CN117025470A - 一种提升香肠发酵品质的复合发酵剂及应用 - Google Patents
一种提升香肠发酵品质的复合发酵剂及应用 Download PDFInfo
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Abstract
本发明涉及肉类发酵剂技术领域,尤其是一种提升香肠发酵品质的复合发酵剂及应用,包括植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18;所述复合发酵剂为液态菌剂;其中,植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18的有效活菌数的比例为1:1:2‑4:2‑4。与传统发酵香肠相比,接种本发明的植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18复合发酵剂可显著快速降低香肠的pH值,抑制其中腐败菌的生长,保证产品的安全性。
Description
技术领域
本发明涉及肉类发酵剂技术领域,具体领域为一种提升香肠发酵品质的复合发酵剂及应用。
背景技术
发酵香肠是指在人工控制或自然环境条件下,利用微生物发酵产生独特风味的一类肉制品。传统发酵香肠主要利用环境中微生物的自然发酵进行生产,产品品质不稳定。而利用人工接种纯种微生物培养物控制发酵,可在很大程度上提高产品的品质和安全性。因此,将纯微生物培养物或制剂(即发酵肉制品发酵剂)人工接种于肉制品中实现其工业化生产已成为一种普遍做法。
乳酸菌和葡萄球菌是发酵肉制品中最为常见的微生物,也通常作为发酵剂在发酵肉制品中使用。接种乳酸菌可快速代谢碳水化合物产酸,降低pH从而抑制腐败、病原微生物的生长,提高产品安全性;葡萄球菌对发酵肉制品的风味和发色品质具有重要作用,一般具有蛋白水解酶、脂肪水解酶和硝酸盐还原酶活性,影响产品的风味特性,促进肉制品成熟阶段的干燥过程,并改善质地,同时促进亚硝基肌红蛋白形成,有利于肉制品良好色泽的形成和稳定。发酵肉制品根据不同的风味需要用到不同的微生物,且在应用上一般不局限于单一菌种,而是多菌种复合以弥补单一菌种的单调性。
定向接种菌株的发酵肉制品在保证口感和质地的前提下,可以解决依靠自然发酵的不稳定性,极大的提升产品的安全性和质量可靠性,并且乳酸菌和葡萄球菌的复合接种比单一接种的效果更可观。
目前,国内生产所用的发酵剂主要为欧美公司生产,如德国科汉森的BactofermTMF-1,尚未有使用国内菌种生产。因此,筛选具有良好发酵和生长性能的乳酸菌凝固酶阴性葡萄球菌应用到发酵香肠中,对香肠及肉制品的加工具有重要意义。
发明内容
本发明的目的在于提供一种提升香肠发酵品质的复合发酵剂及应用。
为实现上述目的,本发明提供如下技术方案:
一种提升香肠发酵品质的复合发酵剂,包括植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18;
所述植物乳杆菌YR07保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,菌种保藏编号为CCTCC NO:M 20221303;
所述清酒乳杆菌L.48保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC NO:M20221306;
所述木糖葡萄球菌S.14保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC M 20221305;
所述松鼠葡萄球菌S.18保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC NO:M20221304。
进一步的,所述复合发酵剂为液态菌剂;其中,植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18的有效活菌数的比例为1:1:2-4:2-4。
本发明所述的复合发酵剂可用于制作发酵肉制品。
其中,所述发酵肉制品为发酵香肠。
一种发酵香肠的制备方法,包括下列步骤:
(1)原料肉中加入调味料和辅料,然后进行腌制,得到预发酵肉;
(2)将权利要求1或2所述的复合发酵剂接种到发酵肉中混匀,灌制香肠;
(3)发酵:25-35℃,RH80-90%条件下发酵20-40h;
(4)风干:10-20℃,RH40-50%条件下发酵10-20d。
其中,所述复合发酵剂为液态菌剂;其中,植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18的有效活菌数的比例为1:1:2-4:2-4。
其中,所述步骤(1)中,所述原料肉为新鲜猪后腿瘦肉和猪背膘,将其表面的筋膜和大的筋腱等部位剔除后进行漂洗,将肉搅碎后按照猪后腿瘦肉与背膘的比例为1-4:9-6进行混合得到。
其中,所述步骤(1)中,腌制温度为3-8℃。
其中,所述步骤(1)中,调味料和辅料按照与原料肉的以下质量百分比添加:2.5%食盐,0.7%葡萄糖,0.02%亚硝酸盐,0.015%大蒜粉,0.05%茴香粉,0.05%黑胡椒粉,0.225%香肠香精。
与现有技术相比,本发明的有益效果是:
(1)采用本发明复合发酵剂制备的发酵香肠与传统发酵香肠相比,接种本发明的植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18复合发酵剂可显著快速降低香肠的pH值,抑制其中腐败菌的生长,保证产品的安全性。
(2)采用本发明的植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18复合发酵剂发酵香肠,相较于自然发酵明显提升了发酵香肠人体必需氨基酸的含量,提升了营养价值。
(3)本发明的植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18复合发酵剂发酵香肠,进行感官评定后,更受消费者喜爱。
附图说明
图1为植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14、松鼠葡萄球菌S.18的电泳图。
图2为植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14、松鼠葡萄球菌S.18的系统发育树。
图3为不同组别发酵组别香肠在发酵过程中pH值图。
生物保藏说明
植物乳杆菌YR07、清酒乳杆菌L.48、木糖葡萄球菌S.14、松鼠葡萄球菌S.18保藏于中国典型培养物保藏中心,保藏地址:湖北省武汉市武昌区八一路299号武汉大学校内,保藏机构简称:CCTCC,保藏日期为2022年8月18日,生物保藏编号为CCTCC NO:M20221303、M20221306、M20221305、M20221304。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
以下实施例所述试剂或材料若无说明,均为市售。
实施例1传统发酵肉制品的采集和乳酸菌、凝固酶阴性葡萄球菌的分离纯化筛选
1.乳酸菌、凝固酶阴性葡萄球菌的分离
从全国各地采集得到陇西腊肉、苏式腊肠、恩施火腿、侗族酸肉、建瓯板鸭和贵州腌肉,在无菌条件下称取10.0g样品,加入90mL无菌生理盐水,均质后进行稀释。
取1.0mL样品稀释液倾注至含2%轻质碳酸钙的MRS固体培养基中,37℃培养48h,挑选具有溶钙圈、形态不一的菌落继续在MRS固体培养基划线培养,重复3~4次,直至得到纯菌落。
取0.1mL样品稀释液涂布于MSA固体培养基中,37℃培养24h,挑取菌落形状不一的菌落继续在MSA固体培养基划线培养,重复3~4次,直至得到纯菌落。
2.乳酸菌、凝固酶阴性葡萄球菌的筛选
对纯化菌株进行革兰氏染色镜检、过氧化氢酶试验,筛选出革兰氏染色阳性、过氧化氢酶阴性的菌株。对获得的菌株进行产黏液、溶血试验、葡萄糖产气试验、产H2S试验、精氨酸产氨试验、氨基酸脱羧酶试验;通过牛津杯扩散平板法对所筛选的乳酸菌进行抑菌特性测定。筛选出两株安全的且发酵性能良好的乳酸菌YR07(植物乳杆菌)、L48(清酒乳杆菌)。
表1乳酸菌安全性能和发酵性能表征
对纯化菌株进行革兰氏染色镜检、过氧化氢酶试验,筛选出革兰氏染色阳性、过氧化氢酶阳性的菌株。对获得的菌株进行蛋白、脂肪水解酶实验、亚硝酸盐还原酶实验、产黏液和溶血试验、葡萄糖产气试验、产H2S试验、精氨酸产氨试验、氨基酸脱羧酶试验。筛选出两株安全的且发酵性能良好具有发色功能的葡萄球菌S.14(木糖葡萄球菌)、S.18(松鼠葡萄球菌)。
表2葡萄球菌的安全性能和发酵性能的表征
安全性能 | S.14 | S.18 | 发酵性能 | S.14 | S.18 |
过氧化氢酶活性 | 阴性 | 阴性 | 耐盐性 | 6% | 6% |
产黏性 | 阴性 | 阴性 | 耐亚硝酸盐性 | 150mg/100ml | 150mg/100ml |
产气 | 阴性 | 阴性 | 脂肪水解能力 | +++ | ++++ |
血平板 | 阴性 | 阴性 | 蛋白质水解能力 | + | ++ |
产H2S | 阴性 | 阴性 | 亚硝酸盐还原酶实验 | + | + |
氨基酸脱羧酶活性 | 阴性 | 阴性 | - | - | - |
产氨 | 阴性 | 阴性 | - | - | - |
注:+表示水解能力的大小,越多水解能力越大。
实施例2乳酸菌和凝固酶阴性葡萄球菌的鉴定
使用细菌基因组DNA提取试剂盒对乳酸菌的基因进行提取,以27F(5'-AGAGTTTGATCCTGGCTCAG-3')和1492R(5'-TACGGYTACCTT-GTTACGACTT-3')上下引物(SEQ IDNO:1-2所示)进行PCR扩增提取的16S rDNA序列,PCR反应条件为95℃初始变性20min,34个循环(95℃ 30s,56℃30s,70℃ 65s)和72℃ 10min最终链延伸。获得的PCR产物进行琼脂糖凝胶电泳,在凝胶成像系统下观察电泳结果,结果如图1所示。
将测序结果在美国国家生物技术信息中心(national center of biotechnologyinformation,NCBI)的GenBank数据中进行局部序列比对基本检索工具(basic localalignment search tool,BLAST)比对搜索,选取同源性较高的模式菌株的16S rDNA序列。构建系统发育树,结果为如图2所示,菌株YR07被鉴定为Lactobacillus plantarum,即植物乳杆菌,菌株L.48被鉴定为Latilactobacillus sakei,即清酒乳杆菌,菌株S.14被鉴定为Staphylococcus xylosus,即木糖葡萄球菌,菌株S.18被鉴定为Mammaliicoccus sciuri,即松鼠葡糖球菌。
实施例3发酵香肠的制备
1.复合发酵剂的制备
将乳酸菌YR07、L48活化后取1%接种量接种至MRS液体培养基中,培养12h,8000×g,4℃离心5min,收集沉淀,用生理盐水洗涤3次。将葡萄球菌S.14、S.18活化后取1%接种至MSA液体培养基培养12h,8000×g,4℃离心5min,收集沉淀,用生理盐水洗涤3次。按1:1:2:2配比将最终菌体浓度调整为1×107CFU/g,体积为1ml,待用。
2.发酵香肠的制备
猪后腿肉按肥瘦比为3:7搅碎混匀,辅料添加比例为:2.5%食盐,0.7%葡萄糖,0.02%亚硝酸盐,0.015%大蒜粉,0.05%茴香粉,0.05%黑胡椒粉,0.225%香肠香精,1mL菌悬液。灌肠时保证肠体饱满、无气泡。随后悬挂于恒温恒湿箱内,30℃,相对湿度85%发酵24h,随后15℃、相对湿度45%风干15天得到成品。试验分组为空白对照组(CK组)、商业发酵组(CS组)接种复合发酵剂组(CH组)。
实施例4发酵香肠指标的测定
1、发酵香肠的pH值测定:
将实施例3中的CK组、CS组和CH组香肠分别在灌肠后、发酵1d、风干1d、风干5d、风干10d和风干15d测定pH值。准确称取10.0g样品,切碎,加入100mL蒸馏水与样品充分混匀,8000×g进行匀浆30s,过滤后取上清液,用pH计测量,重复三次取平均值。
结果如图3所示,发酵前三组的pH相近,发酵1d后,CS和CH组的pH已经降到5.0以下,风干1d时降到最低点,在4.6~4.7之间,之后有缓慢回升,CK组的pH始终在5.0以上,而添加商业发酵剂和复合发酵剂的pH下降的更快,说明添加发酵剂具有快速产酸的能力,缩短香肠的发酵时间,抑制杂菌生长。
3、发酵香肠中氨基酸含量的测定
根据国家标准《GB5009.124-2016-氨基酸的测定》的方法来进行测定,稍作修改。称取适量发酵香肠切碎后冻干,精确称取冻干后的0.1g样品,加入4mL 4%磺基水杨酸溶液,超声浸提30min后,静置10min。待分层后取上清液1.5mL,12 000×g、4℃离心30min,取1mL上清液,用0.22μm水相滤膜过滤,通过全自动氨基酸分析仪进行游离氨基酸测定。
通过表3可以得知,通过本发明中的实施例3中制备的复合发酵剂发酵所得香肠的氨基酸总含量最高,且其中人体8种必需氨基酸含量也较自然发酵和商业发酵组高。
表3香肠中氨基酸含量的测定
注:ND表示未检测到相应化合物。
4、发酵香肠中挥发性化合物含量的测定
将实施例3中的CK组、CS组和CH组香肠成品进行挥发性化合物含量的测定。采用顶空固相微萃取-气质联用的方法分析发酵香肠中的挥发性风味物质。将切碎的香肠样品(2.0g)放入20mL顶空样品瓶中,加入10μL的2,4,6三甲基吡啶(浓度为0.1g/L)作为内标物。将顶空瓶于60℃的温度下平衡5min后,插入事先老化的75μm复合式CAR/PDMS萃取针吸附30min,然后立即将萃取头插入气质进样口,解析5min。
挥发性化合物的测定使用气相色谱-质谱(GC-MS)系统(5977B,安捷伦),气相色谱条件:DB-WAX(30m×0.25mm×0.25μm);进样口温度为250℃;不分流模式进样;以氦气为载体,流速为1mL/min;程序升温:柱初温40℃,保持5min;再以2℃/min升温至90℃,无停留;再以5℃/min升至180℃,无停留,再以10℃/min升至230℃,保留8min。质谱条件:离子源温度230℃;质量扫描范围30~550m/z。
挥发性化合物的鉴定是通过对比NIST 20质谱库,取相似度大于80%作为鉴定结果。挥发性化合物的定量采用内标法,以溶于正己烷的2,4,6-三甲基吡啶溶液作为内标物质,定量结果以μg/kg表示,关键挥发性物质评价方法参考:(刘登勇,周光宏,徐幸莲.确定食品关键风味化合物的一种新方法:“ROAV”法[J].食品科学,2008,344(7):370-374.),采用相对气味活度值(Relative Odor Activity Value,ROAV)分析法确定样品的关键挥发性物质。
结果如表4所示,通过相对气味活度值确定了香肠中关键性挥发性物质共有25种,其中CH组有高含量关键性挥发性物质,含量为216.28μg/kg;3-羟基-2-丁酮是一种特殊的风味化合物,呈令人愉悦的奶油味,在CH组中具有最高的含量。与对照组和商业发酵组相比,接种复合发酵剂能够显著增加发酵香肠中特征性风味物质的含量,因此,复合发酵剂的接种对发酵香肠的风味具有提升作用。
表4接种不同发酵剂组合的发酵香肠特征性挥发性风味物质
5、发酵香肠的感官评定
评定小组由10名经过筛选的食品专业研究生组成,男女各半。香肠煮制20min,切成20mm的厚度。感官评定采用双盲法进行,要求评定成员之间不能相互交流,评定不同样品之前用清水漱口。计分采用10分制,对样品的外观、组织状态、色泽、风味和总体可接受性5个方面进行评定,评定标准如表5所示。
由表6可知,接种本发明中的复合发酵剂改善了香肠的外观完整性、提高了发酵香肠的风味,改善了发酵香肠的质地,提高了发酵香肠的总体可接受性。
表5香肠感官评定标准
表6不同处理组对发酵香肠感官的影响
项目 | CK | CS | CH |
外观 | 7.36±0.53b | 7.65±0.56ab | 8.09±0.49a |
色泽 | 6.68±0.36c | 7.13±0.40b | 8.19±0.30a |
质地 | 6.39±0.27c | 6.92±0.44b | 8.30±0.27a |
风味 | 6.32±0.18c | 7.13±0.32b | 8.37±0.30a |
总体可接受度 | 6.64±0.25c | 7.06±0.20b | 8.36±0.27a |
注:同行中不同小写字母表示样品差异显著(P<0.05)。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.一种提升香肠发酵品质的复合发酵剂,其特征在于:包括植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18;
所述植物乳杆菌YR07保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,菌种保藏编号为CCTCC NO:M 20221303;
所述清酒乳杆菌L.48保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC NO:M20221306;
所述木糖葡萄球菌S.14保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC M 20221305;
所述松鼠葡萄球菌S.18保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2022年8月18日,保藏编号为CCTCC NO:M20221304。
2.根据权利要求1所述的复合发酵剂,其特征在于:所述复合发酵剂为液态菌剂;其中,植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18的有效活菌数的比例为1:1:2-4:2-4。
3.权利要求1或2所述的复合发酵剂在制作发酵肉制品中的应用。
4.根据权利要求3所述复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述发酵肉制品为发酵香肠。
5.根据权利要求4所述复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述发酵香肠的制备方法包括下列步骤:
(1)原料肉中加入调味料和辅料,然后进行腌制,得到预发酵肉;
(2)将权利要求1或2所述的复合发酵剂接种到发酵肉中混匀,灌制香肠;
(3)发酵:25-35℃,RH80-90%条件下发酵20-40h;
(4)风干:10-20℃,RH40-50%条件下发酵10-20d。
6.根据权利要求5所述的复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述复合发酵剂为液态菌剂;其中,植物乳杆菌YR07、清酒乳杆菌L48、木糖葡萄球菌S.14和松鼠葡萄球菌S.18的有效活菌数的比例为1:1:2-4:2-4。
7.根据权利要求6所述的复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述步骤(1)中,所述原料肉为新鲜猪后腿瘦肉和猪背膘,将其表面的筋膜、筋腱部位剔除后进行漂洗,将肉搅碎后按照猪后腿瘦肉与背膘的比例为1-4:9-6进行混合得到。
8.根据权利要求7所述的复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述步骤(1)中,腌制温度为3-8℃。
9.根据权利要求8所述的复合发酵剂在制作发酵肉制品中的应用,其特征在于:所述步骤(1)中,调味料和辅料按照与原料肉的以下质量百分比添加:2.5%食盐,0.7%葡萄糖,0.02%亚硝酸盐,0.015%大蒜粉,0.05%茴香粉,0.05%黑胡椒粉,0.225%香肠香精。
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