CN108531421A - 一种植物乳杆菌及其在羊栖菜脱腥中的应用 - Google Patents
一种植物乳杆菌及其在羊栖菜脱腥中的应用 Download PDFInfo
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Abstract
本发明公开了一种植物乳杆菌及其在羊栖菜脱腥中的应用，该植物乳杆菌，其保藏编号为：CGMCC No.13338，其应用于羊栖菜脱腥包括步骤：(1)斜面培养：将所述的植物乳杆菌接种到斜面培养基上，在35℃条件下培养48小时；(2)种子培养：将斜面培养基上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时，获得植物乳杆菌种子液；(3)发酵脱腥：取待脱腥处理的新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入5‑10wt％的食盐水、1‑5wt％的所述的植物乳杆菌种子液，15‑30℃条件下发酵培养10‑30天，从而完成发酵脱腥操作。本发明的优点是有效地脱除羊栖菜的腥味，促进羊栖菜产业的发展。
Description
技术领域
本发明涉及食品加工领域，具体是指一种植物乳杆菌及其在羊栖菜脱腥中的应用。
背景技术
羊栖菜(Sargassum fusiforme)营养丰富、风味独特，是一种高蛋白、低脂肪的海藻，并含有丰富的多糖、膳食纤维、维生素和矿物质，具有消除大脑疲劳、增进机体免疫力、延缓衰老、降血糖、降血脂等功效(许忠能,王朝晖,孙立.羊栖菜药用价值的研究进展.中草药,2000.31(11):876-878.)。羊栖菜是日本、中国、韩国和朝鲜沿海地区的海洋蔬菜和传统美食。日本人对羊栖菜特别青睐，自古以来就将羊栖菜推崇为益寿食品(张展，刘建国，刘吉东.羊栖菜的研究述评.海洋水产研究,2002.23(3):67-74.)。目前，羊栖菜已实现人工大规模养殖，温州洞头是全国最大的羊栖菜养殖、加工、出口基地，2003年被中国优质农产品开发服务协会命名为“中国羊栖菜之乡”，其生产的羊栖菜产品主要销往日本，年总产量约7万吨原料菜，全县羊栖菜年出口创汇达1.42亿美元(章立浩,章俊.洞头发展羊栖菜养殖业的优势.浙江气象,2009.30(2):31-35.)。
海藻营养丰富，味道鲜美，但腥味重，严重影响了消费者的接受程度和海藻产业的发展。目前人们对食品风味的要求越来越高，如何控制腥味物质的形成及脱除腥味物质越来越受到研究者的重视。针对海藻的腥味，主要的腥味物质脱除技术，包括酸碱处理法、掩盖法、蒸汽脱腥、吸附法、微胶囊法、萃取法、微生物发酵法等。CN101496562公开了一种海带的脱腥方法，采用酸梅汤热烫、茶水浸泡去除海带腥味；CN101218974公开了一种采用二氧化氯漂白液处理对羊栖菜进行护色脱腥的方法；CN102326799公开了一种分步采用甘草液和臭氧水处理脱腥龙须菜的方法；CN104187878公开了一种脱腥海带粉产品及其生产方法，以腌渍海带为原料，采用茶多酚溶液和95％以上的酒精两段处理，制备脱腥海带粉。酸碱处理、掩盖、吸附等传统脱腥方法虽然对海藻有一定的脱腥效果，但对蛋白质等营养成分可能有一定的破坏，从而影响海藻的营养价值，微生物脱腥法是一种绿色环保的脱腥方法，效果好，不但具有改善海藻风味的作用，还可以提高其生物活性(张贤艳，段文杨，邹佳欣，李继丹，杨海龙*.海藻脱腥技术研究进展.食品安全质量检测学报，2017，8(11):4288-4293)。
发明内容
本发明的目的是提供一种植物乳杆菌及其在羊栖菜脱腥中的应用。
为实现上述目的，本发明的技术方案是其保藏编号为：CGMCC No.13338。
本发明的植物乳杆菌(Lactobacillus plantarum)，已于2016年11月24日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编：100101。该菌株的菌株名称是：WZ-01；分类命名是植物乳杆菌(Lactobacillus plantarum)；保藏编号为：CGMCC No.13338。该植物乳杆菌(Lactobacillus plantarum)的核苷酸序列如 SEQ ID NO:1所示。
本发明还提供了一种基于所述的植物乳杆菌在羊栖菜的发酵脱腥中应用。
进一步设置是包括以下步骤：
(1)斜面培养：将所述的植物乳杆菌接种到斜面培养基上，在35℃条件下培养48小时；
(2)种子培养：将斜面培养基上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时，获得植物乳杆菌种子液；
(3)发酵脱腥：取待脱腥处理的新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入5-10wt％的食盐水、1-5wt％的所述的植物乳杆菌种子液，15-30℃条件下发酵培养10-30天，从而完成发酵脱腥操作。
进一步设置是所述的斜面培养基和种子培养基是LB培养基。
本发明的积极进步效果在于：
本发明方法具有工艺简单，成本较低，对羊栖菜的腥味脱除效果好，易于实现工业化生产的特点。而且，经本发明方法处理后，羊栖菜组织质地软硬适中，脆度保持良好，避免了现有脱腥技术中效果不理想或脱腥试剂对羊栖菜成分、组织结构的影响。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1采用MEGA7.0软件，邻位连接法显示菌株CGMCC No.13338与GeneBank 数据库中相关种的16S rDNA序列系统发育树；
图2新鲜羊栖菜的挥发性成分GC-MS总离子流图；
图3羊栖菜经植物乳杆菌CGMCC No.13338发酵后的挥发性成分GC-MS总离子流图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
以下实施例中所用的菌种培养基如下：
乳酸菌筛选培养基(g/L)：酪蛋白胨10，牛肉膏10，酵母提取物5，葡萄糖 5，磷酸氢二钾2，乙酸钠5，柠檬酸三铵2，吐温80 1，硫酸镁0.2，硫酸锰0.05，碳酸钙10，琼脂15，pH6.8，0.6％溴甲酚绿1；
斜面培养基(g/L)：蛋白胨10，酵母提取物5，NaCl 10，琼脂20，pH7.0-7.2；
种子培养基(g/L)：蛋白胨10，酵母提取物5，NaCl 10，pH7.0-7.2；
实施例1植物乳杆菌(Lactobacilus plantarum)WZ-01的筛选分离与鉴定
一、植物乳杆菌(Lactobacilus plantarum)WZ-01是从发酵蔬菜中分离而来的。取发酵蔬菜液，将其稀释10-5，10-6，10-7倍，取100微升涂布在乳酸菌筛选培养基平板，每个浓度重复三次，37℃培养48小时，圆形稍扁平的白色菌落及其周围培养基变为黄色者为产酸菌，挑选产酸菌多次纯化，将纯化后的菌株接种到斜面培养基上，37℃培养48小时后放于4℃冰箱保存待用。
将4℃冰箱保存待用的菌株接种到种子培养基中，37℃培养24小时后，分别取10毫升种子液接种于100克羊栖菜中进行发酵，通过感官评定，从中筛选出一株脱腥效果强的菌株，定名为WZ-01。
二、植物乳杆菌(Lactobacilus plantarum)WZ-01的分子生物学鉴定
将目的菌株在斜面培养基上培养24小时，提取基因组DNA，利用PCR技术扩增该菌株的16S rDNA，所用引物，正向引物 (5'-CAGAGTTTGATCCTGGCT-3')，反向引物 (5'-AGGAGGTGATCCAGCCGCA-3')；
PCR反应体系组成如下表：
PCR条件如下表所示：
对PCR产物进行DNA测序，序列已提交GeneBank，序列登记号为MH016560，与GeneBank数据库中的16S rDNA进行同源性比对，结果见图1，发现与 Lactobacilusplantarum T34(GenBank accession No.MG739430.1)的同源性最高，高达99％，确定该菌株为植物乳杆菌(Lactobacilus plantarum)，已于2016年11 月24日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为：CGMCC No.13338。
实施例2羊栖菜挥发性成分的GC-MS分析
(1)羊栖菜处理
以羊栖菜：蒸馏水＝1:5的比例；把羊栖菜及浸泡水置入均浆机制备均浆液；取均浆液50mL加入25mL乙醚间歇萃取1h。
(2)仪器及测定条件
Agilent气相色谱-质谱联用仪GC 6890-MS5975。
色谱条件：Agilent 6890N气相色谱仪，HP-5MS毛细管色谱柱(30m×250μ m，0.25μm)。柱温按程序升温，初温35℃保持6min，然后以5℃/min升至100℃，再以10℃/min升至230℃。采用不分流进样，进样口温度270℃；载气为He，流量1.0m L/min。
质谱条件：Agilent 5975MSD质谱，电子轰击离子源(EI)，离子源温度230℃，接口温度280℃，扫描质量范围10amu-500amu。环境条件：室温25℃。
(3)数据处理
峰面积归一化法。利用Agilent G1701MSD Productivity Chem Station增强型数据分析工作站Nist标准谱库自动检索各组分质谱数据，按照标准谱图及保留指数对机检结果进行核对和确认。
实施例3
将植物乳杆菌CGMCC No.13338接种到斜面培养基上，在35℃条件下培养48 小时；然后将斜面上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时；取100克新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入5％的食盐水、1％的植物乳杆菌种子液，30℃条件下发酵培养10天。取发酵前后的羊栖菜按照实施例2的方法进行挥发性成分检测，新鲜羊栖菜和植物乳杆菌发酵羊栖菜挥发物质成分的总离子流图如图2、图3所示。由图中可知，植物乳杆菌发酵明显减少羊栖菜的挥发性成分，羊栖菜在低温区的挥发性成分几乎未检测到。新鲜羊栖菜的挥发性成分见表1。植物乳杆菌发酵羊栖菜的挥发性成分见表2。羊栖菜经植物乳杆菌发酵后大部分烃类未检出，所有存在于新鲜羊栖菜中的乙醛、酮类、酚类及胺类成分都未检出。植物乳杆菌发酵显著减少了羊栖菜的挥发性成分，无腥味。
表1新鲜羊栖菜气味成分
表2植物乳杆菌发酵后羊栖菜气味成分
实施例4
将植物乳杆菌CGMCC No.13338接种到斜面培养基上，在35℃条件下培养48 小时；然后将斜面上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时；取1000克新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入8％的食盐水、2％的植物乳杆菌种子液，20℃条件下发酵培养15天。发酵结束后经 GC-MS法分析新鲜和植物乳杆菌发酵羊栖菜的挥发性成分，达到了如实施例3类似的结果，植物乳杆菌发酵后的羊栖菜无腥味。
实施例5
将植物乳杆菌CGMCC No.13338接种到斜面培养基上，在35℃条件下培养 48小时；然后将斜面上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时；取10公斤新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入 10％的食盐水、5％的植物乳杆菌种子液，15℃条件下发酵培养30天。发酵结束后经GC-MS法分析新鲜和植物乳杆菌发酵羊栖菜的挥发性成分，达到了如实施例3类似的结果，植物乳杆菌发酵后的羊栖菜无腥味。
以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。
SEQUENCE LISTING
<110> 温州大学
<120> 一种植物乳杆菌及其在羊栖菜脱腥中的应用
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1308
<212> DNA
<213> 植物乳杆菌（Lactobacillus plantarum）WZ-01
<400> 1
tttacatttg agtgagtggc gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg 60
gggataacac ctggaaacag atgctaatac cgcataacaa cttggaccgc atggtccgag 120
tttgaaagat ggcttcggct atcacttttg gatggtcccg cggcgtatta gctagatggt 180
ggggtaacgg ctcaccatgg caatgatacg tagccgacct gagagggtaa tcggccacat 240
tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg 300
gacgaaagtc tgatggagca acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc 360
tgttgttaaa gaagaacata tctgagagta actgttcagg tattgacggt atttaaccag 420
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 480
ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg 540
gctcaaccga agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa 600
ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 660
gtctggtctg taactgacgc tgaggctcga aagtatgggt agcaaacagg attagatacc 720
ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg agggtttccg cccttcagtg 780
ctgcagctaa cgcattaagc attccgcctg gggagtacgg ccgcaaggct gaaactcaaa 840
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa 900
gaaccttacc aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg 960
acatggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1020
cccgcaacga gcgcaaccct tattatcagt tgccagcatt aagttgggca ctctggtgag 1080
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1140
cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgaactcg cgagagtaag 1200
ctaatctctt aaagccattc tcagttcgga ttgtaggctg caactcgcct acatgaagtc 1260
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttccc 1308
Claims (4)
1.一种对羊栖菜腥味具有脱除作用的植物乳杆菌，其保藏编号为：CGMCC No.13338。
2.一种根据权利要求1所述的植物乳杆菌在羊栖菜的发酵脱腥中应用。
3.根据权利要求2所述的应用，其特征在于包括以下步骤：
(1)斜面培养：将所述的植物乳杆菌接种到斜面培养基上，在35℃条件下培养48小时；
(2)种子培养：将斜面培养基上长好的菌种接种到新鲜的种子培养基中，在35℃条件下静置培养24小时，获得植物乳杆菌种子液；
(3)发酵脱腥：取待脱腥处理的新鲜羊栖菜，洗净后沥干，置于发酵容器中，加入5-10wt％的食盐水、1-5wt％的所述的植物乳杆菌种子液，15-30℃条件下发酵培养10-30天，从而完成发酵脱腥操作。
4.根据权利要求2所述的应用，其特征在于：所述的斜面培养基和种子培养基是LB培养基。
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