酸浆中乳酸菌的分离鉴定及其在大米淀粉提取中的应用
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摘要
酸浆法是生产绿豆和甘薯淀粉的传统方法,在我国已有近千年的历史。为了更好地利用酸浆法,改进我国传统的淀粉提取工艺,拓展酸浆法的应用面,以及解决淀粉生产中工业废水的污染问题,本论文对酸浆法及其在大米淀粉提取中的应用进行了研究。研究内容及结论如下：
(1)采用表型特征鉴定方法,从绿豆、籼米、糯米酸浆中共分离得到了44株菌株,其中12株初步暂定为酵母菌WY,32株乳酸菌鉴定为乳酸乳球菌乳酸亚种和干酪乳杆菌。用通用引物和乳酸菌特异性引物分别对其中6株生理生化特性较典型的乳酸菌菌株进行16S rDNA序列扩增,其序列在GeneBank上的登录号为：HM188409～HM188420;
(2)将酸浆中分离出的乳酸菌菌株制备成菌悬液沉淀绿豆粉乳,筛选沉淀淀粉能力强的菌株。结果：乳酸乳球菌沉淀淀粉能力比干酪乳杆菌强,酵母菌能有效地协同乳酸菌沉淀淀粉,同种菌种之间不同菌株沉淀淀粉能力有显著差异。最终筛选出沉淀淀粉能力强的菌株为乳酸乳球菌MBSL3、干酪乳杆菌LRSL9;不同菌株的最佳复配沉淀淀粉比例为乳酸乳球菌：干酪乳杆菌：酵母菌=3：1：1；
(3)将筛选出的菌株应用于酸浆制备工艺的研究,并以接种量、粉乳初始pH以及粉乳浓度三因素进行L9(33)正交试验,得到酸浆制备最佳工艺：接种量为8%,粉乳初始pH为5.5,粉乳浓度为35%,沉淀温度25℃,酸浆继续养制时间8h。以该工艺制备的酸浆pH约为3.73,菌数约为1.03×109cfu/mL,酸度为15.72%；
(4)将优化后得到的优质酸浆应用于大米淀粉的提取中。以料液比、提取时间、提取温度为单因素,研究了酸浆法提取糯米和籼米两种大米淀粉的工艺。酸浆法提取大米淀粉的最佳工艺为：料液比1：4,提取时间22-24h,提取温度为30℃。采用此法制备的籼米淀粉平均蛋白质残留量约为3.34%,淀粉含量约为87.09%；糯米淀粉平均蛋白质残留量约为3.18%,淀粉含量约为87.48%；
(5)测定了加入酸浆后,淀粉提取过程中的pH变化。结果：加入酸浆能降低粉乳中的pH,且酸浆能有效中和碱法提取淀粉后残留的碱液；
(6)对酸浆法、碱法、酶法、碱-酸浆法、酶-酸浆法等不同方法提取的大米淀粉成分和性质进行比较,测定了淀粉的蛋白质残留量、淀粉含量、水分、灰分、脂肪、酸度等成分指标；研究了淀粉颗粒形态(SEM)、RVA粘度曲线、透明度(OD值)、白度、冻融稳定性、凝沉稳定性、残留蛋白质的SDS-PAGE凝胶电泳等淀粉性质,结果有：酸浆法能应用于大米淀粉提取,且在几种方法中,碱-酸浆法复合提取大米淀粉较好。
The sour liquid method is a traditonal method to produce mung bean and sweet potato starch with nearly one thousand years of history in China. In order to take better advantage of sour liquid method,improve the traditional starch extraction technics,expand the range of application of sour liquid method,as well as solve the pollution problem in starch industry,we studied the sour liquid method and its application to rice starch extaction in this paper.The main contents and results described as follows:
(1) 44 cultures were isolated from mung bean sour liquid,long rice sour liquid and waxy rice sour liquid,and 12 of which were provisionally identified as yeast,3 2 as Lactobacillus casei strain and Lactococcus lactis subsp.lactis strain respectively by phenotypic and molecular identification.At the meantime,16S rDNA of 6 culture s with different physiological and biochemical characteristics were amplified by two pairs of universal primers of 16S rDNA and specific primers of LAB respectively. And 12 accession numbers for 16S rDNA in GeneBank were obtained as follows:H M188409～HM188420;
(2) Screening LAB strains isolated from sour liquids by using the bacterial suspension prepared from LAB strains to deposit the mung bean starch.The results showed that:the Lactococcus lactis subsp.lactis strains had stronger strach deposition capacity than the Lactobacillus casei strains,and yeast strains can be effectively cooperative with LAB strains to starch deposition.There were differences among different strains,so we selected MBSL3 and LRSL9 which were with the strongest starch deposition capacity as well as the yeast strain WY for the further study.The optimal compound proportion of MBSL3,LRSL9 and WY was 3:1:1;
(3) Using the strains MBSL3,LRSL9 and WY to make the sour liquid,this paper studied the effects of bacterium quantity,starch milk initial pH,starch milk concentration, temperature, and keep-standing time on the sour liquid making technique.And the optimal technique of sour liquid making was determined by L9(33) orthogonal test on the 3 factors including bacterium quantity,starch milk initial pH and starch milk concentration.The result showed the optimal conditions were bacterium quantity 8%,starch milk initial pH 5.5,starch milk concentration 35%,temperature 25℃and keep-standing time 8h.Through the optimal technique,we got the high quality sour liquid with pH 3.73, viable amount of about 1.03×109cfu/mL,acidity 15.72%;
(4) The sour liquid made by the optimal technique was applied to rice starch extraction.This paper studied the waxy rice and long rice starch extraction technique.The optimal rice extraction technique was determined by L9(33) orthogonal test on the 3 factors including liquid to material ratio,extraction time and temperature.The result showed the optimal conditions were liquid to material ratio 1:4,extraction time 22～24h and temperature 30℃.By this rice extraction technique,we got the long rice starch with residual protein 3.34% odds,starch amount 87.09%;and the waxy rice starch with residual protein 3.18% odds,starch amount 87.48% in average;
(5) Add the sour liquid made by the optimal technique to the rice starch extracted by the alkali and enzymatic method,then measure the pH change in the rice starch extraction.The result was that,aftter adding sour liquid,the pH decreased and it could neutralize the alkali liquid from alkali method effectively;
(6) Comparing the components and properties of rice starch extracted by differ ent methods,the components indicators including residual protein,starch purity,water,as h,crude fat,acidity;and the properties indicators including starch granules by scanning electron microscopy(SEM),RVA viscosity curve,transparency(OD),whiteness degree,fr eeze-thaw stability,retrogradation stability and SDS-PAGE of residual protein of rice starch were determined.The results were as follows:the sour liquid method can be used for rice starch extraction;and the alkali cooperated with sour liquid method wa s better for rice starch extraction.
(1)采用表型特征鉴定方法,从绿豆、籼米、糯米酸浆中共分离得到了44株菌株,其中12株初步暂定为酵母菌WY,32株乳酸菌鉴定为乳酸乳球菌乳酸亚种和干酪乳杆菌。用通用引物和乳酸菌特异性引物分别对其中6株生理生化特性较典型的乳酸菌菌株进行16S rDNA序列扩增,其序列在GeneBank上的登录号为：HM188409～HM188420;
(2)将酸浆中分离出的乳酸菌菌株制备成菌悬液沉淀绿豆粉乳,筛选沉淀淀粉能力强的菌株。结果：乳酸乳球菌沉淀淀粉能力比干酪乳杆菌强,酵母菌能有效地协同乳酸菌沉淀淀粉,同种菌种之间不同菌株沉淀淀粉能力有显著差异。最终筛选出沉淀淀粉能力强的菌株为乳酸乳球菌MBSL3、干酪乳杆菌LRSL9;不同菌株的最佳复配沉淀淀粉比例为乳酸乳球菌：干酪乳杆菌：酵母菌=3：1：1；
(3)将筛选出的菌株应用于酸浆制备工艺的研究,并以接种量、粉乳初始pH以及粉乳浓度三因素进行L9(33)正交试验,得到酸浆制备最佳工艺：接种量为8%,粉乳初始pH为5.5,粉乳浓度为35%,沉淀温度25℃,酸浆继续养制时间8h。以该工艺制备的酸浆pH约为3.73,菌数约为1.03×109cfu/mL,酸度为15.72%；
(4)将优化后得到的优质酸浆应用于大米淀粉的提取中。以料液比、提取时间、提取温度为单因素,研究了酸浆法提取糯米和籼米两种大米淀粉的工艺。酸浆法提取大米淀粉的最佳工艺为：料液比1：4,提取时间22-24h,提取温度为30℃。采用此法制备的籼米淀粉平均蛋白质残留量约为3.34%,淀粉含量约为87.09%；糯米淀粉平均蛋白质残留量约为3.18%,淀粉含量约为87.48%；
(5)测定了加入酸浆后,淀粉提取过程中的pH变化。结果：加入酸浆能降低粉乳中的pH,且酸浆能有效中和碱法提取淀粉后残留的碱液；
(6)对酸浆法、碱法、酶法、碱-酸浆法、酶-酸浆法等不同方法提取的大米淀粉成分和性质进行比较,测定了淀粉的蛋白质残留量、淀粉含量、水分、灰分、脂肪、酸度等成分指标；研究了淀粉颗粒形态(SEM)、RVA粘度曲线、透明度(OD值)、白度、冻融稳定性、凝沉稳定性、残留蛋白质的SDS-PAGE凝胶电泳等淀粉性质,结果有：酸浆法能应用于大米淀粉提取,且在几种方法中,碱-酸浆法复合提取大米淀粉较好。
The sour liquid method is a traditonal method to produce mung bean and sweet potato starch with nearly one thousand years of history in China. In order to take better advantage of sour liquid method,improve the traditional starch extraction technics,expand the range of application of sour liquid method,as well as solve the pollution problem in starch industry,we studied the sour liquid method and its application to rice starch extaction in this paper.The main contents and results described as follows:
(1) 44 cultures were isolated from mung bean sour liquid,long rice sour liquid and waxy rice sour liquid,and 12 of which were provisionally identified as yeast,3 2 as Lactobacillus casei strain and Lactococcus lactis subsp.lactis strain respectively by phenotypic and molecular identification.At the meantime,16S rDNA of 6 culture s with different physiological and biochemical characteristics were amplified by two pairs of universal primers of 16S rDNA and specific primers of LAB respectively. And 12 accession numbers for 16S rDNA in GeneBank were obtained as follows:H M188409～HM188420;
(2) Screening LAB strains isolated from sour liquids by using the bacterial suspension prepared from LAB strains to deposit the mung bean starch.The results showed that:the Lactococcus lactis subsp.lactis strains had stronger strach deposition capacity than the Lactobacillus casei strains,and yeast strains can be effectively cooperative with LAB strains to starch deposition.There were differences among different strains,so we selected MBSL3 and LRSL9 which were with the strongest starch deposition capacity as well as the yeast strain WY for the further study.The optimal compound proportion of MBSL3,LRSL9 and WY was 3:1:1;
(3) Using the strains MBSL3,LRSL9 and WY to make the sour liquid,this paper studied the effects of bacterium quantity,starch milk initial pH,starch milk concentration, temperature, and keep-standing time on the sour liquid making technique.And the optimal technique of sour liquid making was determined by L9(33) orthogonal test on the 3 factors including bacterium quantity,starch milk initial pH and starch milk concentration.The result showed the optimal conditions were bacterium quantity 8%,starch milk initial pH 5.5,starch milk concentration 35%,temperature 25℃and keep-standing time 8h.Through the optimal technique,we got the high quality sour liquid with pH 3.73, viable amount of about 1.03×109cfu/mL,acidity 15.72%;
(4) The sour liquid made by the optimal technique was applied to rice starch extraction.This paper studied the waxy rice and long rice starch extraction technique.The optimal rice extraction technique was determined by L9(33) orthogonal test on the 3 factors including liquid to material ratio,extraction time and temperature.The result showed the optimal conditions were liquid to material ratio 1:4,extraction time 22～24h and temperature 30℃.By this rice extraction technique,we got the long rice starch with residual protein 3.34% odds,starch amount 87.09%;and the waxy rice starch with residual protein 3.18% odds,starch amount 87.48% in average;
(5) Add the sour liquid made by the optimal technique to the rice starch extracted by the alkali and enzymatic method,then measure the pH change in the rice starch extraction.The result was that,aftter adding sour liquid,the pH decreased and it could neutralize the alkali liquid from alkali method effectively;
(6) Comparing the components and properties of rice starch extracted by differ ent methods,the components indicators including residual protein,starch purity,water,as h,crude fat,acidity;and the properties indicators including starch granules by scanning electron microscopy(SEM),RVA viscosity curve,transparency(OD),whiteness degree,fr eeze-thaw stability,retrogradation stability and SDS-PAGE of residual protein of rice starch were determined.The results were as follows:the sour liquid method can be used for rice starch extraction;and the alkali cooperated with sour liquid method wa s better for rice starch extraction.
引文
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