CN107475166B - Bacillus amyloliquefaciens and application thereof in degrading feather to produce oligopeptide - Google Patents

Abstract

The invention relates to the technical field of microorganisms. Aims to provide a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H5 with high oligopeptide yield in a fermentation product and application of the strain in degrading feathers to produce feather oligopeptide powder. The preservation number of the bacillus amyloliquefaciens H5 is as follows: CGMCC No. 14264. The technical scheme adopted by the invention is as follows: activating and culturing the bacillus amyloliquefaciens H5 to obtain a primary seed solution, and then inoculating the primary seed solution into a seed culture medium to culture to obtain a secondary seed solution; inoculating the secondary seed liquid into a fermentation culture medium for culture, and obtaining a fermentation product which can be used for preparing feather oligopeptide powder or oligopeptide solution. The method can almost completely degrade the complete feather, has high yield of feather oligopeptide in the degradation product, and can be used for producing the easily digestible high-added-value feather oligopeptide powder.

Description

A strain of Bacillus amyloliquefaciens and its application in degrading feathers to produce oligopeptides

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus amyloliquefaciens and its application of degrading feathers to produce oligopeptides.

Background technique

In recent years, with the continuous expansion of livestock and poultry breeding in my country, a large amount of livestock and poultry wastes, such as feathers, hair, horns, hooves, etc., have been produced. Among them, the annual output of waste feathers has reached hundreds of thousands of tons, causing a serious environmental burden. The analysis results show that the main component of feathers is keratin, and its crude protein content can reach more than 80%, and it contains a variety of amino acids, as well as many macro elements, trace elements and some unknown growth factors. High source of dietary protein. However, due to the existence of a large number of disulfide bonds, hydrogen bonds and hydrophobic group interactions between feather keratin molecules, the molecular structure is compact and complex, making it difficult to be degraded by animal-derived proteases such as trypsin and pepsin.

A lot of research has been done on feather degradation protein feed at home and abroad. At present, there are mainly physical methods, chemical methods, enzymatic methods and microbial fermentation methods. Compared with the first three methods, the microbial fermentation method is not only simple in process, but also avoids a lot of power and energy. Energy consumption, and the bacteria itself is a protein source, the nutrients are more balanced, and the palatability is better, so it has been highly valued by researchers at home and abroad, and has a wide range of uses and broad development and application prospects. But in fact, due to the high content of macromolecular protein in fermented products and low digestibility, the amount that can be added to animal feed is less than 10%, resulting in a lot of waste of resources.

In addition to soluble protein, feather keratin degradation products also include small molecular peptides, amino acids, etc. Peptide is the product of dehydration polymerization of amino acids. Usually, the compound formed by dehydration and condensation of 10 to 100 amino acid molecules is called polypeptide, and the peptide composed of 2 to 10 amino acids is called oligopeptide (small molecule peptide). According to modern digestion theory, oligopeptides can be directly absorbed by the body without digestion. It has the characteristics of fast absorption, low energy consumption, and is not easy to be saturated, and it does not compete with the absorption of amino acids, which can greatly improve the absorption and utilization rate of protein. In addition, many studies have confirmed that feeding animals low levels of protein supplemented with synthetic amino acid diets does not result in optimal performance and feed efficiency, both of which require a certain amount of raw protein in the diet and oligopeptides.

In the prior art, the research on feather keratin degradation mostly focuses on improving its degradation efficiency, improving the content and activity of keratinase, for example, the patent application number 2011102816561 provides a kind of monospermia, which is in the preferred The keratinase activity can reach 150U/mL after culturing for 24h under fermentation conditions, which is better than most strains reported at home and abroad. However, the yield of oligopeptides in feather keratin degradation fermentation products, especially in the production of animal feed, has not received much attention, which leads to a large number of renewable resources obtained by efficient degradation and fermentation, which are difficult to be fully utilized and Rapid consumption results in a backlog of renewable resources, which is not conducive to the realization of industrial production of livestock and poultry waste recycling.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to provide a strain of Bacillus amyloliquefaciens H5 with a high yield of oligopeptides in a fermentation product, which was deposited in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection Committee on June 22, 2017, The deposit number is: CGMCC No. 14264, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101.

The Bacillus amyloliquefaciens H5 provided by the present invention is obtained by screening from the soil of long-term stacking of feather waste in chicken farms, and its morphological characteristics are: the cell shape is rod-shaped, 1.12-2.34 μm long, without flagella, and Gram staining is positive The characteristics of the colony are: after culturing on the beef extract peptone plate for 48 hours, the colony is nearly round, flat, with a notch on the edge, opaque, viscous and difficult to pick; the pH value of the culture conditions is in the range of 5.0 to 10.5, the most suitable The pH value is 6-8, the growth temperature range is 15-50 ℃, and the optimum temperature is 28-40 ℃. It grows rapidly in the beef extract peptone medium, and enters the stable period after 10 hours.

Another object of the present invention is to provide the application of Bacillus amyloliquefaciens H5 in degrading feathers to produce feather oligopeptide powder and oligopeptide solution.

In order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is: comprising the following operation steps:

1) Preparation of secondary seed solution: pick a ring of Bacillus amyloliquefaciens H5 preserved on the slant, inoculate it into beef extract peptone test tube medium at 28-40°C and activate for 12h to obtain primary seed solution, then the primary seed solution according to 2% of the inoculum was inoculated into 30ml of beef extract peptone medium and cultured at 28-40°C for 12h to obtain secondary seed liquid.

The beef extract peptone medium comprises: peptone 10g/L, beef extract 5g/L, sodium chloride 5g/L, pH value of 6.0-8.0, and sterilization at 121 DEG C for 20min.

2), degrade feathers by fermentation: insert the secondary seed liquid of Bacillus amyloliquefaciens H5 into the fermentation medium at an inoculum of 2%, and ferment for 72 hours at a temperature of 20 to 45°C and a pH of 6 to 10.5, The fermentation product was collected.

Preferably: the fermented product is dried and pulverized to prepare feather oligopeptide powder.

Preferably: the fermentation product is subjected to solid-liquid separation, ultrafiltration to obtain an oligopeptide solution, which is further dried and pulverized to obtain oligopeptide powder.

Preferably: the fermentation medium comprises: feather 10g, NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, distilled water 1000mL, sterilized at 121°C for 20min.

The fermentation of Bacillus amyloliquefaciens H5 to degrade feathers to produce oligopeptides can be liquid fermentation or solid state fermentation.

Preferably: the fermentation temperature is 40°C.

Preferably: the pH value of the fermentation condition is 9.5.

The invention has the following beneficial effects: the complete feather can be almost completely degraded, the feather oligopeptide content in the degradation product is high, and the feather oligopeptide powder with easy digestion and high added value can be produced. Specifically, the Bacillus amyloliquefaciens H5 provided by the present invention has a strong ability to degrade feather keratin waste, and can degrade it into oligopeptides that are easily absorbed by animals. The absorption and utilization of protein is beneficial to obtain the best performance and feed efficiency. In this way, the problems of high macromolecular protein content and low digestibility in the feather keratin degradation product are solved, the addable amount of the degradation product in animal feed is significantly increased, and the full recycling of resources is realized. The strain provided by the invention has broad development and application prospects in degrading feather keratin to produce feather oligopeptide powder and oligopeptide solution.

Description of drawings

Fig. 1 is the scanning electron microscope picture of Bacillus amyloliquefaciens H5;

Fig. 2 is the Gram-staining diagram of Bacillus amyloliquefaciens H5;

Fig. 3 is the growth curve diagram of Bacillus amyloliquefaciens H5 in beef extract peptone medium;

Fig. 4 is a bar graph of fermentation temperature optimization of oligopeptide produced by Bacillus amyloliquefaciens H5;

Fig. 5 is the fermentation pH optimization bar chart of Bacillus amyloliquefaciens H5 producing oligopeptide;

Fig. 6 is the fermentation time optimization bar chart of Bacillus amyloliquefaciens H5 producing oligopeptide;

Fig. 7 is the bar chart of the shaker rotation speed optimization of oligopeptide produced by Bacillus amyloliquefaciens H5;

Fig. 8 is the contrast photo before and after fermentation of fermentation medium;

Figure 9 shows the preparation process of feather oligopeptide powder, oligopeptide solution and oligopeptide powder.

Detailed ways

Preferred embodiments are specifically provided below to make the technical solutions and effects of the present invention clearer to those skilled in the art.

Example 1: Screening of strains

1. Enrichment and domestication: Take a mixed sample of long-term feather-accumulated soil, fermentation pond biogas residue and sewer sludge, and mix it with water at a mass ratio of 1:5. Take 10ml of it and pack it into a 250ml Erlenmeyer flask containing 80ml No. 1 enrichment medium, shake it at 37°C, pH 7, 150r/min until the feathers are basically rotted; then take the bottle to enrich The solution was inoculated into No. 2 enriched medium at an inoculum of 5%, and cultured under the same conditions as above until the feathers were basically rotted, and passaged twice.

The ingredients of the No. 1 enrichment medium are: NH ₄ Cl 0.5g, NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, MgSO ₄ ·7H ₂ O 0.2g, yeast extract 0.1g , glucose 1g, pH 7.0, add feather 5g, sterilize at 115 ℃ for 30min.

The composition of the No. 2 enrichment medium is: NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, MgSO ₄ 7H ₂ O 0.2g, pH value 7.0, add feather 5g, 121 ℃ Sterilize for 20 minutes.

2. Primary screening and re-screening: take 0.1 ml of the enrichment solution obtained in step 1 and spread it on the primary screening medium, repeat three times or more to purify; pick colonies with transparent circles and inoculate them on the seed medium for overnight culture, Then, it was inoculated into a test tube containing 5 ml of re-screening medium at 20% of the inoculum for re-screening, and the speed of each strain in degrading feathers was recorded, and the screened strains were stored in a refrigerator at 4°C.

The components of the primary screening medium are: NH ₄ Cl 0.5g, NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, MgSO ₄ ·7H ₂ O 0.2g, yeast extract 0.1g, buttermilk Protein 7g/L, pH 7.0, sterilized at 121°C for 20min.

The components of the re-screening medium are: NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, MgSO ₄ 7H ₂ O 0.2g, pH 7.0, feather 6g, sterilized at 121°C for 30min .

The components of the seed medium are: 10 g of peptone, 5 g of beef extract, 5 g of sodium chloride, pH 7.0, and sterilization at 121° C. for 20 minutes.

3. Secondary screening: activate the strains preserved in step 2 in the seed medium for 24 hours, and then insert 2% of the inoculum into a 250ml conical flask containing 50ml of fermentation medium, at 37°C and pH 7. Cultivate for 72h under the condition of 150r/min, and conduct secondary screening according to the degradation of feathers by each strain and the yield of polypeptide and oligopeptide.

The components of the fermentation medium are: feather 10 g, NaCl 0.5 g, K ₂ HPO ₄ 0.7 g, KH ₂ PO ₄ 0.35 g, distilled water 1000 mL, pH value 7, sterilized at 121° C. for 20 min.

Example 2: Strain identification

1. Morphological observation of colonies and bacterial cells: The purified and isolated Bacillus amyloliquefaciens H5 was streaked and cultured on beef extract peptone plate medium, and incubated at 37°C for 48 hours. Raised, notched edges, opaque, sticky and difficult to pick. Observation under a scanning electron microscope, as shown in Figure 1, showed that the bacterial cells were rod-shaped, 1.12-2.34 μm long, without flagella, and were identified as positive by Gram (as shown in Figure 2).

2. Observation of culture characteristics: Bacillus amyloliquefaciens H5 was inoculated into beef extract peptone medium at the same inoculum amount, and placed in a shaker for 18 hours at different temperatures. The strains in the environment of 15-50 ° C can grow, and The strains grow the fastest in the environment of 28-40℃; the strains can grow in the environment with pH values ranging from 5-10, and the strains can grow in the environment with pH values ranging from 6-8. fastest growing.

In addition, Bacillus amyloliquefaciens H5 was inserted into the beef extract peptone medium at 2% inoculum, and cultured at 37°C and pH 7. Every 2h, 0.5ml of the culture solution was taken and placed in 4.5ml of distilled water. In the test tube of , after mixing evenly, measure the absorbance at 600nm wavelength and draw a curve. The drawing result is shown in Figure 3, which is the growth curve of the strain.

3. Molecular biological identification: The whole genome of the pure strain was extracted by using the bacterial whole genome rapid extraction kit, PCR was carried out by selecting bacterial 16SrDNA universal primers 27F and 1492R, and then sequenced and analyzed. The sequencing results were compared by BLAST, and the strain was identified as the preserved strain of the present invention, Bacillus amyloliquefaciens subsp. CGMCC NO. 14264, and the gene sequence is shown in the sequence table.

Example 3: Optimization of fermentation conditions for oligopeptide production

1) Preparation of secondary seed solution: pick a ring of Bacillus amyloliquefaciens H5 preserved on the slant, inoculate it into beef extract peptone test tube medium at 28-40°C and activate for 12h to obtain primary seed solution, then the primary seed solution according to 2% of the inoculum was inoculated into 30ml of beef extract peptone medium and cultured at 28-40°C for 12h to obtain secondary seed liquid.

The beef extract peptone medium comprises: peptone 10g/L, beef extract 5g/L, sodium chloride 5g/L, pH value of 6.0-8.0, and sterilization at 121 DEG C for 20min.

2), degrade feathers by fermentation: the secondary seed liquid of Bacillus amyloliquefaciens H5 is inserted into the fermentation medium at an inoculum size of 2%, and a single factor experiment is adopted to investigate the degradation rate and degradation rate of feathers by different fermentation pH, fermentation temperature and fermentation cycle. For the effect of oligopeptide yield, the initial fermentation conditions of the shake flask were as follows: 2% inoculum size, pH 7, and shaker culture for 72h at 37°C and 180r/min.

Fermentation temperature optimization experiment: other fermentation conditions were initial fermentation conditions, and oligopeptide yields were determined after shaking at 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C for 72 hours. The measurement results are shown in Figure 4. When the fermentation temperature was 40°C, the oligopeptide yield was the highest, which was 59.39%, so the fermentation temperature was selected as 40°C.

The optimal experiment of fermentation pH value: other fermentation conditions are initial fermentation conditions, and the initial pH values are adjusted to 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, respectively, and the yield content is determined after 72 hours of shaking. The measurement results are shown in Figure 5. When the initial pH value is 9.5, the yield of oligopeptide is the highest, which is 64.52%, so the initial pH value is 9.5.

Optimal experiment of fermentation cycle: other fermentation conditions are initial fermentation conditions, sampling every 24h to measure the yield of oligopeptide, to determine the optimal cycle of strain fermentation. The measurement results are shown in Figure 6. The oligopeptide yields of fermentation for 72h and 96h are similar, both reaching more than 54%. From the perspective of practical application, the fermentation period is selected to be 72h.

The optimal experiment of shaking table rotation speed: other fermentation conditions were initial fermentation conditions, shaking table rotation speed was adjusted to 120, 150, 180 and 200 r/min respectively, and oligopeptide yield was measured after shaking table culture for 72 h. The measurement results are shown in Figure 7. When the rotating speed of the shaking table is 180 r/min, the yield of oligopeptide is the highest, which is 57.06%, so the rotating speed of the shaking table is 180 r/min.

Example 4: Degradation of feathers to produce oligopeptides

1) Preparation of secondary seed solution: pick a ring of Bacillus amyloliquefaciens H5 preserved on the slant, inoculate it into beef extract peptone test tube medium at 28-40°C and activate for 12h to obtain primary seed solution, then the primary seed solution according to 2% of the inoculum was inoculated into 30ml of beef extract peptone medium and cultured at 28-40°C for 12h to obtain secondary seed liquid.

2) Fermentation and degradation of feathers: the secondary seed liquid of Bacillus amyloliquefaciens H5 was inserted into the fermentation medium at an inoculum of 2%, and fermented for 72 hours at a temperature of 40° C. and a pH value of 9.5. Feather degradation phenomenon. As the fermentation time increased, the feather twigs fell off, and the medium gradually became cloudy. When the fermentation was over, the complete feather was almost completely degraded, as shown in Figure 8, and the fermentation broth was milky white. The fermentation products were collected, and the degradation rate of feathers was 91.27%, the total peptide yield was 85.09%, and the oligopeptide yield was 68.06%.

Example 5: Effects of different treatments on feather degradation to produce oligopeptides

Three groups of control experiments were set up, and the experimental conditions were set as: 1) natural pH, the medium was not sterilized, and not inoculated; 2) pH was adjusted to 9.5, and sterilized at 121°C for 20 min; no inoculation; 3) pH was adjusted to 9.5, 121 Sterilize at ℃ for 20min, and inoculate Bacillus amyloliquefaciens H5 according to 2% of the inoculum; other conditions are the same as in Example 3, and the effects of different treatments on feather degradation to produce oligopeptides are studied. The results are shown in the following table:

Table 1 Feather degradation rate of oligopeptide under different treatments

Example 6: Preparation of Feather Oligopeptide Powder

On the basis of Example 4, the fermented product was dried and ground to obtain feather oligopeptide powder.

Example 7: Preparation of oligopeptide solution and oligopeptide powder

On the basis of Example 4, the fermentation product was subjected to solid-liquid separation, ultrafiltration to obtain an oligopeptide solution, and further drying and grinding of the oligopeptide solution to obtain oligopeptide powder.

As can be seen from Table 1, the bacterial strain and fermentation method provided by the present invention greatly improve the content of oligopeptide in the degradation product, so that livestock and poultry wastes such as feathers are rapidly degraded, and produce feather oligopeptide powder that can be added in a large amount in animal feed, and then Use this feather oligopeptide meal to produce rations with optimal performance and feed efficiency. In this way, the utilization rate of livestock and poultry waste renewable resources is significantly improved, the backlog of efficiently produced renewable resources is avoided, and the foundation is laid for the industrial production of feather oligopeptide powder.

Finally, it should also be noted that the above enumeration is only a specific implementation example of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

sequence listing

<110> Chengdu Institute of Biology, Chinese Academy of Sciences

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Claims (8)

1. a method utilizing Bacillus amyloliquefaciens H5 to degrade feathers to produce feather oligopeptide powder or oligopeptide solution, is characterized in that: may further comprise the steps: 1) Preparation of secondary seed solution: pick a ring of Bacillus amyloliquefaciens H5 preserved on the slant, inoculate it into beef extract peptone test tube medium at 28-40°C and activate for 12h to obtain primary seed solution, and then the primary seed solution according to 2% of the inoculum was inoculated into 30ml of beef extract peptone medium and cultured at 28-40°C for 12h to obtain secondary seed liquid; The beef extract peptone medium comprises: peptone 10g/L, beef extract 5g/L, sodium chloride 5g/L, pH value of 6.0-8.0, sterilized at 121°C for 20min; 2), degrade feathers by fermentation: insert the secondary seed liquid of Bacillus amyloliquefaciens H5 into the fermentation medium according to the inoculum amount of 2%, and ferment for 72h under the conditions of temperature of 20～45°C and pH value of 6～10.5, collection of fermentation products; The classification name of the Bacillus amyloliquefaciens H5 is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). 2 . The method according to claim 1 , wherein the Bacillus amyloliquefaciens H5 is obtained by screening from the soil where feather wastes are piled up for a long time in chicken farms. 3 . 3 . The method according to claim 1 , wherein the morphological characteristics of the Bacillus amyloliquefaciens H5 are: the cell shape is rod-shaped, 1.12-2.34 μm long, without flagella, and Gram staining is positive; After culturing on beef extract peptone plate for 48 hours, the colonies were nearly round and flat, with notches on the edges, opaque, sticky and not easy to pick. 4. The method according to claim 1, wherein the fermented product is dried and pulverized to prepare feather oligopeptide powder. 5 . The method according to claim 1 , wherein the fermentation product is subjected to solid-liquid separation, ultrafiltration to obtain an oligopeptide solution, which is further dried and pulverized to obtain oligopeptide powder. 6 . 6. method according to claim 1 is characterized in that: described fermentation medium comprises: feather 10g, NaCl 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.35g, distilled water 1000mL, 121 ℃ of sterilization bacteria for 20min. 7. The method according to claim 1, wherein the fermentation temperature is 40°C. 8. The method according to claim 1, wherein the pH value of the fermentation condition is 9.5.

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