CN106434466B - A kind of Rhodococcus ruber and the preparation method and application thereof generating natural red colouring matter - Google Patents
A kind of Rhodococcus ruber and the preparation method and application thereof generating natural red colouring matter Download PDFInfo
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Abstract
The present invention relates to a kind of Rhodococcus rubers and the preparation method and application thereof for generating natural red colouring matter.Wherein microbial fermentation production haematochrome bacterium classification category Rhodococcus ruber (Rhodococcus ruber), deposit number is CGMCC No.12604.The invention further relates to the preparation methods of fermentation natural red colouring matter, it is using fermentation strain Rhodococcus ruber CGMCC No.12604, by 1% ~ 10%(v/v) inoculum concentration by cultured seed access fermentation medium, 30 ~ 40 DEG C of cultivation temperature, fermentation ventilatory capacity be 5 ~ 13m3/ h, tank press 0.02 ~ 0.09 MPa, 120-150 revs/min of mixing speed, fermentation time 120 ~ 144 hours, filtrated air are passed through in fermentation process, maintain fermentation liquid pH 6.5 ~ 7.5 with ammonium hydroxide or liquid alkaline, fermentation results haematochrome yield is 1 ~ 2g/L.The invention also discloses the extracting methods of Rhodococcus ruber haematochrome.
Description
Technical field
The invention belongs to field of biotechnology, it is related to generating natural red colouring matter microorganism Rhodococcus ruber and its haematochrome preparation
Method.In particular be: a kind of strain of fermenting and producing haematochrome Rhodococcus ruber, the strain be present invention discover that one plant of energy
It is good to extract simple and stability with strain fermentation production for the effectively new strains of production haematochrome.
Background technique
Pigment is widely used in food industry, Cosmetic Manufacture, weaving and daily necessities production, is brought preferably for product
Added value.But the potential risk of synthetic dyestuff is carcinogenic, teratogenesis etc., and application is increasingly subject to repel, and natural pigment is more next
More have been favored by people.The source of natural pigment is broadly divided into animal pigment, phytochrome, microorganisms pigments and mineral pigment
Deng.Animal and plant source pigment oneself be not able to satisfy the demand of industrial high speed development.In recent years, it is dedicated to finding for replacement
The research of natural pigment increases, and sight has been invested microorganisms pigments by these research major parts.Some microorganisms were growing
Cheng Zhonghui chromogenesis, and microorganisms pigments growth it is fast, easily culture, can simply large-scale industrial production, microorganisms pigments
Gradually become a kind of important channel for obtaining natural pigment.More importantly its safety, the scope of application and research on maximum utilized quantity be all
There is apparent advantage than synthetic food color.Red pigments as one of three big basic pigments have very high " gold content " it
Multiple color tones can be deployed into blue, yellow strain natural pigment, keep the color of natural pigment more abundant.Natural red pigments are not
Can only assign food in riotous profusion color, and have it is some also there are different physiological roles, can be widely used for medicine, food and change
Cosmetic field.The general trend that nontoxic natural red pigments have become pigment development is researched and developed, oneself is through foring with day
Right pigment is leading market.Therefore the research and development of natural red pigments has wide prospect and development potentiality.
Summary of the invention
The purpose of the present invention is to provide a kind of Rhodococcus ruber (CGMCC No.12604) fermenting and producing natural red colouring matters
The extraction process of culture medium prescription and natural red colouring matter.Prepared natural red colouring matter can be used as food additives, feed addition
As drug or the advanced additive of makeup cosmetic after agent or purification.
It is an object of the present invention to provide a kind of strain of microbial fermentation production natural red colouring matter, classification belongs to crimson
Coccus (Rhodococcus ruber), deposit number is CGMCC No. 12604.
Another object of the present invention be disclose using Rhodococcus ruber (Rhodococcus ruber), deposit number is
The method that 12604 strain of CGMCC No. prepares natural red colouring matter.
The present invention a further object is disclose using Rhodococcus ruber (Rhodococcus ruber), deposit number is
The preparation method of 12604 strain fermentation of CGMCC No. production natural red colouring matter.
To achieve the above object, technology contents disclosed by the invention are as follows:
A kind of Rhodococcus ruber, classification category Rhodococcus ruber (Rhodococcus ruber), deposit number is CGMCC No.
12604。
12604 bacterial strain of CGMCC No. provided by the invention is one plant of new strains that can produce natural red colouring matter, with the bacterium
Strain fermentation saccharine material produces haematochrome.
Antifungal lipopeptid microbial fermentation bacterial strain, classification category Rhodococcus ruber (Rhodococcus ruber), deposit number is
CGMCC No. 12604, preservation date on June 12nd, 2016.Preservation place: China Committee for Culture Collection of Microorganisms is general
Logical microorganism center (China General Microbiological Cultuer Collection Centre) is protected
Hiding, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode:
100101。
The physio-biochemical characteristics of Rhodococcus ruber are as follows:
The bacterium cell be it is rod-shaped, Grain stain is uniform, and for the positive, do not generate gemma.The bacterium on broth bouillon plate
Fall rounded or irregular, bacterial strain colony edge is neatly rounded, the smooth wet red in surface.Bacterial strain atrichia, is not transported
It is dynamic, it does not shine, haematochrome is only produced in intracellular, non-pigment in culture medium.
The present invention further discloses using Rhodococcus ruber (Rhodococcus ruber), deposit number is CGMCC No.
The method that 12604 strains prepare natural red colouring matter:
(1) seed culture and culture medium
The fermentation strain used is CGMCC No. 12604, and seed culture medium forms (g/L): 10 ~ 30g of glucose, albumen
2 ~ 5g of peptone, 5 g/L of yeast powder, sodium chloride 10 g/L, K2HPO42 ~ 6g, pH 7.0~7.2；150 ~ 180 revs/min of shaking table shakes
Swing culture, 30 ~ 40 DEG C, 36 ~ 48h.
(2) for the fermentation strain used for CGMCC No. 12604, fermentation medium group becomes (g/L): saccharine material 20 ~
40g, 2 ~ 15g of peptone, 15 g/L of yeast powder, 15 g/L of corn pulp, 10 g/L of sodium chloride, 1.5 ~ 3g of sodium citrate,
MgSO4·7H20 0.1 ~ 0.2g, K2HPO42 ~ 6g, pH 7.0~7.2；
(3) cultured seed fermented and cultured: is accessed into fermentation medium, culture temperature by the inoculum concentration of 1%-10%(v/v)
30 ~ 40 DEG C of degree, fermentation ventilatory capacity are 5 ~ 13m3/ h, tank press 0.02 ~ 0.09 MPa, 120-150 revs/min of mixing speed, ferment
Time 120 ~ 144 hours, it is passed through filtrated air in fermentation process, maintains fermentation liquid pH 6.5 ~ 7.5 with ammonium hydroxide or liquid alkaline, ferments
It is passed through filtrated air in the process, maintains fermentation liquid pH 6.5 ~ 7.5 with ammonium hydroxide or liquid alkaline, fermentation results haematochrome yield is 1 ~
2g/L。
(4) haematochrome extracting method: multigelation broken wall method and ultrasonic wall-cracking method are used, takes fermentation liquid in centrifuge tube
In, 4000r/min is centrifuged 10min, discards supernatant liquid, is washed with deionized and precipitates to obtain wet thallus, be placed in -20 °C immediately
Refrigerator 12h is continuously repeated twice, and 100% ethyl alcohol is added and mixes, the progress ultrasonic disruption processing on ice bath, ultrasonic power 200w,
Ethyl alcohol pigment extract can be obtained in ultrasonic time 5s, interval time 5s, total time 30min, centrifugation removal thallus.
Preparation method of the present invention, wherein saccharine material includes: glucose, sucrose, fructose, maltose, solubility
Starch saccharificating liquid or corn flour saccharified liquid.
More detailed description of the present invention is as follows:
(1) screening and identification of microbial strains:
Bacterial strain is inoculated into the inorganic salt liquid training of the sugar such as 1% glucose, sucrose, L- arabinose, mannitol respectively
It supports in base, 28 DEG C of 3 ~ 7d of constant temperature incubation, observes strain growth situation.The bacterial strain can utilize dextrin, polysorbate40, Tween 80, D-
The various saccharides acetic acid such as fructose, alpha-D-glucose, α-mannose, D-Psicose, D-ribose, sucrose, β-glycolic acid, α-hydroxyl second
The organic acids such as acid, oxopentanoic acid, lactic acid, propionic acid, pyruvic acid, L- glutamic acid can also utilize methyl pyruvate, mono succinate methyl
Ester, but D-ALPHA-Hydroxypropionic acid methyl esters cannot be utilized.Gas cannot be produced using glucose, can be grown in the environment of NaCl concentration is 2%,
For gram-positive bacteria, the physiological and biochemical property of bacterial strain consistent with the growth characteristics of Rhodococcus ruber is with Buchanan and Ji Bensi etc.
(1984) " the common bacteria identification that the elegant pearl of " Berger bacterial identification manual " write the 8th edition and east, Cai Miaoying (2001) write
Handbook " described in Rhodococcus ruber bacterium it is identical.
(2) 16SrDNA sequencing: gene cloning is carried out to the 16SrDNA sequence of the bacterial strain, amplification obtains
The sequence of 1438bp.The 16SrDNA sequence is carried out in GenBank known to sequence analysis, the bacterial strain and crimson ball
Bacterium (Rhodococcus ruber) affiliation is closest, and similitude is up to 99%.By the strain growth physiological property and
16SrDNA sequencing results judge it for Rhodococcus ruber.The sequence of the 16SrDNA of bacterial strain is as follows:
Rhodococcus sp. (CGMCC No. 12604) 16S rDNA gene
TCGAACGCTGGGGGCGTGCTTAACACATGCAAGTCGAACGGTAAGGCCCTTTCGGGGGTACACGAGTGG
CGAACGGGTGAGTAACACGTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACCGGGTCTAATACCGGATA
TGAGCTCCTGCCGCATGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGTCCGCGGCCTATCAGCTTGTTGGTGG
GGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGACCC
AGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGAT
GACGGTCTTCGGATTGTAAACTCCTTTCAGTAGGGACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCCA
ACTACGTGCCAGCAACCACGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGC
GGTTTGTCACGTCGTCTGTGAAATCCTCCAGCTCAACTGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACA
GGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCT
GGGTAGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCAAACAGGAGCAGATACCCTGGTAAGTCCATGCCGTAAG
CGGTGGGCGCTAGGTGTGGGGTCCTTCCACGGATTCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTA
CGTCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAA
CGCGAAGAACCTTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTGTATACAGG
TGGTGCATGGTTGTCGTCAGCTCGTGTCGTCAGATGCTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCTCATG
TTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATC
ATCATGCCCCTTATGTCTAGGGCTTCACACATGCTACAATGGCTAGTACAGAGGGCTGCGAGACCGCGAGGTGGAGC
GAATCCCTTAAAGCTAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGC
AGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACC
CGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGGTGGGATCTCGCGATTTGA
(3) culture of microbial strains and fermenting and producing haematochrome:
The composition (g/L) on inclined-plane and seed culture medium: glucose 1 ~ 3, tryptone 5 ~ 10, yeast powder 3 ~ 6, NaCl 6 ~
12, deionized water is prepared, pH 7.2~7.5；Solid medium need to add 15g agar powder.
The composition (g/L) of fermentation medium: 20 ~ 40g of saccharine material, 2 ~ 15g of peptone, 15 g of yeast powder, corn pulp 15
G, 10 g of sodium chloride, sodium citrate 1.5 ~ 3g, MgSO47H20 0.1 ~ 0.2g, K2HPO42 ~ 6g, pH 7.0~7.2；Hair
Ferment culture: cultured seed is accessed into fermentation medium by the inoculum concentration of 1% ~ 10%(v/v), 30 ~ 40 DEG C of cultivation temperature, is fermented
Ventilatory capacity is 5 ~ 13m3/ h, tank press 0.02 ~ 0.09 MPa, and 120-150 revs/min of mixing speed, fermentation time 120 ~ 144 is small
When, filtrated air is passed through in fermentation process, maintains fermentation liquid pH to be passed through nothing in 6.5 ~ 7.5, fermentation process with ammonium hydroxide or liquid alkaline
Bacterium air maintains fermentation liquid pH 6.5 ~ 7.5 with ammonium hydroxide or liquid alkaline, and fermentation results haematochrome yield is 1 ~ 2g/L.
The present invention can be glucose, sucrose, fructose, soluble starch, corn flour saccharification for the saccharine material of fermentation
Liquid.Fermentation process is passed through filtrated air, maintains the pH of fermentation liquid bacterium in 6.5 ~ 7.5, fermentation liquid red with ammonium hydroxide or liquid alkaline
Plain yield is 1 ~ 2g/L.
Haematochrome extracting method: using multigelation broken wall method and ultrasonic wall-cracking method, take fermentation liquid in centrifuge tube,
4000r/min is centrifuged 10min, discards supernatant liquid, is washed with deionized and precipitates to obtain wet thallus, is placed in -20 DEG C of refrigerators immediately
12h multigelation twice, is added 100% ethyl alcohol and mixes, and ultrasonic disruption processing, ultrasonic power 200w, ultrasound are carried out on ice bath
5s time, 5s interval time, total time 30min.Centrifugation removal thallus, can be obtained ethyl alcohol pigment extract.
The present invention further investigated using Rhodococcus ruber (Rhodococcus ruber), deposit number is CGMCC No.
The steadiness of the natural red colouring matter of 12604 strains preparation:
(1) it influence of the light to pigment: takes pigment solution to set under indoor ultraviolet light and irradiates, be measured in 0,1,2,3,4,5h
Absorbance simultaneously observes color change situation.Its storage rate is respectively 100%, 97.9%, 97.8%, 97.7%, 91.9%, 88.9%.It should
Pigment is more stable to ultraviolet light.It takes pigment solution to set under indoor white flag light to irradiate, be measured absorbance simultaneously in 0,1,2,3,4,5h
Observe color change situation.Its storage rate is respectively 100%, 100%, 100%, 97.7 %, 97 %, 96.7%.The pigment solution is white
It is very stable under flag light.
(2) influence of the temperature to pigment: take pigment solution in 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100
DEG C, under the conditions of heat 0.5h respectively after, measure its solution absorbance respectively, storage rate is respectively 99.7%, 99.4%, 99%,
98.9%,98.6%,98.1%,94.4%,92.7%.For the pigment solution compared with high temperature resistant, room temperature stability inferior is high.
(3) influence of the oxidant to pigment: pigment solution is taken, 30%H is used2O2Be configured to 0%, 0.1%, 0.2%, 0.3%,
0.4%, the H of 0.5% concentration2O2Pigment solution, avoid light place 1h survey its absorbance, storage rate is respectively 100%, 99.8%,
99.2%,98.1%,97.7%,97.5%.Oxidant H2O2Influence result to pigment stability as it can be seen that pigment in various concentration
Absorbance change very little under oxidant, its corresponding color show that the pigment antioxidant is fine also without significant change
(4) influence of the reducing agent to pigment: pigment solution is taken, Na is used2SO3Be configured to 0,0.1,0.2,0.4,0.8mg/L it is dense
The Na of degree2SO3Pigment solution, avoid light place 1h survey its absorbance, storage rate is respectively 100%, 98.7%, 97.6%, 97.1%,
96.4%.Reducing agent Na2SO3Influence result to pigment stability is as it can be seen that pigment absorbance under the reducing agent of various concentration becomes
Change very little, its corresponding color shows that the resistance to reducing agent of the pigment is fine also without significant change.
(5) influence of food additives: respectively with potassium sorbate, sodium benzoate, vitamin C, sodium citrate, lactic acid configuration
At the pigment solution of 2mg/L, avoid light place 1h surveys its absorbance, storage rate is respectively 100%, 100%, 100%, 100%,
100%.Pigment solution is not any change under the influence of food additives, shows that the pigment is highly stable to food additives.
It is actively imitated possessed by Rhodococcus ruber disclosed by the invention for generating natural red colouring matter and the preparation method and application thereof
Fruit is:
(1) Rhodococcus ruber is a kind of nontoxic microorganism, can carry out industrialization large-scale culture.
(2) pigment extracted by Rhodococcus ruber thallus is good to the stability of light and temperature, can be widely applied to food and
In cosmetics.
(3) it extracts completely, quickly, the technological process of production is simple, product non-toxic residual, highly-safe.
Detailed description of the invention
Fig. 1 be Rhodococcus sp (Rhodococcus ruber)Colonial morphology figure；Because bacterium colony produces haematochrome, in red
Color.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.The raw materials used in the present invention and reagent are commercially available.Such as glucose, sucrose, fructose, solubility
Starch, corn flour etc. are commercially available.
Embodiment 1
Rhodococcus ruber, classification category Rhodococcus ruber (Rhodococcus ruber), deposit number is CGMCC No. 12604.
Screening technique it is as follows:
(1) take 5 grams of pedotheque (rich in humus) in seed culture medium, wherein seed culture medium composition (g/L): Portugal
Grape sugar 10g, peptone 2g, 5 g of yeast powder, sodium chloride 10 g, K2HPO42g, pH 7.0~7.2；150 ~ 180 revs/min are shaken
Bed shake culture, 30 ~ 40 DEG C, 36 ~ 48h enriched microorganism.
(2) microbial inoculum being enriched with the sterile picking of oese (1) forms (g/L) in seed culture medium: glucose
10g, peptone 2g, 5 g of yeast powder, sodium chloride 10 g, K2HPO42g, pH 7.0~7.2, flat lining out are statically placed in culture
30 ~ 40 DEG C in case, 36 ~ 48h is cultivated；
(3) red colonies grown in (2) plate are observed, and its sterile working picking to seed culture medium is formed into (g/
L): glucose 10g, peptone 2g, 5 g of yeast powder, sodium chloride 10 g, K2HPO42 ~ 6g, pH 7.0~7.2, flat lining out
Separation is statically placed in incubator 30 DEG C, cultivates 36h；
(4) Biolog experiment and 16s rDNA sequencing are carried out by (3) isolated single colonie and compared real
It tests.Determine the kind of its bacterial strain:
Biolog system identification result
Embodiment 2
Using Rhodococcus ruber (Rhodococcus ruber), deposit number is that 12604 strain of CGMCC No. prepares neutral red
The method of pigment:
(1) seed culture and culture medium
The fermentation strain used is CGMCC No. 12604, and seed culture medium forms (g/L): glucose 20g, peptone
4g, 5 g of yeast powder, sodium chloride 10 g, K2HPO43g, pH 7.0~7.2；170 revs/min of shaking table shake cultures, 34 DEG C,
45h。
(2) for the fermentation strain used for CGMCC No. 12604, fermentation medium group becomes (g/L): saccharine material 20 ~
40g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, 10 g of sodium chloride, sodium citrate 2.5, MgSO47H20 0.15g,
K2HPO43g, pH 7.0~7.2.
(3) cultured seed fermented and cultured: is accessed into fermentation medium, culture temperature by the inoculum concentration of 1% ~ 10%(v/v)
35 DEG C of degree, fermentation ventilatory capacity are 8m3/ h, tank press 0.05 MPa, and 120 ~ 150 revs/min of mixing speed, fermentation time 134 is small
When, filtrated air is passed through in fermentation process, maintains fermentation liquid pH to be passed through sterile sky in 7.0, fermentation process with ammonium hydroxide or liquid alkaline
Gas maintains fermentation liquid pH 7.0 with ammonium hydroxide or liquid alkaline, and fermentation results haematochrome yield is 1.5g/L.
(4) haematochrome extracting method: multigelation broken wall method and ultrasonic wall-cracking method are used, takes fermentation liquid in centrifuge tube
In, 4000r/min is centrifuged 10min, discards supernatant liquid, is washed with deionized and precipitates to obtain wet thallus, be placed in -20 °C immediately
Refrigerator 12h is continuously repeated twice, and 100% ethyl alcohol is added and mixes, the progress ultrasonic disruption processing on ice bath, ultrasonic power 200w,
Ethyl alcohol pigment extract can be obtained in ultrasonic time 5s, interval time 5s, total time 30min, centrifugation removal thallus.
Saccharine material of the present invention for fermentation can be glucose, sucrose, fructose, soluble starch or corn flour
Culture medium can be (g/L): glucose 40g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, sodium chloride 10
G, sodium citrate 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0~7.2；；
Culture medium can be (g/L): sucrose 25g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, sodium chloride 10
G, sodium citrate 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0~7.2；；
Culture medium can be (g/L): fructose 25g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, sodium chloride 10
G, sodium citrate 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0~7.2；
Culture medium can be (g/L): soluble starch 20g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, chlorination
10 g of sodium, sodium citrate 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0~7.2；；
Culture medium can be (g/L): corn flour 20g, peptone 4g, 15 g of yeast powder, 15 g of corn pulp, sodium chloride 10
G, sodium citrate 2.5, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0~7.2；
Natural red colouring matter can be used for oily food, baste, processing of aquatic products, vegetable product, jelly, ice cream
, cream, margarine, cheese, salad, sauce, rice made products, in the food such as baked goods, being also widely used for
In cosmetic and pharmacy industry.It can directly be used Rhodococcus ruber haematochrome prepared by the present invention as natural red colouring matter, can also be with
It is emulsified or is widely used in every field, such as food, feed, bait and health care product in powder form, especially added in food
Using very extensive in industry.It can be used for cooked meat product, jelly, beverage, cake etc., maximum usage amount is 4g/kg.Rhodococcus ruber
Haematochrome has good tinctorial property to protein, and tone has the sense of reality close to the Natural color of meat.Such as in ham, sausage, cloth
In fourth ice cream, in the food such as Yoghourt, with the additive amount of 0.2 ~ 0.4 g/kg；It can reach ideal coloring effect, tone is certainly
So, highly-safe.
EQUENCE LISTING
<110>University Of Science and Technology Of Tianjin
<120>a kind of Rhodococcus ruber and the preparation method and application thereof for generating natural red colouring matter
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1438
<212> DNA
<213>artificial sequence
<400> 1
tcgaacgctg ggggcgtgct taacacatgc aagtcgaacg gtaaggccct ttcgggggta 60
cacgagtggc gaacgggtga gtaacacgtg ggtaatctgc cctgcacttc gggataagcc 120
tgggaaaccg ggtctaatac cggatatgag ctcctgccgc atggtggggg ttggaaagtt 180
tttcggtgca ggatgagtcc gcggcctatc agcttgttgg tggggtaatg gcctaccaag 240
gcgacgacgg gtagccggcc tgagagggtg atcggccaca ctgggactga gacacgaccc 300
agactcctac gggaggcagc agtggggaat attgcacaat gggcgaaagc ctgatgcagc 360
gacgccgcgt gggggatgac ggtcttcgga ttgtaaactc ctttcagtag ggacgaagcg 420
aaagtgacgg tacctgcaga agaagcaccg gccaactacg tgccagcaac cacggtaata 480
cgtagggtgc aagcgttgtc cggaattact gggcgtaaag agctcgtagg cggtttgtca 540
cgtcgtctgt gaaatcctcc agctcaactg ggggcgtgca ggcgatacgg gcagacttga 600
gtactacagg ggagactgga attcctggtg tagcggtgaa atgcgcagat atcaggagga 660
acaccggtgg cgaaggcggg tctctgggta gtaactgacg ctgaggagcg aaagcatggg 720
gagcaaacag gagcagatac cctggtaagt ccatgccgta agcggtgggc gctaggtgtg 780
gggtccttcc acggattccg tgccgtagct aacgcattaa gcgccccgcc tggggagtac 840
gtccgcaagg ctaaaactca aaggaattga cggggacccg cacaagcggc ggagcatgtg 900
gattaattcg atgcaacgcg aagaacctta cctaggcttg acatatacag gacgacggca 960
gagatgtcgt ttcccttgtg gcttgtatac aggtggtgca tggttgtcgt cagctcgtgt 1020
cgtcagatgc ttgggttaag tcccgcaacg agcgcaaccc ctgtctcatg ttgccagcac 1080
gttatggtgg ggactcgtga gagactgccg gggtcaactc ggaggaaggt ggggatgacg 1140
tcaaatcatc atgcccctta tgtctagggc ttcacacatg ctacaatggc tagtacagag 1200
ggctgcgaga ccgcgaggtg gagcgaatcc cttaaagcta gtctcagttc ggattggggt 1260
ctgcaactcg accccatgaa gtcggagtcg ctagtaatcg cagatcagca ttgctgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacgtc atgaaagtcg gtaacacccg 1380
aagccggtgg cctaaccctt gtggagggag ccgtcgaagg tgggatctcg cgatttga 1438
Claims (3)
1. a kind of Rhodococcus ruber bacterial strain, it is characterised in that it classify belong to Rhodococcus ruber (Rhodococcus ruber), deposit number is
CGMCC No. 12604；Its nucleotide sequence is as shown in SEQ ID No.1.
2. it is a kind of using the method that 12604 Rhodococcus ruber bacterial strain of CGMCC No. prepares natural red colouring matter described in claim 1,
It is characterized in that carrying out by following step:
(1) seed culture and culture medium
The fermentation strain used is CGMCC No. 12604, seed culture medium composition: 10 ~ 30 g/L of glucose, peptone 2 ~ 5
G/L, 5 g/L of yeast powder, sodium chloride 10 g/L, K2HPO42 ~ 6 g/L, pH 7.0~7.2；150 ~ 180 revs/min of shaking table shakes
Swing culture, 30 ~ 40 DEG C, 36 ~ 48h；
(2) fermentation strain used is CGMCC No. 12604, fermentation medium composition are as follows: 20 ~ 40 g/L of saccharine material, egg
White 2 ~ 15 g/L of peptone, 15 g/L of yeast powder, 15 g/L of corn pulp, 10 g/L of sodium chloride, 1.5 ~ 3 g/L of sodium citrate,
MgSO4·7H20 0.1 ~ 0.2 g/L, K2HPO42 ~ 6 g/L, pH 7.0~7.2；
(3) fermented and cultured: press 1% ~ 10%(v/v) inoculum concentration by cultured seed access fermentation medium, cultivation temperature 30 ~
40 DEG C, fermentation ventilatory capacity is 5 ~ 13m3/ h, tank press 0.02 ~ 0.09 MPa, 120-150 revs/min of mixing speed, fermentation time
120 ~ 144 hours, it is passed through filtrated air in fermentation process, maintains fermentation liquid pH 6.5 ~ 7.5 with ammonium hydroxide or liquid alkaline, fermentation results
Haematochrome yield is 1 ~ 2g/L；
(4) haematochrome extracting method: using multigelation broken wall method and ultrasonic wall-cracking method, take fermentation liquid in centrifuge tube,
4000r/min is centrifuged 10min, discards supernatant liquid, is washed with deionized and precipitates to obtain wet thallus, is placed in -20 DEG C of refrigerators immediately
12h multigelation twice, is added 100% ethyl alcohol and mixes, and ultrasonic disruption processing, ultrasonic power 200w, ultrasound are carried out on ice bath
Ethyl alcohol pigment extract can be obtained in time 5s, interval time 5s, total time 30min, centrifugation removal thallus；Wherein saccharic is former
Material refers to: glucose, sucrose, fructose, maltose, soluble starch saccharified liquid or corn flour saccharified liquid.
3. application of the Rhodococcus ruber described in claim 1 in terms of preparing fermenting and producing natural red colouring matter.
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| US20220175852A1 (en) | 2019-04-24 | 2022-06-09 | Liaoning Greatest Bio-Pharmaceutical Co. Ltd. | Use of rhodococcus ruber product in treating thermal injury |
| US20230069441A1 (en) | 2020-01-21 | 2023-03-02 | Liaoning Greatest Bio-Pharmaceutical Co., Ltd. | Use of rhodococcus ruber cell wall skeleton in regenerative medicine |
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