CN116656562A - 一株多功能发酵粘液乳杆菌kd-1及其应用 - Google Patents
一株多功能发酵粘液乳杆菌kd-1及其应用 Download PDFInfo
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- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
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- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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Abstract
本发明提供一株多功能的发酵粘液乳杆菌KD‑1及其应用,该菌株已于2023年4月6日保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2023477。所述发酵粘液乳杆菌KD‑1具有转化制备纳米硒、产胞外多糖和发酵羊乳产抗氧化肽的多种功能,可以用于制备纳米硒、富硒益生菌发酵乳、胞外多糖、抗氧化发酵乳。
Description
技术领域
本发明属于微生物领域,涉及一株多功能发酵粘液乳杆菌KD-1及其应用。
背景技术
发酵粘液乳杆菌是国家允许用于食品的乳酸菌之一,其能够代谢乳糖、半乳糖等多种糖类产生乳酸、乙酸、琥珀酸、乙醇等代谢产物。发酵粘液乳杆菌在传统发酵食品分布较广。由于传统的发酵食品常年被食用,因此发酵粘液乳杆菌作为发酵剂菌种具有安全性及遗传稳定的特点,对传统食品的风味形成具有较大的贡献。
文献报道了发酵粘液乳杆菌具有抗菌、降胆固醇、增强免疫、缓解肝脏和肠道损伤、抗动脉粥状硬化的活性等功能,具有其他功能的发酵粘液乳杆菌需进一步发掘。
硒是生物体不可缺少的一种微量元素,硒的缺乏会伴随着多种疾病的发生,在大多数缺硒地区,硒作为一种营养添加剂添加在人和动物的饮食中,但是硒作为补充剂在食品中的安全阈值很小。部分乳酸菌具有富集硒且还能将低生物利用度和毒性形式的硒转化成高生物利用度和无毒形式的纳米硒。纳米硒和无机硒相比,在抗氧化、抑菌性、免疫调节等方面都有更显著的作用,且其毒性更低、易吸收、生物利用率高。Péter等利用酸奶中的益生菌乳酸链球菌、双歧杆菌BB-12和嗜酸乳杆菌成功的将亚硒酸盐生物转化后并将其分离纯化后得到了(100-500nm)纳米硒。Chun等利用干酪乳杆菌将亚硒酸钠还原成单质纳米硒(50-80nm)且分布在细胞内,并通过体内和体外实验证明,含有纳米硒的干酪乳杆菌的药品能有效地抵抗产生毒素的大肠杆菌引起的肠道功能障碍。目前,国内外对微生物转化制备纳米硒的研究较多,但没见用发酵粘液乳杆菌的报道。
胞外多糖(Extracellular polysaccharide,EPS)是乳酸菌生长代谢过程中分泌的一种碳水化合物,具有独特的流变特性,可作为稳定剂、凝胶剂、增稠剂用于发酵乳制品等食品中。Mende等从嗜热链球菌中分离EPS并将其应用于酸奶。发现EPS可显著增加酸奶的表观粘度。这是因为带负电荷的EPS被静电吸引到牛奶中带正电荷的酪蛋白颗粒上,从而增强多糖-蛋白质网络结构。Purwandari等人也研究表明,EPS可使酸奶具有更好的流变性能。张铁华等将1%的嗜热链球菌ST1产生的EPS加入到酸奶中,发现可以显著改变脱脂乳的粘度。Girard等为酸奶凝胶的形成与菌株酸化的动力学密切相关。由此可以看出在EPS的作用下,可提高产品的粘度。获得产胞外多糖的优良菌株很有必要。
自由基水平的升高导致机体内氧化和抗氧化作用的失衡,从而引起一系列疾病,威胁人们的健康。研究表明,有些乳酸菌能够在发酵过程中水解蛋白质产生抗氧化肽,这些抗氧化肽不仅具有良好的抗氧化性,且安全无毒,因此急需分离筛选获得发酵乳蛋白产抗氧化肽的菌株。
发明内容
为了解决上述现有技术的问题,本发明提供一株多功能的发酵粘液乳杆菌KD-1及其应用,所述发酵粘液乳杆菌KD-1具有转化制备纳米硒、产胞外多糖和发酵羊乳产抗氧化肽的多种功能。
本发明通过以下技术方案实现:
一株多功能发酵粘液乳杆菌(Limosilactobacillus fermentum)KD-1,其特征在于,该菌株已于2023年4月6日保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2023477。
一种纳米硒益生菌的制备方法,包括:
1)将权利要求1所述的发酵粘液乳杆菌KD-1在MRS肉汤培养基中培养至对数期,离心,获得菌泥;
2)所得菌泥中加入磷酸缓冲液制备成菌悬液,并向菌悬液中加入亚硒酸钠溶液,进行转化反应,得到转化液;
3)将转化液离心,弃去上清液,并用无菌水洗涤,获得纳米硒益生菌;
优选的,菌悬液中发酵粘液乳杆菌KD-1浓度为0.02-0.06g/mL,菌悬液中亚硒酸钠的浓度为50-250μg/mL,向菌悬液中加入亚硒酸钠溶液后,调整pH值为6.4-7.5,转化反应时间为26-46h,离心条件为:6000rpm-800rpm,4-10℃离心10-20min。
采用所述的制备方法得到的纳米硒益生菌。
一种纳米硒的制备方法,包括:
将所述的发酵粘液乳杆菌KD-1在MRS肉汤培养基中培养至对数期,离心,获得菌泥;
所得菌泥中加入磷酸缓冲液制备成菌悬液,并向菌悬液中加入亚硒酸钠溶液,进行转化反应,得到转化液;
从所得转化液中提取得到纳米硒。
一种胞外多糖的制备方法,包括:将已活化的发酵粘液乳杆菌KD-1接入产糖培养基,恒温培养后,离心,收集上清液并添加三氯乙酸溶液,离心去除沉淀,再加入乙醇后静置,离心,收集获得粗多糖沉淀,再将粗多糖沉淀溶解于水中,透析,得到胞外多糖。
一种富硒益生菌发酵乳,其发酵原料中添加有所述的发酵粘液乳杆菌和亚硒酸钠。
优选的,所述发酵原料为生牛乳、生羊乳、复原牛乳或复原羊乳。
一种抗氧化益生菌发酵乳,其发酵原料中添加有所述的发酵粘液乳杆菌。
优选的,其DPPH自由基清除率不低于60%,超氧阴离子清除率不低于70%,亚铁离子螯合率不低于65%,肽含量不低于0.50mg/L,活菌数不低于1.0×108CFU/g。
与现有技术相比,本发明具有如下的有益效果:
本发明发酵粘液乳杆菌KD-1具有转化制备纳米硒、产胞外多糖和发酵羊乳产抗氧化肽的多种功能。发酵粘液乳杆菌KD-1能利用菌体细胞转化亚硒酸盐制备纳米硒,转化体系简单,不需要在培养基中进行,避免了培养基成分对制备纳米硒益生菌菌粉和提取纳米硒的影响,该菌株用于制备富硒益生菌发酵乳,可显著加速酸奶发酵剂TW发酵;发酵粘液乳杆菌KD-1在MRS肉汤中能产胞外多糖,提高其抗氧化性,在牛羊乳发酵中能产抗氧化肽,增加发酵乳的功能性。
附图说明
图1购自辽宁抚顺的乳开菲尔粒LF形态及发酵粘液乳杆菌KD-1菌落形态;
图2菌株KD-1的扫描电镜图((a)为转化前的菌株、(b)为转化后的菌株、(c)为EDX分析图谱);
图3时间对发酵粘液乳杆菌KD-1产纳米硒的影响;
图4温度对发酵粘液乳杆菌KD-1产纳米硒的影响;
图5pH对发酵粘液乳杆菌KD-1产纳米硒的影响;
图6浓度对发酵粘液乳杆菌KD-1产纳米硒的影响;
图7菌的添加量对发酵粘液乳杆菌KD-1产纳米硒的影响;
图8发酵粘液乳杆菌KD-1转化制备的纳米硒冻干粉末;
图9纳米硒的透射电镜图;
图10发酵粘液乳杆菌KD-1制备的纳米硒颗粒表面化学性质分析XPS总谱(a)和Se3d图谱(b);
图11发酵时间对富硒益生菌发酵乳pH值的影响。
具体实施方式
为了进一步理解本发明,下面结合实施例对本发明进行描述,这些描述只是进一步解释本发明的特征和优点,并非用于限制本发明的权利要求。
实施例1本发明发酵粘液乳杆菌KD-1的分离、特性比较和菌种鉴定
1、发酵粘液乳杆菌KD-1的获得
将购自辽宁抚顺的开菲尔粒LF(见图1(a))按5%接种于已灭菌冷却的羊乳中,室温发酵24h后以其为样品,以添加亚硒酸钠的Elliker琼脂为分离培养基,采用平板分离法,经过反复分离筛选,获得一株产纳米硒能力强的乳酸菌KD-1,其菌落形态见图1(b)。
2、发酵粘液乳杆菌KD-1的鉴定
采用16S rDNA扩增及序列测定来确定菌株KD-1的种属,通过变性、PCR扩增、PCR产物纯化及测序,测得序列如SEQ ID No.1所示,并在NCBI对其进行搜索和同源性分析,同源性分析结果显示,KD-1为发酵粘液乳杆菌,该发酵粘液乳杆菌已于2023年4月6日保藏于中国典型培养物保藏中心(CCTCC),保藏地点为中国湖北省武汉市武昌区八一路299号武汉大学校内,保藏编号为CCTCC NO:M 2023477。
SEQ ID No.1如下:
GCCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTA
实施例2发酵粘液乳杆菌KD-1转化制备纳米硒
将发酵粘液乳杆菌KD-1的冻干菌粉用MRS肉汤培养基溶解后,在37℃培养箱中培养24h,以5%(v/v)的接种量连续活化三代后,使活菌数均能达到108,将其放置冰箱中4℃备用。
在无菌条件下,以5%(v/v)的接种量将活化好后的发酵粘液乳杆菌KD-1接种在MRS肉汤培养基中并置于培养箱中37℃培养24h,待菌株的生长达到对数期后,将菌悬液摇匀后,取50mL菌悬液8000r/min离心10min,获得菌泥,再用磷酸缓冲液(pH=6.4)洗涤菌泥,重复洗涤三次后,离心获得菌泥,加入10mL磷酸缓冲液制备成菌悬液,并向菌悬液中加入亚硒酸钠溶液,使其终浓度为100μg/mL,然后将其置于37℃培养箱中转化24h,观察转化液颜色,并通过扫描电镜观察转化前后菌体表面的变化和用X射线能谱分析其成分。
实验结果显示,在100μg/mL的亚硒酸钠溶液中转化24h后,转化液中产生红色沉淀,即为纳米单质硒,这表明,本发明发酵粘液乳杆菌KD-1可以将亚硒酸盐还原后转化成为零价的纳米单质硒,菌株KD-1的亚硒酸钠转化率达到85.12%,且其单位菌体转化纳米硒的量达到1.12mg/g。
将发酵粘液乳杆菌KD-1富硒转化后经过处理,进行扫描电镜观察和X射线能谱分析,结果如2所示。
从图2中可知,没有添加亚硒酸钠的发酵粘液乳杆菌KD-1的形态为饱满的棒状(图(a)),将亚硒酸钠还原后的发酵粘液乳杆菌KD-1菌体表面出现内陷空洞,菌体表面有圆形的纳米颗粒((图(b)))。对图(b)中的圆形纳米颗粒进行EDX能谱分析,从图(c)的能谱分析图中可以发现在1.4KeV处有硒的特征峰,说明发酵粘液乳杆菌KD-1将亚硒酸盐还原后产生纳米硒,且分布在菌体表面和菌体外。
实施例3发酵粘液乳杆菌KD-1转化制备纳米硒的工艺优化及纳米硒表征
1、时间对发酵粘液乳杆菌KD-1制备纳米硒的影响分析
将MRS肉汤培养24h的发酵粘液乳杆菌KD-1离心获得菌泥并洗涤3次,然后将其制成菌泥浓度为0.05g/mL,pH为6.4的菌悬液(亚硒酸钠的浓度为100μg/mL),于温度37℃恒温转化不同时间(12、24、36、48和60h),测定亚硒酸钠还原率和转化液a*红色值,结果如图3所示。
由图3可知,亚硒酸钠的转化率随着反应时间的增加先增大后减小,当时间到达36h后,亚硒酸钠的还原速率达到最大值为86.67%,且当时间为48和60h时,亚硒酸钠的转化率分别为86.57%和86.42%接近最大值,说明发酵粘液乳杆菌KD-1和亚硒酸钠反应过程中,在36h时,已基本将亚硒酸钠还原成纳米硒。a*红色值的变化和亚硒酸钠的还原率基本上保持一致,在48小时达到最大值为22.62和在36h的a*红色值22.32较为接近。综合考虑,选择36h为发酵粘液乳杆菌KD-1转化制备纳米硒的最适时间。
2、温度对发酵粘液乳杆菌KD-1制备纳米硒的影响分析
将发酵粘液乳杆菌KD-1转化制备纳米硒的温度分别设置为33℃、35℃、37℃、39℃和41℃,转化时间为48h,结果如图4所示。
由图4可知,亚硒酸钠的转化率和a*红色值都随着温度的增加先增大后减少,当温度为31℃时,发酵粘液乳杆菌KD-1转化率为77.92%,此是a*红色值也相对较低为17.2。而当温度达到37℃时,亚硒酸钠的转化率达到最大值为84.18%,且a*红色值也达到最大为23.14。因此可知发酵粘液乳杆菌产纳米硒的最适温度为37℃。
3、pH对发酵粘液乳杆菌KD-1制备纳米硒的影响分析
研究不同pH值(5.6、6.3、7、7.7、8.4)对发酵粘液乳杆菌KD-1在37℃恒温转化制备36h纳米硒的影响,结果如图5所示。
由图5可知亚硒酸钠的转化率随pH的增加先增加后减少。当pH为6时亚硒酸钠的转化率仅为65.12%,而当pH为6.8时,亚硒酸钠的转化率达到最大为87.23%,此时a*红色值也达到最大为19.5。而当pH逐渐增大,亚硒酸钠的转化率又逐渐减小,说明发酵粘液乳杆菌KD-1在中性条件下更适合转化制备纳米硒,适宜的转化pH为6.4-7.5。
4、亚硒酸钠浓度对发酵粘液乳杆菌KD-1制备纳米硒的影响分析
将发酵粘液乳杆菌KD-1的菌悬液中亚硒酸钠的浓度分别调整为50、100、150、200和250μg/mL在37℃恒温转化48h,结果如图6所示。
由图6可以看出,随着亚硒酸钠浓度的增加,亚硒酸钠的转化率先增加,后减少,而a*红色值则先增加后变化趋于平缓。当亚硒酸钠的浓度为100μg/mL时,亚硒酸钠的转化率为89.48%达到最大,此时a*红色值也达到了最大为21.04。但当亚硒酸钠的浓度超过100μg/mL之后,亚硒酸钠的转化率逐渐降低。这主要是因为,当亚硒酸钠的含量较少时,菌体中的还原酶能较快的将亚硒酸钠还原,但当亚硒酸钠的浓度增高时,亚硒酸盐离子的毒性可能是导致菌株在较高硒浓度下亚硒酸盐的转化率降低的主要原因,适宜的纳米硒浓度为50-150μg/mL。
5、发酵粘液乳杆菌KD-1添加量对制备纳米硒的影响分析
将菌悬液中发酵粘液乳杆菌KD-1的添加量分别调整为0.02、0.03、0.04、0.05和0.06g/mL,添加亚硒酸钠浓度至100μg/mL,将pH调至7.2,37℃恒温转化48h,结果如图7所示。
从图7中可以看出,亚硒酸钠的转化率和a*红色值都随着菌的添加量先达到一定值后就变化较为平稳,当菌的添加量为0.04和0.05g/mL时,亚硒酸钠的转化率为85.51%和86.33%相差较近,此时a*红色值也比较接近为21.84和21.31,说明,当亚硒酸钠的含量一定后,随着菌含量的增加,菌对亚硒酸钠的转化率也逐渐增加,但是当将亚硒酸钠转化完后,纳米硒的量就不再随着菌体的增加而增加了。
6、温度、菌体添加量及转化时间对发酵粘液乳杆菌KD-1转化制备纳米硒的影响
根据单因素试验结果,选取温度、菌的添加量和时间这三个对转化制备纳米硒过程影响较大的因素来优化工艺条件,选取亚硒酸钠转化率(%)、单位菌体纳米硒的含量(mg/g)和a*红色值为指标优化工艺条件。实验设计和结果如表1所示。
表1发酵粘液乳杆菌KD-1制备纳米硒工艺的试验设计及结果
由表1可知,在温度为32-42℃、接种量为0.023-0.057%、转化时间为26-46h范围内,所得亚硒酸钠转化为纳米硒的转化率为78.22-96.88%,单位菌体的纳米硒含量为(0.806-1.759mg)/g菌体,a*红色值为17.73-23.70。
将发酵粘液乳杆菌KD-1转化液离心(10000r/min,4℃,10min),弃去上清液,收集纳米硒益生菌沉淀,对其中的纳米硒进行提取分离和冷冻干燥,获得纳米硒冻干粉,结果如图8所示。对纳米硒冻干粉通过透射电镜(TEM)、X射线光电子能谱(XPS)表征,结果见图9和图10,由图9可知发酵粘液乳杆菌KD-1转化制备的纳米硒粒径约200nm,该纳米硒在水中的分散性较好。Lampis等(Gigiena I Sanitariia,2000(5):32)用Stenotrophomonasmaltophilia SeITE02还原亚硒酸盐制备了纳米硒,并发现了纳米硒呈球形,大小范围为160nm至250nm。和本文中观察到的纳米硒尺寸形状一致。由图10(a)中发现C、O、N、P、S等元素也存在于纳米硒表面,且这些峰的强度比硒的强,说明纳米硒表面存在一些生物大分子将纳米硒包裹起来。可以看到图10(b)中在55eV附近有零价硒的特征峰,证明是纳米硒的表面存在零价硒。
实施例4发酵粘液乳杆菌KD-1在富硒益生菌发酵羊乳中的应用
羊奶粉和水按1:7(w/v)的比例配成复原乳后90℃灭菌5min后,待其冷却至室温后分成2组,第一组加0.05%(w/v)的直投式发酵剂TW和亚硒酸钠(7μg/mL)作为对照,第二组加入0.03%(w/v)的直投式发酵剂TW、0.02%(w/v)发酵粘液乳杆菌KD-1冻干菌粉和亚硒酸钠(7μg/mL),然后于42℃恒温培养不同时间(3、4、5、6、7h),取样测定富硒益生菌发酵羊乳的pH值,结果如图11所示。
由图11中可以看出两种发酵羊乳的pH随着时间的增加先减小后变化趋于平稳,但是添加发酵粘液乳杆菌KD-1的pH下降速度更快,发酵4h时,添加发酵粘液乳杆菌KD-1的pH值为4.52±0.011,而对照组的pH值为4.91±0.013,在发酵6h时其pH值达到了4.52±0.013.工业上生产发酵乳是在pH值达到4.5时停止发酵后转入后熟,可见添加发酵粘液乳杆菌KD-1促进了羊乳发酵,比对照组提前2h到达发酵终点,显著地缩短了发酵时间。
实施例5发酵粘液乳杆菌KD-1产胞外多糖及其抗氧化性
将已活化的发酵粘液乳杆菌KD-1以2%的接种量接入产糖培养基,37℃恒温培养48h,然后在4℃下7500r/min离心12min,收集上清液添加10%三氯乙酸溶液,离心去除沉淀,再加入3倍体积的乙醇后置于4℃过夜,然后于7500r/min离心12min,收集获得粗多糖沉淀,再将其解于蒸馏水中,并置于透析袋中,透析24h,以除去小分子如单糖、色素和肽,测定EPS量、DPPH自由基清除率和亚铁离子螯合率。
表2发酵粘液乳杆菌KD-1胞外多糖产量及其抗氧化性
菌株 | 胞外多糖产量mg/L | DPPH自由基清除率/% | 亚铁离子螯合率/% |
发酵粘液乳杆菌KD-1 | 210.25±1.58 | 70.32±1.86 | 48.39±2.12 |
由表2可知,发酵粘液乳杆菌KD-1的胞外多糖产量为210.25±1.58,DPPH自由基清除率为70.32±1.86%,亚铁离子螯合率为48.39±2.12%,说明本发明发酵粘液乳杆菌KD-1具有产胞外多糖及抗氧化的功能。
实施例6发酵粘液乳杆菌KD-1发酵牛羊乳制备抗氧化发酵乳
将鲜牛乳和鲜羊乳分别于90℃杀菌10min。待冷却至40℃,以5%接种量将已活化的发酵粘液乳杆菌KD-1,37℃培养15h,每组三个平行,取样测定DPPH自由基清除率、超氧阴离子清除率、肽含量、感官评价、活菌数、酸度和pH。其结果平均值见表3。
表3发酵粘液乳杆菌KD-1发酵牛羊乳的抗氧化性
由表3可知,牛羊乳经发酵粘液乳杆菌KD-1发酵后,其DPPH自由基清除率不低于60%,超氧阴离子清除率不低于70%,亚铁离子螯合率不低于65%,肽含量不低于0.50mg/L,活菌数不低于1.0×108CFU/g,牛乳的感官评价和活菌数均高于羊乳,但羊乳的抗氧化性高于牛乳,说明两种发酵粘液乳杆菌KD-1发酵乳各有特色,发酵粘液乳杆菌KD-1可用于制备抗氧化益生菌发酵乳。
Claims (10)
1.一株多功能发酵粘液乳杆菌(Limosilactobacillus fermentum)KD-1,其特征在于,该菌株已于2023年4月6日保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2023477。
2.一种纳米硒益生菌的制备方法,其特征在于,包括:
1)将权利要求1所述的发酵粘液乳杆菌KD-1在MRS肉汤培养基中培养至对数期,离心,获得菌泥;
2)所得菌泥中加入磷酸缓冲液制备成菌悬液,并向菌悬液中加入亚硒酸钠溶液,进行转化反应,得到转化液;
3)将转化液离心,弃去上清液,并用无菌水洗涤,获得纳米硒益生菌。
3.根据权利要求2所述的纳米硒益生菌的制备方法,其特征在于,菌悬液中发酵粘液乳杆菌KD-1浓度为0.02-0.06g/mL,菌悬液中亚硒酸钠的浓度为50-250μg/mL,向菌悬液中加入亚硒酸钠溶液后,调整pH值为6.4-7.5,转化反应时间为26-46h,离心条件为:6000rpm-800rpm,4-10℃离心10-20min。
4.采用权利要求2或3所述的制备方法得到的纳米硒益生菌。
5.一种纳米硒的制备方法,其特征在于,包括:
将权利要求1所述的发酵粘液乳杆菌KD-1在MRS肉汤培养基中培养至对数期,离心,获得菌泥;
所得菌泥中加入磷酸缓冲液制备成菌悬液,并向菌悬液中加入亚硒酸钠溶液,进行转化反应,得到转化液;
从所得转化液中提取得到纳米硒。
6.一种胞外多糖的制备方法,其特征在于,包括:将已活化的发酵粘液乳杆菌KD-1接入产糖培养基,恒温培养后,离心,收集上清液并添加三氯乙酸溶液,离心去除沉淀,再加入乙醇后静置,离心,收集获得粗多糖沉淀,再将粗多糖沉淀溶解于水中,透析,得到胞外多糖。
7.一种富硒益生菌发酵乳,其特征在于,其发酵原料中添加有权利要求1所述的发酵粘液乳杆菌和亚硒酸钠。
8.根据权利要求7所述的富硒益生菌发酵乳,其特征在于,所述发酵原料为生牛乳、生羊乳、复原牛乳或复原羊乳。
9.一种抗氧化益生菌发酵乳,其特征在于,其发酵原料中添加有权利要求1所述的发酵粘液乳杆菌。
10.根据权利要求9所述的抗氧化益生菌发酵乳,其特征在于,其DPPH自由基清除率不低于60%,超氧阴离子清除率不低于70%,亚铁离子螯合率不低于65%,肽含量不低于0.50mg/L,活菌数不低于1.0×108CFU/g。
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