CN109370948A - One plant height produces 6- phosphoric acid-beta galactosidase Lactococcus lactis and its application - Google Patents
One plant height produces 6- phosphoric acid-beta galactosidase Lactococcus lactis and its application Download PDFInfo
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- CN109370948A CN109370948A CN201811414217.1A CN201811414217A CN109370948A CN 109370948 A CN109370948 A CN 109370948A CN 201811414217 A CN201811414217 A CN 201811414217A CN 109370948 A CN109370948 A CN 109370948A
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- lactococcus lactis
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- 235000014897 Streptococcus lactis Nutrition 0.000 title claims abstract description 134
- 241000194035 Lactococcus lactis Species 0.000 title claims abstract 24
- 108010005774 beta-Galactosidase Proteins 0.000 title abstract description 21
- 108010070626 acid beta-galactosidase Proteins 0.000 title abstract description 19
- 102100026189 Beta-galactosidase Human genes 0.000 title abstract description 18
- 235000021001 fermented dairy product Nutrition 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 44
- 235000020167 acidified milk Nutrition 0.000 claims description 24
- 235000020247 cow milk Nutrition 0.000 claims description 20
- 238000000855 fermentation Methods 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 13
- 239000002994 raw material Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
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- 238000001816 cooling Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
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- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
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- 238000004140 cleaning Methods 0.000 claims description 2
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- 239000000463 material Substances 0.000 claims 1
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 abstract description 19
- 239000008101 lactose Substances 0.000 abstract description 19
- 230000008901 benefit Effects 0.000 abstract description 4
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- 238000001514 detection method Methods 0.000 description 10
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-L 2-(carboxymethyl)-2-hydroxysuccinate Chemical compound [O-]C(=O)CC(O)(C(=O)O)CC([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-L 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
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- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 2
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 2
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- MWKAGZWJHCTVJY-UHFFFAOYSA-N 3-hydroxyoctadecan-2-one Chemical compound CCCCCCCCCCCCCCCC(O)C(C)=O MWKAGZWJHCTVJY-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
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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
- 241000194032 Enterococcus faecalis Species 0.000 description 1
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
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- 238000009777 vacuum freeze-drying Methods 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
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- 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
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- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses a plant heights to produce 6- phosphoric acid-beta galactosidase Lactococcus lactis and its application, belongs to microorganisms technical field.This Lactococcus lactis (Lactococcus lactis) CCFM1033 has fast growth rate, 6- phosphoric acid-beta galactosidase yield height and produces the good advantage of fragrant characteristic, lactose content in fermented dairy product can be effectively reduced, and be conducive to the raising of fermented dairy product quality, there is critically important application in the preparation of fermented dairy product.
Description
Technical field
The present invention relates to a plant heights to produce 6- phosphoric acid-beta galactosidase Lactococcus lactis and its application, belongs to microorganism
Technical field.
Background technique
Fermented dairy product is using cow's milk as primary raw material, through lactobacillus-fermented or lactic acid bacteria, saccharomycete co-fermentation system
At acidic dairy prod, a kind of very popular with flavor by unique mouthfeel as milk product, city of being in great demand
Field is for many years.Some researches show that, influencing the principal element that consumer buys fermented dairy product has flavor, price, availability and brand,
Wherein, flavor accounting is most heavy, and therefore, the flavor for promoting fermented dairy product is undoubtedly enterprise and caters to the most important means in market.
Lactose is most important carbohydrate in cow's milk, accounts for 99% or more of carbohydrate total amount in cow's milk, and cream
Sugar can generate glucose 6-phosphate and glyceraldehyde 3-phosphate through preliminary metabolism, and glucose 6-phosphate and glyceraldehyde 3-phosphate can pass through
Glycolytic pathway be further metabolized generate pyruvic acid, and pyruvic acid can finally be metabolized generate lactic acid, acetaldehyde, acetic acid, biacetyl and
Important flavor substance in the fermented dairy products such as 3-hydroxy-2-butanone, therefore, the system of the lactose metabolism characteristic of lactic acid bacteria to fermented dairy product
It is standby most important.
Two lactose metabolism accesses are primarily present in lactic acid bacteria, the key enzyme of this two metabolic pathways is beta galactose respectively
Glycosides enzyme and 6- phosphoric acid-beta galactosidase, wherein beta galactosidase is generally existing in lactic acid bacteria, and 6- phosphoric acid-β-gala
Glycosidase only (mainly includes Lactobacillus casei, lactobacillus paracasei, Lactobacillus rhamnosus, lactic acid cream in a few lactic acid bacteria
Coccus subsp. cremoris, enterococcus faecium and enterococcus faecalis) in complete biochemical characterization, and presently found these can produce 6- phosphoric acid-
Lactic acid bacteria majority 6- phosphoric acid-beta galactosidase yield of beta galactosidase is not high, is not enough to reach the acidified milk system of influence
The stage of product quality.
Therefore, a kind of lactic acid bacteria of high yield 6- phosphoric acid-beta galactosidase is found, to be applied to cream system as leavening
Product fermentation, to reduce lactose content in fermented dairy product, promotes the quality of fermented dairy product, is this field technology urgently to be resolved
Problem.
Summary of the invention
To solve the above problems, the present invention provides one plant of Lactococcus lactis (Lactococcus lactis)
CCFM1033, this Lactococcus lactis (Lactococcus lactis) CCFM1033 have growth rate it is fast (by this bacterial strain with 1 ×
107The inoculum concentration of CFU/mL is seeded in cow's milk the 2h that ferments, can make the viable count of Lactococcus lactis in acidified milk up to 8.37 ×
108CFU/mL), 6- phosphoric acid-beta galactosidase yield is high (is seeded to MRS fluid nutrient medium for this bacterial strain with 2% inoculum concentration
It is middle to cultivate to OD600Up to 1.736,6- phosphoric acid-beta galactosidase enzyme activity can be made up to 96.9U/L) and to produce fragrant characteristic good
(by this bacterial strain with 1 × 107Inoculum concentration be seeded in cow's milk the 12h that ferments, i.e., 10.2% lactose in metabolizable cow's milk, meanwhile,
The content of biacetyl and 3-hydroxy-2-butanone, can be effective respectively up to the advantage of 5.83 μ g/kg and 31.44 μ g/kg) in the acidified milk made
Lactose content in fermented dairy product is reduced, and is conducive to the raising of fermented dairy product quality, is had in the preparation of fermented dairy product
There is critically important application.
Technical scheme is as follows:
The present invention provides one plant of Lactococcus lactis (Lactococcus lactis) CCFM1033, the Lactococcus lactis
(Lactococcus lactis) CCFM1033 was preserved in Guangdong Province's Culture Collection on 09 20th, 2018,
Deposit number is GDMCC No.60450, and preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
Lactococcus lactis (Lactococcus lactis) CCFM1033 is from Chinese Xining, Qinghai area tradition hair
Isolated in kefir milk product Qula, 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 Lactococcus lactis as the result is shown, is named as Lactococcus lactis
(Lactococcus lactis)CCFM1033。
The cell of Lactococcus lactis (Lactococcus lactis) CCFM1033 is in spherical, Gram's staining result
For purple, as gram-positive bacteria, and its bacterium colony in MRS cultured on solid medium is creamy white, glossy, edge
It is smooth, protrusion, median size.
The present invention provides the leavenings for containing above-mentioned Lactococcus lactis (Lactococcus lactis) CCFM1033.
In one embodiment of the invention, the preparation method of the leavening is by above-mentioned Lactococcus lactis
(Lactococcus lactis) CCFM1033 is seeded in culture medium, is cultivated at 30 DEG C to Lactococcus lactis
(Lactococcus lactis) CCFM1033 viable bacteria concentration is not less than 1 × 108CFU/mL obtains culture solution；By culture solution from
The heart obtains thallus；By thallus be resuspended to bacteria concentration not less than 1 with freeze drying protectant after buffer solution for cleaning 2~3 times ×
109CFU/mL obtains bacteria suspension；Suspension is dry, obtain leavening.
In one embodiment of the invention, the culture medium is MRS culture medium or M17 culture medium.
In one embodiment of the invention, the buffer is distilled water, physiological saline or phosphate buffer.
In one embodiment of the invention, the freeze drying protectant is sucrose, trehalose or skimmed milk powder.
In one embodiment of the invention, the drying is vacuum freeze drying.
The present invention provides above-mentioned Lactococcus lactis (Lactococcus lactis) CCFM1033 or above-mentioned leavening to exist
Prepare the application in terms of food.
In one embodiment of the invention, the food is to use above-mentioned Lactococcus lactis (Lactococcus
Lactis) the fermented dairy product that CCFM1033 or above-mentioned leavening produce.
In one embodiment of the invention, the fermented dairy product includes acidified milk, leben, cheese or horse
Fermented milk.
The present invention provides a kind of preparation method of acidified milk, the method is to use above-mentioned Lactococcus lactis
(Lactococcus lactis) CCFM1033 or above-mentioned leavening.
In one embodiment of the invention, the method is that cow's milk is cooling after homogeneous, pasteurize, is sent out
Ferment raw material；Above-mentioned Lactococcus lactis (Lactococcus lactis) CCFM1033 or above-mentioned leavening are inoculated in fermentation raw material
It ferments, obtains acidified milk.
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, the method be by cow's milk pressure 14MPa~21MPa, temperature 40~
Pasteurize, the cow's milk after being sterilized are carried out under conditions of 85 DEG C after homogeneous；Cow's milk after sterilizing is cooled to 21~30 DEG C,
Obtain fermentation raw material；Above-mentioned Lactococcus lactis (Lactococcus lactis) CCFM1033 or above-mentioned is inoculated in fermentation raw material
Ferment at 37 DEG C 12h after leavening, obtains acidified milk；Yoghourt is placed into 24~48h under conditions of 4 DEG C, after obtaining after-ripening
Acidified milk finished product.
In one embodiment of the invention, Lactococcus lactis (Lactococcus lactis) CCFM1033 or
Inoculum concentration of the leavening in fermentation raw material is Lactococcus lactis (Lactococcus lactis) CCFM1033 in fermentation raw material
In viable count up to 1 × 106~1 × 107CFU/mL。
The present invention provides the acidified milks being prepared using above-mentioned preparation method.
The utility model has the advantages that
(1) Lactococcus lactis of the invention (Lactococcus lactis) CCFM1033 has that growth rate is fast, 6- phosphorus
Acid-beta galactosidase yield is high and produces the good advantage of fragrant characteristic, lactose content in fermented dairy product can be effectively reduced, and have
Conducive to the raising of fermented dairy product quality, there is critically important application in the preparation of fermented dairy product；
(2) by Lactococcus lactis (Lactococcus lactis) CCFM1033 of the invention with 1 × 107CFU/mL's connects
Kind amount is seeded in cow's milk the 2h that ferments, and the viable count of Lactococcus lactis in acidified milk can be made up to 8.37 × 108CFU/mL；
(3) by Lactococcus lactis (Lactococcus lactis) CCFM1033 of the invention with the inoculum concentration of 2% (v/v)
It is seeded in MRS fluid nutrient medium and cultivates to OD600Up to 1.736,6- phosphoric acid-beta galactosidase enzyme in culture solution can be made
It is living to reach 96.9U/L；
(4) by Lactococcus lactis (Lactococcus lactis) CCFM1033 of the invention with 1 × 107Inoculum concentration connect
Kind ferment 12h into cow's milk, i.e., 10.2% lactose in metabolizable cow's milk, meanwhile, biacetyl and second in the acidified milk made
The content of acyloin is respectively up to 5.83 μ g/kg and 31.44 μ g/kg；
(5) acidified milk being prepared using Lactococcus lactis (Lactococcus lactis) CCFM1033 of the invention
The volatile flavor substances such as acetaldehyde, biacetyl and 3-hydroxy-2-butanone content is high, and lactose content is low, has preferable quality.
Biomaterial preservation
One plant of Lactococcus lactis (Lactococcus lactis) CCFM1033, taxology are named as Lactococcus
Lactis was preserved in Guangdong Province's Culture Collection, deposit number GDMCC on 09 20th, 2018
No.60450, preservation address are 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: bacterial strain colony characteristics of the present invention；
Fig. 3: upgrowth situation of the bacterial strain of the present invention in skimmed milk；
Fig. 4: the pH situation of change that bacterial strain of the present invention ferments in skimmed milk；
Fig. 5: the acidity situation of change that bacterial strain of the present invention ferments in skimmed milk；
Fig. 6: 6- phosphoric acid-beta galactosidase yield of the bacterial strain of the present invention in MRS culture medium；
Fig. 7: the lactose situation of change that bacterial strain of the present invention ferments in skimmed milk.
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.
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.
6- phosphoric acid-beta galactosidase enzyme activity detection method: 100 μ L logarithmic phase bacterium solutions are drawn, 900 μ L Z is added
10 μ L chloroforms are added in buffer phosphate buffer solution, and be vortexed concussion 10s, and it is molten that 200 μ L 6- phosphoric acid-ONPG substrates are added
Liquid is placed in isothermal reaction in 30 DEG C of metal baths, and starts timing；When OD420When reaching between 0.6~0.9,500 μ L are added
1mol/L Na2CO3Solution terminates reaction, and measurement terminates OD after reaction420, and recording reacting time, maximum response time are
90min；
By enzyme activity=(1000 × OD420)/[OD600×VBacterium solution(mL) × reaction time (min)] calculate 6- phosphoric acid-β-half
The activity of lactoside enzyme.
Volatile matter content detection: the volatile matter content after GC-MS survey fermentation in Yoghourt；
GC conditions and temperature program: selection Rtx-WAX capillary (30m × 0.25mm, 0.25mm), injection port temperature
225 DEG C of degree, split ratio 10, carrier gas (helium) flow velocity 1mL/min.Temperature program is arranged 30 DEG C of initial temperature, 3min is kept, with 15
DEG C/min is warming up to 225 DEG C, keep 5min；
Mass Spectrometry Conditions: Ionization mode EI, emitted energy 70eV, emission current 200 μ A, detector voltage 1.4kV, ion
240 DEG C of source temperature, 230 DEG C of interface temperature, 150 DEG C of level four bars temperature, mass scan range m/z 30~500；
The spectrogram that GC-MS is obtained, it is qualitative in the retrieval in the 2001 standard spectrum library NIST and standard items comparison progress substance,
It is quantitative with areas of peak normalization method.
The detection of lactose content: weighing 1g (being accurate to 0.001g) acidified milk sample in 10mL volumetric flask, is added 70%
(v/v) ethyl alcohol is settled to scale, extracts for 24 hours for 4 DEG C after mixing, and 11000g, 4 DEG C of centrifugation 10min take supernatant to perform the derivatization,
Using high performance liquid chromatography measurement (quantified by external standard method analysis lactose content) after filtering with microporous membrane.
Embodiment 1: the screening and identification of bacterial strain
1, it screens
(1) it prepares suitable sample dilution gradient and cultivates
The Xining, Qinghai traditional zymotic dairy product Qula glycerol sample stored in -80 DEG C of refrigerators is taken out, is placed in and solves on ice
Freeze, take 0.5mL sample to be added in 4.5mL sterile saline after concussion mixing, completes 10 times of dilutions, after oscillation mixing again
It is added in 4.5mL sterile saline from 0.5mL dilution is taken out in the dilution, second of 10 times of dilution is completed, with such
It pushes away, until being diluted to 10-6, 100 μ L are drawn from the dilution of each gradient, are spread evenly across in MRS solid medium tablets,
It is inverted, is put in 36~48h of culture under 37 DEG C of anaerobism, and observe in time.
(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 6000rpm is centrifuged 3min, 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 6000rpm of 1mL and is centrifuged 3min, incline supernatant, and washing twice, is centrifuged the supernatant that inclines, and obtains
Bacterium mud carries out PCR amplification as template, and process is as follows:
1) 20 μ L of amplification system:
Wherein template quantity is 1 μ L (0.5 μ L of 27F, 0.5 1492R μ L), and Taq enzyme MasterMix is 10 μ L, ddH2O is 7 μ
L, the primer are 27F:AGA GTT TGA TCC TGG CCT CA (SEQ ID No 2) and 1492R:GGT TAC CTT
GTT ACG ACT T(SEQ ID No 3)。
2) amplification condition:
Initial denaturation: 95 DEG C of 3min
First step denaturation: 94 DEG C of 1min
Second step annealing: 60 DEG C of 30s
Third step extends: 72 DEG C of 2min
Cycle-index: 30 circulations
4th step finally extends: 72 DEG C of 5min
5th step is kept: 12 DEG C of 10min
(2) agarose gel electrophoresis
It weighs 80mL 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, 4 μ L nucleic acid dyes are added, shakes up, bubble-free, pours into offset plate slot, after cooling 40min solidification, are placed in
In electrophoresis tank, exhaust bubble sequentially adds pcr amplification product, and 2 μ L pcr amplification products are added in each hole, and 120V 15min is run
It is taken out after glue, is placed in gel electrophoresis imager preservation of taking pictures, the number of record PCR success sample produces successful PCR
Object is placed in preservation in -20 DEG C of refrigerators.
(3) strain sequence send survey and identification
The successful sample of PCR is sent to Hua Da gene to detect, according to Hua Da gene feed back sequence results, in conjunction with
NCBI strain sequence database (http://www.ncbi.nlm.nih.gov/blast) carries out BLAST retrieval, chooses matching degree
Highest bacterial strain information carries out result record, and PCR is successful, and two plants of bacterial strains are Lactococcus lactis, is respectively designated as lactic acid cream
Coccus (Lactococcus lactis) CCFM1033 and Lactococcus lactis (Lactococcus lactis) D-XJ 4-12.
Embodiment 2: the culture of bacterial strain
Lactococcus lactis (Lactococcus lactis) CCFM1033 is inoculated in MRS solid medium, is placed in 30 DEG C
It is inverted culture 48h, observes colonial morphology；Picking single bacterium is fallen on glass slide, by smear, drying, fixation, just dye, washing, matchmaker
Microscopy after contaminating, wash, decolourizing, redying, washing, drying records form and Gram's staining result.
For the Lactococcus lactis CCFM1033 observed under oil mirror in spherical, Gram's staining result is purple, and as leather is blue
Family name's positive bacteria (see Fig. 1)；Also, bacterium colony of the bacterial strain on solid MRS culture medium is white, glossy, the smooth of the edge, impermeable
Bright, microprotrusion, median size (see Fig. 2).
Embodiment 3: growth characteristics of the bacterial strain in newborn system
1, Lactococcus lactis grows the growth curve of 12h in skimmed milk
Lactococcus lactis (Lactococcus lactis) CCFM1033 and Lactococcus lactis that -80 DEG C are saved
(Lactococcus lactis) D-XJ 4-12 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 30 DEG C for 24 hours, passage training
It supports 2~3 times, until bacteria concentration is up to 108~109CFU/mL；The bacterium solution after activating in MRS is taken out to be inoculated with by 2%~4% volume ratio
In skimmed milk, the bacterium amount in system is made to reach 107CFU/g；Inoculated sample is put into 37 DEG C of incubator and is fermented, often
It is sampled every 2h, detects the variation of fermentation process bacterium amount, as a result as shown in Figure 3.
From the figure 3, it may be seen that Lactococcus lactis (Lactococcus lactis) CCFM1033 bacterium amount after the 2h that ferments reaches
8.37×108CFU/mL, Lactococcus lactis (Lactococcus lactis) D-XJ the 4-12 bacterium amount after the 2h that ferments reach 7.95
×108Cfu/mL, therefore, growth rate is more in skimmed milk by Lactococcus lactis (Lactococcus lactis) CCFM1033
Fastly.
2, Lactococcus lactis grows pH and the titratable acidity variation of 12h in skimmed milk
Lactococcus lactis (Lactococcus lactis) CCFM1033 and Lactococcus lactis that -80 DEG C are saved
(Lactococcus lactis) D-XJ 4-12 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 30 DEG C for 24 hours, passage training
It supports 2~3 times, until bacteria concentration 108~109CFU/mL；The bacterium solution after activating in MRS is taken out to be inoculated in by 2%~4% volume ratio
In skimmed milk, the bacterium amount in system is made to reach 107CFU/g；Inoculated sample is put into 37 DEG C of incubator and is fermented, every
2h sampling, detects the variation of pH and titratable acidity in fermentation process, and experimental result is as shown in Figure 4.
As shown in Figure 4, Lactococcus lactis (Lactococcus lactis) CCFM1033 after the 12h that ferments pH be 5.46,
Acidity is 45.10 ° of T, and rate of producing acid is faster than Lactococcus lactis (Lactococcus lactis) D-XJ 4-12.
Embodiment 4: 6- phosphoric acid-beta galactosidase yield of bacterial strain
Lactococcus lactis (Lactococcus lactis) CCFM1033 and Lactococcus lactis that -80 DEG C are saved
(Lactococcus lactis) D-XJ 4-12 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 30 DEG C for 24 hours, passage training
It supports 2~3 times, until bacteria concentration 108~109CFU/mL；Bacterium solution after activating in MRS is inoculated in lactose MRS liquid by 2% volume ratio
In body culture medium, culture to logarithmic phase measures OD600Value is 1.736；It draws 100 μ L logarithmic phase bacterium solutions and carries out 6- phosphoric acid-β-half
The detection of lactoside enzyme enzyme activity, as a result as shown in Figure 4.
As shown in Figure 4,6- phosphoric acid-beta galactosidase of Lactococcus lactis (Lactococcus lactis) CCFM1033
Vigor is 96.9U, 6- phosphoric acid-beta galactosidase vigor of Lactococcus lactis (Lactococcus lactis) D-XJ 4-12
For 1.03U, therefore, 6- phosphoric acid-beta galactosidase yield of Lactococcus lactis (Lactococcus lactis) CCFM1033
It is higher.
Embodiment 5: the application of bacterial strain
Lactococcus lactis (Lactococcus lactis) CCFM1033 and Lactococcus lactis that -80 DEG C are saved
(Lactococcus lactis) D-XJ 4-12 is inoculated in respectively in MRS fluid nutrient medium, is cultivated at 30 DEG C for 24 hours, passage training
It supports 2~3 times, until bacteria concentration 108~109CFU/mL；The bacterium solution after activating in MRS is taken out to be inoculated in by 2%~4% volume ratio
In skimmed milk, the bacterium amount in system is made to reach 107cfu/g；Inoculated sample is put into in 37 DEG C of incubator the 12h that ferments,
Obtain acidified milk；It draws acidified milk and carries out volatile matter content and lactose content detection, volatile matter content such as 1 institute of table
Show, lactose content is as shown in Figure 5.
As shown in Table 1, Lactococcus lactis (Lactococcus lactis) CCFM1033 is after the 12h that ferments, in acidified milk
Acetaldehyde, the yield of biacetyl and 3-hydroxy-2-butanone it is all higher, respectively reach 1.07 μ g/kg, 5.83 μ g/kg and 31.44 μ g/kg, and
Lactococcus lactis (Lactococcus lactis) D-XJ 4-12 respectively reaches 0.29 μ g/kg, 0.29 μ g/kg and 0.62 μ g/
Kg, therefore, Lactococcus lactis (Lactococcus lactis) CCFM1033 produce fragrant characteristic good.
As shown in Figure 5, Lactococcus lactis (Lactococcus lactis) CCFM1033 is after the 12h that ferments, in acidified milk
Lactose content it is lower, be 5873.82mg/100g, and Lactococcus lactis (Lactococcus lactis) D-XJ 4-12 is
6470.60mg/100g, therefore, lactose content is significant after Lactococcus lactis (Lactococcus lactis) CCFM1033 fermentation
Lower than Lactococcus lactis (Lactococcus lactis) D-XJ 4-12, Lactococcus lactis (Lactococcus lactis)
CCFM1033 lactose utilization ability is strong.
Volatile matter content after 1 slowly fermenting 12h of table
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
<110>Southern Yangtze University
<120>one plant heights produce 6- phosphoric acid-beta galactosidase Lactococcus lactis and its application
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caatccgaac tgagaatggt tttaagagat tagctaaaca tcactgtctc gcgactcgtt 180
gtaccatcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc 240
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tagcactcat cgtttacggc gtggactacc agggtatcta atcctgtttg ctccccacgc 660
tttcgagcct cagtgtcagt tacaggccag agagccgctt tcgccaccgg tgttcctcca 720
tatatctacg catttcaccg ctacacatgg aattccactc tcctctcctg cactcaagtc 780
taccagtttc caatgcatac aatggttgag ccactgcctt ttacaccaga cttaataaac 840
cacctgcgct cgctttacgc ccaataaatc cggacaacgc tcgggaccta cgtattaccg 900
cggctgctgg cacgtagtta gccgtccctt tctgggtagt taccgtcact tgatgagctt 960
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Claims (10)
1. one plant of Lactococcus lactis (Lactococcus lactis), which is characterized in that the Lactococcus lactis
(Lactococcus lactis) was preserved in Guangdong Province's Culture Collection, deposit number on 09 20th, 2018
For GDMCC No.60450, preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
2. the leavening containing Lactococcus lactis (Lactococcus lactis) described in claim 1.
3. leavening as claimed in claim 2, which is characterized in that the preparation method of the leavening is by claim 1 institute
It states Lactococcus lactis (Lactococcus lactis) to be seeded in culture medium, cultivate at 30 DEG C to Lactococcus lactis
(Lactococcus lactis) viable bacteria concentration is not less than 1 × 108CFU/mL obtains culture solution；By medium centrifugal, bacterium is obtained
Body；Thallus is resuspended to bacteria concentration with freeze drying protectant not less than 1 × 10 with after buffer solution for cleaning 2~3 times9CFU/mL is obtained
Bacteria suspension；Suspension is dry, obtain leavening.
4. leavening described in Lactococcus lactis described in claim 1 (Lactococcus lactis) or Claims 2 or 3 is being made
Application in terms of standby food.
5. application as claimed in claim 4, which is characterized in that the food is to use Lactococcus lactis described in claim 1
The fermented dairy product that leavening described in (Lactococcus lactis) or Claims 2 or 3 produces.
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 or koumiss.
7. a kind of preparation method of acidified milk, is characterized in that, the method is to use Lactococcus lactis described in claim 1
Leavening described in (Lactococcus lactis) or Claims 2 or 3.
8. preparation method as claimed in claim 7, which is characterized in that the method be by cow's milk after homogeneous, pasteurize
It is cooling, obtain fermentation raw material；Lactococcus lactis (Lactococcus lactis) described in claim 1 is inoculated in fermentation raw material
Or leavening described in Claims 2 or 3 ferments, and obtains acidified milk.
9. preparation method as claimed in claim 7 or 8, which is characterized in that the method be by cow's milk 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；By the cow's milk after sterilizing
21~30 DEG C are cooled to, fermentation raw material is obtained；Lactococcus lactis described in claim 1 is inoculated in fermentation raw material
Ferment at 37 DEG C 12h after leavening described in (Lactococcus lactis) or Claims 2 or 3, obtains acidified milk；It will be sour
Milk places 24~48h, the acidified milk finished product after obtaining after-ripening under conditions of 4 DEG C.
10. the acidified milk that any preparation method of application claim 7-9 is prepared.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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