that of Parabacteroides,Escherichia/Shigella and Clostridium_XIVa.The rise of?-linolenic acid can increase the abundance of intestinal probiotics Bacteroidetes,Lactobacillus and Phascolarctobacterium and decrease the quantity of Firmicutes,Enterococcus,and Clostridium_XIVa,as well as make the Bacteroides/Firmicutes ratio higher.The results show that FSO or ALA has the function of adjusting the structure of intestinal flora,besides,it can promote the proliferation of intestinal probiotics,and has obvious recovery effect on abnormal intestinal flora caused by high-fat diet.?3?Through the bacterial adhesion test,the adhesion effect of three doses of ALA on two intestinal probiotics?Lactobacillus acidophilus,Bifidobacterium bifidum?and two colonic epithelial cells?colon adenocarcinoma Caco-2 cells,normal colonic epithelial cells NCM460 cells?were studied.By Gram stain microscopy and bacterial adhesion analysis,the conclusion is that medium and high doses of ALA can promote the adhesion effect of two probiotics to Caco-2 cells and NCM460 cells,which is conducive for the probiotics to continue its probiotic activity.