CN107937286A

CN107937286A - A kind of Pichia anomala and its fermentation culture medium and application

Info

Publication number: CN107937286A
Application number: CN201711298322.9A
Authority: CN
Inventors: 唐志如; 孙志洪; 马娅君; 毛俊霞
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2018-04-20
Anticipated expiration: 2037-12-08
Also published as: CN107937286B

Abstract

Belong to biological technical field herein, disclose a kind of Pichia anomala and its fermentation culture medium and application, the Pichia anomala is：Pichia anomala AR₂₀₁₆, CCTCC NO：M 2017594.Liquid state fermentation is carried out to it using fermentation medium provided by the invention, the biomass of zymotic fluid is 15 g/L, and the yield of the sanguinariness factor is 5.86 mg/L, and the yield of 1,3 dextranases of β is 964 U/L and the yield of phytase is 1675 U/L.The Pichia anomala of the present invention have it is anti-have suppress dysentery Escherichia coli, S. pullonum, sensitive c-type Candida albicans, Actinobacillus pleuropneumoniae, rise yellow sarcine, staphylococcus aureus, the effect of stomach juice-resistant and cholate.Therefore Pichia anomala provided by the invention can be used for the feed addictive of beasts, birds and aquatic products animal, possess wide application prospect.

Description

A kind of Pichia anomala and its fermentation culture medium and application

Technical field

The present invention relates to biological technical field, is more particularly to a kind of Pichia anomala and its fermentation culture medium and answers With.

Technical background

Pichia anomala category basidiomycetes, bacterium colony are milky, are simple pseudohyphas, and cell shape is circle, for battalion Support body cell gemmation.Pichia anomala has anti-candida albicans, the effect of stomach juice-resistant and cholate, its liquid and solid-state Also contain the sanguinariness factor and beta-1,3-glucanase and phytase in fermentate.Beta-1,3-glucanase energy catalyzing hydrolysis cereal is thin Beta glucan in cell wall.Phytase can hydrolyze phosphate moiety from phytic acid, and it is strong to mineral element to destroy phytic acid Affinity, and then increase the nutrition balance of mineral element, improve the nutritive value function of plant feed.Pichia anomala The fermenting and producing sanguinariness factor, β -1,3- dextranases and phytase, cultivation cycle is short, of low cost, and thalline is nontoxic and containing rich Rich protein, has important research significance and development prospect.Subtropic of China area is with heavy rainfall, and air humidity is big, gas Wait the growth of control environment microorganism, barms aboundresources, but Abnormal pichia anomala kind excavating resource is limited, at present I State mainly concentrates research ocean killer yeast physio-biochemical characteristics, and foreign countries report distillery yeast killer system, but just abnormal Pichia pastoris liquid and solid state fermentation culture have no report in the application of beasts, birds and aquatic products aquaculture.It is different in order to enrich based on this Normal Pichia pastoris strain resources bank, it is contemplated that excavating the subtropical zone land Abnormal pichia anomala with economic value Kind resource, studies its liquid and solid state fermentation cultural method, and tunning is applied in beasts, birds and aquatic products aquaculture.

The content of the invention

It is an object of the invention to provide a kind of Pichia anomala AR₂₀₁₆, which is rich in amino acid, can produce sanguinariness because Son, β -1,3- dextranases and phytase, have and resist a variety of fungies or bacterium, the effect of stomach juice-resistant and cholate, security is good, can Cultivated applied to beasts, birds and aquatic products.The bacterial strain is sent to China typical culture collection center preservation on October 20th, 2017, point Class is named：Pichia anomala (Pichia anomala) AR₂₀₁₆, deposit number is：CCTCC NO：M 2017594, address： China, Wuhan, Wuhan University.

It is another object of the present invention to provide a kind of Pichia anomala Pichia anomala AR₂₀₁₆Fermentation Liquid culture and solid state rheology thing, fermentating formula of the invention is simple, of low cost.It is abnormal complete red using zymotic fluid fermentation Yeast Pichia anomala AR₂₀₁₆, barms can be largely produced, culture sanguinariness containing the high yield factor, β -1,3- Portugals gather Carbohydrase and phytase.

Final object of the present invention is the provision of a kind of Pichia anomala AR₂₀₁₆The application of fermentation culture medium, Including the Pichia anomala Pichia anomala AR₂₀₁₆Fermentation liquid culture or solid state rheology thing supported in herding The application in industry is grown, or utilizes Pichia anomala Pichia anomala AR₂₀₁₆Prepare bacteriostatic agent.

In order to achieve the above object, the present invention takes following technical measures：

Pichia anomala AR₂₀₁₆From Chenzhou City Anren County of Hunan Province, township of Longhai City thatch rushes in Deng Mu groups sweet wine distiller's yeast among the people and divides From gained；The bacterial strain is sent to China typical culture collection center preservation, Classification And Nomenclature on October 20th, 2017：It is abnormal complete Red yeast (Pichia anomala) AR₂₀₁₆；Deposit number is：CCTCC NO：M 2017594；Address：Wuhan, China Wuhan is big Learn.

The morphological feature of bacterium colony：Bacterium colony is rounded or oval, thick, rough surface, color are milky, and bacterium colony is big And it is thick, it is easy to provoke.

Pichia anomala Pichia anomala AR₂₀₁₆Physiological and biochemical property

Galactolipin

Sucrose

Fine disaccharides

Raffinose

Sorbierite

Melezitose

Inositol

Trehalose

+

Acetamide

Mannitol

Rhamnose

Melibiose

Xylose

Ribose

Glycerine

I is sugared

+

-

+

+：It is positive；-：It is negative

A kind of Pichia anomala Pichia anomala AR₂₀₁₆Liquid state fermentation culture medium, including：Glucose 2-4%, Peptone 1-2%, dusty yeast 1-2%, pH6.0, remaining is water, is mass ratio above.

In scheme described above, Pichia anomala Pichia anomala AR₂₀₁₆Biomass, the sanguinariness factor, β- When 1,3- dextranase and phytic acid production of enzyme reach maximum, the formula of large-scale fermentation culture includes：Glucose 4.0%, albumen Peptone 1.5%, dusty yeast 2.0%, remaining is water, pH 6.0.

A kind of Pichia anomala Pichia anomala AR₂₀₁₆Solid-state fermentation culture medium, including：Wheat bran 14-28%, Dregs of beans 5-7%, corn flour 2-5%, corn pulp 4-6%, rice bran 3-5%, ammonium sulfate 0.5-0.8%, dipotassium hydrogen phosphate 0.02- 0.07%, magnesium sulfate 0.07-0.10%, remaining is tap water, is mass ratio above, solid-state fermentation culture medium warp 121 DEG C of high-temperature steam sterilization 20min, are cooled to 40 DEG C and can access Pichia anomala Pichia anomala AR₂₀₁₆。

In scheme described above, Pichia anomala Pichia anomala AR₂₀₁₆Biomass, the sanguinariness factor, β- When 1,3- dextranase and phytase reach maximum, the formula of solid-state fermentation culture medium includes：Wheat bran 23.56%, dregs of beans 6.35%, corn flour 4.36%, corn pulp 4.05%, rice bran 3.09%, ammonium sulfate 0.65%, dipotassium hydrogen phosphate 0.052%, sulphur Sour magnesium 0.105%, remaining is tap water, and pH6.0, is mass ratio above.

A kind of Pichia anomala Pichia anomala AR₂₀₁₆Application, including the Pichia anomala Pichia anomala AR₂₀₁₆The application in livestock breeding industry of fermentation liquid culture or solid state rheology thing, including the exception Pichia pastoris can be used for preparation suppression to have suppression dysentery Escherichia coli, S. pullonum, sensitive c-type Candida albicans, chest Film Actinobacillus, the microbial inoculum for rising yellow sarcine or staphylococcus aureus, the feed addictive for fowl poultry kind animal.

Compared with prior art, the present invention has the following advantages：

1st, Pichia anomala of the invention is separating obtained from the tradition sweet wine distiller's yeast sample of Hunan Anren, is pacified by animal Full property experiment confirms such a Pichia anomala without any toxicity and side effects；

2nd, after Pichia anomala of the invention is fermented, biomass is 15g/L in zymotic fluid, and the yield of the sanguinariness factor is The yield of 5.86mg/L, β -1,3- dextranase is 964U/L, the yield 1675U/L of phytase.Contain 68.44% thick egg in bacterium powder White and 47.68% total amino acid.

3rd, Pichia anomala of the invention has the high yield sanguinariness factor, and compared with the strain reported, it is big to be resistant to dysentery Enterobacteria, S. pullonum, sensitive c-type Candida albicans, Actinobacillus pleuropneumoniae, rise yellow sarcine, golden yellow A variety of pathogens such as staphylococcus, have the function of anti-diarrhea.

4th, Pichia anomala of the invention has the function that stomach juice-resistant, cholate and intestinal juice.

5th, the solid state fermentation of current Pichia anomala and its and beasts, birds and aquatic products animal applications have not been reported, the present invention is solid State fermentation gained Pichia anomala can produce beta-1,3-glucanase and phytase.Beta-1,3-glucanase energy catalyzing hydrolysis cereal Beta glucan in cell membrane.Phytase can hydrolyze phosphate moiety from phytic acid, and it is strong to mineral element to destroy phytic acid Affinity, and then increase the nutrition balance of mineral element, the nutritive value function of plant feed improved, beneficial to livestock and poultry water Produce growth of animal.

6th, it is aquatic feed for domestic animal raw material that solid state fermentation culture is raw materials used, of low cost.Contain what is enriched in culture Essential amino acid, β -1,3- dextranases and phytase, growth of animal and product quality can be improved by being produced applied to animal.

Brief description of the drawings

Fig. 1 is Pichia anomala Pichia anomala AR in embodiment 1₂₀₁₆Colonial morphology figure.

Fig. 2 is Pichia anomala Pichia anomala AR in embodiment 1₂₀₁₆Cellular morphology figure.

Fig. 3 is Pichia anomala Pichia anomala AR₂₀₁₆Hot tolerance schematic diagram.

Fig. 4 is Pichia anomala AR₂₀₁₆Sour tolerance schematic diagram.

Fig. 5 is Pichia anomala AR₂₀₁₆To intestinal juice tolerance schematic diagram.

Embodiment

Technical solution of the present invention, is the ordinary skill in the art if not otherwise specified；The reagent or material are such as It is not specified, derive from commercial channel.

Embodiment 1：Strain separating and identification

Strain isolation

Anren sweet wine distiller's yeast sample among the people of 10g is taken in the 250ml triangular flasks equipped with 100mL sterile waters, in vortex instrument A moment is stood after upper vibration, takes the bacteria suspension of 1mL and the physiological saline of 9ml to mix, then take the bacteria suspension of 1ml and the physiology of 9mL Brine mixes, and so on, different concentration gradients, gradient 10 are set^-1、10^-2、10^-3、10^-4、10^-5, 200 μ l are taken successively Bacteria suspension coating be inoculated in separation tablet on, be placed in 28 DEG C culture 2~3d, according to colonial morphology picking typical case single bacterium colony (bacterium Fall form：Rounded, surface is rough in hairy, and color is creamy white opaque, intermediate projections, is not easy to provoke, size 3mm- 5mm), Pichia anomala is gone out by microscopy scalping.

PDA culture medium：Potato 20g, glucose 20g, agar 20g, water 1000ml, natural pH, sterilize by 115 DEG C 20min。

Secondary screening and identification

The Pichia anomala good to Candida albicans growth inhibition performance is screened by bacteriostatic test, passes through Physiology and biochemistry And 26S rDNA D1/D2 identifications, the Pichia anomala of one plant of production sanguinariness factor is finally screened, is named as abnormal complete red ferment Female P ichia anomala AR₂₀₁₆.The bacterium has been sent to China typical culture collection center progress on October 20th, 2017 Preservation, Classification And Nomenclature：Pichia anomala Pichia anomala AR₂₀₁₆, address：Wuhan, China Wuhan University.

AR₂₀₁₆Bacterial strain specifically has the physiological and biochemical property of typical Pichia anomala：The bacterial strain is bred in PDA culture medium The bacterium colony that growth is formed be rounded, the smooth of the edge, and surface is in that hairy is rough, and color is creamy white opaque, intermediate projections, It is not easy to provoke, size 3mm-5mm (Fig. 1, Fig. 2).Physiological and biochemical property is：Gelatin liquefaction.Ferment galactose, sucrose, glucose, Fine disaccharides, raffinose, sorbierite, melezitose, inositol, trehalose, acetamide, mannitol, rhamnose, xylose, ribose, glycerine and Arabinose；Azymic melibiose.28~32 DEG C of optimum temperature, 50 DEG C of maximum temperature.

So far, applicant obtains one plant of Pichia anomala, which send to Chinese allusion quotation on October 20th, 2017 Type culture collection preservation, Classification And Nomenclature：Pichia anomala Pichia anomala AR₂₀₁₆, deposit number：CCTCC NO：M 2017594；Address：China, Wuhan, Wuhan University.

Embodiment 2：

Pichia anomala Pichia anomala AR₂₀₁₆Large-scale fermentation：

By the Pichia anomala Pichia anomala AR of preservation₂₀₁₆It is inoculated into PDA culture medium, is positioned over 28 DEG C Constant incubator culture 36h, one oese Pichia anomala Pichia anomala AR of picking₂₀₁₆In seed culture fluid (matter Measure fraction (%)：Glucose 2%, peptone 1%, dusty yeast 1%, natural pH, remaining is distilled water) in, at 28 DEG C, 200rpm Isothermal vibration device on cultivate 24h.

500L fermentation tanks liquid amount is 65% (V/V), and liquid state fermentation culture medium includes, (mass fraction, %)：Glucose 4.0%, peptone 1.5%, dusty yeast 2.0%, pH6.0, through 121 DEG C of high-temperature steam sterilization 20min, are cooled to 40 DEG C, Xiang Qi The seed liquor of 10% cell age 24h of middle inoculation, at 28 DEG C, after 200rpm fermentations 96h, the biomass of zymotic fluid is 15g/L, sanguinariness because The yield of son is 5.86mg/L, and β -1, the yield of 3- dextranases is 964U/L and the yield of phytase is 1675U/L.

After putting tank, filter plate is concentrated into original volume 1/8 in 0.2~0.3MPa of gauge pressure, is spray-dried, obtains through filter press (effective bacterial content of every gram of dry powder is 3.5 × 10 to active dry yeasr dry powder¹¹Cfu/g), for following embodiments.

Embodiment 3：

Pichia anomala Pichia anomala AR₂₀₁₆The content of protein and amino acid

Bacterium mud is collected by centrifugation in zymotic fluid in embodiment 2, is dried at 55 DEG C and obtains dry bacterium powder.

Determined amino acid：Dry bacterium powder 120mg accurately is weighed in test tube, is added into the test tube for fill sample 10mL16mol hydrochloric acid, concussion mix.With alcohol blast burner drawing and attenuating to 4-6mm at 1/3 under the test tube, sealed after vacuumizing 10min Pipe.Processed test tube puts sand-bath in 110 DEG C of constant temperature ovens and hydrolyzes 22h, takes out and is cooled to room temperature, shake up filtering, takes 1mL filtrates In 50mL beakers, filtrate is evaporated with 60 DEG C of waters bath with thermostatic control, adds 4 times of 0.02mol hydrochloric acids, is used with 0.22 μm of membrane filtration The full-automatic amino-acid analyzer measure of Hitachi's L-8800 types.Contained using Kjeldahl's method (GB5009.5-2003) measure crude protein Amount.

Pichia anomala Pichia anomala AR₂₀₁₆Dry bacterium powder contains 68.44% crude protein, total amino acid 47.68%, 3.84% aspartic acid, 1.59% threonine, 2.07% serine, 6.51% glutamic acid, 7.69% glycine, 4.61% alanine, 0.26% cysteine, 1.90% valine, 0.47% methionine, 1.30% isoleucine, 2.21% is bright Propylhomoserin, 0.99 tyrosine, 1.55% phenylalanine, 2.82% lysine, 0.14% histidine, 1.43% arginine %, 7.73% proline.

Embodiment 4：

Pichia anomala Pichia anomala AR₂₀₁₆Solid state fermentation：

By Pichia anomala Pichia anomala AR₂₀₁₆It is inoculated into PDA culture medium, is positioned over 28 DEG C of constant temperature trainings Support case culture 36h, one oese Pichia anomala Pichia anomala AR of picking₂₀₁₆In seed culture fluid (respectively into sub-prime Measure fraction (%)：Glucose 2%, peptone 1%, dusty yeast 1%, natural pH) in, at 28 DEG C, the isothermal vibration device of 200rpm Upper culture 24h.

Solid-state fermentation culture medium includes (mass fraction, %)：Wheat bran 23.56%, dregs of beans 6.35%, corn flour 4.36%, Corn pulp 4.05%, rice bran 3.09%, ammonium sulfate 0.65%, dipotassium hydrogen phosphate 0.052%, magnesium sulfate 0.105%, remaining is certainly Water, pH 6.0, is mass ratio above.Through 121 DEG C of high-temperature steam sterilization 20min, 40 DEG C are cooled to.Cell age is inoculated with thereto The seed liquor 7.5% (volume ratio) of 24h, in 28 DEG C of constant temperature biochemical cultivation case after fermentation 72h, the solid state fermentation before drying per g Culture contains 10⁹A Pichia anomala Pichia anomala AR₂₀₁₆, the yield of the sanguinariness factor is 3.45mg/g, β -1,3- The yield of dextranase is 732U/g, the yield 1126U/g of phytase.Abnormal complete red ferment is made through 40 DEG C of dryings in solid-state fermentation content Female Pichia anomala AR₂₀₁₆Feed addictive, for embodiment 12.

Embodiment 5：

Pichia anomala Pichia anomala AR₂₀₁₆Resistance detects

Hot tolerance：Take Pichia anomala growth latter stage medium centrifugal, 0.9% brine twice after Again suspend (1~2 × l0 of viable bacteria amount⁹Cfu/mL after) handling 15min in 35,45,55,65,75,85,95 DEG C of water-baths, rapidly Flowing water cools down, and then measures survival rate using dilution-plate method.The result shows that the strain highest tolerable temperature for 55 DEG C (see figure 3)。

Gastric fluid-resistant ability：The hydrochloric acid 16.4mL for taking mass fraction to be 10% adds distilled water to dilute, and it is 2.0 to make pH value, often Add 1g pepsins in 100mL hydrochloric acid solutions, it is fully degerming with 0.22 μm of filtering with microporous membrane after dissolving, obtain artificial stomach Liquid.Take Pichia anomala AR₂₀₁₆Final stage culture centrifugation is grown, 0.9% brine suspends again after twice, with 10⁷Cfu/mL final concentrations are inoculated in simulated gastric fluid, and concussion mixes, and 37 DEG C of water-baths are incubated, and are sampled every 0.5h and are carried out viable bacteria meter Number.The result shows that Pichia anomala, which is resistant to gastric juice, reaches 2h (being shown in Table 1, Fig. 4).

1 Pichia anomala AR of table₂₀₁₆Number of viable in sour environment

Pig cholate tolerance：Extra addition 1.0%, 2.0%, 3.0%, 4.0% is prepared, 5.0% (w/v) Pig cholate Seed culture fluid.1mL is taken to take Pichia anomala AR₂₀₁₆Bacterium solution (1~2 × l0⁸Cfu/mL), it is inoculated in the high courage of 9mL various concentrations In salinity seed culture fluid, after 30 DEG C of culture 12h, the turbidity change of measure nutrient solution, judges thalli growth situation.The result shows that It is 3% (being shown in Table 2) that Pichia anomala, which is resistant to Pig cholate mass fraction,.

The Pig cholate of 2 various concentrations of table is to Pichia anomala AR₂₀₁₆The influence of growth

Note：" ++ " represents well-grown, and "+", which represents, to be grown.

Intestinal juice tolerance

Weigh KH₂PO₄3.4g, adds distillation 250mL dissolvings, with 1mol/L NaOH tune pH value to 6.8, is diluted with water to 500mL, then trypsase is added by 0.1g/L, it is fully degerming with the filtering with microporous membrane in 0.22 μm of aperture after dissolving.Will activation Good strain is inoculated in simulated intestinal fluid respectively by 10% inoculum concentration, is mixed, 37 DEG C of constant-temperature shaking cultures, simulated intestinal fluid respectively at 0th, 2,4,6,8,10h samplings.Bacterium solution does 10 times of gradient dilutions with sterile water after processing, and viable bacteria is carried out using YPD solid mediums Count.The results are shown in Figure 5, Pichia anomala AR₂₀₁₆The digestion of intestinal juice trypsase is resistant to, keeps activity, 6h After reach logarithmic growth.

Embodiment 6：

Pichia anomala bacteriostasis detects

Sterilized NB agar mediums are heated to melt completely, are poured in culture dish, per ware 15mL (lower floor), treat it Solidification.The training that has solidified is added in addition, the NB culture mediums of thawing are cooled to 50 DEG C or so and are mixed into pharmacosensitive test bacteria culture fluid 5mL Support on base (upper strata) to be solidified.With sterile working media surface directly vertically put Oxford cup (internal diameter 6mm, outside diameter 8mm, High 10mm) circular tubule, the both ends of pipe are smooth), gently pressurize, it is contacted tight with culture medium, added in cup Measuring samples (zymotic fluid), Oxford cup can generally fill 200 μ L, not make its excessive.Fill it up with 37 DEG C of culture 16-18h of postposition, observation As a result, inhibition zone is directly measured with ruler.The result shows that Pichia anomala AR₂₀₁₆It is husky with dysentery Escherichia coli, white diarrhea is suppressed Door Salmonella, sensitive c-type Candida albicans, Actinobacillus pleuropneumoniae, the effect for rising yellow sarcine, staphylococcus aureus (being shown in Table 3).

3 Pichia anomala AR of table₂₀₁₆To the antibacterial circle diameter of different pathogenic bacteria

Pathogenic bacteria	Inhibition zone (mm)
		Dysentery Escherichia coli	10
S. pullonum	10
		Sensitive c-type Candida albicans	20
Actinobacillus pleuropneumoniae	20
		Rise yellow sarcine	20
Staphylococcus aureus	15

Embodiment 7：

Mouse safety test

The kunming mice of 60 weight 20g or so is randomly divided into 3 groups, every group 20, half male and half female.One of which does sky White control, experimental group one every press 1 × 10⁹Cfu/kg mouse weights (100 times of routine dose) gavage abnormal complete red for continuous one week Yeast AR₂₀₁₆Zymotic fluid, two disposable celiac of experimental group inject the bacteria suspension (1 × 10 of 0.2mL⁹cfu/mL).Observation one week, note Record growth and the death condition (table 4) of each group mouse.All mouse feeding experiments are in drying, cleaning, draughty interior Complete.The result shows that：After a week, 3 groups without death, and group of having an injection is had an injection the reduction of mouse food-intake for first day, apathetic, and the 3rd It recovers normal, and the results are shown in Table 4.

Table 4：Mouse experiment weighing results

Experimental group one is gavage group, and experimental group two is group of having an injection.

Embodiment 8：

AR₂₀₁₆The early-weaned piglets experiment of active dry yeasr

Choose similar in weight, with 21 age in days piglet of batch 40, be divided into 4 groups, every group 10, one group is control group, real Group A every is tested by percentage of liveweight 10⁸The bacterium amount of cfu/kg is mixed water and is gavaged, and experimental group B every presses percentage of liveweight 10⁹The bacterium amount of cfu/kg is mixed Water gavages, and experimental group C every presses percentage of liveweight 10¹⁰The bacterium amount of cfu/kg mixes water and gavages experimental group filling daily once, continuous to fill 7 days, Observation 21 days.Free choice feeding, per day entry, spirit and feeding situation, weigh at the end of experiment.Germicidal efficacy by 21 days exists Heavy dose continuously gavage it is lower there is no abnormal response, search for food and growing state be normal, AR₂₀₁₆Active dry yeasr group has obvious rush Growth result (table 5).

Table 5：Piglet gavages various dose Pichia anomala AR₂₀₁₆Influence to weightening

Group	First weight kg	Opisthosoma weight kg	Increase weight kg
				Blank group	6.05	9.89	3.83
Experimental group A	6.08	10.97	4.89
				Experimental group B	6.03	11.42	5.39
Experimental group C	6.06	11.32	5.41

Embodiment 9：

AR₂₀₁₆The pregnant sow experiment of active dry yeasr

Same batch pregnant sow 20 is chosen, is divided into 2 groups, every group 10, one group is control group, and another group is experiment Group；Observation period weans for every production previous moon to sucking pig；Control group diet daily ration；Experimental group by pig weight 2 × 10⁹The bacterium amount of cfu/kg is added to feeding in feed diet, daily to observe sow spirit and excrement situation, at the beginning of postpartum claims sucking pig Weight.It was observed that experimental group prevention of sow constipation and the condition of production are obviously improved, the control group sucking pig initial weight that is averaged is 1.29kg, and litter size is 8.5, and experimental group is averaged, sucking pig initial weight is 1.46kg, litter size 9.8.

Embodiment 10：

AR₂₀₁₆Application effect of the active dry yeast in laying hen and laying duck cultivation.

Selection body condition is close, the brown laying hen 200 of 39 week old sea orchids, and test period is 90 days, and first 5 days are preliminary trial period, after 85 days are the positive examination phase.Random to be classified as 2 treatment groups, each 5 repetitions for the treatment of group are each to repeat 20.I.e. 1 control Group, 1 test group.Control group fed basal diet, test group are that with the addition of 1.5% active Pichia anomala AR₂₀₁₆Dry powder Basal diet.

Body condition is close for selection, the gold of 40 week old determine laying duck 200, and test period is 90 days, is within first 5 days preliminary trial period, and rear 85 It is the positive examination phase.Random to be classified as 2 treatment groups, each 5 repetitions for the treatment of group are each to repeat 20.That is 1 control group, 1 A test group.Control group fed basal diet, test group are that with the addition of 2% Pichia anomala AR₂₀₁₆The basal diet of dry powder.

The results are shown in Table 6.

6 Pichia anomala AR of table₂₀₁₆Growth-promoting effect in laying hen and laying duck cultivation

Experimental subjects	Experiment process	Laying rate (g)	Egg size (g/ pieces)	Egg material ratio
					Laying hen	Control group	68.9	62.6	2.45
	Test group	74.1	64.2	2.31
					Laying duck	Control group	66.4	71.9	2.63
	Test group	72.5	75.4	2.42

Embodiment 11：

Application effect of the bacterium powder in Roche natural pond juvenile prawn cultivates.

Experiment select the hatching of same batch, Individual Size uniformly, physical health, body length is about 4.5cm, weight 1.76g left Right 200 tail of Roche natural pond juvenile prawn, is randomly divided into 2 treatment groups, each 5 repetitions for the treatment of group, each to repeat 20 tail shrimps, i.e., 1 Control group, 1 test group.Control group fed basal diet, the abnormal complete red ferment of activity that test group with the addition of 4.0% for feeding are done The basal diet of powder.Weigh and record initial weight, raise in 200L automatic inflating glass fibre buckets.Experimental period 55d.It is fixed during experiment When oxygenation；Dissolved oxygen 4.5-5.0g/L, in due course replacement water source.Water temperature range is at 22~28 DEG C；Water pH value is 7.6 or so.Often It is 08:00、17:00、22:00 bait throwing in, records daily ration, feeding quantity, and according to ingest situation and the water temperature change adjustment in due course of shrimp, day Daily ration, feeding quantity is the 6%~8% of weight, is fed close to being satiated with food, does not stay residual bait as far as possible, the results are shown in Table 7.

7 Pichia anomala AR of table₂₀₁₆Growth-promoting effect in Roche natural pond juvenile prawn cultivates

Experiment process	Specific growth rate (%/d)	Food conversion	Survival rate
				Control group	3.75	1.33	62%
Test group	3.89	1.24	70%

Embodiment 12：

Application effect of the solid-state fermentation content in laying hen and laying duck cultivation.

Selecting that body condition is close, the Roman egghen of 39 week old 200, test period is 90 days, is within first 5 days preliminary trial period, rear 85 It is the positive examination phase.Random to be classified as 2 treatment groups, each 5 repetitions for the treatment of group are each to repeat 20.That is 1 control group, 1 A test group.Control group fed basal diet, test group are that with the addition of 5.0% active Pichia anomala AR₂₀₁₆Solid state fermentation The basal diet of thing.

Body condition is close for selection, the gold of 40 week old determine laying duck 200, and test period is 90 days, is within first 5 days preliminary trial period, and rear 85 It is the positive examination phase.Random to be classified as 2 treatment groups, each 5 repetitions for the treatment of group are each to repeat 20.That is 1 control group, 2 A test group.Control group fed basal diet, test group are that with the addition of 6.0% active Pichia anomala AR₂₀₁₆Solid state fermentation The basal diet of thing.

Root Ju embodiment 10 and 12 is as a result, liquid state fermentation thing and solid-state fermentation content can improve the growth of laying hen and egg laying duck Performance, effect is suitable, but solid-state fermentation content fermentation is raw materials used cheap compared with liquid state fermentation thing for feed cost, therefore solid-state Fermentate feeding cost is lower.

The results are shown in Table 8.

8 Pichia anomala AR of table₂₀₁₆Growth-promoting effect in laying hen and laying duck cultivation

Experimental subjects	Experiment process	Laying rate (g)	Egg size (g/ pieces)	Egg material ratio
					Laying hen	Control group	64.9	61.5	2.54
	Test group	75.1	62.8	2.36
					Laying duck	Control group	63.8	72.1	2.65
	Test group	71.5	74.8	2.46

Claims

A kind of 1. Pichia anomala, it is characterised in that：The Pichia anomala is：Pichia anomala（Pichia anomala）AR₂₀₁₆；Deposit number is：CCTCC NO：M 2017594.
2. the liquid state fermentation culture medium of Pichia anomala described in claim 1, including：Glucose 3-5%, peptone 1-2%, ferment Female powder 0.5-1.5%, pH 6.0, remaining is water, is mass ratio above.
3. the solid-state fermentation culture medium described in claim 1, including：Wheat bran 15-30%, dregs of beans 6-8%, corn flour 1-3% are beautiful Rice ＆ peanut milk 3-5%, rice bran 2-4%, ammonium sulfate 0.5-1%, dipotassium hydrogen phosphate 0.03-0.08%, magnesium sulfate 0.08-0.12%, remaining is certainly Water, is mass ratio above.
4. application of the Pichia anomala in the sanguinariness factor is prepared described in claim 1.
5. application of the Pichia anomala described in claim 1 in bacteriostatic agent is prepared.
6. application of the Pichia anomala in livestock and poultry animal feed addictive is prepared described in claim 1.
7. application of the Pichia anomala in aquatic animal feed additive is prepared described in claim 1.
8. application according to claim 6, it is characterised in that：The Pichia anomala improves laying hen or egg at the same time The laying rate and egg material ratio of duck.
9. application according to claim 7, it is characterised in that：The Pichia anomala is promoting beasts, birds and aquatic products animal Growth.
10. application according to claim 5, the bacterium bag in the bacteriostatic agent includes：Dysentery Escherichia coli, white diarrhea sramana Salmonella, sensitive c-type Candida albicans, Actinobacillus pleuropneumoniae, rise yellow sarcine or staphylococcus aureus.