CN111206001A - Lactococcus lactis subsp lactis and application thereof in preparation of soybean milk - Google Patents
Lactococcus lactis subsp lactis and application thereof in preparation of soybean milk Download PDFInfo
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- CN111206001A CN111206001A CN202010088420.5A CN202010088420A CN111206001A CN 111206001 A CN111206001 A CN 111206001A CN 202010088420 A CN202010088420 A CN 202010088420A CN 111206001 A CN111206001 A CN 111206001A
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- lactococcus lactis
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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
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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
- A23C20/00—Cheese substitutes
- A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
- A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
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- 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
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Abstract
The invention discloses lactococcus lactis subspecies lactis and application thereof in preparing soybean milk, and belongs to the technical field of microbiology. The lactococcus lactis subspecies lactis CCFM1092 has the following properties: (1) the growth rate of the strain is high, the strain enters a stable period within 8 hours in MRS, the increment of the number of viable bacteria within 6 hours in the soybean milk reaches 2 orders of magnitude, and the strain is suitable for industrial fermentation; (2) the acid production rate of the strain is high, the fermented soybean milk is curdled for 6 hours and reaches the fermentation end point, so that curdled milk and flavor substances are generated; (3) the strain has high yield of 3-methylbutanol, and can produce 476.05 mu g/kg in 6 hours. Lactococcus lactis subsp lactis CCFM1092 is used as a leavening agent for leavening food, improves the fruit flavor of the fermented soybean milk, and has very wide application prospect.
Description
Technical Field
The invention relates to lactococcus lactis subspecies lactis and application thereof in preparing soybean milk, belonging to the technical field of microorganisms.
Background
Alcohols are important volatile substances, and in natural foods such as tomato, apple and melon fruits, the alcohols are main flavor substances (glance sideways Lanchun, Sun Seiyun, Huangruing. fruit aroma formation and its influence factor [ J ]. botanicals report, 2004,21(5):631-637.), and in many fermented foods, the alcohols generated by microbial fermentation play an important role in the flavor of the fermented foods. Sour milk is a novel soy product formed by fermenting soy milk with lactic acid bacteria whose metabolism effectively reduces beany flavor and soy oligosaccharides in sour milk, making it more widely accepted than soy milk (Leblanc J G, Silvestroni A, connections C, et al.reduction of non-gelled lactic acid bacteria in soy milk: application of engineered lactic acid bacteria product alpha-galactoside [ J ]. Genet Mol Res,2004,3(3):432 440.), but residual beany flavor (such as 2-pentylfuran) in fermented soy milk still controls its consumption level due to the different metabolic effects of lactic acid bacteria on different beany flavor substances. Although some researches have been made at home and abroad to improve the bad flavor by adding auxiliary materials such as fruits, milk and grains (Coda R, Lanera A, Trani A, et al. Yogurt-like flavors of foods, soy and grain most: Microbiology, texture, nutritional and sensory properties [ J ]. International Journal of Food biology,2012,155(3): 120:. 127.), the reasons for the bad flavor should be fundamentally solved, so as to be more beneficial to the development of the products.
Lactococcus lactis, a traditional cheese starter, has also been increasingly used in fermented milk in recent years as an adjunct starter. Since the lactic acid bacteria are of plant origin, they have a certain potential for use in plant-based systems, but little research interest has been shown. Golomb et al (Golomb B L, Marco M L. Lactococcus lactis metabolism and genexpression during growth of growing plants tissues [ J ]. Journal of bacteriology,2015,197(2): 371-.
In addition, the research on improving the quality of the sour soybean milk by fermenting lactic acid bacteria mainly focuses on reducing beany flavor and improving mouthfeel, and the research on improving the flavor and quality of the sour soybean milk by fermenting microorganisms is less. Part of lactic acid bacteria can generate 3-methylbutanol through leucine metabolism, a fruit flavor substance, which comprises lactococcus lactis, and lactococcus lactis of plant origin has certain application potential in a plant-based system.
In conclusion, research and development of lactococcus lactis capable of producing 3-methylbutanol with high yield, which is used as a leavening agent for fermenting soybean milk to improve the flavor of the fermented soybean milk and improve the quality of the fermented soybean milk, are problems to be solved in the field.
Disclosure of Invention
The invention provides Lactococcus lactis subsp.lactis CCFM1092 which is preserved in Guangdong province microorganism strain preservation center in 2019, 10 and 16 days, wherein the preservation number is GDMCC No. 60807, and the preservation address is No. 59 floor 5 of Zhonglu 100 McJud.
The invention provides a preparation method of soybean milk with high content of 3-methylbutanol, which comprises the following steps: inoculating the preserved lactococcus lactis subsp. lactis CCFM1092 into a culture medium for culture, and then carrying out subculture for 2-3 times until the strain concentration is 108～109cfu/mL is inoculated into soybean milk to make the final concentration of the strain in the system be at least 105cfu/mL, placing the inoculated sample in an incubator at 35-40 ℃ for fermentation for 6-8 h, and then placing in an environment at 2-6 ℃ for mature fermentation.
In one embodiment of the invention, the lactococcus lactis subsp lactis CCFM1092 has a final concentration in soy milk of at least 105～cfu/mL。
The invention provides a starter culture containing lactococcus lactis subsp lactis CCFM1092, and a preparation method of the starter culture comprises the following steps: inoculating 200-600 mu L of lactococcus lactis subspecies lactis CCFM1092 into 10-30 mL of MRS liquid culture medium, activating for 2-3 generations at 30 ℃, and allowing lactococcus lactis subspecies lactis CCFM1092 to reach 108centrifuging at 8000rpm for 15min when viable count is above cfu/mL, removing supernatant, sequentially adding buffer solution and cryoprotectant in sterile environment until cell concentration is not less than 106And (5) performing vacuum freeze drying treatment when cfu/mL is required to obtain the leavening agent.
In one embodiment of the present invention, the cell concentration is not less than 107And (5) performing vacuum freeze drying treatment when cfu/mL is required to obtain the leavening agent.
In one embodiment of the invention, the buffer solution is physiological saline or 0.1-1M phosphate buffer solution with pH value of 5-8, and the protective agent is sucrose solution with concentration of 10-25% (w/v) and/or skim milk powder with concentration of 5-20% (w/v).
In one embodiment of the invention, the buffer solution is double distilled water and/or normal saline or 0.2M phosphate buffer solution with pH value of 7, and the cryoprotectant is 15-20% (w/v) sucrose solution and/or 10-15% (w/v) skim milk powder.
The invention provides a method for preparing soybean milk by using a leavening agent, which comprises the steps of adding 1-10% of the leavening agent into soybean milk, fermenting at 37 ℃ for 6-8 hours, standing overnight at 3-5 ℃, and fermenting for 24-48 hours to obtain the soybean milk.
In one embodiment of the invention, the method is to add the leavening agent into the soybean milk in an addition amount of 2-8%.
In one embodiment of the present invention, the method for preparing soybean milk comprises: weighing 50-200 g of dry soybeans, adding water for overnight soaking, boiling the soaked soybeans in boiling water, continuously boiling for 1-5 min, adding water according to the mass ratio of the water to the dry soybeans of 1: 3-8, grinding the soybeans in water at 50-70 ℃ for 5-10 min, continuously boiling for 3-10 min, cooling, filtering with 100-150 meshes of gauze, sterilizing at 90-120 ℃ for 5-15 min, and cooling to 15-35 ℃.
The invention provides an application of lactococcus lactis subsp lactis CCFM1092 in preparation of sour soybean milk and fermented soybean milk beverages.
The invention provides application of the leavening agent in preparing soybean milk and fermented soybean milk beverage.
The invention has the beneficial effects that:
1. the method is safe and healthy: the lactococcus lactis subspecies lactis CCFM1092 applied in the invention is a safe strain which can be used for food;
2. the lactococcus lactis subsp lactis CCFM1092 of the invention can produce acid quickly, the pH can be reduced to 4.64 after 6 hours, the fermentation end point is reached, the sucrose in the soybean milk can be well utilized, and the utilization rate of the sucrose is 65%;
3. the lactococcus lactis subspecies lactis CCFM1092 can effectively improve the content of 3-methyl butanol in the fermented soybean milk to 476.05 mug/kg, can effectively reduce the content of a main beany flavor substance 2-pentylfuran in the soybean milk, is not detected after the 2-pentylfuran is fermented, and is beneficial to improving the quality and the characteristic of the soybean milk.
Biological material preservation
Lactococcus lactis subsp. lactis provided by the invention is preserved in Guangdong province microorganism strain preservation center in 2019, 10 and 16 months, wherein the preservation number is GDMCC No. 60807, and the preservation address is Michelia Tokyo No. 59 building 5 of Dazhou No. 100, Michelia Tokyo, Guangzhou.
Drawings
FIG. 1 is a photograph of a colony of lactococcus lactis subspecies lactis CCFM1092 on MRS solid medium.
FIG. 2 is a photograph of a gram-stained strain of lactococcus lactis subsp.lactis CCFM1092 taken under a microscope at 1000X.
FIG. 3 shows the growth of lactococcus lactis subsp.
FIG. 4 shows the change of pH during the fermentation of soybean milk by lactococcus lactis subsp.
FIG. 5 shows the sucrose content of lactococcus lactis subsp.
Detailed Description
MRS solid medium: 10.0g/L tryptone, 10.0g/L beef extract, 5.0g/L yeast extract powder, 2.0g/L diammonium hydrogen citrate, 20.0g/L glucose, 801 mL/L Tween, 2.0g/L anhydrous sodium acetate, 0.5g/L magnesium sulfate heptahydrate, 0.25g/L manganese sulfate monohydrate, and 2.6g/L dipotassium hydrogen phosphate trihydrate, wherein after distilled water is added and completely dissolved, purified agar powder is added according to the amount of 1.5% (m/v), and the mixture is sterilized at 115 ℃ for 20 min.
MRS liquid medium: 10.0g/L tryptone, 10.0g/L beef extract, 5.0g/L yeast extract powder, 2.0g/L diammonium hydrogen citrate, 20.0g/L glucose, 801 mL/L Tween, 2.0g/L anhydrous sodium acetate, 0.5g/L magnesium sulfate heptahydrate, 0.25g/L manganese sulfate monohydrate, and 2.6g/L dipotassium hydrogen phosphate trihydrate, wherein the components are sterilized at 115 ℃ for 20min after being completely dissolved by adding distilled water.
The detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
The pH detection method comprises the following steps: measured with a pH meter.
Detection of sucrose content referring to Hati et al (Hati S, Vij S, Mandal S, et al α -Galactosiaactivity and Oligosaccharides Utilization by Lactobacillus fermentation of sucrose content after fermentation of Soy Milk. weighing 2g of Soy Milk sample, adding 1.5mL of ultrapure water for dilution, incubating at 60 ℃ for 10min, adding 0.25mL of 0.5mol/L potassium ferrocyanide solution, 0.25mL of 0.5mol/L zinc solution and 1.0mL of acetonitrile in sequence, standing at room temperature for 4h, 9000g of 8min, taking supernatant, filtering, analyzing sucrose content by using 0.22 μm of filter, performing qualitative analysis on sucrose content by using HPLC (peak area of light intensity: 70 mm, and selecting a standard sample with a peak area of 20 mm, performing quantitative analysis by using HPLC) and a standard sample volume of 30 mm of ammonia flow rate, and performing quantitative analysis by using a standard liquid phase chromatography (HPLC) to obtain a sample with a peak area of 20 mm of ammonia flow rate and a peak area of a sample volume of 20 mm.
Detection of volatile substance content: the content of volatile substances in soymilk is determined by solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) with reference to the method of Li et al (Li C, Li W, Chen X, et al. microbiological, and microbiological properties of compensated soy milk products with a therebetween polymeric lactic acid bacteria strain [ J ]. LWT-food science and Technology,2014,57(2): 477-485.). A sample of 5g of fermented soymilk was weighed into a 20mL extraction flask, and 1g of sodium chloride and 2. mu.L of 0.1mg/mL methanol solution of 2-methyl-3-heptanone were added in this order. Extracting volatile components from fermented soybean milk by solid phase microextraction method, wherein the extraction probe is CAR/PDMS with a thickness of 85 μm, and is inserted into a sealed extraction bottle, and the extraction probe is exposed in air above the sample, and is extracted at 50 deg.C for 30min and desorbed for 5 min. Gas chromatography conditions and temperature program: selecting Rtx-WAX capillary (30m is multiplied by 0.25mm, 0.25mm), sample inlet temperature is 225 ℃, split ratio is 10, and flow rate of carrier gas (helium) is 1 mL/min. The temperature-raising program sets the initial temperature to 30 ℃, keeps the temperature for 3min, raises the temperature to 225 ℃ at the speed of 15 ℃/min, and keeps the temperature for 5 min. Mass spectrum conditions: the ionization mode EI has the emission energy of 70eV, the emission current of 200 muA, the detector voltage of 1.4kV, the ion source temperature of 240 ℃, the interface temperature of 230 ℃, the quadrupole rod temperature of 150 ℃ and the mass scanning range of m/z of 30-500. And (3) performing substance qualitative determination on the spectrogram obtained by GC-MS by searching in a NIST 2001 standard spectrum library and comparing standard substances, and quantifying by using a peak area normalization method.
Example 1: lactococcus lactis subsp lactis CCFM1092 strain separation and identification method
(1) Preparing appropriate sample dilution gradient and culturing
Taking out the Yunnan Dali milk glycerol sample stored in a refrigerator at-80 deg.C, and thawing on ice. After shaking and mixing evenly, 0.5mL of sample is added into 4.5mL of sterile physiological saline, and the dilution is completed by 10 times to 10 times-1After shaking and mixing evenly, 0.5mL of diluent is taken out from the diluent and added into 4.5mL of sterile physiological saline to finish the second 10-fold dilution to 10-2And the like until the dilution is 10-6And sucking 100 mu L of dilution liquid from each gradient, uniformly coating the dilution liquid on an MRS solid culture medium plate, inverting, placing the plate at 37 ℃ for anaerobic culture for 36-48 h, and observing in time.
(2) Streaking separation and purification
Taking out the plate with the grown bacterial colony, selecting a gradient plate with obvious single bacterial colony, selecting bacterial colonies with different bacterial colony forms, carrying out secondary lineation on an MRS solid culture medium plate, and placing the plate at 37 ℃ for anaerobic culture for 36-48 h until the single bacterial colony is grown; the above streaking procedure was repeated until a single colony grew on each plate.
(3) Gram stain and Catalase assay
Picking a single colony on a glass slide, performing smear, drying, fixing, primary dyeing, washing, mordanting, washing, decoloring, counterdyeing, washing, drying and microscopic examination, and recording a gram staining result; picking a single colony on a glass slide, adding a 3% hydrogen peroxide solution, observing whether bubbles are generated or not, and recording a catalase contact result (both gram staining and catalase contact experimental steps can refer to the master code of microbiology, Yankee Bo and the like, and higher education publishers, 2010); screening out bacteria with gram staining positive and catalase experiment negative; the bacteria which are positive in gram staining and negative in catalase experiment are screened out.
(4) Strain preservation
And selecting a single colony of each strain growing on the plate into 5mL of liquid MRS culture medium, placing the culture medium in an anaerobic condition at 37 ℃ for standing culture for 20-24 h, sucking 1mL of bacterial liquid into a bacteria preservation tube, centrifuging at 6000rpm for 3min, pouring out supernatant, adding 1mL of 30% (v/v) sterile glycerol solution, carrying out heavy suspension, and placing the mixture at-80 ℃ for preservation.
(5)16S rDNA sequence amplification
Sucking 1mL of bacterial liquid at 6000rpm, centrifuging for 3min, pouring out supernatant, washing twice, centrifuging and pouring out supernatant to obtain bacterial sludge, taking the bacterial sludge as a template to perform PCR amplification, wherein the process is as follows:
① amplification System 20. mu.L:
wherein the template amount is 1 μ L, the bidirectional primers are 1 μ L each, the Taq enzyme MasterMix is 10 μ L, ddH2O is 7. mu.L.
The primers used were 27F: AGAGTT TGATCC TGG CCT CA, respectively;
1492R：GGT TAC CTT GTT ACG ACT T。
② amplification conditions:
pre-denaturation: 3min at 95 ℃; first-step denaturation: 1min at 94 ℃; and a second step of annealing: 30s at 60 ℃; and a third step of extension: 2min at 72 ℃; the first to third steps were cycled for 30 times; the fourth step is finally extended: 5min at 72 ℃; the fifth step is that: 10min at 12 ℃.
(6) Agarose gel electrophoresis
Weighing 80mL of agarose, adding the agarose into a conical flask, adding 80mL of 1xTAE, heating for 4min in a microwave discontinuous manner until the liquid is clear and transparent, slightly cooling, adding 4 muL of nucleic acid dye, shaking up without bubbles, pouring the mixture into a gel plate groove, cooling for 40min, solidifying, placing the mixture into an electrophoresis groove, exhausting bubbles, sequentially adding PCR amplification products, adding 2 muL of PCR amplification products into each hole, running gel for 15min at 120V, taking out the mixture after the completion, placing the mixture into a gel electrophoresis imager for photographing and storing, recording the serial number of a PCR successful sample, and placing the successful PCR product into a 4 ℃ refrigerator for storage.
(7) Sequencing and identification
And (3) sending the sample with the successful PCR to an England Weiji (Shanghai) trade company Limited for detection, performing BLAST retrieval in a sequence database (http:// www.ncbi.nlm.nih.gov/BLAST) of the National Center for Biotechnology Information (NCBI) according to the fed-back sequence result, and selecting the strain information with the highest matching degree for result recording. The strain provided by the invention is Lactococcus lactis subsp.
(8) Strain preservation
And (4) preserving the strain in the step (7) in Guangdong province microbial strain preservation center in 2019, 10 and 16 days.
Example 2: growth characteristics of lactococcus lactis subsp. lactis CCFM1092 in soybean milk
Inoculating lactococcus lactis subspecies CCFM1092 stored at-80 ℃ in a liquid MRS culture medium, culturing at 30 ℃ for 24 hours, inoculating the lactococcus lactis subspecies CCFM1092 in an inoculation amount of 2% (v/v) in a fresh liquid MRS culture medium, carrying out subculture, and carrying out subculture for 2-3 times until the subculture reaches 10%8～109cfu/mL. Taking out the activated bacteria liquid in MRS and inoculating the bacteria liquid in soybean milk according to the volume ratio of 2-4 percent to ensure that the number of the living bacteria in the system reaches 106～107cfu/mL. Placing the inoculated sample into an incubator at 37 DEG CIn the middle fermentation, samples were taken every 3h, and the change in viable count during the fermentation was detected, with the results shown in FIG. 3. As can be seen from FIG. 3, the increase of viable count of the strain CCFM1092 after fermentation for 6 hours reaches 1.94 orders of magnitude.
Example 3 acid production Properties of lactococcus lactis subsp lactis CCFM1092 in Soybean milk
Inoculating lactococcus lactis subspecies CCFM1092 preserved at-80 ℃ in an MRS culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times to 108～109cfu/mL. Taking out the activated bacteria liquid in MRS and inoculating the bacteria liquid in the soybean milk according to the volume ratio of 2-4 percent to ensure that the number of the living bacteria in the system reaches 107cfu/g. And (3) fermenting the inoculated sample in an incubator at 37 ℃, sampling every 3h, detecting the change of pH in the fermentation process, wherein the experimental result is shown in figure 4, the pH of the strain CCFM1092 is reduced to 4.64 after fermenting for 6h, and the fermentation end point is reached (the fermentation end point is reached when the soybean milk is fermented until the pH is 4.5-4.7), so that the strain CCFM1092 has a faster acid production rate.
Example 4: application of lactococcus lactis subsp lactis CCFM1092 in soybean milk fermentation
The preparation method of the soybean milk comprises the following steps: weighing 100g of dry soybean, adding 1L of deionized water, soaking overnight, boiling the soaked soybean in boiling water, decocting for 2min, adding 600mL of deionized water, grinding at 60 deg.C for 6min, decocting for 5min, cooling, filtering with 120 mesh gauze, sterilizing at 105 deg.C for 10min, and cooling to room temperature.
Inoculating lactococcus lactis subspecies CCFM1092 preserved at-80 ℃ in MRS liquid culture medium, culturing at 30 ℃ for 24h, subculturing for 2-3 times to 108～109cfu/mL. Taking out the activated bacteria liquid in MRS and inoculating the bacteria liquid in soybean milk according to the volume ratio of 2-4 percent to ensure that the number of the living bacteria in the system reaches 106～107cfu/mL, the inoculated sample was placed in an incubator at 37 ℃ for fermentation.
(1) The sugar content of the fermented soybean milk was measured as shown in fig. 5. As can be seen from FIG. 5, the sucrose content in the soybean milk before fermentation is 2.70g/L, the sucrose content in the lactococcus lactis subsp. lactis CCFM1092 is lower and is 0.93g/L after the fermentation end point (6h) is reached, and the sucrose is the main carbohydrate in the soybean milk, so the screened lactococcus lactis CCFM1092 has the potential of fermentation in the soybean milk.
(2) The volatile content after fermentation was determined as shown in table 1. As is clear from Table 1, after fermentation for 6 hours, lactococcus lactis CCFM1092 had no beany flavor substance, namely 2-pentylfuran, in the yogurt, the content of 3-methylbutanol was 476.05. mu.g/kg, and 3-methylbutanol was an important flavor substance, so that the screened lactococcus lactis CCFM1092 had excellent flavor-producing characteristics. In addition, after the lactococcus lactis CCFM1092 is fermented for 6 hours, the 2, 3-butanedione and acetoin contents in the sour soybean milk are 34.89 mu g/kg and 13.93 mu g/kg respectively, and the contents are main substances with milk fragrance and contribute to improving the flavor of the fermented soybean milk; and the contents of acetic acid, caproic acid and caprylic acid are respectively 78.18 mug/kg, 15.31 mug/kg and 4.40 mug/kg, which is beneficial to improving the quality of the fermented soybean milk.
TABLE 1 content of volatile substances in soymilk
Comparative example 1
The specific implementation manner is the same as that in example 2, except that the CCFM1092 is replaced by the selected lactococcus lactis subspecies lactis DQHXNQ05M30 from the Triton sample in Qinghai Xining area of China, and the increment of the viable count after the fermentation of the lactococcus lactis subspecies lactis CCFM1092 is 1.30 orders of magnitude.
Comparative example 2
The specific implementation manner is the same as that in example 3, except that CCFM1092 is replaced by lactococcus lactis subspecies lactis DQHXNQ05M30 which is screened from a sample of Trira in Qinghai Xining area of China, the pH is only reduced to 6.14 after fermentation for 6h, and the pH is only reduced to 6.18 after fermentation for 12h, and the fermentation end point is not reached yet.
Comparative example 3
The specific implementation manner is the same as example 4, except that CCFM1092 is replaced by lactococcus lactis subspecies lactis DQHXNQ05M30 which is screened from Triton samples in Qinghai Xining area of China.
(1) And (4) measuring the sugar content in the fermented soybean milk, wherein the sucrose content is 1.76g/L after fermentation for 6 hours, and the sucrose content is 1.72g/L after fermentation for 12 hours.
(2) The volatile content after fermentation was determined as shown in table 1.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Lactococcus lactis subsp. lactis has been deposited in Guangdong province collection of microorganisms in 2019, 10.16.D., with the deposit number GDMCC No. 60807.
2. A starter culture comprising the lactococcus lactis subsp.
3. The method for preparing the leaven according to claim 2, wherein the method comprises: activating and culturing lactococcus lactis subspecies lactis of claim 1 to obtain a bacterial liquid, cleaning the bacterial liquid by using a buffer solution, adding a freeze-drying protective agent into the cleaned bacterial liquid to obtain a resuspension solution, adjusting the concentration of viable bacteria in the resuspension solution to a certain concentration, and performing vacuum freeze-drying on the resuspension solution to obtain a leavening agent.
4. A method according to claim 3, wherein the adjusted resuspension is at a viable bacteria concentration of no less than 106cfu/mL。
5. The method according to claim 3, wherein the buffer solution is double distilled water, physiological saline, phosphate buffer solution or a mixture of at least two solutions, and the lyoprotectant is sucrose solution and/or skim milk powder.
6. A method for preparing soybean milk containing 3-methylbutanol, characterized in that the lactococcus lactis subsp.
7. The method according to claim 6, wherein the fermentation is performed for 6-8 h at 35-40 ℃, and then the mature fermentation is performed for 24-48 h at 2-6 ℃ after the fermentation is finished.
8. The method of claim 6, wherein the lactococcus lactis subsp5cfu/mL。
9. The method according to claim 6, wherein the addition amount of the leavening agent in the soybean milk is 1-20% of the soybean milk.
10. Use of lactococcus lactis subsp lactis according to claim 1, or a starter according to claim 2, or a method according to any one of claims 6 to 9 for the preparation of soy milk or a fermented soy milk beverage.
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