【作者】 向进乐；

【导师】 李志西；

【作者基本信息】 西北农林科技大学 ， 粮食、油脂及植物蛋白工程， 2012， 博士

【摘要】 枳椇是一种野生或半野生树种，我国大部分地区均有分布，有很高的利用价值。枳椇果梗是枳椇果实的主要组成部分，但是对于枳椇果实的开发研究主要集中在其种子——枳椇子。由于外形扭曲、个体小、残渣多、不耐贮藏、加工适应性差等，枳椇果梗未能得到合理开发和利用，相关的研究报道也较少。枳椇果梗的含糖量极高，其它营养成分也非常丰富，是很好的天然可食资源。食品发酵对原料外形、大小等没有特殊要求，枳椇果梗经微生物发酵酿造果醋，可以充分利用原料中的营养组分，尤其是高糖的特点。采用微生物发酵法加工利用枳椇果梗能达到扬长避短的效果。本文对枳椇果梗原料中多酚含量和性质、有机酸组成分析方法进行研究，对枳椇果梗果醋发酵过程动态变化进行系统研究，并对酿制的果醋预防高脂饮食引起的肥胖、高脂血症以及慢性酒精肝损伤功能特性进行评价。主要研究内容和结果如下：（1）采用Folin-Ciocalteu比色法测定枳椇果梗总酚含量。优化确定了适宜的测定条件：枳椇果梗提取液0-1.0mL加Folin-Ciocalteu试剂0.6mL、20%的Na₂CO₃溶液1.5mL，加水定容至10mL，30℃避光反应60min后在750nm处测定吸光值。方法学评价结果表明，该法能用于枳椇果梗及其加工制品总酚含量测定。（2）通过有机溶剂萃取将枳椇果梗不同极性多酚分离，采用清除DPPH·、ABTS+自由基以及总还原能力体系对不同极性多酚萃取物体外抗氧化能力进行评价。枳椇果梗不同极性萃取物总酚含量、抗氧化能力有显著差异；极性越大，多酚含量越高、抗氧化能力越强。枳椇果梗不同极性多酚萃取物抗氧化能力与其总酚含量极显著相关，而可能与多酚极性无关。（3）通过酸碱水解，有机溶剂萃取的方法分离制备枳椇果梗不同类型酚酸。枳椇果梗游离酚酸占粗提物总酚11.5%；酯型酚酸占12.3%；糖苷型酚酸占10.5%。除含有较高的可溶性多酚，不溶性结合型酚酸含量也很高，不溶性结合型酚酸与可溶性总酚酸的比值约为1：2。枳椇果梗不同类型酚酸体外抗氧化能力与其多酚含量显著相关。（4）采用离子抑制反相高效液相色谱法测定枳椇果梗及其发酵果酒、果醋中的有机酸，优化确定了色谱条件，色谱柱：Wondasil C₁₈（4.6×250mm ID，5μm）；流动相：0.01mol/L KH₂PO₄-H₃PO₄缓冲溶液，pH2.8；流速：0.7ml/min；柱温：25℃；双波长紫外检测器检测波长：210nm（L-抗坏血酸243nm）。建立的检测方法能满足枳椇果梗及其液态发酵制品有机酸组成和含量分析。通过定性和定量分析，枳椇果梗中共检出7种有机酸，分别是草酸、酒石酸、苹果酸、抗坏血酸、醋酸、富马酸、琥珀酸；以酒石酸和苹果酸为主，占其总有机酸含量的94.92%。发酵果酒和果醋中分别检出9种有机酸，果酒中以酒石酸和乳酸为主，占其总有机酸86.52%。果醋以醋酸和酒石酸为主，占92.39%。。酒石酸是枳椇果梗及其液态发酵制品中的特征有机酸。（5）对枳椇果梗酿造果醋的发酵方法和工艺条件进行研究，确定了果醋发酵的合理方式和工艺参数。酒精发酵采用带皮渣发酵方式，加水1.5倍，发酵温度16℃，发酵时间80h。醋酸发酵采用液态深层发酵法，发酵温度34℃，第一轮发酵72h左右；分割留种发酵30h。枳椇果梗果醋酿造过程中，各种有机酸在发酵过程中不断进行转化、积累与代谢。酒精发酵阶段中最显著的特点是由于苹果-乳酸发酵，乳酸大量积累，最高达8937.09mg/L；苹果酸含量显著降低至419.13mg/L。而醋酸发酵阶段最明显的特点是由于醋酸菌代谢酒精而使醋酸含量不断上升，含量最高可达37592.77mg/L，而其它大部分有机酸经醋酸发酵而减少。酿造过程中发酵液抗氧化能力也因相关抗氧化组分的变化而不断变化。枳椇果梗发酵酒酪体外抗氧化能力较发酵前显著提高，可能与发酵液总酚和维生素C含量显著升高有关。液态深层醋酸发酵发酵快，发酵液总酚下降12.7%，抗坏血酸下降31%，抗氧化能力也显著降低。发酵液抗氧化能力与总酚含量、维生素C含量均有显著相关性，说明多酚和维生素C是枳椇果梗液态发酵制品的主要抗氧化成分。（6）对枳椇果梗果醋预防饮食型肥胖、高脂血症以及保护慢性酒精肝损伤功能进行研究。研究结果显示，枳椇果梗果醋能显著抑制高脂饮食小鼠体重增加、脂肪积累、血清甘油三酯（TG）和总胆固醇（TC）升高；能显著降低高脂、高胆盐饮食小鼠血清TC和TG，显著升高血清高密度脂蛋白胆固醇（HDL-C）含量，降低动脉硬化指数（AI）。说明枳椇果梗果醋能预防高脂饮食型肥胖和高脂血症，对动脉粥样硬化有一定的预防效果。枳椇果梗果汁和果醋能显著降低慢性酒精中毒小鼠血清谷丙转氨酶（ALT）、谷草转氨酶（AST）和γ-谷氨酰转肽酶（γ-GT）活性，降低血清和肝组织中TG和TC含量以及肝脏脂质过氧化（LPO），同时能保护小鼠肝组织抗氧化酶系统。肝组织病理检查也显示，枳椇果梗果汁和果醋对慢性酒精肝损伤具有显著的预防效果，作用机制可能与抑制机体脂质过氧化，提高肝脏抗氧化酶和非酶抗氧化能力有关。更多还原

【Abstract】 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.更多还原