CN105385612A - Candida utilis, composition thereof and application - Google Patents
Candida utilis, composition thereof and application Download PDFInfo
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- CN105385612A CN105385612A CN201510952295.7A CN201510952295A CN105385612A CN 105385612 A CN105385612 A CN 105385612A CN 201510952295 A CN201510952295 A CN 201510952295A CN 105385612 A CN105385612 A CN 105385612A
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- candida utilis
- strain
- fermentation
- gossypol
- cotton dregs
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Classifications
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
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Abstract
The invention provides a new candida utilis, a probiotics composition containing the same and application of the candida utilis and the probiotics composition containing the same to fermentation of cottonseed meal and treatment of waste liquid containing ammonia and nitrogen. The invention further provides a method for preparing the fermented feed probiotics composition and a method of obtaining saccharomycete mutant strains with a high ammonia and nitrogen assimilation rate and a high gossypol degradation rate by means of ultraviolet mutation breeding and screening. The candida utilis is high in ammonia nitrogen assimilation rate, strong in gossypol degradation ability and high in fermentation thallus biomass and protein content. By the adoption of the strains for treating the waste liquid containing ammonia and nitrogen, the content of ammonia and nitrogen can be effectively lowered, and forage-use mycoprotein can be formed; by the adoption of probiotics containing the strains and the probiotics composition for fermenting the cottonseed meal, the content of the toxicant gossypol in cottonseed meal can be remarkably lowered, the content of crude protein and water-soluble protein can be increased, and the probiotics composition can partly replace bean pulp to be used for feeding animals.
Description
Field that the present invention belongs to:
The invention belongs to biological fodder and biological environmental production field, specifically, the present invention relates to Candida utilis bacterium, fermenting agent composition containing this bacterium, and this yeast and the application of its composition in feed fermentation and liquid waste disposal.
Background technology:
Candida utilis (Candidautilis) is the safe biologic (GRAS) that can be used as foodstuff additive through U.S. FDA certification, can be used as safe food or activated feed additive use in China.Candida utilis can produce gsh (Wei Gongyuan, Deng. process engineering journal, 2005,5 (3): 327-331), the flavour substances such as yeast extract is (full of leaves, Deng. foodstuffs industry science and technology, 2014,25 (33): 223-225) material such as, is widely used in food and pharmacy industry.Candida utilis rich in proteins and vitamin B group, be also used as livestock, poultry feedstuff protein.
The ability of conversion of saccharomycetes non-protein nitrogen(NPN) (as urea or ammonium salt) embodies the important indicator of yeast biotransformation capacity, and the ability that different strains transforms non-protein nitrogen(NPN) is different.It is the ability of organonitrogen that Candida utilis has stronger conversion inorganic nitrogen, urea and ammonium nitrogen can be utilized as nitrogenous source, can also with various waste for raw material production edible protein, as wheat bran, molasses, potato starch wastewater (Ge1inasetal., BioresourTechnology, 2007,98 (5): 1138-1143), the multiple organic acid (Gao Weiwei such as lactic acid, Deng. biotechnology, 2008 18 (5): 58-60) etc., be all with a wide range of applications in the field such as fermented feed and wastewater treatment.He Dongdong etc. (Food science, 2009,30 (21): 210-213) are by the ultraviolet mutagenesis to Candida utilis bacterium, and it is the mutagenic strain of 33% that screening obtains protein content.Chen Jia, Deng (fodder industry, 2013,34 (5): 53-55) test is starting strain with Candida utilis, gourmet powder waste water, Semen Maydis powder and wheat bran are substrate, and solid state fermentation produces fodder yeast, and result shows: gourmet powder waste water is as the non-protein nitrogen(NPN) raw material of cheapness, can be used for, in the production of Candida utilis solid state fermentation, there is good economic benefit and potentiality to be exploited.
Cotton dregs are second largest plant protein resources that China is only second to soybean meal, but due to serious detrimentally affect can be produced to animal growth and breeding etc. containing main toxic substance gossypol in cotton dregs, also remote effect are to human health, significantly limit the large-scale application of cotton cake dregs on fodder industry.Gossypol poison-removing method mainly contains physics detoxicity method, chemical passivation method, solvent extraction method and biological detoxication method etc.Fermentable cotton dregs were utilized more and more to come into one's own with the poison-removing method reducing its Free Gossypol content in recent years, mainly because microbe fermentation method can also improve the nutritive value of cotton cake dregs while reducing free gossypol content, as improve protein quality and digestibility etc. (Xiaxin becomes. complex ferment Nutritional Value of Cottonseed Meal and active result analysis and research, China's grain and oil journal, 2012,25 (2): 96-100); Nie Cunxi etc. the meta-bolites research of cottonseed meal source fermented proteinaceous feeds, Animal nutrition journal, 2012,24 (8): 1602-1609).
Current report take cotton dregs as dominant fermentation substrate, the microorganism of degradable gossypol mainly comprises yeast, mould and bacterium etc.The microorganism of different investigator's reports is widely different to the clearance of gossypol in cotton dregs, the highest reach more than 90% (Wu little Yue etc. utilize the research of Microbes To Break Down Free-gossypol In Cottonseed Meal, Scientia Agricultura Sinica, 1989,22 (2): 82-86; Vast stretch of wooded country is brilliant. mutagenic and breeding cottonseed meal high-efficiency detoxicating bacterial strain, journal of Zhejiang university (agricultural and life science version), 2011,37 (3): 252-258), low be only about 50% (Liu Jiancheng etc. efficient degradation cotton dregs Free Gossypol bacteria selection, fodder industry, 2012,33 (1): 29-31; Ma Zhen etc. the optimization of process conditions of the raw cotton dregs of Enterococcus faecalis fermentation, food and fermentation industries, 2013,39 (5): 94-98).This main with the front cotton dregs raw material of fermentation whether through high-temperature sterilization process and employing fermented bacterium and combine different relevant.High-temperature sterilization itself can make free gossypol and protein and amino acid etc. combine and form bound gossoypol, and free gossypol content is significantly declined.The effect of different microorganisms bacterial classification detoxification have notable difference (Yang Xia, Weng Xiaoyan, Guo Jianlin etc. the separation of gossypol degradation bacteria strains, screening and qualification, Cotton Science, 2010, (6): 539-546).It is superfine in grandson that (microbiology is circulated a notice of, 2011,38 (8): 1166-1171) report the Pichi strain (Pichiaguilliermondii) of 1 high-efficiency degradation gossypol, under not carrying out wet heat treatment condition, after inoculation fermentation, virus elimination rate reaches 58%.Cheng Fuliang etc. (feed wide-angle, 2012,14:22-23) utilize Candida utilis to carry out solid fermentation detoxification to the cotton dregs after high temperature high pressure process, and result display cotton dregs are before and after fermentation, and gossypol clearance is about 85.7%.Qiao Xiaoyan etc. (Chinese oil, 2013,38 (5): 30-33) utilize candida tropicalis JD-9 and lactobacterium casei MX-48 fermentation cotton dregs, reach the object improving cotton dregs Forage quality.Result shows: after fermentation, cotton dregs Free Gossypol content is reduced to 270mg/kg from the front 520mg/kg of fermentation, and virus elimination rate reaches 48.1%; (the Wuhan Polytechnic University such as Zhou Huilin, 2014,, 33 (1): 14-16) by the upgrowth situation of more different bacto yeast on the solid medium taking gossypol acetate as sole carbon source, filter out a strain in gossypol acetate substratum, grow good Candida utilis 1087.With Candida utilis 1087 for Inoculant, the solid fermentation being main raw material with cotton dregs and sweet potato waste etc. is tested, and result shows, the sample crude protein content after fermentation improves 37.4%, and gossypol content have dropped 55.6%.
Bacillus licheniformis and plant lactobacillus are the microorganism that the Ministry of Agriculture of China can add in " the fodder additives kind catalogue " of announcement in 2013 in feed.The features such as Bacillus licheniformis is high temperature resistant with it, acid and alkali-resistance, strong stress resistance, easily storage are widely used as probiotic bacterium or pathogenic bacterium competitive inhibitor is applied in livestock and poultry breeding industry.This bacterium, can the multiple enzyme contributing to digesting and assimilating such as extracellular proteinase, amylase and lipase in process of growth, reduction and eliminating antinutritional factor also played vital role (Zhang Ju, etc. Chinese feed, 2012 years, 17:9-11).The milk-acid bacterias such as plant lactobacillus have the function such as regulating intestinal canal, promoting digestion as the normal microflora in humans and animals body, be considered to the desirable bacterial classification producing probiotic agent, often be applied to the food such as milk and feed fermenting agent (golden stake etc. grain and feed industries, 2010 (3): 37-40).These two kinds of bacteriums are all in the news and are mixed for other microorganisms the cotton dregs that ferment, but its ferment effect is not quite similar.(the Chinese oil such as Xu Jing, 2012,37 (7): 16-19) have studied the impact that microbe in solid state raw material fermentation changes cottonseed meal Free Gossypol and protein content, result shows, in the experiment of single bacterium cottonseed meal solid state fermentation, Bacillus licheniformis Bl2 detoxification efficiency is best, virus elimination rate is 42.7%, water-soluble protein content 8.2%, in mixed bacterium combined fermentation, Bacillus licheniformis Bl2+ Candida utilis Cu1 is best of breed, its virus elimination rate is 50.03%, and water-soluble protein content is 9.11%, and mixed fungus fermentation effect is better than the fermentation of single bacterium.(the fodder industry such as Zhang Qinghua, 2007,28 (18): 37-38) report 3 bacterial strains and work in coordination with solid state fermentation to cotton cake toxicity removal effect and bioactive impact thereof, result shows, 3 bacterial strain mixed culture significantly can reduce cotton dregs substrate Free Gossypol content, and its optimum process condition is: candida tropicalis: endomycopsi.sp: plant lactobacillus proportioning is 3:2:1; Cotton dregs: Semen Maydis powder: wheat bran is than when being 6:2:2, and the clearance of cotton dregs is 85.30%.And (the hubei agricultural science such as Yang Yu, 2013,52 (15): 3596-3597) report utilize filter out aspergillus niger, yeast, plant lactobacillus and genus bacillus mixing beneficial flora at the fermentation condition bottom fermentation Hubei cotton dregs of maturation and the dish dregs of rice, result shows, after Hubei cotton dregs process by fermentation, the free gossypol content in raw material increases 129.15% on the contrary.Different strain combined fermentation process cottonseed meal, its detoxification efficiency also can produce very large difference (leaf is bright by force etc., Chinese feed, 2009,15:8-14).
Chinese patent CN201010218533 discloses a kind of composite and highly effective microorganism preparation for refractory wastewater; Chinese patent CN200310107801 discloses a kind of microbiological ammonia nitrogen aligning agent; Chinese patent CN201210329933 the invention discloses and a kind ofly improves cottonseed meal content and can the biological fermentation process of detoxification; Chinese patent CN201110102144 discloses the method for decomposing gossypol in cotton dregs with composite bacteria fermentation; Chinese patent CN201310440055 discloses a kind of cotton dregs microbial starter culture and preparation method thereof; Chinese patent CN201410248620 discloses the method adding mixed enzyme fermentation cotton dregs degraded gossypol by composite bacteria; Chinese patent CN200810044216 discloses the method for the feedstuff raw materials such as a kind of mixed fermentation process vinasse; Chinese patent CN201110161862 discloses a kind of ruminating animal micro-ecological additive and preparation technology thereof.
At present, domestic in exploitation unconventional protein feedstuff is as the technique of the cotton dregs that ferment the often non-protein nitrogen(NPN) such as adding portion ammonium sulfate or urea, expect to be translated into the available tropina of animal by saccharomycetes to make fermentation, increase the protein content of fermented feed, meanwhile, the content of toxic substance gossypol in cottonseed meal is reduced by fermentable effect.This yeast strain just requiring that the fermented cotton dregs of rice produce upper application should possess the ability of stronger non-protein nitrogen(NPN) conversion capability and stronger degraded gossypol simultaneously.The weak point that the Candida utilis bacterial strain reported at present exists is that function ratio is more single.(Wuhan Polytechnic University's journal such as Cao Yufei, report from candiyeast and yeast saccharomyces cerevisiae etc. 4 genus totally 56 strain thermotolerant yeast bacterium screening and obtain Candida utilis (C.utilis) SG607 that 1 strain ammonia nitrogen assimilation ratio reach 81.1% 2015:34 (2): 1-5), but the deficiency that this bacterial strain exists is not strong to the degradation capability of gossypol, is in low-level to the assimilation ratio of ammonia nitrogen.Have not yet to see the report simultaneously possessed ammonia nitrogen assimilative capacity and all very high wild-type Candida utilis of gossypol degradation capability or its mutant strain.
Single yeast strain for ferment the protein fodders such as cotton dregs time the water-soluble protein content that produces limited, unfavorable to the nutritive value promoting feed.Combination bacterial classification mixed fermentation Problems existing is that the coupling of bacterial classification is incorrect, single culture functional strong or blending ratio is improper in mixed strains, often causes fermentation cotton dregs poor effect or there is safety issue.
Invention technology contents:
An object of the present invention is to provide a kind of new Candida utilis bacterial strain, the assimilation ratio of this bacterial strain to ammonia nitrogen is high, to the good degrading effect of gossypol, the organic liquid waste using this bacterial strain can process containing ammonia nitrogen produces fodder yeast tropina, and can ferment cotton cake toxicity removal efficiency utilization non-protein nitrogen(NPN) produces high protein feed.
Another object of the present invention is to provide a kind of probiotic agent composition containing Candida utilis.
Another object of the present invention is to provide the application of Candida utilis in the cotton dregs of fermentation containing non-protein nitrogen(NPN) such as ammonia-state nitrogens.
Another object of the present invention is to provide the method for preparation containing the probiotic agent composition of Candida utilis, Bacillus licheniformis and plant lactobacillus.
Another object of the present invention is to provide the application of probiotic agent composition in cotton dregs fermentation containing Candida utilis.
The invention discloses the Candida utilis bacterium that a strain is new, produce by being separated Candida utilis (C.utilis) SG607 mutagenesis through soil, it is characterized in that, this bacterial strain is Candida utilis SG60765 (CandidautilisSG60765), be preserved in China typical culture collection center, deposit number is CCTCCNO:2015718.Preservation date is on December 3rd, 2015.China typical culture collection center is positioned at Wuchang District, Wuhan City, Hubei Province Wuhan University in the school, phone: 027-68752319, EMAIL:cctccwhu.edu.cn., after the present invention is open, third party investigator can obtain bacterial classification from preservation mechanism in accordance with the law reproduce technology contents of the present invention.
Candida utilis SG60765 strain is separated from Hubei soil and obtains through ultraviolet mutagenesis screening.This bacterial strain has following feature:
Candida utilis SG60765 strain morphological specificity is: at YPD cultured on solid medium 1 ~ 2d, bacterium colony is oyster white, cheese shape, smooth surface, more moistening, 12 ~ 14h is cultivated at 37 DEG C in YPD liquid nutrient medium, cell is spherical in shape, avette or ellipse, and size is (2.2 ~ 5.0) μm × (2.5 ~ 5.5) μm.
Candida utilis SG60765 strain biochemical character is: can ferment and utilize sucrose, semi-lactosi, trehalose, maltose, wood sugar, lactose, cellobiose, raffinose.
Candida utilis SG60765 strain assimilation ammonia nitrogen ability characteristics is: at 37 DEG C, in the substratum of the ammonia nitrogen containing 160mg/L, the assimilation ratio of SG60765 to ammonia nitrogen reaches 94.2%, is yeast saccharomyces cerevisiae CCTCCAY92003 and Candida utilis 1807 2.6 times and 1.6 times respectively.Show that yeast SG60765 is good to the assimilative capacity of ammonia nitrogen.
Candida utilis SG60765 strain degraded gossypol ability characteristics is: at 37 DEG C, containing 60.84mgKg
-1in the cotton dregs of gossypol, the degradation rate of SG60765 to gossypol reaches 93.4%, respectively than yeast saccharomyces cerevisiae CCTCCAY92003 and Candida utilis 1807 degradation rate high 54.8% and 23.7%, shows that yeast SG60765 is good to the degradation capability of gossypol.
Pcr amplification Candida utilis SG60765 strain being carried out to 26SrDNA is tested with order-checking, analyze display: the homology of the 26SrDNA of bacterial strain SG60765 and CandidautilisIFO10707 reaches 99%, determines that bacterial strain SG60765 is a kind of Candida utilis.
The invention also discloses a kind of probiotic agent composition containing Candida utilis SG60765 strain, this probiotic agent composition major ingredient is Candida utilis SG60765 strain and extracellular products.
Preferentially, the invention also discloses a kind of containing Candida utilis SG60765 probiotics agent composition, it is characterized in that it contains following component (by viable count ratio):
Candida utilis 60%
Bacillus licheniformis 20%
Plant lactobacillus 20%
Wherein Candida utilis is CCTCCNO:2015718, and bacillus licheniformis is ACCC19372, and plant lactobacillus is CICC20765.
Bacillus licheniformis is ACCC19372 and plant lactobacillus is that CICC20765 is conventional General Microbiological Culture, publish respectively in the bacterial classification catalogue of Chinese agriculture Microbiological Culture Collection administrative center and Chinese industrial Microbiological Culture Collection administrative center, be in open state, scientific worker can ask for these two culture presevation centers.
Probiotic agent composition disclosed by the invention is liquid or solid-state.Preferential, probiotic agent composition disclosed by the invention is solid-state.
The invention also discloses the method for the solid-state SG60765 probiotic agent of preparation and composition thereof, the method comprises the steps:
(1) actication of culture: inoculation Candida utilis SG60765 strain is in sterilising liq substratum, and 18-24 hour cultivated by 28 ~ 37 DEG C of shaking tables, and liquid nutrient medium composition is glucose 3%, yeast extract paste 1%, peptone 1%, and surplus is distilled water;
(2) solid culture: the yeast liquid of activation culture is pressed 10% quality than mounted box after combined inoculation solid medium, keep room temperature about 30 DEG C, product temperature about 35 DEG C, cultivate fermentation in 2 days, the preparation of solid medium is by wheat bran 96%, brown sugar 4%, with water material moistening to water ratio 40%, boiling cold loose after;
(3) cryodrying: at 45 DEG C, cryodrying process is carried out to the solid sample after fermentation, sample water ratio, lower than after 12%, carries out being packaged to be SG60765 probiotic agent;
(4) by the SG6076 probiotic agent for preparing by weight, pack after adding Bacillus licheniformis 20% and plant lactobacillus agent 20%, obtain the combination microbial inoculum containing SG60765 probiotic bacterium, wherein bacillus licheniformis is ACCC19372, and plant lactobacillus is CICC20765.
The invention also discloses the application of Candida utilis SG60765 strain in fermentation cotton dregs.
Invention also discloses Candida utilis SG60765 strain and contain the application in ammonia nitrogen liquid waste disposal.
Advantage of the present invention:
1, Candida utilis SG60765 provided by the invention strain be a strain newly by the mutant strain that selection by mutation obtains, this bacterial strain has very high ammonia nitrogen assimilative capacity, higher 1.6 times than the assimilatory power of industrial 1807 conventional bacterial strain ammonia nitrogens; Higher by 12.1% than the assimilatory power of its starting strain SG607 ammonia nitrogen.
2, Candida utilis SG60765 strain has the ability of very strong degraded gossypol, higher by 36.1% than industrial 1807 conventional bacterial strain gossypol degradation rates; Higher by 13.2% than its starting strain SG607 gossypol degradation rate.
3, Candida utilis SG60765 strain has very high biomass, under liquid culture condi, higher than the biomass of Candida utilis ACCC20060 bacterial strain 1.4 times.
4, when Candida utilis SG60765 strain is used for carrying out raw material fermentation containing the cotton dregs of ammonia nitrogen, the assimilation ratio of this bacterial strain to ammonia nitrogen and the degradation rate to gossypol all reach more than 90%, show excellent over-all properties, overcome the shortcoming that yeast strain function is more single in the market, production has obvious advantage.
5, containing protein candiyeast SG60765 strain combination microbial inoculum for ferment cotton dregs time, fermentation cotton dregs aqueous soluble protein content than being significantly improved with during single bacteria fermentation.Namely Candida utilis SG60765 strain has the ability of stronger ammonia nitrogen assimilation ratio, gossypol degradation rate and higher biomass, the ammonia nitrogen assimilation ratio that its combination microbial inoculum is not only stronger and gossypol degradation capability, also have the ability of stronger raising fermentation cotton dregs water-soluble protein.
Accompanying drawing explanation
Fig. 1. Candida utilis SG60765 strain 26SrDNAD1/D2 region sequence phylogenetic tree
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be appreciated that these embodiments are only for illustration of the present invention, and can not limit the scope of the invention.
Embodiment
The seed selection of embodiment 1 Candida utilis height ammonia nitrogen assimilation ratio, Cambodia Phenol degradation rate mutant strain SG60765 and qualification
The seed selection of 1.1 Candida utilis height ammonia nitrogen assimilation ratio mutant strain SG6076
With Candida utilis SG607 for starting strain, SG607 bacteria suspension after uv irradiating is applied on YPD solid medium after suitably diluting, when its bacterium colony is obvious, therefrom choose full, large and round colony inoculation on YPD solid medium, grow after single bacterium colony until it, be transferred to and be equipped with in the 25mL culturing bottle of 7mLYPD liquid nutrient medium, under 37 DEG C of 170rpm conditions, 5h cultivated by shaking table, cultured each yeast liquid is quantitatively moved in 96 orifice plates, by microplate reader at wavelength 620nm place, measure its OD value.Therefrom choose the 20 strain bacterial strains that OD value is larger, be numbered No. 1-No. 20 bacterial strains, carry out multiple sieve further, each bacterial strain 3 repetition.Obtain the mutant strain that 5 strain ammonia nitrogen assimilation ratiies are significantly increased than starting strain altogether, the ammonia nitrogen assimilation ratio being wherein numbered the mutant strain of No. 6 is up to 93.2%, 12.1% is improve, by this mutant strain called after SG6076 (see table 1) than the ammonia nitrogen assimilation ratio of starting strain.
The measurement result of the ammonia nitrogen assimilative capacity of table 15 plant mutant bacterial strain
The seed selection of 1.2 Candida utilis Cambodia Phenol degradation rate mutant strain SG60765
In order to obtain the quality yeast bacterial strain possessing high ammonia nitrogen assimilation ratio and Cambodia's Phenol degradation rate simultaneously, the present invention to suddenly change separate principle according to microbial gene, the high ammonia nitrogen assimilation ratio mutant strain SG6076 obtained is carried out to second time ultraviolet mutagenesis and adopts high-throughput screening method, therefrom filters out mutant strain gossypol to high degradation rate.Concrete grammar is: take SG6076 as starting strain, by the SG6076 bacterium liquid after uv irradiating through suitable dilution, is then coated on gossypol acetate solid medium, after lucifuge cultivates 24h, choose full, large and round bacterium colony, after activated, be inoculated in and fill 10mL containing 1000mgL
-1in the substratum of gossypol acetate, 37 DEG C, 5h cultivated by shaking table under 170rpm condition, the bacterium liquid getting 200 μ L, in 96 aseptic orifice plates, at wavelength 620nm place, measures OD by microplate reader
620value.Pass through OD
620value size carries out primary dcreening operation, obtains the mutant strain (table 2) of 4 strains increase more obvious than starting strain OD value altogether.After this 4 plant mutant strain and starting strain activation, inoculate with the inoculum size of 10%, fermentation condition is moisture 42%, temperature 37 DEG C, time 36h, carries out the fermentation of solid cotton dregs, carries out multiple sieve by measuring it to the degradation rate of gossypol.As can be seen from Table 3, it is 93.6% that the gossypol degradation rate of No. 65 mutant strains is up to, and improves 11.5% than starting strain.No. 65 mutant strain called afters are numbered SG60765.
The strain of table 24 plant mutant is compared gossypol degradation capability
The Candida utilis SG60765 strain (CandidautilisSG60765) new by a strain of twice ultraviolet mutagenesis seed selection is preserved in China typical culture collection center by the present inventor, and deposit number is CCTCCNO:2015718.Preservation date is on December 3rd, 2015.China typical culture collection center is positioned at Wuchang District, Wuhan City, Hubei Province Wuhan University in the school, phone: 027-68752319, EMAIL:cctccwhu.edu.cn.
The qualification of 1.3 Candida utilis SG60765 strains
By morphological specificity observation, physiological and biochemical test and 26SrDNAD1/D2 regional gene the sequencing results, SG60765 is identified.In authentication, this experimental selection Angel thermotolerant ethanol fermentation yeast TRADY, Candida utilis CICC1807 bacterial strain in contrast.Yeast saccharomyces cerevisiae CCTCCAY92003 is conventional General Microbiological Culture, publishes in China typical culture collection center catalogue; Candida utilis CICC1807, from Chinese industrial Microbiological Culture Collection administrative center, goes up catalogue, and open state, scientific worker can ask for.
1.3.1 Candida utilis SG60765 strain morphological specificity is observed
At YPD cultured on solid medium 1d-2d, bacterium colony is full circle, and center is given prominence to, oyster white, cheese shape, and smooth surface is moistening, adhesion, easy picking.Cultivate 12h-14h at YPD liquid nutrient medium, cell ovalize, slightly circle, part cell is together end to end, and in typical false thread, size is (2.5-3.5) μm × (6.5-8.5) μm.
1.3.2 Phylogenetic qualification is carried out by the analysis of 26SrDNAD1/D2 regional sequence
With the genomic dna of yeast strain for template does pcr amplification reaction, amplification yeast strain 26SrDNAD1/D2 region, amplimer adopts universal primer NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS4 (5'-CTTCCGTCAATTCCTTTAAG-3').
PCR reaction system (25 μ L): 10 × PCR damping fluid is (containing the Mg of 20mmol/L
2+) 2.5 μ L, primer NS1/NS4 (10 μm of ol/L) each 1 μ L, template DNA (about 50ng/mL) 1.8/2.8 μ L, dNTP (10mmol/Leach) 0.5 μ L, Taq enzyme (5U/ μ L) 0.2 μ L, distilled water 18/17 μ L.
Pcr amplification condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 40s, 50 DEG C of annealing 40s, 72 DEG C extend 60s, and after 30 circulations, 72 DEG C extend 5min.Gained PCR primer is served the raw work biotechnology purifying in sea and is checked order, sequencing primer above-mentioned NL1, NL4.
After being checked order by molecule, find that the homology of the 26SrDNA of bacterial strain SG60765 and CandidautilisIFO10707 reaches 99%, meet difference between Kuttzman & Robnett fixed of the same race interior different strains and be no more than the standard of 1%, determine that bacterial strain SG60765 is a kind of Candida utilis.Fig. 1 is the phylogenetic tree done according to 26SrDNAD1/D2 regional sequence.
1.3.3 the bio-chemical characteristics measurement result of Candida utilis SG60765 strain
The utilization of SG60765 bacterial strain to sugar the results are shown in Table 3.SG60765 bacterial strain all can utilize 6 kinds of sugar such as sucrose.
The fermentation test result of table 3 Candida utilis SG60765 different carbon source
Embodiment 2. Candida utilis SG60765 is to the assimilative capacity of ammonia nitrogen and the effect analysis processing high ammonia-nitrogen content waste liquid
The comparative result of 2.1 different yeast strain ammonia nitrogen assimilation ratiies
By the 7 saccharomycete seed liquor such as SG60765 with 5% inoculum size be transferred to 3 respectively and be independently equipped with in the 250mL triangular flask of 100mL saccharomycetes to make fermentation substratum, shaker fermentation 24h under 37 DEG C of 170rpm conditions, measure fermented liquid supernatant ammonia-nitrogen content, more different yeast strain is to the assimilation ratio of ammonia nitrogen, result is as shown in table 4, mutant strain SG6076 improves 13.1% than the ammonia nitrogen assimilation ratio of its starting strain SG607, to ammonia nitrogen assimilation ratio no significant difference between mutant strain SG6076 and SG60765.
Candida tropicalis CICC1254 is the strain that the is purchased from Chinese industrial Microbiological Culture Collection administrative center strain library bacterial strain for fermentation production of fodder yeast.Candida utilis ACCC20060 is the strain that the is purchased from Chinese agriculture Microbiological Culture Collection administrative center bacterial classification bacterial strain for fermentation production of fodder yeast.Yeast saccharomyces cerevisiae CCTCCAY92003 is conventional General Microbiological Culture, and publish in China typical culture collection center catalogue, be in open state, scientific worker can ask for China typical culture collection center.
The comparison of table 4 different yeast strain ammonia nitrogen assimilation ratio
The effect analysis of 2.2 different yeast strain process height ammonia-nitrogen content waste liquids
Candida utilis SG60765, SG607 and ACCC20060 of choosing above 3 plant height ammonia nitrogen assimilation ratiies carry out the fermentation scale-up experiment of high ammonia-nitrogen content waste liquid, and this time fermentation adopts the nutrient solution filling 1L height ammonia-nitrogen content in 3L triangular flask.Each yeast liquid is inoculated respectively in containing ammonia nitrogen 240.0mgL by 10% volume ratio inoculum size
-1waste liquid in, under 37 DEG C of 170rpm conditions, shaking table is cultivated after 24h, centrifugal in 4000rpm, 5min, collects thalline, 105 DEG C of oven dry, weighs, and measures thalline crude protein content.As can be seen from Table 5, SG60765 has had further raising to ammonia nitrogen assimilation ratio under enlarged culturing condition, reaches 96.6%, respectively than the ammonia nitrogen assimilation ratio high 10.2% and 15.9% of SG607 and ACCC20060 bacterial strain.As compared to SG607 with ACCC20060, the biomass of SG60765 and crude protein content are all higher, reach 3.83gL respectively
-1with 51.0%.Under identical fermentation condition, the biomass of SG60765 yeast thalline is 1.4 times of ACCC20060 bacterial strain biomass.
The effectiveness comparison of table 5 different yeast strain process height ammonia-nitrogen content waste liquid
The capability analysis of gossypol and assimilation ammonia nitrogen in embodiment 3.SG60765 strain fermentation degraded cotton dregs
By yeast saccharomyces cerevisiae CCTCCAY92003, candida tropicalis CICC1254; Candida utilis CICC1807, ACCC20060, SG607, SG6076, SG60765 are after totally 7 strain yeast activate respectively, the cotton dregs inoculating interpolation 1% ammonium sulfate respectively by inoculum size 10% carry out the test of solid-state raw material fermentation and compare, and fermentation condition is: fermentation material starting moisture 42%, leavening temperature 37 DEG C, fermentation time 36h.Measure ammonia nitrogen before and after fermentation in cotton dregs and gossypol content respectively by Kjeldahl determination and liquid phase chromatography, and calculate ammonia nitrogen assimilation ratio and the gossypol degradation rate of each bacterial strain.The gossypol content of measuring cotton dregs raw material is 60.84mgKg
-1.As can be seen from Table 6, the degradation rate of Candida utilis SG60765 to gossypol is up to 93.4%, respectively than yeast saccharomyces cerevisiae CCTCCAY92003 and Candida utilis 1807 degradation rate high 54.8% and 23.7%, higher by 10.0% than its starting strain SG6076, and SG60765 mutant strain is similar to SG6076 to the assimilation ratio of ammonia nitrogen, show that this mutant strain still remains the proterties of its high ammonia nitrogen assimilative capacity while obtaining high gossypol degradation capability.As the degradation capability of CICC1807 and ACCC20060 to gossypol also exists significant difference between same Candida utilis different strains.
The different yeast strain of table 6 is to the comparison of ammonia nitrogen assimilation ratio and gossypol degradation rate
Embodiment 4SG60765 bacterial strain and Bacillus licheniformis and plant lactobacillus press different ratios mixed fermentation cotton dregs
Effectiveness comparison
Be after CICC20765 activation by Bacillus licheniformis ACCC19372, Candida utilis SG60765 and plant lactobacillus, respectively with viable count ratio for 3:1:0,2:1:0,1:1:0,1:2:0,1:3:0 and 1:3:1 carry out combined inoculation fermentation, cotton dregs fermentation condition is moisture 42%, temperature 37 DEG C, time 36h.As can be seen from Table 7, along with inoculum size saccharomycetic in mixed strains constantly increases, gossypol degradation rate constantly raises, and when Bacillus licheniformis ACCC19372 and Candida utilis SG60765 inoculative proportion are 1:3, gossypol degradation rate reaches the highest, is 91.3%.Show that the Degradation of Candida utilis SG6076 to gossypol is greater than Bacillus licheniformis.When adding plant lactobacillus in combination microbial inoculum and ACCC19372:SG60765:CICC20765 is 1:3:1, the gossypol degradation rate of fermentation cotton dregs reaches 91.5%, and the pH value of fermentation material is down to 5.0 by 6.0, show that the combination microbial inoculum containing plant lactobacillus does not obviously reduce the degradation rate of gossypol, but significantly can increase the acidity of fermentation cotton dregs, this is conducive to the palatability and the storage time that increase feed.
Table 7SG60765 and Bacillus licheniformis different mixing proportion are on the impact of gossypol degradation rate
The preparation method of embodiment 5SG60765 probiotic agent and composition thereof
5.1 actication of culture: inoculation Candida utilis SG60765 strain is in sterilising liq substratum (glucose 3%, yeast extract paste 1%, peptone 1%), and 18-24 hour cultivated by 28 ~ 37 DEG C of shaking tables;
5.2 solid culture: the batching of solid culture is: wheat bran 96%, brown sugar 4%, with water material moistening to water ratio 40%, boiling cold loose after, by 5.1 activation culture 2 Yeasts liquid by mounted box after 10% inoculum size volume ratio combined inoculation, keep room temperature about 30 DEG C.Product temperature about 35 DEG C, cultivates and ferments complete in 2 days;
5.3 cryodryings: at 45 DEG C, cryodrying process is carried out to the solid sample after fermentation, sample water ratio, lower than after 12%, carries out being packaged to be SG60765 probiotic agent;
5.4 pack after the SG60765 probiotic agent prepared respectively interpolation Bacillus licheniformis and plant lactobacillus agent by a certain percentage, namely obtain the combination microbial inoculum containing SG60765 probiotic bacterium;
5.5 yeast preparation compositions measurement results: in the probiotic agent composition adopting dilution plate determination experiment room to prepare, Candida utilis bacterium viable count reaches about 1,500,000,000/g.Candida utilis SG60765:ACCC19372:CICC20765 viable count ratio is 6:2:2.
Embodiment 6SG60765 individual plant fermenting agent and the application of composition in fermentation cotton dregs thereof
6.1SG60765 individual plant fermenting agent and composition thereof are used for cotton dregs fermentation test result
By SG60765 single fermentation microbial inoculum and composition thereof respectively with 2% inoculum size to be inoculated in moisture be in the solid cotton dregs of the 100Kg of 40%, after raw material fermentation 40h, pulverize after 60 DEG C, baking oven is dried.From data in table 8, after adopting single microbial inoculum and combination bacteria fermentation cotton dregs, crude protein content adds 7.25% and 7.61% respectively, and gossypol degradation rate is respectively 90.16% and 90.21%, and water-soluble protein is respectively 9.89% and 12.45%.Adopt the cotton dregs of combination bacteria fermentation to improve 25.88% than the water-soluble protein content of the cotton dregs of single bacteria fermentation, show that combine microbial inoculum has clear superiority in the water-soluble protein increasing cotton dregs.Cotton dregs after fermentation all have dense yeast fragrance, can increase the palatability of feed.
The effectiveness comparison of the single microbial inoculum of table 8 and combination bacteria fermentation cotton dregs
Test-results and analysis 6.2 fermentation cotton dregs raise table hens
In order to test the feeding effect of the cotton dregs using combination bacteria fermentation, selecting 100 to go out the public young bird of precious 500 broiler chicken of the healthy section of shell, being divided into 5 groups at random, often organizing 4 repetitions, each repetition 5.Five groups of dregs of beans of feeding respectively, 50% cotton dregs Substitution for Soybean Meal, 100% cotton dregs Substitution for Soybean Meal, 50% fermentation cotton dregs Substitution for Soybean Meal, 100% fermentation cotton dregs Substitution for Soybean Meals.Hen house temperature control controls at 35 DEG C for 1 week, reduces by 3 DEG C weekly, to off-test later.Each group of test chicken free choice feeding and drinking-water.Morning every day, 7:00 fed in raw material and water, and trial period is 21 days.Dead without chicken in process of the test.Test-results is in table 9.From table 9, whether cotton dregs ferment and have pole remarkably influenced (p≤0.001), replacement amount (50% and 100%) the pole remarkably influenced food consumption of dregs of beans to average daily gain and feedstuff-meat ratio.Do not make significant difference by the production performance of 50% couple 0 ~ 21 Day-old Broiler Chickens of fermentation cotton dregs Substitution for Soybean Meal.Show to adopt the cotton dregs of combination bacteria fermentation to have the function of good Substitution for Soybean Meal.
Table 9 liang factor (cotton dregs and fermentation cotton dregs) two levels (Substitution for Soybean Meal 50% and 100%) are tested broiler chicken 0 ~ 21 age in days production performance and are compared
Claims (8)
1. a strain Candida utilis, by through soil be separated Candida utilis (
c.utilis) SG607 mutagenesis generation, it is characterized in that, this bacterial strain is Candida utilis SG60765(
candidautilissG60765), be preserved in China typical culture collection center, deposit number is CCTCCNO:M2015718.
2., containing a microorganism probiotic agent for the Candida utilis SG60765 strain described in claim 1, it is characterized in that its activeconstituents is Candida utilis SG60765 strain thalline and meta-bolites thereof.
3. the probiotic agent composition containing bacterial strain described in claim 1.
4., according to the probiotic agent composition described in claim 3, it is characterized in that it contains following component (viable count ratio):
Candida utilis 60%
Bacillus licheniformis 20%
Plant lactobacillus 20%
Wherein Candida utilis is CCTCCNO:2015718, and bacillus licheniformis is ACCC19372, and plant lactobacillus is CICC20765.
5., according to the probiotic agent composition described in claim 3, it is characterized in that described probiotic agent composition is solid-state microbial inoculum.
6. prepare the method for the probiotic agent composition described in claim 4, comprise the steps:
(1) actication of culture: inoculation Candida utilis SG60765 strain is in sterilising liq substratum, and 18-24 hour cultivated by 28 ~ 37 DEG C of shaking tables, and liquid nutrient medium composition is glucose 3%, yeast extract paste 1%, peptone 1%, and surplus is distilled water;
(2) solid culture: the yeast liquid of activation culture is pressed 10% quality than mounted box after combined inoculation solid medium, keep room temperature about 30 DEG C, product temperature about 35 DEG C, cultivate fermentation in 2 days, the preparation of solid medium is by wheat bran 96%, brown sugar 4%, with water material moistening to water ratio 40%, boiling cold loose after;
(3) cryodrying: at 45 DEG C, cryodrying process is carried out to the solid sample after fermentation, sample water ratio, lower than after 12%, carries out being packaged to be SG60765 probiotic agent;
(4) by the SG6076 probiotic agent for preparing by weight, pack after adding Bacillus licheniformis 20% and plant lactobacillus agent 20%, obtain the combination microbial inoculum containing SG60765 probiotic bacterium, wherein bacillus licheniformis is ACCC19372, and plant lactobacillus is CICC20765.
7. the application of the Candida utilis SG60765 described in claim 1 in fermentation cotton dregs.
8. the Candida utilis SG60765 described in claim 1 is containing the application in ammonia nitrogen liquid waste disposal.
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