Research On Fermtntation Characteristics Of Hovenia Dulcis Peduncles And Health Functions Of The Fermented Vinegar

Hovenia dulcis is a wild or half wild tree widely distributed in our country, has highutilizing value. Ripe Hovenia dulcis fruit is composed of rounded seeds and fleshy pedunclesand weight of the fresh fleshy peduncles is about ninety percent of the whole fruit. Butresearch and development of Hovenia dulcis fruit are mainly focused on the seeds for theirextensive pharmaceutical activities. Most of the peduncles containing high cellulose contentare not effectively utilized probably because of their small, irregular profiles, weak storabilityand poor taste. Little research and development work have been reported on the peduncles ofHovenia dulcis.However, high sugar containing peduncles of Hovenia dulcis with abundant nutrients aregood natural resources that can feed and good raw materials for food fermentation. Becausethere are no special requirements on the materials' profiles, sizes, etc in brewing. Processingand utilization through microbial fermentation can make best use of the advantages andbypass the disadvantages of Hovenia dulcis peduncles.Quantitative method for determination of total polyphenols content and qualitative andquantitative analysis method for determination of organic acids in Hovenia dulcis peduncleswere studied in the papaer. Fruit vinegar was produced from Hovenia dulcis peduncles by asimple fermentation method and changes in biochemical parameters, organic acids,polyphenols and antioxidant capacity in the fermentation process were studied. Preventiveeffects of vinegar from Hovenia dulcis peduncles on obesity and hyperlipaemia induced bylong-term high fat diet and protective effects on chronically ethanol-induced biochemicalchanges in male mice were also investigated. The main research contents and results arepresented in the following:(1) Determination of total polyphenol content in Hovenia dulcis peduncles were studiedby using Folin-Ciocalteu colorimetry (FC) with gallic acid as standard. The results showedthat the total polyphenol content could be well calculated according to their colorimetricabsorption at750nm by applying FC reagent0.6mL and20%Na2CO31.5mL, keeping indark place at30℃for60min, and the linear range of standard curve was0⁸.47mg/L. Stability, repetition, precision and recovery rate were good by RSD. Therefore, FC method isconvenient, and accurate to determine the total polyphenol content of Hovenia dulcispeduncles.(2) The different polar polyphenols of Hovenia dulcis peduncles were separated bydifferent polar organic solvents. The total polyphenol content and the antioxidant activity ofdifferent fractions of Hovenia dulcis peduncles were determined by Folin–Ciocalteu reagent,and DPPH·, ABTS+, and Fe3+methods, respectively. Results indicated that liquid-liquidextraction was a successful method for separation of the polyphenols for different polarity.There are significant differences of the total polyphenol contents and antioxidative activitiesamong the crude extraction and its different fractions. The order of total polyphenol contentand antioxidative activities in vitro is WtF> nBF> EaF> CfF, and the correlations betweenantioxidative activity and total polyphenol content are very significant (p <0.01).(3) Four different kinds of phenolic acids of Hovenia dulcis peduncles were prepared byacid or alkaline hydrolysis and organic solvents extraction. And they are soluble free phenolicacids (SFPA), soluble esterified phenolic acids (SEPA), soluble glycoside phenolic acids(SEPA) and methanol-insoluble ester-bound phenolic acids (ISPA). The total phenolic acidscontent of ISPA is striking higher than the three kinds of soluble phenolic acids. The order oftotal phenolic acids content is ISPA> SEPA>SFPA>SGPA, and antioxidative activity ofISPA is also significantly stronger than the three soluble phenolic acids. The correlationsbetween antioxidative activity and phenolic acids content are very significant (p <0.01).(4) Organic acids in Hovenia dulcis peduncles and the fermented wine and vinegar wereidentified and quantified by ion suppression reversed-phase high performance liquidchromatography (IS-RP-HPLC). Separations were performed on a Wondasil C18(Shimadzu,Japan) column (4.6×250mm ID,5μm), column was placed in a column oven (CTO-15C)set at25℃. The LC system consisted of a Shimadzu series pumping system (LC-15C), anautosampler (SIL-10AF), a UV double wavelength detector (SPD-15C) and ShimadzuEmpower chromatography data station software. Detection of L-ascorbic acid was carried outat243nm, and the rest of organic acids at210nm. Mobile phase prepared fresh daily wasused with a constant flow rate of0.7mL/min and consisted of0.01mol/L KH₂PO₄-H₃PO₄inwater (pH2.80). Individual compounds were identified by co-injections of referencecompounds with the samples and also by comparison of their retention times of the standards.Seven organic acids (oxalic acid, tartaric acid, L-malic acid, L-ascorbic acid, acetic acid,fumaric acid, succinic acid) were identified in the fresh Hovenia dulcis peduncles, and tenorganic acids (oxalic acid, tartaric acid, formic acid, L-malic acid, α-ketoglutaric acid,L-ascorbic acid, L-lactic acid, acetic acid, fumaric acid and succinic acid) were found in the derived wine or vinegar. Tartaric acid was found to be the predominant organic acid in theHovenia dulcis peduncles and the fermented products.(5) Fruit vinegar was produced from Hovenia dulcis peduncles, and the simple andpractical fermentation technology was optimized. Skin-on fermentation was carried out in thealcoholic fermentation stage, and semi continuous submerged fermentation style was carriedout in the acetic fermentation stage in the self priming fermentor developed by our laboratory.Organic acids were transformed, metabolized or accumulated in the fermentation process. Inthe alcoholic fermentation, L-lactic acid produced and accumulated with the highestconcentration of8937.09mg/L through malolactic fermentation (MLF), which was resultedfrom the sharp increase of L-lactic acid and severe decrease of L-malic acid. Acetic acid wastransformed from alcohol by acetic acid bacteria with the highest concentration of37592.77mg/L, and formic acid was produced from methanol in the strong oxidation environment ofthe acetic fermentation.Antioxidant activities of the fermentation liquors were continuously changed with therelated antioxidant compositions. Antioxidant capacity was improved significantly by thealcoholic fermentation probably related the higher contents of vitamin C and total polyphenol.In the submerged acetic fermentation, antioxidant capacity decreased with the loss of totalpolyphenol (12.7%) and vitamin C (31%). The antioxidant capacity was significantlycorrelated to the contents of vitamin C and the total polyphenol contents. It could beconcluded that polyphenols and vitamin C are the major antioxidant compositions of theHovenia dulcis peduncles fermentation liquors.(6) Preventive effects of fermented vinegar from Hovenia dulcis peduncles on obesityand hyperlipaemia were investigated. Results indicated that the excessive weight gain and fataccumulation, increases of serum triglyceride (TG) and total cholesterol (TC) contentsinduced by long-term high fat diet could be significantly restrained by administering Hoveniadulcis peduncles vinegar. And Hovenia dulcis peduncles vinegar could significantly decreasedserum TG and TC, improved serum high density lipoprotein cholesterol (HDL-C) andprevented arteriosclerosis induced by long-term high fat and cholesterol diet.The protective effects of juice and fermented vinegar from Hovenia dulcis peduncles onchronically ethanol-induced biochemical changes in male mice were also investigated.Administration of ethanol (50%, v/v,10mL/kg) to mice for6weeks induced liver damagewith a significant increase (p <0.01) of liver index, aspartate transaminase (AST), alaninetransaminase (ALT), gamma glutamyl transferase (γ-GT) in the serum and the hepatic lipidperoxidation (LPO) level. In contrast, administration of juice or fermented vinegar fromHovenia dulcis peduncles (10mL/kg bw) along with alcohol significantly (p <0.05) decreased the activities of enzymes (AST, ALT and γ-GT), liver index, concentrations oftriglyceride (TG) and total cholesterol (TC) in serum and the hepatic TG and LPO levels.Mice treated with juice or fermented vinegar from Hovenia dulcis peduncles showed betterprofiles of the antioxidant systems with relatively higher glutathione (GSH) content, totalsuperoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)activities. All these results were accompanied by histological observations in liver. The resultsdemonstrate that both of the juice and fermented vinegar from Hovenia dulcis peduncles haveprotective effects against chronically alcohol-induced toxicity probably by preventing LPOand elevating levels of the enzymic and nonenzymatic antioxidants in liver.