CN101270336A - Candida tropicalis ZD-3 bacterial strain, uses and method for producing biological protein feedstuff thereof - Google Patents
Candida tropicalis ZD-3 bacterial strain, uses and method for producing biological protein feedstuff thereof Download PDFInfo
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Abstract
The invention discloses a Candida tropicalis ZD-3 bacterial strain, uses of Candida tropicalis ZD-3 bacterial strain and a method for producing bioprotein feedstuffs with Candida tropicalis ZD-3 bacterial strain. The Candida tropicalis ZD-3 bacterial strain is preserved in China Center for Type Culture Collection with the registered number of CCTCC N<0>M208024, which can be used for the detoxication and the fermentation of cotton seed cakes and similar substance to produce high quality bioprotein feedstuffs. Candida tropicalis ZD-3 bacterial strain has the advantages that Candida tropicalis ZD-3 bacterial strain is cultured conveniently, grows fast, and the detoxication efficiency of free gossypol is high; the modified substrate after fermentation contains microzyme with high content; the proteinase activity of the metabolites is high; Candida tropicalis ZD-3 bacterial strain produces no hazardous materials; the modified substrate produced through the fermentation of Candida tropicalis ZD-3 bacterial is a novel microbe bioprotein feedstuff, and can significantly improve the disease resistance of animals and feedstuff utilization efficiency.
Description
Technical field
The invention belongs to biological technical field, specifically a kind of candida tropicalis ZD-3 bacterial strain (Candidatropicalis ZD-3) and uses thereof and with this bacterial strain in order to produce the method for biological protein feedstuff.
Background technology
Cotton cake dregs is the main byproduct of cotton processing industry, and China's annual production is up to more than 400 ten thousand tons.Its rich in proteins (30%～45%) is the important protein feed resource.At home and abroad, but substitute dregs of beans to the cotton cake dregs more amount and be applied to ruminating animal safely, especially in raising dairy cattle, apply in large quantities, but for monogastric animal, when feeding cotton cake dregs, owing to contain toxic substance gossypol (450～1500mg/kg), can produce tangible detrimentally affect to aspects such as growth of animal, growth and breedings, thereby limited that it is safe and effective as the livestock and poultry protein fodder, the serious waste of feed resource is caused in some areas even it is demoted make fertilizer.At this difficult problem, China scientific research personnel (Wu Xiaoyue etc., 1989; Zhong Yingchang etc., 1989) approach with the microbial fermentation detoxification has at first been proposed, specified microorganisms is inoculated in the cotton cake dregs substratum exactly, utilize growth and breeding and the metabolism of microorganism in feedstuff raw material under certain conditions, gossypol is decomposed to utilize or combine with gal4 amino acid and forms in conjunction with gossypol, thereby reaches the purpose of detoxification.
The molecular formula of gossypol is C
30H
30O
8, chemical structure is 8,8 '-dialdehyde-based-1,1 ', 6,6 ', 7,7 '-hexahydroxy--5,5 '-di-isopropyl-3,3 '-dimethyl-2,2 '-dinaphthalene.Its molecular weight is 518.54.The existence form of gossypol has two kinds in the cotton plant body, promptly in conjunction with gossypol (bound gossypol) and free gossypol (free gossypol).It has been generally acknowledged that being the gossypol that unbound state exists in cotton tissue is called free gossypol, combine gossypol and be called with other materials (protein, amino acid and phosphatide) bonded gossypol.It is generally acknowledged to have only free gossypol that people and animals are had toxicity.The cottonseed cakes that contain the high dosage free gossypol of ingesting for a long time such as pig, chicken, rabbit can damage the animal reproduction function, cause breathing with difficulty, difficulty, and the speed of growth reduces and symptom such as apocleisis.Gossypol can cause the egg yolk variable color, becomes pale brown look by faint yellow.Symptom such as the animal toxicity symptom shows as appetite stimulator or apocleisis, day by day becomes thin, myasthenia of the limbs, tic, diarrhoea, stool colour are thin out.
Summary of the invention
The purpose of this invention is to provide a kind of candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) and uses thereof, also provide a kind of simultaneously the method for above-mentioned bacterial strains in order to the production biological protein feedstuff.
Candida tropicalis ZD-3 bacterial strain of the present invention (Candida tropicalis ZD-3) is deposited in Chinese typical culture collection center on March 1st, 2008, and registering on the books is numbered: CCTCC N
0M208024.
Candida tropicalis ZD-3 bacterial strain of the present invention (Candida tropicalis ZD-3) cell is oval, and size is 3～4 * 4～5 μ m, the breeding of sprouting.Cultivate this bacterium with wort liquid, bacterial sediment is in the pipe end.Cultivate 24～48h on wort agar inclined-plane or plate, it is cream-colored that bacterium colony is, matt or glossy slightly, soft and level and smooth, and wrinkle is arranged.Incubation time bacterium colony of a specified duration is harder.Cultivate on the Corn Meal Agar substratum with cover plate, visible a large amount of pseudohyphas comprise the branch pseudohypha of elongation having blastoconidium above.
Above-mentioned candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 can grow on cotton cake dregs and analogue, and free gossypol content is extremely low in the sex change substrate, produces a large amount of meta-bolites proteolytic enzyme in the substrate simultaneously.Therefore, it can be used for the fermentation detoxification of cotton cake dregs and analogue, and then produces the high quality biological protein feed.
Above-mentioned candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 is as follows in order to the method for producing biological protein feedstuff: with candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) CCTCC N
0M208024 is 25～35 ℃ of environment bottom fermentation 30～60h to the cotton cake dregs detoxification of fermenting in temperature.
With above-mentioned candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 produces in the biological protein feedstuff process, is preferably in to add at least a as the fermentation bed material in 10～30% dregs of beans, Semen Maydis powder, the Wheat bran in the cotton cake dregs.
Above-mentioned usefulness candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 produces in the biological protein feedstuff method, and its preferred bacterial classification making processes is as follows:
(1). first order seed (test tube slant seed): be seeded to test tube wort solid medium from original strain, cultivated 16～36 hours for 28～32 ℃;
(2). secondary seed (test tube liquid seeds): be seeded to the test tube malt juice liquid medium from first order seed, cultivated 16～36 hours for 28～32 ℃;
(3). three grades of seeds (triangular flask liquid seeds): be seeded to the triangular flask that contains 50～150ml wort (5～10 ° of Bx) substratum from secondary seed, cultivated 16～36 hours for 28～32 ℃;
Above-mentioned usefulness candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 produces in the biological protein feedstuff method, being formulated as follows of its bed material:
(1). the preparation of cotton cake dregs fermentation bed material: one or more that get dregs of beans, Semen Maydis powder, Wheat bran mix by arbitrary proportion, ratio in solid-to-liquid ratio 1: 0.6～0.8 adds entry again, steam sterilizing 15～25min under 105～115 ℃ of temperature is cooled to 25～35 ℃;
(2). fermentation: cotton cake dregs fermentation bed material inserts three grades of seeds by 1～10% ratio, and the bed material water content is controlled at 45～55%, and the indoor relative humidity that ferments remains on more than 85%, and temperature maintenance is at 25～35 ℃, ventilating fermentation 30～60 hours;
(3). culture is pulverized 35～45 ℃ of dryings, mixes the back packing.
Advantage of the present invention is as follows: it is convenient to cultivate, fast growth, free gossypol detoxification efficient height; Yeast content height in the fermentation back sex change substrate; Meta-bolites protease activity height; This bacterium does not produce objectionable impurities, and the sex change substrate that is produced with its fermentation is novel little ecologic biological protein fodder, can significantly improve animal disease resistant and food utilization efficiency.
Candida tropicalis ZD-3 bacterial strain of the present invention (Candida tropicalis ZD-3) is deposited in Chinese typical culture collection center on March 1st, 2008, and registering on the books is numbered: CCTCC N
0M208024.
Embodiment
Bacterial screening, domestication and mutagenesis:
According to pertinent literature, collect bacterial strain 28 strains that free gossypol potentiality in the elimination cotton cake dregs are arranged in the laboratory, dibbling is to gossypol acetate sugar-free Cha Shi substratum, wherein 8 strains can well be grown, progressively increase the concentration of sugar-free Cha Shi substratum gossypol acetate then, have 3 strain bacteria growings better during maximum concentration.With its dibbling dull and stereotyped step by step domestication breeding to the gossypol acetate malt extract medium, wherein have strain growth the fastest subsequently, the bacterium colony maximum is numbered ZD-3.The ZD-3 inoculation is carried out solid fermentation to the cotton dregs substratum, measure free gossypol content in the sex change substrate after the fermentation ends, this bacterial strain improves 8 percentage points than starting strain virus elimination rate, virus elimination rate is up to more than 90%, free gossypol is no more than 0.1g/kg, extremely significantly is lower than national standard (being no more than 1.2g/kg).
With this ZD-3 strain culturing and make bacteria suspension, carry out ultraviolet (irradiation of 30cm place is 60 seconds under the 15W ultraviolet lamp), radiation (Co respectively
60Irradiation 10s) and chemical reagent (nitrosoguanidine final concentration 1mg/ml, 28 ℃ left standstill 60 minutes) mutagenesis, after mutagenesis, bacteria suspension coated the primary dcreening operation plate, picking forms the bacterial strain than macrocolony, after the rejuvenation it is inoculated into and carries out solid fermentation on the cotton dregs substratum, measure free gossypol content in the sex change substrate after the fermentation ends, find that virus elimination rate significantly improves, and slightly reduce, determine that therefore the mutagenesis bacterium ZD-3 that sets out is an aimed strain.
Strain identification:
(1) morphological specificity: the ZD-3 strain cell is oval, and size is 3～4 * 4～5 μ m, the breeding of sprouting.Cultivate this bacterium with wort liquid, bacterial sediment is in the pipe end.Cultivate 24～48h on wort agar inclined-plane or plate, it is cream-colored that bacterium colony is, matt or glossy slightly, soft and level and smooth, and wrinkle is arranged.Incubation time bacterium colony of a specified duration is harder.Cultivate on the Corn Meal Agar substratum with cover plate, visible a large amount of pseudohyphas comprise the branch pseudohypha of elongation having blastoconidium above." microbial taxonomy ", " saccharomycetic feature and identification handbook " and the Physiology and biochemistry experimental result of this bacterium of J.A. Barney top grade (1991) according to " An Introduction industrial mycology " (George Smith 1954), " fungi identification handbook " (Wei Jing surpasses 1982), " common and commonly used fungi " (institute of microbiology of the Chinese Academy of Sciences 1973), Zhang Jizhong (1990) chief editor, identify that this bacterial strain is: candida tropicalis (Candidatropicalis), name ZD-3 into Candida tropicalis.
(2) cultural characteristic: the optimum temperuture of this bacterial strain solid fermentation cotton cake dregs is 25～35 ℃; Fermentation time 30～60h; Substrate moisture content 45～55%; Nature pH value; Can add an amount of dregs of beans or Semen Maydis powder and Wheat bran in the cotton cake dregs, preparation fermentation bed material.
Solid fermentation production process:
(1). first order seed (test tube slant seed): be seeded to test tube wort solid medium from original strain, cultivated 16～36 hours for 28～32 ℃;
(2). secondary seed (test tube liquid seeds): be seeded to the test tube malt juice liquid medium from first order seed, cultivated 16～36 hours for 28～32 ℃;
(3). three grades of seeds (triangular flask liquid seeds): be seeded to the triangular flask that contains 50～150ml wort (5～10 ° of Bx) substratum from secondary seed, cultivated 16～36 hours for 28～32 ℃;
(4). the preparation of cotton cake dregs fermentation bed material: the cotton cake dregs raw material adds an amount of dregs of beans or Semen Maydis powder and Wheat bran behind crushing screening, the ratio in solid-to-liquid ratio 1: 0.6～0.8 adds entry again, 0.056MPa, 112.6 ℃ of steam sterilizing 20min, cooling;
(5). fermentation: cotton cake dregs fermentation bed material inserts three grades of seeds by 1～10% ratio, and the bed material water content is controlled at 45～55%, and the indoor relative humidity that ferments remains on more than 85%, and temperature maintenance is at 25～35 ℃, ventilating fermentation 30～60 hours;
(6). culture is pulverized 35～45 ℃ of dryings, mixes the back packing.
Embodiment 1:
Cotton cake dregs bed material solid fermentation
(1). first order seed (test tube slant seed): be seeded to test tube wort solid medium from original strain, cultivated 24 hours for 30 ℃;
(2). secondary seed (test tube liquid seeds): be seeded to the test tube malt juice liquid medium from first order seed, cultivated 20～24 hours for 30 ℃;
(3). three grades of seeds (triangular flask liquid seeds): be seeded to the triangular flask that contains 5 ° of Bx malt extract mediums from secondary seed, cultivated 20～24 hours for 30 ℃;
(4). the preparation of cotton cake dregs fermentation bed material: the cotton cake dregs raw material adds 20% dregs of beans behind crushing screening, add entry in 1: 0.8 ratio of solid-to-liquid ratio again, 0.056MPa, 112.6 ℃ of steam sterilizing 20min are cooled to room temperature;
(5). fermentation: cotton cake dregs fermentation bed material inserts three grades of seeds by 5% ratio, and the bed material water content is controlled at 50%, and the indoor relative humidity that ferments remains on more than 85%, and temperature maintenance is at 30 ℃, ventilating fermentation 48 hours;
(6). culture is pulverized 45 ℃ of dryings, crosses aperture 0.6mm sieve, mixes the back packing.
(7). free gossypol is measured: less than 0.1g/kg.
(8). yeast numeration: greater than 2.8 * 10
13Cell/g.
(9). protease activity determination: neutral greater than 130u/g; Alkalescence is greater than 50u/g; Acid greater than 25u/g
Free gossypol is measured: measure by GB GB13086-91 " measuring method of free gossypol in the feed ".
Yeast numeration: adopt colony counting method, State Standard of the People's Republic of China: GB4789.15-84, food sanitation Micro biological Tests--mould and yeast counts.
Protease activity determination: method is a folin's methods, People's Republic of China's specialized standard: ZB X 66030-87.Proteinase activity is measured the mensuration that comprises Sumizyme MP, neutral protease and aspartic protease.Produce 1 μ g tyrosine at 40 ℃ of following per minute caseinhydrolysates, be defined as 1 protease activity unit of force.
Embodiment 2:
Be the to ferment preparation of bed material of the difference of embodiment 1, other step is identical.
The preparation of cotton cake dregs fermentation bed material: the cotton cake dregs raw material added 20% dregs of beans, 5% corn, 5% Wheat bran after pulverizing aperture 0.5mm sieve, add entry in 1: 0.8 ratio of solid-to-liquid ratio again, 0.05MPa 112.6 ℃ of steam sterilizing 20min are cooled to room temperature.
Embodiment 3:
Be the to ferment preparation of bed material of the difference of embodiment 1,2, other step is identical.
The preparation of cotton cake dregs fermentation bed material: the cotton cake dregs raw material added 10% dregs of beans, 10% corn, 10% Wheat bran after pulverizing aperture 0.4mm sieve, add entry in 1: 0.8 ratio of solid-to-liquid ratio again, 0.045MPa 112.6 ℃ of steam sterilizing 20min are cooled to room temperature.
Embodiment 4:
Difference from Example 1 is: in substrate inoculation patent bacterial classification candida tropicalis ZD-3, inoculate common yeast saccharomyces cerevisiae, carry out the binary complex ferment.The making method of common one, two, three seed of yeast saccharomyces cerevisiae and inoculum size are identical with candida tropicalis ZD-3, and other operation steps is identical with embodiment 1.
Claims (7)
1. a candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) is characterized by CCTCC N
0M208024.
2. a candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0The purposes of M208024 is characterized in that being used for cotton cake dregs fermentation detoxification, eliminates free gossypol, produces biological protein feedstuff.
3. one kind with candida tropicalis ZD-3 bacterial strain (Candida tropicalis ZD-3) CCTCC N
0M208024 produces the method for biological protein feedstuff, it is characterized in that with candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) CCTCC N
0M208024 is 25～35 ℃ of environment bottom fermentation 30～60h to the cotton cake dregs detoxification of fermenting in temperature.
4, according to claim 3 with candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) CCTCC N
0M208024 produces the biological protein feedstuff method, it is characterized in that adding in the cotton cake dregs at least a or several as the fermentation bed materials in 10～30% dregs of beans, Semen Maydis powder, the Wheat bran.
5, according to claim 3 or 4 described candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) the CCTCC N that use
0M208024 produces the biological protein feedstuff method, it is characterized in that being produced as follows of bacterial classification:
(1). first order seed (test tube slant seed): be seeded to test tube wort solid medium from original strain, cultivated 16～36 hours for 28～32 ℃;
(2). secondary seed (test tube liquid seeds): be seeded to the test tube malt juice liquid medium from first order seed, cultivated 16～36 hours for 28～32 ℃;
(3). three grades of seeds (triangular flask liquid seeds): be seeded to the triangular flask that contains 50～150ml wort (5～10 ° of Bx) substratum from secondary seed, cultivated 16～36 hours for 28～32 ℃;
6, according to claim 3 or 4 described candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) the CCTCC N that use
0M208024 produces the biological protein feedstuff method, it is characterized in that being formulated as follows of bed material:
(1). the preparation of cotton cake dregs fermentation bed material: getting dregs of beans, Semen Maydis powder, a kind of of Wheat bran can severally mix by arbitrary proportion, ratio in solid-to-liquid ratio 1: 0.6～0.8 adds entry again, steam sterilizing 15～25min under 105～115 ℃ of temperature is cooled to 25～35 ℃;
(2). fermentation: cotton cake dregs fermentation bed material inserts three grades of seeds by 1～10% ratio, and the bed material water content is controlled at 45～55%, and the indoor relative humidity that ferments remains on more than 85%, and temperature maintenance is at 25～35 ℃, ventilating fermentation 30～60 hours;
(3). culture is pulverized 35～45 ℃ of dryings, mixes the back packing.
7, according to claim 5 with candida tropicalis ZD-3 bacterial strain (Candida tropicalisZD-3) CCTCC N
0M208024 produces the biological protein feedstuff method, it is characterized in that being formulated as follows of bed material:
(1). the preparation of cotton cake dregs fermentation bed material: one or more that get dregs of beans, Semen Maydis powder, Wheat bran mix by arbitrary proportion, ratio in solid-to-liquid ratio 1: 0.6～0.8 adds entry again, steam sterilizing 15～25min under 105～115 ℃ of temperature is cooled to 25～35 ℃;
(2). fermentation: cotton cake dregs fermentation bed material inserts three grades of seeds by 1～10% ratio, and the bed material water content is controlled at 45～55%, and the indoor relative humidity that ferments remains on more than 85%, and temperature maintenance is at 25～35 ℃, ventilating fermentation 30～60 hours;
(3). culture is pulverized 35～45 ℃ of dryings, mixes the back packing.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897383A (en) * | 2010-06-29 | 2010-12-01 | 北京金泰得生物科技股份有限公司 | Method for removing cotton rapeseed meal toxicant by fermentation method and enhancing nutrient value thereof, feeding fermented cotton rapeseed meal protein feedstock and applications thereof |
| CN102318737A (en) * | 2011-10-11 | 2012-01-18 | 广东希普生物科技股份有限公司 | Preparation method of nontoxic cotton dreg animal feed |
| CN101805704B (en) * | 2010-02-03 | 2012-05-23 | 中国药科大学 | Candidatropicalis for producing ribonucleic acid with high yield and application thereof |
| CN101805771B (en) * | 2010-02-03 | 2012-10-10 | 南通秋之友生物科技有限公司 | Method for continuously culturing ribonucleic acid of high-yield nucleic acid Candida tropicalis in agitating tank |
| CN103598564A (en) * | 2013-11-14 | 2014-02-26 | 吴基仔 | Method for brewing soy sauce by cottonseed cakes |
| CN104642726A (en) * | 2014-11-05 | 2015-05-27 | 新疆大禾油脂有限公司 | Microbial detoxication method for cotton seed meal |
| CN105274012A (en) * | 2015-11-20 | 2016-01-27 | 江南大学 | Candida tropicalis with function of gossypol degradation and application of candida tropicalis |
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2008
- 2008-05-09 CN CNA2008100728776A patent/CN101270336A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805704B (en) * | 2010-02-03 | 2012-05-23 | 中国药科大学 | Candidatropicalis for producing ribonucleic acid with high yield and application thereof |
| CN101805771B (en) * | 2010-02-03 | 2012-10-10 | 南通秋之友生物科技有限公司 | Method for continuously culturing ribonucleic acid of high-yield nucleic acid Candida tropicalis in agitating tank |
| CN101897383A (en) * | 2010-06-29 | 2010-12-01 | 北京金泰得生物科技股份有限公司 | Method for removing cotton rapeseed meal toxicant by fermentation method and enhancing nutrient value thereof, feeding fermented cotton rapeseed meal protein feedstock and applications thereof |
| CN101897383B (en) * | 2010-06-29 | 2012-10-03 | 北京金泰得生物科技股份有限公司 | Method for removing cotton rapeseed meal toxicant by fermentation method and enhancing nutrient value thereof, feeding fermented cotton rapeseed meal protein feedstock and applications thereof |
| CN102318737A (en) * | 2011-10-11 | 2012-01-18 | 广东希普生物科技股份有限公司 | Preparation method of nontoxic cotton dreg animal feed |
| CN102318737B (en) * | 2011-10-11 | 2013-01-23 | 广东希普生物科技股份有限公司 | Preparation method of nontoxic cottonseed meal animal feed |
| CN103598564A (en) * | 2013-11-14 | 2014-02-26 | 吴基仔 | Method for brewing soy sauce by cottonseed cakes |
| CN104642726A (en) * | 2014-11-05 | 2015-05-27 | 新疆大禾油脂有限公司 | Microbial detoxication method for cotton seed meal |
| CN105274012A (en) * | 2015-11-20 | 2016-01-27 | 江南大学 | Candida tropicalis with function of gossypol degradation and application of candida tropicalis |
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