CN109402019A - One plant height produces Lactobacillus helveticus and its application of lysine aminopeptidase - Google Patents
One plant height produces Lactobacillus helveticus and its application of lysine aminopeptidase Download PDFInfo
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- CN109402019A CN109402019A CN201811453204.5A CN201811453204A CN109402019A CN 109402019 A CN109402019 A CN 109402019A CN 201811453204 A CN201811453204 A CN 201811453204A CN 109402019 A CN109402019 A CN 109402019A
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- lactobacillus helveticus
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- leavening
- koumiss
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- 235000013967 Lactobacillus helveticus Nutrition 0.000 title claims abstract description 157
- 229940054346 lactobacillus helveticus Drugs 0.000 title claims abstract description 157
- 108030000961 Aminopeptidase Y Proteins 0.000 title abstract description 18
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- 235000020247 cow milk Nutrition 0.000 claims description 15
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- 229940039696 lactobacillus Drugs 0.000 claims description 6
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- LRBQNJMCXXYXIU-PPKXGCFTSA-N Chinese gallotannin Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
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- 241000214517 Lactobacillus casei group Species 0.000 description 1
- 235000010727 Lactobacillus helveticus DSM 20075 Nutrition 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
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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
- A23V2400/11—Lactobacillus
- A23V2400/147—Helveticus
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
The invention discloses Lactobacillus helveticus and its applications that a plant height produces lysine aminopeptidase, belong to microorganisms technical field.This Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 is with advantage fast with growth rate, that production acid is fast and lysine aminopeptidase (Lys-pNA) yield is high, the metabolic capability of albumen in fermented food, the formation for reducing fermented food bitter taste, the flavor for improving fermented food can effectively be enhanced, there is critically important application in the preparation of the fermented foods such as acidified milk, leben, cheese, koumiss, koumiss (Koumiss), sour cream, cookies, vinegar or soy sauce.
Description
Technical field
The present invention relates to Lactobacillus helveticus and its applications that a plant height produces lysine aminopeptidase, belong to microbial technique neck
Domain.
Background technique
Free peptide and amino acid content in cow's milk is less, has higher proteolytic activity, and Lactobacillus helveticus is in
The characteristics of revealing amino acids auxotroph, therefore, Lactobacillus helveticus is in the preparation process of the fermented foods such as cheese maturation
With the ability for accelerating flavor formation, it is play an important role in fermented food field.
The proteolytic system of Lactobacillus helveticus mainly includes cell wall protein enzyme (Cell-envelope
Proteinase, CEP), effect is protein to be hydrolyzed into a series of peptide fragments, and the peptide fragment that these hydrolysis obtain can be turned by oligopeptides
Fortune system (Opp), dipeptides and tripeptides movement system (Dpp and DtpT) transport to intracellular, by peptase intracellular (amino acid peptase,
Endopeptidase, Pro- specificity peptase etc.) amino acid or smaller peptides are further hydrolyzed to, meet the nutritional need of thallus
To preferably grow.
Although polypeptide, oligopeptides and a small amount of amino acid that protein hydrolysis generates, dissolubility, nutritive peculiarity and mouthfeel wind
Taste itself can be obviously improved compared with protein, and still, bitter taste, this bitter taste is often presented in these protein hydrolyzates
Caused by being the polypeptide (bitter peptides) by end for hydrophobic amino acid, this undoubtedly greatly limits answering for protein hydrolysate
With.Therefore, the bitter taste of the protolysate of reduction, prevention and removal Lactobacillus helveticus, is just particularly important.
It has been investigated that amino acid peptase belongs to expeptidase in the peptase intracellular of Lactobacillus helveticus, amino can be made
Acid hydrolyzes one by one from the N- terminal order of polypeptide chain, therefore, the not only growth for Lactobacillus helveticus thallus of amino acid peptase
It is most important, hydrophobic amino acid residues can also be cut off from N-terminal to achieve the purpose that de- hardship, therefore, obtain high yield amino acid
The Lactobacillus helveticus of peptase can undoubtedly reduce the bitter taste of protolysate.
But the aminopeptidase type isolated in lactobacillus helveticus strains at present is less, cannot effectively be directed to Leu,
The hydrophobic amino acid residues influential on bitter taste such as Pro, Arg, Lys, also, these Lactobacillus helveticus that can produce aminopeptidase produce
Amount is not high, is not enough to play the effect of the apparent bitter taste for reducing protolysate.
Therefore, a kind of Lactobacillus helveticus of high yield amino acid peptase is found, to be applied to acidified milk, hair as leavening
The hair of the fermented foods such as kefir milk beverage, cheese, koumiss, koumiss (Koumiss), sour cream, cookies, vinegar or soy sauce
Ferment, to enhance the metabolic capability of albumen in fermented food, the formation for reducing fermented food bitter taste, the flavor for improving fermented food,
It is this field technical problem urgently to be resolved.
Summary of the invention
To solve the above problems, the present invention provides one plant of Lactobacillus helveticus (Lactobacillus helveticus)
Growth rate is fast (to incite somebody to action with having by CCFM1035, this Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035
This bacterial strain is with 1 × 106The inoculum concentration of CFU/mL is seeded in skimmed milk the 10h that ferments, and can make Lactobacillus helveticus in acidified milk
Viable count is up to 4.3 × 108CFU/mL), it is fast (by this bacterial strain with 1 × 10 to produce acid6The inoculum concentration of CFU/mL, which is seeded in skimmed milk, to be sent out
Ferment 10h, can make the pH 4.71 of acidified milk, acidity is 50.57 ° of T, until fermentation termination) and lysine aminopeptidase (Lys-pNA)
This bacterial strain (is seeded in MRS fluid nutrient medium with 2% inoculum concentration and cultivates 16h, can make lysine aminopeptidase by yield height
(Lys-pNA) enzyme activity can effectively enhance the metabolic energy of albumen in fermented food up to the advantage of 177.464nmol/min*mg)
Power, reduce fermented food bitter taste formation, improve fermented food flavor, acidified milk, leben, cheese, koumiss,
There is critically important application in the preparations of fermented foods such as koumiss (Koumiss), sour cream, cookies, vinegar or soy sauce.
Technical scheme is as follows:
The present invention provides one plant of Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035, the Switzerland
Lactobacillus (Lactobacillus helveticus) CCFM1035 was preserved in Guangdong Province microorganism on 09 20th, 2018
Culture Collection Center, deposit number are GDMCC No.60452, and preservation address is No. 59 building of the compound of Xianlie Middle Road, Guangzhou City 100
5 buildings.
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 is from Xining, Qinghai Tibetan area tradition
Isolated in yak Qula of fermenting, for the bacterial strain through sequencing analysis, 16S rRNA sequence, will as shown in SEQ ID NO.1
The sequence is compared in GenBank, this bacterial strain is Lactobacillus helveticus as the result is shown, is named as Lactobacillus helveticus
(Lactobacillushelveticus)CCFM1035。
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035's is raw on MRS solid medium
Bacterium colony when long is rounded, smaller, translucent, smooth；The Lactobacillus helveticus (Lactobacillus helveticus)
CCFM1035 grows 14h in skimmed milk and enters stationary phase, and the 10h that ferments in skimmed milk reaches fermentation termination, and growth is fast, produces
It is sour fast.
The present invention provides the hairs for containing above-mentioned Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035
Ferment agent.
In one embodiment of the invention, the leavening is throw type leaven；
The preparation method of the throw type leaven is by Lactobacillus helveticus (Lactobacillus described in claim 1
Helveticus) CCFM1035 is seeded in culture medium and cultivates to Lactobacillus helveticus (Lactobacillus helveticus)
CCFM1035 viable bacteria concentration is not less than 1 × 108CFU/mL obtains culture solution；By medium centrifugal, thallus is obtained；Thallus is used
It is resuspended to bacteria concentration with freeze drying protectant not less than 1 × 10 after buffer solution for cleaning 2~3 times8CFU/mL obtains bacteria suspension；It will hang
Supernatant liquid is dry, obtains throw type leaven.
In one embodiment of the invention, the culture medium is MRS culture medium or LBS culture medium.
In one embodiment of the invention, the buffer is phosphate buffer or borate buffer solution.
In one embodiment of the invention, the freeze drying protectant is glycerol or skimmed milk.
In one embodiment of the invention, the drying is vacuum freeze drying.
The present invention provides above-mentioned Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 or above-mentioned hairs
Application of the ferment agent in terms of preparing fermented food.
In one embodiment of the invention, the fermented food is to use above-mentioned Lactobacillus helveticus
Fermented dairy product that (Lactobacillus helveticus) CCFM1035 or above-mentioned leavening produce, fermented pasta or
Fermented flavoring material.
In one embodiment of the invention, the fermented dairy product includes acidified milk, leben, cheese, horse
Fermented milk, koumiss (Koumiss) or sour cream；The fermented pasta includes cookies；The fermented flavoring material include vinegar or
Soy sauce.
The present invention provides a kind of preparation method of fermented food, the method is to use above-mentioned Lactobacillus helveticus
(Lactobacillus helveticus) CCFM1035 or above-mentioned leavening.
In one embodiment of the invention, the fermented food be cheese when, preparation method be by cow's milk through homogeneous,
It is cooling after pasteurize, obtain fermentation raw material；Above-mentioned Lactobacillus helveticus (Lactobacillus is inoculated in fermentation raw material
Helveticus) CCFM1035 or above-mentioned leavening ferment, and obtain acidified milk；It is inoculated with above-mentioned Switzerland again in acidified milk
Lactobacillus (Lactobacillus helveticus) CCFM1035 or above-mentioned leavening, while curdled milk being added in acidified milk
Enzyme makes acidified milk carry out curdled milk, obtains ziega；Ziega is cut, stir discharging whey, grumeleuse heap make, chopping plus salt,
Squeezing, obtains cheese；Cheese is subjected to maturation, obtains cheese finished product；
Or the fermented food is when being koumiss, preparation method be it is cooling after sterilizing mare's milk, obtain fermentation raw material；It is sending out
Above-mentioned Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 or above-mentioned leavening are inoculated in ferment raw material, together
When, saccharomyces cerevisiae is inoculated in fermentation raw material and is fermented, and koumiss is obtained.
In one embodiment of the invention, the cow's milk includes raw milk, reconstituted milk or skimmed milk.
The skimmed milk refers to the fat in cow's milk is sloughed after obtained lotion；The raw milk refers to from milk cow's milk
Room squeezes out the raw milk without any processing；The reconstituted milk, which refers to, to be concentrated milk, dries as concentrated milk or milk powder, then
Suitable quantity of water is added, is made and water, the comparable lotion of solids ratio in former cream.
In one embodiment of the invention, when the fermented food is cheese, preparation method is by cow's milk in pressure
14MPa~21MPa, pasteurize, the cow's milk after being sterilized are carried out after homogeneous under conditions of 40~85 DEG C of temperature；After sterilizing
Cow's milk be cooled to 21~30 DEG C, obtain fermentation raw material；Above-mentioned Lactobacillus helveticus is inoculated in fermentation raw material
Ferment at 37 DEG C 10~12h after (Lactobacillus helveticus) CCFM1035 or above-mentioned leavening, is fermented
Cream；It is inoculated with above-mentioned Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 or above-mentioned hair again in acidified milk
Ferment agent, while renin being added in acidified milk, so that acidified milk is carried out curdled milk, obtains ziega；Ziega is first cut,
Then it makes to acidity in 39 DEG C of stirring discharging wheys, then grumeleuse heap and reaches 45~55 ° of T, then shred and add salt, finally squeezed in 14 DEG C
18h obtains cheese；Cheese is carried out to maturation under conditions of 8 DEG C of temperature, humidity 85%, obtains cheese finished product；
Or the fermented food, when being koumiss, preparation method is that mare's milk maintained 10 under conditions of 90~95 DEG C of temperature
~15min is cooled to 35 DEG C after being sterilized, obtain fermentation raw material；Above-mentioned Lactobacillus helveticus is inoculated in fermentation raw material
(Lactobacillus helveticus) CCFM1035 or above-mentioned leavening, meanwhile, saccharomyces cerevisiae is inoculated in fermentation raw material
96h is fermented at 37 DEG C to acidity reaches 70~120 ° of T, alcoholic strength reaches 1.0~3.0 ° of P, obtains koumiss.
In one embodiment of the invention, when the fermented food is cheese, Lactobacillus helveticus
The inoculum concentration of (Lactobacillus helveticus) CCFM1035 or leavening in fermentation raw material is Lactobacillus helveticus
Viable count of (Lactobacillus helveticus) CCFM1035 in fermentation raw material is up to 106~108CFU/mL, Switzerland's cream
The bacillus inoculum concentration of (Lactobacillus helveticus) CCFM1035 or leavening in acidified milk is Lactobacillus helveticus
Viable count of (Lactobacillus helveticus) CCFM1035 in fermentation raw material is up to 106~108CFU/mL, renin
Additive amount in acidified milk accounts for the 0.002% of acidified milk gross mass, additive amount of the salt in ziega and accounts for ziega gross mass
3 ‰；
Or the fermented food be koumiss when, Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035
Or inoculum concentration of the leavening in fermentation raw material is that Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 exists
Viable count in fermentation raw material is up to 106~108The inoculum concentration of CFU/mL, saccharomyces cerevisiae in fermentation raw material is that saccharomyces cerevisiae is being sent out
Viable count in ferment raw material is up to 106~108CFU/mL。
The present invention provides the fermented foods being prepared using above-mentioned preparation method.
The utility model has the advantages that
(1) Lactobacillus helveticus of the invention (Lactobacillus helveticus) CCFM1035 is with growth speed
Rate is fast, produces the advantage that acid is fast and lysine aminopeptidase (Lys-pNA) yield is high, can effectively enhance the metabolism of albumen in fermented food
Ability, the formation for reducing fermented food bitter taste, the flavor for improving fermented food, in acidified milk, leben, cheese, mare's milk
There is critically important answer in the preparations of fermented foods such as wine, koumiss (Koumiss), sour cream, cookies, vinegar or soy sauce
With；
(2) by Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 of the invention with 1 × 106CFU/
The inoculum concentration of mL is seeded in skimmed milk the 10h that ferments, can make the viable count of Lactobacillus helveticus in acidified milk up to 4.3 ×
108CFU/mL；
(3) by Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 of the invention with 1 × 106CFU/
The inoculum concentration of mL is seeded in skimmed milk the 10h that ferments, and can make the pH 4.71 of acidified milk, acidity is 50.57 ° of T, until fermentation is eventually
Point；
(4) by Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 of the invention with 2% inoculation
Amount, which is seeded in MRS fluid nutrient medium, cultivates 16h, and the enzyme activity of lysine aminopeptidase (Lys-pNA) can be made to reach
177.464nmol/min*mg。
Biomaterial preservation
One plant of Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035, taxology are named as
Lactobacillushelveticus was preserved in Guangdong Province's Culture Collection, preservation on 09 20th, 2018
Number is GDMCCNo.60452, and preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
Detailed description of the invention
Fig. 1: the Gram's staining feature of bacterial strain of the present invention；
Fig. 2: upgrowth situation of the bacterial strain of the present invention in skimmed milk；
Fig. 3: the pH situation of change that bacterial strain of the present invention ferments in skimmed milk；
Fig. 4: the acidity situation of change that bacterial strain of the present invention ferments in skimmed milk；
Fig. 5: lysine aminopeptidase (Lys-pNA) enzyme activity of bacterial strain of the present invention in MRS culture medium.
Specific embodiment
The present invention will be further elaborated combined with specific embodiments below.
Skimmed milk involved in following embodiments is purchased from bright dairy products limited liability company；The lactobacillus helveticus atcc-
15009 are purchased from Bei Na biotech firm.
Culture medium involved in following embodiments is as follows:
MRS solid medium: tryptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, hydrogen citrate two
Amine 2.0g/L, glucose 20.0g/L, Tween 80 1mL/L, anhydrous sodium acetate 2.0g/L, epsom salt 0.5g/L, a hydration
Manganese sulfate 0.25g/L, three water dipotassium hydrogen phosphate 2.6g/L are added pure by the amount addition of 1.5% (m/v) after distilled water is completely dissolved
Change agar powder, 115 DEG C of sterilizing 20min.
MRS fluid nutrient medium: tryptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, hydrogen citrate two
Amine 2.0g/L, glucose 20.0g/L, Tween 80 1mL/L, anhydrous sodium acetate 2.0g/L, epsom salt 0.5g/L, a hydration
Manganese sulfate 0.25g/L, three water dipotassium hydrogen phosphate 2.6g/L, after addition distilled water is completely dissolved, 115 DEG C of sterilizing 20min.
Detection method involved in following embodiments is as follows:
The detection method of viable count: national standard " GB 4789.35-2016 national food safety standard Food Microbiology is used
Detect lactic acid bacteria detection ".
PH detection method: it is measured using pH meter.
Acidity detection method: national standard GB 431334-2010 is used.
Lysine aminopeptidase (Lys-pNA) Enzyme activity assay method:
1, the drafting of paranitroanilinum standard curve:
The p-nitrophenyl amine aqueous solution for preparing various concentration (20~160 μM) surveys absorbance in 405nm, obtains standard
Curve；
2, the preparation of substrate solution
The chromogenic substrate (H-Lys-pNA) of 1mM is dissolved in the 50mmol L of pH 7.5-1In sodium phosphate buffer；
3, lysine aminopeptidase (Lys-pNA) Enzyme activity assay
By 50 μ L substrate solutions and 50 μ L CFE after 30 DEG C are cultivated 30 minutes, absorbance is measured in 405nm；It is no positive right
According to without commercial enzyme, blank control replaces CFE with buffer；
The calculation formula of lysine aminopeptidase (Lys-pNA) enzyme activity are as follows: enzyme activity=(paranitroanilinum production quantity * 100*
0.001))/(protein concentration * 50*0.001*30)；
(it can refer to document: Stefanovic E, Kilcawley K N, Rea M C, et al.Genetic,
enzymatic and metabolite profiling of the Lactobacillus casei group reveals
strain biodiversity and potential applications for flavour diversification
[J].Journal of Applied Microbiology,2017,122(5).)
Embodiment 1: the screening and identification of bacterial strain
1, it screens
(1) it prepares suitable sample dilution gradient and cultivates
The Xining, Qinghai 0.5g Tibetan area traditional zymotic yak Qula is weighed, is added to equipped in 4.5mL sterile water, then successively
It takes 0.5mL bacterium solution to be diluted in 4.5mL sterile water, makes the sample concentration gradient dilution to 10-4, take 4 dilutions be respectively 10~
104Each 50 μ L of bacteria suspension be respectively coated on MRS solid medium, 46~48h is cultivated at 37 DEG C of temperature, and is seen in time
It examines.
(2) scribing line isolation and purification
After the plate for growing bacterium colony is taken out, the apparent gradient plate of single colonie, the bacterium of picking difference colonial morphology are chosen
It falls, carries out secondary lineation, until being purified into all single colonies.
(3) Gram's staining and catalase experiment
Picking single bacterium is fallen on glass slide, by smear, drying, fixation, first dye, washing, mordant dyeing, washing, decoloration, is answered
Microscopy after dye, washing, drying, records Gram's staining result；And picking single bacterium is fallen on glass slide, and 3% hydrogen peroxide is added
Solution, observation have bubble-free generation, and record catalase contact result to identify whether the bacterial strain selected has lactic acid bacteria
Feature.
(4) culture presevation
The single colonie of every plant of bacterial strain after the completion of purifying is chosen in 5mL MRS fluid nutrient medium, is placed under 37 DEG C of anaerobism
Stationary culture 20~for 24 hours, 1mL bacterium solution is drawn to protecting in tube, and 4000rpm is centrifuged 5min, and incline supernatant, and 30% nothing of 1mL is added
Bacterium glycerite is resuspended, is placed in -80 DEG C of preservations.
2, it identifies
(1) 16S rDNA sequence amplification
It draws the above-mentioned bacterium solution of 1mL and is centrifuged the supernatant that inclines, twice with 1mL sterile water piping and druming cleaning, be centrifuged the supernatant that inclines, obtain
Bacterium mud is obtained, as template, carries out PCR amplification, process is as follows:
1) 50 μ L of amplification system:
Wherein Mix is 25 μ L, 27F (SEQ ID No 2:AGA GTT TGA TCC TGG CCT CA) 1 μ L, 1492R
(SEQ ID No 3:GGT TAC CTT GTT ACG ACT T) 1 μ L, ddH2O is 23 μ L；Expanding fragment length 1500bp.
2) amplification condition:
Lid:105 DEG C, MBY-16s, V:20 μ L；
Cycle-index: 29 circulations
By DNA double chain in 94 DEG C of holding 10min, 94 DEG C of holding 30s, 50 DEG C of holding 30s, 72 DEG C of holding 80s, recycle 29 times
Afterwards, 72 DEG C of holding 7min.
(2) agarose gel electrophoresis
It weighs 0.8g agarose to be added in conical flask, 80mL 1xTAE is added, microwave discontinuous heats 4min, until liquid is clear
It is clear bright, it is cooling slightly, 8 μ LEB dyestuffs are added；Electrophoresis plate cooling half an hour is added, condenses into solid colloidal to it, uses liquid relief
Rifle squeezes into the sample of 3~5 μ L in the aperture of offset plate, and adds a Marker in every row end；Electrode is plugged, voltage is adjusted
120V runs half an hour；Offset plate is taken out, 10s is exposed at UV, saves the image of electrophoretic band；It will obtain clear electrophoretic band
Sample sequencing.
(3) 16S rRNA sequence analyzes and identifies
According to Hua Da gene feed back sequence results, in conjunction with NCBI strain sequence database (http: //
Www.ncbi.nlm.nih.gov/blast BLAST retrieval) is carried out, the highest bacterial strain information of matching degree is chosen and carries out result note
Record, PCR is successful, and two plants of bacterial strains are Lactobacillus helveticus, are respectively designated as Lactobacillus helveticus (Lactobacillus
Helveticus) CCFM1035 and Lactobacillus helveticus (Lactobacillus helveticus) DNM-B1M10.
Embodiment 2: the culture of bacterial strain
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 is cultivated on MRS solid medium
48h, is observed and picking colony carries out microscopy, gram color and growth characteristics measurement.
It is observed that Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 in MRS solid medium
Bacterium colony when upper growth is rounded, smaller, translucent, smooth.
Embodiment 3: growth characteristics of the bacterial strain in newborn system
1, Lactobacillus helveticus grows the growth curve of 14h in skimmed milk
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035, Lactobacillus helveticus that -80 DEG C are saved
(Lactobacillus helveticus) DNM-B1M10 and Lactobacillus helveticus (Lactobacillus helveticus)
ATCC 15009 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 37 DEG C for 24 hours, and secondary culture 2~3 times, until bacteria concentration reaches
108~109CFU/mL；It takes out the bacterium solution after activating in MRS to be inoculated in skimmed milk by 2%~4% volume ratio, make in system
Bacterium amount reach 106CFU/g；Inoculated sample is put into 37 DEG C of incubator and is fermented, sampled every 2h, detection was fermented
The variation of journey bacterium amount, as a result as shown in Figure 2.
From the figure 3, it may be seen that Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 bacterium amount after the 10h that ferments
Reach 4.3 × 108CFU/mL, Lactobacillus helveticus (Lactobacillus helveticus) DNM-B1M10 and Lactobacillus helveticus
(Lactobacillus helveticus) ATCC 15009 bacterium amount after the 10h that ferments respectively reaches 8.3 × 106CFU/mL、3.4
×107CFU/mL, therefore, Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 grow speed in skimmed milk
Rate is faster.
2, Lactobacillus helveticus grows pH and the titratable acidity variation of 10h in skimmed milk
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035, Lactobacillus helveticus that -80 DEG C are saved
(Lactobacillus helveticus) DNM-B1M10 and Lactobacillus helveticus (Lactobacillus helveticus)
ATCC 15009 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 37 DEG C for 24 hours, and secondary culture 2~3 times, until bacteria concentration reaches
108~109CFU/mL；It takes out the bacterium solution after activating in MRS to be inoculated in skimmed milk by 2%~4% volume ratio, make in system
Bacterium amount reach 106CFU/g；Inoculated sample is put into 37 DEG C of incubator and is fermented, sampled every 2h, taken every 2h
Sample, detects the variation of pH and titratable acidity in fermentation process, and experimental result is as shown in Figure 3-4.
As shown in Figure 4, Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 pH after the 10h that ferments is
4.71, acidity is 50.57 ° of T, until fermentation termination, rate of producing acid is than Lactobacillus helveticus (Lactobacillus helveticus)
DNM-B1M10 and Lactobacillus helveticus (Lactobacillus helveticus) ATCC 15009 is fast.
Embodiment 4: lysine aminopeptidase (Lys-pNA) enzyme activity of bacterial strain
Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035, Lactobacillus helveticus that -80 DEG C are saved
(Lactobacillus helveticus) DNM-B1M10 and Lactobacillus helveticus (Lactobacillus helveticus)
ATCC 15009 is inoculated in respectively in 200mL MRS culture medium, makes in MRS culture medium bacteria concentration up to 106CFU/g；After being inoculated with
Sample cultivate 16h at 37 DEG C；The bacterium solution that culture is obtained collects thallus by 12000g, 5min, 4 DEG C of centrifugations；Thallus is used
The phosphate buffer solution of pH7.5 is cleaned 2 times, and liquid nitrogen grinding 5min is added, is dissolved in the phosphate buffer solution of 3ml pH7.5,
With 12000g, 5min, 4 DEG C of centrifugations, supernatant is taken, crude enzyme liquid is obtained.
Lysine aminopeptidase (Lys-pNA) enzyme activity in crude enzyme liquid is detected, as a result as shown in Figure 5.
As shown in Figure 5, after reacting 30min, Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035's
Lysine aminopeptidase (Lys-pNA) vigor is 177.464nmol/min*mg, Lactobacillus helveticus (Lactobacillus
Helveticus) the lysine of DNM-B1M10 and Lactobacillus helveticus (Lactobacillus helveticus) ATCC 15009
Aminopeptidase (Lys-pNA) vigor is respectively 9.131nmol/min*mg, 46.469nmol/min*mg, therefore, Lactobacillus helveticus
Lysine aminopeptidase (Lys-pNA) vigor of (Lactobacillus helveticus) CCFM1035 is high.
Embodiment 5: the application (preparing cheese) of bacterial strain
Cow's milk is subjected to pasteurize after homogeneous under conditions of pressure 14MPa~21MPa, 40~85 DEG C of temperature, is obtained
Cow's milk after sterilizing；Cow's milk after sterilizing is cooled to 21~30 DEG C, obtains fermentation raw material；Switzerland's cream is inoculated in fermentation raw material
Bacillus (Lactobacillus helveticus) CCFM1035 to bacteria concentration be 106~108Ferment 10 after CFU/mL at 37 DEG C
~12h, obtains acidified milk；It is inoculated with Lactobacillus helveticus (Lactobacillus helveticus) again in acidified milk
CCFM1035 to bacteria concentration be 106~108CFU/mL, while 0.002% (w/w) renin being added in acidified milk, make acidified milk
Curdled milk is carried out, ziega is obtained；Ziega is first cut, is then made in 39 DEG C of stirring discharging wheys, then grumeleuse heap to acidity
Reach 45~55 ° of T, then shred and 3 ‰ salt are added, finally in 14 DEG C of squeezing 18h, obtains cheese；By cheese in 8 DEG C of temperature, wet
Maturation is carried out under conditions of degree 85%, obtains cheese finished product.
This cheese finished product is tasted, milk Flavor is full, no bitter taste.
Embodiment 6: the application (preparation Koumiss) of bacterial strain
35 DEG C are cooled to after maintaining 10~15min to be sterilized under conditions of 90~95 DEG C of temperature in mare's milk, is sent out
Ferment raw material；Lactobacillus helveticus (Lactobacillus helveticus) CCFM1035 is inoculated in fermentation raw material to reach to bacteria concentration
106~108CFU/mL, meanwhile, saccharomyces cerevisiae is inoculated in fermentation raw material to bacteria concentration up to 106~108CFU/mL, after inoculation
Fermentation raw material 96h is fermented at 37 DEG C to acidity reaches 70~120 ° of T, alcoholic strength reaches 1.0~3.0 ° of P, obtain koumiss.
This koumiss is tasted, tart flavour is moderate, and aroma, milk Flavor have both, no bitter taste.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
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Claims (10)
1. one plant of Lactobacillus helveticus (Lactobacillus helveticus), which is characterized in that the Lactobacillus helveticus
(Lactobacillus helveticus) was preserved in Guangdong Province's Culture Collection on 09 20th, 2018, protected
Hiding number is GDMCC No.60452, and preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
2. the leavening containing Lactobacillus helveticus (Lactobacillus helveticus) described in claim 1.
3. leavening as claimed in claim 2, which is characterized in that the leavening is throw type leaven；
The preparation method of the throw type leaven is by Lactobacillus helveticus (Lactobacillus described in claim 1
Helveticus) being seeded in culture medium culture, viable bacteria concentration is not to Lactobacillus helveticus (Lactobacillus helveticus)
Lower than 1 × 108CFU/mL obtains culture solution；By medium centrifugal, thallus is obtained；By thallus with after buffer solution for cleaning 2~3 times
It is resuspended to bacteria concentration with freeze drying protectant not less than 1 × 108CFU/mL obtains bacteria suspension；Suspension is dry, obtain direct putting type
Leavening.
4. fermenting described in Lactobacillus helveticus described in claim 1 (Lactobacillus helveticus) or Claims 2 or 3
Application of the agent in terms of preparing fermented food.
5. application as claimed in claim 4, which is characterized in that the fermented food is using the cream of Switzerland described in claim 1
Fermented dairy product that leavening described in bacillus (Lactobacillus helveticus) or Claims 2 or 3 produces, hair
Ferment wheaten food or fermented flavoring material.
6. application as described in claim 4 or 5, which is characterized in that the fermented dairy product includes acidified milk, acidified milk drink
Material, cheese, koumiss, koumiss (Koumiss) or sour cream；The fermented pasta includes cookies；The fermentation seasoning
Material includes vinegar or soy sauce.
7. a kind of preparation method of fermented food, is characterized in that, the method is to use Lactobacillus helveticus described in claim 1
Leavening described in (Lactobacillus helveticus) or Claims 2 or 3.
8. preparation method as claimed in claim 7, which is characterized in that when the fermented food is cheese, preparation method is will
Cow's milk is cooling after homogeneous, pasteurize, obtains fermentation raw material；Switzerland's cream bar described in claim 1 is inoculated in fermentation raw material
Leavening described in bacterium (Lactobacillus helveticus) or Claims 2 or 3 ferments, and obtains acidified milk；It is sending out
Lactobacillus helveticus described in claim 1 (Lactobacillus helveticus) or Claims 2 or 3 are inoculated in kefir milk again
The leavening, while renin being added in acidified milk, so that acidified milk is carried out curdled milk, obtains ziega；Ziega is carried out
Cutting, stirring discharging whey, grumeleuse heap is made, chopping adds salt, squeezing, obtains cheese；Cheese is subjected to maturation, obtains cheese finished product；
Or the fermented food is when being koumiss, preparation method be it is cooling after sterilizing mare's milk, obtain fermentation raw material；In proferment
It is inoculated with described in Lactobacillus helveticus (Lactobacillus helveticus) described in claim 1 or Claims 2 or 3 and sends out in material
Ferment agent, meanwhile, saccharomyces cerevisiae is inoculated in fermentation raw material and is fermented, and koumiss is obtained.
9. preparation method as claimed in claim 7 or 8, which is characterized in that when the fermented food is cheese, preparation method is
Cow's milk is subjected to pasteurize after homogeneous under conditions of pressure 14MPa~21MPa, 40~85 DEG C of temperature, after being sterilized
Cow's milk；Cow's milk after sterilizing is cooled to 21~30 DEG C, obtains fermentation raw material；It is inoculated with described in claim 1 in fermentation raw material
Ferment 10 after leavening described in Lactobacillus helveticus (Lactobacillus helveticus) or Claims 2 or 3 at 37 DEG C
~12h, obtains acidified milk；It is inoculated with Lactobacillus helveticus (Lactobacillus described in claim 1 again in acidified milk
Helveticus) or leavening described in Claims 2 or 3, while renin being added in acidified milk, coagulates acidified milk
Cream obtains ziega；Ziega is first cut, then makes to acidity in 39 DEG C of stirring discharging wheys, then grumeleuse heap and reaches 45
~55 ° of T, then shred and salt is added to obtain cheese finally in 14 DEG C of squeezing 18h；By cheese under conditions of 8 DEG C of temperature, humidity 85%
Maturation is carried out, cheese finished product is obtained；
Or the fermented food is when being koumiss, preparation method be mare's milk maintains to 10 under conditions of 90~95 DEG C of temperature~
15min is cooled to 35 DEG C after being sterilized, obtain fermentation raw material；Switzerland's cream bar described in claim 1 is inoculated in fermentation raw material
Leavening described in bacterium (Lactobacillus helveticus) or Claims 2 or 3, meanwhile, it is inoculated with and makes in fermentation raw material
Brewer yeast ferments 96h to acidity reaches 70~120 ° of T, alcoholic strength reaches 1.0~3.0 ° of P at 37 DEG C, obtains koumiss.
10. the fermented food that any preparation method of application claim 7-9 is prepared.
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| CN201811453204.5A CN109402019B (en) | 2018-11-30 | 2018-11-30 | Lactobacillus helveticus strain capable of highly producing lysine aminopeptidase and application thereof |
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Cited By (1)
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| CN115305225A (en) * | 2022-09-16 | 2022-11-08 | 微康益生菌(苏州)股份有限公司 | Strain combination capable of rapidly producing acid, freeze-dried strain powder containing strain combination and application of strain combination |
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| EP0198322B1 (en) * | 1985-04-11 | 1991-04-10 | BEHRINGWERKE Aktiengesellschaft | Method for the determination of plasminogen activators |
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| EP0198322B1 (en) * | 1985-04-11 | 1991-04-10 | BEHRINGWERKE Aktiengesellschaft | Method for the determination of plasminogen activators |
| JP2006271275A (en) * | 2005-03-29 | 2006-10-12 | Akita Prefecture | D-aspartic acid-specific endopeptidase and microorganism producing the same |
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| CN115305225A (en) * | 2022-09-16 | 2022-11-08 | 微康益生菌(苏州)股份有限公司 | Strain combination capable of rapidly producing acid, freeze-dried strain powder containing strain combination and application of strain combination |
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