【作者】 王瑞；

【导师】 狄建兵；

【作者基本信息】 山西农业大学 ， 农业硕士（专业学位）， 2024， 硕士

【摘要】 番茄中富含多种生物活性物质,将其深加工,制成一种具有功能性的酵素产品,不仅有助于最大程度地保留其天然的营养成分,还能充分发挥酵素在生物活性方面的优越性。本文以新鲜番茄为原料,优化番茄酵素的发酵工艺,对比分析不同发酵方式制备番茄酵素时,发酵过程中的代谢产物变化、体外抗氧化能力及番茄酵素在体外消化过程中的变化情况,最后对番茄酵素的稳定性进行研究。主要研究内容和结果如下:1.番茄酶解的工艺优化。酶解的最佳工艺条件为:果胶酶添加量1.2%,纤维素酶添加量0.6%,酶解温度50℃,酶解时间150 min,此条件下出汁率为78.33%,可溶性固形物含量为8.40%。2.番茄酵素发酵工艺的研究。酵母菌制备番茄酵素的最佳工艺条件为:接种量1.14%,发酵温度27℃,发酵时间为28 h,此条件下SOD活性达139.14 U/g。乳酸菌制备番茄酵素的最佳工艺条件为:接种量0.31%,发酵温度37℃,p H为5.47,发酵时间40 h的条件下SOD活性达147.63 U/g。复合发酵制备番茄酵素最佳工艺条件为:酵母菌接种量1.14%,发酵温度27℃,发酵21 h后,接种0.31%的植物乳杆菌,调节p H为5.50,37℃继续发酵28 h,此条件下,SOD活性148.43 U/g、总酸含量19.20g/kg、还原糖含量3.77 g/kg、乳酸菌活菌数2.76×10~9CFU/m L、酵母菌活菌数1.18×10~8CFU/m L。3.番茄酵素发酵过程中代谢产物的变化规律。在不同菌种发酵番茄酵素的过程中,总酸含量在乳酸菌和复合菌种发酵时上升,酵母菌发酵在24 h时最高为495.85ug/m L;可溶性固形物含量在酵母菌和乳酸菌发酵时均下降,复合发酵时升高;还原糖在酵母菌发酵时含量下降,乳酸菌和复合菌种发酵在24 h时最高为40.35 g/kg、4.28g/kg;抗坏血酸含量在酵母菌和乳酸菌发酵时间12 h时最高为4.61 mg/100g、2.41mg/100g,复合发酵时升高,在48 h时达4.11 mg/100g。番茄红素、总类胡萝卜素含量均先下降后上升;总酚含量、SOD、脂肪酶及蛋白酶活性发酵后整体显著提高。通过相关性分析结果表明,发酵菌种不同,各代谢物质在发酵过程中的变化规律、含量及相互之间的相关性也具有明显差异。以番茄原汁为对照,利用高效液相色谱法(HPLC)对不同发酵方式制备的番茄酵素有机酸进行定量分析,番茄原汁与酵母发酵番茄酵素中检测出8种有机酸,原汁中以柠檬酸、乙酸、琥珀酸为主要有机酸,柠檬酸含量最高为602.62 ug/m L;酵母发酵中以柠檬酸、琥珀酸为主要有机酸,柠檬酸含量最高达528.16 ug/m L。乳酸菌发酵中共5种有机酸主要为乳酸、柠檬酸、乙酸,乳酸的含量最高为3467.35 ug/m L。复合和自然发酵中均有6种有机酸,复合发酵以乳酸、乙酸、琥珀酸为主要有机酸,乳酸含量最高718.29 ug/m L;自然发酵主要为乳酸、琥珀酸、柠檬酸,乳酸含量最高2096.28ug/m L。采用气相色谱-离子迁移谱技术(GC-IMS)分析检测番茄原汁和四种发酵酵素的挥发性风味物质。五组样品中共定性出114种有机物,包括76种已鉴定的有机物和未鉴定出的38种。其中酯类18种,醇类17种,酮类16种,醛类12种,烯类4种,酸类和醚类各2种,酚类和烷烃类各1种,其它和未鉴定出的共41种。用三维差谱图、挥发性物质峰强度及聚类对比分析对五组不同样品进行差异分析,结果显示番茄原汁发酵后,酯类、醇类、酮类和醛类等挥发性有机物质显著增加,使番茄酵素主要呈现果香味、甜香、奶油香、焦香等气味,且复合发酵可以使酵素在口感和香气上更协调。4.番茄酵素体外抗氧化能力及体外模拟消化研究。复合发酵和自然发酵的番茄酵素对DPPH自由基清除力较高,相比番茄原汁分别提高12.28%、18.54%。复合发酵对羟基自由基、超氧阴离子自由基清除力最高,分别为99.60%、79.50%,比番茄原汁分别提高1.16%、4.35%。酵母发酵时还原力为1.89,植物乳杆菌发酵时为1.54,复合菌种发酵后为1.64,比自然发酵时分别提高87.12%、52.47%和62.38%。说明接菌发酵后可显著提升番茄酵素的抗氧化能力,且复合发酵时的抗氧化能力整体优于其他菌种发酵。经体外模拟胃消化后,总酚含量及SOD活性均显著提升,且胃消化组>胃酸组>空白组。在消化120 min后,相比空白组及胃酸组,总酚含量分别提高26.93%、18.61%,说明胃蛋白酶的加入,可以释放出更多酚类物质并激活SOD使其活性增强。体外模拟肠消化后,总酚含量及SOD活性均为肠消化组>空白组。肠消化组中,总酚含量显著上升,消化结束后含量最高为262.37 ug/m L;SOD活性在肠消化30 min时显著上升至67.33 U/g;消化150 min结束后,肠消化组的总酚含量及SOD活性与空白组相比,分别提升10.89%、2.93%,表明胰蛋白酶和牛胆盐的加入对总酚含量和SOD活性的提升具有一定的促进作用。5.番茄酵素的稳定性研究。以稳定系数R和悬浮稳定性为指标,从七种不同稳定剂中筛选得出黄原胶、果胶、瓜尔豆胶的稳定性较好,通过单因素试验和Box-Behnken试验,得到复合稳定剂的最优配比为:黄原胶0.12%,果胶0.11%,瓜尔豆胶0.17%,此条件下稳定系数R为0.757,悬浮稳定性为2.467。更多还原

【Abstract】 Tomatoes are rich in a variety of biologically active substances.Deep processing them into a functional enzyme product not only helps to maximise the retention of their natural nutrients,but also gives full play to the superiority of enzymes in terms of biological activity.In this paper,the fermentation process of tomato enzyme was optimised using fresh tomato as raw material,and the changes of metabolites during the fermentation process,in vitro antioxidant capacity and the changes of tomato enzyme during in vitro digestion were comparatively analysed when tomato enzyme was prepared by different fermentation methods,and finally the stability of tomato enzyme was investigated.The main research content and results are as follows:1. Process optimisation for enzymatic digestion of tomato.The optimum process conditions for enzymatic digestion are as follows:pectinase addition of 1.2%,cellulase addition of 0.6%,enzymatic temperature of 50℃,enzymolysis time of 150 min,under which the juice yield was 78.33%and the content of soluble solids was 8.40%.2. Study of tomato enzyme fermentation process.The optimum process conditions for the preparation of tomato enzyme by saccharomycetes were:inoculum amount 1.14%,fermentation temperature 27℃,fermentation time 28 h.SOD activity under these conditions reached 139.14 U/g.The optimum process conditions for the preparation of tomato enzymes by lactic acid bacteria are as follows:inoculum size of0.31%,fermentation temperature of 37℃,p H 5.47,and fermentation time of 40 h under the conditions of SOD activity up to 147.63 U/g.The optimal process conditions for the preparation of tomato enzyme by composite fermentation are as follows:yeast inoculum of 1.14%,fermentation temperature of 27℃,after21 h of fermentation,inoculation of 0.31%of Lactobacillus plantarum,adjustment of p H to 5.50,and continuation of the fermentation at 37℃for 28 h.Under these conditions,the SOD activity of 148.43 U/g,total acid content of 19.20 g/kg,reducing sugar content of 3.77 g/kg,lactobacilli viable count of 2.76×109CFU/m L,and the number of yeasts is 1.18×108 CFU/m L.3. Patterns of metabolite changes during tomato enzyme fermentation During the fermentation of tomato enzymes by different strains,the total acid content rose during fermentation of lactic acid bacteria and complex strains,and yeast fermentation was the highest at 495.85 ug/m L at 24 h.Soluble solids content decreased during both yeast and lactic acid bacteria fermentation,and elevated during multi fermentation.Reducing sugar content decreased during yeast fermentation and was highest at 40.35 g/kg and 4.28 g/kg for Lactobacillus and composite strain fermentation at 24 h.Ascorbic acid content of yeast and Lactobacillus was highest at 4.61 mg/100g and 2.41 mg/100g at 12 h of fermentation time,respectively,and increased during composite fermentation,reaching 4.11 mg/100 g.Lycopene and total carotenoids content decreased and then increased.Total phenol content,SOD,lipase and protease activities increased significantly after fermentation.The results of correlation analysis showed that the change pattern,content and correlation between each metabolite during the fermentation process were also significantly different for different fermentation strains.The tomato enzymes prepared by different fermentation methods were quantitatively analysed using high performance liquid chromatography(HPLC),and eight organic acids were detected in tomato raw juice and yeast fermented tomato enzymes,citric acid,acetic acid and succinic acid were the main organic acids in tomato juice,with the highest citric acid content of 602.62 ug/m L;citric acid and succinic acid were the main organic acids in the yeast fermentation,and the content of citric acid was up to 528.16ug/m L.A total of 5 organic acids were detected in lactic acid fermentation mainly as lactic acid,citric acid,and acetic acid,with lactic acid having the highest content of 3467.35 ug/m L.Six organic acids were found in both composite and natural fermentations,with lactic acid,acetic acid,and succinic acid were the main organic acids in complex fermentation,with the highest content of lactic acid 718.29 ug/m L,while the natural fermentation is mainly lactic acid,succinic acid,citric acid,lactic acid content is the highest2096.28 ug/m L.Gas chromatography-ion mobility spectrometry(GC-IMS)was used to analysis and detect the volatile flavour substances in raw tomato juice and four fermented enzymes.A total of 114 organics were characterised in the five groups of samples,including 76 identified organics and 38 unidentified organics.Among them,there were 18 esters,17 alcohols,16 ketones,12 aldehydes,4 alkenes,2 each of acids and ethers,1 each of phenols and alkanes,and a total of 41 other and unidentified.Differential analysis of five different groups of samples using three-dimensional difference spectrograms,peak intensities of volatile substances and cluster comparison analysis showed that volatile organic substances such as esters,alcohols,ketones and aldehydes were significantly increased in tomato raw juice after fermentation,which made the tomato enzymes mainly present fruity,sweet,buttery and burnt odours,and that the complex fermentation could make the enzymes more coordinated in terms of taste and aroma.4. In vitro antioxidant capacity of tomato enzymes and in vitro simulation of digestion.Compound fermentation and natural fermentation of tomato enzymes had higher scavenging power for DPPH free radicals,which increased by 12.28%and 18.54%,respectively,compared with the original tomato juice.Composite fermentation had the highest scavenging power for hydroxyl radicals and superoxide anion radicals,99.60%and 79.50%,respectively,which were 1.16%and 4.35%higher than that of raw tomato juice,respectively.The reducing power was 1.89 in yeast fermentation,1.54 in Lactobacillus plantarum fermentation,and 1.64 in composite strain fermentation,which were 87.12%,52.47%,and 62.38%higher than that in natural fermentation,respectively.It indicates that the antioxidant capacity of tomato enzymes can be significantly enhanced after inoculation and fermentation,and the overall antioxidant capacity is better than that of other strains of fermentation in the case of composite fermentation.After simulated gastric digestion in vitro,the total phenol content and SOD activity were significantly increased,and the gastric digestion group>gastric acid group>blank group.After digestion for 120 min,the total phenol content increased by 26.93%and 18.61%,respectively,compared with the blank group and the gastric acid group,indicating that the addition of pepsin could release more phenolics and activate SOD to enhance its activity.After simulated enteric digestion in vitro,the total phenol content and SOD activity were both enteric digestion group>blank group.In the enteric group,the total phenol content increased significantly,and the highest content was 262.37 ug/m L at the end of digestion;the SOD activity increased significantly to 67.33 U/g at 30 min of enteric digestion;at the end of 150 min of digestion,the total phenol content and SOD activity of the enteric group increased by 10.89%and 2.93%,respectively,compared with that of the blank group,indicated that the addition of trypsin and bovine bile salt had a certain promotion effect on the enhancement of total phenol content and SOD activity.5. Stability study of tomato enzyme.Taking the stability coefficient R and suspension stability as the indexes,from seven different stabilizers,we screened that xanthan gum,pectin and guar gum had the best stability,and through the single-factor test and Box-Behnken test,the optimal ratio of compound stabilizers as follows:0.12%xanthan gum,0.11%pectin,0.17%guar gum,and the stability coefficient R under this condition was 0.757,and the suspension stability was 2.467.更多还原