啤酒有害产酸细菌选择性培养基的优化
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摘要
啤酒是以麦芽、水为主要原料,添加啤酒花,经啤酒酵母发酵而成的一种低酒精度、含有泡沫和一定量CO2的发酵酒。由于啤酒中存在乙醇(0.5%-10%,w/v),酒花苦味物质(大约17-55ppm的异α-酸),高浓度的CO2(大约0.5%,w/v),较低的pH值(3.8-4.7),极低的氧含量(<0.1ppm),使得啤酒被认为是一种安全的饮料。但是如果操作不慎或卫生条件不严,仍难免有厌氧菌等有害菌的污染。乳酸菌是啤酒厂中最容易污染,且对啤酒质量危害最大的有害菌之一。
从济南、青岛、北京三地啤酒厂中分离到有害菌18株,经革兰氏染色、过氧化氢酶实验、发酵葡萄糖产乳酸实验等证明,15株为乳酸菌。从15株乳酸菌中选择6株典型的菌株,进行发酵麦汁产酸实验。结果表明,6株乳酸菌均能使麦汁酸度上升1.5°T。
啤酒有害乳酸菌按形态可分为乳酸杆菌和乳酸球菌。采用单因素实验和正交实验优化乳酸杆菌和乳酸球菌的选择性培养基的成分和培养条件。单因素实验确定最优碳源、氮源分别为葡萄糖、胰蛋白胨、大豆蛋白胨,正交实验优化了其用量为:葡萄糖4%、胰蛋白胨2%、大豆蛋白胨0.5 %;选择MgSO4·7H2O、MnSO4·4H2O、K2HPO4作为培养基的无机盐组分,正交实验优化三者的用量为MgSO4·7H2O 0.02%,MnSO4·4H2O 0.005%,K2HPO40.4% ;对供试菌L1、L3、P1、P2的生长因子进行筛选,发现VB5和泛酸钙对4株菌的生长有不同程度的促进作用。选择胡萝卜汁、番茄汁、苹果汁作为乳酸杆菌的天然生长因子,选择酵母粉、豌豆汁作为乳酸球菌的天然生长因子。正交实验优化乳酸杆菌天然生长因子的添加量为胡萝卜汁3%,番茄汁25%,苹果汁3%;乳酸球菌生长因子的用量为酵母粉0.3%,豌豆汁5%。确定了培养基的初始pH值为6.0-6.2,培养温度为25℃。
啤酒生产中很多工序的取样中都存在啤酒酵母,啤酒酵母的存在干扰有害菌的检测。采用纳他霉素作为啤酒酵母的抑制剂。纳他霉素是一种新型抗真菌剂。采用圆滤纸片法测定了纳他霉素对供试酵母206#、308#、303#、354#的抑制效果。实验表明,5ppm纳他霉素对四株菌产生的抑菌圈直径分别为14mm、16mm、16mm、18mm;最低抑制浓度实验表明,纳他霉素对206#、303#、354#的MIC为3ppm,对308#的MIC为4ppm。高温、强酸、强碱影响纳他霉素的抑菌活性。纳他霉素经121℃维持30min后,10 ppm纳他霉素无法抑制供试菌株的生长。当pH在5.0-7.0时,纳他霉素抑菌活性最高;当pH<5.0或>7.0时,溶解度提高,但是活性损失增大。
乳酸菌在培养基中生长代谢,产生乳酸,改变培养基的pH值。单一指示剂存在变色pH范围过宽,或变色不够灵敏等缺点,因此优化了混合指示剂。将氯酚红与溴钾酚紫、溴钾酚绿、溴酚蓝、溴百里香酚蓝四种指示剂按照不同的比例混合组成混合指示剂,实验表明,以氯酚红与溴钾酚紫混合的效果最佳。用pH计测定溶液变色前后的pH值,结果表明,氯酚红与溴钾酚紫按1:3的比例混合时,颜色由紫红变为黄色,且变色pH范围为6.45-5.93,单因素实验确定其在培养基中的最佳添加量为1.0%(v/v)。
Beer is a kind of product which is fermented by Saccharomyces cerevisiae. Its main raw materials are malt、water、hop、Saccharomyces cerevisiae.Beer has been regarded as a safe beverage due to the presence of ethanol (0.5%-10%,w/v)、hop bitter compounds(approx 17-55ppm of iso-α-acids)、the high content of carbon dioxide(approx 0.5%w/v)、the low pH(3.8-4.7)、the extremely reduced content of oxygen(<0.1ppm). However,a few microorganisms still survived in beer as a result of the wrong operation and poor sanitation.These microorganisms are called as beer spoilage bacteria.Lactic acid bacteria are the most polluted and harmful bacteria.
18 strains were isolated from breweries in JiNan, QingDao and BeiJing. 15 strains were determined to be lactic acid bacteria by the Gram staining, catalase test, morphological examination and so on. The test for acid–producing in wort showed that 6 strains from15 strains could make the acidity of wort increase 1.5°T.
Beer spoilage lactic acid bacteria can be divided into lactobacilli and pediococci according to morphology. The composition of lactobacilli and pediococci’selective medium and their conditions for incubation were optimized by single factor experiment and orthogonal experiment.The optimal carbon source and nitrogen source were glucose, typtone, soybean peptone.Their concentrations were 4%, 2%, 0.5% respectively according to orthogonal experiment; MgSO4·7H2O、MnSO4·4H2O、K2HPO4 were used as the mineral salts of the medium and their concentrations were 0.02%, 0.005%, 0.4% respectively. The experiment of growth factors for L1, L3, P1, P2 showed that VB5 and calcium pantothenate could have an incentive effect on strains’growth. Some natural materials could substitute for these Vitamine.Carrot juice,tomato juice,apple juice could be lactobacilli’natural growth factors and their concentrations were 3%,25%,3%;yeast powder,bean juice could substitute for pediococci’natural growth factors and their concentrations were 0.3% and 5%.The original pH of the medium was determined at 6.0-6.2 and the temperature for incubation was at 25℃。
Saccharomyces cerevisiae existed in many processes of brewing.The presence of Saccharomyces cerevisiae disturbed the determination of beer spoilage bacteria during the incubation on plate.Natamycin is a kind of new antibiotics and can be used in the determination of beer spoilage bacteria. It examined the inhibition of Natamycin on Saccharomyces cerevisiae 206#、S. cerevisiae 308#、S. cerevisiae 303#、S. cerevisiae 354# by use of round filter paper. The result showed that the diamters of antimicrobialzone made by 5ppm Natamycin were 14mm、16mm、16mm、18mm respectively.The test for minimal inhibition concentration showed that the MIC on 206#、308#、303#、354#were 3ppm、4ppm、3ppm、3ppm.Temperature、acid、base have an negative effect on Natamycin. The result showed that 10ppm Natamycin lose inhibition effect after the thermal treatment at 121℃for 30min. The inhibition activist of Natamycin was the highest when the pH of solution at 5.0-7.0.The solution of Natamycin in the water was improved when the pH was lower than 5.0 or higher than 7.0, but the activist was lowed at the same time.
Lactic acid bacteria growed in the media,as a result, they produced lactic acid and the pH of the medium was changed. Single indicator was deficited in colour change and sensitivity, so optimized the mixed indicator. Mixed chlorophenol red with bromocresol purple, bromocresol green, bromophenol blue, bromothymol blue respectively according to different proportion .The mixed indicator which changed colour significantly was Chlorophenol red with Bromocresol purple.When mixed Chlorophenol red with Bromocresol purple according to the proportion of 1:3(v/v),the colour was changed from red to yellow and the pH of mixed indicator was determined from 6.45 to 5.93 by pH meter. The best concentration of this mixed indicator used in the medium was 1.0%(v/v) according to single factor experiment.
从济南、青岛、北京三地啤酒厂中分离到有害菌18株,经革兰氏染色、过氧化氢酶实验、发酵葡萄糖产乳酸实验等证明,15株为乳酸菌。从15株乳酸菌中选择6株典型的菌株,进行发酵麦汁产酸实验。结果表明,6株乳酸菌均能使麦汁酸度上升1.5°T。
啤酒有害乳酸菌按形态可分为乳酸杆菌和乳酸球菌。采用单因素实验和正交实验优化乳酸杆菌和乳酸球菌的选择性培养基的成分和培养条件。单因素实验确定最优碳源、氮源分别为葡萄糖、胰蛋白胨、大豆蛋白胨,正交实验优化了其用量为:葡萄糖4%、胰蛋白胨2%、大豆蛋白胨0.5 %;选择MgSO4·7H2O、MnSO4·4H2O、K2HPO4作为培养基的无机盐组分,正交实验优化三者的用量为MgSO4·7H2O 0.02%,MnSO4·4H2O 0.005%,K2HPO40.4% ;对供试菌L1、L3、P1、P2的生长因子进行筛选,发现VB5和泛酸钙对4株菌的生长有不同程度的促进作用。选择胡萝卜汁、番茄汁、苹果汁作为乳酸杆菌的天然生长因子,选择酵母粉、豌豆汁作为乳酸球菌的天然生长因子。正交实验优化乳酸杆菌天然生长因子的添加量为胡萝卜汁3%,番茄汁25%,苹果汁3%;乳酸球菌生长因子的用量为酵母粉0.3%,豌豆汁5%。确定了培养基的初始pH值为6.0-6.2,培养温度为25℃。
啤酒生产中很多工序的取样中都存在啤酒酵母,啤酒酵母的存在干扰有害菌的检测。采用纳他霉素作为啤酒酵母的抑制剂。纳他霉素是一种新型抗真菌剂。采用圆滤纸片法测定了纳他霉素对供试酵母206#、308#、303#、354#的抑制效果。实验表明,5ppm纳他霉素对四株菌产生的抑菌圈直径分别为14mm、16mm、16mm、18mm;最低抑制浓度实验表明,纳他霉素对206#、303#、354#的MIC为3ppm,对308#的MIC为4ppm。高温、强酸、强碱影响纳他霉素的抑菌活性。纳他霉素经121℃维持30min后,10 ppm纳他霉素无法抑制供试菌株的生长。当pH在5.0-7.0时,纳他霉素抑菌活性最高;当pH<5.0或>7.0时,溶解度提高,但是活性损失增大。
乳酸菌在培养基中生长代谢,产生乳酸,改变培养基的pH值。单一指示剂存在变色pH范围过宽,或变色不够灵敏等缺点,因此优化了混合指示剂。将氯酚红与溴钾酚紫、溴钾酚绿、溴酚蓝、溴百里香酚蓝四种指示剂按照不同的比例混合组成混合指示剂,实验表明,以氯酚红与溴钾酚紫混合的效果最佳。用pH计测定溶液变色前后的pH值,结果表明,氯酚红与溴钾酚紫按1:3的比例混合时,颜色由紫红变为黄色,且变色pH范围为6.45-5.93,单因素实验确定其在培养基中的最佳添加量为1.0%(v/v)。
Beer is a kind of product which is fermented by Saccharomyces cerevisiae. Its main raw materials are malt、water、hop、Saccharomyces cerevisiae.Beer has been regarded as a safe beverage due to the presence of ethanol (0.5%-10%,w/v)、hop bitter compounds(approx 17-55ppm of iso-α-acids)、the high content of carbon dioxide(approx 0.5%w/v)、the low pH(3.8-4.7)、the extremely reduced content of oxygen(<0.1ppm). However,a few microorganisms still survived in beer as a result of the wrong operation and poor sanitation.These microorganisms are called as beer spoilage bacteria.Lactic acid bacteria are the most polluted and harmful bacteria.
18 strains were isolated from breweries in JiNan, QingDao and BeiJing. 15 strains were determined to be lactic acid bacteria by the Gram staining, catalase test, morphological examination and so on. The test for acid–producing in wort showed that 6 strains from15 strains could make the acidity of wort increase 1.5°T.
Beer spoilage lactic acid bacteria can be divided into lactobacilli and pediococci according to morphology. The composition of lactobacilli and pediococci’selective medium and their conditions for incubation were optimized by single factor experiment and orthogonal experiment.The optimal carbon source and nitrogen source were glucose, typtone, soybean peptone.Their concentrations were 4%, 2%, 0.5% respectively according to orthogonal experiment; MgSO4·7H2O、MnSO4·4H2O、K2HPO4 were used as the mineral salts of the medium and their concentrations were 0.02%, 0.005%, 0.4% respectively. The experiment of growth factors for L1, L3, P1, P2 showed that VB5 and calcium pantothenate could have an incentive effect on strains’growth. Some natural materials could substitute for these Vitamine.Carrot juice,tomato juice,apple juice could be lactobacilli’natural growth factors and their concentrations were 3%,25%,3%;yeast powder,bean juice could substitute for pediococci’natural growth factors and their concentrations were 0.3% and 5%.The original pH of the medium was determined at 6.0-6.2 and the temperature for incubation was at 25℃。
Saccharomyces cerevisiae existed in many processes of brewing.The presence of Saccharomyces cerevisiae disturbed the determination of beer spoilage bacteria during the incubation on plate.Natamycin is a kind of new antibiotics and can be used in the determination of beer spoilage bacteria. It examined the inhibition of Natamycin on Saccharomyces cerevisiae 206#、S. cerevisiae 308#、S. cerevisiae 303#、S. cerevisiae 354# by use of round filter paper. The result showed that the diamters of antimicrobialzone made by 5ppm Natamycin were 14mm、16mm、16mm、18mm respectively.The test for minimal inhibition concentration showed that the MIC on 206#、308#、303#、354#were 3ppm、4ppm、3ppm、3ppm.Temperature、acid、base have an negative effect on Natamycin. The result showed that 10ppm Natamycin lose inhibition effect after the thermal treatment at 121℃for 30min. The inhibition activist of Natamycin was the highest when the pH of solution at 5.0-7.0.The solution of Natamycin in the water was improved when the pH was lower than 5.0 or higher than 7.0, but the activist was lowed at the same time.
Lactic acid bacteria growed in the media,as a result, they produced lactic acid and the pH of the medium was changed. Single indicator was deficited in colour change and sensitivity, so optimized the mixed indicator. Mixed chlorophenol red with bromocresol purple, bromocresol green, bromophenol blue, bromothymol blue respectively according to different proportion .The mixed indicator which changed colour significantly was Chlorophenol red with Bromocresol purple.When mixed Chlorophenol red with Bromocresol purple according to the proportion of 1:3(v/v),the colour was changed from red to yellow and the pH of mixed indicator was determined from 6.45 to 5.93 by pH meter. The best concentration of this mixed indicator used in the medium was 1.0%(v/v) according to single factor experiment.
引文
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