CN111436203A

CN111436203A - Fermented lactobacillus plantarum and application thereof

Info

Publication number: CN111436203A
Application number: CN201880030861.0A
Authority: CN
Inventors: 李慧; 陈历水; 高嘉星; 刘蕾; 刘洋; 应欣; 史玉莹; 张连慧; 倪伟; 钱承敬; 王勇; 王宇
Original assignee: Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2018-11-12
Filing date: 2018-12-27
Publication date: 2020-07-21
Anticipated expiration: 2038-12-27
Also published as: WO2020098097A1; AU2018423072A1; AU2018423072B2; CN111436203B

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to lactobacillus plantarum and application thereof, wherein the lactobacillus plantarum is classified and named as L actobacillus plantarum and stored in the common microorganism center of China Committee for culture Collection of microorganisms, the storage number of the lactobacillus plantarum is CGMCC No. 16441.

Description

Fermented lactobacillus plantarum and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation, and particularly discloses lactobacillus plantarum, a direct vat set starter containing the lactobacillus plantarum and a preparation method thereof, and application of the lactobacillus plantarum in the fields of preparing fermented dairy products, fermented grain beverages, fermented bean products, fermented fruit and vegetable products and fermented feeds, and also discloses application of the lactobacillus plantarum in preparing live bacterial preparations and the prepared live bacterial preparations.

Background

Lactic acid bacteria (L AB) are a generic name of bacteria capable of utilizing fermentable carbohydrates to generate a large amount of lactic acid, which is a group of rather numerous and complicated bacteria, at least 18 genera can be divided at present, and more than 200 species are provided, so that the lactic acid bacteria have rich species diversity.

Lactobacillus plantarum strains among lactic acid bacteria are widely used in food production, especially in fermented foods. There are studies showing that: the acid production capability and the fermentation flavor characteristics of the lactic acid bacteria play an important role in the production of fermented milk products, fermented cereal beverages, fermented bean products, fermented fruit products and fermented feeds, and can be used alone or in combination with other bacteria. Lactic acid bacteria as potential probiotics need to have the following properties: has outstanding probiotic function, is required to be resistant to gastrointestinal tract acid environment and bile salt environment, and has better cell adhesion.

The main roles of lactic acid bacteria in fermented products are acid production and flavor generation. The acid-producing characteristics of lactic acid bacteria are important characteristics for screening a fermentation product starter. Lactic acid bacteria having a strong acid-producing ability generally have a strong tendency to acidify thereafter. Lactic acid bacteria with fast acid production and large acid production amount can bring lower fermentation acidity of final products, which is an effective measure for ensuring low post-acidification (such as dairy products). As probiotics, gastric juice and intestinal juice form a biological barrier of a human body, and lactobacillus can only pass through the gastrointestinal tract and attach DIAN to epithelial cells of the intestinal tract to realize colonization so as to exert beneficial effects on the human body or animal hosts.

Exopolysaccharides (EPS) of lactic acid bacteria are polysaccharides produced by lactic acid bacteria during the growth metabolism process and secreted outside the cells, released into the growth substrate or capsular polysaccharides tightly bound to the cell surface. The functions of the following aspects are mainly realized: the product quality is improved, the product is fine and uniform, and the smooth mouthfeel is increased; the water retention is increased, the viscosity of the product is improved, the non-specific adhesion of the lactobacillus to the intestinal surface is improved, the chance of field planting of the lactobacillus is increased, and a certain promotion effect on the regulation of intestinal microbial flora is achieved.

Therefore, the compound microbial inoculum has good acid production capacity and extracellular polysaccharide production capacity, can resist gastrointestinal tract stress, has better cell adhesion, and has prominent fermentation sensory characteristics which are necessary factors for screening leavening agents and probiotics.

Disclosure of Invention

In order to solve the technical problems, the invention screens the lactobacillus plantarum which has high acid production speed, stable acid production and stable curd genetic property and unique fermented product flavor, and the lactobacillus plantarum is used for fermented dairy products, grain products, bean products, fruit and vegetable products and feed products to improve the quality of the fermented products. The lactobacillus plantarum can also tolerate the acid environment of the gastrointestinal tract, can produce extracellular polysaccharide at high yield, improves the implantation fixation of cells, is used for preparing a probiotic viable bacteria preparation, and is beneficial to regulating the composition of intestinal microbial flora.

In order to achieve the above object, the invention provides a lactobacillus plantarum, which is classified and named L actinobacillus plantarum and is deposited in China general microbiological culture Collection center with the deposit number of CGMCC No. 16441.

In a second aspect, the present invention provides the use of lactobacillus plantarum as described above in the field of preparing fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit-cooked products, fermented feed.

In a third aspect, the present invention provides the use of lactobacillus plantarum as described above for the preparation of a ready-to-use starter culture.

In a fourth aspect, the invention provides a direct vat set starter prepared by fermenting and culturing lactobacillus plantarum as described above in a fermentation medium.

In a fifth aspect, the present invention provides a process for direct vat set fermentation as described above, the process comprising:

(1) the lactobacillus plantarum is subjected to fermentation culture in a fermentation culture medium, and the viable count reaches 10⁸cfu/m L or more;

(2) centrifuging the zymocyte liquid obtained in the step (1), washing with a buffer solution, adding a freeze-drying protective agent, and adjusting the concentration of viable bacteria to 10¹⁰And (3) more than cfu/m L, uniformly mixing, and performing vacuum freeze drying to obtain the direct vat set starter.

In a sixth aspect, the present invention provides the use of the direct vat set as described above and/or the direct vat set prepared by the method as described above in the field of preparing fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit boiled products, fermented feed.

In a seventh aspect, the present invention provides the use of a lactobacillus plantarum as described above for the preparation of a live bacterial preparation.

In an eighth aspect, the invention provides a viable bacteria preparation, which is prepared by fermenting and culturing the lactobacillus plantarum in a fermentation medium.

The invention has the beneficial effects that:

1. safe and healthy, and low cost: the lactobacillus plantarum CGMCC No.16441 is a safe strain which is screened from the milk cakes made by the traditional method in the herdsman in inner Mongolia and can be used for food, and the method has no chemical addition, is green and natural, and is nutritional and healthy;

2. the lactobacillus plantarum CGMCC No.16441 can effectively improve the fermentation quality of fermented dairy products, cereal products, bean products, fruit and vegetable products and feed products and exert the probiotic function of probiotics; the lactobacillus plantarum CGMCC No.16441 is used as a leavening agent, so that the acidity of the product can be effectively improved, the content of polysaccharide is increased, the nutritional value and flavor characteristics of the product are improved, and the sensory quality characteristics of the product are improved to a certain extent; the lactobacillus plantarum CGMCC No.16441 is used as probiotics, so that the colonization of cells can be effectively improved;

3. the lactobacillus plantarum CGMCC No.16441 is used as a leavening agent and a probiotic viable bacteria preparation, is easy to culture and prepare, and has high viable bacteria concentration of the leavening agent of the lactobacillus plantarum.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The lactobacillus plantarum (L actinobacillus plantarum) provided by the invention is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 2018, 9 and 7, and the preservation number is CGMCC No.16441, the preservation address is No. 3 of Siro No.1 of Beijing city north Chen in the area of rising, and the microorganism research institute of China academy of sciences (CGMCC for short).

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 shows the colony morphology of Lactobacillus plantarum of the invention on a culture medium;

FIG. 2 shows the microscopic morphology of Lactobacillus plantarum in accordance with the present invention;

FIG. 3 is a graph showing the pH value dynamic change of acid produced by different strains according to the present invention;

FIG. 4 shows the analysis and detection results of the fermented flavor of Lactobacillus plantarum in accordance with the present invention.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the invention provides a lactobacillus plantarum, which is classified and named as L actobacillus plantarum and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16441.

The lactobacillus plantarum CGMCC No.16441 has the following properties:

(1) morphological characteristics: the colony on the MRS culture medium is milky white, round, smooth in surface, moist, opaque, rod-shaped under a microscope, and gram-positive;

(2) genetic stability: the acid production and curd performance are stable heredity, the subculture is carried out for 10 generations, the acidity value is 89.74, and the consistency reaches 4721.86/g;

(3) acid generation: acid production capability test is carried out according to the MC liquid culture medium, the obtained strain CGMCC NO.16441 has pH value of 3.76 after fermentation for 24h, and the acid production capability is higher than that of a commercial strain;

(4) fermentation flavor characteristics: besides common flavor substances, the fermented product also contains 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone, ethyl caproate and other components, so that the product is endowed with faint scent flavor;

(5) tolerance in vivo: the survival rate after 3h of treatment in artificial gastric juice with pH3.0 is 62%; survival rate of treatment in simulated intestinal fluid for 24h was 39%; shows good gastrointestinal tract tolerance;

(6) the polysaccharide production capacity is that after the strain is inoculated in an MRS liquid culture medium and cultured for 48 hours at 37 ℃, the extracellular polysaccharide yield is 803 mg/L which is far higher than 150-600 mg/L of the similar strain subjected to the optimized culture condition;

(7) adhesion: up to 1000 bacteria/100 cells (HT-29).

The lactobacillus plantarum CGMCC No.16441 is screened from milk cakes made by traditional methods in herdsmen in inner Mongolia regions.

The lactobacillus plantarum provided by the invention can generate a large amount of live bacteria of lactobacillus plantarum after liquid culture, and the cultureThe method (2) is not particularly limited as long as it can proliferate the Lactobacillus plantarum, and may be, for example, 10^6-8The culture medium of the lactobacillus can be various culture media suitable for culturing the lactobacillus, which are well known in the art, such as milk and/or lactobacillus (MRS) culture medium described in "Lactobacillus-biological basis and applications" (Poplar, light industry Press, published 1996).

In the present invention, the viable cells of lactobacillus plantarum in the culture solution may be further separated, and the method for separating is not particularly limited as long as the cells can be enriched from the culture solution, and for example, the separation can be achieved by centrifugation and/or filtration, and the conditions for centrifugation and filtration may be known conditions, and the present invention is not described herein again.

In a second aspect, the invention provides application of the lactobacillus plantarum CGMCC No. 16441.

The application of the lactobacillus plantarum CGMCC No.16441 disclosed by the invention refers to the application in fermented food in one embodiment of the invention.

The fermented product, in one embodiment of the invention, comprises dairy products, cereal products, bean products, fruit and vegetable products and feed products.

The dairy product, in one embodiment of the invention, may comprise yogurt, a live lactic acid bacteria beverage, or the like.

The cereal product, in one embodiment of the present invention, may include oat fermented beverage, black rice fermented beverage, buckwheat fermented beverage, and the like.

The soy product, in one embodiment of the present invention, may include fermented soy milk or the like.

The fruit and vegetable product, in one embodiment of the present invention, may comprise fermented fruit and vegetable juice, or the like.

The feed product, in one embodiment of the invention, may comprise a solid anaerobic biological feed.

In the embodiment of the invention, in the conventional production process of producing fermented dairy products, grain products, bean products, fruit and vegetable products and feed products, the lactobacillus plantarum CGMCC No.16441 is inoculated into raw materials to be treated according to the conventional use, and is fermented or survived at the temperature and pressure capable of propagating the lactobacillus plantarum CGMCC No. 16441. By adding CGMCC No.16441 into fermentation substrate, the metabolite of the CGMCC No.16441 ensures that the fermentation product has certain excellent characteristics of acidity, fragrance and the like, prolongs the preservation time of the product, and improves the nutritive value and flavor characteristics of the product.

In a third aspect, the invention provides application of the lactobacillus plantarum CGMCC No.16441 in preparation of a direct vat set starter.

In a fourth aspect, the invention provides a direct vat set starter, which is prepared by fermenting and culturing lactobacillus plantarum CGMCC No.16441 in a fermentation medium.

According to the present invention, the fermentation medium may be any of various media conventionally used in the art for fermentation of lactic acid bacteria, for example, MRS medium as described above, and may be any of various media suitable for fermentation of lactic acid bacteria optimized on the basis of MRS medium.

According to the present invention, the fermentation conditions may be conventional conditions for fermentation culture of lactic acid bacteria well known in the art, and for example, the temperature of the fermentation culture may be 30 to 40 ℃.

According to the present invention, the preparation method of the direct vat set starter can be prepared according to the conventional method in the field, and in one embodiment of the present invention, the direct vat set starter can be prepared according to the following method: culturing lactobacillus plantarum CGMCC No.16441 till the viable count reaches 10⁸More than cfu/m L, and vacuum freeze drying after mixing uniformly.

The direct vat set starter is bacterial powder prepared by vacuum freeze drying lactobacillus plantarum CGMCC No.16441 zymocyte liquid, and contains more than 10 hundred million/g of active lactobacillus plantarum CGMCC No. 16441.

In a fifth aspect, the present invention provides a method of preparing a direct vat set as described above, the method comprising:

(1) the lactobacillus plantarum CGMCC No.16441 is subjected to fermentation culture in a fermentation culture medium, and the viable count reaches 10⁸cfu/m L or more;

The method for fermentation culture of Lactobacillus plantarum CGMCC No.16441 according to the present invention has been described in detail above, and will not be repeated herein in order to avoid unnecessary repetition.

According to the present invention, the method for centrifuging the fermentation broth can be performed according to conventional methods in the art, for example, centrifugation at 5000-12000rpm for 5-20min in a refrigerated centrifuge can be used to obtain cell pellet.

According to the present invention, the lyoprotectant may be various cryoprotectants conventional in the art, for example, at least one of skim milk powder, maltodextrin, trehalose, dextran, and glycerin.

According to the present invention, the buffer may be a buffer conventionally used in the art for washing the cells, and may be, for example, physiological saline or PBS buffer.

In a sixth aspect, the invention provides the use of the direct vat set as described above and/or the direct vat set prepared by the method as described above in the field of preparing fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit boiled products, fermented feed.

In a seventh aspect, the invention provides application of lactobacillus plantarum CGMCC No.16441 in preparation of a live bacterial preparation.

According to the present invention, the live bacterial preparation may be in various forms known as such, such as but not limited to capsules, tablets, oral liquid and powders.

According to the present invention, the preparation method of the live bacterial preparation can be performed by referring to the direct vat set starter as described above to avoid unnecessary repetition, and thus, the details are not repeated herein.

Examples

The media formulations referred to in the following examples are as follows:

MRS medium (Man Rogosa sharp Broth): peptone 1 wt%, beef powder 0.5 wt%, yeast powder 0.4 wt%, glucose 2 wt%, Tween 800.1 wt%, and K₂HPO₄.7H₂0.2% by weight of O, sodium acetate 3H₂0.5% by weight of O, 0.2% by weight of triammonium citrate, MgSO₄·7H₂0.02 wt.% of O, MnSO₄·4H₂0.005 percent of O and 1.5 percent of agar powder by weight are heated and dissolved, the pH value is adjusted to 6.2 +/-0.2, and the mixture is sterilized for 15-20min under high pressure at 121 ℃.

MC culture medium: 0.5 weight percent of soybean peptone, 0.3 weight percent of beef powder, 0.3 weight percent of yeast powder, 2 weight percent of glucose, 2 weight percent of lactose, 1 weight percent of calcium carbonate, 1.5 weight percent of agar and 0.5 weight percent of 1 percent neutral red solution. Adding the above 7 components into distilled water, heating to dissolve, adjusting pH to 6.0 + -0.2, adding neutral red solution, packaging, and autoclaving at 121 deg.C for 15-20 min.

Example 1

This example is used to illustrate the isolation, purification and characterization of Lactobacillus plantarum CGMCC No.16441

The strain is as follows: lactobacillus plantarum CGMCC No.16441 is separated from traditional inner Mongolia milk crisps and is preserved by the center for preservation of nutrient and health strains of the Nutrition and health research institute of Chinese food grain.

The inventor collects samples such as traditional milk cakes, milk shreds, milk strips, milk bean curd and the like prepared by a traditional method from herdsmen in places such as Hulunbel of inner Mongolia, Xilinguo union and the like, and dilutes the samples to 10 degrees by using sterile normal saline in a gradient manner^-6Each dilution gradient is coated with MRS plate and MC plate in sequenceAnd cultured at 36 + -1 deg.C for 72 h. And selecting colonies with different colony morphologies by an inoculating needle to an MRS plate and an MC plate for streaking until single colonies are uniform in size and consistent in morphology.

Strains were selected which were purple in gram stain, catalase negative, and non-sporulating. The strain separated to tentatively become lactobacillus is activated for 3 generations in MRS liquid culture medium and then subjected to physiological and biochemical identification and molecular biological identification, acid production and curding genetic stability, fermentation performance, sensory evaluation, fermentation flavor substances, polysaccharide production capacity, gastrointestinal tract acid environment resistance, gastrointestinal tract bile salt environment resistance and other aspects of lactobacillus are researched, and a lactobacillus plantarum CGMCC NO.16441 is finally screened and obtained from a plurality of wild lactobacillus through a plurality of rounds of research and argumentation.

1. Morphological identification

The screened lactobacillus plantarum CGMCC NO.16441 is cultured for 72 hours at 36 +/-1 ℃, and as shown in figure 1, the bacterial colony on the MRS culture medium is milky white, round, smooth in surface, moist and opaque. As shown in FIG. 2, the cell morphology of Lactobacillus plantarum CGMCC NO.16441 is rod-shaped and gram-positive under the microscope.

2. Physiological and biochemical identification

The screened strain CGMCC NO.16441 is subjected to physiological and biochemical identification by utilizing a French Merrielia API identification system, and the identification result is shown in the following table 1. the isolated strain is lactobacillus plantarum (L actinobacillus plantarum) through the physiological and biochemical identification.

TABLE 1 physiological and biochemical identification experiment results of strain CGMCC NO.16441

Item	Results	Item	Results	Item	Results
Control	-	Inositol	-	D-melezitose	+
Mannitol	-	Mannitol	+	D-raffinose	-
Erythritol	-	Sorbitol	-	Starch	-
D-arabinose	-	Methyl- α D-mannopyranoside	-	Glycogen	-
L-arabinose	+	Methyl- α D-glucopyranoside	-	Xylitol, its preparation method and use	-
D-ribose	+	N-acetylglucosamine	+	D-gentiobiose	+
D-xylose	-	Amygdalin	+	D-Talinum sugar	+
L xylose	-	ARBULIN	+	D-lyxose	-
D-adonitol 1	-	Esculin ferric citrate	+	D-tagatose	-
Methyl- β D xylopyranoside	-	Salicin	+	D-fucose	-
D-galactose	+	D-Cellobiose	+	L fucose	-
D-glucose	+	D-maltose	+	D-arabinose	-

D-fructose	+	D-lactose	+	L-arabitol	-
D-mannose	+	D-melibiose	-	Potassium gluconate	+
L-sorbitol	-	D-sucrose	+		2 Keto Potassium gluconate	-
L rhamnose	-	D-trehalose	+		5 Keto-Glucose Potassium	-
Dulcitol	-	Inulin powder	-

[ note ]: in the table, "+" indicates that the biochemical reaction result is positive, and "-" indicates that the biochemical reaction result is negative.

3. Molecular identification

The 16s rDNA of the separated strain is cloned and sequenced, the nucleotide sequence of the 16s rDNA gene is shown as SEQ ID No.1, the 16s rDNA sequence of the strain is compared with the sequence of NCBI lactobacillus plantarum, and the similarity of the 16s rDNA sequence of the strain and the L actinobacillus plantarum sequence reaches 99%.

The strain was identified as Lactobacillus plantarum (L Acrobacillus plantarum) by combining the results of biochemical identification.

The isolated strain was identified as Lactobacillus plantarum (L Acetobacter plantarum), designated L Acetobacter plantarumLCZMicroorganisms already preserved in ChinaGeneral microbiological center of culture collection management committee, collection address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation number of CGMCC No.16441 and a preservation date of 2018, 9 months and 7 days.

Example 2

This example is used to illustrate the genetic stability study of Lactobacillus plantarum CGMCC NO.16441

In order to examine the acid production and curd performance of CGMCC NO.16441, a test strain is inoculated into a liquid MRS culture medium and cultured for 24-48h at 37 ℃, when the strain recovers the activity, the strain is inoculated into a subculture medium according to the proportion of 4 volume percent (full-cream milk powder is prepared into a reconstituted milk solution of 12 weight percent, and then 6 weight percent of white granulated sugar is added, mixed uniformly and boiled for 10min), and the strain is fermented at 37 ℃. After the milk has solidified, the fermentation time is recorded, and the acidity, texture and the like are measured. The results of the experiment after 10 serial passages are shown in tables 2 and 3.

TABLE 2 CGMCC NO.16441 acid production genetic stability test results

Numbering	Fermentation time/h	End of fermentation pH	After-ripening titration acidity
Generation
10	15	4.53±0.5	89.74±0.32
Generation 1	10	4.32±0.67	95.93±0.78

TABLE 3 CGMCC NO.16441 curd performance genetic stability test results

Numbering	Water retention capacity/%)	Consistency per gram	Viscosity of the oil
Generation
10	48.15±0.48	4721.86±15.80	2388±10.55
Generation 1	42.90±0.37	3886.11±12.47	2852±12.36

As can be seen, after the strain CGMCC NO.16441 is continuously passaged for 10 times, the fermentation end point pH of the fermented milk is 4.53, the after-ripening titration acidity is 89.74, the viscosity reaches 2388, the consistency reaches 4721.86/g, and the acid-producing capability and the curd performance are genetically stable.

Example 3

This example demonstrates the study of the determination of acid productivity of lactic acid bacteria

Inoculating the separated and purified lactobacillus in a closed container filled with MC liquid culture medium after activated microscopic examination, culturing at constant temperature of 37 ℃, taking out 15mI culture solution every 2h, measuring pH value with a precision acidity meter, and measuring for 24 h. The time is used as the abscissa, the pH value of the fermentation culture solution is used as the ordinate, and the pH value dynamic change curve of the acid production of each strain is obtained and is shown in figure 3.

As can be seen, the pH value of the strain CGMCC NO.16441 can reach 3.76 after fermentation for 24 hours, and the acid production capability is higher than that of a commercial strain (the commercial strain No.1 is purchased from Jiangsu Microkang Biotechnology Co., Ltd., lactobacillus strain, the strain type is 1807B, Lactobacillus bulgaricus and Streptococcus thermophilus, and the commercial strain No.1 is purchased from Hebei Yiran Biotechnology Co., Ltd., lactobacillus strain, the strain type is Yo-C698-1F, Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus).

Example 4

This example is used to illustrate the study on the production of polysaccharide by Lactobacillus plantarum CGMCC NO.16441

Inoculating the strain into an MRS liquid culture medium, culturing at 37 ℃ for 48h, taking l0m L culture solution, adding 250u L volume percent of 80% trichloroacetic acid (protein removal), centrifuging in a centrifuge at 8000r/min for 20min, discarding the precipitate after centrifugation, putting the supernatant into a dialysis bag, dialyzing with running water for 48h, adding 3 times volume of 95% ethanol after dialysis, refrigerating at 4 ℃ overnight, separating out the obtained polysaccharide in a flocculent precipitate, centrifuging in a centrifuge at l0000r/min for 20min, discarding the supernatant, obtaining the precipitate which is extracellular polysaccharide, and dissolving the precipitate in l0m L distilled water to obtain an extracellular polysaccharide aqueous solution.

The total sugar is obtained by phenol-sulfuric acid method, wherein 2.0m L of sample to be tested is added into a test tube, then 1m L6% phenol and 5m L% concentrated sulfuric acid are sequentially added, standing for l0min, shaking up, standing at room temperature for 20min, and detecting absorbance at 490nm wavelength.

The reducing sugar is prepared by 3, 5-dinitrosalicylic acid method, which comprises adding 3, 5-dinitrosalicylic acid reagent 0.5m L into sample to be detected, mixing, boiling in water bath for 5min, cooling, adding 4m L of distilled water, mixing, and detecting absorbance at 540 nm.

The polysaccharide content was calculated according to the formula "total sugar-reducing sugar", and the calculation results are shown in table 4 below.

TABLE 4 polysaccharide content of Lactobacillus plantarum CGMCC NO.16441

CGMCC NO.	Concentration (mg/L)
Total sugar	803.0±7.5
Reducing sugar	0
Polysaccharides	803.0

Therefore, the extracellular polysaccharide yield of the screened strain CGMCC NO.16441 is 803.0 mg/L, and the extracellular polysaccharide yield is very high and is far higher than 150-600 mg/L of the similar strain subjected to the optimized culture conditions.

Example 5

This example is used to illustrate the study of Lactobacillus plantarum CGMCC NO.16441 on the simulation of gastric juice and intestinal juice tolerance

Simulated gastric fluid is prepared by dissolving pepsin 0.35g in sterile physiological saline 100m L0.2.2 wt%, adjusting pH to 3.0 with concentrated hydrochloric acid, and sterilizing with 0.45um filter membrane.

Simulated intestinal fluid was prepared by dissolving 0.1g trypsin and 1.8g bile salts in sterile solvent containing 1.1g NaHCO3, 0.2g NaCl and 100M L distilled water, adjusting the pH to 8.0 with 0.5M NaOH, and sterilizing the solution through a 0.45 μ M filter.

Inoculating the activated 3 times bacterial liquid into simulated gastric fluid (pH3.0) according to the inoculation amount of 10 volume percent, mixing uniformly, performing anaerobic culture at 37 ℃, counting sampling plates in 0h and 3h respectively, after culturing in the simulated gastric fluid for 3h, sucking 1m L culture liquid, inoculating into 9m L simulated intestinal fluid (pH8), performing anaerobic culture at 37 ℃, and sampling and counting in 24 h.

Survival (%) (logCFU N)₁/logCFU N₀)×100％；

Wherein N is₁Representing the number of lactic acid bacteria after simulated gastrointestinal culture; n is a radical of₀Representing the number of lactic acid bacteria before the simulated gastrointestinal tract culture.

The results are as follows: the survival rate of CGMCC NO.16441 is 62% after 3h of simulated gastric juice with the pH value of 3, the survival rate is maintained at 39% after 24h of intestinal juice, and the isolated strain shows good gastrointestinal tract tolerance performance.

Example 6

This example illustrates the intestinal adhesion of Lactobacillus plantarum CGMCC NO.16441

The cultured HT-29 cells were digested to prepare a cell suspension (about 10)⁵Pieces/m L), 2m L was added to a 24-well plate with cover slips placed thereon, at 37 ℃ and 5% CO₂95% culture in air, the culture medium is 1640 culture solution, 10 wt% inactivated calf serum and L-glutamine, when the cells on the cover glass are basically fully covered, the old cell culture solution is sucked out, sterile PBS is used for washing for 3 times, and 1m L bacterial solution (containing thalli l 0) is added into each hole⁸cfu/m L) and 1m L1640 cell culture medium at 37 deg.C and 5% CO₂And incubating in 95% air for 2 h. The slides were washed with sterile PBS to remove unbound bacteria. Then 10% formaldehyde was fixed for 2h, gram stained, 20 fields were randomly selected under the microscope, the number of adhered bacteria on 100 cells was counted, 3 wells were paralleled for each treatment, and the average number of adhered bacteria per cell was calculated.

The results are as follows: the adhesiveness of CGMCC NO.16441 to HT-29 cells is 1000 bacteria/100 cells, which is higher than 200 bacteria/100 cells of the same strain subjected to optimized culture conditions.

Example 7

This example illustrates the use of Lactobacillus plantarum CGMCC No.16441 in fermented milk

Obtaining raw milk: a qualified standardized raw milk from tribasic group.

Heating raw milk to 60-70 deg.C, adding 6 wt% white sugar, mixing (adding water to initial weight), homogenizing at 65 deg.C and 200bar, and pasteurizing at 85 deg.C for 30 min; cooling to 42 ℃, inoculating with 5 volume percent of inoculum size, stirring and mixing uniformly, standing and culturing at 42 ℃ for fixing and forming, and after aging at 4 ℃ for 24 hours, obtaining the fermented milk.

The main roles of lactic acid bacteria in dairy fermentation are acid production and flavor generation. The acid-producing properties of lactic acid bacteria are important characteristics for screening leavening agents for fermented dairy products. Lactic acid bacteria having a strong acid-producing ability generally have a strong tendency to acidify thereafter. Lactic acid bacteria with fast acid production and large acid production amount can bring lower fermentation acidity of final products, which is an effective measure for ensuring low post-acidification. The results of the tests on the acid production capacity at the fermentation end of different strains in the prior art are shown in Table 5. The fermentation end point pH of the detected strain is 4.32 +/-0.67, the after-ripening and after-titration acidity is 95.93 +/-0.78, the result shows that the acidity value of the strain CGMCC No.16441 is increased by 20 percent compared with that of the control lactobacillus, and the fermentation time is obviously shortened.

TABLE 5 comparison table of acid-producing capacities of different strains at fermentation end

Bacterial strains	LLD	LLL	LLC	CGMCC NO.16441
Acidity/° T	72.7±2.4	64.4±2.9	71.65±3.4	95.93±0.78
pH value	4.39±0.13	4.77±0.08	4.45±0.14	4.32±0.67
Fermentation time/h	6	6.5	6	5

LL D is lactococcus lactis lactonizus LLL, LL C is lactococcus lactis lactonizus;

the research on flavor substances of lactobacillus plantarum CGMCC NO.16441 fermented milk is realized by adding 20ml of set yoghurt into 5m L saturated NaCl solution, shaking and mixing uniformly, transferring 5m L solution into a headspace bottle to be tested, and analyzing the flavor substance components by GC-MS.

The results of the fermentation flavor test are shown in FIG. 4. In fermented milk, 25 main flavors including ketones, alcohols, esters, ethers, etc. were identified. The common yogurt and the CGMCC No. strain fermented yogurt contain characteristic flavor substances, and also contain flavor components such as 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone and ethyl hexanoate, so that the yogurt is endowed with faint scent flavor.

Example 8

This example illustrates the application of Lactobacillus plantarum CGMCC No.16441 in the preparation of active lactobacillus beverage

Heating purified water to 50-55 deg.C, adding whole milk powder, heating to 65-70 deg.C, homogenizing under 180-200 bar. Cooling, adding Lactobacillus plantarum CGMCC No.16441 as starter in 3-5 vol% of the emulsion, and fermenting at 37-42 deg.C to pH4.0-4.5. Demulsifying, cooling, and storing at low temperature. Heating purified water to 65-75 deg.C, adding mixture of white sugar or other sweetener and stabilizer such as pectin, stirring for 15min, cooling, adding yogurt, adjusting pH to 3.5-4.0, adding essence and water to specified amount, heating to 65-70 deg.C, homogenizing at pressure of 180-200bar and 130 deg.C for 10-20s, cooling, and bottling to obtain lactobacillus beverage, and evaluating its organoleptic properties as shown in Table 6.

TABLE 6 organoleptic evaluation table of lactic acid bacteria beverage

The color, taste, smell and tissue state of the lactobacillus fermented beverage are used as scoring standards, the best fermentation strain is selected from different varieties of commercial lactobacillus, and the corresponding sensory evaluation score of the fermentation strain selection is shown in the following table 7.

TABLE 7 sensory evaluation score

Fermentation strain	Color and luster	Nourishing and smelling	Tissue state	Total score
LLD	10	20	12	42
LLL	15	33	20	68
LLC	14	38	20	72
CGMCC No.16441	18	44	22	84

sensory evaluation is carried out on the lactic acid bacteria beverage fermented by different fermenting agents, so that the different fermenting agents influence the experimental result to a great extent under the same fermentation condition, wherein the lactic acid bacteria beverage fermented by CGMCC No.16441 has the fragrance of fermented lactic acid, unique taste, pure white color and uniform uniformity.

Example 9

This example illustrates the application of Lactobacillus plantarum CGMCC No.16441 in fermenting cereal beverages

Selecting a grain raw material which is good in color, fresh and full and free of diseases and insect pests, and mixing the raw materials according to the following ratio of oat: soaking grain material in water in the range of 1:2-2:1 until it is completely water-swollen.

Taking the soaked raw materials, draining off water, adding water with the weight 8 times of that of the raw materials, pulping and boiling. The slurry enters a colloid mill to circulate for 2-4 min. Adding citric acid to adjust pH of the slurry to 4.0-5.0, adding saccharifying enzyme 1-3% of the composite slurry, and saccharifying at constant temperature of 40-60 deg.C for 4-8 hr.

Compounding the saccharified materialCooling the slurry, adjusting pH to 6.5-7.5 with sodium bicarbonate, adding Lactobacillus plantarum CGMCC No.16441 as starter, fermenting and culturing for a period of time to make its concentration reach 10⁸More than cfu/m L, adding white granulated sugar, citric acid, a stabilizer, essence and the like to adjust the flavor, cooling and filling to obtain the product, and evaluating the sense organ of the product, wherein the evaluation standard is shown in Table 8.

TABLE 8 organoleptic evaluation chart of fermented cereal beverages

The color, flavor and state of the composite grain fermented beverage are used as scoring standards, the best fermentation strain is selected from different varieties of commercial lactic acid bacteria, and the corresponding sensory evaluation scores selected by the fermentation strains are shown in the following table 9.

TABLE 9 sensory evaluation score

Fermentation strain	Color and luster	Nourishing and smelling	Appearance of the product	Total score
LLD
	16	20	11	47
LLL	20	33	13	66
LLC	18	27	12	57
CGMCC	26	41	15	82

sensory evaluation is carried out on the grain fermented beverage fermented by different fermenting agents, so that the different fermenting agents influence the experimental result to a great extent under the same fermentation condition, wherein the grain beverage fermented by the CGMCC No.16441 has grain composite aroma and pleasant fermentation flavor, unique taste, light yellow color and uniform color.

Example 10

This example illustrates the application of Lactobacillus plantarum CGMCC No.16441 in the preparation of fermented soymilk

The fermented soybean milk described in this example was prepared by the following method: according to the volume ratio of soft water to soybean of 2: 4 soaking the soybeans in soft water at the temperature of 75-85 ℃ for 1-2h, removing soybean hulls, removing soaking water, and mixing the soybeans with boiling water according to a weight ratio of 1: grinding 6-10 with boiling water for 10-15min at 80-85 deg.C, and filtering with 150 mesh screen to obtain soybean milk; adding sucrose 5-10% of the weight of soybean milk, homogenizing under 15-25Mpa, sterilizing at 95 deg.C for 5-10min, cooling to 28-38 deg.C, inoculating 0.04-0.05% (by weight of soybean milk) of commercial Lactobacillus bulgaricus, Streptococcus thermophilus and CGMCC NO.16441 starter, with the ratio of bacteria number of 1: 1:1, fermenting at the temperature of 32-42 ℃ for 6-8 hours, and then refrigerating and storing at the temperature of 4 ℃ to obtain the fermented soybean milk.

Compared with fermented soybean milk prepared by using commercial lactobacillus bulgaricus and streptococcus thermophilus (the ratio of the number of bacteria is 1:1) as fermenting agents without adding lactobacillus plantarum CGMCC NO. 16441. The result shows that the acidity of the fermented soybean milk added with the lactobacillus plantarum CGMCC NO.16441 is 2.3 times of that of the fermented soybean milk not added, so that the fermentation time can be shortened by 1-1.5 hours by using the lactobacillus plantarum CGMCC NO.16441 to be compounded with commercial strains.

Example 11

This example illustrates the application of Lactobacillus plantarum CGMCC No.16441 in the preparation of fermented fruit and vegetable juice

Selecting fruits and vegetables with uniform and plump particles, cleaning dirt on the surface of the fruits and vegetables with clear water, soaking the fruits and vegetables in 0.005-0.01g/kg of sodium hypochlorite solution for 7-10 minutes for disinfection, washing the fruits and vegetables for standby, and cutting the fruits and vegetables to be cut into pieces; scalding with boiling water for 3-5min, and optionally treating with mixed solution of 0.5% citric acid and 0.3% Vc; pulping fruits and vegetables with tissue masher for 3-5 min; adding 0.006-0.01% of pectinase and 0.001% of cellulase, and performing enzymolysis at 35-52 deg.C for 1-2 hr; filtering with a double-layer 80-mesh filter screen to obtain clear liquid; sterilizing with microwave for 2min or 80 deg.C for 15min to inactivate enzyme; picking lactobacillus plantarum CGMCC NO.16441 from the inclined plane to a broth culture medium, and culturing for 24 h; selecting the strain, culturing in a small amount of fruit and vegetable juice for 20 hr, inoculating the fermented seed liquid into the fruit and vegetable juice, culturing at appropriate temperature for 24 hr until pH reaches 3.8-4.1 and lactobacillus amount reaches (10)⁸-10⁹) cfu/m L, compared with the fermented fruit and vegetable juice using lactobacillus casei as a leaven, the fermented fruit and vegetable juice of lactobacillus plantarum has smaller acidity change value during refrigeration, smaller post acidification and good fermentation stability.

Example 12

This example illustrates the application of Lactobacillus plantarum CGMCC No.16441 in the preparation of fermented feed

Drying crop straws until the water content is less than 15 wt%, and crushing; harvesting herbaceous plants, drying until the water content is less than 15%, and preparing into chopped grass; premixing rice bran, wheat bran, salt, lactobacillus plantarum CGMCC NO.16441 direct vat set starter and cellulase, and then gradually expanding and uniformly mixing with crop straws and herbaceous plant raw materials; compacting, bundling and film sealing the uniformly mixed micro-storage feed to prepare the micro-storage feed with the specification of 30-100 kg/package; naturally fermenting the obtained product for 12-18 days. The crop straw is one or more selected from vegetable straw, peanut vine, corn stalk, etc. The herbaceous plant is one or more selected from common Astragalus sinicus herbaceous plants.

Compared with the feed fermented by the commercial compound probiotics, the lactic acid content in the feed fermented by the lactobacillus plantarum CGMCC NO.16441 is 21.28g/kg, which is 4.4 times of the lactic acid content in the commercial compound probiotics fermented feed.

Example 13

This example illustrates the application of Lactobacillus plantarum CGMCC NO.16441 in the production of viable bacteria preparation

The screened lactobacillus plantarum is prepared into a viable bacteria preparation. Inoculating Lactobacillus plantarum CGMCC NO.16441 into MRS liquid culture medium according to the inoculation amount of 2-4% (v/v), and culturing at 37-42 deg.C for 20-24h to make the viable count of Lactobacillus plantarum CGMCC NO. reach 10⁸centrifuging at more than cfu/m L (4000rpm, 10min), washing precipitate with PBS buffer solution of pH6.5-7.5 twice, adding skimmed milk and trehalose as lyophilized protectant, and adjusting cell concentration to 10⁹cfu/m L, mixing, vacuum freeze drying to obtain viable bacteria preparation containing viable bacteria of 10 counts⁸-10¹⁰cfu/m L, the microbial inoculum can be directly added into fermentation products for fermentation, or added with proper auxiliary materials according to a conventional method to prepare into food industry or clinically acceptable dosage forms, including various dosage forms such as capsules, microcapsules, tablets, powder and the like.

The viable bacteria preparation prepared from the screened lactobacillus plantarum also comprises a product for keeping the activity of the strains by technical means such as domestication and the like.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

The lactobacillus plantarum is characterized in that the lactobacillus plantarum is classified and named as L actobacillus plantarum and is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number of the lactobacillus plantarum is CGMCC No. 16441.
Use of the lactobacillus plantarum of claim 1 in the preparation of fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit delicacies, fermented feed.
Use according to claim 2, wherein the use comprises the step of contacting the lactobacillus plantarum strain of claim 1 with an adapted fermentation substrate.
Use of lactobacillus plantarum as defined in claim 1 for the preparation of a ready-to-use starter culture.
A direct vat set starter prepared by fermenting the Lactobacillus plantarum of claim 1 in a fermentation medium.
A method of preparing the ready-to-use starter culture of claim 5, comprising:

(1) fermenting and culturing the lactobacillus plantarum of claim 1 in a fermentation medium to reach a viable count of 10⁸cfu/m L or more;

(2) separating the zymophyte liquid obtained in the step (1)Treating heart, washing with buffer, adding lyophilized protectant, and adjusting viable bacteria concentration to 10¹⁰And (3) more than cfu/m L, uniformly mixing, and performing vacuum freeze drying to obtain the direct vat set starter.
Use of the ready-to-use starter culture according to claim 5 and/or the ready-to-use starter culture prepared by the method according to claim 6 in the field of preparing fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit cooked products and fermented feed.
Use of lactobacillus plantarum as claimed in claim 1 for the preparation of a live bacterial formulation.
The use according to claim 8, wherein the viable bacteria formulation comprises capsules, tablets, oral liquid and powders.
A live bacterial preparation, which is prepared by fermenting and culturing the Lactobacillus plantarum of claim 1 in a fermentation medium.