CN109370948B - Lactococcus lactis capable of highly producing 6-phosphate- β -galactosidase and application thereof - Google Patents
Lactococcus lactis capable of highly producing 6-phosphate- β -galactosidase and application thereof Download PDFInfo
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- CN109370948B CN109370948B CN201811414217.1A CN201811414217A CN109370948B CN 109370948 B CN109370948 B CN 109370948B CN 201811414217 A CN201811414217 A CN 201811414217A CN 109370948 B CN109370948 B CN 109370948B
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Classifications
-
- 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
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
-
- 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
-
- 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
- A23V2400/157—Lactis
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Abstract
The lactococcus lactis (L galactococcus lactis) CCFM1033 has the advantages of high growth rate, high 6-phosphate- β -galactosidase yield and good aroma production characteristic, can effectively reduce the lactose content in a fermented dairy product, is beneficial to improving the quality of the fermented dairy product, and has very important application in the preparation of the fermented dairy product.
Description
Technical Field
The invention relates to lactococcus lactis capable of highly producing 6-phosphate- β -galactosidase and application thereof, and belongs to the technical field of microorganisms.
Background
The fermented milk product is an acidic milk product prepared by using cow milk as a main raw material and fermenting by using lactic acid bacteria or fermenting by using lactic acid bacteria and yeast together, is deeply favored by people by virtue of unique taste and flavor, and is already sold in the market for many years. Research shows that the main factors influencing consumers to purchase fermented milk products are flavor, price, availability and brand, wherein the flavor accounts for the heaviest part, and therefore, the improvement of the flavor of the fermented milk products is undoubtedly the most important means for enterprises to meet the market.
Lactose is the most important carbohydrate in cow's milk, and accounts for more than 99% of the total carbohydrate in cow's milk, and lactose is primarily metabolized to generate glucose-6-phosphate and glyceraldehyde-3-phosphate, glucose-6-phosphate and glyceraldehyde-3-phosphate are further metabolized to generate pyruvic acid through glycolysis, and pyruvic acid is finally metabolized to generate important flavor substances in fermented dairy products such as lactic acid, acetaldehyde, acetic acid, diacetyl and acetoin, therefore, the lactose metabolism characteristic of lactic acid bacteria is important for preparing fermented dairy products.
Two lactose metabolic pathways exist mainly in lactic acid bacteria, and key enzymes of the two metabolic pathways are β -galactosidase and 6-phospho- β -galactosidase, respectively, wherein β -galactosidase is ubiquitous in lactic acid bacteria, while 6-phospho- β -galactosidase has achieved biochemical characterization in only a few lactic acid bacteria (mainly including lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactococcus lactis subsp.
Therefore, finding a lactic acid bacterium with high 6-phosphate- β -galactosidase yield, which is expected to be applied to dairy fermentation as a leaven to reduce the lactose content in the fermented dairy product and improve the quality of the fermented dairy product, is a technical problem to be solved in the art.
Disclosure of Invention
In order to solve the problems, the invention provides a lactococcus lactis (L actococcus lactis) CCFM1033, and the lactococcus lactis (L actococcus lactis) CCFM1033 has a fast growth rate (the strain is added with 1 × 107The inoculation amount of CFU/m L is inoculated into cow milk and fermented for 2h, and the viable count of lactococcus lactis in the fermented milk can reach 8.37 × 108CFU/m L), 6-phosphate- β -galactosidase yield (the strain is inoculated into MRS liquid medium at an inoculum size of 2 percent and cultured to OD600The enzyme activity of the 6-phosphate- β -galactosidase can reach 96.9U/L when reaching 1.736, and the aroma-producing property is good (the strain is expressed by 1 × 10)7The inoculation amount of the acetolactate is inoculated into cow milk to be fermented for 12 hours, so that 10.2 percent of lactose in the cow milk can be metabolized, meanwhile, the content of diacetyl and acetoin in the obtained fermented milk respectively reaches 5.83 mu g/kg and 31.44 mu g/kg), and the fermented milk can be effectively reducedThe lactose content in the product is beneficial to improving the quality of the fermented milk product, and the product has important application in the preparation of the fermented milk product.
The technical scheme of the invention is as follows:
the invention provides a lactococcus lactis (L actococcus lactis) CCFM1033, wherein the lactococcus lactis (L actococcus lactis) CCFM1033 is preserved in Guangdong province microbial strain preservation center in 20.09.2018, the preservation number is GDMCC No.60450, and the preservation address is No. 59 building 5 of Michelia Torrens 100, Guangzhou city.
The lactococcus lactis (L actococcus lactis) CCFM1033 is obtained by separating from traditional fermented dairy product Trira in Qinghai Xining area of China, the 16S rRNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence is compared in GenBank, and the result shows that the strain is lactococcus lactis and is named as lactococcus lactis (L actococcus lactis) CCFM 1033.
Cells of the lactococcus lactis (L actinococcus lactis) CCFM1033 are spherical, the gram staining result is purple, the gram staining result is gram-positive bacteria, and bacterial colonies of the lactococcus lactis grow on an MRS solid culture medium and are milky white, glossy, smooth in edges, convex and medium in size.
The present invention provides a fermentation agent comprising lactococcus lactis (L actinococcus lactis) CCFM 1033.
In one embodiment of the invention, the preparation method of the leaven is that the lactococcus lactis (L actococcus lactis) CCFM1033 is inoculated into a culture medium and cultured at 30 ℃ until the viable bacteria concentration of the lactococcus lactis (L actococcus lactis) CCFM1033 is not lower than 1 × 108CFU/m L to obtain culture solution, centrifuging the culture solution to obtain thallus, washing the thallus with buffer solution for 2-3 times, and re-suspending with lyophilized protectant until the concentration of thallus is not less than 1 × 109CFU/m L to obtain bacterial suspension, and drying the suspension to obtain the leaven.
In one embodiment of the invention, the medium is MRS medium or M17 medium.
In one embodiment of the present invention, the buffer is double distilled water, physiological saline or phosphate buffer.
In one embodiment of the present invention, the lyoprotectant is sucrose, trehalose, or skim milk powder.
In one embodiment of the invention, the drying is vacuum freeze drying.
The invention provides the application of the lactococcus lactis (L actococcus lactis) CCFM1033 or the leavening agent in preparing food.
In one embodiment of the present invention, the food product is a fermented milk product produced using the lactococcus lactis (L actococcus) CCFM1033 or the fermentation product.
In one embodiment of the invention, the fermented dairy product comprises fermented milk, fermented milk beverage, cheese or marburg.
The invention provides a preparation method of fermented milk, which uses the lactococcus lactis (L actococcus lactis) CCFM1033 or the leavening agent.
In one embodiment of the invention, the method comprises the steps of homogenizing milk, pasteurizing and cooling to obtain a fermentation raw material, and inoculating the lactococcus lactis (L actinococcus lactis) CCFM1033 or the leavening agent into the fermentation raw material to ferment to obtain the fermented milk.
In one embodiment of the invention, the milk comprises raw milk, reconstituted milk or skim milk.
The skim milk is obtained by removing fat in cow milk; the raw milk refers to raw milk extruded from the cow breast without any treatment; the reconstituted milk is prepared by concentrating and drying milk to obtain concentrated milk or milk powder, and adding appropriate amount of water to obtain emulsion with water and solid content ratio equivalent to that of original milk.
In one embodiment of the invention, the method comprises the steps of homogenizing milk under the conditions of pressure of 14MPa to 21MPa and temperature of 40 ℃ to 85 ℃, carrying out pasteurization to obtain sterilized milk, cooling the sterilized milk to 21 ℃ to 30 ℃ to obtain a fermentation raw material, inoculating the lactococcus lactis (L actococcus lactis) CCFM1033 or the leavening agent into the fermentation raw material, fermenting for 12 hours at 37 ℃ to obtain fermented milk, and standing the fermented milk at 4 ℃ for 24 hours to 48 hours to obtain a finished fermented milk product after post-ripening.
In one embodiment of the invention, the inoculation amount of the lactococcus lactis (L actococcus lactis) CCFM1033 or the leavening agent in the fermentation raw material is that the viable count of the lactococcus lactis (L actococcus lactis) CCFM1033 in the fermentation raw material is 1 × 106～1×107CFU/mL。
The invention provides fermented milk prepared by the preparation method.
Has the advantages that:
(1) the lactococcus lactis (L actinococcus lactis) CCFM1033 has the advantages of high growth rate, high 6-phospho- β -galactosidase yield and good aroma production characteristic, can effectively reduce the content of lactose in a fermented dairy product, is beneficial to improving the quality of the fermented dairy product, and has important application in the preparation of the fermented dairy product;
(2) lactococcus lactis (L actinococcus lactis) CCFM1033 of the present invention was added at 1 × 107The inoculation amount of CFU/m L is inoculated into cow milk and fermented for 2h, and the viable count of lactococcus lactis in the fermented milk can reach 8.37 × 108CFU/mL；
(3) The lactococcus lactis (L actinococcus lactis) CCFM1033 of the invention is inoculated into MRS liquid culture medium at an inoculum size of 2% (v/v) and cultured to OD6001.736, the enzyme activity of 6-phosphoric acid- β -galactosidase in the culture solution reaches 96.9U/L;
(4) lactococcus lactis (L actinococcus lactis) CCFM1033 of the present invention was added at 1 × 107The inoculation amount of the acetogenin is inoculated into cow milk and fermented for 12 hours, so that 10.2 percent of lactose in the cow milk can be metabolized, and meanwhile, the content of diacetyl and acetoin in the obtained fermented milk respectively reaches 5.83 mu g/kg and 31.44 mu g/kg;
(5) the fermented milk prepared by the lactococcus lactis (L actococcus lactis) CCFM1033 has high content of acetaldehyde, diacetyl, acetoin and other volatile flavor substances, low content of lactose and better quality.
Biological material preservation
A lactococcus lactis (L actococcus lactis) CCFM1033 is classified and named as L actococcus lactis, is preserved in Guangdong province microorganism strain preservation center in 20.09.2018, and has the preservation number of GDMCCNo.60450 and the preservation address of No. 59 building 5 of Mirabilitum Torreyao No. 100.
Drawings
FIG. 1: gram staining characteristics of the strains of the invention;
FIG. 2: bacterial colony characteristics of the strain of the invention;
FIG. 3: growth conditions of the strains of the invention in skim milk;
FIG. 4: the pH change of the bacterial strain fermented in skim milk;
FIG. 5: the acidity change condition of the bacterial strain fermented in skim milk;
FIG. 6 shows the 6-phospho- β -galactosidase production in MRS medium by the strain of the present invention;
FIG. 7: the strain of the invention has the change condition of lactose fermented in skim milk.
Detailed Description
The invention is further illustrated with reference to specific examples.
The skim milk referred to in the examples below was purchased from Guangming Dairy products GmbH.
The media involved in the following examples are as follows:
MRS solid culture medium, tryptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L0, dihydrogendiamine citrate 2.0 g/L, glucose 20.0 g/L, Tween 801 m L/L, anhydrous sodium acetate 2.0 g/L, magnesium sulfate heptahydrate 0.5 g/L, manganese sulfate monohydrate 0.25 g/L, dipotassium hydrogen phosphate trihydrate 2.6 g/L, adding distilled water to completely dissolve, adding purified agar powder according to the amount of 1.5% (m/v), and sterilizing at 115 ℃ for 20 min.
MRS liquid culture medium comprises tryptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L0, dihydrogendiamine citrate 2.0 g/L, glucose 20.0 g/L, Tween 801 m L/L, anhydrous sodium acetate 2.0 g/L, magnesium sulfate heptahydrate 0.5 g/L, manganese sulfate monohydrate 0.25 g/L and dipotassium hydrogen phosphate trihydrate 2.6 g/L, and is sterilized at 115 ℃ for 20min after being completely dissolved by adding distilled water.
The detection methods referred to in the following examples are as follows:
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.
And (3) an acidity detection method: the national standard GB 431334-.
The method for detecting the enzyme activity of 6-phosphate- β -galactosidase comprises the steps of sucking 100 mu L log-phase bacterial liquid, adding 900 mu L Zbuffer phosphate buffer solution, adding 10 mu L trichloromethane, carrying out vortex oscillation for 10s, adding 200 mu L6-phosphoric acid-ONPG substrate solution, placing in a metal bath at 30 ℃ for constant-temperature reaction, starting timing, and detecting the OD420When the concentration reaches 0.6-0.9, 500 mu L1 mol/L mol of Na is added2CO3The reaction was terminated in a solution, and OD after the termination reaction was measured420Recording the reaction time, wherein the longest reaction time is 90 min;
expressed as enzyme activity (1000 × OD)420)/[OD600×VBacterial liquid(m L) × reaction time (min)]The activity of 6-phospho- β -galactosidase was calculated.
And (4) detecting the content of volatile substances: measuring the content of volatile substances in the fermented yogurt by GC-MS;
selecting Rtx-WAX capillary (30m × 0.25.25 mm, 0.25mm), sample inlet temperature 225 deg.C, split ratio 10, carrier gas (helium) flow rate 1m L/min, temperature-raising program setting initial temperature 30 deg.C, maintaining for 3min, raising temperature to 225 deg.C at 15 deg.C/min, maintaining for 5 min;
mass spectrum conditions: the ionization mode EI is characterized in that the emission energy is 70eV, the emission current is 200 muA, the voltage of a detector is 1.4kV, the temperature of an ion source is 240 ℃, the interface temperature is 230 ℃, the temperature of a quadrupole is 150 ℃, and the mass scanning range m/z is 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.
And (3) lactose content detection, namely weighing 1g (accurate to 0.001g) of fermented milk sample in a 10m L volumetric flask, adding 70% (v/v) ethanol to a constant volume to scale, uniformly mixing, extracting at 4 ℃ for 24h, centrifuging at 11000g and 4 ℃ for 10min, taking supernatant for derivatization, filtering by using a microporous filter membrane, and determining by using high performance liquid chromatography (quantitative analysis of lactose content by an external standard method).
Example 1: screening and identification of strains
1. Screening
(1) Preparing appropriate sample dilution gradient and culturing
Taking out a traganterol sample of a traditional fermented dairy product of Qinghai-Xining stored in a refrigerator at the temperature of-80 ℃, unfreezing the traganterol sample on ice, adding a 0.5m L sample into 4.5m L sterile physiological saline after shaking and uniformly mixing to finish primary 10-time dilution, taking out 0.5m L diluent from the diluent after shaking and uniformly mixing to add into 4.5m L sterile physiological saline to finish secondary 10-time dilution, and so on until the traganterol sample is diluted to 10-6And sucking 100 mu L from each gradient diluent, uniformly coating the solution 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
And after taking out the plate with the grown bacterial colony, selecting a gradient plate with an obvious single bacterial colony, selecting bacterial colonies with different bacterial colony morphologies, and carrying out secondary scribing until all the single bacterial colonies are purified.
(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; and picking a single colony on a glass slide, adding a 3% hydrogen peroxide solution, observing the generation of bubbles, and recording the contact result of catalase so as to identify whether the selected strain has the characteristics of lactic acid bacteria.
(4) Strain preservation
And (3) picking a single colony of each purified strain into a 5m L MRS liquid culture medium, placing the culture medium in an anaerobic static culture at 37 ℃ for 20-24 h, sucking 1m L bacterial liquid into a bacteria preservation tube, centrifuging at 6000rpm for 3min, pouring out a supernatant, adding 1m L30% of sterile glycerol solution, re-suspending, and placing the mixture in a storage at-80 ℃.
2. Identification
(1)16S rDNA sequence amplification
Sucking 1m L of the bacterial liquid, centrifuging at 6000rpm for 3min, pouring off supernatant, washing twice, centrifuging and pouring off supernatant to obtain bacterial sludge, and performing PCR amplification by taking the bacterial sludge as a template, wherein the flow is as follows:
1) amplification system 20 μ L:
wherein the template amount is 1 μ L (27F 0.5 μ L, 1492R 0.5 μ L), the Taq enzyme MasterMix is 10 μ L, ddH2O is 7. mu. L, and the primers used were 27F: AGA GTT TGA TCC TGG CCT CA (SEQ ID No 2) and 1492R: GGT TAC CTTGTT ACG ACT T (SEQ ID No 3).
2) Amplification conditions:
pre-denaturation: 95 ℃ for 3min
First-step denaturation: 94 ℃ for 1min
And a second step of annealing: 30s at 60 DEG C
And a third step of extension: 72 ℃ for 2min
Cycle number: 30 cycles of
The fourth step is finally extended: 5min at 72 DEG C
The fifth step is that: 10min at 12 DEG C
(2) Agarose gel electrophoresis
Weighing 80m L agarose, adding into a conical flask, adding 80m L1 xTAE, heating intermittently with microwave for 4min until the liquid is clear and transparent, slightly cooling, adding 4 mu L nucleic acid dye, shaking up, pouring into a gel plate groove without bubbles, cooling for 40min, solidifying, placing into an electrophoresis groove, discharging bubbles, sequentially adding PCR amplification products, adding 2 mu L PCR amplification products into each hole, taking out after 120V 15min gel running, placing into a gel electrophoresis imager, photographing for storage, recording the serial number of samples with successful PCR, and placing the PCR products in a refrigerator at-20 ℃.
(3) Strain sequence detection and identification
And (2) sending a sample which is successfully subjected to PCR to Huada gene for detection, carrying out B L AST retrieval by combining an NCBI strain sequence database (http:// www.ncbi.nlm.nih.gov/blast) according to a sequence result fed back by the Huada gene, selecting strain information with the highest matching degree for result recording, wherein the two strains which are successfully subjected to PCR are lactococcus lactis which are named as lactococcus lactis (L actococcus lactis) CCFM1033 and lactococcus lactis (L actococcus lactis) D-XJ 4-12 respectively.
Example 2: cultivation of the Strain
Inoculating lactococcus lactis (L actococcus lactis) CCFM1033 to MRS solid culture medium, placing the MRS solid culture medium at 30 ℃ for inverted culture for 48h, observing colony morphology, picking single colony on a glass slide, performing smear, drying, fixing, primary dyeing, water washing, mordant dyeing, water washing, decoloring, counterdyeing, water washing, drying and microscopic examination, and recording morphology and gram dyeing results.
Lactococcus lactis CCFM1033 observed under an oil lens is spherical, and the gram staining result is purple, namely gram positive bacteria (shown in figure 1); moreover, the colony of the strain on the solid MRS medium is white, glossy, smooth in edge, opaque, slightly convex and medium in size (see figure 2).
Example 3: growth characteristics of strains in milk systems
1. Growth curve of lactococcus lactis in skim milk for 12h
Respectively inoculating lactococcus lactis (L actococcus lactis) CCFM1033 and lactococcus lactis (L actococcus lactis) D-XJ 4-12 stored at-80 ℃ into an MRS liquid culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times until the bacterial concentration reaches 108～109CFU/m L, taking out the bacteria liquid activated in MRS, inoculating the bacteria liquid into skim milk according to the volume ratio of 2-4 percent, and leading the bacteria amount in the system to reach 107CFU/g; the inoculated sample is put into an incubator at 37 ℃ for fermentation, samples are taken every 2 hours, and the change of the bacterial load in the fermentation process is detected, and the result is shown in figure 3.
As can be seen from FIG. 3, the bacterial load of lactococcus lactis (L actinococcus lactis) CCFM1033 reached 8.37 × 10 after 2 hours of fermentation8CFU/m L, lactococcus lactis (L actococcus lactis) D-XJ 4-12, the bacterial load reached 7.95 × 10 after 2h of fermentation8cfu/m L, therefore, lactococcus lactis (L actococcus lactis) CCFM1033 grows faster in skim milk.
2. pH and titrated acidity changes of lactococcus lactis in skim milk for 12h
Lactococcus lactis (L actococcus) preserved at-80 deg.Clactis) CCFM1033 and lactococcus lactis (L actococcus lactis) D-XJ 4-12 are respectively inoculated into MRS liquid culture medium, cultured for 24h at 30 ℃, subcultured for 2-3 times until the bacterial concentration is 108～109CFU/m L, taking out the bacteria liquid activated in MRS, inoculating the bacteria liquid into skim milk according to the volume ratio of 2-4 percent, and leading the bacteria amount in the system to reach 107CFU/g; and (3) fermenting the inoculated sample in an incubator at 37 ℃, sampling every 2h, detecting the change of pH and titrated acidity in the fermentation process, and obtaining an experimental result as shown in figure 4.
As can be seen from FIG. 4, lactococcus lactis (L actococcus lactis) CCFM1033 had pH 5.46 and acidity 45.10 ℃ T after 12 hours of fermentation, and the acid production rate was faster than that of lactococcus lactis (L actococcus lactis) D-XJ 4-12.
Example 4 6-phospho- β -galactosidase production of the Strain
Respectively inoculating lactococcus lactis (L actococcus lactis) CCFM1033 and lactococcus lactis (L actococcus lactis) D-XJ 4-12 stored at-80 ℃ into an MRS liquid culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times until the bacterial concentration is 108～109CFU/m L, inoculating the activated bacteria liquid in MRS into lactose MRS liquid culture medium according to the volume ratio of 2%, culturing to logarithmic phase, and measuring OD600The value is 1.736, 100 mu L of the logarithmic phase bacterial liquid is sucked for detecting the enzyme activity of 6-phosphate- β -galactosidase, and the result is shown in figure 4.
As can be seen from FIG. 4, the 6-phospho- β -galactosidase activity of lactococcus lactis (L actoccus lactis) CCFM1033 was 96.9U, and the 6-phospho- β -galactosidase activity of lactococcus lactis (L actoccus lactis) D-XJ 4-12 was 1.03U, so that the 6-phospho- β -galactosidase production of lactococcus lactis (L actoccus lactis) CCFM1033 was higher.
Example 5: application of strain
Respectively inoculating lactococcus lactis (L actococcus lactis) CCFM1033 and lactococcus lactis (L actococcus lactis) D-XJ 4-12 stored at-80 ℃ into an MRS liquid culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times until the bacterial concentration is 108～109CFU/m L, taking out the activated bacteria liquid in MRS, inoculating the bacteria liquid to the bacteria remover according to the volume ratio of 2-4%In the fat milk, the bacterial load in the system is 107cfu/g; putting the inoculated sample into an incubator at 37 ℃ for fermentation for 12h to obtain fermented milk; the fermented milk was aspirated for volatile matter content and lactose content testing, the volatile matter content is shown in table 1, and the lactose content is shown in fig. 5.
As shown in Table 1, after 12 hours of fermentation, the yields of acetaldehyde, diacetyl and acetoin in the fermented milk were all high and reached 1.07. mu.g/kg, 5.83. mu.g/kg and 31.44. mu.g/kg respectively in lactococcus lactis (L actococcus lactis) CCFM1033, while the yields of D-XJ 4-12 in lactococcus lactis (L actococcus lactis) were 0.29. mu.g/kg, 0.29. mu.g/kg and 0.62. mu.g/kg respectively in lactococcus lactis (L actococcus lactis) CCFM1033 were excellent in flavor-producing characteristics.
As can be seen from FIG. 5, the lactose content in the fermented milk is low, 5873.82mg/100g, after 12h of fermentation of lactococcus lactis (L actoccus lactis) CCFM1033, and 6470.60mg/100g of lactococcus lactis (L actoccus lactis) D-XJ 4-12, so that the lactose content after fermentation of lactococcus lactis (L actoccus lactis) CCFM1033 is significantly lower than that of lactococcus lactis (L actoccus lactis) D-XJ 4-12, and lactococcus lactis (L actoccus lactis) CCFM1033 has strong lactose utilization capacity.
TABLE 1 content of volatile substances after 12h of Slow fermentation
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.
Sequence listing
<110> university of south of the Yangtze river
<120> lactococcus lactis capable of highly producing 6-phosphate- β -galactosidase and application thereof
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tagcactcat cgtttacggc gtggactacc agggtatcta atcctgtttg ctccccacgc 660
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tatatctacg catttcaccg ctacacatgg aattccactc tcctctcctg cactcaagtc 780
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Claims (10)
1. A lactococcus lactis (L actococcus lactis) is characterized in that the lactococcus lactis (L actococcus lactis) is preserved in 20 days 09 and 2018 in Guangdong province of microbial strain preservation center, wherein the preservation number is GDMCC No.60450, and the preservation address is No. 59 building 5 of Michelia Torresiana No. 100 of Guangzhou city.
2. A starter culture comprising lactococcus lactis (L actococcus lactis) according to claim 1.
3. The starter according to claim 2, wherein the starter is prepared by inoculating lactococcus lactis (L actococcus lactis) according to claim 1 into a culture medium, and culturing at 30 ℃ until the viable bacteria concentration of lactococcus lactis (L actococcus lactis) is reachedDegree of not less than 1 × 108CFU/m L to obtain culture solution, centrifuging the culture solution to obtain thallus, washing the thallus with buffer solution for 2-3 times, and re-suspending with lyophilized protectant until the concentration of thallus is not less than 1 × 109CFU/m L to obtain bacterial suspension, and drying the suspension to obtain the leaven.
4. Use of lactococcus lactis (L actococcus lactis) according to claim 1 or of a starter according to claim 2 or 3 for the preparation of a food product.
5. The use according to claim 4, wherein the food product is a fermented dairy product produced using lactococcus lactis (L actococcus lactis) according to claim 1 or a starter according to claim 2 or 3.
6. Use according to claim 4 or 5, wherein the fermented milk product comprises fermented milk, fermented milk drink, cheese or a marburg.
7. A method for producing fermented milk, characterized in that the method comprises using lactococcus lactis (L actococcus lactis) as defined in claim 1 or the fermentation agent as defined in claim 2 or 3.
8. The method according to claim 7, wherein the milk is homogenized, pasteurized, and cooled to obtain a fermented material, and the fermented material is inoculated with lactococcus lactis (L actococcus lactis) according to claim 1 or the starter according to claim 2 or 3 and fermented to obtain the fermented milk.
9. The preparation method of claim 7 or 8, wherein the method comprises homogenizing milk under a pressure of 14MPa to 21MPa and at a temperature of 40 ℃ to 85 ℃, pasteurizing to obtain sterilized milk, cooling the sterilized milk to 21 ℃ to 30 ℃ to obtain a fermentation raw material, inoculating lactococcus lactis (L actococcus lactis) according to claim 1 or the leavening agent according to claim 2 or 3 into the fermentation raw material, fermenting for 12 hours at 37 ℃ to obtain fermented milk, and standing the fermented milk at 4 ℃ for 24 hours to 48 hours to obtain a post-cooked fermented milk product.
10. Fermented milk produced by the production method according to any one of claims 7 to 9.
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