CN114214439B - 一种酸奶后酸化生物标志物及其对后酸化抑制措施的筛选 - Google Patents
一种酸奶后酸化生物标志物及其对后酸化抑制措施的筛选 Download PDFInfo
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Abstract
本发明公开了一种酸奶后酸化生物标志物及其对后酸化抑制措施的筛选。所述标志物为LDB_RS00370基因及其表达产物。与传统通过测定酸奶贮藏期间的酸度值变化来评估后酸化程度的方法相比,本发明的后酸化生物标志物基因能够快速准确的对菌株后酸化性能进行预测,省时高效,同时还具备传统方法所不具备的预测性优势,利用本发明的生物标志物基因还可以筛选出更多潜在的后酸化抑制措施。
Description
技术领域
本发明属于酸奶后酸化抑制技术领域,具体涉及一种酸奶后酸化生物标志物及其对后酸化抑制措施的筛选。
背景技术
酸奶后酸化是指酸奶发酵结束后,特别是在贮藏过程中,耐酸性较强的保加利亚乳杆菌不断产酸导致酸奶口感劣变的现象。针对这一问题,许多学者提出了不同的措施来抑制后酸化的发生。例如改变发酵剂中球菌与杆菌的比例(Torriani et al,1996,International Dairy Journal 6,625-636);添加葡萄糖氧化酶(Cruz et al,2013,FoodResearch International 51,723-728)以及过氧化物酶等(Nakada et al,1996,International Dairy Journal 6,33-42)。评价这些措施对后酸化抑制程度的好坏往往需要测定酸奶发酵结束后在整个贮藏过程中的pH或酸度变化。寻求能够快速准确反映酸奶后酸化程度的生物标志物因此显得十分必要。发明人前期体外模拟了酸奶后酸化的发生过程,通过转录组测序结合荧光定量PCR技术揭示了参与后酸化的候选基因。这些基因与后酸化的关系颇为密切,发明人曾在这些基因中发现了一个后酸化特异基因。对剩余基因进行挖掘,寻求基因表达量与后酸化程度有显著相关性的基因作为生物标志物,利用该标志物建立一套汇集弱后酸化菌株筛选、后酸化抑制措施评定的方法显得尤为重要。
当下对菌株后酸化性能以及后酸化抑制措施效果的评定主要依据测定酸奶在整个贮藏过程中的酸度或者pH变化。采用这种传统方法一方面工作量大,繁琐耗时,无法在短期内实现对酸奶后酸化程度的评估;另一方面,传统方法无法预测酸奶后酸化程度变化趋势。目前,从基因角度入手,利用分子生物学手段,筛选能够快速准确反映酸奶后酸化程度的基因作为生物标志物尚未有报道。
发明内容
本发明的目的在于提供一种酸奶后酸化生物标志物及其应用。
一种酸奶后酸化生物标志物,所述标志物为LDB_RS00370基因及其表达产物;其核苷酸序列如序列表SEQ ID NO:1所示,其编码蛋白的氨基酸序列如序列表SEQ ID NO:2所示。
所述LDB_RS00370基因的编码蛋白还包括序列表SEQ ID NO:2所示的氨基酸序列经替换、缺失、或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列,以及编码这些氨基酸序列的核苷酸序列。
LDB_RS00370基因共有858个碱基,可编码具有285个氨基酸的蛋白质。该蛋白质的分子式为C1389H2161N379O421S4,理论等电点5.27,由20种常见的氨基酸组成,其中氨基酸含量最高的是丙氨酸(Ala)12.6%,其次是亮氨酸(Leu)10.2%,甘氨酸(Gly)8.4%,含量最少的是半胱氨酸(Cys)0.4%。不稳定指数为23.48,表明目的蛋白为稳定蛋白。LDB_RS00370平均疏水性为-0.234,表明该蛋白为亲水蛋白。NCBI对该基因的描述为醛/酮还原酶。
所述表达产物为转录或者转录后翻译产物。
所述LDB_RS00370基因的转录或者转录后翻译产物表达量下调抑制酸奶后酸化。
所述酸奶后酸化的发酵菌为保加利亚乳杆菌。
一种抑制酸奶后酸化的方法,抑制酸奶发酵后LDB_RS00370基因的表达。
所述抑制LDB_RS00370基因的表达的方法为加入烟酸或山梨醇或干酪素或β-环状糊精或烟酸与干酪素以及β-环状糊精三种措施的复配。
本发明利用五种后酸化性能不同的保加利亚乳杆菌(Lb-S1,LD,DR,11842,S-1)分别进行酸奶发酵,待酸奶凝乳后置于4℃条件下低温贮藏,2小时后分别取样用于RNA提取和cDNA合成。剩余酸奶继续置于4℃条件下贮藏,每隔3.5天测定酸奶的滴定酸度,测定酸奶在21天低温贮藏过程中的酸度变化。以cDNA为模板,rpob作为内参基因,发明人从前期实验获取的69个后酸化候选基因中选取了14个基因进行相对表达量测定。利用相关性分析,探究了14个基因的表达量分别与3.5天、7天、10.5天、14天、17.5天以及21天的酸度值之差的关系并获得相应的回归公式。利用分离自新疆酸奶中的一株保加利亚乳杆菌(R2-6)对筛选出的呈现显著相关性的基因进行验证。按照上述同样的方法,将R2-6接种到脱脂乳中进行酸奶发酵,凝乳后,4℃贮藏2小时后取样,提取RNA进行反转录合成cDNA。以cDNA为模板,按照上述同样的方法测定目的基因的表达量。将测得的表达量带入相应的回归公式中计算出理论酸度值差值,并与发酵终点的酸度值相加得到理论酸度值。剩余酸奶同样继续放在4℃条件下贮藏并且每隔3.5天测定酸奶在21天贮藏期内的滴定酸度。利用T检验对理论酸度值与实际测量酸度值进行显著性差异分析。对于差异不显著的基因,说明可以根据该基因的表达量预测出菌株发酵过程中的产酸性能,这样的基因正是发明人要筛选的后酸化生物标志物基因。
进一步地,发明人用不同的措施来处理保加利亚乳杆菌,通过测定生物标志物基因的相对表达量来筛选抑制酸奶后酸化的措施。
本发明的有益效果:与传统通过测定酸奶贮藏期间的酸度值变化来评估后酸化程度的方法相比,本发明的后酸化生物标志物基因能够快速准确的对菌株后酸化性能进行预测,省时高效,同时还具备传统方法所不具备的预测性优势。另外,利用本发明的生物标志物基因还可以筛选出更多潜在的后酸化抑制措施。
附图说明
图1为生物标志物基因LDB_RS00370基因表达量与酸度值之差相关性分析。
图2不同处理措施对LDB_RS00370基因表达量以及酸奶贮藏过程中理化性质的影响。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1酸奶发酵和贮藏期间酸度测定
将培养至对数中期的五株不同保加利亚乳杆菌(Lb-S1,LD,DR,11842,S-1)分别按照1%的接种量接种至10mL脱脂乳培养基中,42℃静置培养,以此接种量在脱脂乳培养基中进行传代培养,直至凝乳时间稳定(5代左右)。参照GB5009.239-2016方法分别测定酸奶在发酵过程中的酸度变化,记录发酵终点的酸度值。凝乳后,将酸奶置于4℃冰箱贮藏。贮藏2小时后,每组样品分别吸取2mL酸奶于无RNase的EP管中,-80℃保存,用于后续RNA的提取。剩余酸奶样品继续置于4℃条件下贮藏并分别在贮藏后的第3.5天、7天、10.5天、14天、17.5天以及第21天测定酸奶的滴定酸度并分别计算在这些天数下酸度值之差。
酸度值之差=当天测定的酸度值-发酵终点酸度值
实施例2 RNA提取、cDNA合成以及基因相对表达量的测定
将低温保存的酸奶样品取出分别置于液氮预冷的研钵中。加入液氮,将样品磨成粉末状后转移至无RNase的EP管中。采用Trizol法提取细胞总RNA,同时检测样品RNA的A280/A260值来评估RNA质量。进一步地,对质量达到要求的RNA采用北京全式金生物One-Step gDNA Removal and cDNA Synthesis SuperMix合成cDNA。以cDNA为模板,使用北京全式金生物/>Green qPCR SuperMix对从前期筛选的后酸化候选基因中选取的14个目的基因进行相对表达量的测定。反应在Roche公司生产的Light Cycler96仪器上进行,使用rpob作为内参基因。依据NCBI上公布的保加利亚乳杆菌ATCC 11842基因组序列,采用DNAMAN 6.0软件设计目的基因和内参基因的上下游引物。内参基因和生物标志物基因上下游引物见表1。反应体系为:/>Green qPCR SuperMix 10μL、上下游引物各1μL、模板2μL、Nuclease-free Water 6μL。PCR反应程序如下:94℃预变性300S,94℃变性5S,51℃退火15S,72℃延伸10S,共45个循环。反应结束后,采用2-ΔΔCt法计算目的基因的相对表达量。
表1.生物标志物基因和内参基因上下游引物
实施例3后酸化生物标志物基因的筛选与验证
采用Pearson相关性分析法对目的基因相对表达量与酸度值之差进行相关性分析,结果发现这14个基因中有4个基因的表达量分别在第14天、第17.5天、第3.5天以及第10.5天与酸度值之差存在显著相关性。生物标志物基因LDB_RS00370的基因表达量与酸度值之差关系见附图1。进一步地,利用一株从新疆酸奶中分离出来的保加利亚乳杆菌(R2-6)进行发酵酸奶来验证这几个基因的可靠性。同样,分别在凝乳后的第3.5天、7天、10.5天、14天、17.5天以及第21天测定酸奶的滴定酸度并分别计算在这些天数下酸度值之差。按照上述方法与步骤进行RNA提取、cDNA合成以及qPCR反应。将测得的基因表达量分别代入对应的回归方程获得理论酸度值差值。将理论酸度值差值与发酵终点酸度值相加得到理论酸度值。使用SPSS 19.0软件对理论酸度值与实际酸度值进行独立样本T检验,结果发现只有LDB_RS00370基因预测结果与实际结果差异不显著(表2),表明LDB_RS00370基因可以作为预测酸奶后酸化程度的生物标志物。表2.基于生物标志物基因LDB_RS00370基因表达量预测的酸度值与实际测量酸度值差异显著性分析
注:*表示显著性水平0.05下呈现差异,**表示显著性水平0.01下呈现差异
实施例4LDB_RS00370基因作为酸奶后酸化生物标志物对菌株后酸化性能的判定
通过上述的筛选与验证,最终确定LDB_RS00370基因是一个可靠的后酸化标志物基因。分析显示,该基因在第10.5天的时候基因表达量与酸度值之差呈现出显著正相关。其中,回归方程为Y=0.004538X+26.63,R2=0.8002。LDB_RS00370基因在五株保加利亚乳杆菌(Lb-S1,LD,DR,11842,S-1)中的平均相对表达量分别为614.7、1129.63、5.64、138.09以及3980.08。代入回归方程,计算得出这五株菌在贮藏10.5天中的酸度值差值排名:S-1>LD>Lb-S1>11842>DR。通过测定这五株菌21天贮藏期间产酸变化,发现这五株菌的后酸化性能排名仍然是S-1>LD>Lb-S1>11842>DR。这说明通过LDB_RS00370的基因表达量来预测菌株的后酸化性能的方法是可靠地。
实施例5 LDB_RS00370基因作为酸奶后酸化生物标志物对后酸化抑制措施的筛选
酸奶后酸化生物标志物基因LDB_RS00370的基因表达量与菌株后酸化性能呈显著正相关。按照这一标准,发明人推测使LDB_RS00370表达量下调的措施很有可能具有抑制酸奶后酸化的潜力。将过夜活化好的保加利亚乳杆菌ATCC11842按照1%的接种量接种到100mL MRS培养基中,42℃培养至对数中期(大约9h)后分装至4个50mL离心管中,每管5mL。分别向管中加入终浓度为100mg/kg的烟酸、4mg/kg的Mg2+(MgSO4制得)以及1g/kg的丝氨酸对保加利亚乳杆菌ATCC 11842进行室温处理40min,其中设置一组未添加组作为对照组(CK)。40min后,分别取1mL菌液,使用Hettich公司UNIVERSAL 320R离心机在室温条件下离心收集菌体(12000r.p.m,2min)。按照上述方法对收集的菌体进行RNA提取、cDNA合成以及相对表达量的测定。将培养至对数中期的保加利亚乳杆菌ATCCC 11842按照1%的接种量接种至10mL脱脂乳培养基中,42℃静置培养,按照这一接种量反复传代直至凝乳时间稳定。凝乳后,分别向酸奶中加入终浓度为100mg/kg的烟酸、4mg/kg的Mg2+(MgSO4制得)以及1g/kg的丝氨酸,其中未添加组作为对照组(CK),然后置于4℃贮藏。按照GB5009.239-2016和GB4789.2-2016分别每隔3.5天对各组酸奶在贮藏21天期间的酸度值、菌落总数以及pH值进行测定。结果显示,烟酸处理组的基因表达量较对照组有所下降;Mg2+处理组和丝氨酸处理组的基因表达量与对照组相比呈现上调变化。从产酸变化来看,酸度变化最大的是丝氨酸处理组,其次是Mg2+处理组;酸度变化最小的是烟酸处理组。各组pH值的变化趋势也和产酸变化趋势一致,同样是丝氨酸处理组酸度值变化较大,烟酸处理组酸度变化最小。这说明使LDB_RS00370基因表达量下调的措施更有利于抑制酸奶后酸化且表达量越低,抑制效果越好。另外,从菌落总数变化情况来看,各组菌落数在贮藏期间差异并不是很明显,后期的时候烟酸处理组的活菌数要高于对照组。这说明这一措施并不会抑制菌株的正常生长,添加烟酸可以作为抑制酸奶后酸化的良好措施(图2)。另外,使LDB_RS00370基因表达量下调的措施还包括添加山梨醇、干酪素、β-环状糊精以及烟酸、干酪素与β-环状糊精三种措施的复配组合等,它们均能使LDB_RS00370的基因表达量下调同时具备抑制后酸化的效果。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 河北农业大学
<120> 一种酸奶后酸化生物标志物及其对后酸化抑制措施的筛选
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Claims (1)
1.一种抑制保加利亚乳杆菌ATCC 11842发酵得到的酸奶后酸化的方法,其特征在于,所述方法为在酸奶发酵结束后添加烟酸下调保加利亚乳杆菌ATCC 11842后酸化基因LDB_ RS00370的表达,所述烟酸添加浓度为100 mg/kg。
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