CN114480140B - A kind of Neurospora intertype and its application - Google Patents

Abstract

The invention discloses a alternaria alternate and application thereof, and relates to the technical field of microorganisms. The Neurospora syndecans of the present invention is designated as Neurospora syndecans (Neurospora intermedia) JY-05211, which was deposited at the microorganism strain collection, guangdong province, at the address: the collection number is GDMCC No.62130 from the university of first, guangzhou, city, no. 100, no. 59, no. 5, guangdong national academy of sciences of China. The alternaria alternate disclosed by the invention can produce various digestive enzymes, can degrade substances such as indigestible macromolecular proteins and cellulose in a fermentation substrate, can inhibit the growth of other miscellaneous bacteria in the fermentation process, and can reduce the pollution of harmful microorganisms in fermentation materials.

Description

A kind of Neurospora intertype and its application

technical field

The invention relates to the technical field of microorganisms, in particular to a neurospora intertype and application thereof.

Background technique

Neurospora, also known as Neurospora, was first discovered on baked bread and can produce a large amount of orange-red pigment, so it is also called "red bread mold". Neurospora belongs to Ascomycota, Ascomycetes, Faecaliceta, Faecaliaceae, and is a general term for a group of fungi with vertical stripes on the ascospore wall. Neurospora has a strong ability to produce enzymes, can produce protease, amylase, cellulase, phytase, laccase and other enzymes, and can also produce biologically active substances such as carotenoids, tomato red, melanin, etc. In addition, Neurospora Bacteria itself is rich in protein, amino acids and other nutrients. Neurospora is easy to spread and grows rapidly. It is a competitive miscellaneous bacteria in the production of edible fungi, but it will not cause any infection or disease to humans, animals and plants. It is approved by the U.S. Food and Drug Administration (Food and Drug Administration FDA) ) approved edible safe organisms. Neurospora is widely used in the fields of food, agriculture, scientific research, and medicine: the "red fungus tofu head" commonly eaten in Hakka areas in eastern Guangdong and western Fujian is made from fermented bean curd residue with Neurospora, which has the function of increasing freshness and flavor , Promote the effect of digestion. In terms of agricultural applications, studies have found that Neurospora fermented corn stalks can significantly increase the content of true protein in the straw, reduce the proportion of crude fiber, and significantly increase the feed value of corn stalks. In the field of scientific research, Neurospora is often used as a model organism for the study of the genetic, biochemical and molecular biological characteristics of eukaryotic microorganisms.

The shortage of feed raw materials is one of the important reasons that restrict the development of my country's aquaculture industry and cause the high price of my country's animal husbandry and aquatic products. At the same time, a large number of by-products in the food and agricultural product processing industry have not been fully utilized, resulting in serious waste of resources and environmental pollution. Improving its feed value through microbial fermentation is currently the main technology to promote the feed utilization of food and agricultural product processing by-products. The commonly used fermentation strains such as Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and other fermentation strains have a relatively slow proliferation rate, and are often polluted by other toxin-producing molds during the fermentation process, and these types of bacteria often produce unpleasant odors during the fermentation process And affect the appearance of the product.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a neurospora intertype and its application. Neurospora intertype of the present invention can produce multiple digestive enzymes, can degrade indigestible macromolecular protein, cellulose and other substances in the fermentation substrate, and at the same time, can inhibit the growth of other miscellaneous bacteria in the fermentation process, reduce the Harmful microbial contamination in fermented materials.

In order to achieve the above purpose, the technical solution adopted by the present invention is: a Neurospora intermedia, named as Neurospora intermedia JY-05211, which has been preserved in Guangdong Microbial Bacteria on December 15, 2021. Species Preservation Center, address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No.62130. The Neurospora intertype JY-05211 screened by the present invention can produce protease, cellulase, amylase and carotenoid, and has broad application prospects and economic value.

As a preferred embodiment of the Neurospora intertype in the present invention, the sequence of the Neurospora intertype is shown in SEQ ID NO.1.

As a preferred embodiment of the Neurospora intertype in the present invention, the Neurospora intertype is isolated from Rhodobacter bean curd head. The present invention uses YPD medium to isolate dominant strains from red fungus tofu heads collected from Renju Town, Pingyuan County, and confirms that the strains are intertype through colony and cell morphology observation, physiological and biochemical tests, and ITS conservative sequence amplification and sequencing. Neurospora.

The present invention also provides the application of the Neurospora intertype in the production of protease, carotenoid or cellulase. The inventors of the present application found through tests that the Neurospora intertype of the present invention can produce protease, cellulase, amylase and carotenoid, and has the functions of degrading macromolecular protein, cellulose and antioxidation.

The present invention also provides a culture of Neurospora intertype, which is obtained by fermentation of the Neurospora intertype.

The present invention also provides the preparation method of described Neurospora intertype culture, comprising the following steps:

(1) Preparation of YPD medium: dissolve 20-30g of glucose, 20-25g of peptone, 10-15g of yeast extract and 20-25g of agar powder in 1000ml of water, sterilize at 121°C for 20min, and pour the plates to obtain the YPD Culture medium;

(2) Preparation of spores of Neurospora intertype: Streak inoculation of the Neurospora intertype into the YPD medium, culture at 28°C for 3 to 5 days, collect the spores with an inoculation shovel and rinse with sterile water, Obtain the Neurospora intertype spores;

(3) Inoculate the above-mentioned Neurospora intertype spores into a fermentation medium composed of dry materials and water, and ferment at 25-37° C. for 4-6 days to obtain the Neurospora intertype-type culture.

As a preferred embodiment of the preparation method of the intertype Neurospora culture of the present invention, the dry material is composed of corn cob flour, corn flour, bran, ammonium sulfate and calcium hydroxide; the corn cob flour, The weight ratio of corn flour, bran, ammonium sulfate and calcium hydroxide is corn cob flour: corn flour: bran: ammonium sulfate: calcium hydroxide=6～8:0.5:1～2.5:0.5:0.1～0.5; The weight ratio of dry material to water in the fermentation base is dry material:water=1:0.5-2.5. The inventors of the present application have found through a large number of experiments that by using the above-mentioned dry material and controlling the weight ratio of each component in the dry material, the spore content in the finally prepared Neurospora intertype culture is relatively high.

As a preferred embodiment of the preparation method of the Neurospora intertype culture of the present invention, the inoculation amount of the spores is 1-1.5×10 ⁷ spores/g.

The present invention also provides the application of the Neurospora intertype or the culture of the Neurospora intertype in preparing diet for broilers.

The invention also provides a diet for broilers, which contains the culture of Neurospora intertype.

The inventors of the present application have found through a large number of experiments that adding Neurospora intertype cultures to the diet of broilers can promote the growth of broilers.

Beneficial effects of the present invention: the present invention provides a new Neurospora intertype, which can produce protease, cellulase, amylase and carotenoid. Applying the Neurospora intertype of the present invention to the preparation of feed can improve the digestibility of feed materials; the Neurospora intertype of the present invention has anti-oxidation effect and can improve the quality and appearance of animal products; Neurospora intertype can proliferate rapidly, competitively inhibit the growth of other molds, reduce the growth of toxin-producing molds and the accumulation of toxins in the fermentation process, and the prepared feed has higher safety.

Description of drawings

Fig. 1 is the appearance diagram of the red fungus bean curd head.

Figure 2 is a colony diagram of Neurospora intertype JY-05211.

Fig. 3 is a mycelium morphology diagram of Neurospora intertype JY-05211.

Fig. 4 is a spore morphology diagram of Neurospora intertype JY-05211.

Fig. 5 is the electrophoresis diagram of the PCR product of Neurospora intertype JY-05211.

Fig. 6 is a phylogenetic tree diagram of Neurospora intertype JY-05211 based on ITS sequence.

Detailed ways

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Preservation Information of Example 1 Neurospora intertype

The present invention provides a Neurospora intermedia, named as Neurospora intermedia JY-05211, which was preserved in Guangdong Microbial Culture Collection Center on December 15, 2021 , Address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCCNo.62130.

Screening of embodiment 2 Neurospora intertype and strain identification

In this example, YPD medium was used to isolate the dominant strain from the red fungus bean curd head collected from Renju Town, Pingyuan County, and the basic biological characteristics of the strain were preliminarily determined through colony and cell morphology observation, physiological and biochemical tests, and analyzed by PCR. The ITS conserved sequence of the strain was amplified and sequenced, the sequences were compared on NCBI, and the phylogenetic tree was constructed to determine the taxonomic status of the strain. The specific process includes the following steps:

(1) Isolation and purification of Neurospora intertype: preparation of YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour the plate out and wait Use; take 10g of crushed red fungus bean curd head, add 100ml of sterile water to soak and filter with 4 layers of gauze, centrifuge the filtrate at 5000rpm for 10min in a centrifuge, remove the supernatant and keep the bottom spores, add 20ml of sterile water to suspend the spores. The spore liquid was inoculated on the YPD plate, cultured at 28°C for 2-3 days, and the growth of mycelia and spores was observed. Pick a few spores from the plate and inoculate them into a new YPD plate, incubate at 28°C for 2-3 days, and pick a purified and pollution-free plate for strain identification.

(2) Type identification of Neurospora intertype: a. observe the hyphae and spore morphology of the isolated and purified bacterial strain; sucrose, glucose, lactose, starch and xylose) liquid culture medium, cultured at 30°C for 2 d after 150 rpm shaker, observed the color change of the culture medium and carried out the measurement of sugar fermentation ability; Sucrose, maltose, galactose, ethanol, citric acid and mannitol) and nitrogen source (ammonium sulfate, potassium nitrate) liquid culture medium, after 3 days of constant temperature cultivation at 28 ℃, observe the growth of mycelia and carry out carbon and nitrogen source assimilation ability c. ITS sequencing identification: Use the forward primer 5'-TCCGTAGGTGAACCTGCGG-3' and the reverse primer 5'-TCCTCCGCTTATTGATATGC-3' to analyze the ITS sequence of the isolated and purified strain, and the ITS sequence on the NCBI website Blast comparison was carried out, and the phylogenetic tree of JY-05211 strain was constructed by Neighbor-Joining method.

The test results are shown in Figures 1-6. From Figures 1 to 4, it can be seen that the strain conforms to the morphological characteristics of Neurospora. The colony is rich in a large number of loose reticular mycelium, which is white cotton flocculent. The mycelium has septum, branching, and multinucleation; the strain asexually reproduces to form conidia , generally branched chains are formed at the top of the aerial hyphae. After the formation of conidia, the mycelium becomes orange fluffy, and the spores are clustered, mostly oval, and orange in color; after maturity, the conidia gather into clumps and cover on the colony. Physiological and biochemical analysis results found that the strain can ferment various substrates such as glucose, sucrose, lactose, xylose and starch, and can assimilate various carbon sources such as glucose, sucrose, maltose, galactose, ethanol, citric acid and mannitol, And can assimilate various inorganic nitrogen sources such as potassium nitrate and ammonium sulfate. It was consistent with the physiological and biochemical characteristics of Neurospora, and was initially identified as Neurospora. The nucleotide sequence of the strain obtained by ITS sequence analysis is shown in SEQ ID NO.1. From the electrophoresis of the PCR product of the JY-05211 strain in Figure 5, it can be seen that the length of the amplified fragment is about 500 bp, which is consistent with the expected length. It can be seen from Figure 6 that the strain has the closest genetic relationship with Neurospora intertype. Based on the results of morphological, physiological, biochemical and molecular biological tests, the strain was identified as Neurospora intertype.

Protease, carotenoid, cellulase characteristic assay of embodiment 3 Neurospora intertype JY-05211

1. Determination of protease production characteristics of Neurospora intertype JY-05211, comprising the following steps:

(1) Prepare YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour it into a plate for later use;

(2) Preparation of spore liquid: Streak-inoculate Neurospora intertype onto a YPD plate, culture it statically at 28°C for 3-5 days, collect spores with an inoculation shovel and rinse with sterile water, and inoculate the spore liquid with sterile water. Dilute to 1×10 ⁸ cells/ml for use;

(3) Preparation of YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour the plate for later use;

(4) Solid fermentation: Dry the freshly purchased bean dregs at low temperature, control the water content below 50%, mix the bean dregs, bran, starch and yeast powder according to the ratio in Table 1, and mix the mixed dry materials, water and spore liquid, placed at 28°C for 4 to 6 days of fermentation, that is, the fermentation ended.

(5) Determination of the protease activity of the fermentation product of Neurospora intertype: the fermentation product was poured into a petri dish, dried at 40° C., and crushed for later use. Weigh 1g of the above pulverized materials into a conical flask, add 50ml of phosphate buffer solution, shake at 40°C for 1.5h, take it out, centrifuge at 4°C, 8000rpm for 10min, take the supernatant for the determination of protease activity, determine The method refers to GB/T23527-2009.

The measurement results are shown in Table 1.

Table 1

2. Determination of carotenoid-producing characteristics of Neurospora intertype JY-05211, comprising the following steps:

(1) Prepare YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour it into a plate for later use;

(2) Preparation of spore liquid: Streak-inoculate Neurospora intertype onto a YPD plate, culture it statically at 28°C for 3-5 days, collect spores with an inoculation shovel and rinse with sterile water, and inoculate the spore liquid with sterile water. Dilute to 1×10 ⁸ cells/ml for use;

(3) Solid fermentation: Stir the dry material (the ratio of corncob powder to bean dregs is 4-6:6-4), mix evenly with water and liquid spore liquid, and the spore concentration in the material is 1-1.5×10 ⁷ per g, ferment at 28°C for 4-6 days, collect fungal spores containing carotenoids, and extract crude carotenoids.

(4) Determination of carotenoid content: after the fermentation, the spores were collected to measure the carotenoid content, and the collected spores were placed in a petri dish, dried at 60° C. for later use. Accurately weigh 0.1g of dry weight spores into a 10ml centrifuge tube, add 1-2.5ml of 1-5mol/l hydrochloric acid to each centrifuge tube, leaching at room temperature for 50min, shaking for 30min, boiling water bath for 2-10min, cooling rapidly, Centrifuge at 8000r/min for 5min, discard the supernatant, wash the precipitate twice with distilled water, add 2ml of acetone, shake for 10-50min, and then centrifuge at 8000r/min for 5min, the supernatant is carotenoid extraction liquid. Using an ultraviolet spectrophotometer, set the measurement wavelength to 475nm, and use acetone as a blank control to measure the absorbance of the extract.

The measurement results are shown in Table 2. It can be seen from Table 2 that the carotenoid yield in the spores of Neurospora intertype in this example is 605ug/g-703ug/g.

Table 2

3. Determination of the characteristics of cellulase produced by Neurospora intertype JY-05211, comprising the following steps:

(1) Prepare YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour it into a plate for later use;

(2) Preparation of spore liquid: Streak-inoculate Neurospora intertype onto a YPD plate, culture it statically at 28°C for 3-5 days, collect spores with an inoculation shovel and rinse with sterile water, and inoculate the spore liquid with sterile water. Dilute to 1×10 ⁸ cells/ml for use;

(3) Solid fermentation: Dry the bran at high temperature, control the water content below 13%, then add 1-5% ammonium sulfate and 0.1-0.5% ferrous sulfate respectively, the material: water is 1:1-1:2 , the inoculum amount of the spore liquid is 1-1.5×10 ⁷ /g, and the fermentation is completed at 28°C for 4-6 days.

(4) Determination of cellulase activity of fermented bran: after drying and pulverizing the fermented culture medium, accurately weigh 1 g of the sample, add 40 ml of citrate buffer solution, oscillate and shake well, place in the dark at 4°C for 24 hours and shake well, Centrifuge at 3000r/min for 3min, take the supernatant to measure the cellulase activity, and refer to GB/T 23881-2009 for the determination method.

The measurement results are shown in Table 3. It can be seen from Table 3 that the fermented bran cellulase activity prepared in this example is 500U/g-631U/g.

table 3

Application of embodiment 4 intertype Neurospora culture in broiler breeding

The preparation method of the Neurospora intertype culture of the embodiment of the present invention comprises the following steps:

(1) Prepare YPD medium: mix 25g of glucose, 22.5g of peptone, 12.5g of yeast extract, 22.5g of agar powder and 1000ml of water, sterilize at 121°C for 20min, pour it into a plate for later use;

(2) Preparation of Neurospora intertype spores: Streak-inoculate the Neurospora intertype in Example 2 into the YPD medium, culture it statically at 28°C for 3 to 5 days, collect the spores with an inoculation shovel and rinse with sterile water Wash to obtain the Neurospora intertype spores;

(3) The above-mentioned Neurospora intertype spores were inoculated in a fermentation medium composed of dry materials and water. The components and contents of the dry materials were shown in Table 4, and fermented at 30°C for 5 days to obtain the intertype Type Neurospora culture.

Table 4

After the fermentation, measure the spore content of Neurospora intertype, take 100ml Tween solution with a graduated cylinder and pour it into the bran bottle gently along the bottle wall in a fume hood, fully stir and shake, and filter with 4 layers of gauze. Dispense the liquid into 45ml centrifuge tubes and centrifuge at 5000rpm for 10min at room temperature, discard the supernatant and leave the spore pellet at the bottom. Then use 20ml Tween solution to resuspend the spore pellet as the initial bacterial solution for spore counting. After drawing 100ul counting bacteria solution, dilute the bacteria solution 10 times with 900ul, and then repeat the above operation to dilute to 100 times with the 10 times diluted bacteria solution. The 100-fold diluted bacterial solution was added to the hemocytometer to count the number of spores. The results showed that the spore content of the Neurospora intertype culture prepared in Group 1 in this example was 7.87× ¹⁰⁹ /g, and that in the Neurospora intertype culture prepared by Group 2 was 5.83× ¹⁰⁹ /g. g, the content of spores in the culture of Neurospora intertype prepared in group 3 is 6.55×10 ⁹ pcs/g.

The culture of Neurospora intertype prepared by the above group 1 was added to the broiler basal diet to carry out the broiler breeding test. The specific test method was as follows: select 84 1-day-old Sanhuang chickens with similar body weight, and randomly divide them into 2 groups. 6 replicates, 7 mice per replicate. Among them, the control group was fed with basic diet, and the experimental group was fed with diet supplemented with 1% Neurospora intertype culture, caged in the house, kept warm with heat lamp, free to eat and drink, and observed the feed intake of the chickens every day , Drinking water and health conditions, record material consumption, etc. Determination of growth performance: At 8:00 in the morning on the 7th, 14th, 21d, 28d, 35d and 42d of the test, the 6 broilers in each group were weighed on an empty stomach (the feed was cut off for 8h and the water was cut off for 2h before weighing), and the calculation Net weight gain and feed to meat ratio. The results are shown in Table 5.

table 5

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), no letters or the same letters indicate no significant differences (P>0.05).

The result shows that at the end of the 42nd day test, compared with the control group, the ante-mortem live weight of the three yellow chickens in the Neurospora intertype culture group increased by 9.36% (P＜0.05), and the average daily gain increased by 9.70% (P＜0.05). <0.05), the average daily feed intake increased by 9.17% (P<0.05). It can be seen that the culture of Neurospora intertype is helpful to increase the feed intake and promote the growth of Sanhuang chicken.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that Modifications or equivalent replacements are made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Jiaying College

<120> A kind of Neurospora intertype and its application

<130> 2022.01.13

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<170> PatentIn version 3.3

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Claims (8)

1. Neurospora crassa (L.) KuntzeNeurospora intermedia) Characterized in that the alternaria is named as alternariaNeurospora intermedia) JY-05211, deposited at the Cantonese microbiological strain collection center, app. 12, 15, 2021: microorganism of Guangzhou City, first, china, no. 100, no. 59, 5, guangdong province academy of sciencesThe institute has a deposit number of GDMCC No.62130.

2. Use of a Neurospora crassa according to claim 1 for the production of proteases, carotenoids or cellulases.

3. A culture of Neurospora crassa according to claim 1, wherein the culture is obtained by fermentation of Neurospora crassa.

4. A method for preparing a culture of alternaria alternate according to claim 3, comprising the steps of:

(1) Preparing YPD medium: dissolving 20-30 g of glucose, 20-25 g of peptone, 10-15 g of yeast extract and 20-25 g of agar powder in 1000ml of water, sterilizing at 121 ℃ for 20min, and pouring into a plate to obtain the YPD culture medium;

(2) Preparation of the alternaria alternate spores: inoculating the alternaria alternate of claim 1 to YPD medium, standing at 28 ℃ for 3-5 days, collecting spores by an inoculating shovel, and flushing with sterile water to obtain the alternaria alternate spores;

(3) Inoculating the alternaria alternate spores into a fermentation medium consisting of dry materials and water, and fermenting for 4-6 days at 25-37 ℃ to obtain the alternaria alternate culture.

5. A method of preparing a culture of neurospora of claim 4, wherein the dry material consists of corncob meal, corn flour, bran, ammonium sulfate and calcium hydroxide; the weight ratio of the corncob powder to the corn flour to the wheat bran to the ammonium sulfate to the calcium hydroxide=0.5 to 1 to 2.5 to 0.5 to 0.1 to 0.5; the weight ratio of the dry materials to the water in the fermentation medium is that the dry materials are: water=1, 0.5 to 2.5.

6. A process for producing a culture of Neurospora as claimed in claim 4, wherein the inoculation amount of the spores is 1 to 1.5X10 ⁷ Each/g.

7. Use of a alternaria alternate according to claim 1 or a alternaria alternate culture according to claim 3 for the preparation of a broiler diet.

8. A broiler feed comprising the culture of neurospora crassa of claim 3.