WO2020098097A1 - 一株发酵植物乳杆菌及其用途 - Google Patents

一株发酵植物乳杆菌及其用途 Download PDF

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WO2020098097A1
WO2020098097A1 PCT/CN2018/124327 CN2018124327W WO2020098097A1 WO 2020098097 A1 WO2020098097 A1 WO 2020098097A1 CN 2018124327 W CN2018124327 W CN 2018124327W WO 2020098097 A1 WO2020098097 A1 WO 2020098097A1
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fermented
lactobacillus plantarum
products
cgmcc
starter
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PCT/CN2018/124327
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English (en)
French (fr)
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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
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中粮营养健康研究院有限公司
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Priority to AU2018423072A priority Critical patent/AU2018423072B2/en
Priority to CN201880030861.0A priority patent/CN111436203B/zh
Publication of WO2020098097A1 publication Critical patent/WO2020098097A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the invention relates to the technical field of microbial fermentation, in particular discloses a strain of Lactobacillus plantarum, a direct-injection starter containing the strain of Lactobacillus plantarum, and a preparation method thereof, as well as their preparation of fermented dairy products, fermented cereal drinks, and fermented soybean products ,
  • the use in the field of fermented fruit and vegetable products, fermented feed and also discloses the use of the Lactobacillus plantarum in the preparation of live bacterial preparations and the prepared live bacterial preparations.
  • Lactic acid bacteria (lactic acid bacteria, LAB) is a generic term for a class of bacteria that can use fermentable carbohydrates to produce large amounts of lactic acid. This is a group of quite complex bacteria, currently divided into at least 18 genera, a total of more than 200 species, with rich species diversity.
  • Traditional fermented dairy products in China's Qinghai, Cambodia, Inner Mongolia, Xinjiang and other places contain rich resources of lactic acid bacteria, which is a rich treasure trove of probiotic bacteria.
  • Lactobacillus plantarum strains in lactic acid bacteria are widely used in food production, especially fermented foods.
  • Lactic acid bacteria as potential probiotics need to have the following properties: have outstanding probiotic functions, and need to tolerate the acidic environment of the gastrointestinal tract and bile salt environment, and have good cell adhesion.
  • lactic acid bacteria The main role of lactic acid bacteria in fermentation products is to produce acid and produce flavor substances.
  • the acid-producing characteristics of lactic acid bacteria are important characteristics for screening starters for fermentation products.
  • a lactic acid bacterium with a strong acid-producing ability has a strong tendency to acidify thereafter.
  • Lactic acid bacteria with fast acid production and large acid production can bring about lower final product fermentation acidity, which is an effective measure to ensure low post-acidification (such as dairy products).
  • gastric juice and intestinal juice constitute the human body's "biological barrier". Lactic acid bacteria must pass through the gastrointestinal tract and be attached to the intestinal epithelial cells to achieve colonization in order to exert beneficial effects on the human body or animal host.
  • Exopolysaccharides are produced by lactic acid bacteria during growth and metabolism and secreted to the outside of the cell, released into the growth matrix or capsular polysaccharide tightly bound to the cell surface. Mainly have the following functions: improve product quality, make the product delicate and even, increase the smooth taste; increase water retention, improve product viscosity, help to increase the non-specific adhesion of lactic acid bacteria to the intestinal surface, and increase the colonization of lactic acid bacteria Opportunities, and has a certain role in promoting the regulation of intestinal microflora composition.
  • the present invention screened a plant Lactobacillus plantarum with fast acid production, stable acid production and curd genetic properties, and a unique flavor of the fermented product, and used it in fermented dairy products, cereal products, beans In products, fruit and vegetable products and feed products, improve the quality of fermented products.
  • the Lactobacillus plantarum can also tolerate the acidic environment of the gastrointestinal tract, can produce high extracellular polysaccharides, improve the colonization of cells, and is used to prepare probiotic live bacterial preparations, which helps to regulate the composition of intestinal microflora.
  • the present invention provides a Lactobacillus plantarum strain.
  • the classification of the Lactobacillus plantarum strain is named Lactobacillus plantarum. .
  • the present invention provides the use of Lactobacillus plantarum as described above in the field of preparing fermented dairy products, fermented cereal drinks, fermented soy products, fermented fruit cooked products, and fermented feed.
  • the present invention provides the use of Lactobacillus plantarum as described above in the preparation of a direct-feed starter.
  • the present invention provides a direct-throwing starter, which is prepared by fermenting the Lactobacillus plantarum as described above in a fermentation medium.
  • the present invention provides a method of direct-injection starter as described above, the method comprising:
  • Lactobacillus plantarum is fermented and cultured in a fermentation medium, and the number of viable bacteria is 10 8 cfu / mL or more;
  • step (2) Centrifuge the fermentation broth obtained in step (1), then rinse with buffer, add lyoprotectant, and adjust the concentration of viable bacteria to 10 10 cfu / mL or more, mix evenly, and then freeze-dry in vacuum , To get the direct-injection starter.
  • the present invention provides the direct-injection starter as described above and / or the direct-injection starter prepared by the method as described above. Uses in the field of fermented feed.
  • the present invention provides the use of Lactobacillus plantarum as described above in the preparation of a live bacterial preparation.
  • the present invention provides a live bacterial preparation, which is prepared by fermenting Lactobacillus plantarum as described above in a fermentation medium.
  • the Lactobacillus plantarum CGMCC No. 16441 of the present invention is a safe strain that can be used for food selected from the milk cake made by the traditional method in the herdsman's house in Inner Mongolia.
  • the method of the present invention has no chemical addition and is green Natural, nutritious and healthy;
  • the Lactobacillus plantarum CGMCC No. 16441 of the present invention can effectively improve the fermentation quality of fermented dairy products, cereal products, soy products, fruit and vegetable products, feed products, and play the probiotic function of probiotics; the present invention uses Lactobacillus plantarum CGMCC No. As a starter, 16441 can effectively increase the acidity of the product, increase the content of polysaccharides, improve the nutritional value and flavor characteristics of the product, and have a certain improvement on the sensory quality characteristics of the product. Effectively improve the colonization of cells;
  • the Lactobacillus plantarum CGMCC No. 16441 of the present invention is easy to culture and prepare as a fermentation agent and a probiotic viable bacteria preparation, and has a high concentration of live bacteria in the fermentation agent of Lactobacillus plantarum.
  • the Lactobacillus plantarum provided by the present invention was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 7, 2018. Its deposit number is CGMCC No. 16441, and the deposit address is Beichenxi, Chaoyang District, Beijing No. 3, No. 1 Road, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC).
  • Figure 1 is the colony morphology of Lactobacillus plantarum of the present invention on the culture medium;
  • Figure 2 is the micromorphology of Lactobacillus plantarum of the present invention.
  • 3 is a graph showing the dynamic change of the pH value of acid production by different strains of the present invention.
  • FIG. 4 is the analysis and detection result of the fermentation flavor substance of Lactobacillus plantarum of the present invention.
  • the present invention provides a Lactobacillus plantarum strain.
  • the classification of the Lactobacillus plantarum strain is named Lactobacillus plantarum, which is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the Lactobacillus plantarum CGMCC No. 16441 has the following properties:
  • Morphological characteristics the colonies on MRS medium are milky white, round, smooth, moist, and opaque. The morphology of the cells is rod-shaped under the microscope, and the Gram stain is positive;
  • the fermented product also contains ingredients such as 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone, and ethyl caproate to give the product a fresh flavor;
  • Adhesion reach 1000 bacteria / 100 cells (HT-29).
  • the Lactobacillus plantarum CGMCC No. 16441 described in the present invention is selected from the milk cake made by the traditional method in the herdsmen's home in Inner Mongolia.
  • the Lactobacillus plantarum provided by the present invention can produce a large number of viable Lactobacillus plantarum through liquid culture, and the culture method is not particularly required, as long as it can proliferate the Lactobacillus plantarum, for example, according to 10 6- 8 CFU / mL inoculation volume Inoculate the live bacterial cells of Lactobacillus plantarum in the Lactobacillus culture medium, and under anaerobic or aerobic conditions, cultivate at a temperature of 15-40 ° C for 8-72 hours to obtain Culture medium.
  • the medium for Lactobacillus can be any medium suitable for the cultivation of Lactobacillus known in the art, for example, it can be milk and / or "lactic acid bacteria-biological basis and application" (Yang Jiebin, Light Industry Press, 1996) ) Described in the lactic acid bacteria (MRS) medium.
  • MFS lactic acid bacteria
  • the present invention can further isolate the live bacterial cells of Lactobacillus plantarum in the above-mentioned culture liquid.
  • the method for the separation is not particularly limited, as long as the cells can be enriched from the culture liquid, for example, by centrifugation and / or filtration Method, the conditions of the centrifugation and the filtration may be well-known conditions, and the present invention will not repeat them here.
  • the present invention provides the application of the Lactobacillus plantarum CGMCC No. 16441.
  • Lactobacillus plantarum CGMCC No. 16441 in one embodiment of the present invention, refers to the application in fermented food.
  • the fermented products include dairy products, cereal products, soy products, fruit and vegetable products, and feed products.
  • the dairy product may include yogurt, active lactic acid bacteria beverage, and the like.
  • the cereal product may include oat fermented beverage, black rice fermented beverage, buckwheat fermented beverage, and the like.
  • the soybean product may include fermented soybean milk and the like.
  • the fruit and vegetable product may include fermented fruit and vegetable juice.
  • the feed product may include solid anaerobic biological feed.
  • Lactobacillus plantarum CGMCC No. 16441 in the present invention is in the conventional production process of producing fermented dairy products, cereal products, soy products, fruit and vegetable products, feed products Inoculate Lactobacillus plantarum CGMCC No. 16441 into the raw material to be treated according to conventional use, and ferment or survive under the temperature and pressure that can propagate the plant Bacillus plantarum CGMCC No. 16441.
  • CGMCC No. 16441 By adding CGMCC No. 16441 to the fermentation substrate, its metabolic products make the fermented products have certain advantages such as acidity and aroma, while prolonging the preservation time of the products and improving the nutritional value and flavor characteristics of the products.
  • the present invention provides the use of the Lactobacillus plantarum CGMCC No. 16441 in the preparation of a direct-feeding starter.
  • the present invention provides a direct-throwing starter, which is prepared by fermenting Lactobacillus plantarum CGMCC No. 16441 in a fermentation medium.
  • the fermentation medium may be various conventionally used for fermenting lactic acid bacteria in the art, for example, may be the MRS medium as described above, or may be various suitable for optimization based on the MRS medium.
  • Culture medium for fermenting lactic acid bacteria may be various conventionally used for fermenting lactic acid bacteria in the art, for example, may be the MRS medium as described above, or may be various suitable for optimization based on the MRS medium.
  • the fermentation conditions may be conventional conditions known in the art for lactic acid bacteria fermentation culture, for example, the fermentation culture temperature may be 30-40 ° C.
  • the preparation method of the direct-injection starter can be prepared according to a conventional method in the art.
  • it can be prepared according to the following method: Lactobacillus plantarum CGMCC No. 16441 Until the number of viable bacteria reaches more than 10 8 cfu / mL, it is prepared by vacuum freeze drying after mixing evenly.
  • the direct-injection starter mentioned above in one embodiment of the present invention, is a bacterial powder prepared by vacuum freeze-drying Lactobacillus plantarum CGMCC No. 16441 fermentation broth, which contains more than 1 billion / g of active Lactobacillus plantarum CGMCC No. 16441.
  • the present invention provides a method for preparing the direct-injection starter as described above, the method comprising:
  • Lactobacillus plantarum CGMCC No. 16441 is fermented and cultured in a fermentation medium, and the number of viable bacteria reaches 10 8 cfu / mL or more;
  • step (2) Centrifuge the fermentation broth obtained in step (1), then rinse with buffer, add lyoprotectant, and adjust the concentration of viable bacteria to 10 10 cfu / mL or more, mix evenly, and then freeze-dry in vacuum , To get the direct-injection starter.
  • the method of centrifuging the fermentation bacterial liquid can be performed according to a conventional method in the art.
  • a bacterial pellet can be obtained by centrifuging at a speed of 5000-12000 rpm for 5-20 minutes in a refrigerated centrifuge.
  • the lyoprotectant may be various cryoprotectants conventional in the art, for example, it may be at least one of skim milk powder, maltodextrin, trehalose, dextran, and glycerin.
  • the buffer solution may be a buffer solution conventionally used for washing bacterial cells, for example, it may be physiological saline or PBS buffer solution.
  • the present invention provides the direct-injection starter as described above and / or the direct-injection starter prepared by the method described above in the preparation of fermented dairy products, fermented cereal beverages, fermented soy products, fermented fruit cooked products 1. Use in the field of fermented feed.
  • the present invention provides the use of Lactobacillus plantarum CGMCC No. 16441 in the preparation of a live bacterial preparation.
  • the live bacterial preparation may be in various known forms, such as but not limited to capsules, tablets, oral liquids, and powders.
  • the present invention provides a live bacterial preparation, which is prepared by fermenting the Lactobacillus plantarum as described above in a fermentation medium.
  • the method for preparing the live bacterial preparation can be prepared by referring to the direct-feeding starter as described above. In order to avoid unnecessary repetition, it will not be repeated here.
  • MRS medium Man Rogosa Sharpe Broth: 1 wt% peptone, beef powder 0.5 wt%, 0.4 wt% yeast extract, 2% glucose, 0.1 Tween 80 wt%, K 2 HPO 4 .7H 2 O 0.2 wt% , 0.5% by weight of sodium acetate ⁇ 3H 2 O, 0.2% by weight of triammonium citrate, 0.02% by weight of MgSO 4 ⁇ 7H 2 O, 0.005% by weight of MnSO 4 ⁇ 4H 2 O, 1.5% by weight of agar powder, dissolved by heating, and adjusted to pH 6.2 ⁇ 0.2, autoclaved at 121 °C for 15-20min.
  • MRS medium Man Rogosa Sharpe Broth
  • MC medium soybean peptone 0.5% by weight, beef powder 0.3% by weight, yeast powder 0.3% by weight, glucose 2% by weight, lactose 2% by weight, calcium carbonate 1% by weight, agar 1.5% by weight, 1% neutral red solution 0.5 weight%.
  • Lactobacillus plantarum CGMCC No. 16441 was isolated from Inner Mongolian traditional milk cakes and was preserved by the Nutrition and Healthy Strains Collection Center of COFCO Nutrition and Health Research Institute.
  • the inventors collected samples of traditional puff pastry, milk shreds, milk bars, milk tofu and other samples made by traditional methods from herdsmen's homes in Hulunbeier, Xilinguole League, Inner Mongolia, etc., and diluted them with sterile physiological saline gradient to 10 -6 .
  • MRS plates and MC plates were incubated at 36 ⁇ 1 °C for 72h. Inoculate needles to select colonies with different colony morphology to the MRS plate and MC plate to streak until single colonies are uniform in size and consistent in shape.
  • the strain with Gram staining purple, catalase negative, and no spore formation was selected.
  • the above-mentioned strains tentatively identified as lactic acid bacteria were activated in MRS liquid medium for 3 generations for physiological and biochemical identification and molecular biological identification, and the lactic acid bacteria's acid production and curd genetic stability, fermentation performance, sensory evaluation, fermentation Flavor substances, polysaccharide-producing ability, gastrointestinal tract acidic environment and gastrointestinal tract bile salt environment, etc. were studied. After several rounds of research and demonstration, a strain of Lactobacillus plantarum CGMCC was finally screened from many wild lactic acid bacteria .16441.
  • the selected Lactobacillus plantarum CGMCC No. 16441 was cultured at 36 ⁇ 1 ° C for 72 hours. As shown in Figure 1, the colonies were milky white, round, smooth, moist and opaque on the MRS medium. As shown in Figure 2, the cell morphology of Lactobacillus plantarum CGMCC No. 16441 was rod-shaped under the microscope, and was Gram-positive.
  • Physiological and biochemical identification of the screening strain CGMCC No. 16441 was carried out using the French Merrill API identification system. The identification results are shown in Table 1 below. After physiological and biochemical identification, the isolated strain was Lactobacillus plantarum.
  • the 16s rDNA of the isolated strain was cloned and sequenced.
  • the nucleotide sequence of the 16s rDNA gene is shown in SEQ ID No. 1.
  • the 16s rDNA sequence of the strain of the present invention was compared with the sequence of NCBI Lactobacillus plantarum The 16s rDNA sequence and Lactobacillus plantarum sequence similarity reached 99%.
  • this strain was identified as Lactobacillus plantarum.
  • the isolated strain was identified as Lactobacillus plantarum, named Lactobacillus plantarum LCZ , and has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee. Deposit address: Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing The Institute of Microbiology has the deposit number CGMCC No. 16441 and the deposit date is September 7, 2018.
  • test strains were connected to liquid MRS medium and cultured at 37 °C for 24-48h. Inoculate into the subculture medium (whole milk powder is formulated into 12% by weight reconstituted milk solution, add 6% by weight white sugar and mix well, boil for 10 min), and ferment at 37 °C After the milk solidifies, record the fermentation time, and measure the acidity and texture.
  • the experimental results are shown in Table 2 and Table 3 after 10 consecutive passages.
  • This example is used to illustrate the determination of the acid production capacity of lactic acid bacteria
  • the isolated and purified lactic acid bacteria were inoculated into an airtight container filled with MC liquid medium after microscopic examination, cultured at 37 ° C under constant temperature, 15mI of culture liquid was taken out every 2h, and the pH value was measured with a precision acidity meter for a total of 24h. Taking time as the abscissa and the pH value of the fermentation broth as the ordinate, the dynamic change curve of the pH value of acid production of each strain is shown in Figure 3.
  • strain CGMCC NO.16441 can reach a pH of 3.76 after 24 hours of fermentation, and its acid production capacity is higher than that of commercial strains (commercial strain No. 1: purchased from Jiangsu Weikang Biotechnology Co., Ltd., lactic acid bacteria strain, strain type: 1807B, Bulgarian milk Bacillus and Streptococcus thermophilus; commercial strain No. 1: purchased from Hebei Yiran Biotechnology Co., Ltd., lactic acid bacteria strain, strain model: Yo-C698-1F, Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus).
  • Phenol sulfuric acid method was used for the total sugar: after 2.0mL of the test sample was added to the test tube, 1mL of 6% phenol and 5mL of concentrated sulfuric acid were added in sequence, and then left to stand for 10min, shaken, and the absorbance was detected at a wavelength of 490nm after 20min at room temperature.
  • polysaccharide total sugar-reducing sugar
  • the screened strain CGMCC NO.16441 has an extracellular polysaccharide yield of up to 803.0 mg / L, which has a high polysaccharide production capacity. It is much higher than 150-600mg / L of similar strains after optimized culture conditions.
  • the preparation of simulated intestinal juice 0.1g trypsin, 1.8g bile salt dissolved in a sterile solvent containing 1.1g NaHCO3, 0.2g NaCl and 100mL distilled water, adjust the pH to 8.0 with 0.5M NaOH.
  • the solution was sterilized through a 0.45 ⁇ m filter membrane.
  • the bacterial liquid that has been activated three times is inoculated into the simulated gastric juice (pH 3.0) according to the inoculation amount of 10% by volume, mixed and incubated at 37 ° C anaerobicly, and the plates are counted at 0 and 3h respectively.
  • N 1 represents the number of lactic acid bacteria after simulated gastrointestinal tract culture
  • N 0 represents the number of lactic acid bacteria before simulated gastrointestinal tract culture.
  • This example is used to illustrate the intestinal adhesion of Lactobacillus plantarum CGMCC No. 16441
  • the HT-29 cells cultured good digestion made cell suspension (approximately 10 5 cells / mL), added to 24-well plates of 2mL coverslips have been placed in, 37 °C, 5% CO 2 , 95% Cultivate in the air.
  • the culture medium was 1640 culture solution and 10% by weight of inactivated calf serum and L-glutamine. After the cells on the coverslip are basically covered, aspirate the old cell culture solution, rinse with sterile PBS 3 times, and add a mixture of 1 mL bacterial solution (containing 10 8 cfu / mL of bacterial cells) and 1 mL 1640 cell culture solution to each well. Incubate at 37 ° C, 5% CO 2 , 95% air for 2h.
  • the adhesion 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 with the optimized culture conditions.
  • the main role of lactic acid bacteria in dairy fermentation is to produce acid and produce flavor substances.
  • the acid-generating properties of lactic acid bacteria are important characteristics for screening starters for fermented dairy products.
  • a lactic acid bacterium with a strong acid-producing ability has a strong tendency to acidify thereafter.
  • Lactic acid bacteria that produce acid quickly and produce a large amount of acid can bring about a low final product fermentation acidity, which is an effective measure to ensure low post-acidification.
  • LLD is a variant of Lactococcus lactis subsp. Butadione; LLL is a subspecies of Lactococcus lactis subsp .; LLC is a subspecies of Lactococcus lactis emulsifiable concentrate;
  • Lactobacillus plantarum CGMCC No. 16441 fermented milk flavor substances by taking 20ml coagulated yogurt added 5mL saturated NaCl solution to oscillate and mix, transfer 5mL solution to the headspace bottle to be tested, GC-MS analysis of flavor substances.
  • test results of fermented flavor substances are shown in Figure 4.
  • a total of 25 main flavor substances were identified in fermented milk, including ketones, alcohols, esters, ethers, etc.
  • the ordinary yogurt and CGMCC No. strain fermented yogurt contain the characteristic flavor substances, and also contain 3-hydroxy-2-butanone, 2-heptanone, 2-nonanone and ethyl caproate and other flavor components, which give the yogurt a fragrance Flavor.
  • LLD is a variant of Lactococcus lactis subsp. Butadione; LLL is a subspecies of Lactococcus lactis subsp .; LLC is a subspecies of Lactococcus lactis emulsifiable concentrate;
  • CGMCC No. 16441 fermented lactic acid bacteria beverage has a fermented lactic acid aroma and a unique taste. The color is pure white and uniform.
  • the slurry enters the colloid mill cycle for 2-4 minutes. Add citric acid to adjust the pH of the slurry to 4.0-5.0, add 1-3% saccharification enzyme of the composite slurry, and saccharify at a constant temperature of 40-60 ° C for 4-8h.
  • LLD is a variant of Lactococcus lactis subsp. Butadione; LLL is a subspecies of Lactococcus lactis subsp .; LLC is a subspecies of Lactococcus lactis emulsifiable concentrate;
  • CGMCC No. 16441 fermented grain beverages have a compound aroma and pleasure
  • the fermented flavor has a unique taste, light yellow color and uniformity.
  • the fermented soymilk described in this example is prepared by the following method: according to the volume ratio of soft water to soybean 2: 4, soak the soybean in soft water at a temperature of 75-85 ° C for 1-2 hours, remove the soybean hull, remove the soaking water, and then follow the soybean and
  • the weight ratio of boiling water is 1: 6-10 plus boiling water grinding slurry, the slurry is kept at a temperature of 80-85 ° C for 10-15min, filtered through a 150 mesh screen to obtain soy milk; add 5-10% by weight of soy milk Sucrose, homogenized again, with a homogenization pressure of 15-25Mpa, followed by a sterilization treatment at a temperature of 95 ° C for 5-10min, after its temperature was reduced to 28-38 ° C, inoculated with 0.04-0.05% (by weight of soybean milk) of commercial Using Lactobacillus bulgaricus, Streptococcus thermophilus and CGMCC NO. 16441 starter, the ratio of bacteria is 1: 1:
  • the fermented soybean milk prepared by using only commercial Lactobacillus bulgaricus and Streptococcus thermophilus (the ratio of the number of bacteria is 1: 1) without the addition of Lactobacillus plantarum CGMCC NO.16441 starter was compared.
  • the results show that the acidity of fermented soybean milk added with L. plantarum CGMCC NO. 16441 is 2.3 times higher than that without fermented soybean milk. Therefore, the use of L. plantarum CGMCC NO. 16441 in combination with commercial strains can shorten the fermentation time 1-1.5 hours.
  • the acid value of the fermented fruit and vegetable juices of Lactobacillus plantarum during refrigeration is smaller, the post-acidification is smaller, and the fermentation stability is good.
  • the crop straw is one or more selected from rape stalks, peanut seedlings, corn stalks and the like.
  • the herbaceous plant is a mixture of one or more selected from the common herbs of Astragalus.
  • the lactic acid content in the feed fermented by Lactobacillus plantarum CGMCC No. 16441 is 21.28g / kg, which is 4.4 times the content of lactic acid in the fermented feed for commercial compound probiotic bacteria.
  • Lactobacillus plantarum selected above is used as a live bacterial preparation.
  • Lactobacillus plantarum CGMCC NO. 16441 is inoculated into MRS liquid medium according to the inoculation amount of 2-4% (v / v), and cultivated at 37-42 ° C for 20-24h to make the number of viable bacteria of L. plantarum CGMCC NO. Above 10 8 cfu / mL, centrifuge (4000rpm, 10min), rinse the pellet twice with PBS buffer pH6.5-7.5, add skim milk and trehalose as lyoprotectant, adjust the cell concentration to 10 9 cfu / mL, after mixing, vacuum freeze-drying to obtain a live bacterial preparation.
  • the number of viable bacteria is 10 8 -10 10 cfu / mL.
  • the fungus can be directly added to the fermentation product for fermentation, or it can be added to the food industry or clinically acceptable by conventional methods.
  • the dosage form includes various dosage forms such as capsules, microcapsules, tablets and powders.
  • the live bacterial preparation made from the above-mentioned screened Lactobacillus plantarum also includes products that maintain the activity of strains through technical means such as domestication.

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Abstract

本发明属于微生物发酵技术领域，具体涉及一株植物乳杆菌及其用途，该植物乳杆菌的分类命名为Lactobacillus plantarum，保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.16441。本发明植物乳杆菌具有耐酸性、耐胆盐能力，对肠上皮细胞株HT-29细胞具有很高的粘附性，具有很高的产酸及产多糖能力，较其他乳酸菌产酸速度快，产酸能力强，产多糖能力高。用本发明植物乳杆菌制备的发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果蔬汁、发酵饲料风味独特、质地均一、口感顺滑，具有良好的感官特征。可广泛应用于发酵产品领域，也可用于制备活菌制剂。

Description

一株发酵植物乳杆菌及其用途 技术领域
本发明涉及微生物发酵技术领域，具体公开了一株植物乳杆菌，以及含有该株植物乳杆菌的直投式发酵剂及其制备方法，以及他们在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果蔬制品、发酵饲料领域中的用途，还公开了该植物乳杆菌在制备活菌制剂中的用途及制备的活菌制剂。
背景技术
乳酸菌(lactic acid bacteria，LAB)是一类能利用可发酵碳水化合物产生大量乳酸的细菌的通称。这是一群相当庞杂的细菌，目前至少可分为18个属，共有200多种，具有丰富的物种多样性。我国青海、西藏、内蒙、新疆等地的传统发酵乳制品中蕴藏着丰富的乳酸菌菌种资源，是丰富的益生菌菌种宝库。
乳酸菌中的植物乳杆菌菌种广泛用于食品生产，尤其是发酵食品。有研究表明：乳酸菌的产酸能力及发酵风味特征等在发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料生产中发挥了重要的作用，使用时可以单独使用，也可以结合其它细菌使用。作为潜在益生菌的乳酸菌需要具备以下性能：具有突出的益生功能，而且需要耐受胃肠道酸性环境和胆盐环境，具有较好的细胞黏附性。
乳酸菌在发酵产品中的主要作用是产酸和产生风味物质。乳酸菌的产酸特性是筛选发酵产品用发酵剂的重要特性。一般而言产酸能力强的乳酸菌，其后酸化的趋势也强。产酸快、产酸量大的乳酸菌能够带来较低的最终产品发酵酸度，这是保证低后酸化(如乳制品)的有效措施。作为益生菌，胃液和肠液构成人体的“生物屏障”，乳酸菌必通过胃肠道，并點附于肠道上皮细胞实现定植，才能发挥对人体或动物宿主的有益功效。
乳酸菌胞外多糖(Exopolysaccharides,EPS)是由乳酸菌在生长代谢过程产生并分泌到细胞外，释放到生长基质中的多糖或紧密结合在细胞表面的荚膜多糖。主要有以下几个方面的功能：改善产品品质，使产品细腻均匀，增加爽滑的口感；增加持水性，改善产品的粘性，有助于提高乳酸菌对肠道表面的非特异性黏附，增加乳酸菌定植的机会，并对调节肠道微生物菌群组成具有一定的促进作用。
因此，具有良好的产酸能力、产胞外多糖能力，并且可抗胃肠道胁迫，具有较好的细胞粘附性，发酵感官特征突出是筛选发酵剂及益生菌的必要因素。
发明内容
为解决上述技术问题，本发明筛选了一株产酸速度快、产酸、凝乳遗传性能稳定，制得发酵产品风味独特的植物乳杆菌，并且将其用于发酵乳制品、谷物制品、豆制品、果蔬制品和饲料制品中，提高发酵产品的品质。该植物乳杆菌还能耐受胃肠道酸性环境，能够高产胞外多糖，提高细胞的定 植性，用于制备益生菌活菌制剂，有助于调节肠道微生物菌群组成。
为了实现上述目的，第一方面，本发明提供一株植物乳杆菌，该植物乳杆菌的分类命名为Lactobacillus plantarum，保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.16441。
第二方面，本发明提供如上所述的植物乳杆菌在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料领域中的用途。
第三方面，本发明提供如上所述的植物乳杆菌在制备直投式发酵剂中的用途。
第四方面，本发明提供一种直投式发酵剂，其该直投式发酵剂由如上所述的植物乳杆菌在发酵培养基中进行发酵培养后制得。
第五方面，本发明提供如上所述的直投式发酵剂的方法，该方法包括：
(1)将如上所述的植物乳杆菌在发酵培养基中进行发酵培养，并使活菌数达到10 8cfu/mL以上；
(2)将步骤(1)得到的发酵菌液进行离心处理，之后用缓冲液进行冲洗，加入冻干保护剂，并调整活菌浓度至10 10cfu/mL以上，混合均匀后进行真空冷冻干燥，即得所述直投式发酵剂。
第六方面，本发明提供如上所述的直投式发酵剂和/或如上所述的方法制备的直投式发酵剂在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料领域中的用途。
第七方面，本发明提供如上所述的植物乳杆菌在制备活菌制剂中的用途。
第八方面，本发明提供一种活菌制剂，该活菌制剂由如上所述的植物乳杆菌在发酵培养基中进行发酵培养后制得。
本发明的有益效果是：
1、安全健康、成本低廉：本发明的植物乳杆菌CGMCC No.16441是从内蒙古地区牧民家中传统方法制作的奶酥中筛选出的可用于食品的安全菌株，本发明的方法无化学添加，绿色天然，营养健康；
2、本发明的植物乳杆菌CGMCC No.16441能够有效提高发酵乳制品、谷物制品、豆制品、果蔬制品、饲料制品的发酵品质及发挥益生菌的益生功能；本发明用植物乳杆菌CGMCC No.16441作为发酵剂，可有效提高产品酸度、增加多糖含量，改善了产品营养价值和风味特性，对产品感官品质特性具有一定的改善作用；本发明的植物乳杆菌CGMCC No.16441作为益生菌，可有效提高提高细胞的定植性；
3、本发明的植物乳杆菌CGMCC No.16441作为发酵剂和益生菌活菌制剂，易于培养和制备，植物乳杆菌的发酵剂活菌浓度高。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
生物保藏
本发明提供的植物乳杆菌(Lactobacillus plantarum)，于2018年9月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.16441，保藏地址为北京市朝阳区北 辰西路1号院3号，中国科学院微生物研究所(缩写为CGMCC)。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中，
图1为本发明植物乳杆菌在培养基上的菌落形态；
图2为本发明植物乳杆菌显微形态；
图3为本发明不同菌株产酸的pH值动态变化曲线图；
图4为本发明植物乳杆菌发酵风味物质分析检测结果。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
第一方面，本发明提供了一株植物乳杆菌，该植物乳杆菌的分类命名为Lactobacillus plantarum，保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.16441。
所述的植物乳杆菌CGMCC No.16441具有下述性质：
(1)形态学特征：在MRS培养基上菌落乳白色，圆形，表面光滑，湿润，不透明，显微镜下细胞形态为杆状，革兰氏染色阳性；
(2)遗传稳定性：产酸及凝乳性能遗传稳定，传代接种10代，酸度值为89.74，稠度达到4721.86/g；
(3)产酸性：根据MC液体培养基进行产酸能力测试，得出菌株CGMCC NO.16441发酵24h后pH能达3.76，产酸能力高于商业菌株；
(4)发酵风味特征：发酵产品中除含有共有风味物质外，还含有3-羟基-2-丁酮、2-庚酮、2-壬酮和己酸乙酯等成分，赋予产品清香的风味；
(5)体内耐受性：在pH 3.0的人工胃液中处理3h后的存活率为62％；在模拟肠液中处理24h的存活率为39％；表现出良好的胃肠道耐受性；
(6)产多糖能力：菌株接种于MRS液体培养基中，37℃培养48h后，胞外多糖产量高达803mg/L，远远高于经过优化培养条件的同类菌株的150-600mg/L；
(7)黏附性：达到1000个细菌/100个细胞(HT-29)。
本发明所述的植物乳杆菌CGMCC No.16441，是从内蒙古地区牧民家中传统方法制作的奶酥中筛选出的。
本发明提供的植物乳杆菌经过液体培养能够产生大量植物乳杆菌的活菌体，所述培养的方法没 有特别的要求，只要是能使所述植物乳杆菌增殖即可，例如可以按照10 6-8CFU/mL的接种量将植物乳杆菌的活菌体接种于乳杆菌培养基中，并且在厌氧或好氧的条件下，在15-40℃的温度下培养8-72小时后，得到培养液。所述乳杆菌的培养基可以为本领域公知的各种适合乳杆菌培养的培养基，例如可以为乳汁和/或《乳酸菌——生物学基础及应用》(杨洁彬，轻工业出版社，1996年出版)中所述的乳酸菌(MRS)培养基。
本发明可以进一步分离上述培养液中的植物乳杆菌的活菌体，所述分离的方法没有特别的限制，只要是能从培养液中富集菌体即可，例如可以通过离心和/或过滤的方法实现，所述离心和所述过滤的条件可以为公知的条件，本发明在此不再赘述。
第二方面，本发明提供所述植物乳杆菌CGMCC No.16441的应用。
本发明所述植物乳杆菌CGMCC No.16441的应用，在本发明的一种实施方式中，是指在发酵食品方面的应用。
所述发酵制品，在本发明的一种实施方式中，包括乳制品、谷物制品、豆制品、果蔬制品、饲料制品。
所述乳制品，在本发明的一种实施方式中，可以包括酸奶、活性乳酸菌饮料等。
所述谷物制品，在本发明的一种实施方式中，可以包括燕麦发酵饮料、黑米发酵饮料、荞麦发酵饮料等。
所述豆制品，在本发明的一种实施方式中，可以包括发酵豆乳等。
所述果蔬制品，在本发明的一种实施方式中，可以包括发酵果蔬汁等。
所述饲料制品，在本发明的一种实施方式中，可以包括固态厌氧生物饲料。
本发明所述的植物乳杆菌CGMCC No.16441在发酵食品方面的应用，在本发明的实施方式中，是在生产发酵乳制品、谷物制品、豆制品、果蔬制品、饲料制品的常规生产过程中，把植物乳杆菌CGMCC No.16441按照常规使用接种到待处理的原料中，在能够使所述植物杆菌CGMCC No.16441繁殖的温度、压力下进行发酵或存活。通过在发酵底物中加入CGMCC No.16441，其代谢产物使发酵制品具有一定的酸度、香味等优益特性，同时使产品延长了保藏时间，改善了产品营养价值和风味特性。
第三方面，本发明提供了所述植物乳杆菌CGMCC No.16441在制备直投式发酵剂中的用途。
第四方面，本发明提供了一种直投式发酵剂，该直投式发酵剂由植物乳杆菌CGMCC No.16441在发酵培养基中进行发酵培养后制得。
根据本发明，所述发酵培养基可以为本领域常规的各种用于发酵乳酸菌，例如，可以为如上所述的MRS培养基，也可以为在MRS培养基的基础上优化的各种适合于发酵乳酸菌的培养基。
根据本发明，所述发酵的条件可以为本领域公知的常规的用于乳酸菌发酵培养的条件，例如，所述发酵培养的温度可以为30-40℃。
根据本发明，所述直投式发酵剂的制备方法可以按照本领域常规的方法进行制备，在本发明的一种实施方式中，可以是按以下方法制备得到：培养植物乳杆菌CGMCC No.16441至活菌数达到 10 8cfu/mL以上，混合均匀后进行真空冷冻干燥制得。
所述的直投式发酵剂，在本发明的一种实施方式中，是真空冷冻干燥植物乳杆菌CGMCC No.16441发酵菌液制备的菌粉，它含有10亿/g以上的活性植物乳杆菌CGMCC No.16441。
第五方面，本发明提供制备如上所述的直投式发酵剂的方法，该方法包括：
(1)将植物乳杆菌CGMCC No.16441在发酵培养基中进行发酵培养，并使活菌数达到10 8cfu/mL以上；
(2)将步骤(1)得到的发酵菌液进行离心处理，之后用缓冲液进行冲洗，加入冻干保护剂，并调整活菌浓度至10 10cfu/mL以上，混合均匀后进行真空冷冻干燥，即得所述直投式发酵剂。
根据本发明，如上对植物乳杆菌CGMCC No.16441进行发酵培养的方法已经进行了详细的介绍，为了避免不必要的重复，此处不再赘述。
根据本发明，对所述发酵菌液进行离心的方法可以按照本领域常规的方法进行，例如，可以在冷冻离心机中以5000-12000rpm的速度离心5-20min获得菌体沉淀。
根据本发明，所述冻干保护剂可以为本领域常规的各种冷冻保护剂，例如，可以为脱脂乳粉、麦芽糊精、海藻糖、葡聚糖及甘油中的至少一种。
根据本发明，所述缓冲液可以为本领域常规的用于对菌体进行冲洗的缓冲液，例如，可以为生理盐水或PBS缓冲液。
第六方面，本发明提供了如上所述的直投式发酵剂和/或如上所述的方法制备的直投式发酵剂在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料领域中的用途。
第七方面，本发明提供了植物乳杆菌CGMCC No.16441在制备活菌制剂中的用途。
根据本发明，所述活菌制剂可以为公知的各种形式，例如但不限于胶囊、片剂、口服液和粉剂。
第八方面，本发明提供了一种活菌制剂，该活菌制剂由如上所述的植物乳杆菌在发酵培养基中进行发酵培养后制得。
根据本发明，所述活菌制剂的制备方法可以参照如上所述的直投式发酵剂进行制备为了避免不必要的重复，此处不再赘述。
实施例
下述实施例中涉及的培养基配方如下：
MRS培养基(Man Rogosa Sharpe Broth)：蛋白胨1重量％，牛肉粉0.5重量％，酵母粉0.4重量％，葡萄糖2重量％，吐温80 0.1重量％，K 2HPO 4.7H 2O 0.2重量％，乙酸钠·3H 2O 0.5重量％，柠檬酸三铵0.2重量％，MgSO 4·7H 2O 0.02重量％，MnSO 4·4H 2O 0.005重量％，琼脂粉1.5％，加热溶解，调节pH至6.2±0.2，121℃高压灭菌15-20min。
MC培养基：大豆蛋白胨0.5重量％，牛肉粉0.3重量％，酵母粉0.3重量％，葡萄糖2重量％，乳糖2重量％，碳酸钙1重量％，琼脂1.5重量％，1％中性红溶液0.5重量％。将前面7种成分加入蒸馏水中，加热溶解，调节pH至6.0±0.2，加入中性红溶液，分装后121℃高压灭菌15-20min。
实施例1
本实施例用于说明植物乳杆菌CGMCC No.16441的分离、纯化及其特性鉴定
菌株：植物乳杆菌CGMCC No.16441分离自内蒙古传统奶酥，由中粮营养健康研究院营养健康菌种保藏中心保藏。
发明人从内蒙古呼伦贝尔、锡林郭勒盟等地区的牧民家中采集传统方法制作的传统奶酥、奶碎、奶条、奶豆腐等样品，以无菌生理盐水梯度稀释至10 -6，各稀释梯度依次涂MRS平板和MC平板，并于36±1℃培养72h。接种针挑选菌落形态各异的菌落至MRS平板和MC平板划线直至出现单菌落大小均一、形态一致。
选择革兰氏染色为紫色，过氧化氢酶阴性，且不形成芽孢的菌株。将上述分离到暂定为乳酸菌的菌株在MRS液体培养基中活化3代后进行生理生化鉴定和分子生物学鉴定，并对乳酸菌的产酸及凝乳遗传稳定性，发酵性能，感官品评，发酵风味物质、产多糖能力及抗胃肠道酸性环境及胃肠道胆盐环境能力等多个方面进行研究，经过多轮研究和论证，从众多野生乳酸菌中最终筛选获得一株植物乳杆菌CGMCC NO.16441。
1、形态学鉴定
将筛选的植物乳杆菌CGMCC NO.16441，于36±1℃培养72小时，如图1所示，在MRS培养基上菌落乳白色，圆形，表面光滑，湿润，不透明。如图2所示，植物乳杆菌CGMCC NO.16441的显微镜下细胞形态为杆状，呈革兰氏阳性。
2、生理生化鉴定
利用法国梅里埃API鉴定系统对筛选菌株CGMCC NO.16441进行生理生化鉴定，鉴定结果见下表1。经生理生化鉴定，分离菌株为植物乳杆菌(Lactobacillus plantarum)。
表1 菌株CGMCC NO.16441的生理生化鉴定实验结果
项目 结果 项目 结果 项目 结果
对照 - 肌醇 - D-松三糖 +
甘露醇 - 甘露醇 + D-棉子糖 -
赤藻糖醇 - 山梨醇 - 淀粉 -
D-阿拉伯糖 - 甲基-αD-吡喃甘露糖苷 - 糖原 -
L-阿拉伯糖 + 甲基-αD-吡喃葡萄糖苷 - 木糖醇 -
D-核糖 + N-乙酰葡萄糖胺 + D-龙胆二糖 +
D-木糖 - 苦杏仁苷 + D-土伦糖 +
L-木糖 - ARBULIN + D-来苏糖 -
D-侧金盏花醇1 - 七叶灵柠檬酸铁 + D-塔格糖 -
甲基-βD吡喃木糖苷 - 水杨苷 + D-岩藻糖 -
D-半乳糖 + D-纤维二糖 + L-岩藻糖 -
D-葡萄糖 + D-麦芽糖 + D-阿拉伯糖 -
D-果糖 + D-乳糖 + L-阿拉伯醇 -
D-甘露糖 + D-蜜二糖 - 葡萄糖酸钾 +
L-山梨醇 - D-蔗糖 + 2酮基葡萄糖酸钾 -
L-鼠李糖 - D-海藻糖 + 5酮基葡萄糖酸钾 -
卫矛醇 - 菊粉 -    
[注]：表中“+”代表生化反应结果为阳性，“-”代表生化反应结果为阴性。
3、分子鉴定
对分离菌株的16s rDNA进行克隆和测序，其16s rDNA基因的核苷酸序列如SEQ ID No.1所示，将本发明菌株的16s rDNA序列与NCBI植物乳杆菌的序列比对，本发明菌株的16s rDNA序列与Lactobacillus plantarum序列相似性达到99％。
Figure PCTCN2018124327-appb-000001
结合生化鉴定结果，鉴定出此菌株为植物乳杆菌(Lactobacillus plantarum)。
将分离菌株确定为植物乳杆菌(Lactobacillus plantarum)，命名为Lactobacillus plantarum  LCZ，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，其保藏编号为CGMCC No.16441，保藏日期为2018年9月7日。
实施例2
本实施例用于说明植物乳杆菌CGMCC NO.16441的遗传稳定性研究
为了对CGMCC NO.16441的产酸及凝乳性能进行考察，将试验菌种接到液体MRS培养基中， 37℃培养24-48h，当菌种恢复活力后，将菌种按4体积％比例接种到传代培养基中(全脂奶粉配成12重量％复原乳溶液，加入6重量％白砂糖混匀、煮沸10min)，37℃发酵。牛奶凝固后记录发酵时间，同时测定酸度和质构等。连续传代10次后实验结果如表2、表3所示。
表2 CGMCC NO.16441产酸遗传稳定性试验结果
编号 发酵时间/h 发酵终点pH 后熟滴定酸度
10代 15 4.53±0.5 89.74±0.32
1代 10 4.32±0.67 95.93±0.78
表3 CGMCC NO.16441凝乳性能遗传稳定性试验结果
编号 持水力/％ 稠度/g 粘度
10代 48.15±0.48 4721.86±15.80 2388±10.55
1代 42.90±0.37 3886.11±12.47 2852±12.36
可见，菌株CGMCC NO.16441连续传代10次后，发酵乳的发酵终点pH为4.53，后熟滴定酸度为89.74，粘度达到2388，稠度达到4721.86/g，产酸能力及凝乳性能遗传稳定。
实施例3
本实施例用于说明乳酸菌产酸能力的测定研究
将分离纯化的乳酸菌活化镜检后接种于装有MC液体培养基的密闭容器中，37℃恒温培养，每2h取出15mI培养液，用精密酸度计测pH值，共测24h。以时间为横坐标，发酵培养液pH值为纵坐标，得到各菌株产酸的pH值动态变化曲线见图3。
可见，菌株CGMCC NO.16441发酵24h后pH能达3.76，产酸能力高于商业菌株(商业菌株1号：购自江苏微康生物科技有限公司，乳酸菌菌种，菌种型号：1807B，保加利亚乳杆菌和嗜热链球菌；商业菌株1号：购自河北一然生物科技有限公司，乳酸菌菌种，菌种型号：Yo-C698-1F，德氏乳杆菌保加利亚亚种和嗜热链球菌)。
实施例4
本实施例用于说明植物乳杆菌CGMCC NO.16441产多糖的研究
将菌株接种于MRS液体培养基中，37℃培养48h，取l0mL培养液，加入250uL体积分数为80％的三氯乙酸(脱除蛋白质)，在离心机中以8000r/min离心20min，离心后弃去沉淀，将上清液装入透析袋中，流水透析48h。透析后加入3倍体积95％的乙醇，在4℃下冷藏过夜，得到的多糖呈絮状沉淀析出，在离心机中以l0000r/min离心20min，弃去上清液，得到的沉淀即为胞外多糖，将沉淀溶解于l0mL蒸馏水中，得到胞外多糖水溶液。
总糖使用苯酚硫酸法：待测样2.0mL加入试管中后，依次加入1mL 6％苯酚，5mL浓硫酸，静置l0min，摇匀，室温放置20min后于490nm波长下检测吸光度。
还原糖使用3,5二硝基水杨酸法：待测样0.5mL，加入3,5二硝基水杨酸试剂混匀，沸水浴5min，冷却后加入4mL蒸馏水，混匀，在540nm下检测吸光度。
所述多糖的含量按照公式“多糖＝总糖-还原糖”进行计算，计算结果见下表4所示。
表4 植物乳杆菌CGMCC NO.16441产多糖含量
CGMCC NO. 浓度(mg/L)
总糖 803.0±7.5
还原糖 0
多糖 803.0
可见，筛选的菌株CGMCC NO.16441胞外多糖产量高达803.0mg/L，具有很高的多糖产生能力。远高于经过优化培养条件的同类菌株的150-600mg/L。
实施例5
本实施例用于说明植物乳杆菌CGMCC NO.16441对模拟胃液、肠液耐受性的研究
模拟胃液的配制：0.35g胃蛋白酶溶于100mL 0.2重量％的无菌生理盐水，用浓盐酸调节pH到3.0，过0.45um滤膜除菌。
模拟肠液的配制：0.1g胰蛋白酶，1.8g胆盐溶于无菌溶剂含1.1g NaHCO3、0.2g NaCl及100mL蒸馏水，用0.5M的NaOH调节pH到8.0。溶液过0.45μm滤膜除菌。
将己活化3次的菌液按10体积％的接种量接种到模拟胃液(pH3.0)中，混匀，37℃厌氧培养，分别在0、3h取样平板计数。在模拟胃液中培养3h后，吸取1mL培养液接种到9mL模拟肠液(pH8))中，37℃厌氧培养，在24h取样计数。
存活率(％)＝(logCFU N 1/logCFU N 0)×100％；
其中N 1代表经过模拟胃肠道培养后的乳酸菌数量；N 0代表模拟胃肠道培养前的乳酸菌数量。
结果如下：CGMCC NO.16441在pH＝3的模拟胃液中生存3h后，存活率为62％，在肠液中24h后，存活率维持在39％，分离菌株显示了很好的胃肠道耐受性能。
实施例6
本实施例用于说明植物乳杆菌CGMCC NO.16441的肠粘附性
将培养好的HT-29细胞进行消化，制成细胞悬液(约10 5个/mL)，取2mL添加到已放置盖玻片的24孔板里，37℃，5％CO 2，95％空气中培养。培养基为1640培养液与10重量％的失活的小牛血清、L-谷氨酰胺。待盖玻片上细胞基本铺满后，吸出旧的细胞培养液，无菌PBS冲洗3次，每孔加入1mL菌液(含菌体l0 8cfu/mL)与1mL 1640细胞培养液的混合液在37℃，5％CO 2，95％空气中孵育2h。无菌PBS洗涤载玻片以除去未结合菌。然后10％甲醛固定2h，革兰氏染色，显微镜下随机挑选20个视野，计数100个细胞上黏附的细菌数，每个处理平行3孔，再计算平均每个细胞所黏附的细菌数。
结果如下：CGMCC NO.16441对HT-29细胞的黏附性为1000个细菌/100个细胞，高于经过优化培养条件的同类菌株的200个细菌/100个细胞。
实施例7
本实施例用于说明植物乳杆菌CGMCC No.16441在发酵乳方面的应用
原料乳的获取：购自三元集团的合格的标准化原料乳。
将原料乳加热至60-70℃，加入6重量％白砂糖混匀(补水至初始重量)，于65℃、200bar下均质一次，85℃巴氏杀菌30min；温度降至42℃，以5体积％接种量接种，搅拌混匀，42℃下静置培养固定成型，4℃后熟24h后，即得到所述的发酵乳。
乳酸菌在乳品发酵中的主要作用是产酸和产生风味物质。乳酸菌的产酸特性是筛选发酵乳品用发酵剂的重要特性。一般而言产酸能力强的乳酸菌，其后酸化的趋势也强。产酸快、产酸量大的乳酸菌能够带来较低的最终产品发酵酸度，这是保证低后酸化的有效措施。通过对现有技术不同菌种发酵终点产酸能力进行测试，结果见表5所示。检测菌株的发酵终点pH为4.32±0.67，后熟后滴定酸度为95.93±0.78，结果表明菌株CGMCC No.16441比对照乳酸菌酸度值增加20％，且发酵时间明显缩短。
表5 不同菌种发酵终点产酸能力对比表
菌株 LLD LLL LLC CGMCC NO.16441
酸度/°T 72.7±2.4 64.4±2.9 71.65±3.4 95.93±0.78
pH值 4.39±0.13 4.77±0.08 4.45±0.14 4.32±0.67
发酵时间/h 6 6.5 6 5
注：LLD为乳酸乳球菌乳酸亚种丁二酮变种；LLL为乳酸乳球菌乳酸亚种；LLC为乳酸乳球菌乳油亚种；
植物乳杆菌CGMCC NO.16441发酵乳风味物质的研究，通过取20ml凝固型酸奶加入5mL饱和NaCl溶液振荡混匀，转移5mL溶液至顶空瓶中待测，GC-MS解析风味物质成分。
发酵风味物质检测结果见图4所示。发酵乳中共鉴定出25种主要风味物质，包括酮类、醇类、酯类、醚类等。其中普通酸奶和CGMCC No.菌株发酵酸奶均含有的特征风味物质外，还含有3-羟基-2-丁酮、2-庚酮、2-壬酮和己酸乙酯等风味成分，赋予酸奶清香的风味。
实施例8
本实施例用于说明植物乳杆菌CGMCC No.16441在制作活性乳酸菌饮料方面的应用
加热纯净水至50-55℃，加入全脂奶粉，加热到65-70℃均质，压力为180-200bar。冷却后按照乳液3-5体积％加入植物乳杆菌CGMCC No.16441发酵剂，在37-42℃发酵至pH4.0-4.5。破乳并冷却，低温下保存备用。加热纯净水至65-75℃，加入白砂糖或其他甜味剂和果胶等稳定剂的混合物， 搅拌15min，冷却后加入酸奶，调节pH为3.5-4.0，加入香精和水到规定量，加热至65-70℃均质，压力为180-200bar，115-130℃进行10-20s的灭菌，冷却灌装即得乳酸菌饮料，对其感官性能进行评价，评价标准如表6所示。
表6 乳酸菌饮料感官评价表
Figure PCTCN2018124327-appb-000002
以乳酸菌发酵饮料的色泽、滋、气味、组织状态为评分标准，从不同品种的商业乳酸菌中选择最佳的发酵菌种，发酵菌种选择的对应感官评价得分如下表7所示。
表7 感官评价得分
发酵菌种 色泽 滋、气味 组织状态 总分
LLD 10 20 12 42
LLL 15 33 20 68
LLC 14 38 20 72
CGMCC No.16441 18 44 22 84
注：LLD为乳酸乳球菌乳酸亚种丁二酮变种；LLL为乳酸乳球菌乳酸亚种；LLC为乳酸乳球菌乳油亚种；
通过对不同发酵剂发酵乳酸菌饮料进行感官评分，可知在相同的发酵条件下，不同发酵剂在很大程度上影响着实验结果，其中CGMCC No.16441发酵的乳酸菌饮料具有发酵乳酸香气，口感独特，色泽纯白，均匀一致。
实施例9
本实施例用于说明植物乳杆菌CGMCC No.16441在发酵谷物饮料方面的应用
选择色泽好，新鲜饱满，无病虫害的谷物原料，按照燕麦：大米＝1:2-2:1的范围将谷物原料加水进行浸泡，至其完全吸水膨胀为止。
取浸泡好的原料，沥干水分，加入原料重量8倍的水进行打浆和煮浆。浆料进入胶体磨循环 2-4min。加入柠檬酸调节浆料pH至4.0-5.0，加入复合浆料质量1-3％的糖化酶，40-60℃恒温糖化4-8h。
将糖化好的复合浆料冷却后，用碳酸氢钠调整pH至6.5-7.5，加入植物乳杆菌CGMCC No.16441发酵剂，发酵培养一段时间，使其浓度达到10 8cfu/mL以上，然后加入白砂糖、柠檬酸、稳定剂、香精等调整风味后进行冷却灌装即得，对其感官进行评价，评价标准见表8所示。
表8 发酵谷物饮料感官评价表
Figure PCTCN2018124327-appb-000003
以复合谷物发酵饮料的色泽、风味、状态为评分标准，从不同品种的商业乳酸菌中选择最佳的发酵菌种，发酵菌种选择的对应感官评价得分如下表9所示。
表9 感官评价得分
发酵菌种 色泽 滋、气味 外观 总分
LLD 16 20 11 47
LLL 20 33 13 66
LLC 18 27 12 57
CGMCC 26 41 15 82
注：LLD为乳酸乳球菌乳酸亚种丁二酮变种；LLL为乳酸乳球菌乳酸亚种；LLC为乳酸乳球菌乳油亚种；
通过对不同发酵剂发酵的谷物发酵饮料进行感官评分，可知在相同的发酵条件下，不同发酵剂在很大程度上影响着实验结果，其中CGMCC No.16441发酵的谷物饮料具有谷物复合香气及愉悦的发酵风味，口感独特，色泽淡黄，均匀一致。
实施例10
本实施例用于说明植物乳杆菌CGMCC No.16441在制作发酵豆乳方面的应用
本实施例所述的发酵豆乳采用如下方法制备：按照软水与大豆的体积比2：4在温度75-85℃下用软水浸泡大豆1-2h，去除大豆皮，去掉浸泡水，再按照大豆与沸水的重量比1：6-10加沸水磨浆， 其浆料在温度80-85℃的条件下保持10-15min，150目筛网过滤，得到豆乳；加入以豆乳重量计5-10％的蔗糖，再均质，均质压力为15-25Mpa，随后在温度95℃下进行杀菌处理5-10min，其温度降至28-38℃后，接种0.04-0.05％(以豆乳重量计)的商业用保加利亚乳杆菌、嗜热链球菌和CGMCC NO.16441发酵剂，菌数比例为1：1：1，在温度32-42℃下发酵6-8小时后4℃冷藏保存，得到所述发酵豆乳。
比较了未添加植物乳杆菌CGMCC NO.16441发酵剂，仅利用商业用保加利亚乳杆菌和嗜热链球菌(菌数比例为1:1)为发酵剂制备的发酵豆乳。结果表明，添加了植物乳杆菌CGMCC NO.16441的发酵豆乳中，酸度是未添加发酵豆乳的2.3倍，因此，利用植物乳杆菌CGMCC NO.16441与商业用菌种复配使用，可缩短发酵时间1-1.5小时。
实施例11
本实施例用于说明植物乳杆菌CGMCC No.16441在制作发酵果蔬汁方面的应用
选择颗粒均匀饱满的水果和蔬菜，用清水洗净表面污垢，用0.005-0.01g/kg次氯酸钠溶液浸泡7-10分钟进行消毒，冲洗后待用，并将需要切块的水果或蔬菜进行切块处理；沸水热烫3-5min，必要时可用0.5％柠檬酸和0.3％Vc混合液处理；用组织捣碎机将水果和蔬菜进行打浆3-5min；后添加0.006-0.01％的果胶酶和0.001％的纤维素酶，在35-52℃酶解1-2h；利用双层80目滤网过滤，得清液；利用微波杀菌2min或80℃、15min杀菌灭酶；从斜面上挑取植物乳杆菌CGMCC NO.16441到肉汤培养基中，培养24h；再挑取该菌到少量果蔬汁中，培养20h，随后将发酵种子液接种到果蔬汁中，合适温度下培养24h，至pH值达到3.8-4.1，乳酸菌量达到(10 8-10 9)cfu/mL。比较了利用干酪乳杆菌作为发酵剂的发酵果蔬汁，植物乳杆菌的发酵果蔬汁在冷藏期间的酸度变化值较小，后酸化较小，发酵稳定性好。
实施例12
本实施例用于说明植物乳杆菌CGMCC No.16441在制作发酵饲料方面的应用
将农作物秸秆干燥至水分含量小于15重量％，粉碎处理；收割草本植物，干燥至水分含量小于15％，制成碎草；将米糠、麦麸、盐、植物乳杆菌CGMCC NO.16441直投式发酵剂和纤维素酶预混合，然后与农作物秸秆和草本植物原料逐级扩大混匀；将混匀的微贮饲料压实、打捆、封膜，制备成30-100kg/包的规格；将所得成品自然发酵12-18天。所述农作物秸秆是一种或多种选自菜子杆、花生秧、玉米杆等。所述草本植物是选自紫云英常见草本植物的一种或多种的混合物。
与利用商业复合益生菌发酵的饲料相比，植物乳杆菌CGMCC NO.16441发酵的饲料中乳酸含量为21.28g/kg，是商业复合益生菌发酵饲料中乳酸含量的4.4倍。
实施例13
本实施例用于说明植物乳杆菌CGMCC NO.16441在生产活菌制剂中的应用
以上述筛选出的植物乳杆菌制成活菌制剂。植物乳杆菌CGMCC NO.16441按照2-4％(v/v)的接种量接种于MRS液体培养基中，在37-42℃下培养20-24h，使植物乳杆菌CGMCC NO.活菌数达到10 8cfu/mL以上，离心(4000rpm，10min)，用pH6.5-7.5的PBS缓冲液冲洗沉淀两次后，加入脱脂乳和海藻糖等作为冻干保护剂，调整细胞浓度至10 9cfu/mL，混合均匀后进行真空冷冻干燥得到活菌制剂。作为直活菌制剂含有活菌数在10 8-10 10cfu/mL，该菌剂可直接加入到发酵制品中进行发酵，也可按常规方法加入合适的辅料制成食品工业或临床可接受的剂型，剂型包括胶囊剂、微胶囊剂、片剂、粉剂等各种剂型。
以上述筛选出的植物乳杆菌制成的活菌制剂，还包括通过驯化等技术手段保持菌种活性的产品。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一株植物乳杆菌，其特征在于，该植物乳杆菌的分类命名为Lactobacillus plantarum，保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.16441。
  2. 权利要求1所述的植物乳杆菌在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料领域中的用途。
  3. 根据权利要求2所述的用途，其中，所述用途包括将权利要求1所述的植物乳杆菌与适配的发酵底物相接触的步骤。
  4. 权利要求1所述的植物乳杆菌在制备直投式发酵剂中的用途。
  5. 一种直投式发酵剂，其特征在于，该直投式发酵剂由权利要求1所述的植物乳杆菌在发酵培养基中进行发酵培养后制得。
  6. 一种制备权利要求5所述的直投式发酵剂的方法，其特征在于，该方法包括：
    (1)将权利要求1所述的植物乳杆菌在发酵培养基中进行发酵培养，并使活菌数达到10 8cfu/mL以上；
    (2)将步骤(1)得到的发酵菌液进行离心处理，之后用缓冲液进行冲洗，加入冻干保护剂，并调整活菌浓度至10 10cfu/mL以上，混合均匀后进行真空冷冻干燥，即得所述直投式发酵剂。
  7. 权利要求5所述的直投式发酵剂和/或权利要求6所述的方法制备的直投式发酵剂在制备发酵乳制品、发酵谷物饮料、发酵豆制品、发酵果熟制品、发酵饲料领域中的用途。
  8. 权利要求1所述的植物乳杆菌在制备活菌制剂中的用途。
  9. 根据权利要求8所述的用途，其中，所述活菌制剂包括胶囊、片剂、口服液和粉剂。
  10. 一种活菌制剂，其特征在于，该活菌制剂由权利要求1所述的植物乳杆菌在发酵培养基中进行发酵培养后制得。
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