CN106434466A - Rhodococcus ruber for generating natural haematochrome and preparation method and application thereof - Google Patents
Rhodococcus ruber for generating natural haematochrome and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to rhodococcus ruber for generating natural haematochrome and a preparation method and application thereof, wherein the haematochrome is generated through microbial fermentation; the strain category belongs to rhodococcus ruber; the preservation numbr is CGMCC No.12604. The invention also relates to a preparation method of the fermentation natural haematochrome. The fermentation strain of rhodococcus ruber CGMCC No.12604 is used for inoculating the cultured seeds into a fermentation culture medium according to the inoculation quantity according to the inoculation quantity of 1 percent to 10 percent (v/v), wherein the culture temperature is 30 to 40 DEG C; the fermentation ventilation quantity is 5 to 13m<3>/h; the tank pressure is 0.02 to 0.09 MPa; the stirring speed is 120 to 150 r/min; the fermentation time is 120 to 144h; in the fermentation process, sterile air is firstly introduced; the pH of the fermentation liquid is maintained at 6.5 to 7.5 by ammonium hydroxide or alkali liquor; according to the fermentation result, the yield of the haematochrome is 1 to 2g/L. The invention also discloses an extraction method of the rhodococcus ruber haematochrome.
Description
Technical field
The invention belongs to biological technical field, it is related to produce natural red colouring matter microorganism Rhodococcus ruber and its haematochrome preparation
Method.In particular it is：A kind of bacterial classification of fermenting and producing haematochrome Rhodococcus ruber, this bacterial classification be present invention discover that one plant of energy
Effectively produce the new strains of haematochrome, produced with this strain fermentation and extract simple and good stability.
Background technology
Pigment is to be widely used in food industry, Cosmetic Manufacture, weaving and commodity production, is that product brings preferably
Added value.But the potential hazard of synthetic dyestuff is carcinogenic, teratogenesis etc., its application is increasingly subject to repel, and natural colouring matter is more next
More favored by people.The source of natural colouring matter is broadly divided into animal pigment, phytochrome, microorganisms pigments and mineral pigment
Deng.Animal and plant source oneself warp of pigment can not meet the demand of industry high speed development.In recent years, it is devoted to finding and be available for replacement
The research of natural colouring matter increases, and these research major parts have invested microorganisms pigments sight.Some microorganisms were growing
In journey can chromogenesis, and microorganisms pigments growth fast, easily culture, can simply large-scale industrial production, microorganisms pigments
Gradually become a kind of important channel obtaining natural colouring matter.More importantly its security, the scope of application and research on maximum utilized quantity be all
There is obvious advantage than synthetic food color.Red pigments as one of three basic greatly pigments have very high " gold content " it
Multiple color tones can be deployed into blue, yellow strain natural colouring matter, make the color of natural colouring matter abundanter.Natural red pigments are not
Only can give food in riotous profusion color, and have some also to have different physiological roles, can be widely used for medicine, food and change
Cosmetic field.Research and develop the general trend that nontoxic natural red pigments have become as pigment development, oneself is through defining with sky
So pigment is leading market.The research and development of therefore natural red pigments has wide prospect and development potentiality.
Content of the invention
It is an object of the invention to provide a kind of Rhodococcus ruber（CGMCC No.12604）The culture of fermenting and producing natural red colouring matter
Based formulas and the extraction process of natural red colouring matter.Prepared natural red colouring matter can as food additives, feed addictive or
As the senior additive of medicine or makeup cosmetic after refined.
It is an object of the present invention to provide a kind of fermentable produces the bacterial classification of natural red colouring matter, its classification belongs to Rhodococcus ruber
（Rhodococcus ruber）, preserving number is CGMCC No. 12604.
Another object of the present invention is to disclose to adopt Rhodococcus ruber（Rhodococcus ruber）, preserving number is
The method that CGMCC No. 12604 bacterial classification prepares natural red colouring matter.
The present invention a further object is the employing Rhodococcus ruber disclosing（Rhodococcus ruber）, preserving number is
CGMCC No. 12604 strain fermentation produces the preparation method of natural red colouring matter.
For achieving the above object, technology contents disclosed by the invention are as follows：
A kind of Rhodococcus ruber, its classification belongs to Rhodococcus ruber（Rhodococcus ruber）, preserving number is CGMCC No. 12604.
The new strains of CGMCC No. 12604 bacterial strain that the present invention provides to be one plant can produce natural red colouring matter, use this bacterium
Strain fermentation saccharine material produces haematochrome.
Antifungal lipopeptid fermentable bacterial strain, its classification belongs to Rhodococcus ruber（Rhodococcus ruber）, preserving 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）Protected
Hide, 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：
This bacterium cell is shaft-like, and Grain stain uniformly, and is the positive, does not produce gemma.On broth bouillon flat board, bacterium colony is in
Circular or irregular, bacterial strain colony edge is neatly rounded, and smooth surface moistens redness.Bacterial strain atrichia, does not move, no
Luminous, haematochrome only produces non-pigment in intracellular, culture medium.
The present invention further discloses adopting Rhodococcus ruber（Rhodococcus ruber）, preserving number is CGMCC No.
The method that 12604 bacterial classifications prepare natural red colouring matter：
（1）Seed culture and culture medium
Using fermentation strain be CGMCC No. 12604, seed culture medium form（g/L）：Glucose 10 ~ 30g, peptone 2 ~
5g, dusty yeast 5 g/L, sodium chloride 10 g/L, K2HPO42 ~ 6g, pH 7.0～7.2；150 ~ 180 revs/min of shaking table concussion trainings
Support, 30 ~ 40 DEG C, 36 ~ 48h.
（2）Using fermentation strain be CGMCC No. 12604, fermentation medium consists of（g/L）：Saccharine material 20 ~
40g, peptone 2 ~ 15g, dusty yeast 15 g/L, corn steep liquor 15 g/L, sodium chloride 10 g/L, sodium citrate 1.5 ~ 3g,
MgSO4·7H20 0.1 ~ 0.2g, K2HPO42 ~ 6g, pH 7.0～7.2；
（3）Fermented and cultured：By 1%-10%（v/v）Inoculum concentration cultured seed is accessed fermentation medium, cultivation temperature 30 ~
40 DEG C, fermentation throughput is 5 ~ 13m3/ h, tank pressure 0.02 ~ 0.09 MPa, mixing speed 120-150 rev/min, fermentation time
120 ~ 144 hours, in sweat, it is passed through filtrated air, maintain zymotic fluid pH 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, sweat
In be passed through filtrated air, maintain zymotic fluid pH 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, fermentation results haematochrome yield is 1 ~ 2g/L.
（4）Haematochrome extracting method:Using multigelation broken wall method and supersonic wave wall breaking method, take zymotic fluid in centrifuge tube
In, 4000r/min is centrifuged 10min, abandoning supernatant, is washed with deionized and precipitates to obtain wet thallus, is positioned over -20 °C immediately
Refrigerator 12h continuously repeats twice, adds 100% ethanol to mix, carries out ultrasonic disruption process on ice bath, ultrasonic power 200w,
Ultrasonic time 5s, interval time 5s, total time 30min, centrifugation removes thalline, you can obtain ethanol pigment extract.
Preparation method of the present invention, wherein saccharine material include：Glucose, sucrose, fructose, maltose, solubility
Starch saccharificating liquid or corn flour saccharified liquid.
More detailed description of the present invention is as follows：
（1）The screening of microbial strains and identification：
Bacterial strain is inoculated into respectively the inorganic salt liquid culture medium of the sugar such as 1% glucose, sucrose, L- arabinose, mannitol
In, 28 DEG C of incubated 3 ~ 7d, observe strain growth situation.This bacterial strain can using dextrin, polysorbate40, Tween 80, D-Fructose,
The various saccharides acetic acid such as alpha-D-glucose, α-mannose, D-Psicose, D-ribose, sucrose, β-glycolic, α-glycolic, penta
The organic acids such as ketone 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 can not be utilized.Glucose aerogenesis can not be utilized, can grow in the environment of NaCl concentration is for 2%, blue for leather
Family name's positive bacteria, the physiological and biochemical property of bacterial strain consistent with the growth characteristics of Rhodococcus ruber all with Buchanan and Ji Bensi etc.（1984）
Write《The outstanding Bacteria Identification handbook of uncle》8th edition and east show pearl, Cai Miaoying（2001）Write《Common bacteria identification handbook》In
The Rhodococcus ruber bacterium of description is identical.
（2）16SrDNA sequencing:Gene cloning is carried out to the 16SrDNA sequence of this bacterial strain, amplification obtains
The sequence of 1438bp.This 16SrDNA sequence is carried out sequence analysis in GenBank understand, this bacterial strain and crimson ball
Bacterium (Rhodococcus ruber) affiliation is closest, and similitude reaches 99%.By this strain growth physiological property and
16SrDNA sequencing results judge that it is Rhodococcus ruber.The sequence of the 16SrDNA of bacterial strain is as follows：
Rhodococcus sp. (CGMCC No. 12604) 16S rDNA gene
TCGAACGCTGGGGGCGTGCTTAACACATGCAAGTCGAACGGTAAGGCCCTTTCGGGGGTACACGAGTGGCGAA
CGGGTGAGTAACACGTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACCGGGTCTAATACCGGATATGAG
CTCCTGCCGCATGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGTCCGCGGCCTATCAGCTTGTTGGTGGGGTA
ATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGACCCAGAC
TCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACG
GTCTTCGGATTGTAAACTCCTTTCAGTAGGGACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTA
CGTGCCAGCAACCACGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTT
TGTCACGTCGTCTGTGAAATCCTCCAGCTCAACTGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGG
AGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGT
AGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCAAACAGGAGCAGATACCCTGGTAAGTCCATGCCGTAAGCGGT
GGGCGCTAGGTGTGGGGTCCTTCCACGGATTCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGTC
CGCAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCG
AAGAACCTTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTGTATACAGGTGGT
GCATGGTTGTCGTCAGCTCGTGTCGTCAGATGCTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCTCATGTTGC
CAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCA
TGCCCCTTATGTCTAGGGCTTCACACATGCTACAATGGCTAGTACAGAGGGCTGCGAGACCGCGAGGTGGAGCGAAT
CCCTTAAAGCTAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGAT
CAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAA
GCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGGTGGGATCTCGCGATTTGA
（3）The culture of microbial strains and fermenting and producing haematochrome：
Inclined-plane and the composition of seed culture medium（g/L）：Glucose 1 ~ 3, tryptone 5 ~ 10, dusty yeast 3 ~ 6, NaCl 6 ~ 12, goes
Ionized water is prepared, pH 7.2～7.5；Solid medium need to add 15g agar powder.
The composition of fermentation medium（g/L）：Saccharine material 20 ~ 40g, peptone 2 ~ 15g, dusty yeast 15 g, corn steep liquor 15
G, sodium chloride 10 g, sodium citrate 1.5 ~ 3g, MgSO4 7,H20 0.1 ~ 0.2g, K2HPO42 ~ 6g, pH 7.0～7.2；Send out
Ferment is cultivated：By 1% ~ 10%（v/v）Inoculum concentration by cultured seed access fermentation medium, 30 ~ 40 DEG C of cultivation temperature, fermentation
Throughput is 5 ~ 13m3/ h, tank pressure 0.02 ~ 0.09 MPa, mixing speed 120-150 rev/min, fermentation time 120 ~ 144 is little
When, it is passed through filtrated air in sweat, maintain zymotic fluid pH 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, be passed through in sweat no
Bacterium air, maintains zymotic fluid pH 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, fermentation results haematochrome yield is 1 ~ 2g/L.
The saccharine material that the present invention is used for fermentation can be glucose, sucrose, fructose, soluble starch, corn flour saccharification
Liquid.Sweat is passed through filtrated air, maintains the pH of zymotic fluid 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, in zymotic fluid, bacterium is red
Plain yield is 1 ~ 2g/L.
Haematochrome extracting method:Using multigelation broken wall method and supersonic wave wall breaking method, take zymotic fluid in centrifuge tube,
4000r/min is centrifuged 10min, abandoning supernatant, is washed with deionized and precipitates to obtain wet thallus, is positioned over -20 DEG C of refrigerators immediately
12h multigelation twice, adds 100% ethanol to mix, carries out ultrasonic disruption process on ice bath, and ultrasonic power 200w is ultrasonic
5s time, 5s interval time, total time 30min.Centrifugation removes thalline, you can obtain ethanol pigment extract.
The present invention has investigated further and has adopted Rhodococcus ruber（Rhodococcus ruber）, preserving number is CGMCC No.
The steadiness of the natural red colouring matter of 12604 bacterial classification preparations：
（1）The impact to pigment for the light：Take pigment solution to put to irradiate under indoor ultraviolet light, 0,1,2,3,4,5h be measured extinction
Spend and observe color change situation.Its storage rate is respectively 100%, 97.9%, 97.8%, 97.7%, 91.9%, 88.9%.This pigment
More stable to ultraviolet light.Take pigment solution to put to irradiate under indoor white flag light, 0,1,2,3,4,5h is measured absorbance and observes
Color change situation.Its storage rate be respectively 100%, 100%, 100%, 97.7 %, 97 %, 96.7%.This pigment solution is in white flag light
Under very stable.
（2）The impact to pigment for the temperature：Take pigment solution 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, its storage rate be respectively 99.7%, 99.4%, 99%,
98.9%、98.6%、98.1%、94.4%、92.7%.This pigment solution is more high temperature resistant, and normal temperature stability inferior is high.
（3）The impact to pigment for the oxidant：Take pigment solution, use 30%H2O2Be configured to 0%, 0.1%, 0.2%, 0.3%,
0.4%th, the H of 0.5% concentration2O2Pigment solution, avoid light place 1h surveys its absorbance, its storage rate be respectively 100%, 99.8%,
99.2%、98.1%、97.7%、97.5%.Oxidant H2O2Impact result to pigment stability is visible, and pigment is in variable concentrations
Absorbance change very little under oxidant, its color corresponding also no significant change, show that this pigment antioxidant is fine
（4）The impact to pigment for the reducing agent：Take pigment solution, use Na2SO3Be configured to 0,0.1,0.2,0.4,0.8mg/L concentration
Na2SO3Pigment solution, avoid light place 1h surveys its absorbance, its storage rate be respectively 100%, 98.7%, 97.6%, 97.1%,
96.4%.Reducing agent Na2SO3Impact result to pigment stability is visible, and pigment absorbance under the reducing agent of variable concentrations becomes
Change very little, its color corresponding also no significant change, show that the resistance to reducing agent of this pigment is fine.
（5）The impact of food additives:Use potassium sorbate, Sodium Benzoate, vitamin C, sodium citrate, lactic acid configuration respectively
Become the pigment solution of 2mg/L, avoid light place 1h surveys its absorbance, its storage rate is respectively 100%, 100%, 100%, 100%,
100%.Pigment solution is not any change under the influence of food additives, shows that this pigment is highly stable to food additives.
The positive effect that Rhodococcus ruber producing natural red colouring matter disclosed by the invention and preparation method and application has
Fruit is：
（1）Rhodococcus ruber is a kind of nontoxic microorganism, can carry out industrializing large-scale culture.
（2）The good stability to light and temperature for the pigment being extracted by Rhodococcus ruber thalline, can be widely applied to food and
In cosmetics.
（3）Extract completely, quickly, the technological process of production is simple, product non-toxic residual, safe.
Brief description
Fig. 1 is Rhodococcus sp（Rhodococcus ruber）Colonial morphology figure；Because bacterium colony produces haematochrome, in red
Color.
Specific embodiment
Describe the present invention 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, and the unrestricted present invention
Scope, 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
On the premise of invention spirit and scope, the material component in these embodiments and consumption are carried out various changes or are changed
Belong to protection scope of the present invention.The present invention is raw materials used and reagent is commercially available.Such as glucose, sucrose, fructose, solubility
Starch, corn flour etc. are commercially available.
Embodiment 1
Rhodococcus ruber, its classification belongs to Rhodococcus ruber（Rhodococcus ruber）, preserving number is CGMCC No. 12604.
Screening technique as follows：
（1）Take pedotheque（Rich in humus）5 grams in seed culture medium, wherein seed culture medium composition（g/L）：Glucose
10g, peptone 2g, dusty yeast 5 g, sodium chloride 10 g, K2HPO42g, pH 7.0～7.2；150 ~ 180 revs/min of shaking table shakes
Swing culture, 30 ~ 40 DEG C, 36 ~ 48h enriched microorganism.
（2）With the aseptic picking of oese（1）The microbial inoculum of enrichment, in seed culture medium composition（g/L）：Glucose
10g, peptone 2g, dusty yeast 5 g, sodium chloride 10 g, K2HPO42g, pH 7.0～7.2, flat lining out, it is statically placed in culture
30 ~ 40 DEG C in case, cultivate 36 ~ 48h；
（3）Observe（2）The red colonies of growth in flat board, and its sterile working picking to seed culture medium is formed（g/L）：Portugal
Grape sugar 10g, peptone 2g, dusty yeast 5 g, sodium chloride 10 g, K2HPO42 ~ 6g, pH 7.0～7.2, flat lining out separates,
It is statically placed in 30 DEG C in incubator, cultivate 36h；
（4）Through（3）Separate the single bacterium colony obtaining and carry out Biolog experiment, and 16s rDNA sequencing and control experiment.Really
The kind of its bacterial strain fixed：
Biolog system identification result
Embodiment 2
Using Rhodococcus ruber（Rhodococcus ruber）, preserving number prepares natural red colouring matter for CGMCC No. 12604 bacterial classification
Method：
（1）Seed culture and culture medium
Using fermentation strain be CGMCC No. 12604, seed culture medium form（g/L）：Glucose 20g, peptone 4g, ferment
Female powder 5 g, sodium chloride 10 g, K2HPO43g, pH 7.0～7.2；170 revs/min of shaking table concussion and cultivates, 34 DEG C, 45h.
（2）Using fermentation strain be CGMCC No. 12604, fermentation medium consists of（g/L）：Saccharine material 20 ~
40g, peptone 4g, dusty yeast 15 g, corn steep liquor 15 g, sodium chloride 10 g, sodium citrate 2.5, MgSO4 7H20 0.15g,
K2HPO43g, pH 7.0～7.2.
（3）Fermented and cultured：By 1% ~ 10%（v/v）Inoculum concentration by cultured seed access fermentation medium, culture temperature
35 DEG C of degree, fermentation throughput is 8m3/ h, tank pressure 0.05 MPa, 120 ~ 150 revs/min of mixing speed, fermentation time 134 is little
When, it is passed through filtrated air in sweat, maintain zymotic fluid pH 7.0 with ammoniacal liquor or liquid caustic soda, in sweat, be passed through aseptic sky
Gas, maintains zymotic fluid pH 7.0 with ammoniacal liquor or liquid caustic soda, fermentation results haematochrome yield is 1.5g/L.
（4）Haematochrome extracting method:Using multigelation broken wall method and supersonic wave wall breaking method, take zymotic fluid in centrifuge tube
In, 4000r/min is centrifuged 10min, abandoning supernatant, is washed with deionized and precipitates to obtain wet thallus, is positioned over -20 °C immediately
Refrigerator 12h continuously repeats twice, adds 100% ethanol to mix, carries out ultrasonic disruption process on ice bath, ultrasonic power 200w,
Ultrasonic time 5s, interval time 5s, total time 30min, centrifugation removes thalline, you can obtain ethanol pigment extract.
The saccharine material that the present invention is used for fermentation can be glucose, sucrose, fructose, soluble starch or corn flour
Culture medium can be（g/L）:Glucose 40g, peptone 4g, dusty yeast 15 g, corn steep liquor 15 g, sodium chloride 10 g, lemon
Lemon acid sodium 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0～7.2；；
Culture medium can be（g/L）:Sucrose 25g, peptone 4g, dusty yeast 15 g, corn steep liquor 15 g, sodium chloride 10 g, lemon
Lemon acid sodium 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0～7.2；；
Culture medium can be（g/L）:Fructose 25g, peptone 4g, dusty yeast 15 g, corn steep liquor 15 g, sodium chloride 10 g, lemon
Lemon acid sodium 2.5g, MgSO4·7H20 0.15g, K2HPO43g, pH 7.0～7.2；
Culture medium can be（g/L）:Soluble starch 20g, peptone 4g, dusty yeast 15 g, corn steep liquor 15 g, sodium chloride 10
G, 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, dusty yeast 15 g, corn steep liquor 15 g, 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, milk
In the food such as oil, margarine, cheese, salad, tartar sauce, rice made products, baked goods, it is also widely used for making up
In product and pharmacy industry.The Rhodococcus ruber haematochrome of present invention preparation it can be directly using it is also possible to warp as natural red colouring matter
Emulsify or be widely used in every field in powder form, such as food, feed, bait and health products etc., especially in food processing
Apply quite varied in industry.Can be used for cooked meat product, jelly, beverage, cake etc., maximum usage amount is 4g/kg.Rhodococcus ruber is red
Pigment has good tinctorial property to protein, and tone is pressed close to the Natural color of meat, had the sense of reality.For example in ham, sausage, pudding
In ice cream, in the food such as Yoghourt, with the addition of 0.2 ~ 0.4 g/kg；Can reach preferable coloring effect, tone is natural,
Safe.
EQUENCE LISTING
<110>University Of Science and Technology Of Tianjin
<120>A kind of Rhodococcus ruber producing natural red colouring matter and preparation method and application
<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 (5)
1. a kind of Rhodococcus ruber bacterial strain belongs to Rhodococcus ruber it is characterised in that it is classified（Rhodococcus ruber）, preserving number is
CGMCC No. 12604.
2. the Rhodococcus ruber strain described in claim 1 is it is characterised in that its nucleotide sequence is as shown in SEQ ID No.1.
3. a kind of method that natural red colouring matter is prepared using CGMCC No. described in claim 1 12604 Rhodococcus ruber bacterial strain, its
It is characterised by carrying out by the steps：
（1）Seed culture and culture medium
Using fermentation strain be CGMCC No. 12604, seed culture medium form（g/L）：Glucose 10 ~ 30g, peptone 2 ~
5g, dusty yeast 5 g/L, sodium chloride 10 g/L, K2HPO42 ~ 6g, pH 7.0～7.2；150 ~ 180 revs/min of shaking table concussion trainings
Support, 30 ~ 40 DEG C, 36 ~ 48h；
（2）Using fermentation strain be CGMCC No. 12604, fermentation medium consists of（g/L）：Saccharine material 20 ~ 40g,
Peptone 2 ~ 15g, dusty yeast 15 g/L, corn steep liquor 15 g/L, sodium chloride 10 g/L, sodium citrate 1.5 ~ 3g, MgSO4
7H20 0.1 ~ 0.2g, K2HPO42 ~ 6g, pH 7.0～7.2；
（3）Fermented and cultured：By 1% ~ 10%（v/v）Inoculum concentration cultured seed is accessed fermentation medium, cultivation temperature 30 ~
40 DEG C, fermentation throughput is 5 ~ 13m3/ h, tank pressure 0.02 ~ 0.09 MPa, mixing speed 120-150 rev/min, fermentation time
120 ~ 144 hours, in sweat, it is passed through filtrated air, maintain zymotic fluid pH 6.5 ~ 7.5 with ammoniacal liquor or liquid caustic soda, fermentation results
Haematochrome yield is 1 ~ 2g/L；
（4）Haematochrome extracting method:Using multigelation broken wall method and supersonic wave wall breaking method, take zymotic fluid in centrifuge tube,
4000r/min is centrifuged 10min, abandoning supernatant, is washed with deionized and precipitates to obtain wet thallus, is positioned over -20 DEG C of refrigerators immediately
12h multigelation twice, adds 100% ethanol to mix, carries out ultrasonic disruption process on ice bath, and ultrasonic power 200w is ultrasonic
Time 5s, interval time 5s, total time 30min, centrifugation removes thalline, you can obtain ethanol pigment extract.
4. the preparation method described in claim 3, wherein saccharine material refers to：Glucose, sucrose, fructose, maltose, can
Soluble starch saccharified liquid or corn flour saccharified liquid.
5. application in terms of preparation fermenting and producing natural red colouring matter for the Rhodococcus ruber described in claim 1.
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| WO2020147472A1 (en) | 2019-01-15 | 2020-07-23 | 辽宁格瑞仕特生物制药有限公司 | Product derived from rhodococcus ruber, and pharmaceutical use thereof |
| WO2020216281A1 (en) | 2019-04-24 | 2020-10-29 | 辽宁格瑞仕特生物制药有限公司 | Use of rhodococcus ruber product in treating thermal injury |
| WO2021147899A1 (en) | 2020-01-21 | 2021-07-29 | 辽宁格瑞仕特生物制药有限公司 | Use of rhodococcus ruber cell wall skeleton in regenerative medicine |
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