WO2021248440A1 - 一种母乳源植物乳杆菌及其应用 - Google Patents

一种母乳源植物乳杆菌及其应用 Download PDF

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WO2021248440A1
WO2021248440A1 PCT/CN2020/095771 CN2020095771W WO2021248440A1 WO 2021248440 A1 WO2021248440 A1 WO 2021248440A1 CN 2020095771 W CN2020095771 W CN 2020095771W WO 2021248440 A1 WO2021248440 A1 WO 2021248440A1
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lactobacillus plantarum
derived
breast milk
survival rate
survival
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PCT/CN2020/095771
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English (en)
French (fr)
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陈历俊
刘璐
尹春媚
刘斌
周伟明
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北京三元食品股份有限公司
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Priority to US17/788,549 priority Critical patent/US20230050528A1/en
Priority to PCT/CN2020/095771 priority patent/WO2021248440A1/zh
Priority to JP2022519786A priority patent/JP2022549015A/ja
Priority to EP20940225.4A priority patent/EP4050095A4/en
Publication of WO2021248440A1 publication Critical patent/WO2021248440A1/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
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of microorganisms, in particular to a lactobacillus plantarum derived from breast milk and its application.
  • breast milk as an indispensable source of nutrition in the early stages of infant growth, plays a vital role in the growth and development of infants. It has been confirmed by a large number of studies that there are lactic acid bacteria in breast milk, which has become an important source of lactic acid bacteria, and has the potential to develop probiotics. At present, there are relatively few probiotics from breast milk, such as Lactobacillus gasseri CECT5714, Lactobacillus fermentum CECT5716, Lactobacillus salivarius CECT5713, etc. These strains all show certain probiotic functions. It is of great significance to continuously discover probiotics from breast milk.
  • Lactobacillus plantarum is a kind of lactic acid bacteria. It is widely distributed in meat, milk, vegetables and fermented products. It is also found in the digestive tract of humans and animals. It is a beneficial bacteria in the human intestinal tract.
  • the purpose of the present invention is to provide a lactobacillus plantarum derived from breast milk and an application thereof.
  • the lactobacillus plantarum derived from breast milk provided in the embodiment of the present invention is obtained by screening from healthy breast milk, has strong adhesion ability and has good probiotic potential. And it has inhibitory effect on 8 kinds of common pathogenic bacteria, and has good antibacterial performance; it is sensitive to 8 kinds of antibiotics; it is expected to be developed and applied in the food industry.
  • the breast milk-derived Lactobacillus plantarum also has strong acid resistance and bile salt resistance.
  • the embodiments of the present invention provide a lactobacillus plantarum derived from breast milk.
  • the lactobacillus plantarum derived from breast milk is classified and named as Lactobacillus plantarum, and the preservation number is CGMCC NO.19748.
  • lactobacillus plantarum P6 from breast milk was deposited at the China General Microbial Culture Collection and Management Center on April 27, 2020; the address of the preservation unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China; The serial number is CGMCC NO.19748.
  • the adhesion rate of the breast milk-derived Lactobacillus plantarum to intestinal cells is ⁇ 140%; optionally, the cells are human colon cancer cells (HT-29) .
  • the lactobacillus plantarum derived from breast milk has an inhibitory effect on gram-positive bacteria and gram-negative bacteria; alternatively, the lactobacillus plantarum derived from breast milk has an inhibitory effect on Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Shigella sungii, Salmonella typhi, Enterobacter sakazakii, Salmonella enteritidis have inhibitory effects, and the diameter of the inhibition zone ⁇ 20mm Further optionally, the lactobacillus plantarum derived from breast milk has an inhibitory effect on Escherichia coli, and the diameter of the inhibition zone is ⁇ 28.5mm.
  • the lactobacillus plantarum derived from breast milk is resistant to macrolide antibiotics, penicillins, aminoglycosides, lincosamides, cephalosporins, quinolones, and chloramphenicol.
  • the lactobacillus plantarum derived from breast milk is sensitive to erythromycin, azithromycin, ampicillin, amikacin, clindamycin, ceftriaxone, ciprofloxacin, and chloramphenicol; further
  • the breast milk-derived Lactobacillus plantarum is sensitive to erythromycin, azithromycin, ampicillin, amikacin, clindamycin, ceftriaxone, and ciprofloxacin; further optionally, the breast milk The source Lactobacillus plantarum is sensitive to azithromycin, clindamycin and ceftriaxone.
  • the breast milk-derived Lactobacillus plantarum has a survival rate of ⁇ 107% after 3 hours of survival at pH 2.5, a survival rate of ⁇ 115% after 6 hours of survival, and survival after 12 hours of survival. Rate ⁇ 74%.
  • the breast milk-derived Lactobacillus plantarum has a survival rate of ⁇ 105% after surviving at pH 3.5 for 3 hours, a survival rate of ⁇ 129% after surviving for 6 hours, and a survival rate of ⁇ 129% after surviving for 12 hours.
  • the survival rate is ⁇ 117%
  • the survival rate after 24h is ⁇ 145%.
  • the breast milk-derived Lactobacillus plantarum has a survival rate of ⁇ 132% after 3 hours of survival at pH 4.5, a survival rate of ⁇ 174% after 6 hours of survival, and survival after 12 hours of survival. Rate ⁇ 107%, survival rate ⁇ 68% after 24h survival.
  • the survival rate of the breast milk-derived Lactobacillus plantarum after surviving at a pH of 3.0 for 1 hour is ⁇ 145%.
  • the survival rate of the breast milk-derived Lactobacillus plantarum after survival for 1 hour under the condition of 0.3% bile salt is ⁇ 225%.
  • the survival rate of the lactobacillus plantarum derived from breast milk is ⁇ 42% after surviving under 0.3% bile salt conditions for 3 hours, and the survival rate is ⁇ 42% after survival under 0.3% bile salt conditions for 6 hours. ⁇ 19%.
  • the survival rate of the breast milk-derived Lactobacillus plantarum after 3 hours under 0.15% bile salt conditions is ⁇ 66%, and the survival rate after 6 hours under 0.15% bile salts conditions ⁇ 60%, survival rate ⁇ 40% after 12h under 0.15% bile salt condition, ⁇ 14% after 24h survival under 0.15% bile salt condition.
  • the embodiment of the present invention also provides a preparation method of the aforementioned lactobacillus plantarum derived from breast milk, and the preparation method includes the following steps:
  • the selected strains were tested for acid resistance, bile salt resistance and adhesion, and a Lactobacillus plantarum with the best adhesion and good acid resistance and bile salt resistance was obtained.
  • the embodiment of the present invention also provides the application of the lactobacillus plantarum derived from breast milk in the preparation of health food.
  • the health food includes infant formula food or yogurt.
  • the health effect of the health food includes enhancing immunity or treating constipation.
  • the lactobacillus plantarum derived from breast milk provided in the embodiments of the present invention is a Lactobacillus plantarum with extremely strong adhesion ability (up to 140%) screened from healthy breast milk, and has good probiotic potential.
  • Adhering to and colonizing the intestine is an important prerequisite for lactic acid bacteria to exert their ecological and physiological functions, and is one of the important basis for evaluating their probiotic potential.
  • the adhesion of lactic acid bacteria to epithelial cells is the main basis for its probiotic function.
  • the adhered lactic acid bacteria can exist in the intestinal tract and can resist the invasion of pathogenic bacteria, and produce metabolites such as antimicrobial peptides to help effectively kill pathogenic bacteria. Prevent the intestinal tract from being infected by pathogenic bacteria and other functions, and enhance the body's immune ability.
  • Microbial contamination and growth and reproduction are the main causes of food spoilage.
  • the antibacterial performance of probiotics is one of the safety indicators of whether they can be used in food.
  • the lactobacillus plantarum derived from breast milk provided in the embodiment of the present invention has an inhibitory effect on all 8 common pathogenic bacteria and has good antibacterial performance; it is sensitive to 8 antibiotics; it is expected to be developed and applied in the food industry.
  • the breast milk-derived Lactobacillus plantarum provided in the embodiment of the present invention also has strong acid resistance (survival rate of 145% after 1 hour survival at pH 3.0) and bile salt tolerance (under 0.3% bile salt conditions) The survival rate is 225% after 1 hour of survival) ability.
  • the prerequisite for lactic acid bacteria to exert its probiotic effect is to tolerate the acidic conditions of human gastric juice (normal human gastric juice pH is between 1.5-4.5) and the high bile salt environment of the small intestine (bile salt concentration is 0.03%-0.3%). Under this condition, normal growth and metabolism can colonize the gastrointestinal tract and play its role.
  • Figure 1-1 shows the results of the acid resistance test of Example 1 of the present invention.
  • Figure 1-2 shows the results of the bile salt tolerance test of Example 1 of the present invention.
  • Figures 1-3 are the results of the adhesion test of Example 1 of the present invention.
  • Figure 2 is an annotation result of the NR database in Embodiment 2 of the present invention.
  • Figure 3-1 is a growth curve of Lactobacillus plantarum P6 derived from breast milk in Example 3 of the present invention.
  • Figure 3-2 is the result of in vitro acid tolerance of lactobacillus plantarum P6 derived from breast milk in Example 3 of the present invention.
  • Fig. 3-3 is the result of in vitro bile salt tolerance of breast milk-derived Lactobacillus plantarum P6 in Example 3 of the present invention.
  • Figure 3-4 shows the results of the antibacterial ability of lactobacillus plantarum P6 derived from breast milk in Example 3 of the present invention.
  • the breast milk-derived Lactobacillus plantarum P6 is classified and named as Lactobacillus plantarum (Lactobacillus plantarum), and the preservation date: April 27, 2020; preservation unit: China General Microbial Culture Collection Management Center; preservation address: Chaoyang, Beijing, China District Beichen West Road No. 1, No. 3 Institute of Microbiology, Chinese Academy of Sciences; the deposit number is CGMCC NO.19748.
  • the MRS solid medium was purchased from Beijing Luqiao Technology Co., Ltd., the article number is CM188;
  • the MRS broth culture medium was purchased from Beijing Luqiao Technology Co., Ltd., the product number is CM187;
  • Nutritional agar was purchased from Beijing Luqiao Technology Co., Ltd., the article number is CM107;
  • the MH solid medium was purchased from Beijing Luqiao Technology Co., Ltd., and the article number is CM902.
  • the P3-P13 strain and the control strains R2 and R9 were tested for acid tolerance, bile salt tolerance and adhesion.
  • R2 and R9 are two strains of Lactobacillus plantarum derived from breast milk that were screened by the company before; for details, please refer to the literature: Yin Chunmei, Li Zhen, Jiang Tiemin, etc. Screening and identification of human breast milk-derived lactic acid bacteria and their ability to lower blood pressure Preliminary determination of [J]. Food Science and Technology, 2019(8):18-22.
  • the washed medium was resuspended in MRS broth medium with a pH of 3.0, cultured anaerobic at 37°C, sampled at 0, 1h, diluted gradually, and spread on MRS solid medium plates at 37°C anaerobic Count after 48h incubation.
  • N 0 is the number of viable cells (cfu/mL) at 0 h
  • N 1 is the number of viable cells (cfu/mL) after the strain is acid-resistant for 1 h.
  • the washed medium was resuspended in MRS broth medium containing 0.3% bile salts and cultured, anaerobic cultured at 37°C, samples were taken at 0 and 1 h, respectively, and diluted gradually, and spread on MRS solid medium plates. Count after anaerobic culture at 37°C for 48h.
  • N 0 is the number of viable cells (cfu/mL) at 0 h
  • N 1 is the number of viable cells (cfu/mL) after the strain is bile-resistant for 1 h.
  • HT-29 cells human colon cancer cells
  • fresh DMEM complete medium was added (DMEM medium was purchased from Thermo Fisher Scientific, and 1% penicillin and streptomycin, 1% Glutamine, 10% newborn calf serum), culture in a 37°C, 5% CO 2 incubator, change the cell culture medium every two days, and wait until the adherent growth and fusion state of the cells reaches about 70%-80%.
  • trypsin-EDTA purchased from Biological Industries
  • the last medium used DMEM incomplete medium 1% glutamine was added to DMEM medium).
  • a hemocytometer for the cultured HT-29 cells, adjust the cell concentration to 5 ⁇ 10 5 cells/mL, load 1 mL per well into a 6-well tissue culture plate, and place it in an incubator at 37°C and 5% CO 2 Incubate until the cells grow to a single layer. Discard the DMEM culture medium in the tissue culture plate and wash it 2-3 times with sterile PBS. One of the wells is used for cell counting, and the remaining wells are added with 1 mL of the above-prepared bacterial suspension and placed in a constant temperature incubator at 37°C. Incubate in medium for 2h, and each sample is made in three groups in parallel.
  • Adhesion rate (%) number of adherent bacteria (CFU/well)/HT-29 cells (pieces/well)*100%;
  • CFU colony forming unit: the total number of bacterial communities in a unit volume; in the culture and count of live bacteria, the colony formed by the growth and reproduction of a single bacterial cell or multiple bacterial cells on a solid medium is called a colony Forming unit by which the number of live bacteria is expressed.
  • the cultured P6 bacterial solution was activated for three generations and inoculated into MRS broth medium with pH 2.5, 3.5, 4.5 and bile salt concentration of 0.15%, 0.3%, 0.45%, and placed in an incubator at 37°C. Oxygen culture, samples were taken at 0, 3, 6, 12, and 24 hours for plate count;
  • Figure 3-2 shows that the bacteria grows stably and vigorously when they are cultured in an environment of pH 6.0, pH 4.5, and pH 3.5 for 0-24 hours.
  • number of cells were 10 8 cfu / mL or more, only the number of viable bacteria strain culture at 24h decreased but still in the 10 7 cfu / mL or more at pH2.5 environment; strong acid capacity; the survival of bacteria at all time points Rate data are shown in Table 1.
  • Bile salt concentration 3h 6h 12h 24h 0% 129% 200% 120% 68% 0.15% 66% 60% 40% 14% 0.30% 42% 19% 4% 0% 0.45% 8% 3% 0% 0%
  • FIG. 3-4 The results of the experiment are shown in Figure 3-4. It can be seen from Figure 3-4 that the bacteria is resistant to Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus) and Gram-negative bacteria ( Escherichia coli, Shigella sungii, Salmonella typhi, Enterobacter sakazakii, Salmonella enteritidis) all have a strong inhibitory effect, and the diameter of the inhibition zone is above 20mm. Among them, it has the strongest antibacterial effect on Escherichia coli. The diameters of the inhibition zone of the fermentation broth and supernatant are 30.17 ⁇ 1.58mm and 29.30 ⁇ 0.72mm, respectively.
  • the cultured P6 bacterial solution was activated for three generations, streaked on the MRS solid medium plate, and after anaerobic culture in a 37°C incubator for 24 hours, a single colony was selected and dissolved in physiological saline to prepare a concentration equivalent to 0.5 Suspension, and spread on MH solid medium. After it is dry, stick 10 kinds of E-test test strips on the MH solid medium and incubate at 37°C for 24-48h, and judge the MIC value (minimum inhibitory concentration value) according to the size of the inhibitory ring.
  • Lactobacillus plantarum P6 is resistant to erythromycin and azithromycin (macrolide antibiotics), ampicillin (penicillins), amikacin (aminoglycosides), clin Very sensitive to chloramphenicol (lincosamide), ceftriaxone (cephalosporin), ciprofloxacin (quinolones), moderately sensitive to chloramphenicol (chloramphenicol), and penicillin G (penicillin), Tetracyclines (tetracyclines) are tolerated.
  • breast milk-derived Lactobacillus plantarum P6 has good growth characteristics, strong acid and bile salt tolerance, and strong adhesion; it has an inhibitory effect on 8 common pathogenic bacteria and has good antibacterial properties; and It is sensitive to 8 kinds of antibiotics.
  • the above results indicate that the breast milk-derived Lactobacillus plantarum P6 has excellent probiotic potential and is expected to be developed and applied in the food industry.
  • the breast-milk-derived Lactobacillus plantarum and its application provided in the embodiment of the present invention The breast-milk-derived Lactobacillus plantarum is classified and named as Lactobacillus plantarum, and the preservation number is CGMCC NO.19748, which is screened from healthy breast milk and has an adhesion ability. Very strong, with good probiotic potential. And it has inhibitory effect on 8 kinds of common pathogenic bacteria, and has good antibacterial performance; it is sensitive to 8 kinds of antibiotics; it is expected to be developed and applied in the food industry.
  • the breast milk-derived Lactobacillus plantarum also has strong acid resistance and bile salt resistance.

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Abstract

提供一种母乳源植物乳杆菌及其应用。该母乳源植物乳杆菌，分类命名为植物乳杆菌，保藏编号为CGMCC NO.19748，是从健康母乳中筛选得到的。其粘附能力强；且其对8种常见致病菌都具有抑制作用；并对8种抗生素表现敏感；还具有耐酸和耐胆盐能力。

Description

一种母乳源植物乳杆菌及其应用 技术领域
本发明涉及微生物领域，具体涉及一种母乳源植物乳杆菌及其应用。
背景技术
母乳作为婴儿成长初期必不可少的营养来源，对婴儿的成长发育起着至关重要的作用。现已被大量研究证实母乳中有乳酸菌的存在，现已成为乳酸菌的一个重要来源，具有潜在的益生菌发掘潜力。目前母乳来源的益生菌相对较少，有格氏乳杆菌CECT5714、发酵乳杆菌CECT5716、唾液乳杆菌CECT5713等，这些菌株均表现出一定的益生功能。不断从母乳中发掘益生菌具有重要意义。
植物乳杆菌是乳酸菌的一种，广泛分布于肉类、乳、蔬菜以及发酵制品中，并且在人和动物的消化道中也被发现其存在，是人体肠道内的有益菌。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
为解决上述技术问题，本发明的目的在于提供一种母乳源植物乳杆菌及其应用。本发明实施例中提供的母乳源植物乳杆菌，是从健康母乳中筛选得到的，粘附能力极强，具有良好的益生潜力。且其对8种常见致病菌都具有抑制作用，抑菌性能好；并对8种抗生素表现敏感；有望被开发并应用于食品行业。此外，所述母乳源植物乳杆菌还具有较强的耐酸和耐胆盐能力。
解决方案
为实现本发明目的，本发明实施例提供了一种母乳源植物乳杆菌，所述母乳源植物乳杆菌分类命名为植物乳杆菌，保藏编号为CGMCC NO.19748。
上述母乳源植物乳杆菌P6已于2020年4月27日保藏在中国普通微生物菌种保藏管理中心；保藏单位地址：中国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；保藏编号为CGMCC NO.19748。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳 杆菌对肠道细胞的粘附率≥140％；可选地，所述细胞为人结肠癌细胞(HT-29)。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌对革兰氏阳性菌和革兰氏阴性菌具有抑制作用；可选地，所述母乳源植物乳杆菌对单增李斯特氏菌、金黄色葡萄球菌、蜡状芽孢杆菌、大肠埃希氏菌、宋氏志贺氏菌、伤寒沙门氏菌、阪崎肠杆菌、肠炎沙门氏菌具有抑制作用，抑菌圈直径≥20mm；进一步可选地，所述母乳源植物乳杆菌对大肠埃希氏菌具有抑制作用，抑菌圈直径≥28.5mm。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌对大环内酯类抗生素、青霉素类、氨基糖苷类、林可胺类、头孢类、喹诺酮类、氯霉素类敏感；可选地，所述母乳源植物乳杆菌对红霉素、阿奇霉素、氨苄西林、阿米卡星、克林霉素、头孢曲松、环丙沙星、氯霉素敏感；进一步可选地，所述母乳源植物乳杆菌对红霉素、阿奇霉素、氨苄西林、阿米卡星、克林霉素、头孢曲松、环丙沙星敏感；更进一步可选地，所述母乳源植物乳杆菌对阿奇霉素、克林霉素、头孢曲松敏感。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在pH2.5条件下存活3h后存活率≥107％，存活6h后存活率≥115％，存活12h后存活率≥74％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在pH3.5条件下存活3h后存活率≥105％，存活6h后存活率≥129％，存活12h后存活率≥117％，存活24h后存活率≥145％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在pH4.5条件下存活3h后存活率≥132％，存活6h后存活率≥174％，存活12h后存活率≥107％，存活24h后存活率≥68％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在pH为3.0条件下存活1h后存活率≥145％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在0.3％胆盐条件下存活1h后存活率≥225％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳杆菌在0.3％胆盐条件下存活3h后存活率≥42％，在0.3％胆盐条件下存活6h后存活率≥19％。
上述母乳源植物乳杆菌在一种可能的实现方式中，所述母乳源植物乳 杆菌在0.15％胆盐条件下存活3h后存活率≥66％，在0.15％胆盐条件下存活6h后存活率≥60％，在0.15％胆盐条件下存活12h后存活率≥40％，在0.15％胆盐条件下存活24h后存活率≥14％。
本发明实施例还提供了上述母乳源植物乳杆菌的制备方法，所述制备方法包括下述步骤：
选择新鲜母乳样品，用灭过菌的PBS进行梯度稀释后，涂布于含有1％CaCO 3的MRS培养基上，于37℃厌氧培养箱中培养48h，挑取具有明显透明圈的呈白色或乳白色的、长势良好的单菌落进行革兰氏染色并镜检，选择杆状且为革兰氏阳性菌的菌株，于37℃厌氧培养箱中进行富集培养；将菌液与灭过菌的30％甘油按照1:1的比例进行混合后于-80℃的冰箱保存待用；乳杆菌与其他种类的乳酸菌相比，益生特性相对来讲更具优势，因此，本发明将筛选目标定在乳酸菌中的乳杆菌上；
对筛选出来的菌株进行耐酸、耐胆盐和粘附性测试，筛选得到一株粘附性最优，同时耐酸、耐胆盐性能较好的植物乳杆菌。
本发明实施例还提供了上述母乳源植物乳杆菌在制备保健食品中的应用。
上述应用在一种可能的实现方式中，所述保健食品包括婴幼儿配方食品或酸奶。
上述应用在一种可能的实现方式中，所述保健食品的保健作用包括增强免疫力或治疗便秘。
有益效果
(1)本发明实施例中提供的母乳源植物乳杆菌，是从健康母乳中筛选得到的粘附能力极强(高达140％)的植物乳杆菌，具有良好的益生潜力。粘附并定植于肠道是乳酸菌发挥其生态效应和生理功能的重要前提，是评估其益生潜力的重要依据之一。乳酸菌粘附在上皮细胞是其发挥其益生功能的主要基础，粘附的乳酸菌可以在肠道内存在并且可以抵制致病菌的入侵，产生抗菌肽等代谢产物帮助有效的杀死致病菌，达到避免肠道受致病菌的侵染等功能，增强机体的免疫能力。
(2)微生物的污染及生长繁殖是造成食物腐败变质的主要原因，益生菌的抑菌性能是其能否应用于食品中的安全指标之一。
抗生素在医药行业的过量使用会导致致病菌株的耐药性能增强，人体肠道中的菌群容易出现紊乱、失衡，因此，对抗生素敏感也是益生菌应用于 食品中是否安全的重要指标之一。
本发明实施例中提供的母乳源植物乳杆菌，对8种常见致病菌都具有抑制作用，抑菌性能好；并对8种抗生素表现敏感；有望被开发并应用于食品行业。
(3)本发明实施例中提供的母乳源植物乳杆菌，还具有较强的耐酸(在pH为3.0条件下存活1h后存活率为145％)和耐胆盐(在0.3％胆盐条件下存活1h后存活率为225％)能力。乳酸菌能发挥其益生作用的前提是能够耐受人体胃液的酸性条件(正常人胃液pH在1.5-4.5之间)和小肠的高胆盐环境(胆盐浓度为0.03％-0.3％)，菌株可在该条件下正常生长和代谢，才能在胃肠道内定植并发挥其作用。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1-1是本发明实施例1耐酸实验结果。
图1-2是本发明实施例1耐胆盐实验结果。
图1-3是本发明实施例1粘附性实验结果。
图2是本发明实施例2中NR数据库注释结果。
图3-1是本发明实施例3中母乳源植物乳杆菌P6生长曲线。
图3-2是本发明实施例3中母乳源植物乳杆菌P6体外耐酸能力结果。
图3-3是本发明实施例3中母乳源植物乳杆菌P6体外耐胆盐能力结果。
图3-4是本发明实施例3中母乳源植物乳杆菌P6抑菌能力结果。
本发明提供的母乳源植物乳杆菌P6分类命名为植物乳杆菌(Lactobacillus plantarum)，保藏日期：2020年4月27日；保藏单位：中国普通微生物菌种保藏管理中心；保藏地址：中国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；保藏编号为CGMCC NO.19748。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发 明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
下述实施例中，
MRS固体培养基购自北京陆桥技术股份有限公司，货号为CM188；
MRS肉汤培养基购自北京陆桥技术股份有限公司，货号为CM187；
营养琼脂购自北京陆桥技术股份有限公司，货号为CM107；
MH固体培养基购自北京陆桥技术股份有限公司，货号为CM902。
实施例1
1、母乳源植物乳杆菌P6的分离
选择新鲜母乳样品，用灭过菌的PBS缓冲液(购自广州洁特生物过滤股份有限公司)进行梯度稀释后，涂布于含有1％CaCO 3的MRS固体培养基上，于37℃厌氧培养箱中培养48h，挑取具有明显透明圈的呈白色或乳白色的单菌落进行革兰氏染色并镜检，选择杆状且为革兰氏阳性菌的菌株，从众多菌株中选择长势较好的菌株P3-P13，于37℃厌氧培养箱中进行富集培养；将菌液与灭过菌的30％甘油按照1:1的比例进行混合后于-80℃的冰箱保存待用。
2.菌株筛选
将上述P3-P13菌株在无菌条件下将甘油保存后的菌株接种于MRS肉汤培养基中，37℃厌氧培养24h，连续活化三代；菌液离心后用灭菌后的PBS洗涤培养基2-3次；
对P3-P13菌株以及对照菌株R2和R9进行耐酸、耐胆盐和粘附性测试。
其中，R2和R9为本公司之前筛选得到的两株效果较优的母乳源植物乳杆菌；具体参见文献：尹春媚,李贞,姜铁民,等.人母乳源乳酸菌的筛选、鉴定及其降血压能力的初步测定[J].食品科技,2019(8):18-22。
耐酸、耐胆盐和粘附性的测试步骤具体如下：
(1)耐酸测试
将洗涤后的培养基重悬于pH为3.0的MRS肉汤培养基中，37℃厌氧培养，分别于0，1h取样进行梯度稀释，涂布于MRS固体培养基平板中，37℃厌氧培养48h后计数。
菌株存活率(％)＝N 1/N 0*100；
式中：N 0为0h的活菌数(cfu/mL)；N 1为菌株耐酸1h后的活菌数(cfu/mL)。
实验结果见图1-1。
(2)耐胆盐测试
将洗涤后的培养基重悬于含0.3％的胆盐的MRS肉汤培养基中培养，37℃厌氧培养，分别于0，1h取样进行梯度稀释，涂布于MRS固体培养基平板中，37℃厌氧培养48h后计数。
菌株存活率(％)＝N 1/N 0*100；
式中：N 0为0h的活菌数(cfu/mL)；N 1为菌株耐胆盐1h后的活菌数(cfu/mL)。
实验结果见图1-2。
(3)粘附性测试
将HT-29细胞(人结肠癌细胞)复苏后接种于细胞培养瓶中，加入新鲜的DMEM完全培养基(DMEM培养基购自Thermo Fisher Scientific，其中加入1％的青霉素和链霉素、1％谷氨酰胺、10％新生牛血清)，于37℃、5％CO 2的培养箱中培养，每两天换一次细胞培养液，待细胞贴壁生长融合状态达到70％-80％左右，用0.25％胰酶-EDTA(购自Biological Industries)混合消化液进行传代，最后一次培养基使用DMEM不完全培养基(DMEM培养基中加入1％谷氨酰胺)。
培养好的HT-29细胞用细胞血球计数版，调整细胞浓度至5×10 5个/mL，按每孔1mL装入6孔组织培养板中，置于37℃、5％CO 2的培养箱中孵育至细胞长至单层。弃去组织培养板中的DMEM培养液，用无菌PBS将其冲洗2-3次，其中一孔用于进行细胞计数，其余孔加入1mL上述制备好的菌悬液，于37℃恒温培养箱中孵育2h，每个样品均做三组平行。孵育完成后，弃去培养基，用无菌PBS缓冲液冲洗3-5次以除去未粘附的植物乳杆菌，使用0.7mL 0.25％胰酶-EDTA将细胞消化后，待细胞完全脱落，加入0.3mL DMEM完全培养液结束消化，收集培养液进行平板计数。
粘附率(％)＝粘附细菌数(CFU/孔)/HT-29细胞(个/孔)*100％；
CFU(菌落形成单位)：单位体积中的细菌群落总数；在活菌培养计数时，由单个菌体或聚集成团的多个菌体在固体培养基上生长繁殖所形成的集落，称为菌落形成单位，以其表达活菌的数量。
实验结果见图1-3。
由图1-1、1-2和1-3结果可知，母乳源植物乳杆菌P6粘附能力极强，高达140％，远高于其他母乳源植物乳杆菌；并且其在耐酸和耐胆盐条件下存活1h后，存活率分别达到145％和225％，耐酸和耐胆盐能力较好。
实施例2
母乳源植物乳杆菌P6的鉴定：
对筛选出来的P6进行全基因测序分析，采用Pacbio(10Kb SMRT Bell文库)和Illumina PE150(350bp小片段文库)测序平台对其进行全基因组测序。
对测序结果进行NR(Non-Redundant Protein Database)数据库注释(用于物种信息鉴定，可做为物种分类用)，结果如图2所示。
由图2结果可知，86.81％的基因符合植物乳杆菌属，因此，结合P6的形态特征，将P6鉴定为植物乳杆菌。
实施例3
母乳源植物乳杆菌P6的益生特性测定
1、母乳源植物乳杆菌P6的生长曲线测定
将培养好的P6菌液活化三代，接种于MRS肉汤培养基中，置于37℃培养箱中，厌氧培养，分别于0、2、4、6、8、10、12、14、16、20、24、28、32、36、40以及48h，测定各细菌的活菌数；
绘制母乳源植物乳杆菌P6的生长曲线，结果见图3-1所示。
2、母乳源植物乳杆菌P6的体外耐酸耐胆盐能力测定
将培养好的P6菌液活化三代，分别接种于pH为2.5、3.5、4.5和胆盐浓度为0.15％、0.3％、0.45％的MRS肉汤培养基中，置于37℃培养箱中，厌氧培养，分别于0、3、6、12、24h取样进行平板计数；
实验结果分别见图3-2和图3-3；由图3-2可知，该菌在pH6.0、pH4.5、pH3.5的环境中培养0-24h时，菌体生长稳定，活菌数均在10 8cfu/mL以上，只有在pH2.5的环境下培养24h时菌株的活菌数有所下降但依然在10 7cfu/mL以上；耐酸能力强；各时间点菌的存活率数据见表1。
表1
pH值 3h 6h 12h 24h
pH 6.0 129％ 200％ 120％ 68％
pH 4.5 132％ 174％ 107％ 68％
pH 3.5 105％ 129％ 117％ 145％
pH 2.5 107％ 115％ 74％ 2％
由图3-3可知，该菌在0.15％胆盐浓度的培养基中生长相对稳定；在胆盐含量为0.3％和0.45％的培养基中培养6h后活菌数分别下降了0.71和1.6个对数值；在含0.45％的胆盐中培养24h后，活菌数明显下降至10 5cfu/mL；耐胆盐能力较强；各时间点菌的存活率数据见表2。
表2
胆盐浓度 3h 6h 12h 24h
0％ 129％ 200％ 120％ 68％
0.15％ 66％ 60％ 40％ 14％
0.30％ 42％ 19％ 4％ 0％
0.45％ 8％ 3％ 0％ 0％
3、母乳源植物乳杆菌P6的抑菌能力测定
食品中含有大量的营养，在日常生活中食品伴随着腐败及变质，危害人体的健康也造成了经济损失。微生物的污染及生长繁殖是造成食物腐败变质的主要原因，因此P6的抑菌性能是其能否应用于食品中的安全指标之一。
以大肠埃希氏菌、单增李斯特氏菌、金黄色葡萄球菌、宋氏志贺氏菌、蜡状芽孢杆菌、伤寒沙门氏菌、阪崎肠杆菌、肠炎沙门氏菌8株常见食源性致病菌为指示菌，将指示菌活化三代后，涂布于营养琼脂上，采用双层琼脂扩散方法进行抑菌活性的初步测定。
用镊子将牛津杯(直径8mm)置于平板中，并倒入45℃左右的营养琼脂固定牛津杯，待其凝固后取出牛津杯，并分别吸取150μL植物乳杆菌P6的发酵液和上清液，分别加入至孔中，将平板放入4℃冰箱中扩散3h左右，置于37℃下恒温培养24h，观察抑菌圈直径，并记录大小，每组试验三次平行，结果以x±s表示。
实验结果见图3-4所示，由图3-4可知，该菌对革兰氏阳性菌(单增李斯特氏菌、金黄色葡萄球菌、蜡状芽孢杆菌)和革兰氏阴性菌(大肠埃希氏菌、宋氏志贺氏菌、伤寒沙门氏菌、阪崎肠杆菌、肠炎沙门氏菌)均有很强的抑制作用，抑菌圈直径均在20mm以上。其中对大肠埃希氏菌的抑菌作用最强，发酵液和上清液的抑菌圈直径分别为30.17±1.58mm、29.30±0.72mm。
4、母乳源植物乳杆菌P6的耐药能力测定
抗生素在医药行业的过量使用会导致致病菌株的耐药性能增强，人体肠道中的菌群容易出现紊乱、失衡，因此获得对抗生素敏感的乳酸菌成为了 其应用于食品中是否安全的重要指标之一。
将培养好的P6菌液活化三代，在MRS固体培养基平板上划线，37℃培养箱中厌氧培养24h后，挑取单菌落溶于生理盐水中，配成相当于0.5麦氏浓度的悬液，并涂布于MH固体培养基。待其干燥后，将10种E-test试纸条贴于MH固体培养基上，37℃培养24-48h，根据抑菌环大小判断MIC值(最低抑菌浓度值)。使用大肠埃希氏菌ATCC25922和金黄色葡萄球菌ATCC25923作为质控菌株，每批试验均使用质控菌作为对照，并要求质控菌株的MIC在CLSI(临床和实验室标准协会)规定范围内。使用不加抗生素的MH固体平板做空白对照。
结果如表3所示，由表3可知，植物乳杆菌P6对红霉素和阿奇霉素(大环内酯类抗生素)、氨苄西林(青霉素类)、阿米卡星(氨基糖苷类)、克林霉素(林可胺类)、头孢曲松(头孢类)、环丙沙星(喹诺酮类)极度敏感，对氯霉素(氯霉素类)中度敏感，对青霉素G(青霉素类)、四环素(四环素类)耐受。
表3
Figure PCTCN2020095771-appb-000001
S:susceptible敏感；I:intermediate中度敏感；R：resistance耐受
综上，母乳源植物乳杆菌P6生长特性良好，具有较强的耐酸和耐胆盐能力，以及极强的粘附能力；对8种常见致病菌都具有抑制作用，抑菌性能好；并对8种抗生素表现敏感。上述结果表明，母乳源植物乳杆菌P6具有优异的益生潜力，并有望被开发并应用于食品行业。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明实施例提供的母乳源植物乳杆菌及其应用，所述母乳源植物乳杆菌，分类命名为植物乳杆菌，保藏编号为CGMCC NO.19748，是从健康母乳中筛选得到的，粘附能力极强，具有良好的益生潜力。且其对8种常见致病菌都具有抑制作用，抑菌性能好；并对8种抗生素表现敏感；有望被开发并应用于食品行业。此外，所述母乳源植物乳杆菌还具有较强的耐酸和耐胆盐能力。

Claims (10)

  1. 一种母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌分类命名为植物乳杆菌，保藏编号为CGMCC NO.19748。
  2. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌对肠道细胞的粘附率≥140％。
  3. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌对人结肠癌细胞的粘附率≥140％。
  4. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌对革兰氏阳性菌和革兰氏阴性菌具有抑制作用；可选地，所述母乳源植物乳杆菌对单增李斯特氏菌、金黄色葡萄球菌、蜡状芽孢杆菌、大肠埃希氏菌、宋氏志贺氏菌、伤寒沙门氏菌、阪崎肠杆菌、肠炎沙门氏菌具有抑制作用；进一步可选地，所述母乳源植物乳杆菌对大肠埃希氏菌具有抑制作用。
  5. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌对大环内酯类抗生素、青霉素类、氨基糖苷类、林可胺类、头孢类、喹诺酮类、氯霉素类敏感；可选地，所述母乳源植物乳杆菌对红霉素、阿奇霉素、氨苄西林、阿米卡星、克林霉素、头孢曲松、环丙沙星、氯霉素敏感；进一步可选地，所述母乳源植物乳杆菌对阿奇霉素、克林霉素、头孢曲松敏感。
  6. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌在pH2.5条件下存活3h后存活率≥107％，存活6h后存活率≥115％，存活12h后存活率≥74％；
    和/或，所述母乳源植物乳杆菌在pH3.5条件下存活3h后存活率≥105％，存活6h后存活率≥129％，存活12h后存活率≥117％，存活24h后存活率≥145％；
    和/或，所述母乳源植物乳杆菌在pH4.5条件下存活3h后存活率≥132％，存活6h后存活率≥174％，存活12h后存活率≥107％，存活24h后存活率≥68％；
    和/或，所述母乳源植物乳杆菌在pH为3.0条件下存活1h后存活率≥145％。
  7. 根据权利要求1所述的母乳源植物乳杆菌，其特征在于：所述母乳源植物乳杆菌在0.3％胆盐条件下存活1h后存活率≥225％；
    和/或，所述母乳源植物乳杆菌在0.3％胆盐条件下存活3h后存活率≥42％，在0.3％胆盐条件下存活6h后存活率≥19％；
    和/或，所述母乳源植物乳杆菌在0.15％胆盐条件下存活3h后存活率≥66％，在0.15％胆盐条件下存活6h后存活率≥60％，在0.15％胆盐条件下存活12h后存活率≥40％，在0.15％胆盐条件下存活24h后存活率≥14％。
  8. 权利要求1所述的母乳源植物乳杆菌在制备保健食品中的应用。
  9. 根据权利要求8所述的应用，其特征在于：所述保健食品包括婴幼儿配方食品或酸奶。
  10. 根据权利要求8所述的应用，其特征在于：所述保健食品的保健作用包括增强免疫力或治疗便秘。
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