Study On Preparation And Anti-Inflammatory Activity Of Fermentation Broth Rich In Gamma-Aminobutyric Acid From Moringa Oleifera Leaves

Moringa oleifera Lam.,is a tropical and subtropical plant with many edible and medicinal values,which rich in nutrition,protein,vitamins and many active ingredients.Gamma-aminobutyric acid?GABA?is one of the characteristic active ingredients of Moringa oleifera,which was the main functional factor of antihypertensive,antiviral and anti-inflammatory activities.Lactobacillus plantarum LK-1 was used to improve the content of gamma-aminobutyric acid in the fermentation liquid of Moringa oleifera leaves.In this paper,the changes of nutrient composition,flavor composition and antioxidant capacity during fermentation were studied,and the anti-inflammatory activity of Moringa oleifera leaves was systematically evaluated.This study can provide theoretical basis and technical guidance for the development of a fermented drinks rich in gamma-aminobutyric acid.1.The fermentation strains was screened and the fermentation conditions of the fermentation liquid of Moringa oleifera leaves rich in gamma-aminobutyric acid was determined.Lactobacillus plantarum LK-1 was selected as the fermentation strain by compairing the GABA production of Moringa oleifera leaves fermentation liquids by four strains respectively.Then single factor and orthogonal experiments were carried out to determine the optimal fermentation conditions by taking GABA yield as the index,including the amount of Moringa oleifera leaves,sodium glutamate,initial pH,fermentation temperature and fermentation time.The results showed the Optimum Fermentation Conditions was:Lactobacillus plantarum LK-1 as the fermentation strain,4%of Moringa oleifera leaves,4 g/L of MSG,6.5 of initial pH value,35 C of fermentation temperature and72 h of fermentation time.Under these conditions,the highest GABA content was 209 mg/L,which was 2.45 times higher than that of the unfermented liquid of Moringa oleifera leaves?p<0.05?.2.The changes of nutrient components?soluble protein,flavonoids,polyphenols,free amino acids?,flavor components?organic acids,volatile substances?,antioxidant activities?DPPH scavenging capacity,hydroxyl radical scavenging capacity,superoxide anion scavenging capacity,total antioxidant capacity?of the fermentation liquid of Moringa oleifera leaves during the fermentation were studied.The results showed that the polyphenol content of the fermentation broth of Moringa oleifera leaves increased by about 24%?p<0.05?comparison with unfermented leaves of Moringa oleifera after fermented 72 hours,while the contents of flavonoids,soluble protein and total free amino acids decreased respectively by 24%,47%and 27.5%?p<0.05?.The flavor substances in the fermentation liquid of Moringa oleifera leaves also changed greatly after fermented 72 hours,lactic acid,linalool,2-heptanone,3-hexene-1-alcohol,3-methyl-3-butene-1-alcohol,2-pentanone,2-methylpropanol and ethylidene became the main flavor substances in the fermentation liquid.The total antioxidant capacity and superoxide anion scavenging capacity were significantly improved?p<0.05?,which were 42%and 68%higher than those in unfermented liquid.The scavenging capacity of hydroxyl radicals and DPPH decreased by 2%and 48%?p<0.05?respectively,but the scavenging rate was still over 60%and 40%.3.The expression of various inflammatory mediators in cells was detected by ELISA,RT-PCR and Western Blot,and the anti-inflammatory mechanism of Moringa oleifera leaves fermentation broth was systematically studied.Firstly,MTT assay was used to detect the cytotoxicity of Moringa oleifera leaves fermentation broth.The results showed that the fermentation broth of Moringa oleifera aqueous extract below 1000 ug/mL had no obvious toxic effect on cells.ELISA showed that the fermentation broth of Moringa oleifera leaves at 125-1000?g/mL could inhibit the secretion levels of IL-1?,IL-6,IL-8 and TNF-?in different degrees?p<0.05?,and the maximum inhibition rates were 35.33%,83.5%,77.44%and 43.3%,and enhance the secretion level of IL-10?p<0.05?,the maximum enhancement rate was 76.1%,which indicated that Moringa oleifera leaves fermentation broth could achieve anti-inflammatory effect by inhibiting the secretion of M1 proinflammatory factors and enhancing the content of M2 anti-inflammatory factors.RT-PCR results showed that Moringa oleifera fermentation broth at 125-500?g/mL could effectively inhibit the expression of M1 type pro-inflammatory factors IL-1?,IL-6,TNF-?,NF-KB and promote the expression of M2-type inflammatory factors IL-10 and TGF-?mRNA.This is basically consistent with the results of measuring the secretion level of inflammatory factors by ELISA.In addition,Moringa oleifera leaves fermentation broth can effectively inhibit the expression of PEG₂ and TLR4,suggesting that it may regulate inflammation through TLR-s/NF-KB pathway.Western Blot results showed that Moringa oleifera leaves fermentation broth at 125-500?g/mL could down-regulate the expression of M1 pro-inflammatory factors IL-1?,IL-6,TNF-?,iNOS protein and up-regulate the expression of M2 anti-inflammatory factor IL-10,which was basically consistent with the results of ELISA and RT-PCR.