CN112094754A - Aspergillus oryzae and application thereof in small-tailed han sheep feed - Google Patents

Aspergillus oryzae and application thereof in small-tailed han sheep feed Download PDF

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CN112094754A
CN112094754A CN202010982837.6A CN202010982837A CN112094754A CN 112094754 A CN112094754 A CN 112094754A CN 202010982837 A CN202010982837 A CN 202010982837A CN 112094754 A CN112094754 A CN 112094754A
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谭竹毅
张佳
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Qingdao Probex Biotechnology Co ltd
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Abstract

The invention is suitable for the technical field of microbial breeding, and provides an Aspergillus oryzae, wherein the Aspergillus oryzae is Aspergillus oryzae PLBS013(Saccharomyces cerevisiae PLBS013), is preserved in China general microbiological culture Collection center (CGMCC) for 12-17 th in 2018, and has the preservation number of CGMCC NO. 16970; the invention also provides application of the Aspergillus oryzae in the feed for small tailed han sheep. Therefore, the Aspergillus oryzae PLBS013 can improve the digestion performance of the rumen of the small tailed han sheep, improve the degradation rate of fibers in the forage, effectively regulate the physiological function of the rumen of the ruminant, improve the absorption and utilization rate of nutrient substances in the feed for the ruminant and promote the growth of the ruminant.

Description

Aspergillus oryzae and application thereof in small-tailed han sheep feed
Technical Field
The invention relates to the technical field of microbial breeding, in particular to an aspergillus oryzae strain and application thereof in feed for small-tailed han sheep.
Background
Since the 80 s in the 20 th century, the Ministry of agriculture in China actively encourages and supports the mutton sheep industry, so that the mutton sheep breeding in agricultural and pastoral areas in China is in great development momentum, the predicament that food resources are in short supply and the demand of livestock products is increased in China is solved, and a foundation is laid for the stable development of animal husbandry. However, the mutton sheep production industry in China has many problems, and in order to pursue the production performance of mutton sheep and increase the economic benefit, breeding enterprises select additives such as antibiotics and the like to be added into the feed of the mutton sheep so as to improve the utilization rate of the feed by the mutton sheep, reduce the feed cost, improve the slaughtering performance of the sheep and improve the economic output value; however, the long-term use of antibiotics causes hormone residues in mutton, which results in unsafe mutton.
Aspergillus oryzae is a common strain of Aspergillus fungi, and is an aerobic fungus. The aspergillus oryzae can effectively regulate the physiological function of the rumen of the ruminant, stabilize the pH value of the rumen, promote the growth of fiber decomposing bacteria, improve the digestibility of fiber substances in daily ration and enhance the fermentation function of the rumen. The Aspergillus oryzae culture obtained by the multi-step fermentation method can effectively promote the growth of fungi on the basis of effectively promoting the growth of rumen bacteria, and has the advantage of more remarkably improving the utilization rate of feed compared with yeast products which can only promote the growth of bacteria.
However, the application research of aspergillus oryzae in the feed of small-tailed han sheep is only reported, and the addition amount and the effect of the aspergillus oryzae are different.
In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.
Disclosure of Invention
Aiming at the defects, the invention aims to provide an Aspergillus oryzae strain and application thereof in feed for small-tailed han sheep, and the addition of the Aspergillus oryzae PLBS013 with an appropriate level in the daily ration of the small-tailed han sheep can improve the digestion performance of the rumen of the small-tailed han sheep, improve the degradation rate of fibers in the forage, effectively regulate the physiological function of the rumen of a ruminant, improve the absorption and utilization rate of nutrients in the feed by the ruminant and promote the growth of the ruminant.
In order to achieve the aim, the invention provides an aspergillus oryzae, wherein the aspergillus oryzae is aspergillus oryzae PLBS013(Saccharomyces cerevisiae PLBS013), is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 and 17 days, and has a preservation number of CGMCC No. 16970.
Aspergillus oryzae according to the invention, said Aspergillus oryzae being obtained from rice.
According to Aspergillus oryzae of the present invention, the optimal temperature for Aspergillus oryzae is 50 ℃.
According to Aspergillus oryzae of the present invention, the Aspergillus oryzae has an optimum pH of 7.0.
According to the Aspergillus oryzae, the addition amount of the Aspergillus oryzae in the basic ration of small tailed han sheep is 150-620 mg/kg.
According to the Aspergillus oryzae, the addition amount of the Aspergillus oryzae in the basic ration of small tailed han sheep is 325-471 mg/kg.
The invention also provides an Aspergillus oryzae screening method, which comprises the following steps:
step one, soaking rice
Soaking rice in normal saline to obtain soaking stock solution;
step two separation and purification
Diluting the soaking stock solution to prepare first spore suspension with different dilution concentrations;
sequentially taking the first spore suspension with different dilution concentrations, coating the first spore suspension on a PDA culture medium, and inversely culturing the PDA culture medium in a constant-temperature incubator;
respectively selecting single colony spores growing in a PDA culture medium, inoculating the single colony spores on the inclined plane, and inverting the single colony spores in a constant temperature incubator for continuous culture to obtain a single bacterial strain;
step three primary screen
Carrying out gradient dilution on the single strain to prepare second spore suspensions with different concentrations, sequentially taking the second spore suspensions with different concentrations, coating the second spore suspensions on a purple oil assimilation plate, culturing in a constant temperature incubator, selecting bacterial colonies which generate yellow color change rings, and carrying out streak preservation to obtain a primary screening strain;
step four double sifting
And inoculating the primary screened strain to a purple oil assimilation plate, and placing the plate in a constant-temperature incubator for inverted culture to obtain a secondary screened strain.
The aspergillus oryzae disclosed by the invention is applied to the feed for small tailed han sheep.
The invention aims to provide an Aspergillus oryzae strain and application thereof in feed for small tailed han sheep, and provides the optimum action temperature, the optimum pH value and the heat resistance of the Aspergillus oryzae PLBS013 and the influence of metal ions on the Aspergillus oryzae PLBS; the addition of the proper level of Aspergillus oryzae PLBS013 to the small tailed Han sheep ration can improve the digestion performance of the rumen of the small tailed Han sheep, improve the degradation rate of fibers in the forage, effectively regulate the physiological function of the rumen of the ruminant, improve the absorption and utilization rate of nutrients in the feed by the ruminant and promote the growth of the ruminant.
Drawings
FIG. 1 is a colony morphology of Aspergillus oryzae PLBS013 according to the invention;
FIG. 2 is a graph of the growth of Aspergillus oryzae PLBS013 according to the invention;
FIG. 3 is a graph showing the enzyme activity of Aspergillus oryzae PLBS013 according to the invention at different reaction temperatures;
FIG. 4 is a graph of the thermal stability of Aspergillus oryzae PLBS013 according to the invention at different reaction temperatures;
FIG. 5 is a graph showing the enzymatic activity of Aspergillus oryzae PLBS013 according to the invention under different pH conditions.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides an aspergillus oryzae, the Latin literature name: aspergillus oryzae; the Aspergillus oryzae is Aspergillus oryzae PLBS013(Aspergillus oryzae PLBS013), which is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Ceh 1 Siro. in the morning area of Beijing city) in 17 months in 2018, and the preservation number is CGMCC NO. 16970.
The invention discloses a method for screening Aspergillus oryzae PLBS013, which comprises the following steps:
step one, soaking rice
Taking 10g of rice, adding 50mL of normal saline, and soaking for 30min under an oscillator to obtain a soaking stock solution.
Step two separation and purification
Diluting the soaking stock solution with sterile distilled water to 10% by gradient-1～10-8Preparing first spore suspension with different dilution concentrations;
take 10 in turn-3～10-8Coating 200 mu L of first spore suspension with concentration on a PDA culture medium, inversely culturing the PDA culture medium for 3-4 days in a constant temperature incubator at 25 ℃, and observing and recording the growth condition of colonies;
respectively picking single colony spores growing in a PDA culture medium, inoculating the single colony spores on a slant, inverting the slant into a constant-temperature incubator at 25 ℃, and continuously culturing to obtain a single strain.
Step three primary screen
Diluting single strain to 10 with sterile distilled water-1～10-8Preparing into second spore suspension with different concentrations, sequentially collecting 10-3～10-8And coating 200 mu L of second spore suspension with concentration on a purple grease assimilation plate, culturing in a constant temperature incubator at 30 ℃ for 3-4 d, selecting bacterial colonies which generate yellow discoloration circles, and performing streak preservation to obtain a primary screening strain.
Step four double sifting
And (3) inoculating the primary screened strain to a purple grease assimilation plate, and placing the plate in a constant-temperature incubator at 30 ℃ for inverted culture for 5-7 d to obtain a secondary screened strain.
After the re-screened strains are screened out by the method, the strains are identified, and the invention provides an identification method of the strains, which comprises the following steps:
morphological identification
The cells were observed by an optical microscope and a stereoscope, and the size and morphology of the cells, the colony texture, the colony color, the colony surface characteristics, the colony edge, the reproductive characteristics, and the like were observed to perform morphological evaluation.
The form of the re-screened strain is as follows: the strain is white at the initial culture stage, then turns to beige, the central bulge is green, the edge is beige, and hyphae are easy to pick up and loose in texture. (see FIG. 1)
Identification of Gene sequences
Performing molecular biological identification on the rescreened strain, extracting the genome DNA of the rescreened strain, and performing amplification according to the general primer of the ITS4/5r RNA gene region of the fungus: its5 has a nucleotide sequence of 5'-GGAAGTAAAAGTCGTAACAAGG-3' and a nucleotide sequence of Its4 of 5'-TCCTCCGCTTATTGATATGC-3', and PCR amplification is carried out to sequence the PCR product by Biotechnology engineering (Shanghai) Ltd. The 5 'end and the 3' end are two ends of the gene, and after the DNA template is opened, the primer is combined with the template and is copied from the 5 'end to the 3' end.
Through gene sequence identification, the re-screened strain is classified as Aspergillus oryzae and named Aspergillus oryzae PLBS 013.
To verify the strain properties of Aspergillus oryzae PLBS013 according to the invention, strain property tests were performed according to the invention, including growth curve, optimal temperature for reaction, thermostability, pH stability of the strain, and various metal ions, NH+4Influence on the protease activity of Aspergillus oryzae.
Growth curve of the Strain
Inoculating the strain into a PDA culture medium, carrying out shake culture and fermentation at a constant temperature of 25-35 ℃, taking fermentation liquor at an interval of 2h in the early stage, taking the fermentation liquor at an interval of 4h, 6h and 12h in the later stage, filtering the fermentation liquor taken each time by using filter cloth, collecting mycelium pellets, drying at 60-70 ℃ to constant weight, then weighing, and drawing the variation trend of a strain growth curve (see figure 2).
According to the growth curve, the bacterial quantity is slowly increased after the bacterial strain is cultured for 2-36 h, the bacterial quantity is exponentially increased after 36-54 h, and the bacterial quantity reaches a stable state after 54-72 h.
Optimum reaction temperature of strain
The protease activity of the Aspergillus oryzae PLBS013 was measured at different temperatures (0 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃), the highest enzyme activity was defined as 100%, and the relative enzyme activities of the proteases at different temperatures were calculated. (see FIG. 3)
As can be seen from FIG. 3, the optimum reaction temperature for the protease of Aspergillus oryzae PLBS013 was 50 ℃ at which the catalytic activity of the enzyme was the highest; the activity of the enzyme is inhibited by low temperature at 30 ℃, and the activity of the enzyme can not be fully exerted; above 60 ℃, the enzyme is denatured by an excessively high temperature, and the activity of the enzyme is decreased by inactivation.
Thermostability of the Strain
Respectively placing the enzyme solution of Aspergillus oryzae PLBS013 under different temperature conditions (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃) for different time (0min, 20min, 40min, 60min, 80min, 100min, 120min), immediately cooling in an ice bath at 0 ℃, then measuring the enzyme activity at 40 ℃, defining the highest enzyme activity as 100%, and respectively calculating the ratio of the residual enzyme activity and the highest enzyme activity of the protease under different temperature conditions. (see FIG. 4)
As can be seen in FIG. 4, the protease of Aspergillus oryzae PLBS013 was relatively stable at 20 ℃; after the treatment for 40min at 30 ℃, the enzyme activity is still over 60 percent; after treatment for 20min at 40 ℃, the enzyme activity is kept above 50%; after treatment for 20min at 50 ℃, the enzyme activity is kept above 45%; after the treatment at 60 ℃ for 20min, the enzyme activity is kept above 40%. Indicating that the protease of Aspergillus oryzae PLBS013 is thermolabile.
pH stability of the Strain
Aspergillus oryzae PLBS013 was reacted under different pH conditions (pH3, 4, 5, 6, 7, 8, 9, 10) and the protease activity of Aspergillus oryzae PLBS013 was determined to obtain the pH of Aspergillus oryzae PLBS 013.
Diluting the enzyme solution of the Aspergillus oryzae PLBS013 in buffer solutions (pH 3.0-10.0) with different pH values respectively, keeping the temperature at 40 ℃ for 2 hours, adjusting the pH value of the system to the optimal pH value of the enzyme reaction, measuring the residual enzyme activity, defining the highest enzyme activity as 100%, and calculating the ratio of the residual enzyme activity to the highest enzyme activity of the protease of the Aspergillus oryzae PLBS013 under the conditions of different pH values respectively. (see FIG. 5)
As shown in FIG. 5, when the pH of the protease of Aspergillus oryzae PLBS013 is 3.0-6.0, the residual enzyme activity gradually increased; when the pH value is 6.0-8.0, the stability is relatively high; when the pH value is 7.0, the enzyme activity is highest; when the pH value is 8.0-10.0, the enzyme activity is slowly reduced. Indicating that the protease of Aspergillus oryzae PLBS013 is suitable for storage under neutral conditions.
Various metal ions, NH+4Effect on protease Activity of Aspergillus oryzae PLBS013
Adding different metal ions (Li) into a reaction system of protease of Aspergillus oryzae PLBS013 and a substrate thereof+、Na+、Mg2+、Al3+、K+、Ca2+、Fe2+、Fe3+、Mn2+、Zn2+、Cu2+、Ag+、 NH+4) The relative enzyme activity of the Aspergillus oryzae PLBS013 protease in each reaction system after the addition of the metal ions was determined separately, with the enzyme activity in the reaction system without the addition of the metal ions defined as 100%.
The results show that Ca2+、Mn2+、Zn2+Has activating effect on protease of Aspergillus oryzae PLBS 013; NH (NH)+4、 K+、Ag+The influence on the enzyme activity is small; li+、Na+、Mg2+、Al3+、Fe2+、Fe3+、Cu2+Has inhibitory effect on the protease.
According to the strain property test, the most suitable action temperature of Aspergillus oryzae PLBS013 is 50 ℃, the most suitable pH value is 7.0, the enzyme stability at 20 ℃ is good, the Aspergillus oryzae is not heat-resistant, and the Aspergillus oryzae is not suitable for long-time storage at room temperature. Ca2+、 Mn2+、Zn2+Has activating effect on protease of Aspergillus oryzae PLBS 013; NH (NH)+4、K+、Ag+The influence on the enzyme activity is small; li+、Na+、Mg2+、Al3+、Fe2+、Fe3+、Cu2+Has inhibitory effect on the protease.
After the strain properties of Aspergillus oryzae PLBS013 were identified, the invention applied Aspergillus oryzae PLBS013 to the Douglas lamb feed. In order to verify the application value of the Aspergillus oryzae PLBS013 in the small-tailed Han sheep feed, the invention further provides an application method of the Aspergillus oryzae in the small-tailed Han sheep feed.
First, experimental animal and raising management
Healthy small-tailed Han sheep rams without diseases and with similar birth date and weight are selected, the small-tailed Han sheep are all provided with permanent rumen fistula, and are randomly divided into 4 treatment groups (a control group and three test groups) according to the principle of similar weight. After 14 days of pre-feeding, the control group was fed a basal diet and the test group was supplemented with Aspergillus oryzae PLBS013 on the basal diet. The feeding test period is 7 days.
Thoroughly cleaning and disinfecting sheep sheepcotes before testing, and expelling parasites, performing immunization injection and punching ear numbers on all small-tailed han sheep. The feeding environmental conditions such as illumination, ventilation and the like are consistent. Fed 2 times daily (7:30, 16: 30). Raising in single column, and feeding freely.
The basic ration is prepared according to the formula of the small tailed han sheep farm according to the nutritional requirements of NRC (2012).
Second, feed formula
The Aspergillus oryzae PLBS013 is added into basic ration of small tailed han sheep, and the addition amount is 150-620 mg/kg.
Third, measuring the index
Measuring pH value of rumen fluid and ammonia Nitrogen (NH) obtained by repeatedly extracting rigid plastic tube in rumen for several times3-N), Volatile Fatty Acid (VFA) concentration and degradation rate of Dry Matter (DM), degradation rate of Neutral Detergent Fiber (NDF), degradation rate of Acid Detergent Fiber (ADF) in nylon bag oven dried sample.
In order to further verify the application value of the Aspergillus oryzae PLBS013 in the feed for the small-tailed han sheep, the small-tailed han sheep is fed by the application method, the following embodiments are arranged, and the relevant indexes of the small-tailed han sheep in each embodiment are measured. The method for feeding and managing the small tailed han sheep in each example was the same, and only the amount of the aspergillus oryzae PLBS013 added in each example was changed, and therefore, the same contents in each example are not shown. (the amount of the Aspergillus oryzae PLBS013 added in each example is shown in Table one; the results of the related indices of the small tailed Han sheep in each example are shown in Table two)
Comparative example
The Aspergillus oryzae PLBS013 is added into a conventional feed for small tailed han sheep, and the addition amount is 0.
After the completion of the culture, the pH value of rumen fluid of the small tailed han sheep is measured to be 6.68, the ammonia nitrogen concentration of the rumen fluid is measured to be 17.49mg/100mL, and the total volatile fatty acid concentration of the rumen fluid is measured to be 38.90 mmol/L; the degradation rate of dry substances in rumen is 29.26%, the degradation rate of neutral detergent fiber is 35.60%, and the degradation rate of acidic detergent fiber is 31.26%.
Example 1
The Aspergillus oryzae PLBS013 disclosed by the invention is added into a conventional feed for small tailed han sheep, wherein the addition amount is 325 mg/kg.
After the completion of the culture, the pH value of rumen fluid of the small tailed han sheep is measured to be 6.66, the ammonia nitrogen concentration of the rumen fluid is measured to be 18.55mg/100mL, and the total volatile fatty acid concentration of the rumen fluid is measured to be 50.62 mmol/L; the degradation rate of dry substances in rumen is 39.67%, the degradation rate of neutral detergent fiber is 45.90%, and the degradation rate of acidic detergent fiber is 41.66%.
Example 2
The Aspergillus oryzae PLBS013 disclosed by the invention is added into a conventional feed for small tailed han sheep in an amount of 396 mg/kg.
After the completion of the culture, the pH value of rumen fluid of the small tailed han sheep is measured to be 6.72, the ammonia nitrogen concentration of the rumen fluid is measured to be 18.94mg/100mL, and the total volatile fatty acid concentration of the rumen fluid is measured to be 52.59 mmol/L; the degradation rate of dry substances in rumen is 42.33%, the degradation rate of neutral detergent fiber is 47.51%, and the degradation rate of acidic detergent fiber is 40.33%.
Example 3
The Aspergillus oryzae PLBS013 provided by the invention is added into a conventional feed for small tailed han sheep, and the addition amount is 471 mg/kg.
After the completion of the culture, the pH value of rumen fluid of the small tailed han sheep is measured to be 6.59, the ammonia nitrogen concentration of the rumen fluid is 17.63mg/100mL, and the total volatile fatty acid concentration of the rumen fluid is 52.44 mmol/L; the degradation rate of dry substances in rumen is 40.69%, the degradation rate of neutral detergent fiber is 46.45%, and the degradation rate of acidic detergent fiber is 42.69%.
Because the implementation processes of the embodiments are the same, the specific processes of other embodiments are not listed, and only the three embodiments with better effects are listed.
In each example, the amount of Aspergillus oryzae PLBS013 added is mg/kg
Figure RE-GDA0002754706790000091
Determination of indexes of Diandi small-tailed Han sheep
Figure RE-GDA0002754706790000101
From the above examples, it can be seen that, by adding the aspergillus oryzae PLBS013 of the present invention to the basal diet of the small tailed han sheep, the pH value of the rumen fluid and the concentration of ammonia nitrogen in the rumen fluid of the small tailed han sheep are not affected, the ammonia nitrogen is a main source of nitrogen required for the growth of rumen microorganisms, the rumen microorganisms grow on the basis of the ammonia nitrogen, and the growth of the microorganisms needs to have a proper level, which indicates that the aspergillus oryzae PLBS013 can maintain the normal pH value and the concentration of ammonia nitrogen in the rumen fluid of the small tailed han sheep, and ensure the normal growth of the rumen microorganisms.
The addition of the Aspergillus oryzae PLBS013 provided by the invention to the basic ration of the small-tailed han sheep can improve the concentration of total volatile fatty acids in the rumen fluid of the small-tailed han sheep, so that the Aspergillus oryzae PLBS013 can promote the growth of fiber decomposing bacteria, improve the digestibility of fiber substances in the ration, enhance the rumen fermentation function and further improve the utilization rate of the feed of the small-tailed han sheep.
The addition of the Aspergillus oryzae PLBS013 provided by the invention into the basic ration of the small tailed han sheep can improve the degradation rate of dry substances, neutral detergent fibers and acidic detergent fibers in the rumen of the small tailed han sheep, so that the Aspergillus oryzae PLBS013 can stimulate the growth of cellulolytic bacteria, improve the yield of cellulase, improve the degradation rate of fibers in forage grass, effectively regulate the physiological function of the rumen of a ruminant and improve the absorption of nutrients in the feed by the ruminant.
In addition, it can be seen from the above examples that when the addition amount of the aspergillus oryzae PLBS013 of the present invention to the basal diet of small tailed han sheep was 325 to 471mg/kg, the rumen digestibility of small tailed han sheep was the best, and therefore, the optimal addition amount of the aspergillus oryzae PLBS013 to the basal diet of small tailed han sheep was 325 to 471 mg/kg.
In conclusion, the invention provides the optimum action temperature, the optimum pH value and the heat resistance of the Aspergillus oryzae PLBS013 and the influence of metal ions on the optimum action temperature, the optimum pH value and the heat resistance; the addition of the proper level of Aspergillus oryzae PLBS013 to the small tailed Han sheep ration can improve the digestion performance of the rumen of the small tailed Han sheep, improve the degradation rate of fibers in the forage, effectively regulate the physiological function of the rumen of the ruminant, improve the absorption and utilization rate of nutrients in the feed by the ruminant and promote the growth of the ruminant.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
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<120> Aspergillus oryzae strain and application thereof in small-tailed Han sheep feed
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Claims (8)

1. An Aspergillus oryzae strain, wherein the Aspergillus oryzae is Aspergillus oryzae PLBS013(Saccharomyces cerevisiae PLBS013), is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 and 17 months, and has a preservation number of CGMCC No. 16970.
2. The Aspergillus oryzae of claim 1, wherein the Aspergillus oryzae is derived from rice.
3. The Aspergillus oryzae of claim 1, wherein the Aspergillus oryzae has an optimum working temperature of 50 ℃.
4. The Aspergillus oryzae of claim 1, wherein the Aspergillus oryzae has a pH optimum of 7.0.
5. The Aspergillus oryzae of claim 1, wherein the Aspergillus oryzae is added in a basal diet of small tailed Han sheep in an amount of 150-620 mg/kg.
6. The Aspergillus oryzae of claim 5, wherein the Aspergillus oryzae is added in an amount of 325-471 mg/kg in a basal diet of small tailed Han sheep.
7. A method of screening Aspergillus oryzae of claim 1, comprising the steps of:
step one, soaking rice
Soaking rice in normal saline to obtain soaking stock solution;
step two separation and purification
Diluting the soaking stock solution to prepare first spore suspension with different dilution concentrations;
sequentially taking the first spore suspension with different dilution concentrations, coating the first spore suspension on a PDA culture medium, and inversely culturing the PDA culture medium in a constant-temperature incubator;
respectively selecting single colony spores growing in a PDA culture medium, inoculating the single colony spores on the inclined plane, and inverting the single colony spores in a constant temperature incubator for continuous culture to obtain a single bacterial strain;
step three primary screen
Carrying out gradient dilution on the single strain to prepare second spore suspensions with different concentrations, sequentially taking the second spore suspensions with different concentrations, coating the second spore suspensions on a purple oil assimilation plate, culturing in a constant temperature incubator, selecting bacterial colonies which generate yellow color change rings, and carrying out streak preservation to obtain a primary screening strain;
step four double sifting
And inoculating the primary screened strain to a purple oil assimilation plate, and placing the plate in a constant-temperature incubator for inverted culture to obtain a secondary screened strain.
8. Use of aspergillus oryzae according to claim 1 in a feed for small tailed han sheep.
CN202010982837.6A 2020-09-18 2020-09-18 Aspergillus oryzae and application thereof in small-tailed han sheep feed Pending CN112094754A (en)

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CN106071143A (en) * 2016-06-21 2016-11-09 山东宝来利来生物工程股份有限公司 A kind of granular pattern microbial ecological agent improving ruminant production performance and preparation method thereof
CN110432391A (en) * 2019-08-15 2019-11-12 湖北省农业科学院畜牧兽医研究所 A kind of feed addictive and preparation method of the metabolism of regulation ruminant tumor gastric

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丁洪涛等: "米曲霉对奶牛体外瘤胃发酵的影响", 《中国畜牧杂志》 *
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Application publication date: 20201218