CN109022301A - A kind of curdlan producing bacterial strain and its application - Google Patents
A kind of curdlan producing bacterial strain and its application Download PDFInfo
- Publication number
- CN109022301A CN109022301A CN201810425602.XA CN201810425602A CN109022301A CN 109022301 A CN109022301 A CN 109022301A CN 201810425602 A CN201810425602 A CN 201810425602A CN 109022301 A CN109022301 A CN 109022301A
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- CN
- China
- Prior art keywords
- curdlan
- bacterial strain
- agrobacterium
- method described
- fermentation
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
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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
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
- A23L29/271—Curdlan; beta-1-3 glucan; Polysaccharides produced by agrobacterium or alcaligenes
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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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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Abstract
The invention discloses a kind of curdlan producing bacterial strain and its application, curdlan polysaccharide producing strains HS-5 is agrobacterium (Agrobacterium sp.), and deposit number is CGMCC NO.15477.Agrobacterium (Agrobacterium sp.) bacterial strain HS-5 shows the good characteristics such as yield is high, molecular weight is small, gel brittleness is good by fermenting, extracting curdlan obtained, can preferably meet the industrialized production and the market demand of curdlan.
Description
Technical field
The invention belongs to Microbial Breeding technical fields, and in particular to a kind of curdlan producing bacterial strain and its application.
Background technique
Curdlan generated by rhizobium (Rhizobium sp.) or agrobacterium (Agrobacterium sp.) one
Kind is by glucose building blocks with β -1, and novel biopolymer, is after Huang made of the link of 3-D- glucoside bond
The third microbial fermentation that being approved by the FDA in the United States after virgin rubber, gellan gum can be used in food industry produces extracellular more
Sugar is used in food with the unique good characteristics such as colloidality, frost resistance, anti-dehydrating that are heated into as stabilizer, solidifying
Gu agent and thickener etc. can obviously improve the quality including foods such as bean product, Flour product, meat products, it is deeply loved by the public.
It is studied early in application of the nineteen sixty-eight Japan Takeda company just to the production of curdlan and in food, it 1989, can
Right glue is obtained as a kind of novel food additives to start to use in the country such as Japan, South Korea.1966, U.S. FDA was formally criticized
Quasi- curdlan is as a kind of food additives using in the food industry.Japanese Takeda company is that current production scale is maximum
And produced colloidality can be best company, favor of the curdlan produced by domestic and international market.Its annual output is at 1000 tons
More than, but supply falls short of demand.The excellent performances such as colloidality are heated into since curdlan is good leads to its demand year by year
Increase.
In China, Shandong Zhong Ke Bioisystech Co., Ltd also realizes curdlan industrialized production, but producing can obtain
The performance of right glue has compared biggish gap with Takeda company of Japan.Due to the bacterial strain for being used for curdlan and producing domestic at present
Low output, and producing colloidality can be poor, causes high production cost, application effect poor, it is extensive in the market to significantly limit it
Using.Therefore, breeding high-yield and curdlan bacterial strain best in quality are a very important job.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of curdlan producing bacterial strain and its applications.The bacterial strain is produced
Curdlan performance yield is high, molecular weight is small, gel brittleness well equal good characteristics can preferably meet industrialized production
And the market demand.
In order to solve the above technical problems, the present invention provides a kind of curdlan polysaccharide producing bacterial strain agrobacterium
(Agrobacterium.sp) bacterial strain HS-5 is now preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and number is CGMCC NO.15477, and the preservation time is 2018 3
The moon 22.
The morphological feature of agrobacterium (Agrobacterium.sp) bacterial strain HS-5 of the invention are as follows: colonial morphology is circle
Shape, surface be smooth, micro- grand, neat in edge, and microscope hypothallus is in uniform rod-shaped.The bacterium is Gram-negative bacteria.
The present invention produce curdlan bacterial strain agrobacterium (Agrobacterium.sp) bacterial strain HS-5 acquisition with
Alcaligenes faecalis var.myxogenes 10C3 (IFO13714) is starting strain, through ultraviolet mutagenesis, nitroso
Guanidine mutagenesis, sodium nitrite mutagenesis and ARTP mutagenesis obtain bacterial strain HS-5 by the method for plate primary dcreening operation, shaking flask secondary screening.
In the present invention to the screening and culturing medium prepared in agrobacterium (Agrobacterium.sp) bacterial strain HS-5 method into
Optimization is gone.The ingredient of original screening and culturing medium is glucose 10-20g/L, yeast extract 1-4g/L, aniline blue 0.02-
0.06g/L, agar powder 15-20g/L, pH=6.0-7.0. have found the same of the aobvious blue of bacterial strain during with the Screening of Media
When bottom plate also show blue, influence the screening of superior strain.It is found through correlative study, this is because curdlan producing bacterial strain is in life
Acid can be produced in growth process, and aniline blue shows blue in acid condition and can deepen.In order to improve the acid influence for causing colour developing, this
A certain amount of urea is added in invention into original culture medium, ammonia can be generated after decomposing due to urea, can be adjusted to a certain extent
PH, the results show that the problem of culture medium that a certain amount of urea is added improves bottom plate colour developing well, greatly improves screening
Efficiency.Culture medium after optimization is glucose 10-20g/L, yeast extract 1-4g/L, urea 1-4g/L, aniline blue 0.02-
0.06g/L, agar powder 15-20g/L, pH=6.5-7.4. preferably, glucose 10g/L, yeast extract 2g/L, urea 2g/
L, aniline blue 0.05g/L, agar powder 18g/L, pH=7.2.
The present invention reflects to agrobacterium (Agrobactrim sp.) bacterial strain HS-5 Physiology and biochemistry and 16S rDNA gene
It is fixed, determine that the bacterial strain is Agrobacterium, Physiology and biochemistry qualification result is shown in Table 1.
1 Physiology and biochemistry qualification result of table
Note :+indicate positive ,-indicate negative；
Agrobacterium (Agrobactrim sp.) bacterial strain HS-5 of the present invention 16S rDNA gene order (1,
403bp) as shown in SEQ ID NO:1:
ttcgggtaaaaccaactcccatggtgtgacgggcggtgtgtacaaggccC
GGGAACGTATTCACCGCAGCATGCTGATCTGCGATTACTAGCGATTCCAA
CTTCATGCACTCGAGTTGCAGAGTGCAATCCGAACTGAGATGGCTTTTGG
AGATTAGCTCGACATCGCTGTCTCGCTGCCCACTGTCACCACCATTGTAG
CACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCC
ACCTTCCTCTCGGCTTATCACCGGCAGTCCCCTTAGAGTGCCCAACTAAA
TGCTGGCAACTAAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAAC
ATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTTCTGGGGCCA
GCCTAACTGAAGGACATCGTCTCCAATGCCCATACCCCGAATGTCAAGAG
CTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCT
TGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTC
CCCAGGCGGAATGTTTAATGCGTTAGCTGCGCCACCGAACAGTATACTGC
CCGACGGCTAACATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAAT
CCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAGTAATGGACCAGTA
AGCCGCCTTCGCCACTGGTGTTCCTCCGAATATCTACGAATTTCACCTCT
ACACTCGGAATTCCACTTACCTCTTCCATACTCAAGATACCCAGTATCAA
AGGCAGTTCCGCAGTTGAGCTGCGGGATTTCACCCCTGACTTAAATATCC
GCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAACGCTAGCCCCCTTC
GTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTCCGACT
ACCGTCATTATCTTCATCGGTGAAAGAGCTTTACAACCCTAAGGCCTTCA
TCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTC
CCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTG
GCTGATCATCCTCTCAGACCAGCTATGGATCGTCGCCTTGGTAGGCCTTT
ACCCCACCAACTAGCTAATCCAACGCGGGCTCATCCATCCCCGATAAATC
TTTCCCCCGTAGGGCGTATGCGGTATTAATTCCAGTTTCCCAGagctatt
ccgcagagatgggtagattcccacgcgttactcacccgtctgccactccc
cttgcggg
The present invention also provides a kind of preparation methods of curdlan, specifically comprise the following steps:
Step 1: seed culture: bacterial strain HS-5 described above being linked into seed culture medium, is placed in shaking table, is trained
It supports, obtains seed liquor.
Step 2: fermented and cultured: the seed liquor of above-mentioned acquisition is linked into fermentation medium, is placed in shaking table, is cultivated,
Obtain fermentation liquid.
Step 3: curdlan extracting method: alkali being added into fermentation liquid, dissolves, stands, is centrifuged for the first time；Take supernatant
Acid is added in liquid, and stirring adjusts pH；Then distilled water is added, filters, 95% alcohol is then added into precipitating, second from
Precipitating, is finally placed in baking oven that drying to constant weight to get curdlan by the heart.
In the first step, the seed culture medium are as follows: glucose 10-20g/L, diammonium hydrogen phosphate 2.5-5g/L, biphosphate
Potassium 1.2-2.0g/L, bitter salt 0.5-1.5g/L, corn pulp 1-3g/L, calcium carbonate 2-3g/L, pH=6.0-7.0；It is excellent
Selection of land is glucose 20g/L, diammonium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1.5g/L, bitter salt 1g/L, corn pulp 1g/
L, calcium carbonate 2g/L, pH=7.0.
In the first step, the bacterial strain HS-5 is agrobacterium (Agrobactrim sp.) bacterial strain HS-5.
In the first step, the condition of the shaking table are as follows: 25-35 DEG C, 150-300r/min；It preferably, is 30 DEG C, 200r/
min。
In the first step, the time of the culture is 14-24h；It preferably, is 18h.
In the first step, the bacterial strain is the bacterial strain after activation.
In second step, the seed liquor is linked into fermentation medium by the inoculum concentration of 5%-10%；Preferably, it is
8%.
In second step, the fermentation medium are as follows: carbon source 70-90g/L, diammonium hydrogen phosphate 1.5-2.5g/L, biphosphate
Potassium 1-2g/L, bitter salt 0.5-1.5g/L, nitrogen source 1-2g/L, calcium carbonate 1.5-2.5g/L, pH=6.0-7.0；It is preferred that
Ground is carbon source 70g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, bitter salt 1g/L, nitrogen source 1g/L, carbon
Sour calcium 2g/L, pH=7.0.
Wherein, the carbon source is glucose, maltose, sucrose, fructose, molasses etc.；It preferably, is glucose.
Wherein, the nitrogen source is organic nitrogen source or inorganic nitrogen-sourced, and the organic nitrogen source is peptone, yeast powder, corn pulp
Deng, it is preferable that it is corn pulp；It is described inorganic nitrogen-sourced for diammonium hydrogen phosphate, ammonium chloride, urea and organic ammonium salt etc..
In second step, the condition of the shaking table are as follows: 25-35 DEG C, 150-300r/min；It preferably, is 30 DEG C, 200r/
min。
In second step, the time of the culture is 2d-8d；It preferably, is 4d.
In third step, the alkali is the alkaline solution, including sodium hydroxide, tertiary sodium phosphate, tricalcium phosphate etc. of pH=12-14
One or more of；It preferably, is sodium hydroxide.
In third step, the concentration of the alkali is 0.1-2mol/L；It preferably, is 0.3mol/L.
In third step, the temperature of the standing is preferably room temperature.
In third step, the time of the standing is 30-60min；It preferably, is 30min.
In third step, the revolving speed of the first time centrifugation is 8000-12000rpm；It preferably, is 9000rpm.
In third step, the time of the first time centrifugation is 20-30min；It preferably, is 20min.
In third step, the acid is one or more of HCl, acetic acid, phosphoric acid, citric acid etc.；It preferably, is HCl.
In third step, the concentration of the acid is 0.1-2mol/L；It preferably, is 0.3mol/L.
In third step, the acid is identical as the volume of the alkali.
In third step, the pH value is preferably 7.0.
In third step, the volume of the distilled water is 2-3 times of precipitation volume after the neutralization；It preferably, is 3 times.
In third step, the strainer is the strainer of 80 mesh.
In third step, the volume of the alcohol is 2-3 times of precipitation volume after the distilled water washing；It preferably, is 2
Times.
In third step, the revolving speed of second of centrifugation is 8000-12000rpm；It preferably, is 9000rpm.
In third step, the time of second of centrifugation is 10-20min；It preferably, is 10min.
, can also be without seed culture in the present invention, the directly appropriate bacterial strain HS-5 of scraping is linked into fermentation training
It supports in base, is placed in shaking table, cultivate, obtain fermentation liquid.
The present invention also provides curdlans prepared by the above method.
The preparation and measuring method of 2% gel are as follows: weigh 0.3g curdlan essence produced by the present invention and get sample product in 15ml
In distilled water, liquid processed is transferred in the test tube of 18mmx180cm by the 13000r/min 10min processed under high-speed homogenization machine,
1min is vibrated on turbula shaker, drives bubble therein away, is then placed in 95 DEG C of water-baths and heats 10min, and taking-up is used immediately
Cold water is cooling.
Gel strength measurement: position of the gel away from bottom 10-15mm is cut according to national standard, with physical properties of food analyzer
Carry out the measurement of gel strength.
Curdlan sample molecule quantity measuring method of the present invention are as follows: weigh 15-20mg curdlan powder prepared by the present invention
10mlDMSO is added into sample cell, with pipette in end, is placed in 100 DEG C of boiling water baths and (has small magnetic blender in water-bath)
Stirring and dissolving 4h, during which take out turn upside down 2-3 times, make the sample speckled on wall be dissolved in DMSO take out after completely dissolution it is cold
But to room temperature.The sample solution after cooling membrane filtration of 0.22um is extremely measured in pipe twice, at least 0.5ml in every pipe.
It is put into GPC and carries out molecular weight determination, the molecular size range of sample is finally calculated according to peak area and correlation formula.
The measuring method of total sugar content (i.e. purity) in sample are as follows: Phenol sulfuric acid procedure.
It is 600-800g/ by the gel strength that this fermentation extraction method obtains 2% gel of curdlan of the present invention
cm2, the molecular weight of curdlan of the present invention is 6.5 × 105-8.5×105Da, curdlan total sugar content of the present invention are 85%-
95%.
Application the present invention also provides curdlan as food additives in food production.
Wherein, the food additives include stabilizer, coagulator, thickener, water binding agent, adhesive, film forming agent etc..
Wherein, the food includes meat products (such as sausage, ham, hamburger), Flour product (such as noodles, dumpling wrapper, won ton
Tun skin etc.), aquatic products (surimi product, fish flesh paste, seafood etc.), frozen food (quick-freezing dumpling, burger etc.), fried food, baking
Roasting product (cake, cheese pie etc.), low-calorie diet (cheese product, diet products etc.) etc..
The beneficial effects of the present invention are, the present invention it is innovative a kind of curdlan producing bacterial strain and its application are provided.
The good characteristics such as the produced curdlan performance yield of the bacterial strain is high, molecular weight is small, gel brittleness is good, can be used as stabilization
Agent, coagulator, thickener, water binding agent, adhesive, film forming agent etc. are used in meat products (such as sausage, ham, hamburger), wheat flour
Product (such as noodles, dumpling wrapper, Wantun skin), aquatic products (surimi product, fish flesh paste, seafood etc.), frozen food (quick-freezing dumpling,
Burger etc.), fried food, baked goods (cake, cheese pie etc.), low-calorie diet (cheese product, diet products etc.) etc.
In production, improves the quality of food, preferably meet the market demand.The present invention optimizes original screening and culturing medium, obtains
A kind of screening and culturing medium with higher identification.
Detailed description of the invention
Fig. 1 is the genetic stability figure of agrobacterium (Agrobacterium.sp) bacterial strain HS-5.
Specific embodiment
A kind of curdlan producing bacterial strain, that is, agrobacterium (Agrobacterium.sp) bacterial strain HS-5 of the present invention, by more
Secondary NTG mutagenesis screening obtains, while the curdlan generated to the bacterial strain extracts and carries out performance evaluation.
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail.Implement process of the invention,
Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to what is specifically mentioned below
It is bright that there are no special restrictions to content.
The screening of 1 bacterial strain HS-5 of embodiment
With Alcaligenes faecalis var.myxogenes 10C3 (IFO13714) for starting strain, through ultraviolet
Mutagenesis, nitrosoguanidine mutagenesis, sodium nitrite mutagenesis and ARTP mutagenesis obtain bacterial strain by the method for plate primary dcreening operation and shaking flask secondary screening
HS-5.The wherein condition of ultraviolet mutagenesis are as follows: action time 45s, mutagenesis distance 30cm, dark treatment 1h.Nitrosoguanidine mutagenesis condition
Are as follows: concentration 1mg/ml, action time 30min, action pH 8.5.NIT mutagenic condition are as follows: 1mg/ml, action time 30min make
Use pH5.5.ARTP mutagenic condition are as follows: action time 30s, operating power 100W, operating distance 2mm.
Genetic stability verifying: by superior strain HS-5, in continuous passage 10 times on slant medium, per generation, are respectively connected to
Into fermentation medium, 200r/min, 30 DEG C of culture 96h survey curdlan yield.
The identification of 2 bacterial strain HS-5 of embodiment
Physiology and biochemistry identification: the superior strain HS-5 that screening is obtained carries out Physiology and biochemistry identification, which is gram
Negative bacterium, rod-shaped, colonial morphology is round, surface is smooth, micro- grand, neat in edge on LB solid medium.Other identification knots
Fruit is as described in table 1.
16S rDNA gene magnification: extracting bacterial strain HS-5 genome, carries out 16S rDNA gene magnification.16S rDNA gene
The primer of PCR amplification is 27f and 1492r, and gene order is respectively 27f, 5 '-AGAGTTTGATCCTGGCTCAG-3 '
1492r,5′-CGGTTACCTTGTTACGACTT-3′.The system of PCR amplification is DNA profiling 2ul, Premix Ex Taq enzyme
25ul, Forward Primer (27f) 2ul, Downstream Primer (1492r) 2ul, ddH2O 19ul.PCR amplification
Program is 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min30s carry out 28 circulations； 72
DEG C repair extend 10min, 4 DEG C heat preservation.
16S rDNA gene sequencing and analysis: 16S rDNA gene PCR product is sequenced, sequence is shown in Table 2.It should
Sequence finds that bacterial strain HS-5 and Agrobacterium homology are 100%, it is thus determined that bacterial strain HS-5 is soil after comparing in NCBI
Earth bacillus (Agrobacterium.sp).
The fermentation and extraction of the produced curdlan of 3 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
It is linked into seed culture medium, is placed in shaking table from appropriate HS-5 thallus is scraped with oese on slant medium,
30 DEG C, 200r/min, 18h is cultivated, seed liquor is obtained, is linked into fermentation medium, is placed in shaking table by 8% inoculum concentration,
30 DEG C, 200r/min, 96h is cultivated, fermentation liquid is obtained.
Seed culture medium component are as follows: glucose 20g/L, diammonium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1.5g/L, seven hydration sulphur
Sour magnesium 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, medium pH=7.0.
Fermentation medium component are as follows: glucose 70g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, medium pH=7.0.
A certain amount of fermentation liquid is taken, the NaOH of a certain amount of 0.3mol/L is added thereto, stirring is stored at room temperature to dissolving
30min, 9000rpm are centrifuged 20min.Take supernatant that the HCl of isometric 0.3mol/L is added, it is stirring while adding, adjust pH to 7.0.
3 times of volume distilled water are added thereto, with 80 mesh filter-cloth filterings, 2 times of 95% alcohol of volume are then added into precipitating,
9000rpm is centrifuged 10min, finally takes out precipitating and is placed in baking oven that drying to constant weight.
The fermentation and extraction of the produced curdlan of 4 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
Fermentation medium component are as follows: glucose 90g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, medium pH=7.0.
Other are the same as embodiment 3.
The fermentation and extraction of the produced curdlan of 5 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
Fermentation medium component are as follows: glucose 80g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, medium pH=7.0.
Other are the same as embodiment 3.
The fermentation and extraction of the produced curdlan of 6 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
It is linked into seed culture medium, is placed in shaking table from appropriate HS-5 thallus is scraped with oese on slant medium,
30 DEG C, 200r/min, 18h is cultivated, seed liquor is obtained, is linked into fermentation medium, is placed in shaking table by 8% inoculum concentration,
30 DEG C, 200r/min, 96h is cultivated, fermentation liquid is obtained.
Seed culture medium component are as follows: glucose 20g/L, diammonium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1.5g/L, seven hydration sulphur
Sour magnesium 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
Fermentation medium component are as follows: maltose 70g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
A certain amount of fermentation liquid is taken, the NaOH of a certain amount of 0.3mol/L is added thereto, stirring is stored at room temperature to dissolving
30min, 9000rpm are centrifuged 20min.Take supernatant that the HCl of isometric 0.3mol/L is added, it is stirring while adding, adjust pH to 7.0.
3 times of volume distilled water are added thereto, with 80 mesh filter-cloth filterings, 2 times of 95% alcohol of volume are then added into precipitating,
9000rpm is centrifuged 10min, finally takes out precipitating and is placed in baking oven that drying to constant weight.
The fermentation and extraction of the produced curdlan of 7 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
Fermentation medium component are as follows: maltose 90g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
Other are the same as embodiment 6.
The fermentation and extraction of the produced curdlan of 8 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
Fermentation medium component are as follows: maltose 80g/L, diammonium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 1.5g/L, seven hydrations
Magnesium sulfate 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
Other are the same as embodiment 6.
The fermentation and extraction of the produced curdlan of 9 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
It is linked into seed culture medium, is placed in shaking table from appropriate HS-5 thallus is scraped with oese on slant medium,
30 DEG C, 200r/min, 18h is cultivated, seed liquor is obtained, is linked into fermentation medium, is placed in shaking table by 8% inoculum concentration,
30 DEG C, 200r/min, 96h is cultivated, fermentation liquid is obtained.
Seed culture medium component are as follows: glucose 20g/L, diammonium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1.5g/L, seven hydration sulphur
Sour magnesium 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
Fermentation medium component are as follows: sucrose 70g/L, (NH4)2HPO43g/L, KH2PO41g/L, MgSO4.7H2O 0.5g/
L, FeSO4.7H2O 0.05g/L, MnSO4.7H2O 0.02g/L, ZnCL20.01 g/L, CoCL20.01g/L, CaCO3 3g/
L, pH=7.0.(inorganic ion in the culture medium replaces the corn pulp in above-described embodiment, is the allusion quotation of fermentation curdlan
Type culture medium)
A certain amount of fermentation liquid is taken, the NaOH of a certain amount of 0.3mol/L is added thereto, stirring is stored at room temperature to dissolving
30min, 9000rpm are centrifuged 20min.Take supernatant that the HCl of isometric 0.3mol/L is added, it is stirring while adding, adjust pH to 7.0.
3 times of volume distilled water are added thereto, with 80 mesh filter-cloth filterings, 2 times of 95% alcohol of volume are then added into precipitating,
9000rpm is centrifuged 10min, finally takes out precipitating and is placed in baking oven that drying to constant weight.
The fermentation and extraction of the produced curdlan of 10 agrobacterium of embodiment (Agrobacterium.sp) bacterial strain HS-5
It is linked into seed culture medium, is placed in shaking table from appropriate HS-5 thallus is scraped with oese on slant medium,
30 DEG C, 200r/min, 18h is cultivated, seed liquor is obtained, is linked into fermentation medium, is placed in shaking table by 8% inoculum concentration,
30 DEG C, 200r/min, 96h is cultivated, fermentation liquid is obtained.
Seed culture medium component are as follows: glucose 20g/L, diammonium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1.5g/L, seven hydration sulphur
Sour magnesium 1g/L, corn pulp 1g/L, calcium carbonate 2g/L, pH=7.0.
Fermentation medium component are as follows: glucose 70g/L, (NH4)2HPO4 2.3g/L,KH2PO4 1g/L, MgSO4.7H2O
0.5g/L,FeSO4.7H2O 0.05g/L,MnSO4.7H2O 0.02g/L, ZnCL20.01g/L, CoCL20.01g/L, CaCO3
3g/L, pH=7.0.
A certain amount of fermentation liquid is taken, the NaOH of a certain amount of 0.3mol/L is added thereto, stirring is stored at room temperature to dissolving
30min, 9000rpm are centrifuged 20min.Take supernatant that the HCl of isometric 0.3mol/L is added, it is stirring while adding, adjust pH to 7.0.
3 times of volume distilled water are added thereto, with 80 mesh filter-cloth filterings, 2 times of 95% alcohol of volume are then added into precipitating,
9000rpm is centrifuged 10min, finally takes out precipitating and is placed in baking oven that drying to constant weight.
Curdlan yield obtained by embodiment 3-10 is as shown in table 2
Table 2
The liquid of curdlan is generated in the present invention for agrobacterium (Agrobacterium.sp) bacterial strain HS-5 fermentation
Culture medium forms not special limitation, including carbon source, nitrogen source, inorganic salts, amino acid etc.；
The present invention generates the fermentation training of curdlan for agrobacterium (Agrobacterium.sp) bacterial strain HS-5 fermentation
The carbon source supported in base can be glucose, maltose, sucrose, fructose, molasses (such as beet molasses, corn molasses) and can be used for
Agrobacterium HS-5 fermentation generates other carbohydrates of curdlan.
The present invention generates the fermentation training of curdlan for agrobacterium (Agrobacterium.sp) bacterial strain HS-5 fermentation
The nitrogen source supported in base can be organic nitrogen source, such as peptone, yeast powder, corn pulp；Can be it is inorganic nitrogen-sourced, such as phosphoric acid hydrogen two
A variety of nitrogen sources such as ammonium, ammonium chloride, urea and various organic ammonium salts.
The present invention generates the fermentation training of curdlan for agrobacterium (Agrobacterium.sp) bacterial strain HS-5 fermentation
Some calcium, potassium, magnesium, manganese, iron, the inorganic ions such as zinc, cobalt, copper can be added by supporting in base, and these inorganic ions can be independent
Add, appropriately combined can also add.
Condition of culture of the present invention is generally cultivated under aerobic condition, and cultivation temperature is 20-40 DEG C, it is therefore preferable to 25-35
℃；Culture revolving speed is 150-300r/min, it is therefore preferable to 200-250r/min；Incubation time is 2d-8d, it is therefore preferable to 3d-5d；
The pH of culture medium is 5-10, it is therefore preferable to 5-7.
The curdlan as caused by agrobacterium of the present invention (Agrobacterium.sp) bacterial strain HS-5 can be used as food
Product additive (stabilizer, coagulator, thickener, water binding agent, adhesive, film forming agent etc.) be used in meat products (such as sausage, ham,
Hamburger etc.), Flour product (such as various noodles, dumpling wrapper, Wantun skin), aquatic products (surimi product, fish flesh paste, seafood etc.),
Frozen food (quick-freezing dumpling, burger etc.), fried food, baked goods (cake, puff, cheese pie etc.), low-calorie diet (milk
Junket product, diet products etc.), in the production of candy (cotton candy, chewing gum, jelly etc.) etc..
Curdlan caused by agrobacterium (Agrobacterium.sp) bacterial strain HS-5 can be added individually in the present invention
In different food products, it can also combine with other additives for producing various types of food.
The curdlan as caused by agrobacterium of the present invention (Agrobacterium.sp) HS-5, which removes, to be applied in food,
Can be applied in medicine and and chemical industry in.If curdlan sulfuric ester can be used for treating HIV infection, porous pottery is made
Construction materials such as porcelain etc..
The property analysis of the produced curdlan of agrobacterium (Agrobacterium.sp) bacterial strain HS-5
The measurement of 2% gel strength of curdlan sample:
Curdlan sample obtained by 0.3g embodiment 3-10 is weighed in 15ml distilled water, under high-speed homogenization machine
13000r/min 10min processed, liquid processed is transferred in the test tube of 18mmx180cm, 1min is vibrated on turbula shaker, is caught up with
Bubble therein is walked, is then placed in 95 DEG C of water-baths and heats 10min, is taken out cooling with cold water immediately.It is cut according to national standard
It takes gel away from the position of bottom 10-15mm, measures gel strength with physical properties of food analyzer.The gel strength measured is 600-
800g/cm2。
The measurement of curdlan molecular weight analyte:
Curdlan obtained by 15-20mg embodiment 3-10 is weighed into sample cell, 10ml is added with pipette and contains chlorination
The DMSO of lithium is placed in 100 DEG C of boiling water baths and (has small magnetic blender in water-bath) stirring and dissolving 4h, during which takes out top up and down
2-3 times, so that the sample speckled on wall is dissolved in DMSO taking-up after completely dissolution and be cooled to room temperature.It is obtained after cooling so
Glue sample solution is extremely measured in pipe twice with the filter filtering of 0.22um, at least 0.5ml in every pipe.It is put into GPC and is divided
It is fixed that son measures, and the molecular size range of sample is finally calculated according to peak area and correlation formula.The molecular weight of the curdlan measured
It is 6.5 × 105-8.5× 105Da。
The measurement of total sugar content (i.e. purity) in curdlan sample:
The total sugar content in curdlan sample obtained by embodiment 3-10 is measured with Phenol sulfuric acid procedure, as the result is shown
Total sugar content is 85-95%.
The detection of curdlan genetic stability:
To the HS-5 continuous passage culture of high yield curdlan agrobacterium (Agrobacterium.sp) bacterial strain, its something lost is detected
Stability is passed, as shown in Figure 1, the curdlan coefficient of variation of the yield measured in 10 passages is 1.5%, it is seen that bacterial strain tool
There is good genetic stability.
Protection content of the invention is not limited to above embodiments.Without departing from the spirit and scope of the invention, originally
Field technical staff it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protect
Protect range.
SEQUENCE LISTING
<110>East China Normal University
<120>a kind of curdlan producing bacterial strain and its application
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
cggttacctt gttacgactt 20
<210> 3
<211> 1308
<212> DNA
<213>artificial sequence
<400> 3
ttcgggtaaa accaactccc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat 60
tcaccgcagc atgctgatct gcgattacta gcgattccaa cttcatgcac tcgagttgca 120
gagtgcaatc cgaactgaga tggcttttgg agattagctc gacatcgctg tctcgctgcc 180
cactgtcacc accattgtag cacgtgtgta gcccagcccg taagggccat gaggacttga 240
cgtcatcccc accttcctct cggcttatca ccggcagtcc ccttagagtg cccaactaaa 300
tgctggcaac taagggcgag ggttgcgctc gttgcgggac ttaacccaac atctcacgac 360
acgagctgac gacagccatg cagcacctgt tctggggcca gcctaactga aggacatcgt 420
ctccaatgcc cataccccga atgtcaagag ctggtaaggt tctgcgcgtt gcttcgaatt 480
aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt ttaatcttgc 540
gaccgtactc cccaggcgga atgtttaatg cgttagctgc gccaccgaac agtatactgc 600
ccgacggcta acattcatcg tttacggcgt ggactaccag ggtatctaat cctgtttgct 660
ccccacgctt tcgcacctca gcgtcagtaa tggaccagta agccgccttc gccactggtg 720
ttcctccgaa tatctacgaa tttcacctct acactcggaa ttccacttac ctcttccata 780
ctcaagatac ccagtatcaa aggcagttcc gcagttgagc tgcgggattt cacccctgac 840
ttaaatatcc gcctacgtgc gctttacgcc cagtaattcc gaacaacgct agcccccttc 900
gtattaccgc ggctgctggc acgaagttag ccggggcttc ttctccgact accgtcatta 960
tcttcatcgg tgaaagagct ttacaaccct aaggccttca tcactcacgc ggcatggctg 1020
gatcaggctt gcgcccattg tccaatattc cccactgctg cctcccgtag gagtttgggc 1080
cgtgtctcag tcccaatgtg gctgatcatc ctctcagacc agctatggat cgtcgccttg 1140
gtaggccttt accccaccaa ctagctaatc caacgcgggc tcatccatcc ccgataaatc 1200
tttcccccgt agggcgtatg cggtattaat tccagtttcc cagagctatt ccgcagagat 1260
gggtagattc ccacgcgtta ctcacccgtc tgccactccc cttgcggg 1308
Claims (15)
1. one plant of curdlan producing bacterial strain agrobacterium Agrobacterium sp. bacterial strain HS-5, which is characterized in that its preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, the deposit date is on 03 22nd, 2018, preservation was compiled
Number be CGMCC NO.15477, be Gram-negative bacteria.
2. bacterial strain according to claim 1, which is characterized in that the colonial morphology of the bacterial strain is round, surface is smooth, micro-
Grand, neat in edge, microscope hypothallus is in uniform rod-shaped.
3. bacterial strain according to claim 1, which is characterized in that the 16S rDNA gene order such as SEQ ID of the bacterial strain
Shown in NO:1.
4. a kind of preparation method of curdlan, which is characterized in that specifically includes the following steps:
Step 1: seed culture: will be linked into seed culture medium, and be placed in such as described in any item bacterial strains of claim 1-3
In shaking table, culture obtains seed liquor；
Step 2: fermented and cultured: seed liquor described in the first step being linked into fermentation medium, is placed in shaking table, cultivates, obtains
To fermentation liquid；
Step 3: curdlan extracting method: alkali being added into fermentation liquid described in second step, stirring is stood, first to dissolving
Secondary centrifugation；Take supernatant that acid is added, stirring adjusts pH；Then distilled water is added, filters, 95% wine is then added into precipitating
Precipitating is finally placed in baking oven and dries to get the curdlan is arrived by essence, second of centrifugation.
5. according to the method described in claim 4, it is characterized in that, in the first step, the seed culture medium are as follows: glucose 10-
20g/L, diammonium hydrogen phosphate 2.5-5g/L, potassium dihydrogen phosphate 1.2-2.0g/L, bitter salt 0.5-1.5g/L, corn pulp 1-
3g/L, calcium carbonate 2-3g/L, pH=6.0-7.0.
6. according to the method described in claim 4, it is characterized in that, in the first step, the condition of the shaking table are as follows: 25-35 DEG C,
150-300r/min；And/or the time of the culture is 14-24h.
7. according to the method described in claim 4, it is characterized in that, in second step, the fermentation medium are as follows: carbon source 70-
90g/L, diammonium hydrogen phosphate 1.5-2.5g/L, potassium dihydrogen phosphate 1-2g/L, bitter salt 0.5-1.5g/L, nitrogen source 1-2g/
L, calcium carbonate 1.5-2.5g/L, pH=6.0-7.0；Wherein, the carbon source is glucose, maltose, sucrose, fructose, molasses；Institute
Stating nitrogen source is organic nitrogen source or inorganic nitrogen-sourced, and the organic nitrogen source is peptone, yeast powder, corn pulp, described inorganic nitrogen-sourced to be
Diammonium hydrogen phosphate, ammonium chloride, urea and organic ammonium salt.
8. according to the method described in claim 4, it is characterized in that, in second step, the condition of the shaking table are as follows: 25-35 DEG C,
150-300r/min；And/or the time of the culture is 2d-8d.
9. according to the method described in claim 4, it is characterized in that, the alkalinity that the alkali is pH=12-14 is molten in third step
One or more of liquid, including sodium hydroxide, tertiary sodium phosphate, tricalcium phosphate；And/or the concentration of the alkali is 0.1-2mol/
L；And/or the acid is one or more of HCl, acetic acid, phosphoric acid, citric acid；And/or the concentration of the acid is 0.1-
2mol/L。
10. according to the method described in claim 4, it is characterized in that, in third step, the revolving speed of the first time centrifugation is
8000-12000rpm；And/or the time of the first time centrifugation is 20-30min；And/or the revolving speed of second of centrifugation
For 8000-12000rpm；And/or the time of second of centrifugation is 10-20min.
11. according to the method described in claim 4, it is characterized in that, the temperature of the standing is room temperature in third step；With/
Or, the pH value is 7.0；And/or the strainer is the strainer of 80 mesh.
12. according to the method described in claim 4, it is characterized in that, 2% gel of the curdlan coagulates in third step
Glue intensity is 600-800g/cm2；The molecular weight of the curdlan is 6.5 × 105-8.5×105Da；The curdlan
Total sugar content is 85%-95%.
13. a kind of curdlan that any one method according to claim 4-12 is prepared.
14. application of the curdlan as claimed in claim 13 as food additives in food production.
15. application according to claim 14, which is characterized in that the food additives are stabilizer, coagulator, thickening
Agent, water binding agent, adhesive, film forming agent；And/or the food is meat products, Flour product, aquatic products, frozen food, fried food
Product, baked goods, low-calorie diet.
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| CN110004140A (en) * | 2019-02-25 | 2019-07-12 | 华东师范大学 | A method of improving curdlan Screening of strain with high productivity efficiency |
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| CN107384795A (en) * | 2017-08-28 | 2017-11-24 | 华东师范大学 | A kind of method of high flux screening high yield curdlan bacterial strain |
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| CN107384795A (en) * | 2017-08-28 | 2017-11-24 | 华东师范大学 | A kind of method of high flux screening high yield curdlan bacterial strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110004140A (en) * | 2019-02-25 | 2019-07-12 | 华东师范大学 | A method of improving curdlan Screening of strain with high productivity efficiency |
| CN110004140B (en) * | 2019-02-25 | 2022-03-11 | 华东师范大学 | Method for improving screening efficiency of curdlan high-yield strains |
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