CN113923993A - 一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法 - Google Patents
一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法 Download PDFInfo
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- A—HUMAN NECESSITIES
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- 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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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C2220/00—Biochemical treatment
- A23C2220/20—Treatment with microorganisms
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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
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- A23V2400/165—Paracasei
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,该方法允许通过从特定地理区域的牛奶、绵羊奶或山羊奶中鉴定并分离获得独特脂肪酸组成,并且还允许获得天然的共生微生物群落,其因正常的奶生产和处理过程中的巴氏灭菌而不复存在,其中天然微生物群落被添加至饮用奶,或以添加至奶中或用于制备含有奶原产地区典型的天然微生物群落的乳品饮料的速溶形式储存,而对于斯洛伐克东部和中部地区,已鉴定并分离这些有益微生物:粪肠球菌SP、粪肠球菌Ccj、希氏肠球菌Klk、植物乳杆菌SP1、植物乳杆菌AN、副干酪乳杆菌TD、链状乳杆菌SB和马克斯克鲁维酵母SB,并且最合适的脂肪酸比率是0.81%的ω‑3α‑亚麻酸(C18:3n3)、2.15%的ω‑6亚油酸(C18:2n6c)、0.22%的花生酸(20:0)和0.11%的山嵛酸(22:0)。
Description
技术领域
本发明总体上涉及食品工业,即饮用奶或乳制品补充剂和饮料的生产。
本发明产生了三种涉及不同技术领域的复合的创新途径:
(1)以斯洛伐克(Slovakia)东部和中部山区的野生牧场饲养为基础,用均衡的食物组成(来自符合有机农业规范的农场的不含防腐剂的当地草饲料)对动物进行受控护理;
(2)一种用于制备具有种族制剂的新乳制品,或功能性新一代乳制品的新工艺,该乳制品具有确定的固定比例的原产地典型的种族共生微生物菌群(ethnic commensalmicrobial flora),其与原始的独特的脂肪酸谱和来自遵守有机农业规范的农场的奶的化学成分组合;
(3)有益的健康影响的证据—肠道菌群,和代谢物(短链脂肪酸)的调节以及预防炎症(低度)的SIgA(分泌型免疫球蛋白A),该炎症是非传染性疾病的主要诱因。
背景技术
如今,饮用奶主要产自奶牛的奶,其被称为“饮用奶”。
饮用奶是一种人类健康发展所必需的生物活性营养素的来源,其包含:
-儿童时期身体发育和人体成长的营养和构建原料(building materials);
-确保对生物体能量供应的物质;
-矿物质、维生素、激素和酶;
-人体无法合成的必需元素。
饮用奶含有乳脂、乳的糖-乳糖、乳蛋白和矿物质,其中钙是最重要的。饮用奶的主要部分是通过新鲜牛乳的乳制品处理产生的,该乳制品处理是一套工艺流程,如澄清、离心、热处理(巴氏灭菌)、均质化、冷却和包装。根据饮用奶生产所用的工艺流程和方法区分不同的市场类型。
奶牛通过乳腺中的腺泡单独生产没有微生物的奶。奶在乳房中已经受到污染(初次污染),因此一些非致病性微生物群落可被认为是生理性的。
奶的这些微生物中最重要的群组是以球菌和杆状形式代表的嗜温乳酸发酵菌。这些微生物被分为同型发酵的(它们将乳糖发酵为乳酸)和异型发酵的(除乳酸外,它们将乳糖发酵为其他成分,例如丁酸和乙醇)。特别有用的是具有乳酸链球菌同型酶细菌的链球菌属以及具有乳脂明串珠菌和葡萄聚糖明串珠菌异型酶细菌的明串珠菌属。乳酸发酵菌的杆状形式结合为乳酸菌属。同型酶细菌中,乳酸乳杆菌(L.lactis)、保加利亚乳杆菌(L.bulgaricus)、嗜酸乳杆菌(L.acidophilus)、瑞士乳杆菌(L.helveticus)、干酪乳杆菌(L.casei)和德氏乳杆菌(L.delbrueckii),以及异型酶细菌中,发酵乳杆菌(L.fermentum)和短乳杆菌(L.brevis)是重要的。
挤奶和奶处理、以及未充分冷却的奶的储存导致的奶的二次污染代表奶中微生物的最大比例。据估计,挤奶过程中的污染比乳房中的生理感染高十倍;奶处理导致另一种多重感染。牛棚中常见污物导致的奶污染是严重的。这些微生物的活性不仅表现于健康缺陷,还表现于牛奶的技术加工中。奶中不希望的微生物群落由常见的埃希氏杆菌属和肠杆菌属的肠道菌(大肠杆菌、产气肠杆菌),其次是丁酸发酵菌(如丁酸梭菌)、蛋白分解菌(枯草芽孢杆菌、蕈状芽孢杆菌)形成。各种酵母和酵母微生物(例如酵母菌、念珠菌)和其他微生物也可能存在。在当今的现代制冷技术中,提取和加工奶的过程中最重要的这些群体包括嗜冷微生物,该嗜冷微生物即使在低的奶储存温度(即约5℃)下也可以繁殖。虽然它们可以容易地被巴氏灭菌破坏,但它们产生的脂肪酶和蛋白酶可导致乳制品口味缺陷、奶成分裂解,并且即使在巴氏灭菌后仍保持其活性。最重要的嗜冷微生物属包括:例如假单胞菌属、产碱杆菌属、黄杆菌属、乳杆菌属、链球菌属、葡萄球菌属、微球菌属等。
刚挤完奶后,每毫升奶中包含数百到数千个微生物,其中90%是外源的。特别地,以细菌、少数酵母菌和丝状真菌为代表,但也经常存在病原微生物,因此引入奶的巴氏灭菌。
奶是一种完美且最天然的饮料。它含有最有价值的动物蛋白质、易消化的脂肪和多种重要的矿物质。它含有必需氨基酸、维生素、乳糖和多种人体营养和发育所必需的微量元素,其保证新陈代谢的正常功能并保护人体健康。奶在营养方面的多样性还通过成年人从一升奶中可获取的每日总消耗量生活必需物质的比例得到证实。该比例为40%的蛋白质、32%的脂肪、12%的糖以及,特别地,高达20%的钙、67%的磷、30%的维生素A、25%的维生素B1和高达70%的维生素B2。
由于人们的现代的且过度化学化的、可长期储存的食品,食用富含原始共生微生物群落的奶的理由越来越充分,其有助于在胃肠道建立微生物平衡,同时增强人群的免疫力。
从现有技术的角度来看,至关重要的是,目前通过依次进行以下工艺过程生产饮用奶:
1)澄清;
2)离心;
3)热处理(巴氏灭菌);
4)均质化;
5)冷却和包装。
我们的发明的创新性在于,向饮用奶生产过程中引入新的步骤(工艺流程),如以下突出显示的:
1)澄清;
2)离心;
3)选定的微生物的分离;
4)热处理(巴氏灭菌);
5)奶的富集,即将第3点中分离的微生物返回至经巴氏灭菌的奶中;
6)均质化;
7)冷却和包装。
上述第3)和第5)点是如下所述的本发明的主题。
除了饮用牛奶的生产之外,现有技术不知道任何其他乳制品粉状饮料或任何其他乳制品补充剂(其包含粉末形式的分离的健康有益的细菌)。
目前所有的乳制品粉状饮料都源自巴氏灭菌后的奶,因此不含奶中发现的所有原始有益细菌。我们的发明的创新性在于,在奶巴氏灭菌之前从奶中分离出天然的、有益健康的细菌,从而保护这些细菌免受巴氏灭菌的破坏,并随后将它们保存在粉末中用于食品工业的各种应用,或者将它们返回至奶中并生产富含这些有益健康的细菌的奶,然后出售并食用。
本发明的第二个创新特征在于从特定地区的有机牧场获得的奶中的有益微生物和脂肪酸的鉴定。具体地,我们已经处理了斯洛伐克东部和中部的山区,但这种奶生产过程可以在世界的任何其他地区类似地进行。
通过鉴定和分离有益健康的微生物,然后我们可以人工繁殖它们,并在巴氏灭菌后将其返回至奶中。同时,我们可获得关于奶中这些微生物的正确且适当的比率的信息,这将适合特定地区的人群。通过鉴定有益健康的脂肪酸,我们可以获得关于适合特定地区人群的奶中这些脂肪酸的正确且适当的比率的信息。
如下所述,这一信息在奶和乳制品饮料的生产过程中是有价值的,因为它使生产更适合人类的食品成为可能。目前,这一趋势体现于各种不同类型的功能性食品的生产和推广中。
功能性食品通常称为对这些食品的食用者具有有利影响的产品。
在大多数情况下,它们目前是通过向天然有机产品中添加益生元和益生菌化合物生产的。
功能性食品是通过添加新成分或更多那些现有成分而获得另一种功能的食品。对功能性、强化食品的需求的增长以及消费者的健康和健身意识的增强可预期对功能性食品的需求的增长。对功能性食品的需求不断增长;消费者的健康和健身意识不断增强,儿童人口的增长、生活方式的改变以及城市化的扩大进一步促进了市场的增长(参考文献1)。
欧洲法规中尚未定义功能性食品。它们通常被认为是作为正常饮食的一部分被食用的食品,并且包含具有改善健康或降低疾病风险的潜力的生物活性成分(参考文献2)。
没有完美食品的食谱。然而,我们可以根据现有知识得出结论,除新的营养的、技术的、感觉的和健康的特性之外,新型食品的制备应确保营养物对消费者及其所含微生物的有用性。测定使营养素的生物利用性适应每个个体(特别是我们强大的共生生物)的特定需求的组成和加工技术将是进入个性化营养新时代的起点(参考文献3)。
参考文献1-3
参考文献1
https://www.quora.com/What-are-the-next-generation-functional-foods
参考文献2
https://ec.europa.eu/jrc/en/publication/eur-scientific-and-technical-resear ch-reports/functional-food-european-union
Serafini M,Stanzione A,Foddai S.Functional foods:traditional use andEuropean legislation.Int J Food Sci Nutr.2012 Mar;63 Suppl 1:7-9.doi:10.3109/09637486.2011.637488.Epub 2011 Nov 28
参考文献3
Ercolini D.,Fogliano V.Food Design To Feed The Human Gut Microbiota.JAgric Food Chem.2018 Apr 18;66(15):3754-3758.doi:10.1021/acs.jafc.8b00456.Epub 2018 Apr 5.
目前,基于与以下有关的已公开的信息(参考文献4-7),奶和乳制品被称为新的潜在功能性食品:
抗氧化特性
参考文献4:Khan IT,Nadeem M,Imran M,Ullah R,Ajmal M,JaspalMH.Antioxidant properties of Milk and dairy products:a comprehensive reviewof current knowledge.Lipids Health Dis.2019;18(1):41.PubIished 2019 Feb4.doi:10.1186/s12944-019-0969-8
调节宿主[大鼠]肠道微生物组的潜在能力
参考文献5:Rettedal EA,Altermann E,Roy NC and Dalziel JE(2019)TheEffects of Unfermented and Fermented Cow and Sheep Milk on the GutMicrobiota,Front.Microbiol.10:458.doi:10.3389/fmicb.2019.00458
对人体免疫功能的影响的部分调查
参考文献6:Perdijk O,van Splunter M,Savelkoul HFJ,Brugman S,vanNeerven RJJ.Cow's Milk and lmmune Function in the Respiratory Tract:PotentialMechanisms.Front Immunol.2018;9:143.Published 2018 Feb 12.doi:10.3389/fimmu.2018.00143
脂肪酸含量作为能量指标
参考文献7:Arch.Anim.Breed.,60,205-212,2017 https://doi.org/10.5194/aab-60-205-2017,Milk fatty acid composition as an indicator of energy statusin Holstein dairy cows;Lana Vrankovic',Jasna Aladrovic,Daria Octenjak,DusankaBijelic,Luka Cvetnic',and Zvonko Stojevic
还已知,脂肪酸的组成是动物饲料和动物良好生活条件的结果。
参考文献8:
http://www.megalac.com/about/news/154-fatty-acids-in-feeding-programmes-for-dairy-cows
作为观察性、前瞻性和荟萃分析(meta-analysis)研究的一部分,先前的研究已推测食品中脂肪和血浆脂肪酸的组成造成胰岛素抵抗和代谢综合症的发展。
参考文献9:
Lee YS,Cho Y;Shin MJ.Dietary Very Long Chain Saturated Fatty Acidsand Metabolic Factors:Findings from the Korea National Health and NutritionExamination Survey 2013.Clin Nutr Res.2015;4(3):182-189.doi:10.7762/cnr.2015.4.3.182.
发明内容
我们提出了新的功能性乳制品产品,其特征在于检测和鉴定脂肪酸以及奶原产地的典型的天然共生微生物的特定比率的特定组合。由此确定的脂肪酸和共生微生物的组合相互作用如下:
-长期储存的鲜奶的天然防腐剂。同时,它们为下游乳制品创造营养条件,以及经过改良的巴氏灭菌工艺(温度降低为56℃)后的储存过程和在奶中生长的共生微生物的添加协同—结果1,它们创造了一个新的乳制品生产工艺,其包括向奶生产工艺中添加一个新步骤(步骤3—选定的微生物的分离),该步骤必须在步骤4)之前进行,即在奶的热处理(巴氏灭菌)之前进行。随后,添加新的步骤5)奶的富集,即将步骤3)中分离的微生物返回至经巴氏灭菌的奶中。使用共生微生物进行这些步骤,从而提供协同生长;
-结果2:防止其他潜在致病微生物的繁殖,使奶在冰箱中以鲜活的形式保鲜10天,其然后转化为酸奶/酸味乳制品;
-结果3:恢复奶和乳制品活性对肠道微生物群落(拟杆菌属(主要是脆弱拟杆菌和多形拟杆菌)、副杆菌属(P-distasonis)、柯林斯菌属、脱硫弧菌属、罗斯氏菌属(食葡糖罗斯氏菌、肠道罗斯氏菌)、真杆菌属(霍氏真杆菌)的水平升高)的有益影响;
-结果4:恢复奶和乳制品活性对肠道代谢物谱的有益影响,即如实验小鼠(BALB/c谱系小鼠,即白鼠育种系)中所发现的,实验小鼠肠道中短链脂肪酸的调节与丁酸和丙酸产生增加和乙酸减少的组合,导致肠道微生物的健康组合的调节。
-结果5:新的功能性乳制品(下一代功能性食品)的特征在于,与原始健康的脂肪酸组成相似的组成—其由固定的独特比率的ω-3和ω-6脂肪酸以及长链脂肪酸限定。我们的下一代与斯洛伐克东部和中部相关的种族功能性食品中典型的脂肪酸具有以下脂肪酸组成:0.81%的ω-3c-亚麻酸(C18:3n3)、2.15%的ω-6亚油酸(C18:2n6c)(短链脂肪酸)和0.22%的花生酸(20:0)和0.11%的山嵛酸(极长链脂肪酸),具有针对代谢综合症风险检测的保护作用。
-结果6:通过增加肠内容物中SIgA(分泌型免疫球蛋白A)的产生(高达150mg/g粪便)调节局部粘膜免疫反应;
-结果7:通过上述过程鉴定并从奶中分离的有用的希氏肠球菌和副干酪乳杆菌菌株的组合物替代了与肥胖和心血管疾病相关的机会致病性肠球菌。
总之,本申请提出了一种新一代乳制品的生产工艺,该新一代乳制品具有独特脂肪酸组成和八种原始乳制品共生菌的微生物混合物,其对食用者的肠道微生物及其代谢物(短链脂肪酸—丁酸、丙酸,但不包括乙酸)的产生具有积极影响,同时刺激肠道中SigA(分泌型免疫球蛋白A)的产生。这提供了局部免疫反应;这些过程一起允许预防导致非传染性疾病(例如心血管疾病、肥胖症或2型糖尿病)的低度炎症。
我们提供了新一代功能性产品,该产品基于共生细菌的独特比率和所选择的固定奶脂肪酸成分,其能够调节有用的肠道微生物组,从而调节它们的代谢物和局部粘膜免疫反应。
通过所提出的方法产生的具有天然获得的长链脂肪酸组成与八种选定的共生细菌菌株的比率的结合的奶是一种新型的种族功能性食品。这样的产品已被证明具有通过靶向宿主肠道微生物群落及其代谢物和抗炎局部免疫反应(如通过实验室小鼠所证实)预防非传染性疾病的能力。
乳制品的细菌部分对肠道微生物群落具有有益影响,因为拟杆菌属(主要是脆弱拟杆菌和多形拟杆菌)、副杆状菌属(P.distasonis)、柯林斯菌属、脱硫弧菌属、罗斯氏菌属(Roseburia imulinivorans、肠道罗斯氏菌)、真杆菌属(霍氏真杆菌)细菌水平的升高(调节),导致短链脂肪酸(例如丁酸、丙酸,但不包括乙酸)的产生增加。短链脂肪酸通过一系列与肠道屏障功能、葡萄糖稳态、免疫调节、食欲调节和肥胖相关的组织特异性机制直接调节宿主健康。
短链脂肪酸的最大影响以及分泌型免疫球蛋白A对宿主代谢健康的影响通过它们在组织上皮屏障以维持肠道完整性和防止细菌炎症分子通过肠壁转运中的重要性得到证明。
以下类型的细菌已被研究并鉴定为来自斯洛伐克东部和中部山区的牛奶的共生有益微生物:
编号 | 微生物:名称 | CFU/ml* | CFU/l** |
1 | 粪肠球菌SP | 2x10<sup>5</sup> | 2x10<sup>8</sup> |
2 | 粪肠球菌Ccj | 3x10<sup>5</sup> | 3x10<sup>8</sup> |
3 | 希氏肠球菌Klk | 2x10<sup>5</sup> | 2x10<sup>8</sup> |
4 | 植物乳杆菌SP | 2x10<sup>3</sup> | 2x10<sup>6</sup> |
5 | 植物乳杆菌AN | 1x10<sup>2</sup> | 1x10<sup>5</sup> |
6 | 副干酪乳杆菌TD | 1x10<sup>2</sup> | 1x10<sup>5</sup> |
7 | 链状乳杆菌SB | 4x10<sup>2</sup> | 4x10<sup>5</sup> |
8 | 马克斯克鲁维酵母SB | 7x10<sup>2</sup> | 7x10<sup>5</sup> |
*每毫升奶的菌落形成单位
**每升奶的菌落形成单位
在世界的其他地区形成这种微生物分离,或通过从其他类型的奶(即不是从牛奶中,而是从山羊奶或绵羊奶)中分离的情况下,则具有进行研究和分离的地区特征性的其他有益共生微生物的物种可以被分离。
此外,奶应该获自山区中相似的繁殖和饲养区域,该山区具有经检测的且固定的营养组成以确保奶的适当化学成分和独特的脂肪酸谱。
因此,最终获得的分离微生物的含量和规格取决于获得奶和随后通过本附录中所述方法加工的地点和地区。
上述细菌的回收如下进行:
奶或酸味乳制品的样品是从不同奶生产商原地局部(当地,即从农场)采集。就我们的特殊情况而言,我们从斯洛伐克东部和中部山区的农场收集样品。但是,以下程序可在世界任何其他地区进行。
将奶或酸味乳制品收集至无菌容器中,该无菌容器具有60ml和120ml的不同体积,并按照确定用于实验室分析的每个农场至少3公升以进行一次分析的量收集。
所有农场(农家庭院)的研究重复三次,并进行牛奶或酸味乳制品的多次取样—每个农场至少三个样品。
将天然(未稀释)奶以未稀释的形式分别添加至MRS(http://himedialabs.com/TD/M641.pdf)肉汤培养基以收集有用的微生物(10ml奶或测试的酸味乳制品(kefir)和40ml MRS肉汤培养基)。MRS是指“de Man、Rogosa和Sharpe培养基”。
为了分离乳酸菌:将在试管(Eppendorf)中用盐水溶液连续稀释(从10-1至10-9)的乳酸杆菌,乳球菌,样品接种至MRS琼脂和肉汤中。为了分离其他有用的微生物,将样品接种至其他不同的选择性培养基中(所有培养基均由HIMEDIA品牌制造;http://www.himedialabs.com/intl/en/products/100000027/Microbiology)。
为了分离双歧杆菌(双歧杆菌属菌株)并随后储存它们,使用半液体Blaurock琼脂和bifid琼脂。
http://www.bimedialab.com/TD/M1396.pdf
为了分离肠球菌,肠球菌(Enterococcus)及其后续培养物,使用肠球菌琼脂和胆汁七叶灵琼脂;http://himedialabs.com/TD/M1580.pdf;
为了确保链球菌的生长(分离、培养、再接种),使用链球菌琼脂;http://himedialabs.com/TD/M248.pdf;
为了检测其他有用的微生物和伴生微生物群落,将所有样品类似地采集并接种至培养基,例如Sabourauda琼脂、营养琼脂/肉汤、葡萄球菌琼脂、亚硫酸铋琼脂、沙门志贺氏菌琼脂、Endo或MacConkey琼脂、梭菌肉汤/琼脂、耶尔森氏菌琼脂、碳水化合物肉汤等。
通过16S rRNA基因测序检测所有其他未培养或极敏感的微生物。
使用Illumina测序试剂盒和StrainSeeker程序进行所有分离的共生微生物的基因组测序。
在15℃、37℃和45℃的不同温度模式下培养接种的样品。
24小时后,将100μl的富集样品接种至MRS琼脂上,并在37℃厌氧条件下培养48小时。
此外,进行试验以分离和鉴定乳制品和其他有用微生物以及所有伴生微生物。具体而言,进行:
常规测试,如革兰氏染色和生化测试(过氧化氢酶、氧化酶、吲哚产生),以及生理测试,例如运动研究、不同温度(15-37-45℃)下的生长,以及糖类测试,例如蔗糖、乳糖、麦芽糖、海藻糖和半乳糖的发酵。
因此,使用常规微生物测试进行乳酸菌的初步鉴定,其中包括革兰氏染色用以检测形态、过氧化氢酶和氧化酶测定、运动力测试、吲哚产生、15°℃下的生长和糖发酵(阿拉伯糖、果糖、乳糖、甘露醇、水杨苷、蔗糖和海藻糖)。
使用半自动或自动生化测试系统(由Lachema提供)进行最终确认鉴定。
在该过程和研究中,我们使用了以下测试:
1)ANAEROtest23
https://www.erbalachema.com/attachments/ANAEROtest23_CZ_SK_EN_RU_PL_R.
2)CANDIDA-Screen
https://www.erbalachema.com/attachments/CANDIDA-Screen_CZ_SK_EN_PL_RU_E.pdf
3)EN-OCCUS测试
https://www.erbalachema.com/attachments/EN-COCCUStest_CZ_S K_EN_RU_PL_P.pdf
生化鉴定后,进行MALDI蛋白质组学鉴定。
对用于制备随后以即食形式添加至膳食的有用微生物的混合物的菌株进行测序。为每个菌株准备了一份用于培养的随附文件。同时,研究并确认它们的协同作用。
进行各种体外测定和实验以确认非致病性、无毒性和非产毒性,以及有用的特性的存在。
有用的微生物包括具有以下已证明特征的菌株:
1)它们是非致病性的;
2)它们是无毒的;
3)它们是非产毒的;
4)它们能够发酵奶并有助于有用代谢物(例如乳酸、维生素等)的产生;
5)它们的特征在于针对致病菌和条件致病菌的抗菌和拮抗特性谱;
6)它们能够刺激身体免疫反应;
7)它们对大多数抗生素没有抗性。
根据微生物的分类法以及其他特征被归类为伴生微生物群落的其他微生物仅以卫生评估奶的质量为目的进行分析。
鉴于上述情况,可以简要说明从奶中获得天然共生微生物群落需要的以下步骤:
1)按照卫生要求从奶牛、山羊或绵羊中提取奶;
2)将微生物接种至各自的选择性培养基;
3)所有微生物的定性和定量含量检测;
4)通过接种彻底分离纯培养物;
5)检测它们的生物学特性;
6)测定它们的无害性;
7)通过应用标准常规程序对分离的菌株的鉴定;
8)使用测试系统和MALDI的鉴定;
9)测序;
10)进行冻干。
关于上述获得的细菌的保留(储存)的详细信息:
2)将培养物另外冷冻在具有15%甘油的MRS保护培养基中,-80℃保存;
3)菌株冷冻干燥并保存在安瓿中。每种已鉴定的斯洛伐克东部和中部地区典型的细菌物种(即粪肠球菌SP、粪肠球菌Ccj、希氏肠球菌Klk;植物乳杆菌SP、植物乳杆菌AN、副干酪乳杆菌TD、链状乳杆菌SB和马克斯克鲁维酵母SB)都具有颁发的菌株认证和无害证书。
通过上述方法将获得的天然微生物群落添加至巴氏灭菌后的鲜奶中。如果这些微生物应以粉末形式使用,则必须规定一个程序,通过应用该程序将细菌(通过上述方法获得)即时保存。为了实现预期的目标,需要做到以下几点:
1)制备包含八种有用的微生物菌种之一的纯(预先接种至适合每种细菌和条件的培养基中)液体(肉汤)每日(18-24小时)培养基;
2)通过特定或共同培养,在平板上或储存罐(发酵罐)中同时培育几种菌株(所有乳酸菌一起和所有肠球菌一起,且马克斯克鲁维酵母单独),每种菌株的浓度至少为109-1011CFU/ml;
3)制作1、10l等的必要量的液体混合物;
4)冷冻干燥(通过升华方法:一种过程,其涉及最初冷冻混合物的快速加热,通常从-40℃到+40℃,并添加保护介质)以获得有用微生物的混合物的粉末;
5)包装进袋子,每升饮料的剂量为约1-10g干燥的微生物混合物(取决于每1克它们的量和推荐剂量);
6)将粉末包装入由天然材料制成的防潮、防水包装。袋子需要在无菌条件下包装。
本发明实施方案的实施例
本发明,一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,其可用于生产饮用奶和其他对人体健康有益的乳制品,以获得其中有益微生物被保留且未受到巴氏灭菌破坏的奶和乳制品;此外,此类奶或乳制品含有最适合产奶地区人群的特定脂肪酸比率。
本发明还可用于获得促进人类健康的共生微生物群落,该共生微生物群落随后以粉末形式被储存。这是一种乳制品补充剂。这种粉末可作为单独的产品提供,例如通过将其添加至这些有益物质已耗尽的饮用奶中。含有原始微生物的粉末将在食用前被即时添加至标准(甚至目前)可得的饮用奶中。
本发明还可用于制备复合奶饮料,无论是天然无味奶饮料还是调味奶饮料(例如用可可、香草、草莓等调味)。因此,在这种情况下,分离和繁殖的健康有益细菌将被添加至奶粉中,并且可通过添加水制备得到的速溶饮料。
本发明不仅可以应用于牛奶,而且可在相同条件下和通过相同程序应用于山羊奶或绵羊奶。
Claims (5)
1.一种通过恢复共生天然微生物群落来生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,其特征在于以下事实:所述方法允许通过从来自特定地理区域的牛奶、山羊奶或绵羊奶中鉴定并分离来获得天然共生微生物群落,而这种微生物群落最适合所述奶的原产地区的人群食用,但经常被作为标准的奶生产和加工的一部分的巴氏灭菌破坏;因此,由此获得的天然共生微生物群落包含共生微生物群落的分离菌株,所述分离菌株随后被工业繁殖并添加回到巴氏灭菌后的饮用奶中,或者所述微生物群落被加工并以即食粉末形式与基料结合。
2.一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和奶补充剂的方法,其特征在于以下事实:所述方法允许通过获得在斯洛伐克东部和中部山区的牛奶中典型的以下天然共生物种:粪肠球菌SP、粪肠球菌Ccj、希氏肠球菌Klk、植物乳杆菌SP、植物乳杆菌AN、副干酪乳杆菌TD、链状乳杆菌SB和马克斯克鲁维酵母SB,如从牛奶鉴定并分离的,且随后工业繁殖、加工并添加回到饮用奶中,或者这些细菌随后被工业繁殖、加工并以即食粉末形式与基料结合,其最适合斯洛伐克东部和中部地区的人群食用。
3.一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,其特征在于以下事实:所述方法允许获得斯洛伐克东部和中部山区获得的牛奶中典型的以下天然共生细菌物种:2.105CFU/ml的量的粪肠球菌SP、3x105 CFU/ml的粪肠球菌Ccj、2x105 CFU/ml的希氏肠球菌Klk、2x103 CFU/ml的植物乳杆菌SP、1x102 CFU/ml的植物乳杆菌AN、1x102 CFU/ml的副干酪乳杆菌TD、4x102 CFU/ml的链状乳杆菌SE和7x102 CFU/ml的马克斯克鲁维酵母SB,其自牛奶被鉴定并分离,且随后被工业繁殖、加工并添加回到饮用奶中,和这些细菌随后被工业繁殖、加工并以即食粉末形式与基料结合,其最适合斯洛伐克东部和中部地区的人群食用。
4.一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,其特征在于以下事实:所述方法允许通过从特定地理区域的牛奶、绵羊奶或山羊奶鉴定并分离获得独特脂肪酸组成,并且这类脂肪酸被用于最适合产奶地区人群食用的奶中,并可作为鲜奶被食用,或者这些脂肪酸可被额外添加至饮用奶中,或者这些脂肪酸可被加工并以即食粉末形式与基料结合。
5.一种通过恢复共生天然微生物群落生产具有独特脂肪酸组成的奶和乳制品补充剂的方法,其特征在于以下事实:所述方法允许获得具有特定比率的以下脂肪酸的以下独特脂肪酸组成:0.81%的ω-3α-亚麻酸(C18:3n3)、2.15%的ω-6亚油酸(C18:2n6c)(短链脂肪酸)、0.22%的花生酸(20:0)和0.11%的山嵛酸(22:0)(极长链脂肪酸),所述独特脂肪酸组成是获自斯洛伐克东部和中部山区的牛奶中典型的,且这些脂肪酸被用于奶中,其最适合斯洛伐克东部和中部地区的人群食用,在所述地区其可作为鲜奶食用,或者这些脂肪酸可被额外添加至饮用奶中,或者这些脂肪酸可被加工并以即食粉末形式与基料结合。
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PCT/SK2019/000004 WO2020231347A2 (en) | 2019-05-15 | 2019-07-22 | A method for producing milk and dairy supplements with a unique fatty acid composition, by restoring the commensal natural microbiota |
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EP3738438A1 (en) | 2020-11-18 |
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