CN110004090B - 一种肠膜明串珠菌及其在发酵酸菜中的应用 - Google Patents
一种肠膜明串珠菌及其在发酵酸菜中的应用 Download PDFInfo
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- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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Abstract
本发明公开了一种肠膜明串珠菌及其在发酵酸菜中的应用,涉及发酵食品技术领域,所述肠膜明串珠菌为Leuconostoc mesenteroides DN‑LM‑D2,发酵酸菜的步骤为:1)选择新鲜大白菜,进行前处理;2)清洗:将步骤1)处理后的大白菜用水洗净,沥干水分,摆放置发酵容器中;3)发酵:在装有大白菜的发酵容器中注入纯净水,然后添加肠膜明串珠菌菌液,同时添加食用盐,密闭发酵,得到酸菜。使用本发明提供的方法发酵酸菜,硝酸盐和亚硝酸盐含量低,成菜率高。
Description
技术领域
本发明涉及发酵食品技术领域,具体涉及一种肠膜明串珠菌及其在发酵酸菜中的应用。
背景技术
以大白菜为原料腌制的酸菜是我国北方传统的发酵蔬菜制品,其风味独特、爽口、同时可以增进食欲。酸菜发酵液中具有丰富的乳酸菌,在其生长代谢过程中产生的芳香类物质使酸菜的风味独特。而乳酸菌主要代谢产物包括酸性代谢物、胞外多糖、乳酸菌素、γ-氨基丁酸等,这些物质在调节机体免疫、抗肿瘤、抗菌、防腐方面起到作用。此外酸菜含有丰富的维生素、矿物质、膳食纤维等营养物质,是一类具有降低胆固醇、调节肠胃及排毒功效的食品。发酵酸菜不仅仅是加工食品而且还可以延长大白菜的贮藏期,是一种营养有效的大白菜加工手段。在我国尤其是东北地区深受居民喜欢,市场潜力巨大。
酸菜发酵方式分为自然发酵及人工接种发酵两种。传统自然发酵方法腌制的酸菜口味醇厚、气味浓郁,但耗时较长、产品质量不稳定、容易腐烂变味,而且容易产生对人体有害的亚硝酸盐等物质。而工业发酵虽然耗时短、产品质量均一,但其口味远不如自然发酵酸菜风味醇厚浓郁。
发明内容
为解决上述问题,本发明提供一种肠膜明串珠菌及其在发酵酸菜中的应用。
首先,本发明提供一种肠膜明串珠菌,该肠膜明串珠菌命名为肠膜明串珠菌Leuconostoc mesenteroides DN-LM-D2,分类命名为Leuconostoc mesenteroides,实验室编号为DN-LM-D2,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.16442,保藏日期为2018年9月10日,保藏单位地址为北京市朝阳区北辰西路1号院3号。
肠膜明串珠菌Leuconostoc mesenteroides DN-LM-D2菌落为白色不透明圆形,且革兰氏染色呈阳性,菌体呈球状,直径≤1mm。过氧化氢酶试验、淀粉水解试验、精氨酸产氨试验、产气试验、运动性检查都为阴性,而甲基红试验、产酸反应、氧化酶试验均显示阳性。
第二,本发明还提供上述肠膜明串珠菌发酵酸菜中的应用。
上述应用包括以下步骤:
1)选择新鲜大白菜,进行前处理;
2)清洗:将步骤1)处理后的大白菜用水洗净,沥干水分,摆放置发酵容器中;
3)发酵:在装有大白菜的发酵容器中注入纯净水,然后添加肠膜明串珠菌菌液,同时添加食用盐,密闭发酵,得到酸菜。
步骤1)所述前处理具体为剥去外层腐烂或失水部分,阴凉干燥通风处放置。
所述放置时间为2-3天。
步骤3)所述注入无菌水的量为液面没过大白菜。
步骤3)所述肠膜明串珠菌为上述Leuconostoc mesenteroides DN-LM-D2。
步骤3)所述肠膜明串珠菌菌液中的菌浓度为1×107-1.8×107cfu/ml。
步骤3)所述肠膜明串珠菌菌液的添加量为步骤1)前处理后所得大白菜质量分数的4%。
步骤3)所述食用盐添加量为步骤1)前处理后所得大白菜质量分数的2%。
步骤3)所述密闭发酵,发酵温度为20℃,发酵时间为30天以上。
步骤3)所述肠膜明串珠菌菌液的制备方法为:取肠膜明串珠菌Leuconostocmesenteroides DN-LM-D2菌种菌液在MRS平板上划线,37℃培养48-72h,挑取单菌落接种至MRS液体培养基中,37℃,150rps摇床培养18-24h,作为种子液,将种子液以5%-20%体积比接种至扩大培养基,进行扩大培养后得到肠膜明串珠菌菌液。
所述扩大培养基配方为蛋白胨10g/L,酵母粉5g/L,牛肉膏5g/L,番茄汁100ml/L,其余为水,并以1M的NaOH调整培养基pH为6.2-6.4。所述扩大培养,条件为37℃,150rps摇床培养,扩大培养时间为18-24h。
有益效果
肠膜明串珠菌(Leuconostoc mesenteroides)DN-LM-D2菌,具有较强降解亚硝酸盐的能力,降低发酵液中的亚硝酸亚含量,降低食用隐患,提高蔬菜发酵制品的食用安全性,利用本发明提供的菌株发酵酸菜,硝酸盐和亚硝酸盐含量较同等条件下自然发酵的酸菜分别降低31.9%和38.5%。同时,肠膜明串珠菌(Leuconostoc mesenteroides)DN-LM-D2菌具有较强耐酸性,这利于发酵后期的发酵液维持产酸,并提高风味物质的积累,口味更接近传统发酵酸菜,其pH与自然发酵的酸菜基本一致。另外,由于人工接种肠膜明串珠菌(Leuconostoc mesenteroides)DN-LM-D2菌,成为发酵过程中的优势菌群,抑制杂菌及有害菌的生长,增加出菜率(发酵结束后酸度足够且未见腐烂的酸菜质量/初始大白菜质量),提高发酵蔬菜制品的品质,利用本发明提供的菌株发酵酸菜,成菜率较同等条件下自然发酵的酸菜高22%。
具体实施方式
实施例1菌种的筛选与鉴定过程
菌种分离纯化
选取哈尔滨地区多家市民自制传统发酵酸菜少许,表面经无菌水和70%乙醇交替冲洗3次后,用无菌水冲洗干净,沥干多余水分。取酸菜叶研磨出汁,用无菌水进行梯度稀释(10-1、10-2、10-3、10-4、10-5),取各个稀释浓度培养液50微升涂布于MRS固体培养基上37℃恒温培养48h,挑取溶钙圈较大的单菌落在MRS固体培养基上划线纯化。筛选出11个产生溶钙圈的菌落分别命名为DN-LP-X1、DN-LP-X2、DN-LP-M1、DN-LP-M2、DN-LP-W1、DN-LP-H2、DN-LA-H7、DN-LM-Y2、DN-LM-Y3、DN-LM-Y4、DN-LM-D2。将这11个菌株在MRS液体培养基中,37℃、150rpm/min条件下培养12h的菌悬液与60%的甘油以1:1的比例混合后放入无菌管中,-80℃保存。
生理生化及菌种分子鉴定
经过形态特征鉴定观察发现这11个菌落均为白色不透明圆形,且革兰氏染色呈阳性。其中DN-LP-X1、DN-LP-X2、DN-LP-M1、DN-LP-M2、DN-LP-W1、DN-LP-H2、DN-LA-H7等9株菌呈杆状,直径1-2mm,DN-LM-Y2、DN-LM-Y3、DN-LM-Y4、DN-LM-D2等四株菌呈球状,直径≤1mm。然后通过生理生化特征鉴定我们得知这11个菌株的过氧化氢酶试验、淀粉水解试验、精氨酸产氨试验、产气试验、运动性检查都为阴性,而甲基红试验、产酸反应、氧化酶试验均显示阳性,初步鉴定这11株菌初步鉴定为乳酸菌属。
菌种分子鉴定:利用16S rDNA通用引物,以菌液基因组为模版进行PCR扩增,扩增出的产物经琼脂糖电泳纯化后送北京华大生物有限公司进行测序。利用NCBI数据库比对测序结果发现用DN-LP-X1、DN-LP-X2、DN-LP-M1、DN-LP-M2、DN-LP-W1、DN-LP-H2、DN-LA-H7与Lactobaciliusplantarum序列相似性高达100%,这些菌株是植物乳杆菌。而DN-LM-Y3、DN-LM-Y4与Leuconostoc mesenteroides相似性高达100%,而DN-LM-Y2、DN-LM-D2与Leuconostoc mesenteroides相似性高达99%,说明DN-LM-Y2、DN-LM-Y3、DN-LM-Y4、DN-LM-D2为肠膜明串珠菌。
菌种降解亚硝酸盐和耐酸能力鉴定。
亚硝酸盐降解能力鉴定
将所有菌株活化至11.8×107-1.8×107cfu/ml(11种菌调整相同菌浓度),并按体积的1%接种在含有10mg/L NaNO2的MRS液体培养基中,37℃条件下培养24h后测定NaNO2的降解率。发现降解亚硝酸盐能力最强的菌株为DN-LM-Y4和DN-LM-D2,分别达到95.32%和98.31%。
表1菌株的亚硝酸盐降解率
耐酸能力鉴定
这11个乳酸菌分别在MRS液体培养基生长的最适合pH为6;当pH=3.5时,所有菌株的生长都受到不同程度的抑制,与pH为6的条件下的OD600相比,吸光值降低的最少的菌株耐酸性越强,其中DN-LM-D2的吸光值降低的最少即OD600(pH=6.5)-OD600(pH=3.5)=0.521,其他菌株DN-LP-X1、DN-LP-X2、DN-LP-M1、DN-LP-M2、DN-LP-W1、DN-LP-H2、DN-LA-H7、DN-LM-Y2、DN-LM-Y3、DN-LM-Y4分别为1.452,1.459,1.407,1.369,1.433,1.322,1.389,1.183,1.231,0.846,0.874,0.856,所以DN-LM-D2表明耐酸性较好。
实施例2肠膜明串珠菌Leuconostoc mesenteroides DN-LM-D2菌在腌制酸菜中的应用
挑取甘油管中保存的Leuconostoc mesenteroides DN-LM-D2菌液在MRS平板划线,37℃培养48-72h,挑取单菌落接种至20-30ml的MRS液体培养基中,37℃,150r摇床培养18-,作为种子液,待扩大培养。扩大发酵培养基配方为蛋白胨10g/L,酵母粉5g/L,牛肉膏5g/L,番茄汁100ml/L,并以1M的NaOH调整培养基pH在6.2-6.4之间,培养18h后得到肠膜明串珠菌菌液。菌液用于发酵酸菜:
1)选择新鲜大白菜,剥去外叶(腐烂或失水的叶子),阴凉干燥通风处放置2-3天。
2)清洗:大白菜用清水洗净,沥干水分,摆放置发酵容器中。
3)发酵:在发酵容器中注入纯净水直至液面需没过大白菜,然后按大白菜重量的4%添加肠膜明串珠菌菌液,同时添加大白菜重量的2%的食用盐。20℃密闭发酵(30天),得到酸菜制品。
对比例1自然发酵酸菜
1)选择新鲜大白菜,剥去外叶(腐烂或失水的叶子),阴凉干燥通风处放置2-3天。
2)清洗:大白菜用清水洗净,沥干水分,摆放置发酵容器中。
3)发酵:在发酵容器中注入纯净水直至液面需没过大白菜,然后按大白菜重量的2%添加食用盐。20℃密闭发酵(30天),得到酸菜制品。
对比例2使用其他乳酸菌发酵酸菜
使用同时筛选出来的菌种Leuconostoc mesenteroides DN-LM-Y4替换实施例2中的Leuconostoc mesenteroides DN-LM-D2菌,其余条件与实施例2相同:
挑取甘油管中保存的Leuconostoc mesenteroides DN-LM-Y4菌液在MRS平板划线,37℃培养48-72h,挑取单菌落接种至20-30ml的MRS液体培养基中,37℃,150r摇床培养18-24h,作为种子液,待扩大培养。扩大发酵培养基配方为蛋白胨10g/L,酵母粉5g/L,牛肉膏5g/L,番茄汁100ml/L,并以1M的NaOH调整培养基pH在6.2-6.4之间,培养18h后得到肠膜明串珠菌Leuconostoc mesenteroides DN-LM-Y4菌液。菌液用于发酵酸菜:
1)选择新鲜大白菜,剥去外叶(腐烂或干瘪的叶子),阴凉干燥通风处放置2-3天。
2)清洗:大白菜用清水洗净,沥干水分,摆放置发酵容器中。
3)发酵:在发酵容器中注入纯净水直至液面需没过大白菜,然后按大白菜重量的4%添加肠膜明串珠菌Leuconostoc mesenteroides DN-LM-Y4菌液,同时添加大白菜重量的2%的食用盐。20℃密闭发酵(30天),得到酸菜制品。
分别测定发酵酸菜的pH(发酵液)、硝酸盐含量、亚硝酸盐含量,测定具体结果见表2。
表2 DN-LM-D2接种发酵酸菜和自然发酵酸菜的比较
从表2中的数据可以看出,使用本发明提供的菌株发酵酸菜,与自然发酵相比,硝酸盐含量降低了31.9%,亚硝酸盐含量降低了38.5%,pH值略低于自然发酵酸菜,证明本发明方法发酵的酸菜酸度较自然发酵的酸菜高;另外,从上表还可以看出,使用本发明提供的菌株发酵酸菜,与其他乳酸菌发酵酸菜相比,硝酸盐含量降低了12.4%,亚硝酸盐含量降低了15.8%,使用其他乳酸菌发酵的酸菜pH高于自然发酵酸菜,更远远高于本发明方法发酵的酸菜,酸度过低,严重影响酸菜食感。
分别检测发酵液中除乳酸菌外的杂菌含量(菌个数/发酵液体积),对比例2为实施例2的37倍,对比例1杂菌含量最高,为实施例2的118倍。分别计算出菜率(发酵结束后酸度足够且未见腐烂的酸菜质量/初始大白菜质量),实施例2成菜率较同等条件下自然发酵的酸菜(对比例1)高22%,较对比例2高9%。
Claims (7)
1.一种肠膜明串珠菌,其特征在于:所述肠膜明串珠菌命名为肠膜明串珠菌Leuconostoc mesenteroides DN-LM-D2,分类命名为Leuconostoc mesenteroides,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.16442,保藏日期为2018年9月10日。
2.一种权利要求1所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:包括以下步骤:
1)选择新鲜大白菜,进行前处理;
2)清洗:将步骤1)处理后的大白菜用水洗净,沥干水分,摆放置发酵容器中;
3)发酵:在装有大白菜的发酵容器中注入纯净水,然后添加肠膜明串珠菌菌液,同时添加食用盐,密闭发酵,发酵温度为20℃,发酵时间为30天以上,得到酸菜。
3.根据权利要求2所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:步骤1)所述前处理具体为剥去外层腐烂或干瘪部分,阴凉干燥通风处放置2-3天。
4.根据权利要求2所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:步骤3)所述注入纯净水的量为液面没过大白菜。
5.根据权利要求2所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:步骤3)所述肠膜明串珠菌菌液中的菌浓度为1×107-1.8×107 cfu/ml。
6.根据权利要求2所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:步骤3)所述肠膜明串珠菌菌液的添加量为步骤1)前处理后所得大白菜质量分数的4%。
7.根据权利要求2所述的肠膜明串珠菌在发酵酸菜中的应用,其特征在于:步骤3)所述食用盐添加量为步骤1)前处理后所得大白菜质量分数的2%。
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