CN109402020B - Lactobacillus helveticus strain capable of highly producing arginine aminopeptidase and application thereof - Google Patents
Lactobacillus helveticus strain capable of highly producing arginine aminopeptidase and application thereof Download PDFInfo
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Abstract
The lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 has the advantages of high growth rate, high acid production speed and high arginine aminopeptidase (Arg-pNA) yield, can effectively enhance the metabolic capability of protein in fermented food, reduce the formation of bitter taste of the fermented food and improve the flavor of the fermented food, and has important application in the preparation of fermented foods such as fermented milk, fermented milk beverages, cheese, mare's wine, fermented mare's milk (Koumiss), sour cream, cookies, vinegar or soy sauce.
Description
Technical Field
The invention relates to lactobacillus helveticus capable of highly producing arginine aminopeptidase and application thereof, belonging to the technical field of microorganisms.
Background
The milk has less content of free peptide and amino acid and higher proteolytic activity, and the lactobacillus helveticus has the characteristic of multi-amino acid auxotrophy, so the lactobacillus helveticus has the capability of accelerating the formation of flavor in the preparation process of fermented foods such as cheese ripening and the like, and occupies an important position in the field of fermented foods.
The proteolytic system of lactobacillus helveticus mainly comprises Cell wall protease (CEP) which is used for hydrolyzing protein into a series of peptide fragments, the peptide fragments obtained by hydrolysis can be transported into cells by an oligopeptide transport system (Opp), a dipeptide and tripeptide transport system (Dpp and DtpT), and are further hydrolyzed into amino acids or smaller peptides by intracellular peptidases (amino acid peptidase, endopeptidase, Pro-specific peptidase and the like), and the nutritional requirements of the cells are met, so that the cells can grow better.
Although the solubility, nutritional properties and taste and flavor of the polypeptides, oligopeptides and small amount of amino acids produced by proteolysis are all improved significantly compared to the proteins themselves, these protein hydrolysates often exhibit bitter taste caused by polypeptides terminated with hydrophobic amino acids (bitter peptides), which undoubtedly greatly limits the application of protein hydrolysates. It is therefore important to reduce, prevent and eliminate the bitter taste of protein hydrolysates of lactobacillus helveticus.
It has been found that, among the intracellular peptidases of lactobacillus helveticus, an amino acid peptidase belonging to a peptide chain terminal hydrolase can hydrolyze amino acids one by one from the N-terminal end of the polypeptide chain, and therefore, the amino acid peptidase is not only important for the growth of lactobacillus helveticus bacterial cells, but also can remove hydrophobic amino acid residues from the N-terminal end to achieve the purpose of debittering, and thus, lactobacillus helveticus which produces a high yield of the amino acid peptidase can certainly reduce the bitterness of protein hydrolysates.
However, the currently isolated aminopeptidases are of a small variety in lactobacillus helveticus strains, and cannot effectively target hydrophobic amino acid residues which affect bitterness such as L eu, Pro, Arg, L ys (particularly Arg, a peptide having Arg at the N-terminus is extremely bitter), and the aminopeptidase-producing lactobacillus helveticus strains are not high in yield, and are insufficient to significantly reduce bitterness of protein hydrolysates.
Therefore, finding a lactobacillus helveticus bacterium with high amino acid peptidase yield as a leaven to be applied to the fermentation of fermented foods such as fermented milk, fermented milk drinks, cheese, mare milk wine, fermented mare milk (Koumiss), sour cream, cookies, vinegar or soy sauce and the like so as to enhance the metabolic capability of protein in the fermented foods, reduce the bitter taste of the fermented foods and improve the flavor of the fermented foods is a technical problem to be solved in the field.
Disclosure of Invention
In order to solve the problems, the invention provides a Lactobacillus helveticus (L Acobacter helveticus) CCFM1036, and the Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 has the advantages of high growth rate (the strain is 1 × 106Inoculating the inoculation amount of CFU/m L into skim milk, and fermenting for 10h to make the viable count of Lactobacillus helveticus in the fermented milk reach 5.5 × 108CFU/m L), quick acid production (the strain is 1 × 106The inoculation amount of CFU/m L is inoculated into skim milk and fermented for 10h, so that the pH value of the fermented milk is 4.23, the acidity is 51.43 DEG T, and the fermentation end point is reached), the yield of arginine aminopeptidase (Arg-pNA) is high (the strain is inoculated into an MRS liquid culture medium for culturing for 16h at the inoculation amount of 2 percent, so that the enzyme activity of the arginine aminopeptidase (Arg-pNA) can reach 93.694nmol/min mg), the metabolic capability of protein in the fermented food can be effectively enhanced, the bitter taste of the fermented food is reduced, the flavor of the fermented food is improved, and the strain has important application in the preparation of fermented foods such as fermented milk, fermented milk beverage, cheese, mare wine, sour mare milk (Koumis), sour cream, cookies, vinegar or soy sauce.
The technical scheme of the invention is as follows:
the invention provides a Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036, wherein the Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 is stored in Guangdong province microbial strain collection center in 20.09.2018, the collection number is GDMCC No.60453, and the collection address is Miyaolu 100 Dazhou No. 59 Lou 5 Lou in Guangzhou city.
The Lactobacillus helveticus (L actabacillus helveticus) CCFM1036 is separated from traditional fermented yak yogurt in the Atha Sichuan, 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 the Lactobacillus helveticus and is named as the Lactobacillus helveticus (L actabacillus helveticus) CCFM 1036.
The bacterial colony of the Lactobacillus helveticus (L Acobacillus helveticus) CCFM1036 is round, medium-sized, white and smooth when growing on an MRS solid culture medium, the Lactobacillus helveticus (L Acobacillus helveticus) CCFM1036 grows in skim milk for 14h, namely enters a stable period, and reaches a fermentation end point after fermenting in the skim milk for 10h, so that the growth and acid production are fast.
The invention provides a leaven containing the lactobacillus helveticus (L actinobacillus helveticus) CCFM 1036.
In one embodiment of the invention, the starter is a direct vat set starter;
the preparation method of the direct vat set starter is to inoculate the Lactobacillus helveticus (L Acobacillus helveticus) CCFM1036 of the claim 1 into a culture medium to culture until the concentration of the viable bacteria of the Lactobacillus helveticus (L Acobacillus helveticus) CCFM1036 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 × 108CFU/m L to obtain bacterial suspension, and drying the suspension to obtain the direct vat set starter.
In one embodiment of the invention, the medium is MRS medium or L BS medium.
In one embodiment of the present invention, the buffer is a phosphate buffer or a borate buffer.
In one embodiment of the invention, the lyoprotectant is glycerol or skim milk.
In one embodiment of the invention, the drying is vacuum freeze drying.
The invention provides application of the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the leavening agent in preparing fermented food.
In one embodiment of the present invention, the fermented food is a fermented dairy product, a fermented pasta or a fermented seasoning produced using the above lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the above leavening agent.
In one embodiment of the invention, the fermented dairy product comprises fermented milk, fermented milk drink, cheese, margarine, fermented mare's milk (Koumiss) or sour cream; the fermented pasta comprises cookies; the fermented seasoning comprises vinegar or soy sauce.
The invention provides a preparation method of fermented food, which uses the Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the leaven.
In one embodiment of the invention, when the fermented food is cheese, the preparation method comprises the steps of homogenizing and pasteurizing milk, cooling to obtain a fermented raw material, inoculating the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the leavening agent into the fermented raw material, fermenting to obtain fermented milk, inoculating the lactobacillus helveticus (L actinobacillus helveticus) CCFM or the leavening agent into the fermented milk again, adding rennin into the fermented milk to curd the fermented milk to obtain rennin blocks, cutting the rennin blocks, stirring to discharge whey, performing curdling, cutting, adding salt, squeezing to obtain cheese, and maturing the cheese to obtain a finished cheese product;
or when the fermented food is fermented mare's milk, the preparation method comprises sterilizing mare's milk, cooling to obtain fermented raw material, inoculating Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the above leaven, and inoculating Saccharomyces cerevisiae to the fermented raw material for fermentation to obtain fermented mare's 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, when the fermented food is cheese, the preparation method comprises the steps of homogenizing milk under the conditions of 14 MPa-21 MPa of pressure and 40-85 ℃ and then carrying out pasteurization to obtain sterilized milk, cooling the sterilized milk to 21-30 ℃ to obtain a fermented raw material, inoculating the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the leavening agent into the fermented raw material, fermenting for 10-12 hours at 37 ℃ to obtain fermented milk, inoculating the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the leavening agent into the fermented milk, simultaneously adding rennin into the fermented milk to carry out curdling to obtain a curd block, cutting the curd block, stirring at 39 ℃ to discharge whey, squeezing the curd block to obtain the acidity of 45-55 DEG T, chopping the milk block, adding salt, and finally carrying out 18 hours at 14 ℃ to obtain cheese, and carrying out aging under the conditions of 85% of humidity to obtain finished cheese;
or when the fermented food is the fermented mare's milk, the preparation method comprises maintaining the mare's milk at the temperature of 90-95 ℃ for 10-15 min for sterilization, cooling to 35 ℃ to obtain a fermented raw material, inoculating the lactobacillus helveticus (L Acobacter helveticus) CCFM1036 or the leavening agent into the fermented raw material, and simultaneously inoculating the saccharomyces cerevisiae into the fermented raw material to ferment for 96h at 37 ℃ until the acidity reaches 70-120 DEG T and the alcoholic strength reaches 1.0-3.0 DEG P to obtain the fermented mare's milk.
In one embodiment of the invention, when the fermented food is cheese, the viable count of lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 or the starter in the fermentation raw material is 106～108The inoculation amount of the CFU/m L, the Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 or the leavening agent in the fermented milk is that the viable count of the Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 in the fermented raw materials is 106～108The additive amount of CFU/m L and rennin in the fermented milk accounts for 0.002% of the total mass of the fermented milk, and the additive amount of salt in the curd block accounts for 3 per mill of the total mass of the curd block;
or when the fermented food is mare's milk, the lactobacillus helveticus (L Acobacter helveticus) CCFM1036 or the inoculum size of the leavening agent in the fermentation raw material is that the viable count of the lactobacillus helveticus (L Acobacter helveticus) CCFM1036 in the fermentation raw material reaches 106～108CFU/m L, brewing wine and fermentingThe inoculation amount of the mother culture in the fermentation raw material is that the viable count of the saccharomyces cerevisiae in the fermentation raw material reaches 106～108CFU/mL。
The invention provides a fermented food prepared by the preparation method.
Has the advantages that:
(1) the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 has the advantages of high growth rate, high acid production speed and high arginine aminopeptidase (Arg-pNA) yield, can effectively enhance the metabolic capability of protein in fermented food, reduce the formation of bitter taste of the fermented food and improve the flavor of the fermented food, and has important application in the preparation of fermented food such as fermented milk, fermented milk beverage, cheese, mare's wine, sour mare's milk (Koumiss), sour cream, cookies, vinegar or soy sauce;
(2) the Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 is mixed with the mixture of 1 × 106Inoculating the inoculation amount of CFU/m L into skim milk, and fermenting for 10h to make the viable count of Lactobacillus helveticus in the fermented milk reach 5.5 × 108CFU/mL；
(3) The Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 is mixed with the mixture of 1 × 106Inoculating the inoculation amount of CFU/m L into skim milk, and fermenting for 10h to make the pH of the fermented milk be 4.23 and the acidity be 51.43 DEG T until the fermentation end point;
(4) the lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 is inoculated into an MRS liquid culture medium for culturing for 16h at the inoculum size of 2 percent, so that the enzyme activity of arginine aminopeptidase (Arg-pNA) can reach 93.694nmol/min mg.
Biological material preservation
A strain of Lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 is taxonomically named as L actinobacillus helveticus, is stored in Guangdong province microorganism strain collection center in 20.09.2018, and has the storage number of GDMCC No.60453 and the storage address of No. 59 Lou 5 of Miehuo 100 of Guangzhou city.
Drawings
FIG. 1: gram staining characteristics of the strains of the invention;
FIG. 2: growth conditions of the strains of the invention in skim milk;
FIG. 3: the pH change of the bacterial strain fermented in skim milk;
FIG. 4: the acidity change condition of the bacterial strain fermented in skim milk;
FIG. 5: the bacterial strain has the enzyme activity of arginine aminopeptidase (Arg-pNA) in an MRS culture medium.
Detailed Description
The invention is further illustrated with reference to specific examples.
Skim milk referred to in the following examples was purchased from Guangming Dairy products GmbH; said Lactobacillus helveticus ATCC15009 was purchased from North Nam Bio Inc.
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 arginine aminopeptidase (Arg-pNA) comprises the following steps:
1. drawing a p-nitroaniline standard curve:
preparing p-nitroaniline solutions with different concentrations (20-160 mu M), and measuring the absorbance at 405nm to obtain a standard curve;
2. preparation of the substrate solution
1mM of chromogenic substrate (H-Arg-pNA) was dissolved in 50mmol L at pH7.5-1In a sodium phosphate buffer solution;
3. enzyme activity detection of arginine aminopeptidase (Arg-pNA)
After incubating 50. mu. L substrate solution and 50. mu. L CFE for 30 minutes at 30 ℃, absorbance was measured at 405nm, no positive control, no commercial enzyme, blank control with buffer instead of CFE;
the calculation formula of the enzyme activity of arginine aminopeptidase (Arg-pNA) is as follows: enzyme activity ═ (p-nitroanilide production 100 × 0.001))/(protein concentration 50 × 0.001 × 30);
(see Stefanovic E, Kilcawley K N, Rea M C, et al genetic, enzymatic and metabolic profiling of the L active bacterium species group regenerative biological activity and biological activity for biological activity [ J ] Journal of Applied Microbiology,2017,122 (5))
Example 1: screening and identification of strains
1. Screening
(1) Preparing appropriate sample dilution gradient and culturing
Weighing 0.5m L Sichuan Alba Tibetan traditional fermented yak yoghourt, adding into 4.5m L sterile water, sequentially diluting 0.5m L bacterial liquid into 4.5m L sterile water, and diluting the sample to 10 concentration gradient-4Taking 4 dilutions of 10-104Respectively coating 50 mu L of the bacterial suspension on an MRS solid culture medium, culturing for 46-48 h at 37 ℃, 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 4000rpm for 5min, pouring out a supernatant, adding 1m L30% of sterile glycerol solution, re-suspending, and placing the mixture at-80 ℃ for preservation.
2. Identification
(1)16S rDNA sequence amplification
Sucking 1m L of the bacterial liquid, centrifuging, pouring and cleaning the supernatant by blowing 1m L of sterile water twice, centrifuging, pouring the supernatant to obtain bacterial sludge, and performing PCR amplification by taking the bacterial sludge as a template, wherein the process comprises the following steps:
1) amplification system 50 μ L:
wherein Mix is 25 mu L, 27F (SEQ ID No 2: AGA GTT TGA TCC TGG CCT CA)1 mu L, 1492R (SEQ ID No 3: GGT TAC CTT GTT ACG ACT T)1 mu L, ddH2O23 mu L, and the length of the amplified fragment is 1500 bp.
2) Amplification conditions:
Lid：105℃、MBY-16s、V：20μL；
cycle number: 29 cycles
The DNA double strand was maintained at 94 ℃ for 10min, 94 ℃ for 30s, 50 ℃ for 30s, and 72 ℃ for 80s, and after 29 cycles, the DNA double strand was maintained at 72 ℃ for 7 min.
(2) Agarose gel electrophoresis
Weighing 0.8g of agarose, adding the agarose into a conical flask, adding 80m L1 xTAE, heating intermittently by microwaves for 4min until the liquid is clear and transparent, slightly cooling, adding 8 mu L EB dye, adding an electrophoresis plate, cooling for half an hour until the electrophoresis plate is condensed into solid colloid, driving a sample of 3-5 mu L into a small hole of the gel plate by using a liquid transfer gun, adding a Marker at the end of each row, inserting electrodes, regulating the voltage to 120V, running for half an hour, taking out the gel plate, exposing the gel plate for 10s under UV (ultraviolet), storing images of electrophoresis strips, and sequencing the sample with clear electrophoresis strips.
(3)16S rRNA sequence analysis and identification
According to the sequence result fed back by the Huada gene, B L AST retrieval is carried out by combining an NCBI strain sequence database (http:// www.ncbi.nlm.nih.gov/blast), the strain information with the highest matching degree is selected for result recording, and the two strains successful in PCR are both lactobacillus helveticus and are named as lactobacillus helveticus (L actabacillus helveticus) CCFM1036 and lactobacillus helveticus (L actabacillus helveticus) DNM-B1M10 respectively.
Example 2: cultivation of the Strain
Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 was cultured on MRS solid medium for 48 hours, and colonies were observed and picked for microscopic examination, gram color and growth characteristics.
It was observed that the colony of Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 was round, medium-sized, white, smooth when grown on MRS solid medium.
Example 3: growth characteristics of strains in milk systems
1. Growth curve of Lactobacillus helveticus in skim milk for 14h
Respectively inoculating lactobacillus helveticus (L Acobacter helveticus) CCFM1036, lactobacillus helveticus (L Acobacter helveticus) DNM-B1M10 and lactobacillus helveticus (L Acobacter helveticus) ATCC15009 which are preserved at the temperature of-80 ℃ into an MRS liquid culture medium, culturing for 24 hours at the temperature of 37 ℃, and subculturing for 2-3 times until the bacterium 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 106CFU/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 2.
As can be seen from FIG. 3, the bacterial load of Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 reached 5.5 × 10 after 10 hours of fermentation8CFU/M L, Lactobacillus helveticus (L actinobacillus helveticus) DNM-B1M10 and Lactobacillus helveticus (L actinobacillus helv)eticus) ATCC15009 reached 8.3 × 10 bacterial counts after 10h of fermentation, respectively6CFU/mL、3.4×107CFU/m L, therefore, Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 grows faster in skim milk.
2. pH and titrated acidity changes of Lactobacillus helveticus grown in skim milk for 10h
Respectively inoculating lactobacillus helveticus (L Acobacter helveticus) CCFM1036, lactobacillus helveticus (L Acobacter helveticus) DNM-B1M10 and lactobacillus helveticus (L Acobacter helveticus) ATCC15009 which are preserved at the temperature of-80 ℃ into an MRS liquid culture medium, culturing for 24 hours at the temperature of 37 ℃, and subculturing for 2-3 times until the bacterium 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 106CFU/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 the experimental result is shown in figures 3-4.
As can be seen from FIG. 4, the pH of Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 after 10 hours of fermentation was 4.23, the acidity was 51.43 DEG T, and the acid production rate was faster than that of Lactobacillus helveticus (L Acobacter helveticus) DNM-B1M10 and Lactobacillus helveticus (L Acobacter helveticus) ATCC15009 until the end of the fermentation.
Example 4: arginine aminopeptidase (Arg-pNA) enzyme activity of the strain
Lactobacillus helveticus (L Acobacter helveticus) CCFM1036, Lactobacillus helveticus (L Acobacter helveticus) DNM-B1M10 and Lactobacillus helveticus (L Acobacter helveticus) ATCC15009 stored at-80 ℃ are respectively inoculated into 200M L MRS medium to make the concentration of bacteria in the MRS medium reach 106CFU/g, culturing the inoculated sample at 37 ℃ for 16h, centrifuging the cultured bacterial liquid at 12000g and 5min and 4 ℃, collecting thalli, washing the thalli with phosphate buffer solution with pH7.5 for 2 times, adding liquid nitrogen, grinding for 5min, dissolving in phosphate buffer solution with pH7.5 of 3m L, centrifuging at 12000g and 5min and 4 ℃, and taking the supernatant to obtain crude enzyme liquid.
The enzyme activity of arginine aminopeptidase (Arg-pNA) in the crude enzyme solution was detected, and the results are shown in FIG. 5.
As can be seen from FIG. 5, after 30min of the reaction, the arginine aminopeptidase (Arg-pNA) activity of Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 was 93.694nmol/min mg, and the arginine aminopeptidase (Arg-pNA) activities of Lactobacillus helveticus (L Acobacter helveticus) DNM-B1M10 and Lactobacillus helveticus (L Acobacter helveticus) ATCC15009 were 9.131nmol/min mg and 46.469nmol/min mg, respectively, so that the arginine aminopeptidase (Arg-pNA) activity of Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 was high.
Example 5: application of the Strain (preparation of cheese)
Homogenizing milk under the conditions of 14-21 MPa of pressure and 40-85 ℃ of temperature, carrying out pasteurization to obtain sterilized milk, cooling the sterilized milk to 21-30 ℃ to obtain a fermentation raw material, and inoculating lactobacillus helveticus (L actinobacillus helveticus) CCFM1036 into the fermentation raw material until the bacteria concentration is 106～108Fermenting at 37 deg.C for 10-12 h after CFU/m L to obtain fermented milk, inoculating Lactobacillus helveticus (L Acobacter helveticus) CCFM1036 to the fermented milk until the bacteria concentration is 106～108CFU/m L, simultaneously adding 0.002% (w/w) rennin into the fermented milk to curdle the fermented milk to obtain curdled milk blocks, cutting the curdled milk blocks, stirring at 39 ℃ to discharge whey, stacking the curdled blocks until the acidity reaches 45-55 DEG T, cutting the curdled blocks, adding 3% o salt, squeezing at 14 ℃ for 18h to obtain cheese, and maturing the cheese at the temperature of 8 ℃ and the humidity of 85% to obtain the finished cheese.
The cheese finished product is tasted, has rich milk flavor and no bitter taste.
Example 6: application of the Strain (preparation Koumiss)
Maintaining mare's milk at 90-95 deg.C for 10-15 min for sterilization, cooling to 35 deg.C to obtain fermentation raw material, inoculating Lactobacillus helveticus (L Lactobacillus helveticus) CCFM1036 to the fermentation raw material until the bacteria concentration reaches 106～108CFU/m L, and simultaneously inoculating Saccharomyces cerevisiae into fermentation raw material until the bacteria concentration reaches 106～108CFU/m L, fermenting the inoculated fermentation raw material at 37 ℃ for 96h until the acidity reaches 70-120And (4) reaching the alcohol content of 1.0-3.0 degrees P at the angle T to obtain the fermented mare milk.
The sour mare's milk has moderate sour taste, both wine fragrance and milk fragrance, and no bitter taste.
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
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Claims (10)
1. The lactobacillus helveticus (L actinobacillus helveticus) is characterized in that the lactobacillus helveticus is preserved in Guangdong province microorganism strain preservation center in 20.09.2018, with the preservation number being GDMCC No.60453, and the preservation address being No. 59 floor 5 of Dazhou No. 100 Jie of Xieli Zhoolu, Guangzhou city.
2. A starter culture comprising the Lactobacillus helveticus strain according to claim 1.
3. The starter culture of claim 2, wherein the starter culture is a direct vat set starter culture;
the preparation method of the direct vat set starter is that the lactobacillus helveticus of claim 1 is inoculated into a culture medium and cultured until the concentration of viable bacteria of the lactobacillus helveticus 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 × 108CFU/m L to obtain bacterial suspension, and drying the suspension to obtain the direct vat set starter.
4. Use of a lactobacillus helveticus bacterium according to claim 1 or a starter culture according to claim 2 or 3 for the preparation of a fermented food product.
5. Use according to claim 4, wherein the fermented food product is a fermented dairy product, a fermented pasta or a fermented sauce produced using the Lactobacillus helveticus bacterium according to claim 1 or the leavening agent 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, margarine, fermented mare's milk (Koumiss) or sour cream; the fermented pasta comprises cookies; the fermented seasoning comprises vinegar or soy sauce.
7. A method for producing a fermented food, characterized in that the method comprises using the Lactobacillus helveticus strain of claim 1 or the starter culture of claim 2 or 3.
8. The method of claim 7, wherein when the fermented food is cheese, the method comprises homogenizing milk, pasteurizing, and cooling to obtain a fermented material; inoculating the lactobacillus helveticus bacterium of claim 1 or the leavening agent of claim 2 or 3 into a fermentation raw material to ferment to obtain fermented milk; inoculating the Lactobacillus helveticus strain of claim 1 or the starter of claim 2 or 3 to the fermented milk again, and adding rennet to the fermented milk to curd the fermented milk to obtain a curd; cutting curd block, stirring to remove whey, stacking the curd, cutting, adding salt, and squeezing to obtain cheese; ripening the cheese to obtain a cheese finished product;
or when the fermented food is fermented mare milk, the preparation method comprises sterilizing mare milk, and cooling to obtain fermented raw material; inoculating the lactobacillus helveticus strain of claim 1 or the leavening agent of claim 2 or 3 into a fermentation raw material, and simultaneously inoculating saccharomyces cerevisiae into the fermentation raw material to perform fermentation to obtain the fermented mare milk.
9. The preparation method according to claim 7 or 8, wherein when the fermented food is cheese, the preparation method comprises homogenizing cow milk under a pressure of 14MPa to 21MPa and at a temperature of 40 ℃ to 85 ℃, and then pasteurizing to obtain sterilized cow milk; cooling the sterilized milk to 21-30 ℃ to obtain a fermentation raw material; inoculating the lactobacillus helveticus strain of claim 1 or the leavening agent of claim 2 or 3 into a fermentation raw material, and fermenting at 37 ℃ for 10-12 hours to obtain fermented milk; inoculating the Lactobacillus helveticus strain of claim 1 or the starter of claim 2 or 3 to the fermented milk again, and adding rennet to the fermented milk to curd the fermented milk to obtain a curd; cutting curd blocks, stirring at 39 ℃ to remove whey, stacking the curd blocks until the acidity reaches 45-55 DEG T, chopping, adding salt, and squeezing at 14 ℃ for 18h to obtain cheese; ripening the cheese at the temperature of 8 ℃ and the humidity of 85% to obtain a cheese finished product;
or when the fermented food is the fermented mare's milk, the preparation method comprises maintaining the mare's milk at the temperature of 90-95 ℃ for 10-15 min for sterilization, and cooling to 35 ℃ to obtain a fermented raw material; inoculating the lactobacillus helveticus strain of claim 1 or the leavening agent of claim 2 or 3 into a fermentation raw material, and simultaneously inoculating saccharomyces cerevisiae into the fermentation raw material to ferment for 96 hours at 37 ℃ until the acidity reaches 70-120 degrees T and the alcoholic strength reaches 1.0-3.0 degrees P, so as to obtain the mare's milk.
10. A fermented food product produced by the production method according to any one of claims 7 to 9.
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| WO2012005578A1 (en) * | 2010-07-06 | 2012-01-12 | Lanthiopep B.V. | Bacterial surface display and screening of thioether-bridge-containing peptides |
| CN106795484A (en) * | 2014-05-15 | 2017-05-31 | 格里康辛有限责任公司 | For α (1,2) the fucosyltransferase mutation used when fucosylation oligosaccharide is produced |
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