CN108185352B - 一种低生物胺发酵泡菜的制作方法 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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Abstract
本发明属于食品领域,尤其是食品行业中的泡菜制备技术领域,具体为一种低生物胺发酵泡菜的制作方法。该方法中采用了一株命名为乳酸乳球菌Lactococcus lactis PCYTY‑13,保藏日期为2017年11月20日,保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNo.14932的乳酸乳球菌。筛选出的发酵菌株PCYTY‑13,不产生物胺,而且能抑制其他能产生生物胺的乳酸菌生长,阻断了生物胺的形成,从而解决了泡菜存在生物胺积累的问题,且产酸能力强,缩短了泡菜的成熟时间。
Description
技术领域
本发明属于食品领域,尤其是食品行业中的泡菜制备技术领域,具体为一种低生物胺发酵泡菜的制作方法。
背景技术
生物胺是一类具有生物活性、含氨基的低分子质量化合物。生物胺普遍存在于食品尤其是发酵食品中,在发酵肉制品、发酵海产品、发酵乳制品和发酵蔬菜等发酵食品中均有生物胺产生的报道。适量摄入生物胺能促进生长、增强代谢活力、增强免疫力和清除自由基等,但是过量摄入生物胺会使人体中毒,引起头疼、血压变化、呼吸紊乱、心悸、呕吐等严重反应。
我国泡菜通常采用老盐水发酵以及循环利用盐水等,可能存在一些生物胺的积累问题。而生物胺与泡菜中的亚硝酸类物质在一定条件下反应产生强致癌类物质——亚硝胺,因此降低泡菜中的生物胺是有意义的。
发明内容
本发明的目的是针对上述技术问题,提供一种低生物胺发酵泡菜的制作方法。本方法中采用筛选出的发酵菌株PCYTY-13,不产生物胺,而且能抑制其他能产生生物胺的乳酸菌生长,阻断了生物胺的形成,从而解决了泡菜存在生物胺积累的问题,且产酸能力强,缩短了泡菜的成熟时间。
为了实现上述目的,本发明的具体技术方案为:
一种乳酸乳球菌,其命名为乳酸乳球菌Lactococcus lactis PCYTY-13,保藏日期为2017年11月20日,保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNo.14932。
一种低生物胺发酵泡菜的制作方法,其于包括以下步骤:
(1)原辅料预处理:将发酵用的蔬菜及辅料进行筛选,除去坏烂部分,选取新鲜、成熟适度,无杂质杂物的原辅料清洗干净,修整、切分;
(2)泡菜坛的消毒处理:将泡菜坛清洗干净,然后加入煮沸开水进行热烫杀菌20min,重复3次;
(3)泡菜制作:按质量比1:1的比例加入蔬菜和冷开水(0-4℃),再以蔬菜和冷开水的总质量计,向其中加入2~4%的食盐,0.1~0.5%老姜,0.5~1%大蒜头,0.02%的八角、0.02%的桂皮、0.05%的花椒、0.01%茴香,0.5~2%的红辣椒;
(4)乳酸乳球菌PCYTY-13菌悬液的制备和添加:将乳酸乳球菌PCYTY-13加入到MRS培养基中进行活化,于30℃下培养24h,将培养液倒入灭菌离心管中,4000r/min离心20min,去除上清液,加入10mL生理盐水,轻微震荡均匀,乳酸乳球菌的活菌数为107cfu/mL,按(蔬菜和冷开水质量)2~4wt%的比例量接入泡菜中;
(5)接菌后水封泡菜坛,30℃发酵3天即得。
本发明的积极效果为:
(一)、食品中生物胺的形成主要是由微生物所产生的氨基酸脱羧酶对游离氨基酸进行脱羧作用产生的。因此本申请以发酵蔬菜汁培养基为基质,定向筛选不产生物胺的菌株,而且该菌株随着发酵的进行快速生长、抑制其他产生氨基酸脱羧酶的微生物的生长,继而降低泡菜中的生物胺含量。尤其是泡菜在长时间发酵过程中,乳杆菌属(植物乳杆菌、短乳杆菌)等占据优势,所以需要从四川泡菜中筛选出一株产酸能力强,且不产生物胺的发酵乳酸菌,并完成专利菌株保藏,该菌株能抑制产生物胺的乳酸菌生长,阻断生物胺的形成;
(二)、应用于泡菜发酵,解决了生物胺积累的安全隐患。
(三)、制备得到的泡菜口感脆甜爽口,风味俱佳。
附图说明
图1为本发明中所述PCYTY-13的16S鉴定结果
具体实施方式
为了使本发明的发明目的、技术方案及优点更加清楚明白,下面结合具体实施方式对本发明作进一步的详细描述,但不应将此理解为本发明上述主题的范围仅限于下述实施例。
乳酸乳球菌PCYTY-13,分类命名为乳酸乳球菌Lactococcus lactis,保藏号为CGMCCNo.14932。该菌株已于2017年11月20日在中国微生物菌种保藏管理委员会普通微生物中心进行保藏,保藏地址:北京市朝阳区大屯路甲3号,中国科学院微生物研究所,邮编100101。
不产生物胺的乳酸乳球菌PCYTY-13筛选方法:
不产生物胺的乳酸菌初筛
取传统四川泡菜样品,无菌条件下剪碎后置于装有180mL灭菌生理盐水的三角瓶中,室温230转摇床震荡培养10分钟,取1mL于MRS液体培养基中,30℃培养24h。从富集培养液中取出1mL培养液,逐级稀释后,涂布于对应的产胺菌下层分离培养基上,30℃培养72小时后,在无茵条件下,在下层培养基上倒一层对应的上层培养基(50℃)显色,并在5分种内记录实验结果,显紫色的为阳性,不变色的为阴性(黄色)。共筛选获得50株乳酸菌,其中3株乳酸菌不产生物。其中,产生物分离下层培养基的配置方法为:蛋白胨5g,酵母膏5g,牛肉膏5g,氯化钠2.5g,葡萄糖0.5g,吐温1g,硫酸镁0.4g,硫酸猛0.03g,磷酸氢二钾2g,柠檬酸三铵2g,碳酸钙0.1g,硫酸亚铁0.4g,维生素B1 0.04g,磷酸吡哆醛0.5g,琼脂18g。色氨酸,精氨酸,赖氨酸,酪氨酸,苯丙氨酸,组氨酸,各5g,1000mL蒸馏水。pH5.2,115℃髙压灭菌15分钟。产生物胺菌分离上层培养基:溴甲酚紫0.06g,琼脂20g,1000mL蒸馈水,pH5.2,121℃灭菌15min。
2.抑制产胺菌的乳酸菌复筛
将步骤1中获得的3株不产生物胺的乳酸菌与泡菜发酵后期的常见优势菌植入乳杆菌、短乳杆菌、肠膜明串珠菌两两组合,以相同的量(107cfu/mL)接种至蔬菜汁培养基中,30℃培养24h。根据菌落形态的不同,分别采用MRS培养基对同一体系的两种菌进行计数,结果如表1所示:
表1 不产生物筛选菌与产胺乳酸菌的相互作用关系
注:表中数据为平均值±标准偏差。
从表1可以得出,以相同含量接种在同一培养体系中,和PCYTY-25能抑制植物乳杆菌、短乳杆菌、肠膜明串珠菌这三种产生物胺乳酸菌的生长,其中PCYTY-13这株菌抑制能力较强。
3.复筛菌株产酸能力测试
将这3株复筛菌活化后接种于蔬菜汁培养基中,30℃条件下静置培养48h,测定其pH和总酸含量,测定结果如表2所示:
表2 筛选菌的产酸能力测试
菌株编号 | pH | 总酸含量(g/100mL) |
PCYTY-13 | 3.68±0.01 | 0.78±0.03 |
PCYTY-19 | 3.82±0.05 | 0.58±0.12 |
PCYTY-25 | 3.73±0.06 | 0.62±0.01 |
注:表中数据为平均值±标准偏差。
从表2中可以得出,PCYTY-13这株菌的产酸能力最强。
4.筛选菌分子鉴定
菌株活化后参照Bacterial DNA Isolation Kit进行DNA的提取。DNA经过16SrDNA扩增,PCR反应体系:25ul 2×PCR Mix(Loading Buffer、Taq DNA Polymerase、dNTPs、Tris-HCl、KCl、MgCl2),1ul总DNA模板,引物27F和1492R各2ul,19.5ul ddH2O。反应条件为:95℃预变性10min,93℃30s,65℃30s,72℃1min,10个循环;93℃30s,60℃30s,72℃1min,10个循环;93℃30s,55℃30s,72℃1min,10个循环;72℃保持5min。扩增反应完毕后,利用2.0%的琼脂糖凝胶电泳进行检测。将PCR扩增产物送测序公司测序,利用BLAST软件将测定的各菌株16SrDNA与GenBank数据库中已知的16S rDNA基因序列进行比对,鉴定结果如图1所示。对PCYTY-13这株菌测序获得的序列在数据库上进行比对,发现其与乳酸乳球菌的同源性高达100%,因此鉴定该菌为乳酸乳球菌。
乳酸乳球菌菌悬液的制备可以按照现有技术进行制备。
实施例1:
一种低生物胺发酵泡菜的制作方法,包括以下步骤:
(1)原辅料预处理:将发酵用的蔬菜及辅料进行筛选,除去坏烂部分,选取新鲜、成熟适度,无杂质杂物的原辅料清洗干净,修整、切分。
(2)泡菜坛的消毒处理:将泡菜坛清洗干净,然后加入煮沸开水进行热烫杀菌20min,重复该步骤3次。
(3)泡菜制作:按质量比1:1的比例加入蔬菜和冷开水,再向其中加入(占蔬菜和冷开水的总质量)4%的食盐,0.5%老姜,0.5%大蒜头,0.02%的八角、0.02%的桂皮、0.05%的花椒、0.01%茴香,2%的红辣椒。
(4)乳酸乳球菌PCYTY-13菌悬液的制备和添加:将乳酸乳球菌PCYTY-13加入到MRS培养基中进行活化,30℃下培养24h,将培养液倒入灭菌离心管中,于4000r/min离心20min,去除上清液。再加入10mL生理盐水,轻微震荡均匀,乳酸乳球菌的活菌数为107cfu/mL,按(占蔬菜和冷开水的总质量)2%的比例量接入泡菜中。
(5)接菌后水封泡菜坛,于30℃发酵3天,定期检查坛内pH变化,当泡菜水pH降至3.5左右,即可成熟。
实施例2:
一种低生物胺发酵泡菜的制作方法,包括以下步骤:
(1)原辅料预处理:将发酵用的蔬菜及辅料进行筛选,除去坏烂部分,选取新鲜、成熟适度,无杂质杂物的原辅料清洗干净,修整、切分。
(2)泡菜坛的消毒处理:将泡菜坛清洗干净,然后加入煮沸开水进行热烫杀菌20min,重复3次。
(3)泡菜制作:按质量比1:1的比例加入蔬菜和冷开水,再向其中加入(占蔬菜和冷开水的总质量)3%的食盐,0.1%老姜,0.5%大蒜头,0.02%的八角、0.02%的桂皮、0.05%的花椒、0.01%茴香,2%的红辣椒。
(4)乳酸乳球菌PCYTY-13菌悬液的制备和添加:将乳酸乳球菌PCYTY-13加入到MRS培养基中进行活化,于30℃下培养24h,将培养液倒入灭菌离心管中,于4000r/min离心20min,去除上清液。加入10mL生理盐水,轻微震荡均匀,乳酸乳球菌的活菌数为107cfu/mL,按(占蔬菜和冷开水的总质量)3%的比例量接入泡菜中。
(5)接菌后水封泡菜坛,于30℃发酵3天,定期检查坛内pH变化,当泡菜水pH降至3.5左右,即可成熟。
实施例1和实施例2中制备得到的泡菜脆甜爽口,口感俱佳。
低生物胺发酵泡菜的理化指标检测
分别将实施例1中采用PCYTY-13制备得到的发酵泡菜和传统发酵泡菜,分别对发酵第1、5、10、15、20、30天的泡菜样品进行pH、总酸、生物胺含量进行测定,具体结果见表3。
表3 低生物胺泡菜与传统泡菜的理化指标比较
从表3我们可以得出,接PCYTY-13发酵泡菜成熟较快,在发酵5天前就完全成熟,而传统泡菜发酵第10天才能成熟。接菌发酵泡菜中生物胺总量整体低于传统泡菜。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种乳酸乳球菌,其特征在于命名为乳酸乳球菌Lactococcus lactis PCYTY-13,保藏日期为2017年11月20日,保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCCNo.14932,菌株PCYTY-13不产生物胺,能抑制其它能产生生物胺的乳酸菌生长,阻断了生物胺的形成,产酸能力强,能缩短了泡菜的成熟时间。
2.一种利用如权利要求1所述乳酸乳球菌进行低生物胺发酵泡菜的制作方法,其特征在于包括以下步骤:
(1)原辅料预处理:将发酵用的蔬菜及辅料进行筛选,除去坏烂部分,选取新鲜、成熟适度,无杂质杂物的原辅料清洗干净,修整、切分;
(2)泡菜坛的消毒处理:将泡菜坛清洗干净,然后加入煮沸开水进行热烫杀菌20min,重复3次;
(3)泡菜制作:按质量比1:1的比例加入蔬菜和冷开水,再加入适量的辅料;
(4)乳酸乳球菌PCYTY-13菌悬液的制备和添加:将乳酸乳球菌PCYTY-13加入到MRS培养基中进行活化,于30℃下培养24h,将培养液倒入灭菌离心管中,4000r/min离心20min,去除上清液,加入10mL生理盐水,轻微震荡均匀,按2~4wt%的比例量接入泡菜中;
(5)接菌后水封泡菜坛,30℃发酵3天即得。
3.如权利要求2所述低生物胺发酵泡菜的制作方法,其特征在于:所述的辅料包括食盐、老姜、大蒜、八角、桂皮、花椒、茴香和红辣椒,其中,以蔬菜和冷开水的总质量计,食盐的添加量为2~4%,老姜0.1~0.5%,大蒜0.5~1%,八角0.02%、桂皮0.02%、花椒0.05%、茴香0.01%,红辣椒0.5~2%。
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