AU2018423072A1

AU2018423072A1 - Strain of Lactobacillus plantarum for fermenting and use thereof

Info

Publication number: AU2018423072A1
Application number: AU2018423072A
Authority: AU
Inventors: Lishui CHEN; Jiaxing GAO; Hui Li; Lei Liu; Yang Liu; Wei Ni; Chengjing QIAN; Yuying SHI; Yong Wang; Yu Wang; Xin YING; Lianhui Zhang
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: 2020-05-28
Anticipated expiration: 2038-12-27
Also published as: WO2020098097A1; CN111436203B; CN111436203A; AU2018423072B2

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Lactobacillus plantarum and use thereof. The Lactobacillus plantarum is classified and designated as Lactobacillus plantarum, preserved in China General Microbiological Culture Collection Center, and assigned with a preservation number CGMCC No. 16441. The Lactobacillus plantarum provided in the present invention is acid-resistant and bile salt-resistant, has high adhesion to intestinal epithelial cell line HT-29, has very high acid production and polysaccharides production capability, and can produce acids more rapidly and better and produce more polysaccharides than other lactic acid bacteria. Fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable juice products, and fermented feed products prepared with the Lactobacillus plantarum in the present invention have unique flavors, uniform texture, smooth taste and favorable sensory features. The Lactobacillus plantarum provided in the present invention can be widely used in the field of fermented products, and can also be used to prepare viable bacterial preparations. 11877756_1 (GHMaers) P112431.AU

Description

Strain of Lactobacillus plantarum for Fermenting and Use Thereof

CROSS REFERENCE TO RELATED APPLICARTIONS

The application claims priority to Chinese Application No. 201811341522.2, filed on November 12, 2018, entitled “ Strain of Lactobacillus plantarum for Fermenting and Use Thereof” , which is specifically and entirely incorporated by reference.

FIELD

The invention relates to the technical field of microbial fermentation, and particularly discloses a strain of Lactobacillus plantarum, a ready-to-use starter containing the strain of Lactobacillus plantarum and a preparation method thereof, and use of the strain of Lactobacillus plantarum and the starter in the field of preparing fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products, and further discloses use of the strain of Lactobacillus plantarum in preparation of a viable bacterial preparation and a viable bacterial preparation prepared from the strain of Lactobacillus plantarum.

BACKGROUND

Lactic acid bacteria (LAB) is a generic term for bacteria that produce a large amount of lactic acid by utilizing fermentable carbohydrates. LAB is a group of bacteria that are quite numerous and jumbled, and may be divided into at least 18 genera, and more than 200 species totally at present, with rich species diversity. Traditional fermented dairy products in China's Qinghai, Tibet, Inner Mongolia, Xinjiang and other places contain abundant lactic acid bacteria strain resources, and are treasures with rich probiotic bacteria strains.

Strains of Lactobacillus plantarum in lactic acid bacteria are widely used in

11877756_1 (GHMatters) P112431 .AU food production, especially production of fermented foods. Researches have demonstrated that the acid production capability and fermentation flavor features of lactic acid bacteria play an important role in the production of fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products. They can be used separately or in combination with other bacteria. As potential probiotic bacteria, lactic acid bacteria require the following features: outstanding probiotic functions, high tolerance to the acidic environment and the bile salt environment of gastrointestinal tract, and excellent cell adhesion.

The main function of lactic acid bacteria in fermented products is to produce acid and flavor substances. The acid production feature of lactic acid bacteria is an important feature for screening starters for fermented products. In general, lactic acid bacteria with stronger acid production capability have a stronger tendency to post-acidification. Lactic acid bacteria capable of producing acid in a larger amount and rapidly can lead to lower fermentation acidity of the final product, which is an effective measure to ensure low post-acidification (e.g., dairy products). Gastric fluid and intestinal fluid constitute biological barriers in human body, and as probiotic bacteria, lactic acid bacteria have to pass through the gastrointestinal tract and adhere to the intestinal epithelial cells to achieve colonization, so as to they can exert beneficial effects on human or an animal host.

Lactic acid bacteria exopolysaccharides (LAB EPS) are produced by lactic acid bacteria in their growth and metabolic process and secreted out of the cell, and released into the growth matrix as polysaccharides or tightly bound to the cell surface as capsular polysaccharides. They mainly have the following functions: improving product quality, making the product fine and uniform, improving smooth taste, improving water binding capacity, improving product viscosity, promoting non-specific adhesion of lactic acid bacteria to intestinal surface, increasing the opportunity of lactic acid bacteria colonization, and

11877756_1 (GHMatters) P112431 .AU promoting regulation of intestinal micro flora composition to a certain degree.

Therefore, outstanding acid production capability, high exopolysaccharides production capability, strong resistance to gastrointestinal stress, excellent cell adhesion, and prominent sensory features of fermentation are essential factors for screening starters and probiotic bacteria.

SUMMARY

In order to solve the above technical problems, the present invention screens out a strain of Lactobacillus plantarum that can produce acids rapidly, has genetic stability on acid production and milk curdling properties, and can be used to produce fermented products with unique flavors. Applying the strain of Lactobacillus plantarum in fermented dairy products, cereal products, soybean products, fruit and vegetable products and feed products can improve the quality of the fermented products. Moreover, the strain of Lactobacillus plantarum can resist the acidic gastrointestinal environment, produce exopolysaccharides in high yield, improve cell colonization, can be used to prepare active probiotic bacterial preparations, and is helpful for regulating the composition of intestinal microflora.

In order to attain the above objects, in a first aspect, the present invention provides a strain of Lactobacillus plantarum, which is classified and designated as Lactobacillus plantarum, and is preserved in China General Microbiological Culture Collection Center, assigned with a preservation number CGMCC No. 16441.

In a second aspect, the present invention provides a use of the Lactobacillus plantarum described above in the field of preparing fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products.

In a third aspect, the present invention provides a use of the Lactobacillus plantarum described above for preparing a ready-to-use starter.

11877756_1 (GHMatters) P112431 .AU

In a fourth aspect, the present invention provides a ready-to-use starter prepared by fermenting and culturing the Lactobacillus plantarum described above in a fermentation medium.

In a fifth aspect, the present invention provides a method for preparing the ready-to-use starter described above, comprising:

(1) fermenting and culturing the Lactobacillus plantarum described above in a fermentation medium, till the viable count reaches 10⁸ cfu/mL or above to obtain a fermentation broth;

(2) centrifuging the fermentation broth obtained in the step (1), then rinsing with a buffer solution, adding a lyoprotectant, and adjusting the concentration of viable bacteria to 10¹⁰ cfu/mL or above, mixing to a homogeneous state, and then performing vacuum freeze-drying, so as to obtain the ready-to-use starter.

In a sixth aspect, the present invention provides a use of the ready-to-use starter described above and/or a ready-to-use starter prepared with the method described above in the field of preparing fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products.

In a seventh aspect, the present invention provides a use of the Lactobacillus plantarum described above for preparing a viable bacterial preparation.

In an eighth aspect, the present invention provides a viable bacterial preparation prepared by fermenting and culturing the Lactobacillus plantarum described above in a fermentation medium.

The present invention attains the following beneficial effects:

1. Safe, healthy, and low cost: the Lactobacillus plantarum CGMCC No. 16441 in the present invention is a safe strain that is screened out from butter biscuits prepared with the traditional method in herdsmen's homes in Inner Mongolia and can be used in foods; the method of the

11877756_1 (GHMatters) P112431 .AU present invention involves no chemical additive, and is green, natural, nutritional, and healthy;

2. The Lactobacillus plantarum CGMCC No. 16441 in the present invention can effectively improve the fermentation quality of fermented dairy products, cereal products, soybean products, fruit and vegetable products, and feed products, and play a role of the probiotic function of probiotic bacteria; the invention utilizes the Lactobacillus plantarum CGMCC No. 16441 as a starter, which can effectively improve the acidity and increase the content of polysaccharides of the products, can improve the nutritional value and flavor features of the products, and have certain improvement effect on the sensory quality and features of the products; as probiotic bacteria, the Lactobacillus plantarum CGMCC No. 16441 in the present invention can effectively improve cell colonization;

3. The Lactobacillus plantarum CGMCC No. 16441 in the present invention can be easily cultured and prepared as a starter and an active probiotic bacterial preparation, and the concentration of viable bacteria in the starter prepared from Lactobacillus plantarum is very high.

Other features and advantages of the present invention will be further detailed in the embodiments hereunder.

BIOLOGICAL PRESERVATION

The Lactobacillus plantarum provided in the present invention was preserved and assigned with a preservation number CGMCC No. 16441 on Sept. 7, 2018 in China General Microbiological Culture Collection Center, in Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), Building 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

BRIEF DESCRITION OF THE DRAWINGS

To make the content of the present invention understood more clearly and

11877756_1 (GHMatters) P112431 .AU easily, hereunder the present invention will be further detailed in some embodiments, with reference to the accompanying drawings. In the figures:

Fig. 1 shows the colony morphology of the Lactobacillus plantarum in the present invention in a culture medium;

Fig. 2 shows the microscopic morphology of the Lactobacillus plantarum in the present invention;

Fig. 3 shows dynamic change curves of pH values of the acids produced by different strains;

Fig. 4 shows a result of analysis and detection of the fermentation flavor substances produced by the Lactobacillus plantarum in the present invention.

DETAILED DESCRPTION

The end points and any value in the ranges disclosed in the present invention are not limited to the exact ranges or values; instead, those ranges or values shall be comprehended as encompassing values that are close to those ranges or values. For numeric ranges, the end points of the ranges, the end points of the ranges and the discrete point values, and the discrete point values may be combined with each other to obtain one or more new numeric ranges, which shall be deemed as having been disclosed specifically in this document.

In a first aspect, the present invention provides a strain of Lactobacillus plantarum, which is classified and designated as Lactobacillus plantarum, and is preserved in China General Microbiological Culture Collection Center, assigned with a preservation number CGMCC No. 16441.

The Lactobacillus plantarum CGMCC No. 16441 has the following features:

(1) Morphological features: in MRS broth, the colony is in milky white color and in a circular shape, has a smooth surface, is moist and opaque, is rod-shaped cell morphology as observed under a microscope, and is Gram-positive;

11877756_1 (GHMatters) P112431 .AU (2) Genetic stability: the acid-producing and milk curdling properties are genetically stable; after sub-culturing for 10 generations, the acidity value is 89.74, and the consistency is as high as 4,721.86g/s;

(3) Acid production capability: according to the result of acid production capability tested in a MC liquid culture medium, the pH is as high as 3.76 after the strain CGMCC NO. 16441 ferments for 24h, which indicates the acid production capability of the strain CGMCC NO. 16441 is higher than that of commercial strains;

(4) Fermentation flavor features: in addition to common flavor substances, the fermented product also contains components such as 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone and ethyl hexanoate, which impart a cleaning flavor to the product;

(5) In vivo tolerance: the survival ratio is 62% after treatment for 3h in artificial gastric fluid at pH=3.0; the survival ratio is 39% after treatment for 24h in simulated intestinal fluid; the strain exhibits excellent gastrointestinal tolerance;

(6) Polysaccharides production capability: after the strain is inoculated into a MRS liquid medium and cultured at 372 for 48h, the yield of exopolysaccharides is as high as 803 mg/L, which is much higher than the 150-600 mg/L yield attained by strains of idem genus cultured under optimized culture conditions;

(7) Adhesion: as high as 1,000 bacterial / 100 cells (HT-29).

The Lactobacillus plantarum CGMCC No. 16441 in the present invention is screened out from butter biscuits prepared with the traditional method in herdsmen's homes in Inner Mongolia.

The Lactobacillus plantarum provided in the present invention can produce a large quantity of viable Lactobacillus plantarum bacteria through liquid culturing. There is no particular requirement for the culturing method, as long as the Lactobacillus plantarum can be proliferated with the culturing method. For

11877756_1 (GHMatters) P112431 .AU example, viable Lactobacillus plantarum bacteria may be inoculated in IO⁶’⁸CFU/mL inoculation amount into a Lactobacillus culture medium, and cultured under an anaerobic or aerobic condition at 15-40°C temperature for 8-72h to obtain a culture broth. The Lactobacillus culture medium may be any well-known culture medium suitable for culturing Lactobacilli in the art. For example, the Lactobacillus culture medium may be milk and/or the lactic acid bacteria broth (MRS) described in Lactic Acid Bacteria - Biological Basis and Application (Yang Jiebin, China Light Industry Press, 1996).

In the present invention, the viable Lactobacillus plantarum bacteria in the above-mentioned culture broth may be further separated. There is no particular restriction on the separation method, as long as the bacteria can be concentrated from the culture broth with the separation method. For example, the separation may be achieved by centrifugation and/or filtration, and the conditions of the centrifugation and filtration may be well-known conditions. That aspect will not be further detailed here.

In a second aspect, the invention provides a use of the Lactobacillus plantarum CGMCCNo. 16441.

In one embodiment of the present invention, the use of the Lactobacillus plantarum CGMCC No. 16441 in the present invention refers to the use in fermented products.

In one embodiment of the invention, the fermented products may include dairy products, cereal products, soybean products, fruit and vegetable products, and feed products.

In one embodiment of the present invention, the dairy products may include yogurts, active yoghurt beverages, and the like.

In one embodiment of the present invention, the cereal products may include fermented oat beverages, fermented black rice beverages, fermented buckwheat beverages, and the like.

In one embodiment of the present invention, the soybean products may

11877756_1 (GHMatters) P112431 .AU include fermented soymilk and the like.

In one embodiment of the present invention, the fruit and vegetable products may include fermented fruit and vegetable juice and the like.

In one embodiment of the present invention, the feed products may include solid anaerobic biochemical feed.

In embodiments of the present invention, the use of the Lactobacillus plantarum CGMCC No. 16441 in the present invention in fermented products is to inoculate the Lactobacillus plantarum CGMCC No. 16441 into the raw material to be treated with a conventional method in a conventional production process of a fermented dairy products, cereal products, soybean products, fruit or vegetable products, or feed products, and allow the Lactobacillus plantarum CGMCC No. 16441 to ferment or survive at a temperature and a pressure that allow the Lactobacillus plantarum CGMCC No. 16441 to propagate. By adding the Lactobacillus plantarum CGMCC No.16441 to the fermentation substrate, the metabolites of the Lactobacillus plantarum CGMCC No. 16441 make the fermented product have certain acidity, fragrance and other advantageous properties, and at the same time extend the preservation time of the product and improve the nutritional value and flavor features of the product.

In a third aspect, the present invention provides a use of the Lactobacillus plantarum CGMCC No. 16441 for preparing a ready-to-use starter.

In a fourth aspect, the present invention provides a ready-to-use starter prepared by fermenting and culturing the Lactobacillus plantarum CGMCC No. 16441 in a fermentation medium.

According to the present invention, the fermentation medium may be any conventional fermentation medium for fermenting lactic acid bacteria in the art. For example, the fermentation medium may be the MRS broth described above, or may be various fermentation medium optimized based on the MRS broth and suitable for fermenting lactic acid bacteria.

According to the present invention, the conditions of the fermentation may

11877756_1 (GHMatters) P112431 .AU be conventional well-known conditions for fermenting and culturing lactic acid bacteria in the art. For example, the temperature for fermenting and culturing may be 30-40°C.

According to the present invention, the preparation of the ready-to-use starter may be carried out with a conventional method in the art, and, in one embodiment of the present invention, the ready-to-use starter may be prepared as follows: culturing the Lactobacillus plantarum CGMCC No. 16441 till the viable count reaches to 10⁸ cfu/mL or above, mixing the fermentation products obtained to a homogeneous state, and then performing vacuum freeze-drying.

In one embodiment of the present invention, the ready-to-use starter is powder of Lactobacillus plantarum CGMCC No. 16441 prepared by performing vacuum freeze-drying of Lactobacillus plantarum CGMCC No. 16441, which contains active Lactobacillus plantarum CGMCC No. 16441 higher than 1 billion/g.

(1) fermenting and culturing the Lactobacillus plantarum CGMCC No. 16441 in a fermentation medium, till the viable count reaches 10⁸cfu/mL or above;

According to the present invention, the method for fermenting and culturing Lactobacillus plantarum CGMCC No. 16441 is described above in detail, and will not be further detailed here to avoid unnecessary repetition.

According to the present invention, the centrifugation of the fermentation broth may be carried out with a conventional method in the art. For example, the

11877756_1 (GHMatters) P112431 .AU fermentation broth may be centrifuged in a refrigerated centrifuge at 5,000-12,OOOrpm speed for 5-20min. to obtain bacteria precipitate.

According to the present invention, the lyoprotectant may be any conventional cryoprotectant in the art. For example, the lyoprotectant may be at least one selected from the group consisting of skim milk powder, maltodextrin, trehalose, dextran, and glycerin.

According to the present invention, the buffer solution may be any conventional buffer for rinsing bacteria in the art. For example, the buffer solution may be physiological saline or PBS buffer solution.

In a seventh aspect, the present invention provides a use of the Lactobacillus plantarum CGMCC No. 16441 for preparing a viable bacterial preparation.

According to the present invention, the viable bacterial preparation may be in any well-known form, including, but not limited to, capsule, tablet, oral liquid, and powder.

According to the present invention, the viable bacterial preparation may be prepared with reference to the preparation method of the ready-to-use starter described above, and the preparation method will not be further detailed here to avoid unnecessary repetition.

11877756_1 (GHMatters) P112431 .AU

Examples

The formulations of the culture medium involved in the following examples are as follows:

Man Rogosa Sharpe (MRS) broth: lwt% peptone, 0.5wt% beef powder, 0.4wt% yeast powder, 2wt% glucose, 0.1 wt% Tween 80, 0.2wt% K2HPO47H2O, 0.5wt% sodium acetate· SELO, 0.2wt% triammonium citrate, 0.02wt% MgSO4’7H2O, 0.005wt% MnSOrTEEO, and 1.5wt% agar powder, dissolved by heating, adjusted to pH=6.2±0.2, and sterilized at a high pressure at 121°C for 15-20min.

MC broth: 0.5wt% soybean peptone, 0.3wt% beef powder, 0.3wt% yeast powder, 2wt% glucose, 2wt% lactose, lwt% calcium carbonate, 1.5wt% agar, and 0.5wt% 1% neutral red solution. The first seven components are added into distilled water, dissolved by heating, and adjusted to pH=6.0±0.2, then the neutral red solution is added, and the broth is sub-packed and then sterilized at 121 °C at a high pressure for 15-20min.

Example 1

This example is provided to describe the separation, purification and characterization of the Lactobacillus plantarum CGMCC No. 16441.

Strain: Lactobacillus plantarum CGMCC No. 16441, separated from traditional butter biscuits in Inner Mongolia, and preserved in Culture Collection Center for Nutrition and Health, COFCO Institute of Nutrition and Health.

Traditional butter biscuits, milk pieces, milk bars, dried milk cakes and other samples prepared with traditional methods are collected by the inventor from the herdsmen's homes in Hulunbeir and Xilin Gol League in Inner Mongolia, and gradient diluted to 10^-6 level with sterile physiological saline. The samples diluted at each gradient are spreading on MRS plates and MC plates, and are cultured at 36±1°C for 72h. Colonies in different colony morphologies are selected with inoculating needles onto MRS plates and MC plates and then

11877756_1 (GHMatters) P112431 .AU streaked and cultured, till the individual colonies are in uniform size and uniform morphology.

Strains that are Gram-stained to purple, catalase-negative, and don't form spores are selected. The above strains that are tentatively designated as lactic acid bacteria are activated for 3 generations in MRS broth, and then physiological and biochemical identification and molecular biological identification are carried out for them; in addition, the genetic stability of acid production and milk curdling properties, fermentation property, sensory quality, fermentation flavor substances, polysaccharide production capability, and tolerance to gastrointestinal acidic environment and gastrointestinal bile salt environment, etc. of the lactic acid bacteria are studied. After several rounds of study and proofing, a strain of Lactobacillus plantarum CGMCC NO. 16441 is finally screened out from a variety of wild lactic acid bacteria.

1. Morphological identification

The selected Lactobacillus plantarum CGMCC No. 16441 is cultured at 36±1°C for 72h. As shown in Fig. 1, the colony is in milky white color and in a circular shape, has a smooth surface, and is moist and opaque on the MRS medium. As shown in Fig. 2, the cell morphology of the Lactobacillus plantarum CGMCC No. 16441 is rod-shape under microscope and is Gram-positive.

2. Physiological and biochemical identification

Physiological and biochemical identification is carried out for the selected strain CGMCC NO. 16441 with BioMerienx API identification system. The identification result is shown in the following Table 1. Through physiological and biochemical identification, the separated strain is identified as Lactobacillus plantarum.

Table 1 - Result of Physiological and Biochemical Identification of the Strain CGMCC

NO. 16441

11877756_1 (GHMatters) P112431 .AU

Item	Result	Item	Result	Item	Result
Control	-	Inositol	-	D-melizitose	+
Mannitol	-	Mannitol	+	D-raffinose	-
Erythritol	-	Sorbitol	-	Starch	-
D-arabinose	-	Methyl-aD-mannopyranoside	-	Glycogen	-
L-arabinose	+	Methyl-aD-glucopyranoside	-	Xylitol	-
D-ribose	+	N-acety Iglucosamine	+	D-gentiobiose	+
D-xylose	-	Amygdaloside	+	D-Toulon sugar	+
L-xylose	-	ARBULIN	+	D-lyxose	-
D-adonit 1	-	Esculin ferric citrate	+	D-tagatose	-
Methyl-3D-xyl opyranoside	-	Salicin	+	D-fucose	-
D-galactose	+	D-cellobiose	+	L-fucose	-
D-glucose	+	D-maltose	+	D-arabinose	-
D-fructose	+	D-lactose	+	L-arabitol	-
D-mannose	+	D-melibioze	-	Potassium gluconate	+
L-sorbitol	-	D-sucrose	+	2-keto-potassium gluconate	-
L-rhamnose	-	D-trehalose	+	5 -keto-potassium gluconate	-
Galactitol	-	Inulin	-

[Note]: In the table, + represents a positive result of biochemical reaction, and represents a negative result of biochemical reaction.

3. Molecular identification

The 16s rDNA of the separated strain is cloned and sequenced, and the nucleotide sequence of the 16s rDNA gene is as shown in SEQ ID No. 1. The 16s rDNA sequence of the strain in the present invention is compared with the sequence Lactobacillus plantarum from NCBI. The 16s rDNA sequence of the strain in the present invention has 99% similarity to that of the Lactobacillus plantarum.

GGGGGCCTAATACATGCCAAGTCGAACGAACTCTGGTATTGATTG

GTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTA ACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACA

11877756_1 (GHMatters) P112431 .AU

GATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAA GATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGA TGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAG AGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACG GGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGA GCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTT GTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTAT TTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA CGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGC AGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGA AGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGA ACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGT GGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGT ATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACG ATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAA CGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTC AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA TTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAA TCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCA TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGG GGGAGACTGCCGGTGACAAACCGGAAGAAAGGGGGGGGATGAAGTT CAA

With reference to the biochemical identification result, the strain is identified as Lactobacillus plantarum.

The separated strain is identified as Lactobacillus plantarum and designated as Lactobacillus plantarum LCZ, and has been preserved in China General Microbiological Culture Collection Center, in Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), Building 3, Yard 1,

11877756_1 (GHMatters) P112431 .AU

Beichen West Road, Chao yang District, Beijing. The preservation number is CGMCC No. 16441, and the preservation date is Sept. 7, 2018.

Example 2

This example is provided to describe the study on genetic stability of the Lactobacillus plantarum CGMCC NO. 16441.

To study the acid production and milk curdling properties of CGMCC NO. 16441, the strain to be tested is inoculated into MRS broth and cultured at 37°C for 24-48h. After the strain is rejuvenated, it is inoculated at 4 vol% into a subculture medium (whole fat milk powder is prepared into 12wt% reconstituted milk solution, 6wt% white granulated sugar is added into the solution, and then the solution is mixed to a homogeneous state, and boiled for lOmin.), and ferments at 37°C. The fermentation time is recorded after the milk is coagulated, and the acidity and texture are measured. The results of the experiment after serial passage for 10 times are shown in Table 2 and Table 3.

Table 2 - Test Result of Genetic Stability of Acid Production Property of CGMCC

NO. 16441

No.	Fermentation time / h	pH at the end of fermentation	After-ripening titration acidity
GENERATION 10	15	4.53±0.5	89.74i0.32
GENERATION 1	10	4.32±0.67	95.93i0.78

Table 3 - Test Result of Genetic Stability of Milk Curdling Property of CGMCC NO. 16441

No.	Water retentivity / %	Consistency / g	Viscosity/
GENERATION 10	48.15i0.48	4721.86M5.80	2388il0.55
GENERATION 1	42.90i0.37	3886.11il2.47	2852M2.36

It can be seen, after the serial passage of the strain CGMCC NO. 16441 for times, the pH at the end of fermentation of the fermented milk is 4.53, the

11877756_1 (GHMatters) P112431 .AU after-ripening titration acidity is 89.74, the viscosity is 2388, and the consistency is 4721.86g, indicating that the acid production and milk curdling properties are genetically stable.

Example 3

This example is provided to describe the measurement of acid production capability of lactic acid bacteria.

Separated and purified lactic acid bacteria are activated, microscopically examined, and inoculated into a hermetically sealed container containing MC broth, and cultured at 37°C constant temperature; 15 mL culture solution is taken out and the pH value is measured with a precision acidimeter in every 2h; the measurement lasts 24h totally. Plotted with the time as the x coordinate and the pH value of the fermentation broth as the y coordinate, a dynamic change curve of pH representing the acid production capability of each strain is obtained, as shown in Fig. 3.

It can be seen, the pH reaches 3.76 after the strain CGMCC NO. 16441 ferments for 24h, and the acid production capability is higher than that of commercial strains (commercial strain No. 1: from Jiangsu Weikang Biotechnology Co., Ltd., lactic acid bacteria strain, strain designation: 1807B, Lactobacillus bulgaricus and Streptococcus thermophilus', commercial strain No. 2: from Hebei Yiran Biotechnology Co., Ltd., lactic acid bacteria strain, strain designation: Yo-C698-1F, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus).

Example 4

This example is provided to describe the study on polysaccharide production capability of the Lactobacillus plantarum CGMCC NO.16441.

The strain is inoculated into MRS broth, and cultured at 37°C for 48h; then lOmL culture medium is taken, and added 250pL 80 vol% trichloroacetic acid

11877756_1 (GHMatters) P112431 .AU (to remove proteins); the obtained mixture is centrifuged at 8,000r/min. for 20min. in a centrifuge, then the precipitate is discarded, and the supernatant is placed in a dialysis bag and dialyzed for 48h in running water. After dialysis, 95vol % ethanol in volume equal to 3 times of the volume of the supernatant is added, and the mixture is stored at 4°C overnight. The obtained polysaccharides are precipitated in a flocculent state; the mixture is centrifuged at 10,000r/min. for 20min. in a centrifuge, and then the supernatant is discarded, and the obtained precipitate is exopolysaccharides; then the precipitate is dissolved in lOmL distilled water to obtain an water solution of exopolysaccharides.

Testing total sugar with a phenol-sulfuric acid method: 2.0mL sample to be tested is loaded into a test tube, ImL 6% phenol and 5mL concentrated sulfuric acid are added sequentially, and the mixture is held still for lOmin., then is shaken up, kept at room temperature for 20min., and then the absorbance is measured at 490nm wavelength.

Testing reducing sugar with a 3,5-dinitrosalicylic acid method: 0.5mL sample to be tested is taken, 3,5-dinitrosalicylic acid reagent is added into the sample, the mixture is mixed to a homogeneous state, kept in boiling water bath for 5min., and cooled down, and then 4mL distilled water is added and mixed to a homogeneous state, and the absorbance is measured at 540nm wavelength.

The content of the polysaccharides is calculated according to a formula polysaccharides = total sugar - reducing sugar, and the calculation result is shown in the following Table 4.

Table 4 - Content of Polysaccharides Produced by Lactobacillus plantarum CGMCC

NO. 16441

CGMCC NO. 16441	Concentration (mg/L)
Total sugar	803.0±7.5
Reducing sugar	0
Polysaccharides	803.0

It can be seen, the yield of exopolysaccharides produced by the selected

11877756_1 (GHMatters) P112431 .AU strain CGMCC NO.16441 is as high as 803.0mg/L, indicating that the strain has high polysaccharides production capability. The yield of polysaccharides is much higher than the 150-600mg/L yield attained by strains of idem genus under optimized culture conditions.

Example 5

This example is provided to describe the study on the tolerance of Lactobacillus plantarum CGMCC NO. 16441 to simulated gastric fluid and intestinal fluid.

Preparation of simulated gastric fluid: 0.35g pepsin is dissolved in lOOmL 0.2wt% sterile physiological saline, the pH is adjusted to 3.0 with concentrated hydrochloric acid, and then the obtained solution is filtered through a 0.45pm filter membrane to sterilize.

Preparation of simulated intestinal fluid: O.lg trypsin and 1.8g bile salt are dissolved in a sterile solvent containing l.lg NaHCOs, 0.2g NaCl and lOOmL distilled water, and the pH is adjusted to 8.0 with 0.5M NaOH. The obtained solution is filtered through a 0.45pm filter membrane to sterilize.

The bacteria solution activated for three times is inoculated at 10vol% inoculum size into the simulated gastric fluid (pH=3.0), the mixture is mixed to a homogeneous state, and anaerobically cultured at 37°C; a sample is taken and loaded on a plate for counting at Oh and 3h, respectively. After the strain is cultured for 3h in the simulated gastric fluid, 1 mL culture broth is inoculated into 9mL the simulated intestinal fluid (pH=8), and anaerobically cultured at 37°C, and samples are taken for counting at 24h.

Survival ratio (%) = (logCFU Ni/logCFU No) x 100%;

Where, Ni represents the quantity of lactic acid bacteria after culturing in the simulated gastrointestinal fluid; No represents the quantity of lactic acid bacteria before culturing in the simulated gastrointestinal fluid.

The result is as follows: the survival ratio of CGMCC NO. 16441 is 62%

11877756_1 (GHMatters) P112431 .AU after the strain cultured in simulated gastric fluid at pH=3 for 3h, and the survival ratio is 39% after the strain cultured in the intestinal fluid for 24h. The separated strain exhibits excellent gastrointestinal tolerance.

Example 6

This example is provided to describe the study on intestinal adhesion of the Lactobacillus plantarum CGMCC NO. 16441.

Cultured HT-29 cells are digested to prepare a cell suspension (about 10⁵cells/mL), and 2mL cell suspension is taken and loaded into a 24-well plate with a cover slip, and cultured at 37°C in atmosphere composed of 5% CO2 and 95% air. The culture medium is composed of cell culture broth 1640, 10wt% inactivated calf serum and L-glutamine. After the cells essentially spread over the entire cover slip, the old cell culture medium is sucked out, the wells are washed with sterile PBS for 3 times, and a mixed solution of 1 mL bacterial solution (containing 10⁸ cfu/mL bacteria) and 1 mL cell culture broth 1640 is added into each well, and incubated at 37°C in atmosphere composed of 5% CO2 and 95% air for 2h. The glass slide is washed with sterile PBS to remove unbound bacteria. Then the strain remained on the glass slide is fixed with 10% formaldehyde for 2h and Gram-stained; 20 viewing fields are randomly selected under the microscope, and the number of bacteria adhering to 100 cells is counted; each treatment is made for 3 wells in parallel, and the average number of bacteria adhering to each cell is calculated.

The result is as follows: the adhesion of CGMCC NO.16441 to HT-29 cells is 1,000 bacterium/100 cells, which is higher than the value of 200 bacterium/100 cells attained by strains of idem genus under optimized culture conditions.

Example 7

This example is provided to describe the use of the Lactobacillus

11877756_1 (GHMatters) P112431 .AU plantarum CGMCC No. 16441 in fermented milk.

Acquisition of raw milk: qualified standard raw milk from Sanyuan Group.

The raw milk is heated up to 60-70°C, 6wt% white granulated sugar is added in the raw milk, and the mixture is mixed to a homogeneous state (water is replenished to recover the initial volume), homogenized once at 65°C and 200bar, and pasteurized at 85°C for 30min.; the milk is cooled to 42°C, the strain is inoculated at 5vol% inoculum size into the cooled milk, then the milk is stirred and mixed to a homogenous state, and cultured in a still state to cure shape at 42°C, and then after-ripened at 4°C for 24h; thus, the fermented milk is obtained.

The main function of lactic acid bacteria in milk fermentation is to produce acid and produce flavor substances. The acid production property of lactic acid bacteria is an important property for screening starters for fermented milk products. In general, lactic acid bacteria with stronger acid production capability have a stronger tendency to post-acidification. Lactic acid bacteria capable of producing acid in a larger amount and rapidly can lead to lower fermentation acidity of the final product, which is an effective measure to ensure low post-acidification. The acid production capabilities of different strains in the prior art at the end of fermentation are tested, and the results are shown in Table

5. The pH attained by the Lactobacillus plantarum CGMCC No. 16441 at the end of fermentation is 4.32±0.67, and the titration acidity after after-ripening is 95.93±0.78. The results indicate that the acidity value of the strain CGMCC No. 16441 is 20% higher than that of the control lactic acid bacteria, and the fermentation time is significantly shorter.

Table 5 - Comparison between Acid Production Capabilities of Different Strains at the

End of Fermentation

Strain	LLD	LLL	LLC	CGMCC NO. 16441
Acidity / °T	72.7±2.4	64.4±2.9	71.65±3.4	95.93±0.78

11877756_1 (GHMatters) P112431 .AU

pH	4.39±0.13	4.77±0.08	4.45±0.14	4.32±0.67
Fermentation time / h	6	6.5	6	5

Note: LLD is a dimethylglyoxal variant of Lactococcus lactis subsp lactis; LLL is Lactococcus lactis subsp lactis; LLC is a Lactococcus lactis subsp cremoris.

To study the flavor substances produced by the Lactobacillus plantarum CGMCC NO.16441 in fermented milk, mix 20ml set yogurt and 5mL saturated NaCl solution, the obtained mixture is shaken and mixed to a homogeneous state; then 5mL solution is transferred to a head-space bottle, and the components of the flavor substances are analyzed by GC-MS.

The detection result of the fermentation flavor substances is shown in Fig.

4. Altogether 25 major flavor substances are identified in fermented milk, including ketones, alcohols, esters, and ethers, etc. In addition to the characteristic flavor substances contained in ordinary yogurts and the yogurt fermented with CGMCC No. 16441, the yogurt fermented with CGMCC No. 16441 also contains flavor components such as 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone and ethyl hexanoate, which impart a cleaning flavor to the yoghurt.

Example 8

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in preparing active lactic acid bacteria beverages.

Purified water is heated up to 50-55°C, whole milk powder is added into the purified water, the mixture is heated up to 65-70°C, and is homogenized at a pressure of 180-200 bar. After the mixture is cooled down, a Lactobacillus plantarum CGMCC No. 16441 starter is added in volume equal to 3-5vol% of the mixture (emulsion), and then the emulsion is fermented at 37-42°C, till the pH is 4.0-4.5. The obtained product is demulsified and cooled down to obtain yogurt, and stored at a low temperature for later use. Purified water is heated up

11877756_1 (GHMatters) P112431 .AU to 65-75 °C, a mixture of white granulated sugar or other sweetener and a stabilizer (e.g., pectin) is added into the purified water, the obtained mixture is stirred for 15min., cooled down, and then adding the obtained yogurt, adjusting the pH to 3.5-4.0, and adding essences and water to a specified amount, heating up to 65-70°C and homogenizing, sterilizing at 180-200bar pressure at 115-130°C for 10-20s, cooling down and bottling; thus, a lactic acid bacteria beverage is obtained. The sensory features of the beverage are evaluated according to the evaluation criteria shown in Table 6.

Table 6 - Sensory Evaluation of the Lactic Acid Bacteria Beverage

Item	Color (20)	Taste and smell (50)	Structural state (30)	Total score (100)
Grade I	15-20 points White, uniform	40-50 points The taste is soft, lubrication in mouth, with rich fermentation fragrance, and unique taste.	20-30 points Uniform structural state, without stratification	>80
Grade II	10-20 points Dark yellow color	30-39 points The taste is relatively soft, with obvious fermentation fragrance, and is acidic or sweet.	15-20 points Relatively uniform structural state, with slight stratification	>70
Grade III	<10 points Light yellow color	<30 points Poor taste, without obvious fermentation fragrance	<10 points Obvious stratification	>60

Through evaluation on the basis of the color, taste, smell and structural state of the beverage fermented with lactic acid bacteria, the best fermentation strains are selected from different strains of commercial lactic acid bacteria. The corresponding sensory evaluation scores of the selected fermentation strains are shown in the following Table 7.

Table 7 - Sensory Evaluation Scores

Fermentation strain	Color	Taste and smell	Structural state	Total score
LLD	10	20	12	42
LLL	15	33	20	68

11877756_1 (GHMatters) P112431 .AU

LLC	14	38	20	72
CGMCC No. 16441	18	44	22	84

Note: LLD is a dimethylglyoxal variant of Lactococcus lactis subsp lactis; LLL is Lactococcus lactis subsp lactis; LLC is Lactococcus lactis subsp cremoris.

By scoring the lactic acid bacteria beverages fermented with different starters, it can be seen that different starters affect the experimental results significantly under the same fermentation conditions, wherein the lactic acid bacteria beverage fermented with CGMCC No. 16441 has fermented lactic acid fragrance, unique taste, pure white color, and uniform structural state.

Example 9

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in fermented cereal beverages.

Cereal raw materials that are in good color, fresh and full, free of pest damage and disease are selected, blended at a ratio of oat to rice within a range of 1:2-2:1, and soaked in water, until they water swell fully.

Taking the soaked raw materials and draining off the water, adding water in weight equal to 8 times of the weight of the raw materials to mash and boil. Loading the obtained slurry into a colloid mill and circulated for 2-4min. Adding citric acid to adjust the pH of the slurry to 4.0-5.0 to obtain a composite slurry, then adding a saccharifying enzyme in weight equal to 1-3% of the weight of the composite slurry to saccharify the composite slurry at 40-60°C for 4-8h.

Cooling down the saccharified composite slurry, adjusting the pH to 6.5-7.5 with sodium bicarbonate, and adding the Lactobacillus plantarum CGMCC No. 16441 starter; fermenting and culturing for a period of time till the concentration of Lactobacillus plantarum CGMCC No. 16441 reaches 10⁸ cfu/mL or above, and then adding white granulated sugar, citric acid, stabilizer, and essences, etc. to adjust the flavor; then cooling down and bottling the obtained product, and evaluating the sensory features of the product according to the evaluation

11877756_1 (GHMatters) P112431 .AU criteria shown in Table 8.

Table 8 - Sensory Evaluation of the Fermented Cereal Beverage

Item	Color (30)	Taste and smell (50)	Structural state (20)	Total score (100)
Grade I	26-30 points Pleasant yellowish, uniform	40-50 points The taste is soft, lubrication in mouth, with rich fermentation fragrance and unique taste.	17-20 points Uniform structural state without delamination	>80
Grade II	20-25 points Yellowish, slightly dark, slightly brown	30-39 points The taste is relatively soft, with obvious fermentation fragrance, and is acidic or sweet.	14-16 points Relatively uniform structural state,, with slight delamination	>70
Grade III	<20 points Light brown	<30 points Poor taste, without obvious fermentation fragrance	<14 points Obvious stratification	>60

Through evaluation on the basis of the color, flavor and structural state of the composite cereal fermented beverage, the best fermentation strains are selected from different strains of commercial lactic acid bacteria. The corresponding sensory evaluation scores of the selected fermentation strains are shown in the following Table 9.

Table 9 - Sensory Evaluation Scores

Fermentation strain	Color	Taste and smell	Structural state	Total score
LLD	16	20	11	47
LLL	20	33	13	66
LLC	18	27	12	57
CGMCC	26	41	15	82

By scoring the cereal fermented beverages fermented with different starters, it can be seen that different starters affect the experimental results significantly under the same fermentation conditions, wherein the cereal beverage fermented with CGMCC No. 16441 has composite fragrance from cereal and pleasant

11877756_1 (GHMatters) P112431 .AU fermentation flavor, unique taste, yellowish color, and uniform structural state.

Example 10

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in preparing fermented soybean milk.

The fermented soybean milk described in this example is prepared with the following method: soaking soybeans in soft water at volume ratio of soft water to soybean equal to 2:4 at 75-85°C temperature for l-2h, removing the soybean hulls and the soaking water, then adding boiling water at a weight ratio of soybean to boiling water equal to 1:6-10 to perform grinding, keeping the obtained slurry at 80-85°C temperature for 10-15min., and filtering the slurry through a 150-mesh screen to obtain soybean milk; adding 5-10wt% sucrose into the soybean milk based on the weight of the soybean milk, and then homogenizing at 15-25Mpa pressure, then sterilizing at 95°C temperature for 5-10min., cooling down the temperature to 28-38°C; inoculating with commercial Lactobacillus bulgaricus, Streptococcus thermophilus and CGMCC NO. 16441 starter in 0.04- 0.05wt% (based on the weight of the soymilk) with a ratio of bacterial count equal to 1:1:1, then fermenting the soybean milk at 32-42°C temperature for 6-8h, and then storing at 4°C low temperature; thus, obtaining the fermented soybean milk.

Comparing the fermented soybean milk fermented only with a starter composed of Lactobacillus bulgaricus and Streptococcus thermophilus (at 1:1 ratio of bacterial count), i.e., without the Lactobacillus plantarum CGMCC No. 16441 starter, the results indicates that the acidity of fermented soybean milk fermented with a starter including Lactobacillus plantarum CGMCC No. 16441 is 2.3 times of the acidity of fermented soybean milk fermented with a starter without Lactobacillus plantarum CGMCC No. 16441. Therefore, by using Lactobacillus plantarum CGMCC NO. 16441 in combination with commercial strains, the fermentation time can be shortened by 1 -1,5h.

11877756_1 (GHMatters) P112431 .AU

Example 11

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in preparing fermented fruit and vegetable juice. Selecting fruits and vegetables that are uniform and full in shape, washing with clean water to remove any dirt on the surface, soaking with 0.005-0.0Ig/kg sodium hypochlorite solution for 7-10min. for disinfection, and then rinsing, and dicing if required; blanching with boiling water for 3-5min., and treating with 0.5% citric acid and 0.3% Vc if necessary; mashing the fruits and vegetables with a tissue masher for 3-5min.; then adding 0.006-0.01% pectinase and 0.001% cellulase, and hydrolyzing at 35-52°C for l-2h; filtering through a double-layer filter with 80-mesh to obtain a clear liquid; sterilizing with microwave for 2min. or sterilizing bacteria and inactivating enzymes at 80°C for 15min. to obtain fruit or vegetable juice; picking the Lactobacillus plantarum CGMCC NO. 16441 from a slant into a broth culture medium to culture for 24h; then picking the strain into a small amount of the fruit or vegetable juice to culture for 20h; then inoculating the fermented seed solution into the prepared fruit or vegetable juice to culture at an appropriate temperature for 24h, till the pH value reaches 3.8-4.1 and the amount of lactic acid bacteria reaches (10⁸-10⁹) cfu/mL. Compared with fermented fruit or vegetable juice prepared with Lactobacillus casei as a starter, the fermented fruit and vegetable juice prepared with Lactobacillus plantarum had less acidity change and less post-acidification during cold storage, and has better fermentation stability.

Example 12

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in preparing fermented feed products.

Drying crop straws till the moisture content is lower than 15wt%, and then pulverizing; harvesting herbaceous plans, drying till the moisture content is

11877756_1 (GHMatters) P112431 .AU lower than 15wt%, and then chopping; premixing rice bran, wheat bran, salt, Lactobacillus plantarum CGMCC NO. 16441 ready-to-use starter and cellulase, and then mixing with the crop straw and the herbaceous plan raw materials progressively to a homogeneous state; compacting, bundling, and sealing the mixed microbial feed product to form a scale in size of 30-100kg/pack; fermenting the resultant product naturally for 12-18 days. The crop straws are selected from one or more of rape stalks , peanut stalks , and com stalks, etc. The herbaceous plans are a mixture of herbaceous plan selected from one or more of common herbaceous plan, such as Chinese milkvetch, etc.

Compared with the feed products fermented with commercial complex-probiotic-preparation, the lactic acid content in feed product fermented with the Lactobacillus plantarum CGMCC NO.16441 is 21.28g/kg, which is 4.4 times of that in the feed products fermented with commercial complex-probiotic-preparation.

Example 13

This example is provided to describe the use of the Lactobacillus plantarum CGMCC No. 16441 in preparing viable bacterial preparations.

Preparing the Lactobacillus plantarum selected above into a viable bacterial preparation. Inoculatomg the Lactobacillus plantarum CGMCC No. 16441 in 2-4% (v/v) inoculum size into MRS liquid culture medium, and culturing at 37-42°C for 20-24h to allow viable count of the Lactobacillus plantarum CGMCC NO. 16441 to reache 10⁸ cfu/mL or above; then, centrifuging the fermentation broth (at 4,000rpm speed, for lOmin.), washing the obtained precipitate twice with PBS buffer at pH=6.5-7.5, then adding skim milk and trehalose, etc. as a lyoprotectant, and adjusting the cell concentration to 10⁹cfu/mL; mixing the obtained mixture to a homogeneous state, and then performing vacuum freeze-drying to obtain a viable bacterial preparation. As a direct viable bacterial preparation, it contains 10⁸-10¹⁰ cfu/mL viable bacteria. It

11877756_1 (GHMatters) P112431 .AU may be directly added into fermented products for fermentation; alternatively, appropriate excipients may be added to the viable bacterial preparation with a conventional method to prepare formulations that are acceptable in the food industry or acceptable clinically, including various dose forms such as capsule, micro-capsule, tablet, and powder, etc.

The viable bacterial preparations prepared with the above-mentioned selected Lactobacillus plantarum may further comprise products in which the activity of the strain is maintained by technical means such as domestication, etc.

While the present invention is described above in detail in some preferred embodiments, the present invention is not limited to those embodiments. Various simple variations, including combinations of the technical features in any other appropriate way, can be made to the technical scheme of the present invention within the scope of the technical concept of the present invention, but such variations and combinations shall be deemed as disclosed content in the present invention and falling in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum, classified and designated as Lactobacillus plantarum, and preserved in China General Microbiological Culture Collection Center, assigned with a preservation number CGMCC No. 16441.

2. Use of the Lactobacillus plantarum according to claim 1 in the field of preparing fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products.

3. The use according to claim 2, comprising a step of allowing the Lactobacillus plantarum according to claim 1 to contact with an adapted fermentation substrate.

4. Use of the Lactobacillus plantarum according to claim 1 in preparing a ready-to-use starter.

5. A ready-to-use starter prepared by fermenting and culturing the Lactobacillus plantarum according to claim 1 in a fermentation medium.

6. A method for preparing the ready-to-use starter according to claim 5, comprising:

(1) fermenting and culturing the Lactobacillus plantarum according to claim 1 in a fermentation medium, till a viable count reaches 10⁸ cfu/mL or above to obtain a fermentation broth;

(2) centrifuging the fermentation broth obtained in the step (1), then rinsing with a buffer solution, adding a lyoprotectant, and adjusting the

11877756_1 (GHMatters) P112431 .AU concentration of viable bacteria to IO¹⁰ cfu/mL or above, mixing to a homogeneous state, and then performing vacuum freeze-drying, so as to obtain the ready-to-use starter.

7. Use of the ready-to-use starter according to claim 5 and/or a ready-to-use starter prepared with the method according to claim 6 in the field of preparing fermented dairy products, fermented cereal beverages, fermented soybean products, fermented fruit and vegetable products, and fermented feed products.

8. Use of the Lactobacillus plantarum according to claim 1 for preparing a viable bacterial preparation.

9. The use according to claim 8, wherein, the viable bacterial preparation comprises capsule, tablet, oral liquid, and powder.

10. A viable bacterial preparation prepared by fermenting and culturing the Lactobacillus plantarum according to claim 1 in a fermentation medium.