WO2020083119A1 - 一种哈尔滨乳杆菌及其应用 - Google Patents

一种哈尔滨乳杆菌及其应用 Download PDF

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WO2020083119A1
WO2020083119A1 PCT/CN2019/112046 CN2019112046W WO2020083119A1 WO 2020083119 A1 WO2020083119 A1 WO 2020083119A1 CN 2019112046 W CN2019112046 W CN 2019112046W WO 2020083119 A1 WO2020083119 A1 WO 2020083119A1
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lactic acid
acid bacteria
harbin
strain
lactobacillus
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WO2020083119A8 (zh
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李理
郑茵
费永涛
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华南理工大学
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    • 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
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
    • A23C11/106—Addition of, or treatment with, microorganisms
    • 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
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • 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
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A23L11/65—Soy drinks
    • 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
    • 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 application field of probiotics, in particular to a Harbin lactic acid bacterium (Lactobacillus harbinensis M1) and its application.
  • the Harbin lactic acid bacterium can be used as a probiotic bacterium and can be used as a fermentation agent in fermented soybean milk.
  • Probiotics refer to live microorganisms that can give a beneficial effect on host health when given a certain amount (usually above 10 6 CFU / g). Only those microorganisms that can tolerate digestion in the gastrointestinal tract, adhere to and colonize epithelial cells of the small intestine, and inhibit the growth of pathogenic microorganisms can play a beneficial role in the health of the host. Commonly used probiotics mainly include Lactobacillus, Bifidobacterium and Gram-positive cocci, such as Streptococcus thermophilus. Its probiotic functions are mainly reflected in strengthening immunity, regulating intestinal flora and improving gastrointestinal function, resistance Oxidation and delay aging, lower cholesterol and improve blood lipids.
  • Soybeans are rich in high-quality protein, unsaturated fatty acids, oligosaccharides, isoflavones, saponins and other functional ingredients. They are environmentally friendly, healthy, and economical. They are an important plant protein resource in the traditional sense of China. They have important economic value and Social value.
  • the soybean protein drink prepared by fermenting soybeans with probiotics not only contains a lot of live probiotics, but also makes isoflavones into easily absorbed aglycones through biotransformation, degrades soybean proteins to form peptides and amino acids, and generates ⁇ -aminobutan Functional factors such as acids and B vitamins, so probiotic fermented soy milk is receiving widespread attention from food scientists and modern consumers.
  • probiotic fermented soy milk still has a big gap in taste and flavor. Among them, the beany smell is more prominent, which seriously affects the product's quality. Acceptability restricts its industrial application.
  • the beany smell of soybean food mainly comes from the oxidative degradation products of n-hexanal, nonanal, 1-octen-3-ol and other volatile fats catalyzed by lipoxygenase.
  • the effect of applying probiotic fermentation to reduce the beany smell is closely related to the strain. Since most of the commercially available lactic acid bacteria are derived from fermented dairy products, the adaptability of these strains when applied to fermented soybean milk is often poor, mainly reflected in poor fermentability and poor product flavor.
  • the purpose of the present invention is to provide a Harbin lactic acid bacteria (Lactobacillus harbinensis) M1 suitable for fermented foods, especially fermented soybean foods according to the problems existing in the prior art.
  • the strain has good safety and probiotic functions and can be effective
  • the use of various oligosaccharides in soybeans significantly improves and enhances the sensory flavor of legume foods.
  • Tofu yellow syrup water is the yellow drain water produced in the process of making tofu. Since the yellow syrup water is still rich in nutrients, it is very suitable for the growth and reproduction of microorganisms, so it will naturally acidify during storage. In some parts of China, there is a habit of making tofu with physalis water as a coagulant. Because of its delicious taste and unique flavor, this physalis tofu has been loved by more and more consumers in recent years. Microorganisms in physalis water have a strong ability to utilize legume components such as raffinose, stachyose and isoflavones due to long-term training, and have a good flavor. The present invention selects one strain of Lactobacillus harbini with excellent fermentation performance in soybean matrix on the basis of in-depth study of tofu acid pulp water. This strain has potential probiotic function and important application value.
  • a strain of Lactobacillus harbinensis (M1). The strain's deposit number is GDMCC No. 60305. It is deposited at the Guangdong Provincial Microbial Culture Collection Center. The place of preservation is Guangdong Microbiology, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou. Bacterial species collection center, the preservation date is December 20, 2017.
  • Harbin lactic acid bacteria as probiotics.
  • Harbin lactic acid bacteria are sensitive to the antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol, and the safety meets the requirements of EFSA.
  • the supernatant of the Harbin lactic acid bacteria has significant inhibitory effects on Listeria monocytogenes, Salmonella typhimurium, E. coli O157, Enterobacter sakazakii, Streptococcus hemolyticus, and Staphylococcus aureus.
  • Harbin lactic acid bacteria to gastric juice, intestinal juice and bile salts can be colonized on the small intestine epithelial cells.
  • Harbin lactic acid bacteria in fermented soybean milk.
  • the utilization rate of the Harbin lactic acid bacteria for sucrose, stachyose and raffinose is comparable to the utilization rate for glucose.
  • Harbin lactic acid bacteria completely eliminated the beany taste component n-hexanal in soybean milk.
  • Harbin lactic acid bacteria increased the milk aroma components diacetyl and acetoin.
  • Harbin lactic acid bacteria (Lactobacillus harbinensis M1) is collected on-site by using a sterile sampling bottle to collect naturally fermented acid pulp water samples, and is obtained by screening by dilution plate method.
  • the biological activity characteristics of this strain are as follows: the colony protrusions on the MRS solid medium plate are round, generally 1-3mm in diameter, off-white, opaque, moist and smooth; the bacteria are Gram-positive non-Bacillus sp.
  • Lactobacillus Harbin M1 meets the characteristics of probiotics.
  • the strain shows good tolerance in simulated gastric fluid and simulated intestinal fluid, and can adhere to small intestinal epithelial cells Caco-2.
  • This strain can effectively inhibit the propagation of food-borne pathogenic bacteria such as Listeria monocytogenes, Salmonella typhimurium, E. coli O157, Enterobacter sakazakii, Streptococcus hemolyticus and Staphylococcus aureus.
  • the growth of this strain can effectively utilize sucrose, stachyose and raffinose, and is suitable for the production of fermented plant oligosaccharide foods.
  • the fermented soymilk prepared by the strain has higher viable cell count and acidity, and the volatile flavor component of the beany flavor component is reduced, and the milk aroma components diacetyl and acetoin are increased, which effectively improves the sensory flavor of the product.
  • the present invention Compared with the existing commercial strains, the present invention has the following advantages and beneficial effects:
  • the strain Lactobacillus harbinensis M1 of the present invention has a good tolerance to the gastrointestinal tract, can reach the small intestine alive and adhere to and colonize epithelial cells, and has a strong inhibition on a variety of food-borne pathogenic bacteria It has the potential to become a dominant bacteria in the intestinal tract and can provide a good probiotic effect for the host.
  • the strain of the present invention has a very high utilization rate of oligosaccharides such as raffinose, stachyose, sucrose, etc., and can be grown and propagated in plant materials rich in such oligosaccharides, such as soybeans, and fermented soybeans and other plants The production of similar foods provides new adaptable strains.
  • oligosaccharides such as raffinose, stachyose, sucrose, etc.
  • the strain of the present invention can reduce the content of beany odorous substances such as n-hexanal, nonanal, 1-octen-3-ol in fermented soybean milk, increase the aroma components such as diacetyl and acetoin, and impart unique milk to fermented soybean milk
  • the fragrance significantly improves the sensory flavor of fermented soybean milk.
  • the strain of the present invention has a wide growth temperature range, fast growth speed, simple culture conditions, easy industrial production and management, and broad development and application prospects.
  • 1 is a colony morphology diagram of the Lactobacillus harbinensis M1 strain of the present invention
  • Lactobacillus harbinensis M1 strain of the present invention is a morphology diagram of the Lactobacillus harbinensis M1 strain of the present invention.
  • Modified MRS liquid medium (g / L) (for the cultivation of lactic acid bacteria):
  • antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, For tetracycline and chloramphenicol, prepare an antibacterial drug stock solution with a concentration of 5120 ⁇ g / mL, dilute to the required concentration according to the dilution solution before use, and add to the corresponding medium in proportion. 9.0 mL of the modified MRS agar medium was divided into large test tubes and sterilized at 121 ° C. for 15 min, then placed in a water bath (50 ° C.) to keep warm for use.
  • Gastrointestinal fluid tolerance Take a 100mL Erlenmeyer flask, add 50mL of modified MRS liquid medium, sterilize at 121 ° C for 15min, inoculate lactic acid bacteria after cooling, incubate at 37 ° C overnight, and use it as a seed culture solution.
  • Simulated gastric juice 0.27g pepsin was diluted in 90mL sterile phosphate buffer solution PBS, adjusted to pH 3 with hydrochloric acid, filtered and sterilized for use with a 0.22 ⁇ m disposable needle filter.
  • Simulated bile salt tolerance 0.3g swine bile salt was added to 100mL modified MRS liquid medium, sterilized at 121 °C for 15min, and cooled to 37 °C.
  • the cultured lactic acid bacteria were inoculated into MRS liquid medium containing 0.3% pig bile salt at 10 9 CFU / mL, and samples were taken every hour from 0h to 11h and the absorbance was read at 620nm.
  • the strain was activated with MRS medium for 2-3 generations.
  • the pathogenic bacteria were activated with LB medium for 2-3 generations and then used.
  • the MRS liquid medium containing the strain was centrifuged at 10000r / min and 4 ° C for 5min. The supernatant was taken and divided into two equal portions, one portion was not processed, and the other was adjusted to pH 7.0. The supernatant was filtered through a 0.22 ⁇ m disposable needle filter, and the bacteriostatic activity of the filtrate was tested by Oxford cup diffusion method. Pour a suspension (10 8 CFU / mL) containing 1% pathogenic bacteria (Listeria monocytogenes, Salmonella typhimurium, E.
  • Lactic acid bacteria were inoculated into MRS medium containing only glucose, sucrose, stachyose, and raffinose, and cultured at 37 ° C for 24 hours. The absorbance was read at 600 nm, and three parallel experiments were set up. The formula for calculating the relative growth rate of lactic acid bacteria is as follows:
  • X the acidity of the sample, the unit is ° T;
  • m The mass of the sample in g.
  • SPME-GC / MS analysis of volatile aromatic substances 50 / 30 ⁇ m DVB / CAR / PDMS extraction fiber head (SPME) was aged at the inlet (270 °C) for 30min. Weigh 5.0g of fermented soymilk 1: 1 with distilled water and add it to a 25mL headspace bottle. After heating at 40 ° C and equilibrating for 10 minutes, insert the SPME needle, and after 30 minutes of adsorption, insert it into the GC / MS chromatograph sample inlet for analysis.
  • Gas chromatographic conditions inlet temperature, inlet temperature 300 °C; heating program: keep at 35 °C for 2 minutes, then program to raise the temperature at 5 °C / min, 110 °C, keep 8min, 15 °C / min, program to 240 °C, hold for 5 minutes.
  • Split ratio 30 1; MS conditions: MS interface temperature 250 °C; ion source temperature 230 °C; quadrupole temperature 150 °C; ionization mode: EI; scan mode: full scan, mass range: 33 ⁇ 400m / z; Solvent delay: 0.1min.
  • Lactobacillus casei for positive control was Lactobacillus casei-01, a sample of Danish Cohensen Co., Ltd.
  • the first step of sampling and plate separation use a sterile sampling bottle to collect acidic water from natural fermentation on site and bring it back at low temperature. Immediately use sterile water to dilute them to 10 -3 , 10 -4 , and 10 -5 times, and spread them on the modified MRS solid medium. Then put it in a 37 ° C constant temperature incubator and incubate for 48 hours. Pick the suspected colonies and carry out the streak separation of the plate, repeat 4 to 5 times in this way, until a pure single colony is obtained. The purified single colony was punctured into MRS semi-solid medium and stored in a refrigerator at 4 ° C.
  • the third step of molecular identification the activated strain M1 is cultured and sent to a professional testing organization for sequencing to obtain the 16S rDNA sequence (sequence is SEQ.ID.NO1, see the sequence table). The results are compared and found on the NCBI gene library.
  • the standard strains KT897917.1 (Lactobacillus harbinensis strain LH-1), KF312693.1 (Lactobacillus harbinensis strain TCP001) and NR_113969.1 (Lactobacillus harbinensis strain NBRC100982), which were closely related to this strain, were obtained.
  • M1 and Lactobacillus harbinensis strain LH-1 sequence homology exceeds 98% (see Table 3), should be the same kind.
  • the strain was identified as Lactobacillus harbinensis based on the colony, bacterial morphology and physiological and biochemical characteristics.
  • Table 3 is a BLAST sequence alignment table of the Lactobacillus harbinensis M1 strain according to the 16S rDNA sequence of the present invention.
  • the strain was deposited in the Guangdong Microbial Strains Collection Center, the deposit location is the Guangdongzhou Microbial Strains Collection Center, 5th Floor, Building 59, Building No. 100, Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No. 60305.
  • the deposit date is 2017.12 On the 20th.
  • the first step is to prepare a lactic acid bacteria suspension: take a 100mL Erlenmeyer flask, add 50mL modified MRS liquid medium, sterilize at 121 ° C for 15min, inoculate Lactobacillus harbinensis M1 and Lactobacillus casei-01 strains respectively, incubate at 37 ° C overnight, store in a refrigerator at 4 ° C spare.
  • Step 2 Preparation of resistant plates: antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol, prepared at a concentration of 5120 ⁇ g / mL of antibacterial drug storage solution, dilute to the required concentration according to the diluent preparation method before use. Pipette 1.0mL of antimicrobial drug diluent into a large test tube of 9.0mL modified MRS agar medium, immediately vortex to mix, pour into a sterile plate, and wait for coagulation, that is, a certain drug concentration ( ⁇ g / mL) of bacteriostatic flat. At the same time, a control plate without drug was prepared.
  • the third step antibiotic sensitivity analysis take 1 ⁇ L of bacterial suspension (10 9 CFU / mL) to inoculate the surface of the plate and incubate for 16-20h at 37 ° C incubator. At the same time, use a blank plate without inoculation as a control. Observe the critical concentration (MIC value) of the strain not growing on the agar plate and make a record. The results showed that, as with the positive control strain Lactobacillus casei-01, the MIC values of Lactobacillus harbinensis M1 against the above nine antibiotics are all less than the resistance inflection point (see Table 4), which meets EFSA safety standards.
  • Table 4 shows the analysis results of drug resistance of the Lactobacillus harbinensis M1 strain of the present invention
  • the first step is to prepare a lactic acid bacteria suspension: take a 100mL Erlenmeyer flask, add 50mL modified MRS liquid medium, sterilize at 121 ° C for 15min, inoculate Lactobacillus harbinensis M1 and Lactobacillus casei-01 strains respectively, incubate at 37 ° C overnight, store in a refrigerator at 4 ° C spare.
  • Step 3 Tolerance to bile salts: Inoculate the cultured lactic acid bacteria at 10 9 CFU / mL into MRS liquid medium containing 0.3% porcine bile salts, and sample at 0h-11h every hour and read at 620nm Absorbance value, calculate the bacterial survival rate. The results showed that the survival rate of Lactobacillus harbinensis M1 in the bile salt tolerance test was 94.3%, and the survival rate of the positive control strain Lactobacillus casei-01 was 98.1%, both of which had a higher tolerance to bile salts.
  • the first step is to prepare the cell-free supernatant of lactic acid bacteria: take a 100mL Erlenmeyer flask, add 50mL modified MRS liquid medium, sterilize at 121 °C for 15min, inoculate LactobacillusharbinensisM1 and Lactobacilluscasei-01 strains respectively, and incubate at 37 °C for 24 hours.
  • the bacterial suspension prepared above was centrifuged at 10000r / min and 4 ° C for 5min, and the supernatant was divided into two equal parts, one part was not processed, the other was adjusted to pH 7.0, and the resulting supernatant was used once every 0.22 ⁇ m Filtered with a sexual needle filter and placed in a refrigerator at 4 ° C for later use.
  • Table 5 shows the analysis results of the antibacterial activity of the Lactobacillus harbinensis M1 strain of the present invention.
  • the second step of antibacterial activity analysis the Oxford Cup diffusion method was used to test the cell-free supernatant of lactic acid bacteria for Listeria monocytogenes, Salmonella typhimurium, E. coli O157, Enterobacter sakazakii, Streptococcus hemolyticus and Staphylococcus aureus Compared with the positive control strain, Lactobacillus harbinensis M1 supernatant has stronger ability to inhibit the pathogenic bacteria tested at pH, and may produce bacteriocin-like bacteriostatic substances (Table 5).
  • the first step is to prepare a lactic acid bacteria suspension: take a 100mL Erlenmeyer flask, add 50mL modified MRS liquid medium, sterilize at 121 ° C for 15min, inoculate Lactobacillus harbinensis M1 and Lactobacillus casei-01 strains respectively, incubate at 37 ° C for 24 hours, use DMEM without double antibodies + 10% bovine serum adjusted the bacterial concentration to 10 9 CFU / mL.
  • the second step of cell adhesion experiment Caco-2 cells were seeded into cell culture plates and cultured at 37 ° C and 5% CO 2 for 10 days. Wash three times with sterile PBS, add 1 mL of the above lactic acid bacteria suspension (10 9 CFU / mL), and continue culturing for 2 hours at 37 ° C, 5% CO 2. Wash three times with sterile PBS, digest with 1 mL pancreatin, and sample Calculate the number of viable cells by dilution coating method.
  • the first step is to prepare a lactic acid bacteria suspension: take a 100mL Erlenmeyer flask, add 50mL modified MRS liquid medium, sterilize at 121 ° C for 15min, inoculate Lactobacillus harbinensis M1 and Lactobacillus casei-01 strains respectively, incubate at 37 ° C overnight, store in a refrigerator at 4 ° C spare.
  • the second step is the use of oligosaccharides: the activated strains are inoculated into MRS medium containing only glucose, sucrose, stachyose, and raffinose, incubated at 37 ° C for 24 hours, and the absorbance is read at 600 nm.
  • Example 7 Live bacteria count and volatile components of fermented soybean milk
  • the first step is the preparation of fermented soybean milk and analysis of the number of viable bacteria: the pure soy milk filtered through a 180-mesh sieve is divided into 10ml large test tubes and sterilized at 100 ° C for 15 minutes. Add a single strain at 10 6 CFU / mL, ferment in a 37 ° C incubator for 24h, then place in a 4 ° C refrigerator for 24h, and analyze the number of viable bacteria and titratable acidity in the sample.
  • the second step analysis of volatile components of fermented soymilk solid phase microextraction-mass spectrometry (SPME-GC / MS) analysis of volatile aromatic substances in fermented soymilk, the results are shown in Table 6. It can be seen from the results in the table that (1) Lactobacillus harbinensis M1 fermented soymilk can significantly reduce the content of beany odor substances such as n-hexanal (100%), nonanal (100%), 1-octen-3-ol (31%) However, the control strain Lactobacillus casei-01 fermented soymilk has a significantly weaker ability to reduce n-hexanal (61%), 1-octen-3-ol (13%) and other bean-flavored substances, and even improves some bean-flavored components Such as the content of n-hexanol, 2-ethylfuran.
  • SPME-GC / MS solid phase microextraction-mass spectrometry
  • Lactobacillus harbinensis M1 strain fermented soymilk can produce rich aroma components.
  • the characteristic flavor components of milk, butadione and acetoin are 7 times and 202 times that of the control Lactobacillus casei-01 fermented soymilk, respectively.
  • the product presents a unique milk flavor.
  • Lactobacillus harbinensis producing milk-flavored volatile substances There are no reports of Lactobacillus harbinensis producing milk-flavored volatile substances.
  • Table 6 shows the effect of the Lactobacillus harbinensis M1 strain on the volatile components of fermented soybean milk.
  • the third step of sensory evaluation the sensory evaluation test of two kinds of fermented soybean milk showed that the overall acceptability of Lactobacillus harbinensis M1 strained soybean milk sensory evaluation score was 8.13, of which smell 8.38, appearance 7.38, taste 8.63, texture 8.13;
  • the Lactobacillus casei-01 fermented soybean milk sensory evaluation score has an overall acceptability of 6.5, including odor 6.00, appearance 6.13, taste 6.25, and texture 6.75.
  • the scores of Lactobacillus harbinensis M1 fermented soymilk were significantly higher than those of the control strain Lactobacillus casei-01 fermented soymilk.
  • the strain Lactobacillus harbinensis M1 isolated from tofu acid pulp water has the characteristics of wide growth temperature range and easy cultivation (Example 2). Like Lactobacillus casei-01, it has good probiotic tolerance to digestive juice (Example 3), adhesion to small intestinal epithelial cells (Example 4), and inhibition against pathogenic bacteria (Example 5), etc. Characteristics, in which the strain has a good inhibitory effect on pathogenic bacteria, and may produce bacteriocin-like bacteriostatic substances.
  • the strain has a high utilization rate of oligosaccharides and can grow in a matrix rich in oligosaccharides such as sucrose, stachyose, raffinose, etc. It is an ideal starter for fermenting plant-based foods such as soybean milk (Example 6) ).
  • the fermented soymilk prepared with this strain has a higher number of viable bacteria and acidity; the bacteria can significantly reduce the beany taste component in soymilk and add a special milk aroma component to significantly improve the flavor and taste of soymilk (Example 7), has a good application prospect.
  • the Lactobacillus harbini of the present invention is Gram-positive non-Bacillus, glucose heterotypic fermentation, wide growth temperature range (20-45 °C can be normal growth), fast growth rate; the results of conventional physiological and biochemical experiments show that strain M1 is Gram positive , Catalase negative, non-exercise.
  • the 16S rDNA sequence was aligned by BLAST, and the standard strains KT897917.1 (Lactobacillus harbinensis strain LH-1), KF312693.1 (Lactobacillus harbinensis strain TCP001) and NR_113969.1 (Lactobacillus harbinensis strain NBRC100982), which were closely related to this bacterium Partial sequence of rDNA was analyzed by similarity with standard bacteria to identify it as Lactobacillus harbinensis.
  • the strain is sensitive to antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol, and its safety meets the requirements of EFSA.
  • the supernatant of this strain has significant inhibitory effect on pathogenic bacteria such as Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157, Enterobacter sakazakii, Streptococcus hemolyticus and Staphylococcus aureus, and the pH value of the supernatant is adjusted After 7.0, it still has strong bacteriostatic ability, indicating that this strain may produce bacteriocin-like bacteriostatic substances.
  • pathogenic bacteria such as Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157, Enterobacter sakazakii, Streptococcus hemolyticus and Staphylococcus aureus
  • the utilization rate of the strain on sucrose, stachyose and raffinose is significantly higher than that of the control bacteria, and the fermented soybean milk prepared by using the strain has higher viable bacterial count and acidity value.
  • the strain can produce rich aroma components when fermenting soy milk.
  • the characteristic aroma components of milk, butadione and acetoin are significantly higher than the fermented soybean milk of the control bacteria, and the content of beany flavor components is significantly reduced.
  • the results of sensory evaluation show that this strain can significantly improve the flavor and taste of fermented soybean milk, and is an ideal starter for fermented soybean milk and other plant raw materials, with a very broad application prospect.

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Abstract

提供了一种哈尔滨乳杆菌(Lactobacillus harbinensis) M1及其应用，该菌株保藏号为GDMCC No.60305，对抗生素氨苄西林、四环素和氯霉素等敏感，安全性符合EFSA的要求，其上清液对单增李斯特菌、溶血链球菌以及金黄色葡萄球菌等抑制效果显著。还提供了所述哈尔滨乳酸菌在发酵豆乳中的应用，利用该菌株单独发酵豆乳产酸能力强、活菌数高，豆腥味物质含量降低甚至完全消失，发酵牛奶特征性香气成分含量明显提升，极大地改善了发酵豆乳的感官风味。

Description

一种哈尔滨乳杆菌及其应用 技术领域
本发明涉及益生菌应用领域，具体涉及一种哈尔滨乳酸菌(Lactobacillus harbinensis M1)及其应用，该哈尔滨乳酸菌可以作为益生菌，并可以作为发酵剂应用于发酵豆乳中。
背景技术
益生菌是指给予一定数量(通常在10 6CFU/g以上)时能对宿主健康产生有益作用的活的微生物。只有那些能耐受胃肠道消化、能粘附和定植在小肠上皮细胞，并能抑制病源微生物生长的微生物才能对宿主健康发挥有益作用。常用益生菌主要包括乳杆菌类、双歧杆菌类和革兰阳性球菌类，如嗜热链球菌等，其益生功能主要体现在增强免疫力、调节肠道菌群和改善胃肠道功能、抗氧化和延缓衰老、降低胆固醇和改善血脂等方面。
大豆含有丰富的优质蛋白、不饱和脂肪酸以及低聚糖、异黄酮和皂苷等功能性成分，具有环境友好、健康经济等特点，是我国传统意义上重要的植物蛋白资源，具有重要的经济价值和社会价值。应用益生菌发酵大豆所制备的大豆蛋白饮品不仅含有大量活的益生菌，还通过生物转化作用使异黄酮成为易于吸收的苷元型、使大豆蛋白降解形成多肽和氨基酸，同时生成γ-氨基丁酸、B族维生素等功能因子，因此益生菌发酵豆乳正受到食品科学工作者和现代消费者的广泛关注。但该类产品目前还存在一些感官品质上的缺陷，如与发酵牛乳相比，益生菌发酵豆乳在口感和风味方面还有较大的差距，其中豆腥味比较突出，严重影响了产品的可接受性，制约了其产业化应用。
大豆食品的豆腥味主要来自于脂肪氧化酶催化生成的正己醛、壬醛、1-辛烯-3-醇等挥发性的脂肪氧化降解产物，目前主要有灭酶、氧隔离和生物转化等处理方法来降低该不良风味。其中，应用益生菌发酵降低豆腥味的效果与菌种关系密切。由于现有商业化应用的乳酸菌大多数来源于发酵乳制品，将这些菌种应用于发酵豆乳时往往适应性不佳，主要体现在发酵性不好和产品风味不良。
发明内容
本发明的目的在于根据现有技术存在的问题，提供一种适用于发酵食品，特别是发酵大豆食品的哈尔滨乳酸菌(Lactobacillus harbinensis)M1，该菌株具有很好的安全性和益生功能，并能 够有效利用大豆中的各种低聚糖，显著改善和提高豆类食品的感官风味。
豆腐黄浆水是豆腐制作过程中产生的黄色沥水，由于黄浆水中仍然含有丰富的营养物质，非常适宜于微生物的生长和繁殖，因此在存放的过程中会自然酸化。我国部分地方，有利用酸浆水作凝固剂制备豆腐的习惯，由于这种酸浆豆腐滋味鲜美、风味独特，近年来受到了越来越多消费者的喜爱。酸浆水中的微生物由于长期的训化，对豆类成分如棉子糖、水苏糖和异黄酮等具有很强的利用能力，且具有良好的风味。本发明在深入研究豆腐酸浆水的基础上，筛选到1株在大豆基质中发酵性能优良的哈尔滨乳杆菌，该菌株具有潜在益生功能及重要的应用价值。
本发明目的通过以下技术方案实现：
一种哈尔滨乳酸菌(Lactobacillus harbinensis M1)，该菌株的保藏号为GDMCC No.60305，保藏于广东省微生物菌种保藏中心，保藏地点为广州市先烈中路100号大院59号楼5楼广东省微生物菌种保藏中心，保藏日期为2017年12月20日。
所述哈尔滨乳酸菌作为益生菌的应用。
所述哈尔滨乳酸菌对抗生素氨苄西林、万古霉素、庆大霉素、卡那霉素、链霉素、红霉素、克林霉素、四环素和氯霉素敏感，安全性符合EFSA的要求。
所述哈尔滨乳酸菌的上清液对单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌抑制效果显著。
所述哈尔滨乳酸菌对胃液、肠液及胆盐的耐受，可定植在小肠上皮细胞上。
所述哈尔滨乳酸菌在发酵豆乳中的应用。
所述哈尔滨乳酸菌对蔗糖、水苏糖和棉籽糖的利用率与对葡萄糖的利用率相当。
所述哈尔滨乳酸菌使豆乳中的豆腥味成分正己醛完全消失。
所述哈尔滨乳酸菌增加了牛奶香气成分丁二酮和乙偶姻。
本发明中哈尔滨乳酸菌(Lactobacillus harbinensis M1)是使用无菌采样瓶现场采集自然发酵而成的酸浆水样品，通过稀释平板法筛选获得。该菌株的生物活性特征如下：在MRS固体培养基平板上菌落突起，呈圆形，直径一般为1-3mm，灰白色，不透明，湿润光滑；菌体为革兰氏染色阳性无芽孢杆菌，呈细短杆状，成对或堆状排列；葡萄糖异型发酵，生长温域宽(20-45℃可正常生长)，生长速度快；常规生理生化实验结果表明菌株M1为革兰氏阳性、过氧化氢酶阴性、非运动性。进一步对16S rDNA序列进行分析，并通过BLAST比对，鉴定其为哈尔滨乳酸菌(Lactobacillus harbinensis)。
本发明中哈尔滨乳杆菌M1符合益生菌特性，该菌株在模拟胃液以及模拟肠液中显示出较好的耐受性，并且能够粘附于小肠上皮细胞Caco-2。该菌株能够有效抑制食源性致病菌如单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌等的繁殖。同时，该菌株的生长能够有效利用蔗糖、水苏糖和棉籽糖，适用于发酵富含植物低聚糖食品的生产。利用该菌株制备的发酵豆乳具有较高的活菌数和酸度，且挥发性风味成分中豆腥味成分降低，牛奶香气成分丁二酮和乙偶姻增加，有效地提升了产品的感官风味。
本发明相对于已有的商业菌株，具有以下的优点和有益效果：
(1)本发明菌株Lactobacillus harbinensis M1对胃肠道有很好的耐受性，能活着到达小肠并在上皮细胞上粘附与定植，且对多种食源性致病菌有很强的抑制作用，具有在肠道中成为优势菌的潜力，可以为宿主提供良好的益生效应。
(2)本发明菌株对棉子糖、水苏糖、蔗糖等低聚糖的利用率非常高，能够在大豆等富含此类低聚糖的植物性原料中生长繁殖，为发酵大豆等植物类食品的生产提供了新的适应性菌种。
(3)本发明菌株能够降低发酵豆乳中正己醛、壬醛、1-辛烯-3-醇等豆腥味物质含量，增加丁二酮和乙偶姻等香气成分，赋予发酵豆乳特有的奶香味，显著改善发酵豆乳感官风味。
(4)本发明菌株生长温域宽，生长速度快，培养条件简单，易于工业化生产和管理，开发应用前景广阔。
附图说明
图1为本发明Lactobacillus harbinensisM1菌株的菌落形态图；
图2为本发明Lactobacillus harbinensisM1菌株的菌体形态图。
具体实施方式
为更好地理解本发明，下面结合附图和实施例对本发明作进一步说明，但实施例不对本发明保护范围构成任何限定。
以下实施例中：
(1)革兰氏染色、形状观察及常用生理生化鉴定培养基参考凌代文主编的《乳酸细菌分类鉴定及试验方法》(1999年版)。
(2)改良MRS液体培养基(g/L)(用于乳酸菌的培养)：
牛肉膏10g，蛋白胨10g，酵母浸粉5g，葡萄糖20g，吐温-80 1mL，K 2HPO 4·3H 2O 2g，NaAc·3H 2O 5g，柠檬酸三铵2g，MgSO 4·7H 2O 0.58g，MnSO 4·H 2O 0.25g；蒸馏水1L，pH 6.4 ±0.2(MRS固体培养基在液体培养基的基础上添加0.7-2％的琼脂)，121℃灭菌15min。
(3)耐药性分析
根据欧洲食品安全局规定的细菌耐药性判定标准(2012版)，采用抗生素氨苄西林、万古霉素、庆大霉素、卡那霉素、链霉素、红霉素、克林霉素、四环素和氯霉素，配制浓度为5120μg/mL的抗菌药物贮存液，临用时按稀释液配制法稀释至所需浓度的使用液，并按比例加入到相应培养基中。将改良MRS琼脂培养基取9.0mL分装于大试管中在121℃灭菌15min后，置于水浴锅(50℃)中保温备用。吸取1.0mL抗菌药物稀释液于该大试管中，立即涡旋混匀，倒入无菌平皿中，待凝固，即制得一定药物浓度(μg/mL)的抑菌平板。每种药物都按照二倍稀释法制得8个浓度梯度的抑菌平板。同时制备不加药物的对照平板。分别蘸取菌悬液1μL(10 9CFU/mL)接种至平板表面于37℃恒温箱培养16-20小时，同时，用不接种的空白平皿作对照。观察琼脂平板上菌株不生长的临界浓度(MIC值)并做记录，实验设置3个平行。乳酸菌药敏结果的判定参照相关标准如下表1：
表1
Figure PCTCN2019112046-appb-000001
(4)对胃液、肠液和胆盐的耐受性分析
胃肠液耐受性：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，冷却后接种乳酸菌，37℃培养过夜，作为种子培养液备用。模拟胃液：0.27g胃蛋白酶稀释于90mL无菌磷酸缓冲溶液PBS中，用盐酸调整pH至3，0.22μm一次性针头滤器过滤除菌备用。模拟肠液：0.1g胰蛋白酶稀释于100mL无菌磷酸缓冲溶液PBS中，采用氢氧化钠调整pH至8.0， 0.22μm一次性针头滤器过滤除菌备用。把培养好的乳酸菌以10 9CFU/mL接种到模拟胃液(pH＝3)中，分别在0h-3h每小时取样用稀释涂布法计算活菌数；3小时后转接到模拟肠液(pH＝8)中，分别在0h-12h每小时取样用稀释涂布法计算活菌数，实验设置3个平行。
模拟胆盐耐受性：0.3g猪胆盐加入100mL改良MRS液体培养基，121℃灭菌15min，冷却至37℃。把培养好的乳酸菌以10 9CFU/mL接种到含0.3％猪胆盐的MRS液体培养基中，分别在0h-11h每小时取样在620nm下读取吸光值，实验设置3个平行。细菌的存活率计算公式如下：X(％)＝*100％
(5)牛津杯扩散法测试抑菌活性
将菌株用MRS培养基活化2-3代。致病菌用LB培养基活化2-3代后备用。10000r/min、4℃离心含菌株的MRS液体培养基5min，取上清液，分成两等份，一份不处理，另一份调pH至7.0。上清液均经0.22μm一次性针头滤器过滤，滤液抑菌活性检测采用牛津杯扩散法。将含1％致病菌(单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌)悬液(10 8CFU/mL)倾倒入灭菌的平皿中，待其凝固后，用灭菌的牛津杯放置在琼脂平板上。将滤液60μL注射入牛津杯中，37℃培养24小时，记录抑菌圈大小，实验设置3个平行。
(6)对Caco-2细胞的粘附作用
将菌株接种到MRS培养基中，37℃培养24小时，离心收集菌体后，用无菌溶液洗涤两次，最后再重悬于溶液中，于600nm下测量吸光度，调整菌体浓度为10 9CFU/mL(OD 600＝0.57)，离心(6000g,5min)，弃去上清液，加入与弃去等体积的无双抗的DMEM+10％牛血清，混匀。Caco-2细胞使用DMEM+10％胎牛血清为基础培养基，接种至细胞培养板中，于37℃、5％CO 2培养10天。当细胞聚合度达90％-100％，用无菌PBS洗涤三次，加入上述1mL乳酸菌悬浮液(10 9CFU/mL)，于37℃、5％CO 2培养2小时，用无菌PBS洗涤三次，用1mL胰酶进行消化，取样用稀释涂布法计算活菌数，实验设置3个平行。
(7)对低聚糖的利用
将乳酸菌分别接种到只含葡萄糖、蔗糖、水苏糖、棉籽糖的MRS培养基中，37℃培养24小时，在600nm下读取吸光值，实验设置3个平行。乳酸菌的相对生长率计算公式如下：
X(％)＝*100％
(8)发酵豆乳可滴定酸度分析
挑选无破损、未霉变的大豆，加入6倍质量的水，然后加入0.5％NaHCO 3，搅匀，在常温 下浸泡14h，加入85℃水，按照豆水比1:8(g:mL)进行热磨浆，经180目筛过滤得到纯豆浆，并于100℃下灭菌15min。按10 6CFU/mL加入单菌株发酵剂，放在37℃的恒温培养箱中发酵24h，然后于4℃下后熟24h，取样用稀释涂布法计算活菌数。酸度值按GB 5413.34—2010《乳和乳制品酸度的测定》将样品用玻璃棒搅拌均匀，精确称取10g样品，再加入无CO 2的蒸馏水20mL，混匀后加入0.5mL酚酞指示剂，用0.1N NaOH标准溶液滴定至溶液呈微红色，且30s内不褪色，记录消耗氢氧化钠的体积，实验设置3个平行。酸度的计算按照下面的公式：
X＝(C×V×100)/(m×0.1)
X——样品的酸度，单位°T；
C——氢氧化钠标液的浓度，单位mol/L；
V——消耗氢氧化钠标液的体积，单位mL；
m——样品的质量，单位g。
(9)固相微萃取-气质联用(SPME-GC/MS)分析挥发性芳香物质：50/30μm DVB/CAR/PDMS萃取纤维头(SPME)于进样口(270℃)老化30min。称取5.0g发酵豆乳1:1与蒸馏水混合加入25mL顶空瓶中。加热温度40℃，加热平衡10min后，插入SPME针，吸附30min后插入GC/MS色谱仪进样口解析。气相色谱条件：进样口温度进样口温度300℃；升温程序：35℃保持2min后以5℃/min程序升温110℃，保8min，15℃/min程序升温至240℃，保持5min。分流比30:1；质谱条件：质谱接口温度250℃；离子源温度230℃；四极杆温度150℃；离子化模式：EI；扫描模式：全扫描，质量数范围：33～400m/z；溶剂延迟：0.1min。
(10)感官风味评价：按9分制进行打分，1分为最差，9分表示最好。邀请30位品评员在20℃环境下对样品的气味、外观、滋味、质构和总体可接受性打分。
(11)阳性对照用干酪乳杆菌为Lactobacillus casei-01，丹麦科汉森股份有限公司样品。
实施例1：菌种筛选和鉴定
第一步采样和平板分离：使用无菌采样瓶，现场采集自然发酵而成的酸浆水，低温带回。立即使用无菌水分别稀释至10 -3、10 -4、10 -5倍，涂布于改良的MRS固体培养基上。然后放入37℃的恒温培养箱中，培养48小时。挑取疑似菌落，进行平板划线分离，如此重复4～5次，直至获得纯的单菌落。将纯化的单菌落穿刺接种到MRS半固体培养基，于4℃冰箱内保存。
第二步形态观察和生理生化实验：该菌株在MRS培养基上的菌落呈圆形、灰白色、半透明状，湿润光滑(图1)；进行革兰氏染色和细胞形状观察，菌株M1的菌体呈短杆状，成对或堆生(见图2)。常规生理生化实验结果(见表2)表明菌株M1为革兰氏阳性、过氧化氢酶阴性、 异型发酵的无芽孢杆菌，能在20～45℃内的温域范围内生长。表2为本发明Lactobacillus harbinensisM1菌株的生理生化特性；
表2
Figure PCTCN2019112046-appb-000002
第三步分子鉴定：将所得菌株M1活化培养，送专业的检测机构测序，获得16S rDNA序列(序列为SEQ.ID.NO1，见序列表)，结果在NCBI的基因库上进行比对，找出与此菌亲缘相近的标准菌株KT897917.1(Lactobacillus harbinensisstrain LH-1)，KF312693.1(Lactobacillus harbinensisstrain TCP001)和NR_113969.1(Lactobacillus harbinensisstrain NBRC100982)，将菌株M1的16S rDNA的部分序列与标准菌进行相似度分析，M1与哈尔滨乳杆菌Lactobacillus harbinensisstrain LH-1序列同源性超过98％(见表3)，应为同一种。结合菌落、菌体形态，生理生化特征将菌株鉴定为哈尔滨乳酸菌(Lactobacillus harbinensis)。
表3为本发明Lactobacillus harbinensisM1菌株根据16S rDNA序列所做的BLAST序列比对情况表。
表3
Figure PCTCN2019112046-appb-000003
该菌株保藏于广东省微生物菌种保藏中心，保藏地点为广州市先烈中路100号大院59号楼5楼广东省微生物菌种保藏中心，保藏号为GDMCC No.60305，保藏日期为2017年12月20日。
实施例2：抗生素敏感性
第一步乳酸菌菌悬液制备：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，分别接种Lactobacillus harbinensisM1和Lactobacillus casei-01菌株，37℃培养过夜，置于4℃冰箱保存备用。
第二步抗性平板制备：采用抗生素氨苄西林、万古霉素、庆大霉素、卡那霉素、链霉素、红霉素、克林霉素、四环素和氯霉素，配制浓度为5120μg/mL的抗菌药物贮存液，临用时按稀释液配制法稀释至所需浓度的稀释液。吸取1.0mL抗菌药物稀释液于9.0mL改良MRS琼脂培养基的大试管中，立即涡旋混匀，倒入无菌平皿中，待凝固，即制得一定药物浓度(μg/mL)的抑菌平板。与此同时，制备不加药物的对照平板。
第三步抗生素敏感性分析：取菌悬液1μL(10 9CFU/mL)接种至平板表面于37℃恒温箱培养16-20h，同时，用不接种的空白平皿作对照。观察琼脂平板上菌株不生长的临界浓度(MIC值)并做记录。结果表明，跟阳性对照菌株Lactobacillus casei-01一样，Lactobacillus harbinensisM1对上述9种抗生素的MIC值均小于耐药性拐点(见表4)，符合EFSA安全性标准。
表4为本发明Lactobacillus harbinensisM1菌株的耐药性分析结果；
表4
Figure PCTCN2019112046-appb-000004
实施例3：对消化液的耐受性
第一步乳酸菌菌悬液制备：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，分别接种Lactobacillus harbinensisM1和Lactobacillus casei-01菌株，37℃培养过夜，置于4℃冰箱保存备用。
第二步对模拟胃液肠液的耐受性：把培养好的乳酸菌以10 9CFU/mL接种到模拟胃液(pH＝3)中，3小时后转接到模拟肠液(pH＝8)中，分别在0h-12h每小时取样用稀释涂布法计算活菌数。结果表明，Lactobacillus harbinensisM1在模拟胃液、肠液中剩余活菌数为2.76log CFU/mL，阳性对照菌Lactobacillus casei-01的剩余活菌数为2.98log CFU/ml,两者没有显著性差异。
第三步对胆盐的耐受性：把培养好的乳酸菌以10 9CFU/mL接种到含0.3％猪胆盐的MRS液体培养基中，分别在0h-11h每小时取样在620nm下读取吸光值，计算细菌存活率。结果表明，Lactobacillus harbinensisM1在耐胆盐测试中存活率为94.3％，阳性对照菌Lactobacillus casei-01的存活率为98.1％，两者对胆盐都有较高的耐受性。
实施例4：对致病菌的抑制效果
第一步乳酸菌无细胞上清液制备：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，分别接种Lactobacillus harbinensisM1和Lactobacillus casei-01菌株，37℃培养24小时。将上述制备的菌悬液以10000r/min、4℃离心5min，取上清液，分成两等份，一份不处理，另一份将pH调至7.0，所得上清液均用0.22μm一次性针头滤器过滤，置于4℃冰箱保存备用。
表5为本发明Lactobacillus harbinensisM1菌株的抑菌活性分析结果。
表5
Figure PCTCN2019112046-appb-000005
第二步抑菌活性分析：采用牛津杯扩散法测试乳酸菌无细胞上清液对病源菌单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌的抑制效果， 结果表明，跟阳性对照菌株相比，Lactobacillus harbinensisM1上清液在pH性时对测试病源菌的抑制能力更强，有可能产生了细菌素类的抑菌物质(表5)。
实施例5：对小肠上皮细胞Caco-2的粘附性
第一步乳酸菌菌悬液制备：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，分别接种Lactobacillus harbinensisM1和Lactobacillus casei-01菌株，37℃培养24小时，用无双抗的DMEM+10％牛血清调整菌体浓度为10 9CFU/mL。
第二步细胞粘附实验：将Caco-2细胞接种至细胞培养板中，于37℃、5％CO 2的条件下培养10天。用无菌PBS洗涤三次，加入上述乳酸菌悬浮液(10 9CFU/mL)1mL，于37℃、5％CO 2条件下继续培养2小时，无菌PBS洗涤三次，用1mL胰酶进行消化，取样用稀释涂布法计算活菌数。结果显示，Lactobacillus harbinensisM1对小肠上皮细胞Caco-2的粘附菌数为5.21log CFU/mL，阳性对照菌Lactobacillus casei-01的粘附菌数为5.22log CFU/mL，表明两者均对小肠上皮细胞均有良好的粘附性。
实施例6：低聚糖利用特性
第一步乳酸菌菌悬液制备：取100mL三角瓶，加入50mL改良MRS液体培养基，121℃灭菌15min，分别接种Lactobacillus harbinensisM1和Lactobacillus casei-01菌株，37℃培养过夜，置于4℃冰箱保存备用。
第二步对低聚糖的利用：将经过活化的菌种接种到只含葡萄糖、蔗糖、水苏糖、棉籽糖的MRS培养基中，37℃培养24小时，在600nm下读取吸光值。结果显示，Lactobacillus harbinensisM1在蔗糖、水苏糖和棉籽糖中的相对生长率分别为121.59％，94.20％，103.08％，而阳性对照菌株Lactobacillus casei-01在蔗糖、水苏糖棉籽糖中的相对生长率分别为98.27％，48.25％，91.91％，表明该菌株对蔗糖、水苏糖和棉子糖有很高的利用率，与对葡萄糖的利用率相当，适宜于在豆类基质或植物性原料中生长。
实施例7：发酵豆乳的活菌数及挥发性成分
第一步发酵豆乳的制备及活菌数分析：取经180目筛过滤得到的纯豆浆分装于10ml大试管中，于100℃下灭菌15min。按10 6CFU/mL加入单菌种，在37℃的恒温培养箱中发酵24h，再置于4℃冰箱后熟24h，分析样品中的活菌数和可滴定酸度。结果显示，Lactobacillus harbinensisM1发酵豆乳的活菌数达到9.08log CFU mL -1，酸度值为44.35°T，而阳性对照菌Lactobacillus casei-01发酵豆乳的活菌数为8.42log CFU mL -1，酸度值为36.91°T，两者有显著性差异，表明该菌株更适合在豆浆中发酵。
第二步发酵豆乳的挥发性成分分析：利用固相微萃取-气质联用(SPME-GC/MS)分析发酵豆乳的挥发性芳香物质，结果见表6。从表中结果可知，(1)Lactobacillus harbinensis M1发酵豆乳能够显著降低正己醛(100％)、壬醛(100％)、1-辛烯-3-醇(31％)等豆腥味物质的含量，而对照菌株Lactobacillus casei-01发酵豆乳降低正己醛(61％)、1-辛烯-3-醇(13％)等豆腥味物质的能力明显偏弱，甚至提高了某些豆腥味成分如正己醇、2-乙基呋喃的含量。(2)Lactobacillus harbinensis M1菌株发酵豆乳能够产生丰富的香气成分，其中牛奶的特征性风味成分丁二酮和乙偶姻的含量分别是对照Lactobacillus casei-01发酵豆乳的7倍和202倍，从而使产品呈现特有的奶香味。目前尚未有关于Lactobacillus harbinensis产生奶香味挥发性物质的报道。
表6为本发明Lactobacillus harbinensis M1菌株对发酵豆乳挥发性成分的影响结果。
表6
Figure PCTCN2019112046-appb-000006
第三步感官品评：对两种发酵豆乳进行感官评价试验，结果显示，Lactobacillus harbinensis M1株发酵豆乳感官评价得分总体可接受性为8.13，其中气味8.38、外观7.38、滋味8.63、质构8.13； 对照菌Lactobacillus casei-01发酵豆乳感官评价得分总体可接受性为6.5，其中气味6.00、外观6.13、滋味6.25、质构6.75。Lactobacillus harbinensis M1发酵豆乳的各项评分均显著高于对照菌Lactobacillus casei-01发酵豆乳的评分。
由以上实施例可见，从豆腐酸浆水分离出来的菌株Lactobacillus harbinensis M1具有生长温域宽、易于培养等特点(实施例2)。与Lactobacillus casei-01一样，具有良好的对消化液的耐受性(实施例3)、对小肠上皮细胞的粘附性(实施例4)、对病源菌的抑制性(实施例5)等益生特性，其中该菌株对病源菌的抑制效果很好，可能产生了细菌素类的抑菌物质。同时，该菌株对低聚糖利用率很高，能够在富含蔗糖、水苏糖、棉子糖等低聚糖的基质中生长，是发酵豆乳等植物基食品的理想发酵剂(实施例6)。利用该菌株制备的发酵豆乳具有更高的活菌数和酸度；该菌能够显著降低豆乳中的豆腥味成分，并增加了特殊的牛奶香气成分，显著提高酸豆乳的风味与滋味(实施例7)，有很好的应用前景。
本发明哈尔滨乳杆菌为革兰氏染色阳性无芽孢杆菌，葡萄糖异型发酵，生长温域宽(20-45℃可正常生长)，生长速度快；常规生理生化实验结果表明菌株M1为革兰氏阳性、过氧化氢酶阴性、非运动性。16S rDNA序列通过BLAST比对，与此菌亲缘相近的标准菌株KT897917.1(Lactobacillus harbinensisstrain LH-1)，KF312693.1(Lactobacillus harbinensisstrain TCP001)和NR_113969.1(Lactobacillus harbinensisstrain NBRC100982)，将菌株M1的16S rDNA的部分序列与标准菌进行相似度分析，鉴定其为哈尔滨乳酸菌Lactobacillus harbinensis。
该菌株对抗生素氨苄西林、万古霉素、庆大霉素、卡那霉素、链霉素、红霉素、克林霉素、四环素和氯霉素敏感，安全性符合EFSA的要求。
该菌株对模拟胃液(pH＝3，3h)、肠液(pH＝8，12h)及胆盐的耐受性、以及对小肠上皮细胞Caco-2的粘附能力均与对照菌株Lactobacillus casei-01类似，可定植在小肠上皮细胞并发挥益生功效。
该菌株的上清液对单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌等病源菌的抑制效果显著，将其上清液pH值调整到7.0后仍具有很强的抑菌能力，表明该菌株有可能产生细菌素类的抑菌物质。
该菌株对蔗糖、水苏糖和棉籽糖的利用率显著高于对照菌，应用该菌株制备的发酵豆乳具有更高的活菌数和酸度值。不仅如此，该菌株发酵豆乳时还能够产生丰富的香气成分，其中牛奶特征性香气成分丁二酮和乙偶姻的含量显著高于对照菌发酵豆乳，并显著降低豆腥味成分含量，其中正己醛完全消失。感官品评的结果显示，该菌株可明显改善发酵豆乳的风味和滋味，是发酵豆 乳等植物原料的理想发酵剂，应用前景十分广阔。
本发明不受上述实施例约束，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的替代方式，都包含在本发明的保护范围之内。
Figure PCTCN2019112046-appb-000007
Figure PCTCN2019112046-appb-000008

Claims (9)

  1. 一种哈尔滨乳酸菌(Lactobacillus harbinensis M1)，其特征在于：该菌株的保藏号为GDMCC No.60305。
  2. 权利要求1所述哈尔滨乳酸菌作为益生菌的应用。
  3. 根据权利要求2所述哈尔滨乳酸菌作为益生菌的应用，其特征在于，所述哈尔滨乳酸菌对抗生素氨苄西林、万古霉素、庆大霉素、卡那霉素、链霉素、红霉素、克林霉素、四环素和氯霉素敏感，安全性符合EFSA的要求。
  4. 根据权利要求2所述哈尔滨乳酸菌作为益生菌的应用，其特征在于，所述哈尔滨乳酸菌的上清液对单增李斯特菌、鼠伤寒沙门氏菌、大肠杆菌O157、阪崎肠杆菌、溶血链球菌以及金黄色葡萄球菌抑制效果显著。
  5. 根据权利要求2所述哈尔滨乳酸菌作为益生菌的应用，其特征在于，所述哈尔滨乳酸菌对胃液、肠液及胆盐的耐受，可定植在小肠上皮细胞上。
  6. 权利要求1所述哈尔滨乳酸菌在发酵豆乳中的应用。
  7. 根据权利要求6所述哈尔滨乳酸菌在发酵豆乳中的应用，其特征在于，所述哈尔滨乳酸菌对蔗糖、水苏糖和棉籽糖的利用率与对葡萄糖的利用率相当。
  8. 根据权利要求6所述哈尔滨乳酸菌在发酵豆乳中的应用，其特征在于，所述哈尔滨乳酸菌使豆乳中的豆腥味成分正己醛完全消失。
  9. 根据权利要求6所述哈尔滨乳酸菌在发酵豆乳中的应用，其特征在于，所述哈尔滨乳酸菌增加了牛奶香气成分丁二酮和乙偶姻。
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