CN110016443B - 一种罗伊氏乳杆菌及其在富硒鸡蛋生产中的应用 - Google Patents
一种罗伊氏乳杆菌及其在富硒鸡蛋生产中的应用 Download PDFInfo
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Abstract
本发明公开了一种罗伊氏乳杆菌及其在富硒鸡蛋生产中的应用,其特征在生物保藏信息为CGMCC No.16721。本发明是一种安全可靠、高产量、低成本的富硒鸡蛋生产途径。通过本发明获得的富硒鸡蛋不会对蛋鸡和人体产生毒副作用,还能提高蛋鸡的抵抗力、免疫力及生产性能,提高经济效益。
Description
技术领域
本发明涉及一种乳酸菌,具体涉及一种罗伊氏乳杆菌及其在富硒鸡蛋生产中的应用。
背景技术
硒是人体必需的微量元素之一,与机体正常生理功能的维持和疾病的发生发展有密切的关系。适量的硒可以清除体内过多的活性氧自由基,保护细胞膜的结构和功能免受过度氧化损伤,从而延缓人体衰老,预防肿瘤、心血管疾病等。此外,硒能刺激人体免疫球蛋白和抗体的产生,增强人体免疫力。人体缺硒可引起克山病和大骨节病等地方性疾病的发生,还会增加癌症、心血管疾病、白内障和男性不育等疾病的发病率,但过度摄入硒也会产生中毒症状,故对硒的补充利用需谨慎。2000年,中国营养学会推荐成人膳食摄入量(RNI)为50μg/d,可耐受最高摄入量(UL)为400μg/d。
生物体内的硒大多源于蔬菜、水果和肉类等食物。天然食物含硒量低,而且我国地区性缺硒的情况普遍存在,因此人体适量补硒是十分必要的,通过食品途径强化硒元素是现在功能性食品研究热点之一。由于无机硒(如亚硒酸钠)毒性强,吸收率低,不适合直接添加到食品中,有机硒被视为是人工补硒最有效最安全的手段。通过生物学的方法将无机硒转变为有机硒是生产富硒食品和添加剂的最好方法。
目前,有机硒的获得运用最多的方法是微生物转化法。微生物转化法运用较多的是培养富硒酵母,所用的菌种一般是啤酒酵母。随着生物化学技术的不断发展,乳酸菌成为有机硒的一个新型来源。乳酸菌是一种可以调节机体内异常菌群,控制毒素扩散的益生菌,是目前发酵食品生产中普遍使用的菌种,其发酵食品风味独特,营养丰富,安全可靠,具有众多保健功能。1995年,Calomme等首先揭示了乳酸菌具有富集转化硒的能力。利用乳酸菌富集合成有机硒,开发富硒食品具有重要的研究意义和广阔的开发前景。
鸡蛋富含蛋白质、脂肪、卵磷脂、微量元素和各种维生素等,营养价值高,是我国重要的农产品,也是居民常食用的食品之一。虽然现有的鸡蛋营养价值高,但是硒元素的含量并不高。现有技术中,一般都采用饲料中拌入亚硒酸钠喂养蛋鸡生产高硒鸡蛋,但亚硒酸钠是无机硒,毒性强,家禽耐受程度低,容易引起母鸡中毒,造成产蛋下降,母鸡死亡,存在很大的隐患。因此,通过单纯饲喂硒元素获得富硒鸡蛋安全性得不到保障。因此,发明一种全新的富硒鸡蛋生产工艺具有重要意义,近年来,代替亚硒酸钠的硒源逐渐出现,如酵母硒。
发明内容
针对上述问题,本发明提供了一种罗伊氏乳杆菌及其在富硒鸡蛋生产中的应用,本发明的罗伊氏乳杆菌具有较好的富硒能力,并且本发明的罗伊氏乳杆菌可应用于富硒鸡蛋的生产,是一种安全可靠、高产量、低成本的富硒鸡蛋生产途径。
本发明通过下述技术方案实现。
一方面,本发明提供了一种罗伊氏乳杆菌,其特征在于其生物保藏信息为CGMCCNo.16721;
该菌株于2018年11月8日保藏于为中国微生物菌种保藏管理委员会普通微生物中心,保藏单位地址为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,该菌株的分类学命名为:罗伊氏乳杆菌(Lactobacillus reuteri),株名为zym-YiAn-1;该菌株单菌落呈圆形,中等大小,凸起,边缘齐整,微白色,具有较强富硒能力。
作为具体技术方案,该菌株通过如下方法获得:
(1)取猪场粪污堆肥用无菌水适当稀释,取一定量涂布于固态筛选培养基上,于37℃倒置培养72h,待长出菌落后挑取单菌落进行转接,经平板划线法得到单菌落;
(2)将步骤(1)筛选出的单菌种分别接入装有5mL发酵培养基的试管,于37℃过夜培养24h;
(3)将步骤(2)过夜培养获得的试管菌液涂布于不同Na2SeO3含量的平板培养基上,于37℃倒置培养48h,观察平板菌落颜色变化,选出Na2SeO3抗性强的菌株;
(4)菌株16S rDNA分析
将步骤(3)获得的Na2SeO3抗性强的菌株利用细菌基因组DNA提取试剂盒提取菌株的DNA;将DNA送检测序,通过BLAST方法比对,鉴定筛选出本发明所得的罗伊氏乳杆菌;
作为优选技术方案,该罗伊氏乳杆菌的最大耐硒量为55μg/mL;
一方面,本发明提供了一种富硒罗伊氏乳杆菌,其特征在于通过下述方法获得:
采用前述的罗伊氏乳杆菌,选用MRS液体培养基,在接种量10%、Na2SeO3添加量58μg/mL、pH 6.2、温度37℃的条件下培养36h进行富硒,之后将含富硒罗伊氏乳的培养基进行离心,收集沉淀进行干燥,即得富硒罗伊氏乳杆菌;
作为具体技术方案,所述MRS液体培养基的配方为蛋白胨10.0g、牛肉膏10.0g、酵母提取物5.0g、葡萄糖20.0g、吐温-80 1.0mL、K2HPO4·3H2O 1.52g、醋酸钠5.0g、柠檬酸三铵2.0g、MgSO4·7H2O 0.58g、MnSO4·4H2O 0.25g、蒸馏水1000mL。
另一方面,本发明还提供了上述富硒罗伊氏乳杆菌在富硒鸡蛋生产中的应用;在蛋鸡的饲料中添加一定剂量的上述硒罗伊氏乳杆菌制剂能够提高蛋鸡的生产性能,并改善鸡蛋的品质,提高鸡蛋蛋白质和硒元素含量。
作为具体技术方案,上述富硒罗伊氏乳杆菌在富硒鸡蛋生产中的应用,其特征在于应用方法为:
(1)制备复合微生态制剂:以复合微生物菌粉、低聚异麦芽糖、菜籽饼粉和食用香精为原料制备复合微生态制剂;所述复合微生物菌粉由权利要求4所述的富硒罗伊氏乳杆菌和芽孢杆菌组成;
(2)蛋鸡的养殖:选取健康的蛋鸡,预试周只饲喂基础日粮,之后在基础日粮中添加复合微生态制剂进行养殖。
作为优选技术方案,所述复合微生态制剂由以下质量百分比的原料制备而成:复合微生物菌粉20%~30%,低聚异麦芽糖1.0%~5.0%,菜籽饼粉65%~80%,食用香精0.1%~0.2%。
本发明具有以下有益技术效果:
本发明在基础日粮中添加了复合微生物制剂即一定剂量的乳酸杆菌制剂,利用原生态益生菌作为蛋鸡饲料的重要添加成分。具有富硒转化功能的罗伊氏乳杆菌可以在生物体内将无机硒转变为有机硒,供蛋鸡更好地吸收利用,提高了蛋鸡对微量元素硒的摄取,也增加了鸡蛋中的硒元素及蛋白质含量,同时脂肪、胆固醇和有害细菌的含量得到了控制。由于蛋鸡吸收利用的是由富硒乳酸菌转化而来的有机硒,通过本发明获得富硒鸡蛋不会对蛋鸡和人体产生毒副作用,还能提高蛋鸡的抵抗力、免疫力及生产性能,提高经济效益。此外,该方法得到的富硒鸡蛋质量可靠,营养丰富,极大的改善了鸡蛋的品质和风味。
附图说明
图1为硒标准曲线见图。
具体实施方式
本发明的以下实施方式仅用来说明实现本发明的具体实施方式,这些实施方式不能理解为是对本发明的限制。其他的任何在未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均视为等效的置换方式,落在本发明的保护范围之内。
实施例1
本发明菌株16S rDNA分析结果
采用细菌基因组DNA提取试剂盒(离心柱型)试剂盒提取菌株的DNA;向无菌EP管中加入9.5μL超纯水、12.5μL 2×PCR Master Mix、上下游引物各1μL、DNA模板1μL,瞬间离心后置于荧光定量PCR仪中,于如下PCR扩增条件中扩增:94℃预变性5min,一次循环;94℃变性1min,65℃复性1min,72℃延伸20s,经30个循环后,72℃继续延伸10min,一次循环;扩增后的DNA在1%琼脂糖凝胶板上于80V电泳50min,再凝胶成像;将DNA送检测序,通过BLAST方法比对,鉴定出所得菌株,具体结果见表1;
表1菌株zym-YiAn-1的BLAST同源性比较结果
由系统发育树和对比结果(表1)可知,被测菌株zym-YiAn-1与Lactobacillusreuteri CICC 6226的匹配一致性达到99%,同时结合生化鉴定结果来看,可以确定被测菌株zym-YiAn-1即为一株罗伊氏乳杆菌。
实施例2
本发明罗伊氏乳杆菌菌株的耐硒性能测试
将预先灭菌的Na2SeO3浓溶液,制成含Na2SeO3不同浓度梯度的筛选培养基平板,平板中Na2SeO3的含量分别为10μg/mL,20μg/mL,30μg/mL,40μg/mL,50μg/mL,60μg/mL,70μg/mL,80μg/mL,90μg/mL,100μg/mL;将本发明的罗伊氏乳杆菌涂布于上述平板培养基上,37℃倒置培养48h,观察平板菌落颜色变化,确定菌株耐硒大致浓度区间,再在浓度区间内划分更细的Na2SeO3浓度,重复上述筛选过程,确定菌株耐硒浓度,具体检测结果见表2;
表2 Na2SeO3平板中菌株的耐硒性能检测结果(单位:μg/mL)
注:“+”代表有红色单质硒出现,“-”代表无红色单质硒出现。
由表2可知,当发酵培养基中Na2SeO3浓度为50μg/mL,55μg/mL,60μg/mL时,该株细菌都可以正常生长,且液体培养24h后高速离心不出现红色菌体,即说明该株细菌的耐硒浓度大约在50~60μg/mL之间;后经过梯度实验,表明当培养基平板加入Na2SeO3 56μg/mL时,菌体出现红色,说明菌体把一部分无机硒还原成单质硒,而不是转化成有机硒,故Na2SeO3平板上菌种最大的耐硒量为55μg/mL。
实施例3
本发明罗伊氏乳杆菌的富硒条件的确定
1、硒含量标准曲线的绘制
硒含量的测定选用的是3,3’-二氨基联苯胺比色法,具体如下;取浓度1μg/mL的亚硒酸钠标准液0mL,2mL,4mL,6mL,8mL和10mL,分别移入125mL分液漏斗,加水至35mL,加入5%的DETA-2Na溶液1mL,摇匀后调节pH值2~3,各加0.5%3,3’-二氨基联苯胺溶液4mL,摇匀,置于暗处半小时;调节pH值至中性,加入10mL甲苯振荡2min,静置分层,弃水层,甲苯层通过棉花栓过滤于比色皿中,置于420nm波长处测定吸光度,并绘制标准曲线,硒标准曲线见图1;
2、残留无机硒含量的测定
发酵液以6000rpm离心20min,取20mL上清液加水至35mL,加入2mL5%的DETA-2Na溶液,以下同标准曲线;根据样品测得的吸光度,从标准曲线中查得相应的硒含量,此为残留无机硒含量;
有机硒含量的计算:有机硒含量=总硒含量-残留无机硒含量;
富硒能力(有机硒转化率)的计算:有机硒转化率=有机硒含量/总硒含量×100%;
3、不同条件下菌株富硒能力测定
由菌株耐硒性能确定其液体发酵时Na2SeO3的大致添加量,配制Na2SeO3浓度为50μg/mL,52μg/mL,54μg/mL,56μg/mL,58μg/mL,60μg/mL的发酵培养基,100mL液体培养基装于250mL三角瓶中,10%接种量,37℃,200rpm条件下培养,每隔2h测pH值的变化,每隔12h取10mL发酵液以6000rpm离心15min,观察菌体颜色变化,并测定发酵液含硒量,具体结果见表3;
表3不同条件下菌株富硒能力
根据上述结果初步得出菌株zym-YiAn-1选用MRS液体培养基富硒时,接种量10%,Na2SeO3的添加量58μg/mL,pH 6.2,37℃培养36h,富硒效果较好,残留无机硒含量24μg/mL,有机硒含量34μg/mL,富硒能力为58.62%。
实施例4
本发明的微生态制剂作为饲料添加剂对鸡蛋硒含量和营养价值的影响试验。
试验选取健康海兰/罗曼,刚开产的蛋鸡800只,随机分为2组(一组为对照组,另一组试验组),每组含有8个重复,每个重复50只鸡。对照组只饲喂基础日粮,试验组在基础日粮中添加4kg/t微生物制剂。各组自由采食和饮水,用药和防疫按鸡场常规程序进行。试验组在饲喂基础日粮预试周后,开始饲喂相应的试验日粮,试验期8周。试验期末,每个重复随机选取10枚鸡蛋,测定鸡蛋蛋白质、脂肪、胆固醇和硒元素的含量,结果见表4.
表4微生态制剂作为添加剂对鸡蛋营养价值和硒元素含量的影响
检测项目 | 对照组 | 试验组 | 变化情况 |
蛋白质 | 11.8g/100g | 14.3g/100g | +21.2% |
脂肪 | 15g/100g | 7.7g/100g | -48.7% |
胆固醇 | 680mg/100g | 432mg/100g | -36.5% |
硒 | 50ug/kg | 146ug/kg | +192% |
本试验结果表明,在蛋鸡基础日粮中添加一定剂量的富硒罗伊氏乳杆菌制剂,可以增强蛋鸡对营养物质的吸收。对营养物质吸收的增强可以从试验组鸡蛋蛋白质含量和硒含量中看出来,养殖户在没有额外添加硒元素的前提下,试验组硒元素的含量比对照组提高了近两倍,蛋白质含量则增加了21.2%,同时罗伊氏乳杆菌作为有益菌,能产生的大量生长因子,促进动物对营养物质的吸收利用,并转化为蛋白质,减少脂肪的沉积和胆固醇的产生。此外,试验组与对照组相比,脂肪降低了48.7%,胆固醇降低36.5%。据美国食物经协会中心的资料记载,每日每人摄入胆固醇300毫克就完全满足人体需要了,对照组普通鸡蛋胆固醇含量高达630mg/100g,试验组鸡蛋较低的脂肪和胆固醇含量更有利于人体健康。
另外,试验组蛋鸡生产破壳蛋和软壳蛋数量大大减少,每颗鸡蛋的重量也有所增加,对照组一筐鸡蛋45斤,试验组鸡蛋不用拾满一筐就能达到45斤。试验组在夏季高温高湿的环境下产蛋率依然能够保持较高的水平。
因此,在基础日粮中添加一定浓度的富硒罗伊氏乳杆菌制剂,不仅可以提高蛋鸡的生产性能,还可以提高鸡蛋品质,增加蛋白质和硒元素含量,减少脂肪和胆固醇的积累。
实施例5
本发明的微生态制剂作为饲料添加剂对鸡蛋中有害细菌的影响试验。
试验选取健康海兰/罗曼,刚开产的蛋鸡800只,随机分为2组(一组为对照组,另一组试验组),每组含有8个重组,每个重复50只鸡。对照组只饲喂基础日粮,试验组在基础日粮中添加4kg/t亿安奇乐。各组自由采食和饮水,用药和防疫按鸡场常规程序进行。试验组在饲喂基础日粮预试周后,开始饲喂相应的试验日粮,试验期8周。在试验期末,检测鸡蛋中大肠杆菌和沙门氏菌数量的变化,结果见表5。
表5微生态制剂作为添加剂对鸡蛋有害细菌的影响
检测项目 | 普通鸡蛋 | 亿安鸡蛋 | 变化情况 |
大肠杆菌 | ≤100 | <3 | 大肠杆菌减少30倍 |
沙门氏菌 | 不得检出 | 未检出 |
试验结果表明,试验组鸡蛋大肠杆菌数量比对照组鸡蛋降低了33倍,沙门氏菌没有检出。大肠杆菌数量大大降低,意味着鸡蛋的保鲜期会大大延长,鸡蛋长时间存放直到风干都不会坏掉。
本发明制备的微生态制剂富含了多种有益微生物,通过蛋鸡的采食或饮水作用在肠道内,使大量有益菌在肠道占位定植,并在肠道内产生大量消化酶、代谢酶和纤维素酶能分解饲料中的粗纤维和提高饲料的消化利用率;同时产生生理性物质、抗菌物质,可提高畜禽免疫力,防治畜禽疾病;还能消除圈舍粪便恶臭,改善生态环境。蛋鸡的肠道得到了净化,肠道内的有害大肠杆菌和沙门氏菌几乎没有生存的空间,使得鸡蛋中大肠杆菌和沙门氏菌数量大大降低,蛋鸡的机体对营养物质消化吸收能力加强,得到大量的营养,提高了机体的健康度,蛋鸡的疾病和抗生素的使用大大减少。
综上所述,本发明在蛋鸡饲料中添加一定剂量的乳酸杆菌制剂能提高蛋鸡的生产性能,改善鸡蛋的品质,提高蛋鸡对无机硒源的利用效率及硒元素在鸡蛋中的沉积效率,帮助养殖场,饲料厂选择利用高性价比的硒源,提高经济效益,打造安全健康的品牌蛋。乳酸杆菌等益生菌还可以促进蛋鸡生长,增强蛋鸡免疫机能,可有效替代抗生素,减少抗生素的使用,有效防治畜禽疾病,适合在饲料中广泛应用。
Claims (6)
1.一种罗伊氏乳杆菌(Lactobacillus reuteri),其特征在于其生物保藏信息为CGMCCNo.16721。
2.一种富硒罗伊氏乳杆菌,其特征在于通过下述方法获得:
采用权利要求1所述的罗伊氏乳杆菌,选用MRS液体培养基,在接种量10%、Na2SeO3添加量58 µg/mL、pH 6.2、温度37 ℃的条件下培养36 h进行富硒,之后将含富硒罗伊氏乳的培养基进行离心,收集沉淀进行干燥,即得富硒罗伊氏乳杆菌。
3.如权利要求2所述一种富硒罗伊氏乳杆菌,其特征在于所述MRS液体培养基的配方为蛋白胨10.0 g、牛肉膏10.0 g、酵母提取物5.0 g、葡萄糖20.0 g、吐温-80 1.0 mL、K2HPO4·3H2O 1.52 g、醋酸钠5.0 g、柠檬酸三铵2.0 g、MgSO4·7H2O 0.58 g、MnSO4·4H2O 0.25 g、蒸馏水1000 mL。
4.如权利要求2所述的富硒罗伊氏乳杆菌在富硒鸡蛋生产中的应用。
5.如权利要求4所述的富硒罗伊氏乳杆菌在富硒鸡蛋生产中的应用,其特征在于应用方法为:
(1)制备复合微生态制剂:以复合微生物菌粉、低聚异麦芽糖、菜籽饼粉和食用香精为原料制备复合微生态制剂;所述复合微生物菌粉由权利要求4所述的富硒罗伊氏乳杆菌和芽孢杆菌组成;
(2)蛋鸡的养殖:选取健康的蛋鸡,预试周只饲喂基础日粮,之后在基础日粮中添加复合微生态制剂进行养殖。
6.如权利要求5所述的富硒罗伊氏乳杆菌在富硒鸡蛋生产中的应用,其特征在于所述复合微生态制剂由以下质量百分比的原料制备而成:复合微生物菌粉20%~30%,低聚异麦芽糖1.0%~5.0%,菜籽饼粉65%~80%,食用香精0.1%~0.2%。
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