CN103444988B - Aspergillus oryzae degrading straw produces the method for protein feed - Google Patents

Abstract

Aspergillus oryzae degrading straw of the present invention produces the method for protein feed, relates to bioengineering field.Relate to and utilize stalk for primary raw material, fermented by aspergillus oryzae (Aspergillus oryzae), produce the method for protein feed, its technological process of production is that stalk is through pretreating process such as pulverizing, prewet, aspergillus oryzae is cultivated through liquid submerged culture, first order seed, is mixed carry out solid state fermentation with stalk, obtains the protein feed of fermented stalk.This zymotechnique is easy, to environment, also can not can not cause waste to resource.Adopt the dietary protein level 15 ~ 30% that the method is obtained, Lignin degradation rate is 34 ~ 76%, feed yield 45 ~ 60%.Feed, due to effect lignin degrading, the cellulose of aspergillus oryzae, thus improves the utilization rate of stalk.

Description

Method for producing protein feed by aspergillus oryzae degrading straw

technical field

The present invention relates to the field of bioengineering, and in particular refers to a process for producing protein feed by degrading lignin and cellulose in the straw without high temperature and high pressure treatment by Aspergillus oryzae.

Background technique

my country is a large agricultural country, with an annual output of more than 600 million tons of crop straw, except for a small amount used in the textile and papermaking industries, but most of it is dumped into the environment in the form of accumulation and incineration, which wastes resources and causes great harm to the environment .

Straw contains more than half of the energy accumulated by plant photosynthesis. It is rich in raw sugar, crude protein and other ingredients that are beneficial to livestock and poultry. As a non-competitive feed resource, as long as it can be processed and prepared reasonably, it can be used to feed livestock and poultry. Poultry, especially for feeding ruminant livestock such as cattle and sheep, may become a high-quality feed resource. At the same time, my country is a big country with a serious shortage of feed (especially protein feed), and a large amount of fish meal needs to be imported every year to make up for the shortage of protein feed. It is particularly important to be able to convert straw fiber into protein feed that can be used by animals and has high nutritional value to solve the serious shortage of feed demand.

Plant straw is mainly composed of lignin and cellulose. Lignin is a kind of natural polymer with aromatic properties, which is composed of phenol and non-phenol structural units connected by ether bonds and carbon-carbon bonds. Its diversity in component types and linkage types means that its The heterogeneity and irregularity of the structure also determine the complexity and specificity of the biodegradation of lignin, and after it is covalently bonded with hemicellulose in the plant cell wall, it embeds the cellulose molecules Among them, a strong natural barrier is formed, which makes it difficult for ordinary microorganisms to enter and decompose cellulose. Therefore, the degradation of lignin is also a key factor for the effective use of straw resources.

Currently, the main methods for degrading straw typically include five methods: steam explosion pretreatment, sulfuric acid pretreatment, sodium hydroxide pretreatment, ammonia water pretreatment, and biological pretreatment. Steam explosion is to use high-pressure steam to infiltrate the interior of the fiber and release it from the closed space in the form of air flow. The fiber undergoes a certain mechanical fracture, and most of the hemicellulose and a small amount of lignin and cellulose in the raw material are degraded and dissolved. The composition of the method is relatively high, large-scale equipment is required, and lignin needs to be treated; the acid treatment method uses sulfuric acid to hydrolyze glycosidic bonds, ester bonds, etc., so that the network structure of corn stalk fibers is loose, but this method consumes a large amount of organic acid or Inorganic acid and lignin still need to be further processed and are not suitable for large-scale industrial production. Alkali treatment and ammonia water treatment use lignin to dissolve in alkaline solution, and there are also environmental pollution problems like acid treatment. This is due to the fact that these methods generally have problems such as difficult control of reaction conditions, high reaction temperature, long treatment time, large space occupation, unsatisfactory treatment effect, and inability to remove lignocellulose.

Aspergillus oryzae can secrete a variety of enzymes such as protease, amylase, lipase, pectinase, phytase, glucoamylase, etc., so the application field of this fungus in modern industry continues to expand. Tejomyee S. B. et al. isolated Aspergillus oryzae from soil contaminated with monocrotophos, and studied its biodegradation effect on the pesticide monocrotophos. sex. Truong Q.T. et al. studied the treatment of cassava starch processing wastewater containing high-concentration suspensions with Aspergillus oryzae, and improved its degradation efficiency of wastewater by changing a variety of factors that affect the growth of Aspergillus oryzae, and its degradation rate can be as high as 90%. . Xiang J.M. et al. isolated Aspergillus oryzae capable of degrading nicotine from tobacco leaves, and studied the degradation pathway of nicotine using resting cell analysis of Aspergillus oryzae. Through the analysis of the intermediate products in the degradation process, a new way of using fungi to degrade nicotine was finally determined. Parshetti G.K. et al studied the biodegradation of reactive blue-25 by Aspergillus oryzae NCIM-1146. Studies have found that the decolorization of reactive blue is absorbed by fungi and then degraded, and it has faster absorption and decolorization under shaking culture conditions than static culture. Addition of glucose enhanced the degradation of reactive blue by Aspergillus oryzae. This shows that Aspergillus oryzae has the ability to degrade organic matter that is difficult to degrade.

Contents of the invention

After in-depth research, the present inventor has explored a process of using straw as the main raw material to degrade lignin and cellulose in the straw through solid culture of Aspergillus oryzae to produce protein feed. This process is different from the previous method in that the strain used is the rice koji that can be directly fed to animals as a feed-grade microbial additive recognized by the U.S. Food and Drug Administration (FDA), the American Feed Association (AAFCO), and the Ministry of Agriculture of my country. The mold species uses straw as the main raw material and obtains protein feed through solid-state fermentation.

The purpose of the present invention is to provide a process for producing protein feed using Aspergillus oryzae as the strain and carrying out solid-state fermentation with stalks as the raw material, including taking Aspergillus oryzae as the starting strain, carrying out test tube expansion culture, liquid shake flask culture, first-class seed Cultivate and solid ferment culture, obtain the protein feed of degraded straw.

The bacterial classification used in the present invention is any bacterial classification of Aspergillus oryzae, such as China Common Microorganism Culture Collection Management Center (CCGMC), China Agricultural Microbiology Culture Collection Management Center (ACCC), China Industrial Microbiology Culture Collection Management Center (CICC) ), China Medical Microbial Culture Collection Center (CMCC), National Veterinary Microbiological Culture Collection Center (CVCC) and Antibiotic Culture Collection Management Center, etc.

The raw material used in the present invention is any kind of straw, such as corn straw, rice straw, wheat straw, sorghum straw, soybean straw, cotton straw, etc., which contain lignin. The straw is first crushed and passed through 20-100 mesh screen.

In the fermentation process of the present invention, its test tube expansion medium is potato sucrose medium.

In the fermentation process of the present invention, its liquid shake flask medium and primary seed medium are both (g/L): glucose 5-30, ammonium tartrate 0.1-5, benzyl alcohol 0.2-3, magnesium sulfate 0.2- 1. Tween 80 0.5-10, potassium dihydrogen phosphate 2-9, phthalic acid 3-18, pH 5-8, sterilized at 120-140°C for 20-40 minutes;

The shake flask culture process conditions described therein are: inoculate a ring of Aspergillus oryzae test tube slant spores in a conical flask containing 16-48% (volume ratio) medium, at a speed of 150 rpm, 25-35°C, Cultivate for 24-72 hours;

The first-level seed cultivation process described therein is to inoculate the shake flask culture seed liquid into the first-level seed medium at an inoculum amount of 1-20% (volume ratio), at a temperature of 25-35°C, and a stirring speed of 50-200 RPM, ventilation rate 0.2-2:1 volume/volume/minute (ventilation gas volume/fermentation broth volume/minute), culture for 18-72 hours;

In the solid fermentation culture described in the present invention, its solid fermentation medium is composed of: 10 kilograms of straw, 20-60 kilograms of water, 0.5-3 kilograms of glucose, 0.05-0.2 kilogram of urea; 5-15 grams of copper sulfate, ferrous sulfate 5-15 grams, 5-20 grams of manganese sulfate, the components of the medium can be enlarged in the same proportion;

The solid fermentation culture fermentation process described therein is: according to 2-20% (seed liquid volume/fermentation medium weight), the thickness of the compost is 0.1-0.4 meters, the width of the material is 0.5-2 meters, the length is not limited, and the ventilation rate is 0.3～1.5:1 (gas volume/weight of fermentation medium)/per minute, the culture time is 3 days to 23 days, and the material is turned 1 or 2 times in the middle.

In the fermentation process of the present invention, before carrying out shake flask culture, at first the Aspergillus oryzae strain is inserted in the potato sucrose culture medium of new configuration and cultivated, culture temperature 25～35 ℃, culture time 48～144 hours; Store at 4-10°C for later use.

The straw fermented product obtained by the present invention contains a large amount of yellow Aspergillus oryzae spores, the color of the fermented product is beige to brown, has a wine aroma, the lignin degradation rate is 34-76%, the protein content is 15-40%, and the feed yield is 45-40%. 60%.

According to the fermentation and cultivation process of the present invention, combined with basic knowledge in the field, secondary, tertiary or even quaternary seed tanks can be added according to production needs, and the production scale of solid-state fermentation can be further expanded for industrialized production. The medium used in the secondary, tertiary and even quaternary seed tanks is the same as the shake flask medium and the primary seed medium, the inoculation amount is 5-20%, and the cultivation conditions are the same as the primary seed cultivation.

The advantage of adopting this process is that it adopts solid-state fermentation and does not undergo high-temperature and high-pressure treatment, so that it will not pollute the environment and waste resources.

In order to further illustrate the materials and processes involved in the technical solution of the present invention, the following examples are given, but these examples do not limit the scope of the present invention in any form.

Description of drawings

Fig. 1 is the process flow chart of the method for producing protein feed by aspergillus oryzae degrading stalks of the present invention.

Detailed ways

The protein determination method adopted according to this process is the Coomassie Brilliant Blue kit production method produced by Nanjing Jiancheng Bioengineering Company. Accurately weigh 1 gram of protein feed, dissolve it in 50mL of water, shake at room temperature for 2 hours, filter with suction, and dilute the filtrate to 50mL, which is the sample solution for protein determination. The assay operation steps were carried out according to the kit instructions. The protein content is calculated according to the following formula:

According to this process, the following method is used for the determination of lignin: Weigh 100 mg of the sample and place it in a 15 mL reaction flask, add 3 mL of 72% H ₂ SO ₄ , hydrolyze for 3 hours at 25 ° C, and keep shaking, transfer the hydrolyzed product into the Erlenmeyer flask, add 190 mL of supernatant Pure H ₂ O was reacted at 121°C for 1 hour, cooled, and filtered through a sand core crucible with constant weight (W1). Calculate the acid-insoluble lignin content (W2-W1)/W. Fill the filtrate to 500mL, and measure the absorbance of the filtrate at a wavelength of 205nm. When the absorbance (A) of the sample solution is between 0.2 and 0.7, the acid-soluble lignin content ρ (mg/g) is calculated according to formula (1). Each sample was repeated 6 times, and the average value was taken.

In the formula, D is the dilution factor of the sample solution.

Example 1

1. Production of test tube slant strains

Insert a ring of Aspergillus oryzae into the newly configured potato sucrose medium ("Industrial Microbiology Experimental Technology Manual" edited by Zhuge Jian and Wang Zhengxiang, 1994, page 367), at 26°C, and cultivate for 50 hours ; Store at 4°C for later use.

2. Production of cultured strains in liquid shake flasks

Accurately weigh 5 grams of glucose, 0.1 grams of ammonium tartrate, 0.2 grams of benzyl alcohol, 0.2 grams of magnesium sulfate, 0.5 grams of Tween 80, 2 grams of potassium dihydrogen phosphate, 3 grams of phthalic acid buffer, 1000 mL of water, pH 5 , divided into 250mL triangular flasks, 50 grams per bottle, 20 bottles in total, sterilized at 120°C for 40 minutes; minutes, cultivated for 72 hours;

3. Production of first-level liquid strains

Accurately weigh 50 grams of glucose, 1 gram of ammonium tartrate, 2 grams of benzyl alcohol, 2 grams of magnesium sulfate, 5 grams of Tween 80, 20 grams of potassium dihydrogen phosphate, 30 grams of phthalic acid, 10L of water, and put them in 15L of seeds In the tank, sterilize at 120°C for 20 minutes; after sterilization and cooling, add 20% of the inoculum to the above liquid shake flask strains, at a temperature of 25°C, a stirring speed of 50 rpm, and an air flow of 0.2:1 volume/volume /per minute (ventilation gas volume/fermentation liquid volume/per minute), cultivated for 72 hours;

4. Solid fermented protein feed

Rice straw is crushed and passed through a 20-mesh sieve. Weigh 750 kg of straw powder, 1500 kg of water, 37.5 kg of glucose, 3.75 kg of urea; 375 grams of copper sulfate, 375 grams of ferrous sulfate, and 375 grams of manganese sulfate. Add 20% of the inoculum to the above-mentioned first-level seed culture solution, mix evenly, and make a stockpile with a thickness of 0.1 m, a width of 0.5 m, and a length of 37.5 m. Control the temperature at 25°C for 23 days, and turn the stock 2 in the middle. Second, after the cultivation, the fermented product is dried and crushed to be protein feed.

The obtained straw fermented product contains a large amount of yellow Aspergillus oryzae spores, and the fermented product is orange in color and has a wine aroma. The lignin degradation rate is 42.2%, the protein content is 16.7%, and the feed yield is 49.1%.

Example 2

1. The production of bacteria on the inclined plane of the test tube

Insert a ring of Aspergillus oryzae into the newly configured potato sucrose medium ("Industrial Microbiology Experimental Technology Manual" edited by Zhuge Jian and Wang Zhengxiang, 1994, page 367), at 30°C, and cultivate for 100 hours ; Store at 7°C for later use.

2. Production of cultured strains in liquid shake flasks

Accurately weigh 150 grams of glucose, 250 grams of ammonium tartrate, 15 grams of benzyl alcohol, 0.4 grams of magnesium sulfate, 4 grams of Tween 80, 5 grams of potassium dihydrogen phosphate, 20 grams of phthalic acid buffer, 10 L of water, pH 6, Divide into 250mL triangular flasks, 100 grams per bottle, 100 bottles in total, sterilize at 130°C for 30 minutes; after cooling, put a ring of Aspergillus oryzae strains stored at 7°C in each bottle, at 30°C, 150 rpm , cultivated for 50 hours;

3. Production of first-level liquid strains

Accurately weigh 2000 grams of glucose, 200 grams of ammonium tartrate, 150 grams of benzyl alcohol, 60 grams of magnesium sulfate, 60 grams of Tween 80, 400 grams of potassium dihydrogen phosphate, 1000 grams of phthalic acid, 100 L of water, and put them in 130 L of seeds In the tank, sterilize at 130°C for 30 minutes; after cooling, add the strains in the above-mentioned liquid shake flask at a volume ratio of 10%, at a temperature of 30°C, a stirring speed of 125 rpm, and an aeration rate of 1:1 volume/volume /per minute (aeration gas volume/fermentation broth volume/minute), cultivated for 50 hours;

4. Solid fermented protein feed

Corn stalks are pulverized and passed through a 60-mesh sieve. Weigh 1,000 kg of the straw powder, 3,000 kg of water, 150 kg of glucose, and 10 kg of urea; 600 grams of copper sulfate, 600 grams of ferrous sulfate, and 1,000 grams of manganese sulfate; Add 10% (volume/weight) of the above-mentioned first-level seed culture solution, mix evenly, and make a stockpile with a thickness of 0.2 meters, a width of 1 meter and a length of 225 meters. Control the temperature at 30°C for 15 days, and turn over halfway. Feed 2 times. After the cultivation, the fermented product is dried and crushed to become protein feed.

The obtained straw fermented product contains a large amount of yellow Aspergillus oryzae spores, and the fermented product is yellowish brown and has a wine aroma. The lignin degradation rate is 58.4%, the protein content is 31.2%, and the feed yield rate is 59.2%.

Example 3

1. The production of bacteria on the inclined plane of the test tube

Insert a ring of Aspergillus oryzae into the newly configured potato sucrose medium ("Industrial Microbiology Experimental Technology Manual" edited by Zhuge Jian and Wang Zhengxiang, 1994, page 367), at 35°C, and cultivate for 144 hours ; Store at 10°C for later use.

2. Production of cultured strains in liquid shake flasks

Accurately weigh 300 grams of glucose, 50 grams of ammonium tartrate, 30 grams of benzyl alcohol, 10 grams of magnesium sulfate, 100 grams of Tween 80, 90 grams of potassium dihydrogen phosphate, 180 grams of phthalic acid buffer, 10 L of water, pH7.5 , divided into 250mL triangular flasks, 120 mL per bottle, 80 bottles in total, sterilized at 140°C for 20 minutes; minutes, cultivated for 72 hours;

3. Production of first-level liquid strains

Accurately weigh 1,500 grams of glucose, 250 grams of ammonium tartrate, 150 grams of benzyl alcohol, 50 grams of magnesium sulfate, 500 grams of Tween 80, 450 grams of potassium dihydrogen phosphate, 900 grams of phthalic acid buffer, 50L of water, and put it in a 70L In the first-class seed tank, sterilize at 140°C for 40 minutes; after sterilization and cooling, insert the above-mentioned strains into the liquid shake flask at a temperature of 35°C with a stirring speed of 200 rpm and a ventilation volume of 2: 1 volume/volume/minute (aeration gas volume/fermentation broth volume/minute), cultivated for 72 hours;

4. Production of secondary liquid strains

Accurately weigh 15,000 grams of glucose, 2,500 grams of ammonium tartrate, 1,500 grams of benzyl alcohol, 500 grams of magnesium sulfate, 5,000 grams of Tween 80, 4,500 grams of potassium dihydrogen phosphate, 9,000 grams of phthalic acid buffer, 500 L of water, and put it in a 700 L container. In the first-class seed tank, sterilize at 140°C for 40 minutes; after sterilization and cooling, add the above-mentioned first-class liquid bacteria at a ratio of 5%, at a temperature of 35°C, a stirring speed of 200 rpm, and an air flow of 2:1 Volume/volume/minute (aeration gas volume/fermentation broth volume/minute), cultivated for 18 hours;

5. Solid fermented protein feed

Wheat straw is crushed and passed through a 100-mesh sieve. Weigh 3,500 kg of straw powder, 21,000 kg of water, 525 kg of glucose, 52.5 kg of urea; 5,000 grams of copper sulfate, 5,000 grams of ferrous sulfate, and 6,000 grams of manganese sulfate. Add 5% of the above-mentioned secondary seed culture solution, mix evenly, and make a compost pile with a thickness of 0.3 meters, a width of 2 meters, and a length of 300 meters. The temperature is controlled at 35°C for 6 days, and the material is turned once in the middle. After the cultivation, the fermented product is dried and crushed to be protein feed.

The obtained straw fermented protein feed contained a large amount of yellow Aspergillus oryzae spores, and the fermented product was brown with wine aroma. The lignin degradation rate was 69.7%, the protein content was 37.3%, and the feed yield was 51.6%.

Claims (1)

1. aspergillus oryzae degrading straw produces the method for protein feed, it is characterized in that carrying out according to following step: take stalk as the technique that raw material carries out the production protein feed of solid state fermentation, comprise with aspergillus oryzae is starting strain, carry out test tube and expand cultivation, liquid submerged culture, first order seed cultivation and solid fermentation cultivation, obtain the protein feed of degrading straw;

Bacterial classification used is aspergillus oryzae any one bacterial classification;

The raw material used is any one stalk, and stalk is first through pulverizing, and crosses 20 ~ 100 object sieves;

It is potato sucrose culture medium that described test tube expands culture medium;

In described zymotechnique, its liquid submerged culture base and primary-seed medium are: glucose 5 ~ 30 grams per liter, ammonium tartrate 0.1 ~ 5 grams per liter, phenmethylol 0.2 ~ 3 grams per liter, magnesium sulfate 0.2 ~ 1 grams per liter, Tween 80 0.5 ~ 10 grams per liter, potassium dihydrogen phosphate 2 ~ 9 grams per liter, phthalic acid 3 ~ 18 grams per liter, pH 5 ~ 8,120 ~ 140 DEG C of sterilizings 20 ~ 40 minutes;

Wherein said Shaking culture process conditions are, inoculate a ring aspergillus oryzae test tube slant spore in the triangular flask that volume ratio 16 ~ 48% culture medium is housed, and at rotating speed: 150 revs/min, 25 ~ 35 DEG C, cultivate 24 ~ 72 hours;

Wherein said first order seed culture process is, Shaking culture seed liquor is inoculated in primary-seed medium by the inoculum concentration of 1 ~ 20% by volume, temperature 25 ~ 35 DEG C, speed of agitator 50 ~ 200 revs/min, throughput 0.2 ~ 2:1 ventilation gas volume/fermentating liquid volume/per minute, cultivates 18 ~ 72 hours;

During described solid fermentation is cultivated, its solid fermentation culture medium consists of: stalk 10 kilograms, 20 ~ 60 kilograms, water, glucose 0.5 ~ 3 kilogram, 0.05 ~ 0.2 kilogram, urea; 5 ~ 15 grams, copper sulphate, 5 ~ 15 grams, ferrous sulfate, manganese sulfate 5 ~ 20 grams, each composition of this culture medium can by this scale amplifying equal;

Wherein said solid fermentation cultivation and fermentation technique is: be 2 ~ 20% by the ratio of seed liquor volume/fermentation medium weight, compost thickness is 0.1 ~ 0.4 meter, expect wide 0.5 ~ 2 meter, length is not limit, throughput is 0.3 ~ 1.5:1(gas volume/fermentation medium weight)/per minute, incubation time is 3 days ~ 23 days, midway stirring 1 ~ 2 time; Described test tube expands cultivation, before carrying out Shaking culture, first cultivates in the potato sucrose culture medium of new for the access of aspergillus oryzae bacterial classification preparation, cultivation temperature 25 ~ 35 DEG C, incubation time 48 ~ 144 hours; 4 ~ 10 DEG C save backup.