CN103642695A - Strain of Aspergillus oryzae and applications in preparation of feed additives through microbial fermentation - Google Patents

Abstract

The invention provides a new bacterial strain-Aspergillus oryzae 2013-DP1, and applications in preparation of feed additives through microbial fermentation. The strain is preserved in China Center for Type Culture Collection. The address is Wuhan University, Wuhan, China. The postcode is 430072. The preservation date is June 21th, 2013. The preservation number is CCTCC No: M2013276. Through fermentation and enzymatic hydrolysis conversion of low-value cellulose substances of straws and the like, the strain raises the protein content properly, raises nutritive values, can promote development of feed sources, raises the utilization rate of waste biological resources, and has obvious economic and ecological significances.

Description

A strain of Aspergillus oryzae and its application in the preparation of feed additives by microbial fermentation

(1) Technical field

The invention relates to a new bacterial strain obtained through screening and mutagenesis—Aspergillusoryzae (Aspergillusoryzae) 2013-DP1, and its application in the preparation of feed additives by microbial fermentation.

(2) Background technology

my country has a lot of people and little land. In recent years, with the rapid development of animal husbandry, the contradiction between humans and animals competing for food is quite prominent. my country has become a country with very tight feed resources, especially protein feed resources are very scarce. Since protein is one of the main nutritional components to meet the growth and development of livestock and poultry and improve nutrition and quality, and energy components constitute the main body of the feed, the protein content and quality of the feed determine the nutritional value of the feed. According to the best breeding technology, the amount of protein resources in my country's feed will be short of more than half. With the sustainable development of animal husbandry, protein feed continues to be in short supply and seriously restricts the further development of the breeding industry. At present, the main feed proteins in my country are fishmeal and soybean meal, etc., which are imported in large quantities every year. Due to the gradual deterioration of marine resources and overfishing by humans, the supply of fishmeal is becoming less and less, and the price continues to increase. According to the analysis of relevant experts from the Feed Research Institute of the Chinese Academy of Agricultural Sciences, by 2020, the gap between the supply and demand of protein feed in my country will be 48 million tons. Therefore, only relying on the existing conventional protein feed can not meet the needs of my country's feed industry, and it is urgent to develop more new protein feed sources.

Microorganisms have the advantages of many types, fast growth, and simple nutritional requirements. They can use a variety of waste organic matter to grow and reproduce and can be converted into organic nutrients that animals can use. For example, the use of microorganisms to ferment organic matter such as straw rich in cellulose can degrade the difficult-to-use macromolecular cellulose into usable small-molecular organic matter. Organic substances, and rich in amino acids, vitamins and so on. Therefore, the use of microorganisms to ferment organic matter is a very potential way to develop new protein feed additives.

Meals such as soybean meal are the by-products of extracting fatty substances from soybeans. Soybean meal has been successfully developed into a plant protein feed material that can partially replace fish meal. In recent years, soybean meal has been widely used in aquaculture. However, soybean meal contains a large number of anti-nutritional factors, such as soybean lectin, trypsin inhibitor, soybean oligosaccharides, phytic acid, gossypol oxalate, glucosinolates, tannins, etc. Soybean meal also contains antigenic proteins such as glycinin. The existence of these anti-nutritional factors will affect the health of animals by interfering with the digestion and absorption of nutrients by animals, disrupting normal metabolism and causing adverse physiological reactions, which will affect the utilization of soybean meal to a large extent. Studies have shown that after soybean meal is treated by microbial fermentation, anti-nutritional factors and antigenic proteins can be degraded to a certain extent by the protease system secreted by microbial growth, reducing the level of these undesirable substances in soybean meal, and significantly improving the nutritional quality of soybean meal. Efficient utilization of soybean meal.

In addition, my country's agricultural production, agricultural products and light industrial food processing will produce a large amount of organic matter rich in cellulose, protein and other substances, such as distiller's grains, straw, cotton meal, industrial waste organic matter, rice bran, etc. These organic matters are Good feed raw materials provide sufficient raw materials for the production of cell protein. As long as the microbial species that utilize these raw materials can be screened out, it is expected to develop protein feed additives. Take cellulose as an example. my country has abundant cellulose resources. The annual output of straw alone is about 570 million tons, but less than 10% of the amount is due to feed production. Due to the high content of crude fiber in straw feed and low protein and mineral content, The low digestibility and poor palatability of livestock and poultry limit its application. After microbial treatment and processing, its quality can be improved, including degrading macromolecular cellulose and other substances into small molecular substances that are easy to digest and absorb, increasing protein content, increasing nutritional value, and producing a large number of beneficial microorganisms at the same time, improving animal intestinal health and improving animal health. Quality of animal products. Therefore, using microbial fermentation technology to develop unconventional feed resources to produce protein feed additives can effectively alleviate the serious shortage of protein feed resources.

(3) Contents of the invention

The purpose of the present invention is to provide a new strain obtained through screening and mutagenesis—Aspergillus oryzae (Aspergillus oryzae) 2013-DP1, and its application in the preparation of feed additives by microbial fermentation. The fungus can use organic matter such as soybean meal, bran, and rice straw as a medium for growth, and the fermented organic matter can be used as a feed additive.

The technical scheme adopted in the present invention is:

Aspergillus oryzae (Aspergillus oryzae) 2013-DP1, deposited in China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, postcode: 430072, date of deposit: June 21, 2013, deposit number: CCTCC No: M2013276.

The bacterial strain of the present invention is initially screened under natural conditions to produce complex enzymes, including protease, cellulase, amylase and the like. In the process of long-term fermentation and cultivation of organic matter, it is continuously purified and rejuvenated for improvement. In addition, several rounds of ultraviolet mutation breeding were carried out on the strain, and the strain 2013-DP1 with improved cellulase and protease production ability was obtained through screening.

Aspergillus oryzae strain 2013-DP1 culture and propagation method:

Aspergillus oryzae can be cultured on PDA medium (potato, agar medium) or on Chase medium. It can be used for the growth and reproduction of Candida utilis KF1-1. The culture temperature is 30°C. Aspergillus oryzae grows vigorously on this medium, producing abundant hyphae and a large number of spores.

Preparation of PDA medium: 200 grams of potatoes (fresh potatoes are peeled, cut into pieces, boiled for 15 minutes, and then filtered to obtain juice), 20 grams of glucose, 15-20 grams of agar, 1000 mL of tap water, natural pH.

Chase medium preparation: NaNO ₃ , 2g; K ₂ HPO ₄ , 1g; KCl, 0.5g; MgSO ₄ , 0.5g; FeSO ₄ , 0.01g; sucrose, 30g; nature.

The above two media were sterilized at 121°C for 20 minutes.

Colony and morphological characteristics: Aspergillus oryzae colonies are loose in texture, initially white to light yellow, with the extension of culture time, the middle part of the colony begins to produce a large number of conidia, the front of the colony turns light greenish brown, and the back is colorless. There are fluffy hyphae on the surface of the colony, which are easy to stir up. The conidiophores grow on the subspherical apical capsule with a diameter of 150-300 μm. Conidia are oval or oval, with a diameter of 3.5-5 μm. When cultured in liquid shake flasks, mycelia tend to form balls and produce a large number of small mycelium balls.

Physiological and metabolic characteristics: Aspergillus oryzae strain 2013-DP1 can utilize common monosaccharides, such as glucose, galactose, fructose, etc. It can also ferment and utilize disaccharides, such as sucrose and maltose. Mixtures such as molasses are also good carbon sources for the growth of Aspergillus oryzae. It can also ferment and utilize organic substances containing a large amount of polysaccharides and proteins, such as straw, sugar residue, soybean meal, bran, corn flour, and sawdust, to transform and produce bacterial protein, feed additives, cellulase, amylase, etc.

The present invention also relates to the application of the Aspergillus oryzae 2013-DP1 in the preparation of feed additives by microbial fermentation.

Specifically, the application is: take one or more of soybean meal, bran, and straw, add water until the water content is 40-50% (w/w), and then sterilize at high temperature to make a fermentation medium. Inoculate Aspergillus oryzae 2013-DP1, and carry out aerobic fermentation for 48-64 hours. After the fermentation, the fermentation product is dried and pulverized to make a feed additive.

Usually, before the strains are fermented and cultivated, they generally need to be expanded by seeds. After the strains stored in ultra-low temperature preservation or freeze-dried powder are inoculated and activated on the PDA medium plate, they are inoculated into the seed shake flask for cultivation to obtain the seed liquid, and then inoculated Go to the fermentation shaker flask or fermenter for amplified culture of bacteria. After culturing a large number of bacteria, mix them with soybean meal, bran, straw powder and other organic substrates according to a certain proportion of the inoculation amount (generally 10% volume ratio) and then culture and ferment. The fermentation substrate contains an appropriate amount of water (40-50%). After a certain period of fermentation, microorganisms degrade and transform macromolecules such as polysaccharides and proteins. During the fermentation process, measures such as material turning, temperature control, and air permeability adjustment are taken to ensure the normal progress of fermentation. The fermented substrate is dried and pulverized to make a finished feed additive.

Aspergillus oryzae prepares organic matter such as fermented soybean meal to prepare the technological process of feed additive as follows:

Sterilization and aging treatment of medium materials - material-water ratio, strain inoculation - solid-state fermentation - material turning and ventilation - drying - crushing - finished product.

Specifically, the Aspergillus oryzae strain 2013-DP1 can be cultured and activated on PDA medium (containing 200 grams of potato, 20 grams of glucose, 15-20 grams of agar, 1000 ml of tap water, and natural pH per liter). The low-temperature-preserved strains were inoculated on PDA medium, placed in a 30°C incubator, and activated for 72 hours. Inoculate 3 to 5 single colonies of the activated strain into the seed shake flask (PDA medium, without agar), and prepare the seed solution on a shaking table with a rotation speed of 200 rpm and 30°C. After 24 hours of culturing in the seed shake flask, inoculate 10% of the inoculum into the fermentation shake flask or fermenter (medium as above) for amplified culture of bacteria. After 24 hours of cultivation, inoculate and ferment according to 10% of the weight of the material.

During the fermentation process, the material is turned over every 24 hours to allow sufficient ventilation and facilitate sufficient aerobic fermentation for microorganisms. At the same time, the temperature change of the fermentation material is monitored, and when the internal temperature of the material pile is too high (greater than 50°C), the material is turned over to cool down. When there is no heating equipment and fermentation is carried out under natural environmental conditions, if the ambient temperature is low, the fermentation time can be extended appropriately. During the fermentation process, it is also necessary to observe the changes in the condition of the fermented materials, including changes in color appearance and smell, and whether there is blackening, mildew, and agglomeration in the fermented products.

The protein feed additive rich in acid-soluble protein (small peptide) and other active peptides can be produced after the soybean meal is fermented by the bacterial strain of the invention. The bacterial strain secretes a large amount of highly active cellulase, and the bacterial strain can efficiently ferment straw and increase its protein content. Due to the large amount of abandoned agricultural and industrial by-products in my country, using the microbial strains in the present invention to develop new protein feed resources that can partially replace and supplement existing feed varieties and reduce costs has extremely important implications for the feed industry meaningful and highly feasible.

The beneficial effects of the present invention are mainly reflected in: in the present invention, an Aspergillus oryzae bacterial strain 2013-DP1 has been obtained through screening and breeding. Fermentative degradation and transformation of feed raw materials. Using this strain to ferment soybean meal can significantly increase the protein content, increase the crude protein content of raw soybean meal by more than 6%, and produce a large amount of soluble small peptides (acid-soluble protein content is more than 12%). In addition, the soybean meal fermented by the bacteria can effectively degrade the anti-nutritional factors and antigenic proteins in the soybean meal, significantly improve the nutritional quality of the soybean meal, improve the utilization efficiency of the soybean meal by animals, and improve animal health. The strain can ferment and enzymatically convert low-value cellulose substances such as rice straw, appropriately increase the protein content, improve its nutritional value, promote the development of feed sources, improve the utilization rate of waste biological resources, and have obvious economic benefits. , Ecological significance.

(4) Description of drawings

Fig. 1 is the colony morphology of Aspergillus oryzae 2013-DP1 on the PDA medium;

Fig. 2 is the cellulase activity identification of aspergillus oryzae fermented liquid crude enzyme liquid;

Figure 3 is the hydrolysis circle of Aspergillus oryzae on soybean meal medium.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Embodiment 1: Screening and selection of strains

Isolation of Aspergillus oryzae with high activity of cellulase and protease

Isolation of Aspergillus oryzae from discarded plant samples such as straw. Prepare PDA medium (200 grams of potatoes, 20 grams of glucose, 15-20 grams of agar, 1000 mL of tap water, natural pH, sterilized at 121 °C for 20 min). Take a sample that may contain Aspergillus oryzae, grind it, add 1 gram to 99 mL of water, shake and mix well for 10 minutes, then take 100 μL and spread it on a PDA plate. Placed at 25°C for 48 hours and observed the colonies, selected candidate Aspergillus oryzae strains, isolated and purified single spores, and preserved the strains for further experiments after further microscope observation and identification.

After multiple times of culture and mutation breeding, the screened strains were screened to obtain the strain 2013-DP1 with fermentation ability for soybean meal and so on. The cellulase and protease activities of the strain were identified as follows:

Identification of cellulase by Congo red chromogenic method. Congo red is a dye that can form red complexes with polysaccharides such as cellulose, but cannot react with hydrolyzed cellobiose and glucose. When Congo red is added to a medium containing cellulose Congo red will combine with cellulose to form Congo red-cellulose red complex. When the cellulose in the medium is decomposed, the Congo red-cellulose complex cannot be formed. Congo red, which is not firmly bound to the protein, will appear in the medium as a transparent circle centered on cellulose-degrading enzymes or bacteria.

Aspergillus oryzae was cultured in liquid culture solution (the above potato medium without agar, supplemented with 10g/L rice straw powder) and then centrifuged to remove mycelia, and the supernatant (crude enzyme solution) was placed on a petri dish containing cellulose substrate identified on the plate. Specifically: use an aqueous solution containing 0.5% CMC-Na substrate and 20g/L agar to make a solid petri dish, drop 10 μL of crude enzyme solution on the petri dish, cover it, and react in a 37°C incubator for 3 to 4 hours. Dye with 0.1% Congo red staining solution for 30min, rinse with water, and decolorize with 1M NaCl solution for 15min. Obvious yellow transparent circles appear, indicating that the fermentation broth of Aspergillus oryzae has cellulase activity. Congo red stained plate is shown in Figure 2.

Identification of protease activity of Aspergillus oryzae strain 2013-DP1. The hydrolysis circle of Aspergillus oryzae on casein plate and soybean meal plate can be used to judge whether protease is produced and its activity is high or low.

Casein medium plate, beef extract: 3g, casein: 10g, agar: 20g, Nacl: 5g, water: 1000mL, disodium hydrogen phosphate: 2g, final pH=7.4. Dissolve 2g of casein in 50mL of buffer solution (0.1mol/l disodium hydrogen phosphate-disodium hydrogen phosphate pH=8) and slightly heat to dissolve. Soybean meal medium plate, with 1000mL medium as the standard, add about 20g of soybean meal powder, then boil for 1 hour, filter the soybean meal powder residue, add 20g of agar powder after it cools down, mix and stir, sterilize at 121℃ for 15 minutes, wait Pour onto plate after cooling. After the strain was inoculated on the above medium and cultured for 48 hours, the size of the hydrolysis circle was observed, and it was found that there was a hydrolysis circle at the edge of the colony (Figure 3).

The above results show that the Aspergillus oryzae strain 2013-DP1 of the present invention has a strong ability to produce protease and cellulase.

Example 2:

Preparation of PDA medium: 200 grams of potatoes, 20 grams of glucose, 15-20 grams of agar, 1000 ml of tap water, natural pH, sterilized at 121°C for 20 minutes.

The cryopreserved Aspergillus oryzae strain CCTCC No: M2013276 was inoculated on PDA medium, placed in a 30°C incubator, and activated for 72 hours.

Inoculate 3 to 5 single colonies of the activated strain into seed shake flasks (PDA medium, without agar), and culture them on a shaker at 200 rpm at 30°C for 24 hours to obtain seed liquid.

Take soybean meal, add water to 45% water content, sterilize at 121°C for 20 minutes to make fermentation medium, inoculate Aspergillus oryzae seed liquid at a volume ratio of 10%, and carry out aerobic fermentation for 64 hours (turn over once every 24 hours), After the fermentation is over, take a little fermentation product and dry it until the water content is about 10%, crush it and pass it through a 90-mesh sieve to test the content of each index.

The crude protein content of the raw soybean meal is 45.93%, and the acid-soluble protein content is 4.58%.

Among the fermentation products: water 11.00%, crude protein 53.63%, acid-soluble protein 12.93%, volatile basic nitrogen 35.23mg/100g.

Example 3:

Aspergillus oryzae strain CCTCC No: the activation and seed culture of M2013276 are the same as in Example 2.

Take a mixture of soybean meal: bran: rice straw powder with a mass ratio of 1:1:1, add water to a water content of 40%, sterilize at 121°C for 20 minutes to make a fermentation medium, and inoculate the Aspergillus oryzae seed solution at a volume ratio of 10%. Carry out aerobic fermentation for 48 hours (turn the material once every 24 hours). After the fermentation is over, take a little fermentation product and dry it until the water content is about 10%.

Crude protein content of each raw material: soybean meal, 45.93%; bran, 14.57%; straw powder, 2.89%.

Among the fermentation products: water 11.00%, crude protein 25.63%, acid-soluble protein 4.89%.

Claims (3)

1. A strain of Aspergillus oryzae (Aspergillus oryzae) 2013-DP1, deposited in the China Center for Type Culture Collection, address: China, Wuhan, Wuhan University, postcode: 430072, date of deposit: June 21, 2013, deposit number: CCTCC No: M2013276. 2. the application of aspergillus oryzae 2013-DP1 in microbial fermentation preparation feed additive as claimed in claim 1. 3. The application according to claim 2, characterized in that the application is: take one or more of soybean meal, bran, and straw, add water until the water content is 40-50%, and then sterilize at high temperature Prepare a fermentation medium, inoculate Aspergillus oryzae 2013-DP1, and carry out aerobic fermentation for 48-64 hours. After the fermentation, the fermentation product is dried and pulverized to make a feed additive.

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