Preventive Effects Of Fermented Kiwifruit Pulp On The Damage Of Intestinal Mucosal Mechanical Barrier And Its Mechanism

Objective:The results showed that lactic acid bacteria fermentation could improve the antioxidant activity of fruits and vegetables.However,most of the existing studies are based on the changes of antioxidant content and antioxidant capacity in vitro.Whether fermented fruits and vegetables can prevent intestinal injury by improving the antioxidant capacity of experimental animals is unknown.In order to clarify the effect of fermentation on the activity of kiwifruit pulp,simulated digestion was used to study the antioxidant activity of kiwifruit pulp fermented by different lactic acid bacteria in vitro.And the fermented kiwifruit pulp with strong antioxidant capacity and large number of live bacteria was selected;The non-targeted metabonomics was used to explore the difference of material composition of kiwifruit pulp before and after fermentation;The preventive effect of fermented kiwifruit pulp on intestinal injury was studied in animals with acute lipopolysaccharide stress,and its possible mechanism was studied from the perspective of mitochondrial injury.This study provides theoretical support for further clarifying the health effects of fermented fruit and vegetable products on the body.Methods:1.The effects of simulated digestion on the antioxidant capacity of kiwifruit:(1)The contents of polyphenols(total phenolic acids and total flavonoids)in kiwifruit pulp fermented by Lactobacillus plantarum(FKP-LP),Lactobacillus acidophilus(FKP-LA)and Lactobacillus casei(FKP-LC)were detected by Folin phenol method and aluminum nitrate colorimetry respectively,and the number of viable bacteria was measured by plate counting method;(2)DPPH,ABTS,hydroxyl radical and superoxide anion radical scavenging capacity were used to determine the changes of antioxidant capacity of kiwifruit pulp fermented by lactic acid bacteria during the simulated digestion;(3)The methods of ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS)was established to determine the changes of polyphenol compounds in kiwifruit pulp fermented by lactic acid bacteria during the simulated digestion,and to clarify the effects of lactic acid bacteria fermentation on polyphenols in kiwifruit pulp.The fermented kiwifruit pulp with more viable bacteria,the highest polyphenol content and the strongest free radical scavenging ability after the simulated digestion was selected for animal experiment.2.The effects of Lactobacillus plantarum fermentation on the material composition of kiwifruit pulp: the material composition of kiwifruit pulp before and after Lactobacillus plantarum fermentation was determined by non-targeted metabonomics technology,and the changes of differential metabolites of kiwifruit before and after Lactobacillus plantarum fermentation were determined by multivariate statistical analysis method.3.Preventive effect of fermented kiwifruit pulp on the damage of intestinal mucosal mechanical barrier: 40 male SD rats were randomly divided into 5 groups according to body weight: the control group(Control),Lipopolysaccharide group(LPS),kiwifruit pulp with Lactobacillus plantarum powder group(KP+LP),Lactobacillus plantarum fermented kiwifruit pulp group(FKP-LP)and Lactobacillus plantarum bacterial powder group(LP).Gavage was performed once a day for 4weeks,and body weight and food intake were recorded:(1)After the experiment,the DAO content in plasma and intestine was detected by spectrophotometry.(2)Morphological changes of colon of different groups were observed by pathological sections,and tight junctions of intestinal epithelial cells were observed by transmission electron microscopy to determine the effect of fermented and unfermented kiwifruit pulp on LPS-damaged intestine of rats.(3)Real-time PCR was used to detect tight junction protein gene expression(Claudin,Occludin and ZO-1)in the intestinal mucosa to further clarify the preventive effect of kiwifruit pulp on the damage of intestinal mucosal mechanical barrier in fermented and unfermented kiwifruit pulp.4.Preventive effects of fermented kiwifruit pulp on intestinal mitochondrial damage:(1)The activities of MDA,SOD,GSH and CAT in intestine and intestinal mitochondria were detected by spectrophotometry to clarify the damage mitigation effect of kiwifruit pulp on intestinal mucosal mechanical barrier before and after fermentation.(2)The morphological changes of mitochondria in intestinal epithelial cells were observed by transmission electron microscope.(3)The contents of ATP,ADP and AMP in intestine were detected by high performance liquid chromatography(4)The related genes of mitogenesis and mitophagy(AMPK,ULK-2,NRF-1,PGC-1α and TFAM)m RNA were detected by real time PCR,to explore the mechanism of kiwifruit pulp on the remission of LPS injury in rats before and after fermentation.Results :1.Effects of the simulated digestion on antioxidant activity of kiwifruit pulp:(1)Compared with undigested kiwifruit pulp(fermented and unfermented),after simulated gastric digestion and intestinal digestion,the content of total phenols and flavonoids increased(P<0.05).Compared with different strains,it was found that FKP-LP and FKP-LA showed higher antioxidant content in the simulated digestion process.(2)The fermentation of lactic acid bacteria can improve the scavenging activities of DPPH,ABTS and superoxide anion radical in kiwifruit pulp,but has little effect on the scavenging activity of hydroxyl radical.After simulated gastric and intestinal digestion,compared with FKP-LC and FKP-LA,FKP-LP fermented kiwifruit pulp showed stronger free radical scavenging ability(P<0.05).(3)The composition of polyphenols during simulated digestion was determined by UPLCMS.And it was found that FKP-LP showed higher contents of protocatechuic acid,chlorogenic acid and caffeic acid after simulated gastric and intestinal digestion,followed by FKP-LA.(4)The number of viable bacteria in kiwifruit pulp fermented by different strains decreased in varying degrees in gastric and intestinal digestion,and FKP-LP fermented fruit pulp showed more viable bacteria after simulated digestion(P<0.05).2.Effects of Lactobacillus plantarum fermentation on the composition of kiwifruit pulp: after the fermentation of Lactobacillus plantarum,the substances of kiwifruit pulp changed greatly.Compared with unfermented kiwifruit pulp,the peptides,hormones and vitamins in kiwifruit pulp increased,while nucleic acids,organic acids and carbohydrates decreased;In univariate analysis,we found that under positive ion mode,the contents of ammonium acetate,artemisinin and 10 hydroxydecanoic acid increased,while the contents of dextromethorphan hydrobromide,tenemycin and 16-heptyne-1,2,4-triol decreased;In the negative ion mode,methoxy ester alcohol,hydroxydecenoic acid and 3-methoxycarbonylamino-2,2,3-trimethylbutyric acid increased,while n-acetyl-l-tryptophan,uridine and pantothenic acid decreased.3.Preventive effects of kiwifruit pulp on the injury of intestinal mucosal mechanical barrier:(1)LPS stress caused the increase of plasma DAO content,which decreased in varying degrees after kiwifruit pulp intervention(P<0.05).(2)The results showed that in the normal group,the structure of colonic mucosa was clear and the glands were arranged in order.In rats which were injected intraperitoneally with LPS,the glands were disordered and accompanied by a large number of inflammatory cell infiltration;Disorder of gland arrangement can be seen occasionally in LP group,while the morphology of KP + LP group and FKP-LP group is equivalent to that of normal diet group,and FKP-LP group is better than KP + LP group;The observation of tight junction of intestinal epithelial cells by transmission electron microscope was consistent with the results of intestinal pathology.(3)Fermented kiwifruit pulp can relieve the injury of the intestinal mucosa mechanical barrier by increasing the expression of Clauding-1,ZO-1 and Occludin.The expression of tight junction protein in Normal group was at a high level.After LPS injury,the damage of intestinal tight junction led to the decrease of gene expression.After kiwifruit intervention in different groups,the expression level increased to varying degrees.4.Preventive effects of kiwifruit pulp on the damage of intestinal mitochondrial:(1)The activities of MDA,SOD,GSH and CAT in intestinal tract and intestinal mitochondria were detected by spectrophotometry.The content of MDA increased significantly and the activities of GSH-Px,SOD and CAT decreased significantly in LPS group(P < 0.05).The intervention group could effectively inhibit the expression of MDA in intestinal tract and intestinal mitochondria after LPS injury,and increase the expression of GSH-Px,SOD and CAT(P<0.05).(2)The morphological changes of mitochondria in intestinal epithelial cells were observed by transmission electron microscope.The morphology of mitochondria in the normal group was complete,and the outer membrane of mitochondria was deformed and broken in the LPS intervention group.After kiwifruit pulp intervention,the mitochondrial damage was alleviated in varying degrees,and the mitochondrial structure of FKP-LP was clearer,which was close to the morphology of mitochondria in the normal group.(3)The contents of ATP,ADP and AMP in intestine were detected by HPLC-MS.The results showed that the mitochondrial energy metabolism was stable in the normal group.After LPS injury,mitochondrial compensatory changes occurred,and the contents of ATP,ADP and AMP increased.After kiwifruit intervention,they decreased in varying degrees(P< 0.05).(4)Acute LPS injury causes the expression of steady-state regulation related genes(AMPK,PGC-1α,NRF-1,ULK-2 and TFAM)increased,while the expression decreased in different degrees after kiwifruit pulp intervention(P<0.05).Conclusions :1.Effects of the simulated digestion on antioxidant activity of kiwifruit pulp:compared with unfermented kiwifruit pulp,kiwifruit pulp fermented by lactic acid bacteria showed stronger antioxidant capacity in the process of the simulated digestion;Kiwifruit pulp fermented by Lactobacillus plantarum was superior to Lactobacillus acidophilus and Lactobacillus casei in content of polyphenol,the antioxidant capacity and the number of viable bacteria.2.Effects of Lactobacillus plantarum fermentation on the material composition of kiwifruit pulp: Lactobacillus plantarum fermentation has a great impact on the material composition of kiwifruit pulp,and the peptides,hormones and vitamins of kiwifruit pulp increase after fermentation.3.Preventive effects of kiwifruit pulp on intestinal mucosal mechanical barrier injury: unfermented kiwifruit pulp,Lactobacillus plantarum powder and Lactobacillus plantarum fermented kiwifruit pulp can relieve the increase of intestinal permeability caused by acute LPS stress;However,compared with the intervention of unfermented kiwifruit pulp and Lactobacillus plantarum powder,Lactobacillus plantarum fermented fruit pulp showed better effects of preventing intestinal injury.4.Preventive effect of kiwifruit pulp on intestinal mitochondrial damage: LPS acute stress will activate mitochondrial compensatory pathways,such as promoting mitogenesis and mitophagy,and promoting the production of energy substances.The intervention of unfermented kiwifruit pulp,fermented kiwifruit pulp and Lactobacillus plantarum powder can improve the antioxidant capacity of intestinal tract,and reduce the compensatory response caused by acute LPS,and fermented kiwifruit pulp group had a better the remission effect.In conclusion,compared with unfermented kiwifruit pulp,Lactobacillus plantarum fermentation can improve the antioxidant capacity of kiwifruit pulp and produce some new physiologically active substances;Kiwifruit pulp fermented by Lactobacillus plantarum can alleviate the damage of intestinal mechanical barrier caused by acute LPS stress and down-regulate the compensatory response of intestinal mitochondria.