A method for preparing antibacterial protein feed powder using composite probiotics and puffed feathers

Abstract

The invention discloses a method for preparing antibacterial protein feed powder by using composite probiotics and puffed feathers. Step 1: Wash and dry the discarded feathers, process them with a twin-screw extruder, and then filter them through a filter screen to obtain uniform puffed feather powder; Step 2: Mix the prepared puffed feather powder with glucose, dissolve them in deionized water, sterilize them with ultra-high pressure, and prepare a fermentation medium; Step 3: Inoculate the activated composite bacterial liquid into the fermentation medium, place it in a shaking table for constant temperature fermentation and culture; Step 4: After the fermentation is completed, collect the fermentation products to obtain protein feed powder; the composite bacterial liquid is a composite bacterial liquid of Lactobacillus paracasei and Bacillus subtilis. The present invention utilizes Lactobacillus paracasei and Bacillus subtilis for synergistic fermentation, and the utilization rate of puffed feathers reaches 65%. It not only improves the palatability and nutritional value of puffed feather powder, but also increases the product added value of puffed feather powder.

Description

Technical Field

[0001] The invention discloses a method for preparing antibacterial protein feed powder by utilizing puffed feathers with composite probiotics, and belongs to the technical field of animal nutrition and feed. Background Art

[0002] With the intensive development of my country's breeding industry, millions of tons of feather waste are generated every year. This not only causes serious environmental pollution, but also is a huge waste of protein resources. The protein content in feathers is as high as 85%-90%, which is considered to be a natural high-protein resource. However, due to the presence of hydrogen bonds, hydrophobic effects and a large number of disulfide bonds in the feather structure, the feather structure in the natural state is stable and difficult to degrade, which limits the full utilization of feathers. The processed feather feed contains rich trace elements and amino acids, especially the level of cysteine ​​ranks first among natural feeds, which makes feather processed feed a high-protein feed additive. However, the economic value of existing feather powder additives has not been fully developed, and the scope of use is limited. In addition, the existing high-protein feed products are susceptible to microbial contamination during storage, which affects their shelf life. To this end, the present invention proposes a method for preparing antibacterial protein feed powder using puffed feathers with a composite probiotic, which is used to increase the economic value and scope of application of feather feed. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing antibacterial protein feed powder by using composite probiotics and puffed feathers, so as to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for preparing antibacterial protein feed powder by using composite probiotics and puffed feathers, comprising the following steps:

[0005] Step 1: Wash and dry the discarded feathers, process them with a twin-screw extruder, and then filter them through a filter screen to obtain uniform puffed feather powder;

[0006] Step 2: mixing the prepared puffed feather powder with glucose, dissolving the mixture in deionized water, and sterilizing the mixture by ultra-high pressure to prepare a fermentation medium;

[0007] Step 3: inoculating the activated composite bacterial solution into the fermentation medium and placing it in a shaking incubator for constant temperature fermentation culture;

[0008] Step 4: After the fermentation is completed, the fermentation product is collected to obtain protein feed powder;

[0009] The composite bacterial liquid is a composite bacterial liquid of Lactobacillus paracasei and Bacillus subtilis.

[0010] Preferably, the Lactobacillus paracasei is Lactobacillus paracasei 85, and the Bacillus subtilis is Bacillus subtilis HC2.

[0011] Preferably, the puffed feather powder in step 1 is carried out as follows: the collected discarded feathers are washed, dried, and processed with a twin-screw extruder to obtain coarse feather powder. The puffing conditions are: speed 180-200r / min, temperature 150-160°C, pressure 0.40-0.50Mpa, and the coarse feather powder is filtered through a 200-mesh filter screen to obtain uniform puffed feather powder.

[0012] Preferably, in step 2, the prepared puffed feather powder is mixed with glucose and dissolved in deionized water, and the specific addition amount is: the mass volume ratio of puffed feather powder added to deionized water is 5% g / ml, and the added amount of glucose is 5g / L.

[0013] Preferably, in step three, before inoculation, Lactobacillus paracasei is revived in MRS agar medium and inoculated in MRS broth medium for activation for 36-48 hours, Bacillus subtilis is revived in LB agar medium and inoculated in LB broth medium for activation for 12-24 hours, and then the live bacteria of Lactobacillus paracasei and Bacillus subtilis after activation are counted, and the inoculation amounts are compounded in a ratio of 1:1 to obtain a composite bacterial solution.

[0014] Preferably, the formula of the fermentation medium is: 2.0 g / L sodium chloride, 5.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 20.0 g / L glucose, 5-10% g / ml puffed feather powder by mass volume fraction, 1000 mL deionized water, and pH 6.0-9.0.

[0015] Preferably, the waste feathers in step one include duck feathers, chicken feathers and goose feathers.

[0016] Preferably, the fermentation product produced by collecting in step 4 is first filtered to separate the fermentation liquid and the remaining puffed feather solids; the fermentation liquid is centrifuged to collect the bacterial precipitate, and the bacterial precipitate is dried to remove moisture; the fermentation supernatant is spray-dried to obtain the fermentation product, and the bacterial powder and the fermentation liquid powder are combined to obtain the protein feed powder.

[0017] The present invention also provides a protein feed powder with antibacterial ability obtained by any of the above methods.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. Compared with single strain fermentation, compound probiotic fermentation makes the protein feed powder contain high density of compound active probiotics (Lactobacillus paracasei 4.95×10 7 CFU / g, Bacillus subtilis 1.02×10 8 CFU / g).

[0020] 2. Lactobacillus paracasei and Bacillus subtilis produce a variety of metabolites during the culture process and have antifungal activity, ensuring the safety of the feed preservation process.

[0021] 3. Lactobacillus paracasei and Bacillus subtilis utilize the fermentation process of puffed feathers, accompanied by the production of a variety of acidic metabolites, which enrich the types of amino acids in feather feed, improve the amino acid ratio, and make its nutrition more balanced.

[0022] 4. The present invention utilizes Lactobacillus paracasei and Bacillus subtilis for synergistic fermentation, and the utilization rate of puffed feathers reaches 65%, which not only improves the palatability and nutritional value of puffed feather powder, but also increases the product added value of puffed feather powder.

[0023] 5. High probiotic active protein feed powder has the potential to regulate the stability of animal intestinal flora and provide poultry with resistance to pathogenic bacteria risks.

[0024] Lactobacillus paracasei 85 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on April 26, 2023. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is GDMCC No: 63407.

[0025] Bacillus subtilis HC2, deposit number GDMCC No: 62466, was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 12, 2022. It was disclosed in CN202310774131.4. The invention name is Application of feather degradation liquid in citrus preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the degradation rate of puffed feather meal by three treatments.

[0027] Figure 2 It is the live bacteria count before and after fermentation of the compound probiotic fermentation system.

[0028] Figure 3 It is the inhibitory ability of protein feed powder on Escherichia coli. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0030] Lactobacillus paracasei can be selected from conventional Lactobacillus paracasei. In this embodiment, Lactobacillus paracasei 85 is selected, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on April 26, 2023. The deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is GDMCC No: 63407. It has antifungal ability.

[0031] The Bacillus subtilis can be selected from conventional Bacillus subtilis. In this embodiment, Bacillus subtilis HC2 is selected, with a preservation number of GDMCC No: 62466, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 12, 2022, and is disclosed in CN202310774131.4. The invention name is Application of Feather Degradation Liquid in Citrus Preservation.

[0032] The preparation method of puffed feather powder used in the following examples is: collect discarded chicken feathers, wash them, dry them, and process them with a twin-screw extruder to obtain coarse feather powder. Puffing conditions: speed 180-200r / min, temperature 150-160°C, pressure 0.45-0.50Mpa. The coarse feather powder is filtered through a 200-mesh filter screen to obtain a uniform puffed feather powder raw material for standby use.

[0033] Example 1 Compound probiotics using puffed feather powder

[0034] Lactobacillus paracasei 85 was revived and activated in MRS agar medium, and a single colony was picked and inoculated in MRS broth medium for activation for 48 hours. Bacillus subtilis HC2 was revived in LB agar medium and inoculated in LB broth medium for activation for 24 hours. Puffed feather powder was added to double distilled water containing 5.0 g / L glucose at a mass volume ratio of 5% (g / mL) and sterilized at 121°C for 20 minutes. A mixed bacterial solution of Lactobacillus paracasei liquid, Bacillus subtilis liquid and the bacterial solution of the two strains in a volume ratio of 1:1 was inoculated into a medium containing puffed feather powder, and the inoculation amount was 1×10 6 CFU / mL, cultured at 37℃ for 30h. Filter the fermentation liquid, collect the remaining puffed feathers, determine the dry weight by differential weight method, and calculate the degradation capacity of the two strains of bacteria on feather powder. Figure 1 As shown, among the three combined fermentation schemes, the mixed fermentation of two strains of bacteria significantly improved the degradation ability, and the utilization rate of puffed feather powder under synergistic effect could reach 58%, indicating that the synergistic utilization of the two strains of bacteria accelerated the degradation of puffed feather powder.

[0035] Example 2 Preparation of protein feed powder by mixed fermentation of composite probiotics

[0036] The mixed bacterial seed solution in Example 1 was added to the seed fermentation solution (2.0 g / L sodium chloride, 5.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 20.0 g / L glucose, 5-10% g / mL puffed feather powder). Lactobacillus paracasei and Bacillus subtilis were mixed in an inoculum ratio of 1:1 and cultured at 37°C for 30 h. The fermentation solution was filtered to remove the remaining puffed feather powder, and the number of live bacteria of the two strains in the fermentation system was determined by the live bacteria counting method. Figure 2 As shown in the figure, after the fermentation was completed, 2.05×10 Lactobacillus paracasei in the fermentation system 7 CFU / g, Bacillus subtilis 0.96×10 8 After fermentation is completed, the fermentation liquid is centrifuged to collect the bacterial precipitate, and the bacterial precipitate is dried to remove moisture; the fermentation supernatant is spray-dried to obtain the fermentation product, and the bacterial powder and the fermentation liquid powder are combined to form the protein feed (microbial protein powder).

[0037] Example 3 Inhibitory effect of protein feed powder on Escherichia coli

[0038] The bacterial protein powder collected in Example 2 was dissolved in sterile water at a concentration gradient of 1:10, 1:100, 1:1000, and 1:10000 to prepare a protein suspension. Enterohemorrhagic Escherichia coli (ACCC 11222, purchased from the China Agricultural Microbial Culture Collection Center) was revived and activated in LB medium, and the bacterial population density was adjusted to 1×10 5 CFU / mL. The protein suspension and E. coli suspension were inoculated into a 96-well plate at a volume ratio of 1:1 and monitored continuously for 24 hours at 37°C. The final OD value was deducted from the initial OD value to calculate the antibacterial ability of the protein feed powder. Figure 3 As shown, the protein feed powder diluted less than 1000 times has a significant inhibitory effect on Escherichia coli (inhibition rate of 44%), and can significantly inhibit the proliferation of Escherichia coli.

[0039] LB medium: 5g yeast powder, 10g tryptone, 10g sodium chloride, dilute to 1000mL with ultrapure water, adjust pH to 7.0. Sterilize at 121℃ for 20min after mixing well. 15g agar powder should be added to LB agar medium.

[0040] MRS medium: peptone 10g, beef extract powder 5g, yeast extract powder 4g, glucose 20g, ammonium citrate 2g, K2HP04 4g, sodium acetate 5g, MgS04 0.4g, MnS04 0.05g, 0.1% Tween 80, ultrapure water to 1000mL, adjust pH to 6.2. Sterilize at 121℃ for 20min. MRS agar medium needs to add 15g agar powder.

Claims

1. A method for preparing antibacterial protein feed powder using composite probiotics and puffed feathers, characterized in that: The following steps are involved: Step 1: Wash and dry the discarded feathers, process them with a twin-screw extruder, and then filter them through a filter screen to obtain uniform puffed feather powder; Step 2: mixing the prepared puffed feather powder with glucose, dissolving the mixture in deionized water, and sterilizing the mixture by ultra-high pressure to prepare a fermentation medium; Step 3: inoculating the activated composite bacterial solution into the fermentation medium and placing it in a shaking incubator for constant temperature fermentation culture; Step 4: After the fermentation is completed, the fermentation product is collected to obtain protein feed powder; The composite bacterial liquid is a composite bacterial liquid of Lactobacillus paracasei and Bacillus subtilis.

2. The method according to claim 1, characterized in that The Lactobacillus paracasei is Lactobacillus paracasei 85, and the Bacillus subtilis is Bacillus subtilis HC2.

3. The method according to claim 1, characterized in that The puffed feather powder in step 1 is carried out as follows: the collected discarded feathers are washed, dried, and processed with a twin-screw extruder to obtain coarse feather powder. The puffing conditions are: speed 180-200r / min, temperature 150-160°C, pressure 0.40-0.50Mpa, and the coarse feather powder is filtered through a 200-mesh filter screen to obtain uniform puffed feather powder.

4. The method according to claim 1, characterized in that: In step 2, the prepared puffed feather powder is mixed with glucose and dissolved in deionized water. The specific addition amount is: the mass volume ratio of the puffed feather powder added to the deionized water is 5% g / ml, and the glucose addition amount is 5g / L.

5. The method according to claim 1, characterized in that Step 3: Before inoculation, Lactobacillus paracasei is revived in MRS agar medium and inoculated in MRS broth medium for activation for 36-48 hours; Bacillus subtilis is revived in LB agar medium and inoculated in LB broth medium for activation for 12-24 hours; then, the live bacteria of the activated Lactobacillus paracasei and Bacillus subtilis are counted, and the inoculation amounts are compounded in a 1:1 ratio to obtain a composite bacterial solution.

6. The method according to claim 1, characterized in that The formula of the fermentation medium is: 2.0 g / L sodium chloride, 5.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 20.0 g / L glucose, 5-10% g / ml puffed feather powder by mass volume fraction, 1000 mL deionized water, and pH of 6.0-9.

0.

7. The method according to claim 1, characterized in that The waste feathers in step 1 include duck feathers, chicken feathers and goose feathers.

8. The method according to claim 1, characterized in that The collection of the fermentation product described in step 4 is first performed by filtering the fermentation liquid and the remaining puffed feather solids to separate; the fermentation liquid is centrifuged to collect the bacterial precipitate, and the bacterial precipitate is dried to remove moisture; the fermentation supernatant is spray-dried to obtain the fermentation product, and the bacterial powder and the fermentation liquid powder are combined to obtain the protein feed powder.

9. A protein feed powder with antibacterial ability prepared according to any one of the methods of claims 1-8.

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