耐酸醋酸菌的选育及高酸度发酵果醋的研究
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摘要
高酸度醋具有杀菌力强、存储和运输成本低等诸多优点，除了用作调味品以外，在食品加工、医药、家居、美容等领域均发挥着重要作用。果醋是以果品或果品加工的下脚料为原料，经微生物发酵而成的集食醋与水果的营养、保健及食疗于一体的新型黄金饮料。利用我国丰富的水果资源生产高酸度果醋不仅能解决水果运输难、存储期短等问题，有效提高水果种植和加工的经济效益，而且以果代粮酿醋可节约大量粮食。本文通过诱变筛选获得高酸度果醋醋酸菌株、并对果醋发酵条件及补料分批发酵高酸度果醋进行研究。
从自然发酵的杏果醋醪中分离醋酸菌株，并对其进行耐酒精、耐酸、耐温、产酯性能及遗传稳定性等试验，从而获得醋酸菌AFA-01，初步考察该菌株的产酸能力为5.00g/100mL。通过形态特征、培养特征观察及生理生化试验，初步确定AFA-01菌株为醋酸杆菌属(Acetobacter Beijerinck)。
使用醋酸菌AFA-01进行葡萄皮渣的醋酸发酵。使用Plackett-Burman设计筛选出影响葡萄皮渣醋酸发酵的三个主要因素，根据Box-Behnken中心组合试验设计原理，采用响应面法优化生产工艺并进行回归分析。结果表明，起始酒精度、摇床转速和发酵时间是影响醋酸发酵的重要因素。在起始酒精度7%，摇床转速147r/min，发酵时间132h的最佳工艺条件下，葡萄皮渣发酵醋醪中总酸(以醋酸计)可达5.11g/100mL。
以醋酸菌AFA-01为出发菌株，微波结合盐酸羟铵法诱变菌株，获得遗传性能稳定的突变株AFA-WH3，检测诱变前后酶活性发现，诱变后乙醇脱氢酶活力增加了94.83%，达2786u/mL，利用该诱变菌株可发酵酸度为7.35g/100mL的醋酸，产酸水平提高了43.55%。经16S rDNA保守序列分析鉴定AFA-WH3为醋酸杆菌属(Acetobacter Beijerinck)巴氏醋酸杆菌(Acetobacter pasteurianus)。
以杏果为原料，研究两步法发酵生产果醋的生产工艺，并对发酵条件进行优化。在单因素试验基础上，确定酒精发酵基质的最适料水比为1:1.5，以及果胶酶在44℃、pH4.4酶解4h的最适添加量为0.5g/L。通过L9(34)正交试验确定杏果酒精发酵的最优组合：发酵基质糖度16°Bx、酵母接种量3%、发酵温度30℃。使用AFA-WH3醋酸菌进行杏果醋的液体发酵。研究了影响醋酸发酵的诸多因素，并采用四因素二次通用旋转组合试验设计优化发酵条件，得出最优组合：初始酒精度6.7%、醋酸菌接种量13%、摇床转速153r/min、发酵温度34℃，此条件下发酵132h，总酸(以醋酸计)含量可达7.11g/100mL，较优化前提高13.8%，挥发酸(以醋酸计)为6.23g/100mL。
使用反相高效液相色谱法对果酒、果醋中草酸、酒石酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、富马酸、琥珀酸9种有机酸进行定性和定量分析。结果表明，不同的代谢类型和不同原料均会影响有机酸的种类和含量。杏果酒中除含有9种有机酸外，还含有多种未知有机酸，其中柠檬酸含量最高。杏果醋和葡萄皮渣果醋中均不含丙酮酸，乙酸为主要有机酸，两种果醋中有机酸组成的差别主要体现在酒石酸、乳酸和柠檬酸的含量上，杏果醋中的乳酸、柠檬酸含量较高，而葡萄皮渣果醋中酒石酸含量较高。
在摇瓶分批发酵杏果醋的试验基础上，采用摇瓶补料分批发酵法研究不同初始酒精浓度、不同补料方式对杏果醋酸发酵产酸的影响。试验结果表明，通过调整初始酒精浓度为4%，控制发酵液总乙醇浓度为7%，在发酵第48h、60h、72h分三次补加剩余杏果酒醪，醋酸发酵最终酸度可达9.02g/100mL，相比分批发酵提高了8.4%。
The high acidity of vinegar has so many advantages, such as strongbactericidal power, low cost of storage and transportation. The vinegar not onlywas used as condiment, but also played an important role in food processing,medicine, household and cosmetic fields. The fruit vinegar was fermented withfruit or the by-product of fruit processing as main materials. The fruit vinegarincluded the nutrition of vinegar and fruit, so is a new and health beverage.Compared with conventional vinegar, high acidity fruit vinegar was made of fruitinstead of grain, which could not only make full use the abundant fruit resourcesto solve the difficult of fruit transportation and storage, but also save a largeamount of grain. In this paper, we mainly separated and improved the acidtolerant acetic acid bacteria, optimized the conditions of fruit vinegarfermentation and high-acid fruit vinegar flask-shaking fed batch fermentation.
A bacterial strain was isolated from the natural fermented vinegar in theliquid of apricot, namely Acetic bacteria AFA-01. The characters wereinvestigated include ethanol resistance, acid resistance, thermo-resistant,easter-producing, genetic stability. The acid producing ability of AFA-01wasabout5.00g/100mL. According to the morphology, physiological andbiochemical characteristics, AFA-01was preliminarily identified as AcetobacterBeijerinck.
Plackett-Burman design and response surface methodology were employedto optimize the acetic fermentation conditions for production of acetic acid byAFA-01using grape pomace as material. A Plackett-Burman design of fivefactors was applied to evaluate the effects of different fermentation conditions.Initial alcohol concentrarion, shaker rotate speed and fermentation time werefound to be significant components influencing the acetic acid production. The optimal levels of three factors were investigated by response surfacemethodology using Box-Benhnken design. The final fermentation conditionsoptimized was as follows: initial alcohol concentrarion7%, shaker rotate speed147r/min and fermentation time132h. Under these conditions.The maximumtotal acidity (measured by the amount of acetic acid) obtained was5.11g/100mL.
The compound mutation of hydroxylamine hydrochloride and microwaveirradiation was performed to improve the acetic acid-producing ability ofAFA-01. The mutant strain AFA-WH3was obtained with higher yeild of aceticacid and alcohol-dehydrogenase(ADH) activity. Using the strain, total acidity ofproduced vinegar was increased by43.55%, up to a concentration of7.35g/100mL. Moreover ADH activity reached2786u/mL, increased by94.83%.Biochemical tests followed by16S rDNA were employed for identification ofAFA-WH3and phylogenic tree was constructed. In phylogenetic trees based on16S rDNA gene sequences, WFA-WH3was located in the lineage of Acetobacterpasteurianus and had100%sequence similarity to the type strain of Acetobacterpasteurianus.
The technology for making apricot vinegar was researched via alcoholicfermentation and then acetic fermentation. The best processing condition wasthat apricot with the material and water ratio of1:1.5was enzyme-treated by0.5g/L pectase concentration at44℃, pH4.4for four hours. The alcoholicfermentation was optimized by means of L49(3) orthogonal test based onmono-factor experiments. The optimal conditions were set at the Brix16°Bx,inoculation volume of yeast3%and fermentation temperature30℃for threedays. These factors that influenced liquid fermentation of AFA-WH3aceticfermentation were analyzed, and technological parameters were optimized byusing the four factors quadratic currency rotational composite experiment. Theoptimal fermentation parameters were obtained as follows: initial alcoholconcentrarion6.7%,13%inoculation volume of acetic bacteria, fermentationtemperature34℃and shaker rotate speed153r/min for132hours. Under theseconditions, total acidity of apricot vinegar was up to7.11g/100mL, more than13.8%compared to the unoptimization, and volatile acid was up to6.23g/100mL.
Reversed-phase high performance liquid chromatography was used to takequalitative and quantitative analysis of oxalic acid, tartaric acid, pyruvic acid,malic acid, lactic acid, acetic acid, citric acid, fumaric acid and succinic acid infruit wine and fruit vinegar. The experiment results demonstrated that the abovenine kind of organic acids and unknown organic acids were contained in apricotwine, among which the content of citric acid was highest. Acetic acid was themajor acid in fermented apricot vinegar and grape pomace vinegar. There was nopyruvic acid in the above fruit vinegars. The difference between apricot vinegarand grape pomace vinegar lied in the content of tartaric acid, lactic acid, citricacid. So kinds and contents of organic acids were different owing to differentfermented materials and different metabolism.
Based on batch fermentation of apricot vinegar, flask-shaking fed batchfermentation adjusting initial alcohol concentration to4%was carried out byrespectively supplementing apricot wine with1%alcohol concentration at48h,60h,72h. It was indicated that final acidity reached9.02g/100mL, increased by8.4%contrasted to that in batch fermentation.
从自然发酵的杏果醋醪中分离醋酸菌株，并对其进行耐酒精、耐酸、耐温、产酯性能及遗传稳定性等试验，从而获得醋酸菌AFA-01，初步考察该菌株的产酸能力为5.00g/100mL。通过形态特征、培养特征观察及生理生化试验，初步确定AFA-01菌株为醋酸杆菌属(Acetobacter Beijerinck)。
使用醋酸菌AFA-01进行葡萄皮渣的醋酸发酵。使用Plackett-Burman设计筛选出影响葡萄皮渣醋酸发酵的三个主要因素，根据Box-Behnken中心组合试验设计原理，采用响应面法优化生产工艺并进行回归分析。结果表明，起始酒精度、摇床转速和发酵时间是影响醋酸发酵的重要因素。在起始酒精度7%，摇床转速147r/min，发酵时间132h的最佳工艺条件下，葡萄皮渣发酵醋醪中总酸(以醋酸计)可达5.11g/100mL。
以醋酸菌AFA-01为出发菌株，微波结合盐酸羟铵法诱变菌株，获得遗传性能稳定的突变株AFA-WH3，检测诱变前后酶活性发现，诱变后乙醇脱氢酶活力增加了94.83%，达2786u/mL，利用该诱变菌株可发酵酸度为7.35g/100mL的醋酸，产酸水平提高了43.55%。经16S rDNA保守序列分析鉴定AFA-WH3为醋酸杆菌属(Acetobacter Beijerinck)巴氏醋酸杆菌(Acetobacter pasteurianus)。
以杏果为原料，研究两步法发酵生产果醋的生产工艺，并对发酵条件进行优化。在单因素试验基础上，确定酒精发酵基质的最适料水比为1:1.5，以及果胶酶在44℃、pH4.4酶解4h的最适添加量为0.5g/L。通过L9(34)正交试验确定杏果酒精发酵的最优组合：发酵基质糖度16°Bx、酵母接种量3%、发酵温度30℃。使用AFA-WH3醋酸菌进行杏果醋的液体发酵。研究了影响醋酸发酵的诸多因素，并采用四因素二次通用旋转组合试验设计优化发酵条件，得出最优组合：初始酒精度6.7%、醋酸菌接种量13%、摇床转速153r/min、发酵温度34℃，此条件下发酵132h，总酸(以醋酸计)含量可达7.11g/100mL，较优化前提高13.8%，挥发酸(以醋酸计)为6.23g/100mL。
使用反相高效液相色谱法对果酒、果醋中草酸、酒石酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、富马酸、琥珀酸9种有机酸进行定性和定量分析。结果表明，不同的代谢类型和不同原料均会影响有机酸的种类和含量。杏果酒中除含有9种有机酸外，还含有多种未知有机酸，其中柠檬酸含量最高。杏果醋和葡萄皮渣果醋中均不含丙酮酸，乙酸为主要有机酸，两种果醋中有机酸组成的差别主要体现在酒石酸、乳酸和柠檬酸的含量上，杏果醋中的乳酸、柠檬酸含量较高，而葡萄皮渣果醋中酒石酸含量较高。
在摇瓶分批发酵杏果醋的试验基础上，采用摇瓶补料分批发酵法研究不同初始酒精浓度、不同补料方式对杏果醋酸发酵产酸的影响。试验结果表明，通过调整初始酒精浓度为4%，控制发酵液总乙醇浓度为7%，在发酵第48h、60h、72h分三次补加剩余杏果酒醪，醋酸发酵最终酸度可达9.02g/100mL，相比分批发酵提高了8.4%。
The high acidity of vinegar has so many advantages, such as strongbactericidal power, low cost of storage and transportation. The vinegar not onlywas used as condiment, but also played an important role in food processing,medicine, household and cosmetic fields. The fruit vinegar was fermented withfruit or the by-product of fruit processing as main materials. The fruit vinegarincluded the nutrition of vinegar and fruit, so is a new and health beverage.Compared with conventional vinegar, high acidity fruit vinegar was made of fruitinstead of grain, which could not only make full use the abundant fruit resourcesto solve the difficult of fruit transportation and storage, but also save a largeamount of grain. In this paper, we mainly separated and improved the acidtolerant acetic acid bacteria, optimized the conditions of fruit vinegarfermentation and high-acid fruit vinegar flask-shaking fed batch fermentation.
A bacterial strain was isolated from the natural fermented vinegar in theliquid of apricot, namely Acetic bacteria AFA-01. The characters wereinvestigated include ethanol resistance, acid resistance, thermo-resistant,easter-producing, genetic stability. The acid producing ability of AFA-01wasabout5.00g/100mL. According to the morphology, physiological andbiochemical characteristics, AFA-01was preliminarily identified as AcetobacterBeijerinck.
Plackett-Burman design and response surface methodology were employedto optimize the acetic fermentation conditions for production of acetic acid byAFA-01using grape pomace as material. A Plackett-Burman design of fivefactors was applied to evaluate the effects of different fermentation conditions.Initial alcohol concentrarion, shaker rotate speed and fermentation time werefound to be significant components influencing the acetic acid production. The optimal levels of three factors were investigated by response surfacemethodology using Box-Benhnken design. The final fermentation conditionsoptimized was as follows: initial alcohol concentrarion7%, shaker rotate speed147r/min and fermentation time132h. Under these conditions.The maximumtotal acidity (measured by the amount of acetic acid) obtained was5.11g/100mL.
The compound mutation of hydroxylamine hydrochloride and microwaveirradiation was performed to improve the acetic acid-producing ability ofAFA-01. The mutant strain AFA-WH3was obtained with higher yeild of aceticacid and alcohol-dehydrogenase(ADH) activity. Using the strain, total acidity ofproduced vinegar was increased by43.55%, up to a concentration of7.35g/100mL. Moreover ADH activity reached2786u/mL, increased by94.83%.Biochemical tests followed by16S rDNA were employed for identification ofAFA-WH3and phylogenic tree was constructed. In phylogenetic trees based on16S rDNA gene sequences, WFA-WH3was located in the lineage of Acetobacterpasteurianus and had100%sequence similarity to the type strain of Acetobacterpasteurianus.
The technology for making apricot vinegar was researched via alcoholicfermentation and then acetic fermentation. The best processing condition wasthat apricot with the material and water ratio of1:1.5was enzyme-treated by0.5g/L pectase concentration at44℃, pH4.4for four hours. The alcoholicfermentation was optimized by means of L49(3) orthogonal test based onmono-factor experiments. The optimal conditions were set at the Brix16°Bx,inoculation volume of yeast3%and fermentation temperature30℃for threedays. These factors that influenced liquid fermentation of AFA-WH3aceticfermentation were analyzed, and technological parameters were optimized byusing the four factors quadratic currency rotational composite experiment. Theoptimal fermentation parameters were obtained as follows: initial alcoholconcentrarion6.7%,13%inoculation volume of acetic bacteria, fermentationtemperature34℃and shaker rotate speed153r/min for132hours. Under theseconditions, total acidity of apricot vinegar was up to7.11g/100mL, more than13.8%compared to the unoptimization, and volatile acid was up to6.23g/100mL.
Reversed-phase high performance liquid chromatography was used to takequalitative and quantitative analysis of oxalic acid, tartaric acid, pyruvic acid,malic acid, lactic acid, acetic acid, citric acid, fumaric acid and succinic acid infruit wine and fruit vinegar. The experiment results demonstrated that the abovenine kind of organic acids and unknown organic acids were contained in apricotwine, among which the content of citric acid was highest. Acetic acid was themajor acid in fermented apricot vinegar and grape pomace vinegar. There was nopyruvic acid in the above fruit vinegars. The difference between apricot vinegarand grape pomace vinegar lied in the content of tartaric acid, lactic acid, citricacid. So kinds and contents of organic acids were different owing to differentfermented materials and different metabolism.
Based on batch fermentation of apricot vinegar, flask-shaking fed batchfermentation adjusting initial alcohol concentration to4%was carried out byrespectively supplementing apricot wine with1%alcohol concentration at48h,60h,72h. It was indicated that final acidity reached9.02g/100mL, increased by8.4%contrasted to that in batch fermentation.
引文
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