CN103484395B - Bacterial strain used for preparing single-cell protein from methanol, and applications of bacterial strain - Google Patents
Bacterial strain used for preparing single-cell protein from methanol, and applications of bacterial strain Download PDFInfo
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- CN103484395B CN103484395B CN201310284159.6A CN201310284159A CN103484395B CN 103484395 B CN103484395 B CN 103484395B CN 201310284159 A CN201310284159 A CN 201310284159A CN 103484395 B CN103484395 B CN 103484395B
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Abstract
The invention discloses a bacterial strain used for preparing single-cell protein from methanol, and applications of the bacterial strain. The bacterial strain is Methylovorusglucosotrophus M25, and the preservation number of the bacterial strain is CGMCC No.7036. Methylovorusglucosotrophus M25 (CGMCC No.7036) is capable of preparing single-cell protein by using methanol, and can be used for production of animal feed additive.
Description
Technical field
The present invention relates to the bacterial strain of a strain converting methanol manufacture order cell protein and the application in single cell protein is produced thereof.Concrete finger is utilizing the methyl alcohol resource of China's abundant, when forming new Downstream Products of Methanol production chain, methanol conversion is generated single cell protein product, have production energy consumption lower, do not affect by natural environment and climate, do not need rely on plough, can continuous seepage, be easy to control and pollute the advantages such as little.Solve traditional single cell protein to produce and limit by raw material sources, can not the shortcoming of scale operation, thus realize the new alternate source of general proteins feed.
Background technology
Take methyl alcohol as raw material, the microorganism that application is suitable for is that the novel single cell protein that culture propagation is produced is nutritious, containing rich in protein, amino acid and multivitamin, as feed, Production of Livestock and Poultry can be promoted and improve efficiency of feed utilization, there is higher added value, the Protein supplements such as fish meal, soybean, bone meal, meat and skim-milk can be replaced.This single cell protein safety non-toxic, be can be used as animal-feed by state's government permissions such as Britain, Germany, Belgium, Holland, Denmark, Russia, Mexico and uses, used for more than 20 years abroad, be proved to be a kind of safe animal feedstuff additive.
Be larger to the consumption of methyl alcohol in the novel single cell protein produce market of raw material production and production process with methyl alcohol.In feed, protein content is generally 15%-20%, by bacterium with methyl alcohol be the protein content of the novel single cell protein of raw material production generally higher than 70%, if by 1 ton preparation 10 tons of feeds, wherein protein content can be made to reach more than 7%.The output of domestic annual feed is about 200,000,000 tons, if wherein the albumen of 7% is provided by this novel single cell protein, then the demand of this product is 2,000 ten thousand tons; If need consumption 2 tons of methyl alcohol to calculate by production 1 ton of protein product, methyl alcohol about 4,000 ten thousand tons can be consumed.Therefore, be that the novel single cell protein produce market prospect of raw material production is better with methyl alcohol, can be the product form that added value is higher by the methanol conversion of part producing surplus simultaneously.
Summary of the invention
An object of the present invention is to provide the bacterial strain of a strain converting methanol manufacture order cell protein and the application in single cell protein is produced thereof.Be application methyl alcohol minimal medium domestication Central Plains, Puyang great Hua plant area mud sample, bacterial strain separation and purification obtained, is obtained by vacuum mutagenic and breeding, and this bacterium is food grape confectionery methyl bacterium M25, Latin name:
methylovorus glucosotrophus); Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution is called for short: CGMCC, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on December 24th, 2012, deposit number CGMCC No. 7036.
Food grape confectionery methyl bacterium (
methylovorus glucosotrophus) bacterium colony of M25 presents lightpink, neat in edge; Cellular form and physical and chemical experiment result as shown in table 1; 16S rRNA gene order is as shown in SEQ ID No.1.
Food grape confectionery methyl bacterium provided by the present invention (
methylovorus glucosotrophus) M25 can be used for the production of single cell protein.
Another object of the present invention be to provide this bacterial strain single cell protein produce in application, comprise the steps: by described food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 joins in the culture system containing methyl alcohol
In above-mentioned single cell protein production application, described production is carried out according to the method comprised the steps: in minimal medium, add yeast extract paste or corn steep liquor, add trace element, 121 DEG C of sterilizing 20min, add the methyl alcohol that filtering with microporous membrane through 0.22 μm of aperture is degerming, mix rear inoculation described food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036.
Above-mentioned minimal medium for add 1.5g K in 1L water
2hPO
4, 0.5g KH
2pO
4, 0.2g MgSO
47H
2o, 1g NaCl and 1g(NH
4)
2sO
4, pH nature.
Above-mentioned trace element for add 1.69g MnSO in 1L water
4h
2o, 0.24g CoCl
26H
2o, 1.16g H
3bO
3, 0.024g Na
2moO
42H
2o, 2.78g Fe SO
47H
2o, 1.15g Zn SO
47H
2o and 0.38g CuSO
45H
2o, pH7.0.
In above-mentioned single cell protein production application, the temperature of described fermentation is 25 DEG C ~ 37 DEG C, preferably 30 DEG C ~ 37 DEG C; The time of described fermentation is 12 ~ 24h; Initial methanol concentration is 0.1 ~ 10%, preferably 0.2%; Organic nitrogen source is corn steep liquor 0.1 ~ 0.3%, or yeast extract paste 0.3 ~ 0.5%; Trace element is 0.1%; Liquid amount is 70%; Every liter capacity air flow quantity is 60 ~ 80L/h.
The described novel single cell protein that method described above prepares belongs to protection scope of the present invention.
The single cell protein product protein content that application aforesaid method prepares is 71.53% ~ 77.92%, and methyl alcohol is residual not to be detected.Product animal toxicity test result is actual nontoxic level.Salmonella reversion test, Micronucleus test and mouse inbred strain result are negative.Microorganism detection result shows, product is not containing pathogenic bacterium such as Salmonellass.
Use food grape confectionery methyl bacterium provided by the present invention (
methylovorus glucosotrophus) M25 CGMCC No. 7036 converting methanol manufacture order cell protein have with low cost, be easy to advantages such as controlling, be produced on a large scale, can be used for the production of animal feedstuff additive.
preservation explanation
Strain name: food grape confectionery methyl bacterium
Latin name:
methylovorus glucosotrophus
Strain number: M25
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on December 24th, 2012
Register on the books numbering: CGMCC No. 7036 at preservation center.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Inorganic salt liquid fermentation base: solvent is water, and solute is K
2hPO
41.5g/L, KH
2pO
40.5g/L, MgSO
47H
2o 0.2g/L, NaCl 1g/L, (NH
4)
2sO
41g/L, pH nature.
Solid medium: often go up the substratum stated inorganic salt liquid substratum interpolation 15g agar and prepare.
Trace element solution for add 1.69g MnSO in 1L water
4h
2o, 0.24g CoCl
26H
2o, 1.16g H
3bO
3, 0.024g Na
2moO
42H
2o, 2.78g Fe SO
47H
2o, 1.15g Zn SO
47H
2o and 0.38g CuSO
45H
2o, pH7.0
Embodiment 1
Food grape confectionery methyl bacterium (
methylovorus glucosotrophus) mutagenic and breeding of M25 CGMCC No. 7036 and qualification.
Be inoculated in by Central Plains, 5g Puyang great Hua plant area mud sample respectively and 100mL is housed contains in the 250mL triangular flask of the minimal medium of 5% methyl alcohol, 180rpm, 30 DEG C of domestications cultivate 15d, interval 5d supplemented medium.Liquid after domestication obtains single bacterium colony through solid plate separation and purification, and wherein bacterial strain M growth performance in the inorganic salt liquid substratum containing 5% methyl alcohol is best.With bacterial strain M for starting strain, join in 5mL water or 5% aqueous trehalose with the bacterial strain M that enlarged culturing that solid medium is rule grows by aseptic cotton carrier, vibration, get 0.1mL and be coated with 60mm plate, carrying out dosage after drying is respectively 1 × 10
15n
+/ cm
2, 5 × 10
15n
+/ cm
2with 1 × 10
16n
+/ cm
2ion implantation, the vacuum-treat group under various dose is set simultaneously, with under 3mL washing after mutagenesis, joins 100mL and to contain in the minimal medium of 5% methyl alcohol shaking table and cultivate 24h, respectively at separation and purification on solid medium.To be separated the single bacterium colony enlarged culturing on solid medium obtained, the bacterium colony grown under wiping with aseptic cotton carrier, washes and is dissolved in sterilized water.By above-mentioned bacterium liquid by 1% inoculum size be inoculated in respectively and 100mL is housed contains in the 250mL triangular flask of 5% or 0.2% methyl alcohol minimal medium, 180rpm, 30 DEG C of condition bottom fermentations.Compared with starting strain M, fermentation 24h, is separated the bacterial strain M25 fermented liquid OD obtained after the mutagenesis of 10min vacuum
600value improves 79.6% and 34.54% respectively.
The bacterium colony of bacterial strain M25 on solid medium presents lightpink, neat in edge; Cellular form and physical and chemical experiment result as shown in table 1; 16S rRNA gene order is as shown in SEQ ID No.1.According to cell microscopic morphology, physical and chemical experiment result and 16S rRNA gene sequence data, bacterial strain M25 is accredited as food grape confectionery methyl bacterium (
methylovorus glucosotrophus), and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on December 24th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation accession designation number is CGMCC No.7036.
table 1 cellular form and physical and chemical experiment result
Embodiment 2, application food grape confectionery methyl bacterium (
methylovorus glucosotrophus) single cell protein that carries out of M25 CGMCC No. 7036 produces
One, eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) the single cell protein production of M25 CGMCC No. 7036 under condition of different temperatures
With aseptic cotton carrier by solid plate grows food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 wipe under, wash and be dissolved in sterilized water, inoculum size by 1% is inoculated in and 100ml is housed contains in the 250ml triangular flask of the minimal medium of 5% methyl alcohol, be put on shaking table, 180rpm, 24h is cultivated under 30 DEG C of conditions, contain in the 250ml triangular flask of the minimal medium of 5% methyl alcohol by 20% inoculum size transferred species in being equipped with 100ml, be put on the shaking table of 25 DEG C, 30 DEG C and 37 DEG C respectively, 180rpm, fermentation 12h fermented liquid is as shown in table 2 at the light absorption value at 600nm place, food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 can grow in 25 DEG C ~ 37 DEG C temperature ranges, fermentation 12h, 30 DEG C with 37 DEG C of condition hypothallus density indifferences, all cultivate higher than under 25 DEG C of conditions.Therefore, preferably cultivate under 30 DEG C ~ 37 DEG C conditions.
The thalli growth situation of table 2 condition of different temperatures bottom fermentation 12h
Two, eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 applies the single cell protein that different substratum carries out in shaking flask and produce
Method I eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 shake flask fermentation test in the substratum containing 5% ~ 10% methyl alcohol
In the scope that initial methanol concentration is 5% ~ 10%, with liquid amount and nutrient media components methyl alcohol, ammonium sulfate and yeast extract paste for factor, design 4 factor 3 horizontal quadratures experiments, experiment is divided into 9 groups, often organizes 3 repetitions.With aseptic cotton carrier by solid plate grows food grape confectionery methyl bacterium (
methylovorus glucosotrophus) under M25 CGMCC No.7036 wipes, wash and be dissolved in sterilized water, be inoculated in each experimental group by 1% inoculum size, often group designs the control group do not inoculated, experimental design and result as shown in table 3.
The orthogonal experiment of table 3 initial methanol concentration in 5% ~ 10% scope
This bacterial strain can tolerate and can grow in containing the substratum of 10% methyl alcohol, and in 5% ~ 10% scope, increase with methanol concentration, thalline yield declines.In the substratum containing 5% methyl alcohol, 0.3% ammonium sulfate and 0.3% yeast extract paste, liquid amount is that the condition hypothallus yield of 100mL is higher, with this understanding, the experimental group inoculated and the control group do not inoculated remain at similarity condition bottom fermentation 24h methyl alcohol and are respectively 3.87% and 4.37%, namely the transformation efficiency of methyl alcohol is only 11.44%, illustrate to also have most of methyl alcohol not to be utilized.Extreme difference data presentation, in this experimental concentration scope, in 4 factors, the influence of methyl alcohol is maximum.
Method II eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 shake flask fermentation test in the substratum containing 1% ~ 5% methyl alcohol
In the scope that initial methanol concentration is 1% ~ 5%, with nutrient media components methyl alcohol, glucose, ammonium sulfate and yeast extract paste for factor, design 4 factor 3 horizontal quadrature experiments, experiment is divided into 9 groups, often organizes 3 repetitions.With aseptic cotton carrier by solid plate grows food grape confectionery methyl bacterium (
methylovorus glucosotrophus) under M25 CGMCC No.7036 wipes, wash and be dissolved in sterilized water, be inoculated in each experimental group by 1% inoculum size, often group designs the control group do not inoculated, experimental design and result as shown in table 4.In 1% ~ 5% scope, increase with methanol concentration, thalline yield declines; Increase with concentration in glucose 0 ~ 1%, yeast extract paste 0 ~ 0.5% scope, thalline yield raises; Ammonium sulfate is all applicable to thalli growth in 0.1% ~ 0.3% scope.Extreme difference data presentation, in this experimental concentration scope, in 4 factors, the influence of yeast extract paste is maximum.
The orthogonal experiment of table 4 initial methanol concentration in 1% ~ 5% scope
Method III eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 shake flask fermentation test in the substratum containing 0.2% methyl alcohol
With aseptic cotton carrier by solid plate grows food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 wipe under, wash and be dissolved in sterilized water, be inoculated in by 1% inoculum size in the minimal medium not adding or add the micro-containing 0.1% of 0.1% corn steep liquor and 0.2% methyl alcohol, shake flask fermentation result is as shown in table 5, relatively the growing state of bacterial strain in formula A and B is known, when not adding 0.1% corn steep liquor, appropriate increase is inorganic nitrogen-sourced is conducive to thalli growth, but thalline yield is not high.Formula C and D adds 0.1% corn steep liquor on the basis of formula A and B, and by contrast, bacterial strain is that in the substratum of C and D, cell density significantly increases at formula, and the visible appropriate organic nitrogen source that adds contributes to thalli growth.
Table 5 does not add or adds the impact of micro-organic nitrogen source on thalli growth
Three, eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 applies the single cell protein that different substratum carries out in 10L fermentor tank and produce
Method I is ferment in the minimal medium containing 5% methyl alcohol at basic medium
With aseptic cotton carrier by solid plate grows food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 wipe under, wash and be dissolved in sterilized water, be inoculated in minimal medium (A) containing 5% methyl alcohol respectively by 1% inoculum size, add the minimal medium (B) of 5% methyl alcohol of 0.1% trace element, add the minimal medium (C) of 5% methyl alcohol of 0.3% corn steep liquor and 0.1% trace element, add in the minimal medium (D) of 5% methyl alcohol of 0.3% yeast extract paste and 0.1% trace element, shake-flask culture 24h, then be inoculated in the 10L fermentation cylinder for fermentation of 4 kinds of substratum that above-mentioned correspondence is housed respectively by the inoculum size of 14.28%.
Result shows, the application A substratum thalline color obtained of ferment is redder, and impurity is less, but cell density is lower, and ferment 24h fermented liquid OD
600be only 0.4 ~ 0.5; The application B substratum thalline color obtained of ferment is also redder, and impurity is also less, and cell density increases, and ferment 24h fermented liquid OD
600reach 0.50 ~ 0.6; The fermentation of application C substratum, due to the initial OD of this substratum
600be worth higher, fermentation 24h fermented liquid OD
600>=1, but centrifugal after and the impurity that simultaneously precipitates of thalline more; The fermentation of application D substratum, fermentation 24h fermented liquid OD
600>=1, compared with fermenting with application C substratum, less with the impurity of thalline co-precipitation.
Method II is ferment in the minimal medium containing 0.2% methyl alcohol at basic medium
To eat grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 is inoculated in shake-flask culture 24h in the minimal medium of interpolation 0.2% methyl alcohol, 0.1% trace element and 0.3% yeast extract paste, then is inoculated in above-mentioned substratum ferments by the inoculum size of 14.28%, fermentation 24h fermented liquid OD
600>=1, show, food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25 CGMCC No.7036 carbon source only containing 0.2% methyl alcohol culture system in also can grow preferably.
<110> Henan Coal Chemical Industry Group Institute Co., Ltd., Zhengzhou University
The bacterial strain of <120> converting methanol manufacture order cell protein and application thereof
<160> 1
<210> 1
<211> 1407
<212> DNA
<213> food grape confectionery methyl bacterium (
methylovorusglucosotrophus) M25
<400> 1
ccagtcgacg gtaacagaga gaagcttgct tctctgctga cgagtggcga acgggtgagt 60
aatatatcgg aacgtaccgt attgtggggg ataactagtc gaaagattag ctaataccgc 120
atacgccctg agggggaaag taggggatct tcggacctta cgcagaacga gcggccgata 180
tctgattagc tagttggtgg ggtaaaggcc caccaaggcg acgatcagta gctggtctga 240
gaggacgacc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 300
ggggaatttt ggacaatggg cgcaagcctg atccagccat gccgcgtgag tgaagaaggc 360
cttcgggttg taaagctctt tcgcaaggaa agaaaactta ctcgctaata ccgggtgagg 420
atgacggtac cttgataaga agcaccggct aactacgtgc cagcagccgc ggtaatacgt 480
agggtgcgag cgttaatcgg aattactggg cgtaaagcga gcgcaggcgg ttttgtaagt 540
cagatgtgaa agccccgggc tcaacctggg aactgcgttt gaaactgcaa ggctagagta 600
tgggagaggg gggtagaatt ccacgtgtag cagtgaaatg cgtagagatg tggaggaata 660
ccaatggcga aggcagcccc ctggcctaat actgacgctc atgctcgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccctaaacg atgtctacta gttgttggtg 780
gagtaaaatc cattagtaac gcagctaacg cgtgaagtag accgcctggg gagtacggtc 840
gcaagattaa aactcaaagg aattgacggg ggcccgcaca agcggtggat tatgtggatt 900
aattcgatgc aacgcgaaaa ccttacctgg ccttgacatg ctactaacga agcagagatg 960
cattaggtgc ccgaaaggga aagtagacac aggtgctgca tggctgtcgt cagctcgtgt 1020
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgccaataat tgccatcatt 1080
tagttgggca ctttattggg actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140
agtcctcatg gcccttatgg ccagggcttc acacgtaata caatggtcgg tacagagggt 1200
tgccaacccg cgagggggag ccaatcccag aaagccgatc gtagtccgga ttgcagtctg 1260
caactcgact gcatgaagtc ggaatcgcta gtaatcgcgg atcagcatgt cgcggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttc taccagaagt 1380
agttagtcta accgcaaggg gacgata 1407
Claims (7)
1. the bacterial strain of converting methanol manufacture order cell protein, is characterized in that: this bacterial strain for food grape confectionery methyl bacterium (
methylovorus glucosotrophus) M25, its preservation registration number is CGMCC No.7036.
2. the application of bacterial strain in single cell protein is produced of converting methanol manufacture order cell protein according to claim 1.
3. the application of bacterial strain in single cell protein is produced of converting methanol manufacture order cell protein according to claim 2, it is characterized in that: the method that described single cell protein is produced, is be inoculated into by food grape confectionery methyl bacterium M25 in the system containing methyl alcohol to ferment.
4. the application of bacterial strain in single cell protein is produced of converting methanol manufacture order cell protein according to claim 3, is characterized in that: described leavening temperature is 25 DEG C ~ 37 DEG C.
5. the application of bacterial strain in single cell protein is produced of the converting methanol manufacture order cell protein according to any one in claim 3 ~ 4, is characterized in that: described is 0.1 ~ 10% containing the concentration of methyl alcohol in the system of methyl alcohol.
6. the application according to any one in claim 3 ~ 4, is characterized in that: described is 0.1 ~ 0.3% corn steep liquor or 0.3 ~ 0.5% yeast extract paste containing adding organic nitrogen source in the system of methyl alcohol.
7. according to the application of bacterial strain in single cell protein is produced of the converting methanol manufacture order cell protein in claim 3 ~ 4 described in any one, it is characterized in that: the described system containing methyl alcohol refers to, in minimal medium, add yeast extract paste or corn steep liquor, the mass percent that corn steep liquor accounts for system be 0.1 ~ 0.3% or the yeast extract paste mass percent that accounts for system be 0.3 ~ 0.5%, add trace element solution, 121 DEG C of sterilizing 20min, add the methyl alcohol that filtering with microporous membrane through 0.22 μm of aperture is degerming
Above-mentioned minimal medium for add 1.5g K in 1L water
2hPO
4, 0.5g KH
2pO
4, 0.2g MgSO
47H
2o, 1g NaCl and 1g(NH
4)
2sO
4, pH nature;
Above-mentioned trace element solution for add 1.69g MnSO in 1L water
4h
2o, 0.24g CoCl
26H
2o, 1.16g H
3bO
3, 0.024g Na
2moO
42H
2o, 2.78g Fe SO
47H
2o, 1.15g Zn SO
47H
2o and 0.38g CuSO
45H
2o, pH7.0, the volume percent accounting for total system at the described system medium trace element solution containing methyl alcohol is 0.1%.
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