CN105274012A - Candida tropicalis with function of gossypol degradation and application of candida tropicalis - Google Patents
Candida tropicalis with function of gossypol degradation and application of candida tropicalis Download PDFInfo
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- CN105274012A CN105274012A CN201510812724.0A CN201510812724A CN105274012A CN 105274012 A CN105274012 A CN 105274012A CN 201510812724 A CN201510812724 A CN 201510812724A CN 105274012 A CN105274012 A CN 105274012A
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Abstract
The invention discloses candida tropicalis with a function of gossypol degradation and application of the candida tropicalis. The candida tropicalis is preserved in China General Microbiological Culture Collection Center on June 23, 2015, and a culture preservation number of the candida tropicalis is CGMCC No.11000. The candida tropicalis is applied to cottonseed meal to realize solid-state anaerobic fermentation, gossypol degradation rate is up to 82.63% after fermentation, growth of contaminating microorganisms can be inhibited effectively to prevent secondary contamination, and safety and nontoxicity of cottonseed meal fodder can be truly realized. The fermented cottonseed meal fodder has a unique flavor, the utilization rate of the cottonseed meal used as protein fodder is greatly increased, great significance to relief of protein fodder shortage, reduction of import of fish meal, soybean meal and the like, reduction of fodder cost and increase of stock farming benefits can be achieved, and the candida tropicalis can be truly and widely applied to industrial production.
Description
Technical field
The present invention relates to a kind of candida tropicalis and the application thereof with the effect of degraded gossypol, belong to microbial technology field.
Background technology
Cotton dregs (CottonseedMeal, CSM) are cottonseeds through shelling, heating, flatten after flakiness solvent hexane leaches oil, remaining byproduct.Cotton dregs are one of important protein feed resources, and its crude protein content is about 40%, is only second to dregs of beans, and China is Chan Mian big country of the world, and output of cotton ranks first in the world, and annual cotton cake dregs output is about 400-500 ten thousand tons.But cotton dregs but can not get efficiency utilization, trace it to its cause, be because the existence of the toxic substances such as gossypol, its application is restricted greatly.In recent years along with the development of livestock industry, the shortage of world wide internal protein feed becomes increasingly conspicuous.Cotton cake dregs is the vegetable protein source of Substitution for Soybean Meal desirable under existing situation, and cotton cake dregs is cheap, current selling price, less than the half of soybean cake dregs price, to the exploitation of cotton cake dregs, brings significant economic benefit can to undoubtedly China's livestock industry.
Gossypol (Gossypol) is commonly called as cotton toxin, and be the polyphenol pigment of a kind of yellow in cottonseed body of gland, molecular formula is C
30h
30o
8, molecular weight 518.5.The toxic action main manifestations of free gossypol (FreeGossypol, FG) is as follows: destroy Testicular Seminiferous Epithelium, affect the Reproductive Performance of animal; There is hormesis to gastrointestinal mucosa, easily cause gastrointestinal mucosa inflammation hemorrhage; Body nerve is sustained damage and suppression etc.Feed Free Gossypol content exceedes safety limit, appetite of livestock and poultry decline, growth retardation will be caused, lose weight, suffer from diarrhoea, trichomadesis, sterile etc., serious meeting causes myocardial damage, causes visceral congestion and oedema, and thoracic cavity and abdominal cavity body fluid leach, hemorrhage, even death.
At present, some patent documents relate to method and the application thereof of gossypol degraded, and such as CN104146166A discloses a kind of cotton dregs feed adding iron vitriol and crystal Methionin, effectively can reduce the content of its Free Gossypol; CN103766597A discloses a kind of new feed processing technology---Steam explosion treatment technology, the free gossypol in cotton dregs can be removed quickly and efficiently; CN102178030A discloses a kind of NaOH solution of adding 1% and carries out the method for cotton byproduct removing cotton phenol.Such as, but relate at present and utilize the mode of anaerobically fermenting efficiently to reduce the patent application document of gossypol content in cotton dregs seldom, CN104031852A discloses a kind of Candida utilis bacterium solid state fermentation cotton stalk, and degradation rate reaches 53.9%; CN102154120A is 61.38% to the degradation rate of gossypol after disclosing a kind of rhizopus oryzae fermentation cotton dregs.
The multiplex mould of forefathers carries out aerobic fermentation cotton dregs, in this process, owing to fully contacting with air, easily cause living contaminants, other miscellaneous bacterias are and then fermented together, cause the easy secondary pollution of leavened prod, cotton dregs after such fermentation not only do not accomplish safe detoxification, likely increase its toxicity on the contrary.And the candida tropicalis that the present invention filters out, not only for the degradation rate of free gossypol up to more than 80%, and fermentation mode adopts anaerobically fermenting, compared to the aerobic fermentation of forefathers, has many advantages: effectively can suppress miscellaneous bacteria, prevents secondary pollution; In tunning, the content of other nutritive substances such as crude protein rises, and makes cotton dregs while detoxification, can not destroy again nutritive ingredient wherein, make cotton dregs feed really accomplish safety non-toxic; Cotton dregs feed after fermentation is rich in flavour, effectively can increase palatability, greatly improves the utilization ratio of cotton dregs as animal and fowl fodder.
Therefore, filter out a kind of bacterial strain by anaerobically fermenting Highly effected degrading cotton phenol, and practical application just seems very necessary in cotton dregs and other cotton byproducts.The present invention, on the basis of summing up prior art, by lot of experiments, completes the present invention finally.
Summary of the invention
The object of the invention is to for the practical problems in production practice and demand, probe into and filter out a kind of novel free gossypol degradation bacteria, use candida tropicalis of the present invention that the free gossypol clearance in high gossypol content cotton dregs can be made to reach 82.63%, and have good strain stability.Use this degradation bacteria strains can free gossypol in efficient degradation cottonseed meal, make it to reach use standard, cottonseed meal is become available feedstuff protein source, to improving the efficiency utilization rate of cottonseed meal and promoting that the development of China's animal husbandry has great importance.
The candida tropicalis (Candidatropicalis) that the present invention obtains, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on 06 23rd, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11000.
Described candida tropicalis CGMCCNo.11000 has following characteristic:
(1) colony characteristics: well-grown on YPD substratum, bacterium colony is in cream-colored, and slightly gloss, soft and level and smooth, have fold, basis of microscopic observation cellular form is oval.
(2) growth characteristics: candida tropicalis CGMCCNo.11000 is good with the growth temperature of 28-35 DEG C, and cultivate under 30 DEG C of conditions, this bacterial strain is cultivated about 8h and entered logarithmic phase in liquid YPD medium, cultivate about 20h and enter stationary phase.Growth curve is as Fig. 1.
(3) candida tropicalis CGMCCNo.11000 can be that sole carbon source grows with free gossypol, is in the sole carbon source substratum of 0.1g/L, can reach 82.56% to the degradation rate of FG in free gossypol (FG) concentration.The degradation rate of free gossypol is along with the change curve of time is as Fig. 2.
Candida tropicalis CGMCCNo.11000 provided by the invention can be used for biological degradation free gossypol, can apply in the biological treatment of the cottonseed meal of high density free gossypol content.Effectively can reduce the content of cotton dregs Free Gossypol after fermentation, be widely used prospect.
Biomaterial preservation
Candida tropicalis (Candidatropicalis), this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 23rd, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11000.
Accompanying drawing explanation
Fig. 1 is the growth curve of candida tropicalis CGMCCNo.11000.
Fig. 2 be under different time candida tropicalis CGMCCNo.11000 to the degradation effect figure of free gossypol.
Fig. 3 is the impact of temperature on candida tropicalis CGMCCNo.11000 degraded free gossypol ability.
Fig. 4 is the impact of pH value on candida tropicalis CGMCCNo.11000 degraded free gossypol ability.
Fig. 5 is the content figure utilizing cotton dregs Free Gossypol before and after candida tropicalis CGMCCNo.11000 fermentation.
Embodiment
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
The configuration of embodiment 1 free gossypol reference liquid
Described free gossypol is free gossypol standard substance, is provided, be mixed with the reference liquid of 1000mg/L during use by Southern Yangtze University's oils and vegetable protein engineering research centre.Concrete compound method is: the free gossypol standard substance accurately taking 100mg, are dissolved in the acetonitrile of 100mL, constant volume in brown volumetric flask, after fully mixing is dissolved, is transferred in brown reagent bottle, saves backup at being placed in-20 DEG C.
Embodiment 2 is degraded the saccharomycetic screening of free gossypol
Preparation take free gossypol as the solid medium of sole carbon source, and wherein carbon-free basal medium formulation is as follows: ammonium sulfate 5g, potassium primary phosphate 1g, sodium-chlor 0.1g, magnesium sulfate 0.5g, calcium chloride 0.1g, yeast extract paste 0.2g, tween 80 7.5ml, agar 20g.Add deionized water to 1000mL, 115 DEG C of moist heat sterilization 20min.Under aseptic condition, in sterilized carbon-free basic medium, add free gossypol reference liquid, make its concentration reach 0.1g/L.
By be separated from the different yeast strains of traditional fermented food coat above-mentioned with free gossypol be sole carbon source solid medium on, meanwhile, not add the substratum of carbon source as negative control, glucosyl group basal culture medium is as positive control.Leave standstill constant temperature culture at 30 DEG C, the growing state of routine observation bacterial strain, carries out preliminary screening.By the part bacterial strain just filtered out, be inoculated in free gossypol be sole carbon source liquid nutrient medium in, high performance liquid chromatography (HPLC) is utilized to detect the change of its Free Gossypol content, thus filter out the yeast strain of energy efficient degradation free gossypol, obtain candida tropicalis CGMCCNo.11000.
The mensuration of embodiment 3 candida tropicalis CGMCCNo.11000 growth curve
Get the candida tropicalis CGMCCNo.11000 that-20 DEG C of glycerine pipes are preserved, it is streak culture on YPD solid medium.After cultivating 24 hours at 30 DEG C, from well-grown candida tropicalis (Candidatropicalis) single bacterium colony, picking yeast cell is inoculated into shaking table in YPD liquid nutrient medium and cultivates 24 hours, culture temperature is 30 DEG C, and rotating speed is 150r/min.Then by 2% inoculum size, the candida tropicalis CGMCCNo.11000 nutrient solution after this activation is inoculated in fresh YPD liquid nutrient medium, it is 30 DEG C in temperature, rotating speed is shaking culture 24 hours in the shaking table of 150r/min, OD value under getting 5mL nutrient solution use ultraviolet spectrophotometer survey 600nm every 2 hours also carries out plate count, the growth curve of candida tropicalis CGMCCNo.11000 is drawn according to result, its result as shown in Figure 1, as can be seen from Figure 1: candida tropicalis CGMCCNo.11000 grows comparatively rapid in YPD substratum, logarithmic phase is entered at about 8h, about 20h enters stationary phase.
The cultivation of embodiment 4 candida tropicalis CGMCCNo.11000, for the preparation of cotton dregs fermented bacterium
Slant strains: make YPD slant medium, streak inoculation candida tropicalis CGMCCNo.11000, cultivates 20h, slant strains preserved in-20 DEG C of refrigerators at 30 DEG C.
First order seed: picking colony is forwarded in fresh YPD slant medium from slant strains preservation pipe, and streak inoculation, cultivates 20h at 30 DEG C.
Secondary seed: be seeded in fresh YPD liquid nutrient medium by 2% inoculum size from first order seed, cultivate 20h at 30 DEG C, namely make fermented bacterium.
Under embodiment 5 different time, candida tropicalis CGMCCNo.11000 to degrade situation analysis to free gossypol
Learnt from else's experience and went down to posterity for twice, be cultured to the candida tropicalis CGMCCNo.11000 of logarithmic phase, inoculum size according to 2% be inoculated in gossypol be sole carbon source liquid nutrient medium in, in 30 DEG C, cultivate in the shaking table of 150r/min, interval 24h samples, by the nutrient solution of taking-up in 4 DEG C, the centrifugal 10min of rotating speed of 10000rpm gets supernatant liquor, with 0.22 μm of filtering with microporous membrane, high performance liquid chromatography is adopted to detect its Free Gossypol residual quantity.3 Duplicate Samples are done in the process of each time.Meanwhile, arranging not heating zone candiyeast CGMCCNo.11000 is control group.
The results are shown in Figure 2, the speed of candida tropicalis CGMCCNo.11000 degraded free gossypol is comparatively rapid, and along with the growth degradation rate of time constantly raises, substantially tended towards stability at about 9 days, degradation rate can reach 82.56%.
Embodiment 6 temperature is on the impact of candida tropicalis CGMCCNo.11000 degraded free gossypol ability
According to the method for embodiment 5, candida tropicalis CGMCCNo.11000 is inoculated in free gossypol be sole carbon source substratum in, arrange different group, cultivate at being placed on 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C respectively, other conditions are all consistent.Cultivate after 9 days, measure candida tropicalis CGMCCNo.11000 in each group according to the method for embodiment 5 and, to the degradation rate of free gossypol, the results are shown in Figure 3.As seen from the figure: the optimum temperature range of candida tropicalis CGMCCNo.11000 degraded free gossypol is between 25-40 DEG C, and in this temperature range, degradation rate reaches about 80% substantially.
Embodiment 7pH value is on the impact of candida tropicalis CGMCCNo.11000 degraded free gossypol ability
According to the method for embodiment 5, candida tropicalis CGMCCNo.11000 is inoculated in free gossypol be sole carbon source substratum in, arrange different group, being placed on pH value is respectively cultivate under the condition of 2.0,3.0,4.0,5.0,6.0,7.0,8.0, and other conditions are all consistent.Cultivate after 9 days, measure candida tropicalis CGMCCNo.11000 in each group according to the method for embodiment 5 and, to the degradation rate of free gossypol, the results are shown in Figure 4.As seen from the figure: the optimum pH scope of candida tropicalis CGMCCNo.11000 degraded free gossypol is between 4-7, and in this pH value range, degradation rate reaches about 80% substantially.
The application of embodiment 8 candida tropicalis CGMCCNo.11000 in cotton cake toxicity removal
The preparation of fermention medium: cotton dregs were pulverized 50 mesh sieves, by itself and Semen Maydis powder, the ratio of wheat bran mixes according to 7:2:1, composition fermentation substrate.By substrate at 115 DEG C after sterilizing 20min, according to moisture content 40%, inoculum size 10%, in valve bag, closing anaerobic fermentation 48h, measures the content of cotton dregs Free Gossypol before and after fermentation respectively, the results are shown in Figure 5.
Utilize candida tropicalis CGMCCNo.11000 of the present invention to be inoculated in cotton dregs substrate to ferment, by the content of its Free Gossypol before and after the fermentation of contrast cotton dregs, after can drawing 48h, the virus elimination rate of this bacterial strain to cotton dregs Free Gossypol reaches 82.63%, effectively can reduce the free gossypol content in cotton dregs, there is good effect.
The application of embodiment 9 candida tropicalis CGMCCNo.11000 in production high-quality cotton dregs
According to the method for embodiment 8, after utilizing candida tropicalis CGMCCNo.11000 to carry out solid anaerobic digestion to cotton dregs, the cotton dregs feeds product obtained, has special fermenting aroma, can improve the food consumption of livestock and poultry, increases palatability.The process of anaerobically fermenting suppresses living contaminants effectively, and the cotton dregs feed organoleptic quality after fermentation is good, the pollutions such as mould do not detected.In cotton dregs feed after fermentation, crude protein content improves 20.56%, enhances the nutritive value of cotton dregs.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (7)
1. one kind has the candida tropicalis (Candidatropicalis) of degraded gossypol effect, this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 23rd, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11000.
2. the application of candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 in biological degradation free gossypol.
3. candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 fermentation cotton dregs to reduce the application in its Free Gossypol content.
4. the application of candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 in cotton cake toxicity removal.
5. candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 is effectively suppressing varied bacteria growing, prevents secondary pollution, improves palatability, obtains the application in safety non-toxic cotton dregs feed.
6. the application of candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 in the utilization ratio improving protein feed.
7. candida tropicalis according to claim 1 (Candidatropicalis) CGMCCNo.11000 is reducing the application in feed cost.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109810956A (en) * | 2019-02-19 | 2019-05-28 | 中国农业科学院饲料研究所 | Gossypol degrading enzyme HIGD and its encoding gene and application |
| CN114680230A (en) * | 2022-04-01 | 2022-07-01 | 播恩集团股份有限公司 | Low-gossypol high-nutrition fermented cottonseed meal, preparation method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109810956A (en) * | 2019-02-19 | 2019-05-28 | 中国农业科学院饲料研究所 | Gossypol degrading enzyme HIGD and its encoding gene and application |
| CN109810956B (en) * | 2019-02-19 | 2021-01-15 | 中国农业科学院北京畜牧兽医研究所 | Gossypol degrading enzyme HIGD and coding gene and application thereof |
| CN114680230A (en) * | 2022-04-01 | 2022-07-01 | 播恩集团股份有限公司 | Low-gossypol high-nutrition fermented cottonseed meal, preparation method and application |
| CN114680230B (en) * | 2022-04-01 | 2022-10-04 | 播恩集团股份有限公司 | Low-gossypol high-nutrition fermented cottonseed meal, preparation method and application |
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