CN108220201A - Streptococcus thermophilus streptococcus thermophilus benshit - Google Patents
Streptococcus thermophilus streptococcus thermophilus benshit Download PDFInfo
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- CN108220201A CN108220201A CN201810161956.8A CN201810161956A CN108220201A CN 108220201 A CN108220201 A CN 108220201A CN 201810161956 A CN201810161956 A CN 201810161956A CN 108220201 A CN108220201 A CN 108220201A
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- streptococcus thermophilus
- benshit
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- gained
- thermophilus
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- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 90
- 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 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 239000008101 lactose Substances 0.000 claims abstract description 15
- 235000013365 dairy product Nutrition 0.000 claims abstract description 10
- 241000194017 Streptococcus Species 0.000 claims abstract description 5
- 239000012530 fluid Substances 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 239000006228 supernatant Substances 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 19
- 235000013618 yogurt Nutrition 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 17
- 229910021641 deionized water Inorganic materials 0.000 claims description 17
- 239000002054 inoculum Substances 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 16
- 239000012141 concentrate Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 235000020183 skimmed milk Nutrition 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 13
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- 230000001954 sterilising effect Effects 0.000 claims description 7
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 10
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- 230000006920 protein precipitation Effects 0.000 description 3
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
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- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
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- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 229960002518 gentamicin Drugs 0.000 description 1
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 1
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
- 229960001180 norfloxacin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
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- 238000013456 study Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical class S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
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- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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
- A23C19/00—Cheese; Cheese preparations; Making thereof
-
- 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
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
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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)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
Abstract
The present invention discloses a kind of streptococcus thermophilusStreptococcus thermophilusBenshit and its application.The streptococcus thermophilusStreptococcus thermophilus Benshit is sifted out from Xinjiang Dairy pimple, and deposit number is CGMCC No.12098.Streptococcus thermophilusStreptococcus thermophilusFor Benshit when lactose is primary carbon source, EPS yield can reach 160 mg/L；It is used to individually produce EPS as leavening, or it is used to produce streptococcus thermophilus fermentation dairy products as leavening, due to EPS yield height, therefore when it is as fermented dairy product leavening, the viscosity of fermented dairy product can be significantly improved, improve the structural state of fermented dairy product, make the fermented dairy product obtained by fermentation that there is good flavor.
Description
Technical field
The present invention relates to a kind of streptococcus thermophilus and application thereof, more particularly to a kind of streptococcus thermophilus
Streptococcus thermophilus Benshit and leavening is utilized it as producing exocellular polysaccharide, thermophilus
Bacterium fermented dairy product or streptococcus thermophilus cheese etc..
Background technology
Streptococcus thermophilus (Streptococcus thermophilus) is a kind of important industrial lactic acid bacteria (Lactic
Acid Bacteria, LAB), unique generally recognized as safe strain is determined as in 93 streptococcuses by USA and EU.As one
The important commercial fermentation agent of kind, is widely used in the production of the fermented dairy products such as Yoghourt and cheese.
Streptococcus thermophilus can synthesize exocellular polysaccharide (Extrapolysaccharide, hereinafter referred to as EPS), and EPS is in life
The water-soluble polysaccharide that cell wall is outer, is secreted into environment is secreted into long metabolic process.In dairy products production, streptococcus thermophilus
And its viscosity, the stability that the EPS generated has, it can effectively improve the rheological properties and matter of streptococcus thermophilus fermentation breast
Structure characteristic, avoids whey from being precipitated；Meanwhile lubrication sense and flavor, still, current streptococcus thermophilus can be improved
(Streptococcus thermophilus) is highest in the exocellular polysaccharide of synthesis under conditions of carbon source is fermented in lactose
Only~90mg/L[1]。
And the problems such as domestic prepared to streptococcus thermophilus fermentation agent is still not perfect, and the fermented dairy product quality of production is not high
Cause commercial application degree not high.With the development of China's dairy industry, the demand to streptococcus thermophilus fermentation agent is more next
Bigger, application prospect is boundless.Become a heat of current research in relation to correlative studys such as screening high yield EPS streptococcus thermophilus
Point has very important economic and social benefit.
In conclusion current streptococcus thermophilus produces the low technical problem of EPS amounts due to existing, so that thermophilus
The performance of viscosity, texture and the mouthfeel of fermented dairy product of final gained etc. is influenced when bacterium produces for fermented dairy product,
Therefore, active demand is a kind of produces the high streptococcus thermophilus of EPS amounts, it is enable effectively to change when being produced for fermented dairy product
Viscosity, texture and mouthfeel of kind fermented dairy product etc..
Bibliography：
1.Li,D.；Li,J.；Zhao,F.；Wang,G.；Qin,Q.；Hao,Y.,The influence of
fermentation condition on production and molecular mass of EPS produced by
Streptococcus thermophilus 05-34 in milk-based medium.Food Chem 2016,197,367-
72.
2. Feng is small gentle, Xia Yongjun, Wang Guangqiang, the screening of the extracellular polysaccharide vegetable lactobacillus of and the work of Thick many candies in Ai Lian
Journal of Sex Research [J] Food Sciences, 2016,37 (13):125-129.
Invention content
One of the object of the invention is carried to solve the streptococcus thermophilus production low technical problem of EPS amounts of the prior art
The high streptococcus thermophilus Streptococcus thermophilus Benshit of EPS amounts are produced for a kind of.
The second purpose of the present invention is utilize a kind of above-mentioned streptococcus thermophilus Streptococcus thermophilus
Benshit is used to individually produce EPS as leavening.
The three of the object of the invention are to utilize a kind of above-mentioned streptococcus thermophilus Streptococcus thermophilus
Benshit is used to produce streptococcus thermophilus fermentation dairy products as leavening, due to streptococcus thermophilus Streptococcus
Thermophilus Benshit ferment under conditions of lactose is carbon source, and production EPS reaches as high as 160mg/L, so as to be conducive to
Improve the viscosity and quality of streptococcus thermophilus fermentation dairy products, in the food industry with good value.
Technical scheme of the present invention
A kind of streptococcus thermophilus Streptococcus thermophilus Benshit are that one kind belongs to bacterium class hammer
The bacterial strain of Pseudomonas, which is isolated from Chinese hotan milk pimple, on January 22nd, 2016 in the micro- life of China
Object culture presevation administration committee's common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) preservation,
Number is CGMCC No.12098.
Above-mentioned streptococcus thermophilus Streptococcus thermophilus Benshit bacterial strains have following microorganism
Learn feature：
(1), morphological feature
Streptococcus thermophilus Streptococcus thermophilus Benshit CGMCC No.12098 bacterial strain grams
It dyes and is positive for purple, it is that chain is spherical to observe cellular morphology under the microscope, no sporulation；
(2), feature is learned in culture
In the flat lining out separation of LM7,42 DEG C of anaerobism cultivate 24-48h, and strain growth is good, and bacterium colony is oval
Or similar round, neat in edge, bacterium colony size are generally 2~3mm, milky；
(3), physiological and biochemical property
Catalase and oxydase reaction are negative, glucose fermentation not aerogenesis；
(4), drug resistance
To ampicillin, Amoxicillin, tetracycline, erythromycin, minocycline, vancomycin, Compound New Nomin and crin
Mycin medicaments insensitive, it is insensitive to gentamicin, kanamycins, acidum nalidixicum and Norfloxacin drug, specifically it see the table below：
Streptococcus thermophilus Benshit drug-resistant tests
It is noted in table：Represent drug resistance；+ expression medium sensitivity；++ represent sensitive
(5), carbon assimilation
It is positive：Lactose, galactolipin, glucose；
It is negative：L- xyloses, D- xyloses；
(6), motility：Without motion；
(7), optimal temperature is grown：37℃；
(8), stability：The strain still keeps character to stablize after passage 20 times, therefore stablizes with good passage
Property.
It is used to give birth to by the use of above-mentioned streptococcus thermophilus Streptococcus thermophilus Benshit as leavening
The method for producing EPS, specifically comprises the following steps：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% (v/v) inoculum concentration in sterilized non-fat cow's milk culture medium, and 42 DEG C of quiescent culture 10h are activated, and are obtained
The streptococcus thermophilus Streptococcus thermophilus Benshit activated；
The streptococcus thermophilus Streptococcus thermophilus being preserved in skimmed milk culture medium
Benshit, by every liter of calculating, the quantity containing streptococcus thermophilus Streptococcus thermophilus Benshit is at least
For 1*1010Cfu (Colony-Forming Units refer to the bacterial community sum in unit volume)；
(2), by the streptococcus thermophilus Streptococcus thermophilus Benshit activated by 3% (v/v)
In inoculum concentration access LM17 fluid nutrient mediums, 37 DEG C of quiescent culture 12-24h obtain seed liquor；
(3), fermented and cultured
Seed liquor obtained by step (2) is inoculated in the inoculum concentration of 3% (v/v) in LM17 fluid nutrient mediums, at 37 DEG C
Under the conditions of quiescent culture for 24 hours, obtain zymotic fluid；
(4), the zymotic fluid obtained by step (3) is subjected to centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, goes to sink
It forms sediment, collects the supernatant 1 of gained；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 1 of above-mentioned gained is carried out being concentrated into original volume
1/3, obtain concentrate 1, by the concentrate 1 of gained after 10min is to inactivate the enzyme of degradable polysaccharide in boiling water bath, Ran Hou
The aqueous solution for the trichloroacetic acid that concentration of volume percent is 80% (w/v) is added in concentrate 1 to trichloroacetic acid final concentration of 4%
(w/v), it stands overnight, carrying out centrifugation 20min for 9000g again controlled at 4 DEG C, rotating speed removes protein precipitation, collects institute
The supernatant 2 obtained；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 2 of above-mentioned gained is carried out being concentrated into original volume
1/3, obtain concentrate 2, in the concentrate 2 of gained add in concentration of volume percent be 95% (v/v) ethanol solution extremely
Final concentration of 75% (v/v) of ethyl alcohol, 4 DEG C stand for 24 hours, carry out centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, receive
Collect the precipitation of gained；
By the precipitation deionized water dissolving of above-mentioned gained, centrifugation 20min is carried out for 8000g controlled at 4 DEG C, rotating speed
It goes to precipitate, collects the supernatant 3 of gained；
The dosage of above-mentioned precipitation and deionized water by weight calculating, precipitates：Deionized water is 1：30-50；
The supernatant 3 of above-mentioned gained with deionized water is dialysed 72h, it is primary to change deionized water per 4h, after dialysis, obtains
Obtain Thick many candies solution；
(5), by step (4) obtained by Thick many candies solution, controlled at -40 DEG C progress pre-freeze 3-5h, then control temperature
It spends and carries out first time dry 20h for -15 DEG C -20 DEG C, then carry out second of dry 5-10h again controlled at 4 DEG C, it is final to obtain
To EPS.
Above-mentioned streptococcus thermophilus Streptococcus thermophilus Benshit pass through routine as leavening
Method prepares streptococcus thermophilus fermentation dairy products, particularly thermophilus bacteria yoghurt and streptococcus thermophilus cheese.
Beneficial effects of the present invention
A kind of streptococcus thermophilus Streptococcus thermophilus Benshit of the present invention are carbon source in lactose
Under conditions of ferment, production EPS amounts reach as high as 160mg/L, and EPS extraction process is simple, can realize heavy industrialization answer
With.
Further, a kind of streptococcus thermophilus Streptococcus thermoph ilus Benshit of the invention make
For leavening for producing fermented dairy product, when as leavening for producing thermophilus bacteria yoghurt, due to its generation
The EPS of high yield can significantly improve the texture characteristics such as the structural state of thermophilus bacteria yoghurt, the thermophilus for the gained that ferments
Bacteria yoghurt viscosity is good, makes product that need not add stabilizer, meets demand of the consumer to additive-free low fat sour milk.
Description of the drawings
The growth of Fig. 1, streptococcus thermophilus Streptococcus thermophilus Benshit under different carbon source is bent
Line schematic diagram；
The EPS yield of Fig. 2, streptococcus thermophilus Streptococcus thermophilus Benshit under different carbon source
Schematic diagram.
Specific embodiment
Below by specific embodiment and with reference to attached drawing, the present invention will be further described, but is not intended to limit this hair
It is bright.
Experimental method used in following embodiments is conventional method unless otherwise specified；Used material,
Reagent etc., is commercially available unless otherwise specified.
Used in various embodiments of the present invention：
LM17 fluid nutrient mediums：By every liter of calculating, 5g containing tryptone, soy peptone 5g, yeast extract powder 2.5g, ox
Meat extract 5g, lactose 20g, β-phosphoglycerol disodium five hydrate 19g, epsom salt 0.58g, vitamin C 0.5g, surplus
For water；115 DEG C of sterilizing 20min；
LM17 solid mediums, by every liter of calculating, 5g containing tryptone, soy peptone 5g, yeast extract powder 2.5g, ox
Meat extract 5g, lactose 20g, β-phosphoglycerol disodium five hydrate 19g, epsom salt 0.58g, vitamin C 0.5g, agar
Powder 20g, surplus are water, 115 DEG C of sterilizing 20min；
GM17 fluid nutrient mediums：Lactose in LM17 fluid nutrient mediums is replaced with into glucose；
GM17 solid mediums：Lactose in LM17 solid mediums is replaced with into glucose；
GalM17 fluid nutrient mediums：Lactose in LM17 fluid nutrient mediums is replaced with into galactolipin；
GalM17 solid mediums：Lactose in LM17 solid mediums is replaced with into galactolipin.
Sterilized non-fat cow's milk culture medium：The skimmed milk powder aqueous solution of 120g/L, 115 DEG C of sterilizing 15min.
The assay method of EPS in the embodiment of the present invention：Using Phenol sulfuric acid procedure, it is specifically shown in bibliography 1.
The assay method of Thick many candies solution：Using Phenol sulfuric acid procedure, it is specifically shown in bibliography 2.
Embodiment 1
The cultural method of streptococcus thermophilus Streptococcus thermophilus Benshit, is as follows：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% (v/v) inoculum concentration in sterilized non-fat cow's milk culture medium, and 42 DEG C of quiescent culture 10h are activated, and are obtained
The streptococcus thermophilus Streptococcus thermophilus Benshit activated；
The streptococcus thermophilus Streptococcus thermophilus being preserved in skimmed milk culture medium
Benshit, by every liter of calculating, the quantity containing streptococcus thermophilus Streptococcus thermophilus Benshit is at least
For 1*1010cfu；
(2), by the streptococcus thermophilus Streptococcus thermophilus activated obtained by step (1)
Benshit is respectively connected to by 3% (v/v) inoculum concentration in LM17, GM17 and GalM17 fluid nutrient medium, 37 DEG C of quiescent cultures for 24 hours,
Respectively obtain the culture solution corresponding to LM17, GM17 and GalM17 fluid nutrient medium.
Every streptococcus thermophilus of the 2h using spectrophotometer in 600nm measures culture solution in above-mentioned incubation
The concentration OD of Streptococcus thermophilus Benshit600, with streptococcus thermophilus Streptococcus
The concentration OD of thermophilus Benshit600Incubation time is mapped to obtain streptococcus thermophilus Streptococcus
Growth curves of the thermophilus Benshit in LM17, GM17 and GalM17 as shown in Figure 1, from figure 1 it appears that
Streptococcus thermophilus Streptococcus thermophilus Benshit are grown rapidly in LM17 fluid nutrient mediums, on a 4h left sides
The right logarithmic phase, 10h or so that enters enters stationary phase, but slow-growing in GM17 and GalM17 fluid nutrient mediums, thus says
Bright lactose is the most suitable carbon source of hot streptococcus Streptococcus thermophilus Benshit growths.
Embodiment 2
Fermenting and producing EPS is carried out using streptococcus thermophilus Streptococcus thermophilus Benshit, specifically
Step is as follows：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% inoculum concentration in the LM17 solid mediums of sterilizing, and 37 DEG C of quiescent cultures are activated for 24 hours, continuous living
Changed for two generations, obtain the streptococcus thermophilus Streptococcus thermophilus Benshit activated；
(2), by the streptococcus thermophilus Streptococcus thermophilus for obtaining having activated obtained by step (1)
Benshit；It is inoculated in the triangular flask equipped with LM17 fluid nutrient mediums by the inoculum concentration of 3% (v/v), 37 DEG C of quiescent culture 12h,
Obtain seed liquor；
(3), fermented and cultured：By the seed liquor obtained by step (2) with the inoculum concentration of 3% (v/v) be inoculated in respectively LM17,
In GM17 and GalM17 fluid nutrient mediums, quiescent culture for 24 hours, obtains corresponding to LM17, GM17 and GalM17 under the conditions of 37 DEG C
The zymotic fluid of fluid nutrient medium；
(4), temperature will be controlled respectively corresponding to LM17, GM17 and GalM17 solution culture fermentation liquid obtained by step (3)
It is that 8000g carries out centrifugation 20min to spend for 4 DEG C, rotating speed, collects supernatant 1a, 1b, 1c of gained respectively, respectively in boiling water bath
After 10min, respectively in supernatant 1a, 1b, 1c add in concentration of volume percent be 80% (w/v) trichloroacetic acid solution extremely
Final concentration of 4% (w/v) of trichloroacetic acid, stands overnight respectively, is carried out respectively controlled at 4 DEG C, rotating speed for 9000g again
20min is centrifuged, removes protein precipitation, collects supernatant 2a, 2b, 2c of gained respectively；
Then supernatant 2a, 2b, 2c of above-mentioned gained are changed into deionized water one respectively with deionized water dialysis 72h per 4h
It is secondary, after dialysis, obtain Thick many candies solution；
The Thick many candies solution of above-mentioned gained is taken respectively, and using UV-1600PC types ultraviolet-uisible spectrophotometer, (Shanghai U.S. composes
Up to Instrument Ltd.), use Phenol sulfuric acid procedure[2]EPS contents are measured, measuring system, (described treats test sample for 1mL samples to be tested
Product are the Thick many candies solution that obtains after above-mentioned dialysis), the phenol solution of 0.5mL 3% (v/v) and the 5mL concentrated sulfuric acids, gained
Corresponding to the EPS contents schematic diagram in the zymotic fluid of LM17, GM17 and GalM17 fluid nutrient medium as shown in Fig. 2, can from Fig. 2
To find out, streptococcus thermophilus Streptococcus thermophilus Benshit grow best, EPS when carbon source is lactose
Yield can reach 160mg/L；
(5), by step (4) obtained by Thick many candies solution, controlled at -40 DEG C progress pre-freeze 3-5h, then control temperature
It spends and carries out first time dry 20h for -15 DEG C -20 DEG C, then carry out second of dry 5-10h again controlled at 4 DEG C, it is final to obtain
To EPS.
Embodiment 3
Streptococcus thermophilus Streptococcus thermophilus Benshit fermenting and producing EPS, are as follows：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% inoculum concentration in the LM17 solid mediums of sterilizing, and 37 DEG C of quiescent cultures are activated for 24 hours, continuous living
Changed for two generations, obtain the streptococcus thermophilus Streptococcus thermophilus Benshit activated；
(2), by the streptococcus thermophilus Streptococcus thermophilus for obtaining having activated obtained by step (1)
Benshit；It is inoculated in the triangular flask equipped with LM17 fluid nutrient mediums by the inoculum concentration of 3% (v/v), 37 DEG C of quiescent culture 12h,
Obtain seed liquor；
(3), the seed liquor obtained by step (2) is inoculated in the inoculum concentration of 3% (v/v) in LM17 fluid nutrient mediums,
Quiescent culture for 24 hours, obtains zymotic fluid under the conditions of 37 DEG C；
(4), the zymotic fluid obtained by step (3) is subjected to centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, goes to sink
It forms sediment to remove thalline and coagulated protein, collects the supernatant 1 of gained；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 1 of above-mentioned gained is carried out being concentrated into original volume
1/3, obtain concentrate 1, by the concentrate 1 of gained after 10min is to inactivate the enzyme of degradable polysaccharide in boiling water bath, Ran Hou
The aqueous solution for the trichloroacetic acid that concentration of volume percent is 80% (w/v) is added in concentrate 1 to trichloroacetic acid final concentration of 4%
(w/v), it stands overnight, carrying out centrifugation 20min for 9000g again controlled at 4 DEG C, rotating speed removes protein precipitation, collects institute
The supernatant 2 obtained；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 2 of above-mentioned gained is carried out being concentrated into original volume
1/3, obtain concentrate 2, in the concentrate 2 of gained add in concentration of volume percent be 95% (v/v) ethanol solution extremely
Final concentration of 75% (v/v) of ethyl alcohol, 4 DEG C stand for 24 hours, carry out centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, receive
Collect the precipitation of gained；
By the precipitation deionized water dissolving of above-mentioned gained, centrifugation 20min is carried out for 8000g controlled at 4 DEG C, rotating speed
It goes to precipitate, collects the supernatant 3 of gained；
The dosage of above-mentioned precipitation and deionized water by weight calculating, precipitates：Deionized water is 1：30-50；
The supernatant 3 of above-mentioned gained with deionized water is dialysed 72h, it is primary to change deionized water per 4h, after dialysis, obtains
Obtain Thick many candies solution；
(5), by step (4) obtained by Thick many candies solution, controlled at -40 DEG C progress pre-freeze 3-5h, then control temperature
It spends and carries out first time dry 20h for -15 DEG C -20 DEG C, then carry out second of dry 5-10h again controlled at 4 DEG C, it is final to obtain
To EPS.
Embodiment 4
Streptococcus thermophilus Streptococcus thermophilus Benshit are used to prepare thermophilic chain as leavening
The method of coccus Yoghourt, is as follows：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% (v/v) inoculum concentration in sterilized non-fat cow's milk culture medium, and 42 DEG C of quiescent culture 10h are activated, and are obtained
The streptococcus thermophilus Streptococcus thermophilus Benshit activated；
The streptococcus thermophilus Streptococcus thermophilus being preserved in skimmed milk culture medium
Benshit, by every liter of calculating, the quantity containing streptococcus thermophilus Streptococcus thermophilus Benshit is at least
For 1*1010cfu；
(2), by the streptococcus thermophilus Streptococcus thermophilus for obtaining having activated obtained by step (1)
Benshit；It is inoculated in the triangular flask equipped with fresh milk by the inoculum concentration of 3-5% (v/v), 40-42 DEG C of quiescent culture to ox
Curdling is consolidated, and is then controlled 4 DEG C of progress after-ripening 16h of temperature, is obtained thermophilus bacteria yoghurt.
The thermophilus bacteria yoghurt quality of above-mentioned gained is sticky, and few whey is precipitated, in good taste.It is surveyed using Ubbelohde viscometer
Fixed Yoghourt viscosity is 4570mPas, and Yoghourt retention ability is 50.8%.
In conclusion the streptococcus thermophilus Streptococcus thermophilus Benshit of the present invention, in lactose
Best to be grown during carbon source, EPS yield reaches as high as 160mg/L, which significantly improves as ferment agent for sour milk
Yoghourt viscosity, Yoghourt viscosity improve the structural state of Yoghourt up to 4570mPas, and Yoghourt retention ability is 50.8%, so as to
Make the Yoghourt obtained by fermentation that there is good flavor
Above said content is only the basic explanation under present inventive concept, and what technical solution according to the present invention was made appoints
What equivalent transformation, is within the scope of protection of the invention.
Claims (6)
1. a kind of streptococcus thermophilus Streptococcus thermophilus Benshit, deposit number CGMCC
No.12098。
2. streptococcus thermophilus Streptococcus thermophilus Benshit as described in claim 1 are as leavening
For producing EPS or for producing streptococcus thermophilus fermentation dairy products.
3. streptococcus thermophilus Streptococcus thermophilus Benshit as claimed in claim 2 are as leavening
For producing the method for EPS, it is characterised in that specifically comprise the following steps：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% (v/v) inoculum concentration in sterilized non-fat cow's milk culture medium, and 42 DEG C of quiescent culture 10h are activated, and are obtained
The streptococcus thermophilus Streptococcus thermophilus Benshit activated；
The streptococcus thermophilus Streptococcus thermophilus being preserved in skimmed milk culture medium
Benshit, by every liter of calculating, the quantity containing streptococcus thermophilus Streptococcus thermophilus Benshit is at least
For 1*1010cfu；
(2), the streptococcus thermophilus Streptococcus thermophilus Benshit activated are inoculated with by 3% (v/v)
In amount access LM17 fluid nutrient mediums, 37 DEG C of quiescent culture 12-24h obtain seed liquor；
(3), fermented and cultured
Seed liquor obtained by step (2) is inoculated in the inoculum concentration of 3% (v/v) in LM17 fluid nutrient mediums, in 37 DEG C of conditions
Lower quiescent culture for 24 hours, obtains zymotic fluid；
(4), the zymotic fluid obtained by step (3) is subjected to centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, collects gained
Supernatant 1；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 1 of above-mentioned gained is carried out being concentrated into the 1/ of original volume
3, obtain concentrate 1；
It is 80% (w/v) that the concentrate 1 of gained is added in concentration of volume percent in concentrate 1 after 10min in boiling water bath
Trichloroacetic acid aqueous solution to trichloroacetic acid final concentration of 4% (w/v), stand overnight, then again controlled at 4 DEG C, turns
Speed carries out centrifugation 20min for 9000g, collects the supernatant 2 of gained；
Using the method for spin concentration, controlled at 50 DEG C, the supernatant 2 of above-mentioned gained is carried out being concentrated into the 1/ of original volume
3, obtain concentrate 2；
The ethanol solution that addition concentration of volume percent is 95% (v/v) in the concentrate 2 of gained is final concentration of to ethyl alcohol
75% (v/v), 4 DEG C stand for 24 hours, carry out centrifugation 20min for 8000g controlled at 4 DEG C, rotating speed, collect the precipitation of gained；
By the precipitation deionized water dissolving of above-mentioned gained, centrifugation 20min is carried out for 8000g controlled at 4 DEG C, rotating speed and go to sink
It forms sediment, collects the supernatant 3 of gained；
The dosage of above-mentioned precipitation and deionized water by weight calculating, precipitates：Deionized water is 1：30-50；
The supernatant 3 of above-mentioned gained with deionized water is dialysed 72h, it is primary to change deionized water per 4h, after dialysis, obtains thick
Polysaccharide solution；
(5), by step (4) obtained by Thick many candies solution, controlled at -40 DEG C progress pre-freeze 3-5h, then controlled at -
15 DEG C -20 DEG C carry out dry 20h for the first time, then carry out second of dry 5-10h controlled at 4 DEG C again, finally obtain
EPS。
4. streptococcus thermophilus Streptococcus thermophilus Benshit as claimed in claim 3 are as leavening
For producing the method for EPS, it is characterised in that the sterilized non-fat cow's milk culture medium described in step (1)：The skimmed milk of 120g/L
Amidin, 115 DEG C of sterilizing 15min；
LM17 fluid nutrient mediums described in step (2), step (3)：By every liter of calculating, 5g containing tryptone, soy peptone
5g, yeast extract powder 2.5g, beef extract 5g, lactose 20g, β-five hydrate 19g of phosphoglycerol disodium, epsom salt
0.58g, vitamin C 0.5g, surplus are water；In use, 115 DEG C of sterilizing 20min.
5. streptococcus thermophilus Streptococcus thermophilus Benshit as claimed in claim 2 are as leavening
For producing streptococcus thermophilus fermentation dairy products, the streptococcus thermophilus fermentation dairy products are thermophilus bacteria yoghurt or thermophilic
Streptococcus cheese.
6. streptococcus thermophilus Streptococcus thermophilus Benshit as claimed in claim 5 are as leavening
It is used to prepare the method for thermophilus bacteria yoghurt, it is characterised in that be as follows：
(1), the streptococcus thermophilus Streptococcus thermophilus that will be preserved in skimmed milk culture medium
Benshit is accessed by 3% (v/v) inoculum concentration in sterilized non-fat cow's milk culture medium, and 42 DEG C of quiescent culture 10h are activated, and are obtained
The streptococcus thermophilus Streptococcus thermophilus Benshit activated；
The streptococcus thermophilus Streptococcus thermophilus being preserved in skimmed milk culture medium
Benshit, by every liter of calculating, the quantity containing streptococcus thermophilus Streptococcus thermophilus Benshit is at least
For 1*1010cfu；
(2), by the streptococcus thermophilus Streptococcus thermophilus for obtaining having activated obtained by step (1)
Benshit；It is inoculated in the triangular flask equipped with fresh milk by the inoculum concentration of 3-5% (v/v), 40-42 DEG C of quiescent culture to ox
Curdling is consolidated, and is then controlled 4 DEG C of progress after-ripening 16h of temperature, is obtained thermophilus bacteria yoghurt.
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| CN110408663A (en) * | 2019-08-01 | 2019-11-05 | 浙江一鸣食品股份有限公司 | A kind of lactic acid bacteria of high-yield extracellular polysaccharide and preparation method and application |
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| CN111411114B (en) * | 2019-12-30 | 2022-09-06 | 上海理工大学 | Method for regulating content of exopolysaccharide of streptococcus thermophilus |
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