CN105995785B - 一种使用复合菌快速发酵生产萍乡擦菜的方法 - Google Patents
一种使用复合菌快速发酵生产萍乡擦菜的方法 Download PDFInfo
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- CN105995785B CN105995785B CN201610493529.0A CN201610493529A CN105995785B CN 105995785 B CN105995785 B CN 105995785B CN 201610493529 A CN201610493529 A CN 201610493529A CN 105995785 B CN105995785 B CN 105995785B
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- lactic acid
- fermentation
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- acid bacteria
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- 241000894006 Bacteria Species 0.000 title claims abstract description 61
- 238000000855 fermentation Methods 0.000 title claims abstract description 39
- 230000004151 fermentation Effects 0.000 title claims abstract description 39
- 150000001875 compounds Chemical class 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 29
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound 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CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 26
- 239000004310 lactic acid Substances 0.000 claims abstract description 26
- 230000001954 sterilising Effects 0.000 claims abstract description 20
- 235000013311 vegetables Nutrition 0.000 claims abstract description 19
- 241000235342 Saccharomycetes Species 0.000 claims abstract description 15
- 230000002269 spontaneous Effects 0.000 claims abstract description 12
- 238000004140 cleaning Methods 0.000 claims abstract description 7
- 239000002054 inoculum Substances 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 3
- 239000002609 media Substances 0.000 claims description 25
- 239000012530 fluid Substances 0.000 claims description 15
- 239000001963 growth media Substances 0.000 claims description 15
- 235000015097 nutrients Nutrition 0.000 claims description 15
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 12
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
一种使用复合菌快速发酵生产萍乡擦菜的方法，从自然发酵搓菜中筛选并鉴定出两类优势菌种：乳酸菌和酵母菌。并以这两类菌作为初始发酵菌种，将洗净的蔬菜干燥至水分含量15~65%，将纯化得到或购买的优势乳酸‑优势酵母，以乳酸菌:酵母菌=1~6:1~6接种至蔬菜表面，接种量为蔬菜总量的0.5~5%，均匀搅拌后压实，并置于已灭菌容器中，封口后避光阴凉保存，20~30天后即可取出食用。本发明发酵时间只需自然发酵的1/3，大大提高了生产效益；口感、色泽等均优于或接近自然发酵搓菜；相对传统发酵泡菜，亚硝酸盐含量明显减少，提高了食品的安全性；人工发酵菌群可控，乳酸‑酵母复合菌在协同共生中提高了对腐败菌的抑制作用。
Description
技术领域
本发明属于食品加工领域。
背景技术
传统萍乡擦菜的制作过程是将新鲜的蔬菜（白菜、青菜、黄瓜、萝卜、豇豆、芥菜等）晒干揉擦后，置于密闭缸内。蔬菜分层而放，在缸口处放置一些洗净的稻草，或用干净的大石头压住，赶尽缸内空气，自然发酵，一般三个月后即可食用。
萍乡的擦菜制作已有上百年历史，擦菜口感独特，干香爽口，深受当地居民的喜爱。目前，大部分发酵蔬菜含盐量高、含发酵母液、亚硝酸盐含量偏高，保质期不长。而擦菜制作过程不加盐，亚硝酸盐含量低；且没有发酵母液，能较好地保留蔬菜中的营养成分；同时擦菜含水量低，利于保存和运输，保质期可长达两年。可见，萍乡擦菜有不可比拟的优点。然而，传统方法加工萍乡擦菜存在着杂菌不易控制、发酵菌群代谢产物复杂、蔬菜的腌制时间较长等问题。
近年来，发酵蔬菜的加工技术已由传统的纯手工制作向全自动机器化方向转变。人工发酵可以提高生产效率，稳定产品质量、提高经济效益。与此同时，现代微生物发酵技术在蔬菜发酵过程中的应用，可使蔬菜的发酵时间大大缩短，同时还可以提高成品的质量。
发明内容
本发明的目的是使用乳酸-酵母复合菌，对新鲜蔬菜进行人工发酵，快速生产萍乡擦菜。
本发明首先从传统萍乡擦菜中提取纯化出优势菌种，并分别对其进行鉴定（形态观察、革兰氏染色、生理生化特性、分子生物学鉴定等将菌落鉴定至属，再用现代分子实验技术如16SrDNA测序分析及26SrDNAD1/D2 区基因克隆序列分析将微生物鉴定至种）。鉴定后的优势菌，依据其生长特性，产酸和产醇能力以及菌种间拮抗试验，筛选出优势乳酸菌有：植物乳杆菌、布氏乳杆菌、戊糖乳杆菌、短乳杆菌、粪肠球菌；筛选出优势酵母菌有：东方伊萨酵母、西方伊萨酵母、酿酒酵母、鲁氏酵母、毕赤酵母。利用筛选鉴定出的乳酸-酵母复合菌作为初始发酵菌种，对蔬菜进行人工发酵。乳酸-酵母复合菌适用于食品蔬菜加工行业，能为食品提供风味物质和良好的质地；还能够降低发酵蔬菜的pH值，抑制腐败微生物和病原菌的生长；大大缩短蔬菜的发酵时间，延长保藏期；改善肠道微生物菌群的平衡，增强机体的免疫力；大幅度降低腌菜的亚硝酸盐含量，提高食品安全性。
本发明是通过以下技术方案实现的。
本发明所述的一种使用复合菌快速发酵生产萍乡擦菜的方法，包括以下步骤。
（1）选择适合的乳酸菌培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌10 ~ 40 min，备用；所述的乳酸菌培养基可以为MRS、LBS等固体或液体培养基。
选择适合的酵母菌培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌10 ~ 40 min，备用；所述的酵母菌培养基可以为YPD、PDA等固体或液体培养基。
（2）将步骤（1）所述的乳酸菌培养基和酵母菌培养基移植到平板上，再将优势乳酸菌和优势酵母菌按定期移植法、平板划线法或斜面法进行菌种活化，接种到有培养基的平板上，30~37℃恒温培养2-3天。
（3）将活化后的优势乳酸菌和优势酵母菌菌种分别转接至按步骤（1）所述的相应的液体培养基中，30~37℃恒温培养2-3天，得到优势乳酸菌和优势酵母菌纯种菌液，混合成乳酸-酵母复合菌溶液，混合比例为乳酸菌总量:酵母菌总量=1~6:1~6。
（4）将洗净的蔬菜干燥至水分含量为15~65%，然后，将步骤（3）的乳酸-酵母复合菌溶液喷洒接种于蔬菜表面，最终接种量为蔬菜总质量的0.5~5%，拌匀后置于密闭灭菌容器中厌氧阴凉避光发酵，一个月后取出，即可食用。
本发明所述的优势乳酸菌为东方伊萨酵母、西方伊萨酵母、酿酒酵母、鲁氏酵母或毕赤酵母中的一种或多种；所述的优势酵母菌为植物乳杆菌、布氏乳杆菌、戊糖乳杆菌、短乳杆菌、粪肠球菌中的一种或多种。
本发明所述的优势乳酸菌和优势酵母菌，可以是市售纯菌。也可以是从自然发酵的擦菜中筛选纯化得到。
本发明所述优势乳酸菌和优势酵母菌的筛选纯化过程可按以下方法操作。
称取1~50质量份数的自然发酵擦菜，在无菌环境下剪碎后，浸泡于10~50倍体积灭菌水中，震荡0.5~2 h后，再加无菌水制成10倍稀释液。在此基础上，再加无菌水制成10-2稀释液，以此类推，直至将样品稀释到10-7倍。从中选择适宜稀释液，涂布在初筛平板培养基上，30~37 ℃倒置厌氧培养24~48 h，取平板上形态不同的菌落，分别在与初筛培养基相同成分的培养基上划线分离，重复分离3次后，-4 ℃斜面保藏，筛选出优势酵母菌：东方伊萨酵母、西方伊萨酵母、酿酒酵母、鲁氏酵母、毕赤酵母和乳酸菌：植物乳杆菌、布氏乳杆菌、戊糖乳杆菌、短乳杆菌、粪肠球菌。依据所选菌种，分别纯化。
本发明的主要特点是。
1、乳酸-酵母复合菌可以为擦菜提供良好的风味和质地，并能够降低发酵蔬菜的pH值，抑制腐败微生物的生长，进而起到防腐的作用。
2、采用纯菌发酵，发酵过程中乳酸菌产酸，亚硝酸盐在酸性的环境中能被迅速降解，减少擦菜中亚硝酸盐含量。
3、酵母菌和乳酸菌有其各自的生理生化特性，当这两种微生物一起培养时，可以发挥互惠、协同、加强等优势。使发酵环境达到最优，在协同共生中提高对腐败菌的抑制作用。
4、使用直投式复合菌发酵，可以将发酵时间缩短至自然发酵所需时间的1/3，大大提高了生产效益。
具体实施方式
下面结合实施例对本发明作进一步详细说明，但应理解本发明的范围非仅限于这些实施例的范围。
实施例1。
（1）取自然发酵擦菜10 g，在无菌环境下剪碎后，浸泡于90 mL灭菌水中，震荡30min后，加无菌水制成10倍稀释液。再取1体积 10倍稀释液加入9 体积灭菌水中，混匀，制成10-2稀释液，以此类推，将样品稀释至10-7倍。选择10-3稀释度，涂布在初筛平板培养基上，36℃ 倒置厌氧培养48 h，取平板上形态不同的菌落，分别在与初筛培养基相同成分的培养基上划线分离，重复分离3次后，筛选出纯种植物乳杆菌、布氏乳杆菌、西方伊萨酵母和东方伊萨酵母的菌种，-4℃斜面保藏，以此作为初始发酵菌种。
（2）分别配制MRS固体和液体培养基，分装至锥形瓶内，塞上棉塞， 121℃高压蒸汽灭菌，灭菌20 min，备用。
（3）分别配制YPD固体和液体培养基，分装至锥形瓶内，塞上棉塞， 121℃高压蒸汽灭菌，灭菌20 min，备用。
（4）将培育出的植物乳杆菌、布氏乳杆菌、西方伊萨酵母和东方伊萨酵母的菌种按定期移植法分别进行菌种活化，并接种到平板内的备用固体培养基上，35℃恒温培养2天。
（5）活化后的植物乳杆菌和布氏乳杆菌转接至MRS液体培养基中，东方伊萨酵母和西方伊萨酵母菌种转接至YPD液体培养基中，35℃恒温培养2天，得植物乳杆菌、布氏乳杆菌、西方伊萨酵母和东方伊萨酵母纯种菌液，备用。
（6）洗净的芥菜鼓风干燥至水分含量为55 %，取出冷却后，将植物乳杆菌-布氏乳杆菌-西方伊萨酵母-东方伊萨酵母复合菌液接种至芥菜表面，配比为植物乳杆菌:布氏乳杆菌:西方伊萨酵母:东方伊萨酵母=1:1:2:2，接种量为芥菜总量的1%，拌匀后置于密闭灭菌容器中厌氧阴凉避光发酵，一个月左右取出，即可食用。风味、色泽、口感等与自然发酵擦菜一致。
实施例2。
（1）配制LBS液体培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌30min，备用。
（2）配制PDA液体培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌30min，备用。
（3）购买市售植物乳杆菌、短乳杆菌、毕赤酵母和酿酒酵母，按平板划线法分别进行菌种活化，分别接种到平板内的备用液体培养基上，32℃恒温培养3天。
（4）活化后的植物乳杆菌及短乳杆菌转接至LBS液体培养基中，毕赤酵母和酿酒酵母菌种转接至PDA液体培养基中，32℃恒温培养3天，得植物乳杆菌、短乳杆菌、毕赤酵母和酿酒酵母纯种菌液，备用。
（2）将洗净的包心菜自然晒干至水分含量为50 %，将乳酸-酵母复合菌液接种至芥菜表面，配比为植物乳杆菌:短乳杆菌:毕赤酵母:酿酒酵母=2:1:1:3，接种量为包心菜总量的2%，拌匀后置于密闭灭菌容器中厌氧阴凉避光发酵，一个月左右取出，即可食用。风味、色泽、口感等与自然发酵擦菜一致。
实施例3。
（1）配制MRS液体培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌20min，备用。
（3）配制马铃薯葡萄糖液体培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌20 min，备用。
（4）购买市售戊糖乳杆菌、粪肠球菌、东方伊萨酵母和鲁氏酵母，按平板划线法分别进行菌种活化，分别接种到平板内的备用液体培养基上，32℃恒温培养3天。
（5）活化后的戊糖乳杆菌和粪肠球菌转接至MRS液体培养基中，东方伊萨酵母和鲁氏酵母菌种转接至马铃薯葡萄糖液体培养基中，35℃恒温培养3天，得到戊糖乳杆菌、粪肠球菌、东方伊萨酵母和鲁氏酵母纯种菌液，备用。
（6）洗净的萝卜菜鼓风干燥至水分含量为30 %，取出冷却后，将戊糖乳杆菌-粪肠球菌-东方伊萨酵母-鲁氏酵母复合菌液接种至芥菜表面，配比为戊糖乳杆菌:粪肠球菌:东方伊萨酵母:鲁氏酵母=3:1:5:2，接种量为萝卜菜总量的3%，拌匀后置于密闭灭菌容器中厌氧阴凉避光发酵，一个月左右取出，即可食用。风味、色泽、口感等与自然发酵擦菜一致。
Claims (1)
1.一种使用复合菌快速发酵生产萍乡擦菜的方法，其特征是包括以下步骤：
（1）选择适合的乳酸菌培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌10 ~ 40 min，备用；所述的乳酸菌培养基为MRS或LBS固体或液体培养基；
选择适合的酵母菌培养基，分装至锥形瓶内，塞上棉塞，121℃高压蒸汽灭菌，灭菌10 ~40 min，备用；所述的酵母菌培养基为YPD或PDA固体或液体培养基；
（2）将步骤（1）所述的乳酸菌培养基和酵母菌培养基移植到平板上，再将优势乳酸菌和优势酵母菌按定期移植法、平板划线法或斜面法进行菌种活化，接种到有培养基的平板上，30~37℃恒温培养2-3天；
（3）将活化后的优势乳酸菌和优势酵母菌菌种分别转接至按步骤（1）所述的相应的液体培养基中，30~37℃恒温培养2-3天，得到优势乳酸菌和优势酵母菌纯种菌液，混合成乳酸-酵母复合菌溶液，混合比例为乳酸菌总量:酵母菌总量=1~6:1~6；
（4）将洗净的蔬菜干燥至水分含量为15~65%，然后，将步骤（3）的乳酸-酵母复合菌溶液喷洒接种于蔬菜表面，最终接种量为蔬菜总质量的0.5~5%，拌匀后置于密闭灭菌容器中厌氧阴凉避光发酵，一个月后取出，即可食用；
所述优势乳酸菌和优势酵母菌的筛选纯化过程按以下方法操作：
称取1~50质量份数的自然发酵擦菜，在无菌环境下剪碎后，浸泡于10~50倍体积灭菌水中，震荡0.5~2 h后，再加无菌水制成10倍稀释液；在此基础上，再加无菌水制成10-2稀释液，以此类推，直至将样品稀释到10-7倍；从中选择适宜稀释液，涂布在初筛平板培养基上，30~37 ℃倒置厌氧培养24~48 h，取平板上形态不同的菌落，分别在与初筛培养基相同成分的培养基上划线分离，重复分离3次后，-4℃斜面保藏；
其中，筛选出纯种菌种为植物乳杆菌、布氏乳杆菌、西方伊萨酵母和东方伊萨酵母的菌种作为初始发酵菌种。
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