CN115181682A - 一株高富集有机锌的发酵乳杆菌 - Google Patents
一株高富集有机锌的发酵乳杆菌 Download PDFInfo
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- CN115181682A CN115181682A CN202210124428.1A CN202210124428A CN115181682A CN 115181682 A CN115181682 A CN 115181682A CN 202210124428 A CN202210124428 A CN 202210124428A CN 115181682 A CN115181682 A CN 115181682A
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- Prior art keywords
- zinc
- lactobacillus fermentum
- ccfm1196
- rich
- lactobacillus
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- 239000011701 zinc Substances 0.000 title claims abstract description 192
- 229910052725 zinc Inorganic materials 0.000 title claims abstract description 192
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 title claims abstract description 191
- 241000186840 Lactobacillus fermentum Species 0.000 title claims abstract description 67
- 229940012969 lactobacillus fermentum Drugs 0.000 title claims abstract description 67
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
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- JBQYATWDVHIOAR-UHFFFAOYSA-N tellanylidenegermanium Chemical compound [Te]=[Ge] JBQYATWDVHIOAR-UHFFFAOYSA-N 0.000 description 1
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- WGIWBXUNRXCYRA-UHFFFAOYSA-H trizinc;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [Zn+2].[Zn+2].[Zn+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O WGIWBXUNRXCYRA-UHFFFAOYSA-H 0.000 description 1
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- 235000021340 zinc rich food Nutrition 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Nutrition Science (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
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Abstract
本发明公开了一株高富集有机锌的发酵乳杆菌,属于微生物技术领域。本发明筛选得到的发酵乳杆菌(Lactobacillus fermentum)CCFM1196能够将无机锌高效富集,转化为有机锌,被机体更好的吸收利用,具体体现在:(1)发酵乳杆菌(Lactobacillus fermentum)CCFM1196可将无机锌高效富集,转化为有机锌,有机锌含量为90.0%;(2)富集的锌含量可达到11.6mg/g菌粉,活菌数可达到7×1010CFU/g。使用本发明的乳杆菌CCFM1196可对无机锌进行高效富集,在食品和医药领域具有巨大的应用前景。
Description
技术领域
本发明涉及一株高富集有机锌的发酵乳杆菌,属于微生物技术领域。
背景技术
锌(Zn)动植物及人类重要的必须微量元素,参与体内多种生化反应,如蛋白质的合成、DNA和RNA的代谢、信号传导、基因表达以及调节细胞凋亡。不仅如此,锌还在促进动物的生长、免疫、繁殖及脑神经等方面起着重要的作用。缺锌会引起生长发育迟缓,此外,锌缺乏还与睾丸体积/重量减少、性腺机能减退、性腺功能障碍、人类第二性征发育不良、生精小管萎缩等有关。目前在我国一些贫困地区,由于动物性食物的摄入量低,对富锌的食物获取有限,出生后以植物性食物为主,植物性饮食中的植酸盐等会抑制锌的吸收,造成生长发育期长期锌缺乏,影响生长发育。
健康成人每天从膳食中摄入约10-15mg锌,吸收率一般为20-30%。目前人工添加的各类补锌产品主要分为无机锌、有机锌、人工合成锌和生物活性锌四代,无机锌主要为氧化锌、硫酸锌等,其吸收利用率低且与胃酸结合产生氯化锌,刺激胃肠道,引起恶心呕吐,副作用大。由于无机锌价格低廉,硫酸锌等无机锌是常用的饲料锌营养源。有机锌如柠檬酸锌、葡萄糖酸锌等吸收率较高于无机锌,但依然会对肠胃产生刺激,有一定副作用。且含锌量较高,能拮抗钙、铁等其他微量元素的吸收,长期服用会导致缺钙、贫血等症状。人工合成锌如蛋白锌是蛋白质和锌的结合物,是国内比较先进的一种补锌制剂,其无副作用且吸收比较好。生物活性锌如酵母锌是生物态锌,具有生物转化过程,吸收利用率高,无副作用可有效促进人体对各种营养素的吸收和利用,且不会拮抗钙铁等营养素的吸收,能够更好的达到补充各种营养素的效果。
通过微生物转化有机锌是近十几年的研究的热门,益生菌作为一类对宿主健康有益的活的微生物,具有极多的健康功效,被广泛地用于富锌研究。富锌益生菌具有有机锌和益生菌的双重功效,如毒性低、吸收利用率高、抗氧化、环境污染小等特点,因此发现能高效转化无机锌为有机锌的益生菌是目前研究的热点。目前的报道研究,在富锌微生物方面研究较多的是酵母锌,然而与酵母菌相比,益生菌在许多生物活性方面更加具有优势,例如增加肠道内有益微生物的数量、调节肠道菌群、促进营养吸收等。富锌益生菌作为一种新型的含有活性益生菌的膳食锌源,其较益生菌及其他锌补充剂的优势值得探索。无机锌作为锌源,其吸收利用率低,且易造成环境的污染,所以寻找新的锌源迫在眉睫。因此,利用率较高的微生物锌受到了广泛的重视,与单独补充无机锌相比,有必要对富锌益生菌高效富集无机锌,转化为有机锌进行研究,开发出一种价格低廉、制备方法简单、安全可靠且补锌更高效的锌补充剂。
通过在培养过程中添加无机锌(非吸附),最终筛选出一株富锌量高且有机生物态含量高的发酵乳杆菌CCFM1196,其干菌粉锌含量能够达到11.6mg/g且有机锌含量可达到90.0%。其余菌株的锌含量均不超过2mg/g。张青松等人对24株乳杆菌进行了筛选,对锌的富集量在0.064mg/g~1.76mg/g之间不等。在公开号为CN101971921B的专利申请文本中,胡文锋等人所述的嗜酸乳杆菌锌含量为0.57mg/g,唾液乳杆菌锌含量为0.2mg/g,锌含量较低。周海泳等人对唾液乳杆菌富锌工艺优化后,唾液乳杆菌L3的锌含量为1.18mg/g。在公开号为CN108220208B的专利申请文本中,王宝维等人所述的枯草芽孢杆菌锌含量为2.51mg/g。在公开号为CN108841763A的专利申请文本中A,方曙光等人将植物乳杆菌在含锌离子的改良MRS培养基中驯化培养40-60代,筛选出耐高锌植物乳杆菌菌种,其锌含量也仅为1mg/g~4mg/g。刘文群等人对保加利亚乳杆菌富集锌进行了研究,在最优的培养条件下,其富集的锌含量为0.25mg/g,有机态锌占总量的80%以上。以上所述文献中乳杆菌对锌的富集含量均较低,无法达到本专利菌株高富锌量、高有机锌含量的理想效果。
发明内容
为解决上述问题,本发明提供了一株高富集有机锌的发酵乳杆菌。该菌株结果富锌培养后,每克菌粉有机锌含量能够达到达到11.6mg以上,有机锌含量可达到90.0%,活菌数能够达到7×1010CFU/g以上。
本发明提供了一株高富集有机锌的发酵乳杆菌,已于2021年09月28日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61959,保藏地址为广州市先烈中路100号大院59号楼5楼。
本发明提供发酵乳杆菌CCFM1196通过本发明提供的培养方法培养后具有以下特征:
(1)该菌株在MRS培养基上培养48h后呈小的、白色、不透明菌落;
(2)该菌株可将无机锌富集,转化为生物态锌,能够被机体更好的吸收利用,较无机锌具有更好的调节肠道菌群的功能;
(3)该菌株生产的有机锌能够在远低于日常膳食补充剂量的情况下,发挥较无机锌更高的生物活性,达到补充锌的生理要求;
(4)该菌株经过富锌发酵后,每克菌粉中锌含量可达到11.6mg以上,有机锌含量可达到90.0%,活菌数能够达到7×1010CFU/g以上。
本发明还提供了含发酵乳杆菌CCFM1196的益生菌制剂。
在一种实施方式中,每g或每mL益生菌制剂中含有≥1×1010CFU所述发酵乳杆菌CCFM1196。
本发明还提供了含有经富锌培养的发酵乳杆菌CCFM1196细胞,或含有机锌的发酵乳杆菌CCFM1196细胞裂解物的益生菌制剂。
在一种实施方式中,每g或每mL所述益生菌制剂中的有机锌含量≥10.44mg。
在一种实施方式中,所述富锌培养是将所述发酵乳杆菌CCFM1196在富锌培养基中培养至菌体数量≥1×108CFU/mL。
在一种实施方式中,所述富锌培养基是含锌离子浓度200~500mg/L的培养基。
在一种实施方式中,所述富锌培养基含有葡萄糖20-30g/L,氮源15-25g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、硫酸锌(按锌离子浓度为200~500mg/L换算添加);所述氮源包括质量比为1:2的酵母浸粉和蛋白胨。
在一种实施方式中,所述富锌培养的发酵乳杆菌CCFM1196是在MRS培养基中培养获得种子液,再将培种子液以2%的接种量转接至富锌培养基中,于30~37℃培养12~18h。
在一种实施方式中,收集富锌培养后的菌体细胞,洗涤,加入冻干保护剂,冻干后获得菌粉。
在一种实施方式中,所述富锌培养的发酵乳杆菌CCFM1196通过下述步骤制备:
(1)将发酵乳杆菌在MRS固体培养基上划线,平板于37℃倒置培养36~48h。挑取单菌落接入到MRS液体培养基中于37℃培养24h。再以2%(v/v)的接种量接入到MRS液体培养基中37℃培养12~18h作为后续培养的种子菌液;
(2)将发酵乳杆菌的种子菌液以2%(v/v)的接种量接入发酵瓶中培养12~18h,发酵瓶中为富锌液体培养基;
(3)发酵完成后将菌液在4℃条件下6000g/min离心15min,取湿菌体用纯水漂洗2次,得到高富集有机锌的发酵乳杆菌菌泥,经冷冻干燥,得到高富集有机锌的发酵乳杆菌菌粉。
在一种实施方式中,步骤(2)所述的富锌液体培养基中锌离子浓度为200~500mg/L。
在一种实施方式中,所述富锌发酵乳杆菌CCFM1196菌泥还经过干燥处理,所述干燥包括但不限于:真空冷冻干燥、喷雾干燥、真空干燥或流化床干燥。
在一种实施方式中,所述富锌发酵乳杆菌CCFM1196菌泥经灭活、干燥处理,得到无细胞活性的高富集有机锌的发酵乳杆菌CCFM1196菌粉;所述干燥使用蛋白或糊精作填充剂或不使用填充剂。
本发明还提供所述发酵乳杆菌CCFM1196,或所述益生菌制剂,或生产富锌益生菌的方法在食品、药品或保健品领域的应用。
在一种实施方式中,所述应用包括但不限于制备用于补充锌元素的食品、药品或保健品。
有益效果:本发明提供了一株高富集有机锌的发酵乳杆菌CCFM1196,该菌株能够将无机锌富集吸收并在菌体内转化为生物态锌,有机锌含量可达到90.0%,有助于锌被机体更好的吸收利用。本发明提供的发酵乳杆菌CCFM1196经过富锌发酵后制成菌粉,每克菌粉中锌含量可达到11.6mg以上,活菌数能够达到7×1010CFU/g以上。
生物材料保藏
一株发酵乳杆菌CCFM1196,分类命名为Lactobacillus fermentum,已于2021年09月28日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61959,保藏地址为广州市先烈中路100号大院59号楼5楼。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的硫酸锌(产品货号:10024018、CAS:7446-20-0)购自国药集团化学试剂有限公司;硝酸(产品货号:yb2-308、CAS:7697-37-2)购于国药集团化学试剂有限公司。
下述实施例中涉及的培养基如下:
MRS液体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、蒸馏水1000g/L。
MRS固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、MnSO4·7H2O 0.25g/L、吐温-80 1g/L、琼脂20g/L、蒸馏水1000g/L。
富锌液体培养基(g/L):葡萄糖20-30g/L,氮源15-25g/L(酵母浸粉、蛋白胨质量比为1:2)、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、蒸馏水1000g/L、硫酸锌(按锌离子浓度为200~500mg/L换算添加)。
实施例1:发酵乳杆菌的筛选、菌种鉴定及保存
1、筛选
以来源于安徽省芜湖市的老年人粪便为样本,用无菌生理盐水进行10倍梯度稀释至10-6,然后分别取100μL稀释倍数为10-4、10-5、10-6的稀释液于改良MRS固体培养基上进行平板涂布,37℃培养48h,观察并记录菌落形态;挑取改良MRS固体培养基上不同形态的菌落进行划线分离,经37℃培养48h后,再次挑取改良MRS固体培养基上不同形态的单菌落进行划线分离,直至得到形态一致的纯的单菌落;挑取改良MRS固体培养基上的纯菌落接种于含有硫酸锌的富锌液体培养基中,37℃培养18h;将菌液转移至无菌离心管中,6000g/min离心10min后弃去上层培养基,得到的菌泥漂洗2遍后冷冻干燥,得到富锌菌粉。使用原子吸收分光光度计检测菌粉中的锌含量,选出富集锌能力较强的菌株;
2、鉴定
将分离得到的富集锌能力较强的菌株进行PCR扩增16S rDNA,PCR产物送至英潍捷基(上海)贸易有限公司进行测序,将测序得到的结果在NCBI中进行核酸序列比对,最终得到1株发酵乳杆菌,命名为发酵乳杆菌(Lactobacillus fermentum)CCFM1196。
3、保存
将发酵乳杆菌(Lactobacillus fermentum)CCFM1196接种于MRS液体培养基中,37℃培养18h;取1mL菌液于无菌离心管中,6000g/min离心10min后弃去上层培养基,菌泥重悬于30%甘油溶液中置于-80℃中保藏。
实施例2:富锌发酵乳杆菌的制备方法
(1)将实施例1筛选获得的发酵乳杆菌CCFM1196在MRS固体培养基上划线,平板于37℃倒置培养48h;挑取单菌落接入MRS液体培养基中于37℃培养24h;以2%(v/v)的接种量接入到改良MRS液体培养基中37℃培养12-18h作为后续培养的种子菌液。
(2)将发酵乳杆菌的种子菌液以2%(v/v)的接种量接入到含富锌液体培养基的发酵瓶中,于37℃培养12-18h。
(3)将步骤(2)培养完成后将菌液在4℃条件下6000g/min离心15min,取湿菌体用纯水洗涤2次,以质量分数13%的脱脂乳作为冻干保护剂,将洗涤后的湿菌体与冻干保护剂以质量比1:1混匀,冷冻干燥,得到高富集锌的发酵乳杆菌菌粉,活菌数为7×1010CFU/g菌粉。
实施例3:富锌发酵乳杆菌的制备方法
将富锌发酵乳杆菌灭活、经干燥处理制备菌粉,干燥方式可选用喷雾干燥、真空干燥、流化床干燥或真空冷冻干燥。
具体步骤为:按照实施例2步骤(1)~(2)的方法进行富锌培养,将步骤(2)发酵完成后将菌液在4℃条件下8000g/min离心20min,取湿菌体用纯水漂洗2次,将洗涤后的湿菌体进行喷雾干燥或真空干燥或流化床或真空冷冻干燥,使用蛋白或糊精作填充剂(菌泥与填充剂溶液质量比为1:1,填充剂溶液为质量分数13%的乳清蛋白、胶原蛋白、大豆蛋白或糊精溶液),得到无活性的高富集锌发酵乳杆菌菌粉,菌粉中的有机锌含量可达10440.09μg(约10.44mg)以上。
实施例4:富锌发酵乳杆菌锌含量及有机锌检测
1、富锌发酵乳杆菌锌含量检测
(1)微波消解
称取实施例2或实施例3制备的发酵乳杆菌CCFM1196菌粉样品0.1g~0.15g于微波消解罐中,加入5mL硝酸后,进行微波消解。冷却后取出消解罐,在电热板上于140℃~160℃赶酸至1mL左右。消解罐放冷后,将消化液转移至25mL容量瓶中,用少量水洗涤消解罐2次~3次,合并洗涤液于容量瓶中,用水定容至刻度,混匀备用。同时做试剂空白试验。
(2)标准溶液配制
①锌标准储备液(1000mg/L):准确称取1.2447g(精确至0.0001g)氧化锌,加少量体积分数50%的硝酸溶液,加热溶解,冷却后移入1000mL容量瓶,加水至刻度,混匀。
②锌标准中间液(10mg/L)准确吸取锌标准储备液(1000mg/L)0.5mL于50mL容量瓶中,加体积分数为5%的硝酸溶液至刻度,混匀。
③锌标准系列溶液:分别准确吸取锌标准中间液0mL、0.5mL、1mL、2mL、4mL、和5mL于50mL容量瓶中,加硝酸溶液(5+95)至刻度,混匀。此锌标准系列溶液的质量浓度分别为0mg/L、0.1mg/L、0.2mg/L、0.4mg/L、0.8mg/L、1mg/L。
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量样品中的锌含量,其检测结果为11.6mg/g。
2、富锌发酵乳杆菌有机锌分析
准确称取0.5g实施例2或实施例3制备的富锌发酵乳杆菌CCFM1196菌粉于烧杯中,加入45ml的蒸馏水,然后用稀酸或稀碱调节蒸馏水pH值至6.5,调好后用50ml容量瓶定容,定容蒸馏水的pH值也调至6.5,定容毕,将富锌发酵乳杆菌溶液转移至烧杯中,用玻璃棒缓慢匀速搅拌5~10min,搅拌毕,8000r/min,室温离心15min。上清液用于测量富锌发酵乳杆菌细胞表面水溶性锌(即无机锌)含量。沉淀中加入45ml的10mmol/L EDTA溶液,然后用稀酸或稀碱调节EDTA溶液pH值至6.5,调好后用50ml容量瓶定容,定容的EDTA溶液pH值也调至6.5,定容毕,将富锌发酵乳杆菌溶液转移至烧杯中,用玻璃棒缓慢匀速搅拌5~10min,搅拌毕,8000r/min,室温离心15min。上清液用于测量富锌发酵乳杆菌细胞壁多糖及蛋白质络合的锌含量。第二次离心沉淀用于测量富锌发酵乳杆菌细胞内有机大分子或小分子结合锌含量。
有机化程度=(细胞壁多糖及蛋白质络合的锌含量+细胞内有机大分子或小分子结合锌含量)/总锌含量。
参照中华人民共和国国家标准GB 5009.14-2017中的第一种方法火焰原子吸收光谱法测量各组分中的锌含量,其检测结果如下:
表1富锌发酵乳杆菌CCFM1196菌粉锌含量分析
由此可见,富锌发酵乳杆菌CCFM1196菌粉总锌含量达11.6mg/g,表明该发酵乳杆菌对锌的富集能力较强,且无机态锌含量为10.0%,说明该发酵乳杆菌CCFM1196对无机锌的同化效果较好。有5.9%的锌以有机体的形式结合在细胞壁的多糖和蛋白质等大分子上;84.2%的锌与发酵乳杆菌细胞内有机大分子或小分子结合。采用相同方法检测无活性的高富集锌发酵乳杆菌菌粉中有机锌含量,结果显示灭活后的菌泥制备的菌粉中总锌含量为11.6mg/g,无机态锌含量为10.0%,有机态锌含量为90.0%。
以上结果表明,无论发酵乳杆菌是否具有细胞活性,均可作为负载有机锌的细胞载体用于富锌产品的制备。
对比例1:不同富锌乳杆菌的富锌量和有机化程度
检索并收集不同来源的益生菌富锌培养后的锌含量。其中,表2中鼠李糖乳杆菌CCFM237、罗伊氏乳杆菌CCFM795、植物乳杆菌CCFM8610、植物乳杆菌CCFM8661、植物乳杆菌CCFM242、植物乳杆菌CCFM259、干酪乳杆菌CCFM30、乳酸片球菌CCFM18、发酵乳杆菌5-1L、保加利亚乳杆菌MJ-1、消化乳杆菌CCFM636、清酒乳杆菌RS70-4、格氏乳杆菌AH-WH-7-4、嗜酸乳杆菌GuXi-8-2-GMM、嗜热链球菌1M7公开于论文《乳酸菌对锌的富集特性及富锌乳酸菌对小鼠结肠炎的缓解作用》中;枯草芽孢杆菌NZ56公开于公开号为CN 108220208 B的专利中,嗜酸乳杆菌(自命名为7L5)和唾液乳杆菌(自命名为FXJCJ7M2)公开于公开号为CN101971921 B的专利中,唾液乳杆菌L3公开于论文《唾液乳杆菌富锌工艺优化》中。
表2不同富锌乳杆菌的锌含量对比
按照实施例2相同的方法培养表3所示的菌株,并检测培养18h后的锌含量。其中,短双歧杆菌F-JS-ZJ-1-M5、鼠李糖乳杆菌DG11-1、植物乳杆菌NFM11、干酪乳杆菌RS-2-1、发酵乳杆菌NT65-2为自行筛选的富锌菌株。
表3不同富锌乳杆菌有机化程度对比
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.发酵乳杆菌CCFM1196,已于2021年09月28日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61959,保藏地址为广州市先烈中路100号大院59号楼5楼。
2.益生菌制剂,其特征在于,含有(a)、(b)或(c):
(a)权利要求1所述的发酵乳杆菌CCFM1196;
(b)权利要求1所述发酵乳杆菌CCFM1196经富锌培养后的细胞;
(c)权利要求1所述发酵乳杆菌CCFM1196经富锌培养得到的含有机锌的细胞裂解物。
3.根据权利要求2所述的益生菌制剂,其特征在于,每g或每mL益生菌制剂中含有≥1×1010CFU所述发酵乳杆菌CCFM1196。
4.根据权利要求2或3所述的益生菌制剂,其特征在于,所述富锌培养是将权利要求1所述的发酵乳杆菌CCFM1196在富锌培养基中培养至菌体数量≥1×108CFU/mL。
5.根据权利要求4所述的益生菌制剂,其特征在于,所述富锌培养基是含锌离子浓度200~500mg/L的培养基。
6.根据权利要求2所述的益生菌制剂,其特征在于,所述细胞包括但不限于活细胞、自然失活的细胞或经灭活处理的细胞。
7.一种富锌发酵乳杆菌的制备方法,其特征在于,将权利要求1所述的发酵乳杆菌CCFM1196在富锌培养基中培养至菌体数量≥1×108CFU/mL;所述富锌培养基是含锌离子浓度200~500mg/L的培养基。
8.根据权利要求7所述的方法,其特征在于,所述富锌培养基含有葡萄糖20-30g/L,氮源15-25g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O0.1g/L、MnSO4·7H2O 0.05g/L、吐温-80 1g/L、半胱氨酸0.5g/L、硫酸锌;所述氮源包括质量比为1:2的酵母浸粉和蛋白胨。
9.根据权利要求7或8所述的方法,其特征在于,所述方法是将发酵乳杆菌CCFM1196是在MRS培养基中培养获得种子液,再将培种子液转接至富锌培养基中,于30~37℃培养12~18h。
10.权利要求1所述的发酵乳杆菌CCFM1196,或权利要求2~6任一所述的益生菌制剂,或权利要求7~9任一所述方法在生产食品、药品或保健品中的应用。
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