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CN102399730A - A kind of lactic acid bacteria with high exopolysaccharide production and its use and exopolysaccharide produced - Google Patents

A kind of lactic acid bacteria with high exopolysaccharide production and its use and exopolysaccharide produced Download PDF

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CN102399730A
CN102399730A CN2011104029503A CN201110402950A CN102399730A CN 102399730 A CN102399730 A CN 102399730A CN 2011104029503 A CN2011104029503 A CN 2011104029503A CN 201110402950 A CN201110402950 A CN 201110402950A CN 102399730 A CN102399730 A CN 102399730A
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lactic acid
acid bacteria
exopolysaccharide
yogurt
milk
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CN102399730B (en
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王昌禄
高鑫
陈勉华
王玉荣
李风娟
李贞景
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Tianjin University of Science and Technology
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Abstract

The invention relates to a lactobacillus for high yield of exopolysaccharide, an application thereof and the exopolysaccharide produced by the lactobacillus, wherein the WG3-9 strain has the classification name: lactococcus lactis subsp cremoris, with a deposit number: CGMCC No. 5260. The high-yield exopolysaccharide lactobacillus WG3-9 screened by the invention can produce exopolysaccharide with high yield, and the content of the exopolysaccharide reaches 434.3mg/L, which is obviously higher than that of other lactobacillus. The experiment proves that the acidity of the yogurt fermented by the strain WG3-9 is 78 DEG T, the viscosity of the yogurt is 8600mPa & s, the curding time is 5.5h, the water holding capacity of the yogurt is 29.5%, and the syneresis sensitivity is 20%.

Description

一种高产胞外多糖的乳酸菌及其用途及所产生的胞外多糖A kind of lactic acid bacteria with high exopolysaccharide production and its use and exopolysaccharide produced

技术领域 technical field

本发明属于微生物领域,涉及一种新筛选的微生物,尤其是一种高产胞外多糖的乳酸菌及其用途及所产生的胞外多糖。The invention belongs to the field of microorganisms, and relates to a newly screened microorganism, in particular to a lactic acid bacterium with high production of extracellular polysaccharides, its use and the produced extracellular polysaccharides.

背景技术 Background technique

发酵乳是指用脱脂乳或全脂乳经特殊微生物发酵而成的制品,在销售前必须保持微生物的活性,不得含有任何致病菌。美国、日本、欧洲及我国食品市场的信息显示,酸乳正朝着保健功能强、风味更饱满、柔和的方向发展。具有功能性的乳酸菌应用广泛,但我国具有自主知识产权的菌株较少,因此,我国急需开发风味优良、功能性强的乳酸菌。Fermented milk refers to products fermented with skim milk or whole milk by special microorganisms, which must maintain the activity of microorganisms before sale and must not contain any pathogenic bacteria. Information from the food markets in the United States, Japan, Europe and my country shows that yogurt is developing in the direction of strong health care functions, fuller and softer flavors. Functional lactic acid bacteria are widely used, but there are few strains with independent intellectual property rights in my country. Therefore, there is an urgent need to develop lactic acid bacteria with excellent flavor and strong functionality in our country.

乳酸菌胞外多糖(Exopolysaccharides,EPS)是乳酸菌在生长代谢过程中分泌到细胞壁外的一类糖类化合物,有的依附于微生物细胞壁形成荚膜,称为荚膜多糖;有的则进入培养基形成粘液,称为粘液多糖,是微生物适应环境的产物。乳酸菌EPS具有显著的生理功能:(1)抗癌、抗肿瘤作用;(2)促进益生菌在肠道粘膜上的吸附。(3)对免疫系统的调节有促进作用。(4)对细胞的保护作用。另外,乳酸菌EPS能够显著改善乳制品的结构与质地。在酸乳生产过程中,常出现粘度低、凝块破碎和大量乳清析出等问题,这些问题可以通过使用产EPS的乳酸菌解决。利用高产EPS的乳酸菌可以提高酸乳粘度,减少乳清析出和颗粒感,在大规模生产搅拌型酸乳时,有利于保持产品的均匀性。含有EPS的酸乳在经泵抽、搅拌和灌装时,可降低机械破坏程度,对热和物理应激的抵抗能力增强,使酸乳生产不添加或少添加稳定剂,满足消费者对纯天然食品的需求。此外,乳酸菌胞外多糖还可作为增稠剂、稳定剂、乳化剂、胶凝剂、填充剂和持水剂,广泛用于食品、制药、石油化工以及生化产品等领域。Lactic acid bacteria exopolysaccharides (Exopolysaccharides, EPS) are a type of carbohydrate compound secreted by lactic acid bacteria outside the cell wall during their growth and metabolism. Some of them attach to the microbial cell wall to form capsules, called capsular polysaccharides; Mucus, called mucopolysaccharides, is a product of microbial adaptation to the environment. Lactic acid bacteria EPS has significant physiological functions: (1) anti-cancer and anti-tumor effects; (2) promote the adsorption of probiotics on the intestinal mucosa. (3) It has a promoting effect on the regulation of the immune system. (4) Protective effect on cells. In addition, lactic acid bacteria EPS can significantly improve the structure and texture of dairy products. During the production of yogurt, problems such as low viscosity, broken clots, and a large amount of whey precipitation often occur. These problems can be solved by using lactic acid bacteria that produce EPS. The use of lactic acid bacteria with high EPS production can increase the viscosity of yogurt, reduce whey precipitation and graininess, and help maintain the uniformity of the product when producing stirred yogurt on a large scale. When yogurt containing EPS is pumped, stirred and filled, the degree of mechanical damage can be reduced, and the resistance to heat and physical stress can be enhanced, so that yogurt can be produced without adding or adding less stabilizer, satisfying consumers' desire for pure milk. Natural food needs. In addition, lactic acid bacteria exopolysaccharides can also be used as thickeners, stabilizers, emulsifiers, gelling agents, fillers and water-holding agents, and are widely used in food, pharmaceutical, petrochemical and biochemical products and other fields.

发酵酸乳按发酵温度可分为两个主要类型:嗜热型乳酸菌发酵和嗜温型乳酸菌发酵。目前,在市售酸乳中,主要以嗜热链球菌和保加利亚乳杆菌作为主要发酵剂,在40℃左右进行发酵的高温发酵型酸乳,而由嗜温型乳酸菌发酵的酸乳主要以手工作坊的形式流传于民间,尤其在少数民族聚居的偏远牧区,因此,嗜温型乳酸菌发酵产品亟待开发。嗜温型乳酸菌最适生长温度介于20~30℃之间,常用菌种包括乳球菌和明串珠菌等,与高温发酵酸乳相比,中温发酵酸乳的发酵时间较长,酸度温和,挥发性风味物质更多,表观粘度更大,脱水收缩作用敏感性小,这就决定了中温发酵乳具有广阔的发展前景。Fermented yogurt can be divided into two main types according to the fermentation temperature: fermented by thermophilic lactic acid bacteria and fermented by mesophilic lactic acid bacteria. At present, among the commercially available yogurts, Streptococcus thermophilus and Lactobacillus bulgaricus are mainly used as the main starters, and high-temperature fermented yogurts are fermented at about 40°C, while yogurts fermented by mesophilic lactic acid bacteria are mainly fermented by hand. The form of workshops has spread among the people, especially in remote pastoral areas where ethnic minorities live in compact communities. Therefore, mesophilic lactic acid bacteria fermentation products need to be developed urgently. The optimum growth temperature for mesophilic lactic acid bacteria is between 20°C and 30°C. The commonly used strains include Lactococcus and Leuconostoc, etc. Compared with high-temperature fermented yogurt, medium-temperature fermented yogurt has a longer fermentation time and mild acidity. There are more volatile flavor substances, higher apparent viscosity and less sensitivity to syneresis, which determine that medium-temperature fermented milk has broad development prospects.

发明内容Contents of the invention

本发明的目的在于克服现有技术的不足之处,提供一种高产胞外多糖的乳酸菌及其用途及所产生的胞外多糖,本菌种在中温条件下发酵能够延缓乳酸菌产酸过程,发酵后所得发酵乳的酸度柔和,产生更多典型酸乳挥发性风味物质,产品风味饱满;此外,中温发酵工艺可降低生产能耗。The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a lactic acid bacterium with high exopolysaccharide production and its use and the exopolysaccharide produced. Fermentation of this strain at medium temperature can delay the acid production process of lactic acid bacteria. The acidity of the obtained fermented milk is mild, and more typical yogurt volatile flavor substances are produced, and the product flavor is full; in addition, the medium temperature fermentation process can reduce production energy consumption.

本发明的目的是通过以下技术方案实现的:The purpose of the present invention is achieved through the following technical solutions:

一种高产胞外多糖的乳酸菌,名称为WG3-9,菌株的分类名称:乳酸乳球菌乳脂亚种Lactococcus.Lactis subsp cremaris,保存编号为:CGMCC No.5260,保藏日期为:2011年09年20日,保藏单位为:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号。A lactic acid bacterium with high exopolysaccharide production, the name is WG3-9, the taxonomic name of the strain: Lactococcus. Today, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

而且,所述乳酸菌高产胞外多糖,其胞外多糖产量为:434.3mg/L。Moreover, the lactic acid bacteria highly produce exopolysaccharide, and the exopolysaccharide output is: 434.3mg/L.

而且,所述乳酸菌发酵酸乳的最适发酵温度为30℃。Moreover, the optimal fermentation temperature of the lactic acid bacteria fermented yoghurt is 30°C.

一种高产胞外多糖的乳酸菌作为酸乳发酵剂的用途。The use of a lactic acid bacterium with high exopolysaccharide production as a yogurt starter.

一种高产胞外多糖的乳酸菌发酵酸乳的方法,方法如下:将乳酸菌按5%--10%的接种量接种于含全脂牛乳的容器中,30℃静置培养至牛乳凝固,4℃保持16h,得到酸乳。A method for fermenting yoghurt with lactic acid bacteria with high production of extracellular polysaccharides, the method is as follows: inoculate lactic acid bacteria in a container containing whole milk according to the inoculum amount of 5%-10%, culture at 30°C until the milk coagulates, and 4°C Keep it for 16 hours to get yoghurt.

而且,所述乳酸菌为先活化的乳酸菌。Moreover, the lactic acid bacteria are first activated lactic acid bacteria.

而且,所述乳酸菌的活化方法为:将保藏于脱脂牛乳培养基中的乳酸菌,按5%接种量接入灭菌脱脂牛乳培养基中,30℃静置培养48h进行活化。Moreover, the activation method of the lactic acid bacteria is as follows: the lactic acid bacteria preserved in the skim milk medium are inserted into the sterilized skim milk medium according to the inoculation amount of 5%, and the lactic acid bacteria are statically cultured at 30° C. for 48 hours for activation.

一种胞外多糖,由上述的高产胞外多糖的乳酸菌发酵得到。An exopolysaccharide obtained by fermentation of the above-mentioned high-yield exopolysaccharide lactic acid bacteria.

本发明的优点和积极效果如下:Advantage of the present invention and positive effect are as follows:

1.本发明筛选出的高产胞外多糖乳酸菌WG3-9能够高产胞外多糖,其含量达434.3mg/L,显著高于其它乳酸菌。实验测得菌株WG3-9发酵所得的酸乳酸度为78°T,酸乳粘度为8600mPa·s,凝乳时间为5.5h,酸乳持水力为29.5%,脱水收缩敏感性为20%。1. The exopolysaccharide-producing lactic acid bacteria WG3-9 screened out by the present invention can produce exopolysaccharides at a high rate, and its content reaches 434.3 mg/L, which is significantly higher than other lactic acid bacteria. The acidity of the yogurt fermented by the strain WG3-9 was 78°T, the viscosity of the yogurt was 8600mPa·s, the curdling time was 5.5h, the water holding capacity of the yogurt was 29.5%, and the syneresis sensitivity was 20%.

2.本发明筛选出的高产胞外多糖乳酸菌WG3-9,可在30℃发酵酸乳,是一种中温型发酵菌株,在中温条件下发酵能够延缓乳酸菌产酸过程,发酵后所得发酵乳的酸度柔和,产生更多典型酸乳挥发性风味物质,产品风味饱满;此外,中温发酵工艺可降低生产能耗。2. The high-yield exopolysaccharide lactic acid bacteria WG3-9 screened out by the present invention can ferment yogurt at 30°C. It is a mesophilic fermentation strain. Fermentation under mesophilic conditions can delay the acid production process of lactic acid bacteria, and the fermented milk obtained after fermentation The acidity is mild, and more typical yogurt volatile flavor substances are produced, and the product has a full flavor; in addition, the medium temperature fermentation process can reduce production energy consumption.

3.本发明筛选出的高产胞外多糖乳酸菌WG3-9发酵所得发酵酸乳粘稠,本菌株产生的胞外多糖能够显著改善乳制品的结构与质地,使产品无需添加稳定剂,满足消费者对纯天然食品的需求。3. The fermented yogurt obtained from the fermentation of the high-yielding exopolysaccharide lactic acid bacteria WG3-9 screened out by the present invention is viscous. The exopolysaccharide produced by this strain can significantly improve the structure and texture of dairy products, so that the product does not need to add stabilizers and satisfies consumers. Demand for all-natural foods.

附图说明: Description of drawings:

图1为本发明乳酸菌WG3-9的菌落照片;Fig. 1 is the colony photo of lactic acid bacteria WG3-9 of the present invention;

图2为本发明乳酸菌WG3-9的电子显微镜照片。Fig. 2 is an electron micrograph of lactic acid bacteria WG3-9 of the present invention.

具体实施方式 Detailed ways

下面结合实施例,对本发明进一步说明,下述实施例是说明性的,不是限定性的,不能以下述实施例来限定本发明的保护范围。Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

下述实施实例中所使用的实验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。The experimental methods used in the following implementation examples are conventional methods unless otherwise specified; the materials and reagents used, etc., can be obtained from commercial sources unless otherwise specified.

M17固体培养基:多聚蛋白胨5g/L,植物蛋白胨5g/L,牛肉膏5g/L,酵母膏2.5g/L,β-甘油磷酸二钠19g/L,抗坏血酸0.5g/L,MgSO4·7H2O 0.25g/L,乳糖5g/L,琼脂15g/L,调节pH至7.1。M17 solid medium: polypeptone 5g/L, plant peptone 5g/L, beef extract 5g/L, yeast extract 2.5g/L, β-glycerophosphate disodium 19g/L, ascorbic acid 0.5g/L, MgSO 4 · 7H 2 O 0.25g/L, lactose 5g/L, agar 15g/L, adjust the pH to 7.1.

MRS固体培养基:蛋白胨10g/L,牛肉膏10g/L,酵母提取物5g/L,K2HPO4 2g/L,柠檬酸二铵2g/L,乙酸钠5g/L,葡萄糖20g/L,吐温80 1mL,MgSO4·7H2O 0.58g/L,MnSO4·4H2O 0.25g/L,琼脂20g/L,调节pH至6.4。MRS solid medium: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, K 2 HPO 4 2g/L, diammonium citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, Tween 80 1mL, MgSO 4 ·7H 2 O 0.58g/L, MnSO 4 ·4H 2 O 0.25g/L, agar 20g/L, adjust the pH to 6.4.

牛乳营养琼脂培养基:蛋白胨10g/L,牛肉膏10g/L,NaCl 5g/L,牛乳200g/L,琼脂25g/L,调节pH至5.5。Milk nutrient agar medium: peptone 10g/L, beef extract 10g/L, NaCl 5g/L, milk 200g/L, agar 25g/L, adjust the pH to 5.5.

全脂牛乳:全脂乳粉溶于蒸馏水中,浓度为100g/L。Whole milk: Whole milk powder is dissolved in distilled water with a concentration of 100g/L.

Sevage试剂:氯仿与正丁醇以4∶1混合(体积比)。Sevage reagent: Chloroform and n-butanol were mixed in a ratio of 4:1 (volume ratio).

一、菌株的分离与鉴定1. Isolation and identification of strains

1.样品稀释液制备及菌株分离1. Sample dilution preparation and strain isolation

菌株分离样品为新疆伊犁地区哈萨克族牧民自制传统发酵乳制品,酸奶酪。The isolated samples of strains were traditional fermented dairy products and yogurt made by Kazak herdsmen in Ili area, Xinjiang.

以无菌操作称取25g样品,放入装有225mL生理盐水的无菌锥形瓶中(瓶内预置适当数量的无菌玻璃珠),充分振摇,制成1∶10的样品悬浮液。Weigh 25g of sample by aseptic operation, put it into a sterile Erlenmeyer flask filled with 225mL of physiological saline (preset an appropriate number of sterile glass beads in the bottle), shake it fully, and make a sample suspension of 1:10 .

吸取1∶10样品悬浮液1mL,沿管壁缓慢注于装有9mL生理盐水的无菌试管中,振摇试管或换用1支无菌吸管反复吹打使其混合均匀,制成1∶100的样品悬浮液。另吸取1mL1∶10样品悬浮液液,按上述操作顺序,做10倍递增样品悬浮液。Draw 1mL of the 1:10 sample suspension, slowly pour it into a sterile test tube containing 9mL of normal saline along the tube wall, shake the test tube or use a sterile straw to blow repeatedly to mix evenly, and make a 1:100 sample suspension. Take another 1mL of 1:10 sample suspension, and make 10 times incremental sample suspension according to the above operation sequence.

根据待检样品活菌总数的估计,选择2~3个稀释度样品,每个稀释度吸取0.1mL样品悬浮液分别置于MRS固体培养基、M17固体培养基、牛乳营养琼脂培养基进行表面涂布,30℃培养48h。According to the estimation of the total number of viable bacteria in the sample to be tested, select 2 to 3 dilution samples, draw 0.1mL sample suspension for each dilution, and place them on MRS solid medium, M17 solid medium, and milk nutrient agar medium for surface coating. Cloth, cultured at 30°C for 48h.

2.菌株初筛2. Primary screening of strains

用接种针挑取上述平板上的单菌落,再进行划线或涂布分离纯化,对单菌落顺序编号,分别接入牛乳营养琼脂培养基/全脂牛乳培养基中,30℃培养24-48h。Use an inoculation needle to pick a single colony on the above-mentioned plate, then streak or smear to separate and purify, number the single colony sequentially, insert them into milk nutrient agar medium/whole milk medium, and incubate at 30°C for 24-48h .

形态观察:观察平板上菌落形态,挑取少量菌泥进行革兰氏染色,进行显微镜观察。Morphology observation: Observe the colony shape on the plate, pick a small amount of bacteria sludge for Gram staining, and observe under the microscope.

3.菌株复筛3. Strain re-screening

3.1凝乳实验3.1 Curd experiment

将保藏于脱脂牛乳培养基中的乳酸菌,按5%接种量接入灭菌脱脂牛乳培养基中,30℃培养48h进行活化,活化后的菌株接种于含5mL全脂牛乳培养基试管中,30℃静置培养至牛乳凝固,按5%--10%的接种量接种于含100mL全脂牛乳三角瓶中,30℃静置培养至牛乳凝固,观察并记录凝乳状态。Put the lactic acid bacteria preserved in the skim milk medium into the sterilized skim milk medium according to the inoculation amount of 5%, and activate it by culturing at 30°C for 48 hours. Cultivate statically at ℃ until the milk coagulates, inoculate at 5%-10% inoculum size into 100mL whole milk Erlenmeyer flasks, culture at 30℃ until the milk coagulates, observe and record the curdling state.

3.2粘度的测定3.2 Determination of viscosity

粘度的测定:采用流变仪(博力飞公司DV-III+型)。在4℃下测试粘度,转子型号为RV5,分别在16r/min的速度下测定三次,每15s取值一次,每个样品做两个平行试样,结果计算算术平均值。Determination of viscosity: adopt a rheometer (DV-III+ type of Bolifei Company). Viscosity was tested at 4°C, the rotor type was RV5, and the viscosity was measured three times at a speed of 16r/min, and the value was taken every 15s. Two parallel samples were made for each sample, and the arithmetic mean value was calculated.

3.3感官评定3.3 Sensory evaluation

如表1所示,通过感官评定,得到一株凝乳快、质地粘稠并且风味优良的菌株,编号为WG3-9。As shown in Table 1, through sensory evaluation, a strain with fast curdling, viscous texture and excellent flavor was obtained, numbered WG3-9.

表1复筛菌株特性评价结果Table 1 Evaluation results of re-screening strain characteristics

Figure BDA0000116851460000041
Figure BDA0000116851460000041

注:感官评价总分为口感,气味和滋味,组织状态评分之和,感官评价值为10名评价员评价的平均值。粘度值为三次测定的平均值。Note: The total sensory evaluation is divided into mouthfeel, smell and taste, and the sum of organizational state scores, and the sensory evaluation value is the average value evaluated by 10 evaluators. Viscosity values are the average of three determinations.

乳酸乳球菌乳脂亚种Lactococcus.Lactis subsp cremaris,保存编号为:CGMCC No.5260,保藏日期为:2011年09年20日,保藏单位为:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号。Lactococcus Lactis subspecies Lactococcus.Lactis subsp cremaris, preservation number: CGMCC No.5260, preservation date: September 20, 2011, preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation address: : No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

4.菌株的鉴定4. Identification of strains

4.1形态观察4.1 Morphological observation

观察平板上菌落形态,挑取少量菌泥进行革兰氏染色,用光学显微镜及电子显微镜进行观察。结果显示:菌落为乳白色,圆形,有拉丝感,边缘光滑(图1),光学显微镜观察显示:革兰氏染色呈阳性球菌,菌体成链或片状,电子显微镜显示:WG3-9为球菌,链状(图2)。Observe the colony morphology on the plate, pick a small amount of bacteria sludge for Gram staining, and observe with an optical microscope and an electron microscope. The results showed that: the colony was milky white, round, with a sense of drawing, and the edges were smooth (Figure 1). Optical microscope observation showed: Gram-positive cocci, and the bacteria were in chains or flakes. Electron microscope showed: WG3-9 was Cocci, chain-like (Fig. 2).

4.2生理生化鉴定4.2 Physiological and biochemical identification

(1)葡萄糖产气实验(1) Glucose gas production experiment

在装有MRS液体培养基的试管中倒置一个杜氏管,接入WG3-9,37℃培养24h,观察杜氏管中有无气泡生成。结果显示,WG3-9为葡萄糖产气,阴性。Invert a Duchenne tube in the test tube containing the MRS liquid medium, connect it to WG3-9, incubate at 37°C for 24 hours, and observe whether there are bubbles in the Duchenne tube. The results showed that WG3-9 was negative for glucose gas production.

(2)葡萄糖产酸实验(2) Glucose acid production experiment

在装有MRS液体培养基的试管中加入1.4mL浓度为1.6g/100mL的溴甲酚紫作为指示剂,接入WG3-9,37℃培养24h,观察培养基颜色变化。Add 1.4mL of bromocresol purple with a concentration of 1.6g/100mL in the test tube containing MRS liquid medium as an indicator, insert into WG3-9, culture at 37°C for 24h, and observe the color change of the medium.

结果显示,WG3-9为葡萄糖产酸,阳性。The results showed that WG3-9 was positive for glucose acid production.

(3)过氧化氢酶实验(3) Catalase test

用接种环挑取M17培养基上的WG3-9,涂于干净的载玻片上,在其上滴加3%的过氧化氢溶液2mL,观察有无气泡生成。Use an inoculation loop to pick up WG3-9 on M17 medium, spread it on a clean glass slide, add 2 mL of 3% hydrogen peroxide solution dropwise on it, and observe whether there are bubbles formed.

结果显示,WG3-9为过氧化氢酶阴性。The results showed that WG3-9 was negative for catalase.

4.316s rDNA鉴定4. 316s rDNA identification

挑取WG3-9菌适量,加入M17培养基中,37℃培养过夜,取1mL菌液,5000×g离心5min,弃上清液,在沉淀中加入300μL细胞裂解液,加入10μL蛋白酶K,56℃水浴1h。100℃煮10min后,12000×g离心10min,取上清液进行PCR反应。引物由英潍捷基(上海)贸易有限公司合成。引物序列如下:Pick an appropriate amount of WG3-9 bacteria, add them to M17 medium, and culture overnight at 37°C, take 1 mL of bacterial liquid, centrifuge at 5000×g for 5 min, discard the supernatant, add 300 μL of cell lysate to the pellet, and add 10 μL of proteinase K, 56 ℃ water bath for 1h. After cooking at 100°C for 10 minutes, centrifuge at 12000×g for 10 minutes, and take the supernatant for PCR reaction. Primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd. The primer sequences are as follows:

上游引物:AGAGTTTGATCCTGGCTCAGUpstream primer: AGAGTTTGATCCTGGCTCAG

下游引物:GGTTACCTTGTTACGACTTDownstream primer: GGTTACCTTGTTACGACTT

PCR产物纯化后送交英潍捷基(上海)贸易有限公司进行测序。After purification, the PCR products were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing.

测得菌株WG3-9的16s rDNA序列如序列3所示。The 16s rDNA sequence of strain WG3-9 is shown in sequence 3.

测序结果用BLAST程序与美国国立生物技术信息中心数据库中已报道的细菌16s rDNA进行比对,结果显示,WG3-9菌株的16s rDNA序列与乳酸乳球菌乳脂亚种Lactococcus.Lactissubsp cremaris的同源性大于99%。The sequencing results were compared with the reported bacterial 16s rDNA in the database of the National Center for Biotechnology Information using the BLAST program. The results showed that the 16s rDNA sequence of the WG3-9 strain was homologous to Lactococcus.Lactissubsp cremaris Greater than 99%.

由以上鉴定结果可得,菌株WG3-9为乳酸乳球菌乳脂亚种(Lactococcus.Lactis subspcremaris),该菌已于2011年09月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏号:CGMCC No.5260。From the above identification results, it can be concluded that the strain WG3-9 is Lactococcus.Lactis subspcremaris, which was preserved in the General Microorganism Center (CGMCC) of the China Committee for Culture Collection of Microorganisms on September 20, 2011. , deposit number: CGMCC No.5260.

二、乳酸菌WG3-9制备功能性酸乳2. Preparation of functional yogurt by lactic acid bacteria WG3-9

1.功能性酸乳的制备1. Preparation of functional yoghurt

将保藏于脱脂牛乳培养基中的乳酸菌,按5%接种量接入灭菌脱脂牛乳培养基中,30℃培养48h进行活化。活化后的菌接种入5mL全脂牛乳试管中,30℃静置培养至牛乳凝固,按5%--10%的接种量接种于100mL全脂牛乳三角瓶中,30℃静置培养至牛乳凝固,4℃保持16h,得到不需添加增稠剂的酸乳。The lactic acid bacteria preserved in the skimmed milk medium were inserted into the sterilized skimmed milk medium according to the inoculum amount of 5%, and cultured at 30° C. for 48 hours for activation. Inoculate the activated bacteria into a 5mL whole milk test tube, culture at 30°C until the milk coagulates, inoculate 5%--10% inoculum into a 100mL whole milk triangular flask, and culture at 30°C until the milk coagulates , kept at 4°C for 16 hours to obtain yoghurt without adding thickener.

2.酸乳理化指标测定2. Determination of physical and chemical indicators of yogurt

2.1酸乳凝乳时间的测定2.1 Determination of yogurt curdling time

活化后的菌接种于含5mL全脂牛乳的试管中,30℃静置培养至牛乳凝固,记录凝乳时间,做三组平行实验,取平均值。The activated bacteria were inoculated into a test tube containing 5mL of whole milk, and cultured at 30°C until the milk coagulated, and the curdling time was recorded, and three parallel experiments were performed, and the average value was taken.

实验测得菌株WG3-9凝乳时间为5.5h。The curd time of the strain WG3-9 was measured to be 5.5h.

2.2酸乳粘度的测定2.2 Determination of Yogurt Viscosity

采用流变仪(博力飞公司DV-III+型)。在4℃下测试粘度,转子型号为RV5,分别在16r/min的速度下测定三次,每15s取值一次,每个样品做两个平行试样,结果计算算术平均值。A rheometer (DV-III+ type of Bolifei Company) is used. Viscosity was tested at 4°C, the rotor type was RV5, and the viscosity was measured three times at a speed of 16r/min, and the value was taken every 15s. Two parallel samples were made for each sample, and the arithmetic mean value was calculated.

实验测得菌株WG3-9发酵所得的酸乳粘度为8600mPa·s。The viscosity of yoghurt fermented by strain WG3-9 was measured to be 8600mPa·s.

2.3酸乳滴定酸度的测定2.3 Determination of titrated acidity of yogurt

根据GB/T5009.46-1996,称量10g酸乳样品,置于100mL三角瓶中,加入20mL水,再加入0.5mL 0.5%的酚酞乙醇溶液,小心摇匀,用0.1mol/L的氢氧化钠标准溶液滴定至微红色在1min内不消失为止。消耗的0.1mol/L的氢氧化钠标准溶液的毫升数乘以10,即得酸度(°T),取三次测定的算术平均值。According to GB/T5009.46-1996, weigh 10g of yoghurt sample, put it in a 100mL conical flask, add 20mL of water, then add 0.5mL of 0.5% phenolphthalein ethanol solution, shake carefully, and oxidize with 0.1mol/L hydrogen Sodium standard solution is titrated until the reddish color does not disappear within 1 min. Multiply the milliliters of the consumed 0.1mol/L sodium hydroxide standard solution by 10 to get the acidity (°T), and take the arithmetic mean of three determinations.

实验测得菌株WG3-9发酵所得的酸乳酸度为78°T。The acidity of the yogurt fermented by the strain WG3-9 was measured to be 78°T.

2.4持水力的测定2.4 Determination of water holding capacity

取酸乳样品20g,在10℃下,10000g离心30min,倾去上清物,使离心管保持倒置状态10min,结束后立即称重,每个样品做三个平行试样,结果取算术平均值。Take 20g of yoghurt sample, centrifuge at 10000g for 30min at 10°C, pour off the supernatant, keep the centrifuge tube upside down for 10min, weigh immediately after the end, make three parallel samples for each sample, and take the arithmetic mean of the results .

酸乳的持水力=离心沉淀物重量/样品重量×100%Water holding capacity of yogurt = centrifuge sediment weight/sample weight × 100%

实验测得菌株WG3-9发酵所得的酸乳持水力为29.5%。The water holding capacity of the yoghurt fermented by the strain WG3-9 was measured to be 29.5%.

2.5酸乳脱水收缩敏感性的测定2.5 Determination of the sensitivity of yoghurt to syneresis

在6℃下,将50g凝固成熟后的酸乳小心倾入带有120目不锈钢丝网的大漏斗中,用100mL量筒收集沥出的乳清。收集时间为2h,最后对收集的乳清称重,每个样品做三个平行试样,结果取算术平均值。At 6°C, carefully pour 50 g of solidified and matured yogurt into a large funnel with a 120-mesh stainless steel wire mesh, and collect the leached whey with a 100 mL measuring cylinder. The collection time is 2 hours, and finally the collected whey is weighed, and three parallel samples are made for each sample, and the arithmetic mean value is taken for the results.

酸乳胶体脱水收缩作用敏感性=乳清析出量/样品重量×100%。Syneresis sensitivity of yoghurt colloid = whey precipitation/sample weight × 100%.

实验测得菌株WG3-9发酵所得的酸乳脱水收缩敏感性为20%。The experiment shows that the sensitivity of the yogurt fermented by the strain WG3-9 is 20%.

三、乳酸菌WG3-9的胞外多糖的制备3. Preparation of exopolysaccharide of lactic acid bacteria WG3-9

1.酸乳的制备1. Preparation of yoghurt

见实施例步骤二“1.功能性酸乳的制备”部分的记载。See the description in the part "1. Preparation of functional yoghurt" in the second step of the embodiment.

2.胞外多糖的提取2. Extraction of Exopolysaccharides

(1)等电点法除酪蛋白:用饱和NaOH调样液pH至8.0,4000r/min,离心15min,上清液用HCL调节pH至4.6,3000×g离心15min。(1) Casein removal by isoelectric point method: adjust pH to 8.0 with saturated NaOH, centrifuge at 4000r/min for 15min, adjust pH of supernatant to 4.6 with HCL, centrifuge at 3000×g for 15min.

(2)酶解:调pH至7.5,加入1/10体积的胰酶液(浓度为3g/L,现配现用),40℃水浴,酶解2.5h后,2000×g离心15min。(2) Enzymolysis: adjust the pH to 7.5, add 1/10 volume of trypsin solution (concentration: 3g/L, ready-to-use), 40°C water bath, enzymolysis for 2.5h, centrifuge at 2000×g for 15min.

(3)Sevag脱蛋白:加入1/3体积的Sevag液振荡30min,3000×g离心15min,去除沉淀,重复三次。(3) Sevag deproteinization: add 1/3 volume of Sevag solution, shake for 30 minutes, centrifuge at 3000×g for 15 minutes, remove the precipitate, repeat three times.

(4)乙醇沉淀:加入3-5倍体积的95%的乙醇,充分振荡(多糖称絮状沉淀析出),3000×g离心15min,收集沉淀。(4) Ethanol precipitation: add 3-5 times the volume of 95% ethanol, shake fully (polysaccharide is called flocculent precipitation), centrifuge at 3000×g for 15 minutes, and collect the precipitate.

(5)洗涤干燥:将沉淀用丙酮洗涤脱水两次,真空干燥得粗多糖。(5) Washing and drying: the precipitate was washed and dehydrated twice with acetone, and dried in vacuum to obtain crude polysaccharide.

3.粗多糖纯度测定3. Determination of the purity of crude polysaccharides

采用苯酚-浓硫酸法测定EPS含量,用葡萄糖作标准曲线。The EPS content was determined by the phenol-concentrated sulfuric acid method, and glucose was used as the standard curve.

准确吸取葡萄糖标准溶液(1mg/mL),0.05,0.06,0.07,0.08,0.09,0.1,0.12mL(相当于葡萄糖0.0,50,60,70,80,90,100,120μg),及各样品备用液0.1mL置于10mL比色管中,用蒸馏水补足1.0mL,分别加入1.0mL 5.0%苯酚水溶液,摇匀后静止2min,迅速加入5mL浓硫酸,静止10min后置于30℃水浴中加热15min,取出,置冷水中冷却,以空白管为对照,用紫外分光光度计在波长490nm处测定光密度。以葡萄糖浓度为横坐标,光密度值为纵坐标绘制标准曲线,计算胞外多糖含量。Accurately draw glucose standard solution (1mg/mL), 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12mL (equivalent to glucose 0.0, 50, 60, 70, 80, 90, 100, 120μg), and each sample for use Place 0.1mL of solution in a 10mL colorimetric tube, make up 1.0mL with distilled water, add 1.0mL 5.0% phenol aqueous solution respectively, shake well and stand still for 2min, then quickly add 5mL concentrated sulfuric acid, place it in a 30°C water bath for 15min after standing still for 10min, Take it out, cool it in cold water, and measure the optical density at a wavelength of 490nm with a UV spectrophotometer with a blank tube as a control. Draw a standard curve with the glucose concentration as the abscissa and the optical density as the ordinate to calculate the exopolysaccharide content.

测得该菌株胞外多糖产量为434.3mg/L。The exopolysaccharide output of the strain was measured to be 434.3mg/L.

Figure IDA0000116851560000011
Figure IDA0000116851560000011

Figure IDA0000116851560000031
Figure IDA0000116851560000031

Claims (8)

1.一种高产胞外多糖的乳酸菌,其特征在于:名称为WG3-9,菌株的分类名称:乳酸乳球菌乳脂亚种Lactococcus.Lactis subsp.cremaris,保存编号为:CGMCC No.5260,保藏日期为:2011年09年20日,保藏单位为:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号。1. A lactic acid bacterium with high production of extracellular polysaccharides, characterized in that: the name is WG3-9, the classification name of the strain: Lactococcus.Lactis subsp.cremaris, the preservation number is: CGMCC No.5260, and the preservation date For: September 20, 2011, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. 2.根据权利要求1所述的高产胞外多糖的乳酸菌,其特征在于:所述乳酸菌高产胞外多糖,其胞外多糖产量为:434.3mg/L。2. The lactic acid bacteria with high exopolysaccharide production according to claim 1, characterized in that: said lactic acid bacteria have high exopolysaccharide production, and the exopolysaccharide output is 434.3mg/L. 3.根据权利要求1所述的高产胞外多糖的乳酸菌,其特征在于:所述乳酸菌发酵酸乳的最适发酵温度为30℃。3. The lactic acid bacteria with high exopolysaccharide production according to claim 1, characterized in that: the optimum fermentation temperature of the yogurt fermented by the lactic acid bacteria is 30°C. 4.一种如权利要求1所述的高产胞外多糖的乳酸菌作为酸乳发酵剂的用途。4. a kind of lactic acid bacterium that produces exopolysaccharide as claimed in claim 1 is as the purposes of sour milk starter. 5.一种如权利要求1所述的高产胞外多糖的乳酸菌发酵酸乳的方法,其特征在于:方法如下:将乳酸菌按5%--10%的接种量接种于含全脂牛乳的容器中,30℃静置培养至牛乳凝固,4℃保持16h,得到酸乳。5. A method for lactic acid bacteria fermenting yogurt with high exopolysaccharide production as claimed in claim 1, characterized in that: the method is as follows: the lactic acid bacteria are inoculated in the container containing whole milk by the inoculum size of 5%--10% In 30 ° C static culture until the milk coagulation, 4 ° C for 16 hours to obtain yoghurt. 6.根据权利要求5所述的高产胞外多糖的乳酸菌发酵酸乳的方法,其特征在于:所述乳酸菌为先活化的乳酸菌。6 . The method for fermenting yoghurt with lactic acid bacteria with high exopolysaccharide production according to claim 5 , characterized in that: the lactic acid bacteria are first activated lactic acid bacteria. 7 . 7.根据权利要求6所述的高产胞外多糖的乳酸菌发酵酸乳的方法,其特征在于:所述乳酸菌的活化方法为:将保藏于脱脂牛乳培养基中的乳酸菌,按5%接种量接入灭菌脱脂牛乳培养基中,30℃静置培养48h进行活化。7. The method for lactic acid bacteria fermenting yogurt with high exopolysaccharide production according to claim 6, characterized in that: the activation method of the lactic acid bacteria is: the lactic acid bacteria preserved in the skimmed milk medium are inoculated with 5% inoculum Put into sterilized skimmed milk medium, and culture at 30°C for 48h to activate. 8.一种胞外多糖,其特征在于:由权利要求1所述的高产胞外多糖的乳酸菌发酵得到。8. An exopolysaccharide, characterized in that: it is obtained by fermentation of the high-yield exopolysaccharide lactic acid bacteria according to claim 1.
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CN104489085A (en) * 2014-12-31 2015-04-08 临沂格瑞食品有限公司 Fermented milk rich in beta-glucan and preparation process thereof
CN108072741A (en) * 2016-11-14 2018-05-25 中国农业科学院原子能利用研究所 A kind of method for measuring curdled milk bleed shrinkage
CN110050815A (en) * 2019-05-13 2019-07-26 沈阳师范大学 A kind of production method of low GI high dietary-fiber whole-wheat bread
CN110408663A (en) * 2019-08-01 2019-11-05 浙江一鸣食品股份有限公司 A kind of lactic acid bacteria with high exopolysaccharide production and its preparation method and application
CN110408663B (en) * 2019-08-01 2021-04-06 浙江一鸣食品股份有限公司 Lactobacillus with high extracellular polysaccharide yield, preparation method and application thereof
CN117062905A (en) * 2021-03-02 2023-11-14 富吉高株式会社 Novel lactic acid bacteria strain and composition comprising the same
CN117062905B (en) * 2021-03-02 2025-03-18 富吉高株式会社 New lactic acid bacteria strains and compositions containing the same

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