CN114631601B - Production method and application of peanut meal by virtue of step-by-step fermentation of bacillus belgii and pediococcus acidilactici - Google Patents

Abstract

The application relates to a production method of peanut meal by virtue of step-by-step fermentation of bacillus belgii and pediococcus acidilactici, belongs to the technical field of biology, and mainly aims to provide a fermentation process for improving functional characteristics and nutritional level of peanut meal, fermented peanut meal prepared by the process and application of the fermented peanut meal in reducing the abdominal fat percentage of broiler chickens and improving meat quality. The fermentation method of the peanut meal comprises the following steps: pretreating raw materials, fermenting in a first stage, adjusting discharging, fermenting in a second stage, adjusting discharging and the like; the fermented peanut meal prepared by the fermentation process has the advantages that the total flavone content, the antioxidant performance, the crude protein content, the acid soluble protein content, the total acid content, the total unsaturated fatty acid content and the partial essential amino acid content are obviously increased, the arginine/lysine ratio is closer to the ideal amino acid ratio, and the AFB (amino acid binding protein) ₁ And phytic acid is significantly degraded. The fermented peanut meal is added into the feed for the broiler chickens, so that the abdominal fat rate of the broiler chickens of 42 days can be obviously reduced, the flavor characteristics of breast muscle are improved, the contents of intramuscular protein, total amino acid, flavor amino acid and monounsaturated fatty acid are obviously improved, the pH is reduced and slowed down, and the shelf life is improved.

Description

Production method and application of peanut meal by virtue of step-by-step fermentation of bacillus belgii and pediococcus acidilactici

Technical Field

The application belongs to the technical field of biology, and particularly relates to a production method for peanut meal by virtue of step-by-step fermentation of bacillus beiLeisi and pediococcus acidilactici.

Background

At present, along with the popularization of genetic germplasm improvement and intensive feeding modes, fast large-scale broiler chicken strains and a large-scale breeding mode are favored, the slaughtering period of broilers is further shortened, the feed conversion rate is continuously improved, however, the reduction of the quality of the poultry meat is brought while the production performance is improved, and the problems of reduction of flavor characteristics, pale or hard meat (PSE-like meat, lignified meat, white streaky meat and the like), reduction of intramuscular protein or water retention capacity, reduction of container period and the like are solved, so that the purchase expectation of consumers is reduced, and therefore, how to produce products with higher quality and higher safety on the premise of ensuring high yield is one of important research directions of current meat poultry researchers.

The Peanut meal (PNM) is a byproduct of Peanut kernel after oil extraction, mainly comprises broken Peanut kernels mixed with a small amount of seed coats, is generally in a powder or small block shape, is brown, and has strong Peanut characteristic fragrance. According to statistics, the global peanut meal yield in 2021/2022 years is 799 ten thousand tons, and the peanut meal belongs to the fifth global meal. Is an important vegetable protein raw material. The content of the crude protein of the peanut meal is higher than that of the soybean meal, generally more than 46 percent, in addition, the peanut meal is rich in glutamic acid, arginine and aspartic acid, wherein the content of the glutamic acid and the content of the arginine are respectively as high as 7.52 percent and 5.2 percent, and the peanut meal has good taste and is suitable for being used in poultry feed. Meanwhile, the total flavone content in the peanut meal is about 1.15mg/g, and the flavone has the function of regulating lipid metabolism, can reduce the accumulation of fat in the liver, and has important significance for regulating the lipid metabolism of the broiler chicken if the content of the fat can be improved through biotransformation. However, the starting materialThere are some limiting factors in the application: one, susceptibility to harmful microorganisms, such as opportunistic pathogens and Aflatoxins (AFB) ₁ ) Aspergillus flavus and Aspergillus parasiticus of Aspergillus; secondly, the high-quality protein is insufficient, and the digestion and absorption rate is low; thirdly, the amino acid distribution is unbalanced, the lysine content is only 50 percent of that of the soybean meal, the methionine content is also lower, and the ratio of arginine to lysine is 3.0-4.0 and is far higher than the ideal ratio of 1.0; fourthly, the fatty acid spectrum is single, most of the fatty acid spectrum is oleic acid, and the oxidation rancidity is easy; fifthly, the product is rich in anti-nutritional factors such as phytic acid, and phosphorus exists in the form of phytate phosphorus. Researches show that the average daily gain of the peanut meal is reduced when the addition amount is more than 6-8% in the whole period of raising broiler chickens, so that the peanut meal can be improved according to the nutritional characteristics, the addition proportion in a formula system is increased while the functional characteristics are improved, and the peanut meal feed has great significance for relieving the increase of the breeding cost caused by the high-price operation of the soybean meal.

The chicken quality is influenced by multiple aspects such as genetic breeding, feeding management, nutrition regulation and the like, the mechanism is complex, and the improvement of the chicken quality through the nutrition regulation and the regulation is a relatively safe and effective way. There are many reports on the regulation of chicken quality by nutrition: patent CN104922373B discloses a traditional Chinese medicine preparation for improving chicken quality, which contains nearly twenty traditional Chinese medicines, the addition proportion in the embodiment is 0.035%, and the application cost of adding traditional Chinese medicines in chicken feed is high. Secondly, the improvement on meat quality only shows an increase in the content of crude protein and crude fat and a reduction in moisture and drip loss in chicken; the patent CN103478465B also provides a feed containing Chinese herbal medicines for improving the fat metabolism and carcass quality of broiler chickens, the application effect of the feed is definite, the breast muscle rate is improved, the leg muscle rate is reduced, the abdominal fat rate is reduced, the lipid metabolism is influenced, but other aspects of improving the specific chicken quality are not disclosed; the patent CN104277999B discloses a novel multi-effect clostridium butyricum and application thereof in the aspects of improving the oxidation resistance of animals and improving the meat quality, the clostridium butyricum preparation increases the content of functional fatty acid in chicken, but does not definitely improve the flavor characteristics of the chicken and the content change of amino acid; patent CN102008030B discloses a feed containing about 10% of unfermented peanut meal for improving pork quality, which can make the pork color fresh and meat quality good when fed to fattening pigs 30 days before slaughtering, but the influence on chicken quality is not reported.

Therefore, the prior art lacks a feed processing technology which can reduce the abdominal fat rate of broiler chickens and improve the meat quality, such as increasing the contents of intramuscular protein, total amino acid, flavor amino acid and monounsaturated fatty acid, so as to achieve the effects of producing broiler chickens on a large scale and keeping the chicken quality consistent.

Disclosure of Invention

Aiming at the problems and defects in the prior art, the application aims to provide a fermentation process for improving the functional characteristics and the nutrient level of peanut meal, fermented peanut meal prepared by the process and application of the fermented peanut meal in reducing the abdominal fat rate of broiler chickens and improving the meat quality. The fermentation method of the peanut meal in the application comprises the following steps: pretreating raw materials, fermenting in a first stage, adjusting discharging, fermenting in a second stage, adjusting discharging and the like; the fermented peanut meal prepared by the fermentation process has the advantages that the total flavone content, the antioxidant performance, the crude protein content, the acid soluble protein content, the total acid content, the total unsaturated fatty acid content and the partial essential amino acid content are obviously increased, arginine and lysine are closer to ideal amino acids relatively to Lily, and AFB (amino acid Bromide) ₁ And phytic acid is significantly degraded. The fermented peanut meal is added into the feed for the broiler chickens, so that the abdominal fat rate of the broiler chickens of 42 days can be obviously reduced, the flavor characteristics of breast muscle are improved, the contents of intramuscular protein, total amino acid, flavor amino acid and monounsaturated fatty acid are obviously improved, the pH is reduced and slowed down, and the shelf life is improved.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

a fermentation process for improving the functional characteristics and the nutritional level of peanut meal comprises the following steps:

(1) pretreatment of raw materials: crushing and sieving the peanut meal, sterilizing the peanut meal for 15min by high-pressure steam at 121 ℃ with 10-20 meshes, specifically 18 meshes, and cooling the peanut meal to room temperature;

(2) stage one fermentation: preparing an activated starting strain, namely Bacillus belgii LB-Y-1 fermentation inoculation liquid, and inoculating the activated starting strain into sterilized peanut meal, wherein the final process comprises the following steps: the inoculation amount is 2.0-4.0%, specifically 2.0%, the water content is 36.0-42.0%, specifically 37.38%, the materials are fully and uniformly mixed, the fermentation temperature is 36.0-39.0 ℃, specifically 38.18 ℃, the fermentation time is 48-60 hours, specifically 54.41 hours, the materials are turned over once every 4 hours, the aerobic environment is kept, and the first-stage fermentation is carried out;

(3) adjusting discharging: uniformly mixing the fermented materials, and discharging;

(4) and (3) stage two fermentation: preparing an activated starting strain, namely pediococcus acidilactici LC-9-1 fermentation inoculation liquid to be inoculated on the produced material, and finally: the inoculation amount is 1.8-3.3 percent, specifically 1.89 percent, the water content is 39.0-46.0 percent, specifically 40.00 percent, the mixture is fully and uniformly mixed, the fermentation temperature is 35.0-39.0 ℃, specifically 37.11 ℃, the fermentation time is 10-16 hours, specifically 12.42 hours, the mixture is subpackaged into breathing bags, the anaerobic environment is kept, and the second stage fermentation is carried out;

(5) adjusting discharging: after fermentation, uniformly mixing, drying at a low temperature, controlling the drying temperature to be 45-50 ℃, specifically 48 ℃, ensuring that the moisture of a final product is controlled to be 9.5-10.5%, specifically 10.0%, crushing the material, sieving the crushed material by a 18-mesh sieve, uniformly mixing, and discharging to obtain a final fermentation product;

the strain in the step (2) of the application is a strain capable of degrading aflatoxin B ₁ And high-performance spore strain for high-yield complex enzyme (protease and cellulase), specifically Bacillus velezensis (LB-Y-1), which is preserved in China general microbiological culture Collection center (CGMCC), with the preservation address: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and having a collection number of CGMCC No.21344, as a stage-one fermentation strain;

the strain in the step (4) is a lactobacillus which can produce acid at high level and has inhibitory effect on various pathogenic bacteria (escherichia coli, ATCC25922, salmonella mirabilis, CMCCB, 49005 and staphylococcus aureus, ATCC6538), specifically Pediococcus acidilactici (Pediococcus acidilactici) LC-9-1, is preserved in CGMCC, and is registered with the number of CGMCC No.21345 by a preservation center as a second-stage fermentation bacterium;

on the basis of the scheme, the inoculation amounts of the Bacillus belgii LB-Y-1 and Pediococcus acidilactici LC-9-1 are the percentage (w/w) of the mass of the inoculated bacterial liquid and the mass of the inoculated culture medium;

on the basis of the scheme, the activation method of the Bacillus belgii LB-Y-1 comprises the following steps: inoculating LB-Y-1 strain preserved in glycerol on an LB solid culture medium plate, carrying out inverted culture at 37 ℃ for 18h, picking out a single colony by using a disposable inoculating loop, inoculating the single colony in an LB liquid culture medium, carrying out culture at 37 ℃ and 160rmp/min for 24h to obtain a seed solution, continuously inoculating the seed solution into a new LB liquid culture medium according to the inoculation amount of 0.5 percent of the seed solution, carrying out amplification culture at 37 ℃ and 160rmp/min, and obtaining a liquid LB-Y-1 strain with the concentration of more than or equal to 3.0 x 10 ⁸ CFU/mL is the first stage fermentation inoculation liquid.

On the basis of the scheme, the method for activating pediococcus acidilactici LC-9-1 comprises the following steps: inoculating the LC-9-1 strain preserved in glycerol on an MRS solid culture medium plate, performing inversion culture at 37 ℃ for 24h, picking out a single colony by using a disposable inoculating loop, inoculating the single colony in an MRS liquid culture medium, performing static culture at 37 ℃ for 24h to obtain a seed solution, continuously inoculating the seed solution to a new MRS liquid culture medium according to the inoculation amount of 1.0 percent of the seed solution, performing static culture at 37 ℃ for expansion culture, and obtaining liquid LC-9-1 strain with the concentration of more than or equal to 1.0 multiplied by 10 ⁸ CFU/mL is the second stage fermentation inoculation liquid.

On the basis of the scheme, the functional characteristics of the fermented peanut meal prepared by the process are improved, and the process specifically comprises the following steps: the total flavone content, the antioxidant performance, the crude protein content, the acid soluble protein content, the total acid content, the total unsaturated fatty acid content and the partial essential amino acid content are obviously increased, the relative proportion of arginine and lysine is closer to the ideal amino acid, AFB ₁ And phytic acid is significantly degraded.

On the basis of the scheme, the fermented peanut meal prepared by the process reduces the fat percentage of broiler chickens, and the method is to add the fermented peanut meal into feed in a proportion of 5-15 g/100g of feed, in particular 10g/100g of feed.

On the basis of the scheme, the fermented peanut meal prepared by the process improves the chicken quality, and the method is to add the fermented peanut meal into feed in a proportion of 5-15 g/100g of feed, in particular 10g/100g of feed.

The beneficial effect of this application:

1. the application makes creative research on exploring the application of different fermentation strains to improving the functional characteristics and the nutritional components of the peanut meal. In the first stage of solid state fermentation process, the content of total flavonoids in the peanut meal is obviously improved through the biotransformation effect of microorganisms, the functional characteristics are improved, meanwhile, by means of a strong extracellular enzyme secretion system of Bacillus belgii LB-Y-1, the protease effectively degrades macromolecules/denatured proteins to generate micromolecule peptides and free amino acids, the phytase degrades phytic acid which is not easy to digest and utilize, the utilization rate of phosphorus is increased, and AFB in the peanut meal is degraded through the phytase ₁ And (4) realizing degradation. Through the second stage of solid state fermentation, pediococcus acidilactici LC-9-1 can further degrade phytic acid, regulate fatty acid spectrum, reduce raw material pollution risk through secreted organic acid and bacteriostatic components on the one hand, and improve the flavor characteristic and functional characteristic of peanut meal at the same time. The functional characteristics and the nutritional characteristics of the peanut meal after the step-by-step fermentation of the Bacillus belgii LB-Y-1 and Pediococcus acidilactici LC-9-1 are obviously improved, and the peanut meal has irreplaceability compared with the step-by-step fermentation under other combination modes of two strains.

2. The functional characteristics and the nutritional characteristics of the peanut meal fermented by the special process are obviously improved, and the method specifically comprises the following steps: the content of the total flavone is increased from 1.15mg/g to 1.97mg/g, the increase is 71.30 percent, and the lipid regulation capability is enhanced; the oxidation resistance is evaluated by DPPH scavenging capacity and hydroxyl radical scavenging capacity, and the performance is respectively improved by 95.63 percent and 102.02 percent; the content of crude protein is increased from 47.55 percent to 52.25 percent and is increased by 9.88 percent; with the prolonging of the fermentation time, the macromolecular protein with the KD of more than 35.0 is obviously degraded, and after the fermentation is started for 24 hours, the protein molecules with the KD of about 17 are also obviously degraded; the content of acid soluble protein is increased from 3.09% to 20.39%, and increased by 559.87%; the total acid content is increased from 0.69% to 3.58%, and increased by 418.84%; lysine, histidine, phenylalanine, leucine, alanine, glycine and proline are all obviously increased in different degrees, and the ratio of arginine to lysine is adjusted from 3.07 to 1.52, which is closer to the ideal amino acid ratio; the total unsaturated fatty acid is increased by 10.17 percent; from specific fatty acid type analysis, excessive C14:0 is not beneficial to health, and is remarkably reduced after fermentation; c14:1 is obviously reduced, C16:1 and C18:1n9C are obviously increased, which is beneficial to positively regulating blood lipid metabolism and protecting vascular endothelium; c18:2n6C (linoleic acid) and C18:3n6 (linolenic acid) are obviously increased, and the body needs fatty acid.

3. The application also finds that the degradation of the toxin mainly occurs in the first stage fermentation aiming at the step-by-step fermentation process of the two bacteria, namely the strain Bacillus beleisis LB-Y-1 plays a main role, and through the treatment process, the aflatoxin B in the peanut meal ₁ The initial concentration is reduced from 84.94 mu g/kg to 44.67 mu g/kg, and the degradation efficiency is 47.41 percent; the phytic acid is used as an anti-nutritional factor with higher content in the peanut meal raw material, and the degradation efficiency of the phytic acid by the process is 45.49%.

4. The abdominal fat percentage of the broiler chickens at 42 days old is reduced by 15.12% compared with that of the group without the fermented feed by adding 10% of the fermented peanut meal into the feed for the broiler chickens; the flavor intensity of the breast muscle is enhanced, the content of intramuscular protein is improved by 6.18 percent compared with that of an unfermented group, the content of total amino acid is improved by 8.81 percent compared with that of an unfermented group, the content of monounsaturated fatty acid is improved by 15.81 percent, and the pH value is reduced and slowed down. In addition, according to the research on the influence of the fermented peanut meal on the breast muscle quality, the pH value of the fermented peanut meal group is normally reduced and stabilized to about 5.80 after being refrigerated for 24 hours, the delta pH value is obviously reduced compared with that of an unfermented peanut meal group and a corn bean meal type daily ration group, and the fermented peanut meal group has a positive effect on prolonging the shelf life. The problem of chicken quality deterioration is improved, and no antibiotic is added in the whole feeding process, so that the feed has good ecological benefit and economic value.

5. It is worth emphasizing that in the prior art group, the addition of the unfermented peanut meal in the daily ration generally recognized by the technical personnel in the field to significantly reduce the content of the breast muscle protein of the broiler chicken, while the addition after the fermentation by the process of the application significantly increases the content of the breast muscle protein; the moisture content and ash content in the meat have no obvious difference in each treatment group; the unfermented peanut meal group with intramuscular fat content is obviously higher than the control group and the fermented peanut meal group, and in poultry, the deposition of excessively high intramuscular fat easily causes lignified meat and white streaked meat, so that the appearance and the taste are greatly influenced, and the trend is relieved by fermentation treatment. Therefore, the application solves the technical difficulties which are always forced to be solved in the technical development of the field, and obtains unexpected technical effects.

6. The fermentation process parameters and the numerical range of the strain are optimized, and are also made by a large number of creative repeated tests of scientific researchers, and the process parameters of the inoculation amount, the water content, the fermentation temperature and the fermentation time are determined mainly through a single-factor experiment, a Plackett-Burman experiment and a Box-Behnken experiment; inoculating a fermentation inoculation liquid into sterilized peanut meal, taking the water content, the inoculation amount, the fermentation time and the fermentation temperature as independent variables, taking the TCA-N content (stage one) and the total acid content (stage two) as dependent variables, firstly determining the independent variable selection range of the subsequent test and the extreme value of a Plackett-Burman test through a single-factor experiment, fully mixing according to various parameters, and fermenting under aerobic or anaerobic conditions; and designing various parameters of Plackett-Burman according to the results of the single-factor test, performing the fermentation operation flow and the single-factor test, analyzing and judging independent variables which have obvious influence on the content of TCA-N or total acid according to the standard normal effect and the pareto chart, and only considering the independent variables which have obvious influence in the design of the Box-Behnken test.

7. The stepwise fermentation of the application has irreplaceability, and scientific researchers try to change the sequence of strains for stepwise fermentation or simultaneously use two strains for fermentation together, which have great influence on the final fermentation depth. The effect is better after the stepwise fermentation process is combined, the Bacillus beilaisi LB-Y-1 is inoculated in the first stage, the Pediococcus acidilactici LC-9-1 is inoculated in the second stage, and finally the TCA-N yield is higher and the reaction depth is optimal.

Drawings

FIG. 1A stage-fermentation Single-factor test of the Effect of different fermentation parameters (inoculum size, fermentation time, moisture content, fermentation temperature) on the TCA-N content

FIG. 2 Effect of different fermentation parameters of stage one fermentation Plackett-Burman on the TCA-N content (residual and normalized Effect plot)

FIG. 3 Effect of different fermentation parameters of the stage-one fermentation Box-Behnken on the TCA-N content

FIG. 4 shows the influence of different fermentation parameters (inoculum size, fermentation time, moisture content, fermentation temperature) on the total acid content in a two-stage fermentation single-factor test

FIG. 5 Effect of different fermentation parameters of the stage two fermentation Plackett-Burman on the Total acid content (residual and normalized Effect plot)

FIG. 6 Effect of different fermentation parameters of Box-Behnken on Total acid content in stage two fermentation

FIG. 7 Effect of fermentation Process on Total Flavonoids content in peanut meal (PNM: unfermented peanut meal, FPNM 1: fermentation stage one, FPNM 2: fermentation stage two)

FIG. 8 shows the effect of fermentation on the antioxidant activity of peanut meal (A: DPPH scavenging ability, B: hydroxyl radical scavenging ability)

FIG. 9 Effect of fermentation Process on peanut meal protein molecular weight

FIG. 10 Effect of fermentation Process on amino acid profiles of peanut meal (A: free amino acids, B: hydrolyzed amino acids, PNM: peanut meal, FPNM: fermented peanut meal)

FIG. 11 fermentation Process on AFB in peanut meal ₁ And influence of phytic acid content

FIG. 12 shows the effect of fermented peanut meal on the flavor profile of breast muscle (CON-basic ration group, PNM-peanut meal group, FPNM-fermented peanut meal group, AGPs-antibiotic control group)

FIG. 13 shows the effect of fermented peanut meal on the flavor profile of breast muscle (CON: basic ration group, PNM: peanut meal group, FPNM: fermented peanut meal group; DM: dry matter, Total protein: crude protein, IMF: intramuscular fat, Ash; none of the common superscripts in rows a-d indicates significant difference (P < 0.05))

FIG. 14 Effect of fermented peanut meal on thoracic muscle pH (CON-basal diet group, PNM-peanut meal group, FPNM-fermented peanut meal group; none of the common superscripts in rows a-d indicates significant differences (P < 0.05))

Deposit description

The name of Chinese: bacillus belgii

Latin name: bacillus velezensis

The strain number is as follows: LB-Y-1

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 12 month and 10 days of 2020

Registration number of the preservation center: CGMCC No. 21344.

The name of Chinese: pediococcus acidilactici

Latin name: pediococcus acidilactaciii

The strain number is as follows: LC-9-1

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 12 month and 10 days of 2020

Registration number of the preservation center: CGMCC No. 21345.

Detailed Description

The following examples are disclosed to facilitate an understanding of the present application and are not intended to limit the present application, and persons skilled in the art may make appropriate optimization and adjustment of process parameters according to the purpose of the application, and it is to be particularly understood that all such modifications and substitutions will be apparent to those skilled in the art, and that the methods and applications of the present application are described in the examples and can be fully adjusted or appropriately substituted by those skilled in the art without departing from the scope, spirit and scope of the present application.

Culture medium referred to in this application (sterilized at 121 ℃ for 20min before use):

LB liquid Medium (/ L): 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride, and the pH is adjusted to 7.2-7.4.

LB solid Medium (/ L): 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride and 15.0g of agar powder, and the pH value is adjusted to 7.2-7.4.

MRS liquid medium (/ L): 10.0g of peptone, 5.0g of beef powder, 20.0g of glucose, 4.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 0.2g of magnesium sulfate, 2.0g of triammonium citrate, 0.05g of manganese sulfate and 801 ml of Tween, and the pH value is adjusted to 7.2-7.4.

MRS solid medium (/ L): 10.0g of peptone, 5.0g of beef powder, 20.0g of glucose, 4.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 0.2g of magnesium sulfate, 2.0g of triammonium citrate, 0.05g of manganese sulfate, 801 ml of Tween and 15.0g of agar powder, and the pH value is adjusted to 7.2-7.4.

The following examples will further illustrate the present application:

example 1

A production method of peanut meal by stepwise fermentation of Bacillus belgii and Pediococcus acidilactici comprises the following steps,

(1) pretreatment of raw materials: pulverizing peanut meal, sieving with 18 mesh sieve, sterilizing with high pressure steam at 121 deg.C for 15min, and cooling to room temperature;

(2) b, activating the Bacillus beiLeisi LB-Y-1: inoculating LB-Y-1 strain preserved in glycerol on an LB solid medium plate, performing inverted culture at 37 ℃ for 18h, picking out a single colony by using a disposable inoculating loop, inoculating the single colony in an LB liquid medium, performing culture at 37 ℃ and 160rmp/min for 24h to obtain a seed solution, continuously inoculating the seed solution in a new LB liquid medium according to 0.5 percent of inoculation amount, performing amplification culture at 37 ℃ and 160rmp/min, and obtaining a liquid LB-Y-1 strain with the concentration of more than or equal to 3.0 x 10 ⁸ CFU/mL, namely the fermentation inoculation liquid of the first stage;

activation of Pediococcus acidilactici LC-9-1: inoculating the LC-9-1 strain preserved in glycerol on an MRS solid culture medium plate, performing inverted culture at 37 ℃ for 24h, selecting a single colony by using a disposable inoculating loop, inoculating the single colony in an MRS liquid culture medium, performing static culture at 37 ℃ for 24h to obtain a seed solution,continuously inoculating to new MRS liquid culture medium at 1.0%, standing at 37 deg.C for amplification culture to obtain liquid LC-9-1 with thallus concentration of 1.0 × 10 ⁸ CFU/mL is the second stage fermentation inoculation liquid;

(3) stage one fermentation: preparing an activated starting strain fermentation inoculation liquid to be inoculated into sterilized peanut meal, and the final process comprises the following steps: the inoculation amount is 2.0 percent, the water content is 37.38 percent, the materials are fully and uniformly mixed, the fermentation temperature is 38.18 ℃, the fermentation time is 54.41 hours, the stirring is carried out once every 4 hours, the aerobic environment is kept, and the first-stage fermentation is carried out;

(4) adjusting discharging: uniformly mixing the fermented materials, and discharging;

(5) and (3) stage two fermentation: preparing an activated starting strain fermentation inoculum, inoculating the activated starting strain fermentation inoculum to the produced material, wherein the inoculum size is 1.89%, the water content is 40.00%, fully and uniformly mixing, the fermentation temperature is 37.11 ℃, the fermentation time is 12.42h, subpackaging to breathing bags, keeping an anaerobic environment, and carrying out second-stage fermentation.

(6) Adjusting discharging: and after the fermentation is finished, uniformly mixing, drying at a low temperature, controlling the drying temperature to be 48 ℃, ensuring that the moisture of the final product is controlled to be 10.0%, crushing the materials, sieving the crushed materials by a 18-mesh sieve, uniformly mixing, and discharging to obtain the final fermentation product.

Example 2

The fermented peanut meal prepared by the method for producing the stepped fermented peanut meal by using the bacillus belgii and pediococcus acidilactici of example 1.

Example 3

The fermented peanut meal of example 2 is applied to improving the content of total flavonoids, the antioxidant performance, the content of crude protein, the content of acid-soluble protein and the content of total acids in the peanut meal, and optimizing the amino acid spectrum and the arginine/lysine ratio.

Example 4

The fermented peanut meal of embodiment 2 is used for optimizing a fatty acid spectrum and improving the contents of linoleic acid and linolenic acid.

Example 5

Example 2 fermentation of peanut meal in degrading AFB in peanut meal ₁ Or in phytic acidThe use of (1).

Example 6

The use of the fermented peanut meal of example 2 to reduce the fat content of broiler belly and/or improve chicken quality.

Example 7

The fermented peanut meal of embodiment 2 is applied to improve the flavor characteristics of the breast muscles of broiler chickens, the contents of intramuscular proteins and amino acids, optimize fatty acid spectra or prolong the counter period of the breast muscles.

Example 8

The feed prepared from the fermented peanut meal in the embodiment 2 is prepared by adding the fermented peanut meal into the feed in a ratio of 10g/100 g.

Comparative example 1 transverse comparison experiment of different preparation methods of fermented peanut meal, i.e. comparison of two strains of simultaneous/stepwise fermentation effects

Activated starting strains, Bacillus belgii LB-Y-1 and Pediococcus acidilactici LC-9-1, were prepared as in example 1. Synchronous fermentation, namely inoculating two fermentation strains at the same time, and fractional fermentation, namely sequentially inoculating the two fermentation strains at different stages of fermentation. The specific scheme is that the method is operated according to the table 1, the moisture content, the inoculation amount, the fermentation time and the fermentation temperature are set according to the process parameters of the table 1, the raw materials are fully mixed, the materials are turned once every 4 hours, the materials are uniformly mixed and discharged after the fermentation reaches the preset time, the acid soluble protein (TCA-N) content after the fermentation is finished is used as an evaluation index, and the fermentation method adopted in the next step is judged.

TABLE 1 influence of different fermentation methods on TCA-N content of peanut meal

According to the fermentation result, the sequence of synchronous/step-by-step fermentation and strain inoculation has great influence on the final TCA-N yield. The method adopts step-by-step fermentation, and the Bacillus beiLeisi LB-Y-1 is inoculated in the first stage, and the Pediococcus acidilactici LC-9-1 is inoculated in the second stage, so that the yield of TCA-N is higher and the reaction depth is optimal finally, and the preparation method is adopted in the subsequent process.

Experiment one: preparation method of fermented peanut meal and staged fermentation process

The experimental method comprises the following steps: in the first stage of fermentation, an activated starting strain is prepared, Bacillus beiLeisi LB-Y-1 fermentation inoculum is inoculated into sterilized peanut meal, the water content, the inoculation amount, the fermentation time and the fermentation temperature are used as independent variables, the TCA-N content is used as a dependent variable, firstly, the independent variable selection range of the subsequent test and the extreme value of the Plackett-Burman test are determined through a single-factor experiment, the materials are fully mixed according to the parameters in the table 2, the fermentation is carried out under the aerobic condition, the materials are turned over once every 4h, and the materials are uniformly mixed and adjusted to be discharged after the fermentation reaches the preset time.

TABLE 2 stage-fermentation single factor test parameter design

The experimental results are as follows: the results of the single factor test are shown in FIG. 1: the range of the inoculation amount is 2.0-6.0%, and the content of TCA-N at the fermentation end point among different inoculation amounts does not fluctuate greatly; along with the extension of the fermentation time, the content of TCA-N in the peanut meal is obviously increased after 12h and tends to be stable after 48 h; with the increase of the moisture content, the TCA-N content is increased and then decreased, and reaches the highest point at 40 percent of moisture content; the TCA-N content peaks at a fermentation temperature of 37 ℃.

Various parameters of Plackett-Burman are designed according to the single-factor test result, the range of the inoculation amount parameter is 2.0-6.0%, the water content is 32.5-40.0%, the fermentation temperature is 34.0-40.0 ℃, the fermentation time is 38-58 h, the fermentation operation flow is the same as the single-factor test, the result is shown in Table 3, the influence of the inoculation amount on the yield of TCA-N is not significant (P is more than 0.05), and the influence of the variation of the other three factors on the content of TCA-N is large.

TABLE 3 screening of the Effect of variables on TCA-N production Using Plackett-Burman

Similarly, the independent variable that significantly affects the TCA-N content is the moisture content (X) as determined by standard normal effect and pareto chart analysis (FIG. 2) ₂ ) Fermentation temperature (X) ₃ ) And fermentation time (X) ₄ ) And the amount of inoculation (X) ₁ ) The effect of (a) was not significant, so only three other factors were considered in the Box-Behnken test design.

Comparative example 2 preparation method of fermented peanut meal, stage one fermentation Process

Based on the results of the experiment, the three independent variables of moisture content, fermentation temperature and fermentation time were performed according to the process parameters in table 4, and the results are also shown in table 4, with the TCA-N content as the response value.

TABLE 4 Effect of different fermentation methods on TCA-N content of peanut meal

And (3) performing regression fitting analysis on the data in the table 4 by adopting Design-Expert 7.0 statistical analysis software to obtain final response results of the response values, namely influence factors of TCA-N content (Y) in the peanut meal, namely fermentation temperature (A), moisture content (B) and fermentation time (C) (shown in figure 3). Predicting the optimal fermentation process of the first stage: inoculation amount: 2.0%, fermentation temperature: 38.18 ℃, moisture content: 37.38%, fermentation time: 54.41h, under which conditions the TCA-N yield is predicted to be: 20.55 percent.

Stage one verification test:

after inoculation amount: 2.0%, fermentation temperature: 38.18 ℃, moisture content: 37.38%, fermentation time: 54.41h, the TCA-N content after the first stage actual fermentation is: 19.98. + -. 0.35%, close to the predicted value of the Box-Behnken test.

Experiment two: preparation method of fermented peanut meal and stage two fermentation process

The experimental method comprises the following steps: after the first fermentation stage is finished, inoculating the activated Pediococcus acidilactici LC-9-1 fermentation inoculum into the peanut meal fermented in the first fermentation stage, taking the water content, the inoculum size, the fermentation time and the fermentation temperature as independent variables, taking the total acid content as a dependent variable, firstly determining the independent variable selection range of the subsequent test and the extreme value of the Plackett-Burman test through a single-factor experiment, fully mixing according to the parameters in the table 5, weighing 2.5kg, putting the weighed materials into a 5kg breathing bag, sealing for the second time to ensure no air circulation, and fermenting under the anaerobic condition. Taking out the fermented product from the fermentation bag after the fermentation reaches the preset time, uniformly mixing the fermented product after the fermentation is finished, drying the fermented product at a low temperature, controlling the drying temperature to be 48 ℃ under the condition of 45-50 ℃, ensuring that the moisture of the final product is controlled to be 9.5-10.5 percent, specifically 10.0 percent, crushing the material, sieving the crushed material by a 18-mesh sieve, uniformly mixing the crushed material and the sieved material, and then discharging the material to obtain the final fermented product.

Table 5 design of two-stage fermentation single-factor test parameters

The experimental results are as follows: the results of the single factor test are shown in FIG. 4: the range of the inoculation amount is 0.5-3.0%, the total acid content of the fermentation end point between different inoculation amounts is increased along with the increase of the inoculation amount and is stable after 2.0%; along with the prolonging of the fermentation time, the total acid content in the peanut meal is obviously increased after 4 hours and tends to be stable after 12 hours; with the increase of the moisture content, the total acid content has no obvious fluctuation; at a fermentation temperature of 37 ℃, the total acid content reaches the highest point, and above or below the temperature, the total acid content is reduced to different degrees at the end of the fermentation.

Various parameters of Plackett-Burman are designed according to the single-factor test results, the range of the inoculation amount parameter is 0.5-2.0%, the water content is 40.0-47.5%, the fermentation temperature is 33.0-39.0 ℃, the fermentation time is 8-14 h, the two-stage single-factor test is carried out in the same fermentation operation flow, the results are shown in Table 6, the influence of the water content on the yield of the final total acid is not significant (P is more than 0.05), and the influence of the variation of the other three factors on the total acid content in the peanut meal is large.

TABLE 6 screening of the Effect of variables on Total acid production Using Plackett-Burman

Similarly, the independent variable that significantly affected total acid content was the inoculum size (X) as determined by standard normal effect and pareto chart analysis (fig. 5) ₁ ) Fermentation temperature (X) ₃ ) And fermentation time (X) ₄ ) And water content (X) ₂ ) The effect of (a) was not significant, so only three other factors were considered in the Box-Behnken test design.

Comparative example 3 preparation method of fermented peanut meal, stage two Process

Based on the results of the second experiment, the water content, fermentation time and fermentation temperature were varied according to the process parameters in table 7, and the total acid content was also used as a response value, and the results are also shown in table 7.

TABLE 7 influence of different fermentation methods on the total acid content of peanut meal

And (3) performing regression fitting analysis on the data in the table 7 by adopting Design-Expert 7.0 statistical analysis software to obtain final response results of the response values, namely the influence factors of the total acid content (Y) in the peanut meal, namely the fermentation temperature (A), the inoculation amount (B) and the fermentation time (C) (shown in figure 6). Predicting the optimal fermentation process in the second stage: inoculation amount: 1.89%, temperature: 37.11 ℃, moisture content: 40.00%, time: 12.42h, prediction of total acid yield under the condition: 3.71 percent.

And (3) stage two verification test:

in the inoculation amount: 1.89%, temperature: 37.11 ℃, moisture content: 40.00%, time: under the process of 12.42h, the total acid content after the actual fermentation of the second stage is as follows: 3.58 +/-0.25%.

Experiment three: influence of fermentation process on functional components and oxidation resistance of peanut meal

The experimental method comprises the following steps: fermented peanut meal prepared by the final process identified in comparative example 2 and comparative example 3. In the determination of the content of the total flavonoids, according to a method of aged sea arone (sea buckthorn leaf total flavonoids extraction process research [ J ], northwest agriculture report, 2006, 15 (1): 33-36.), 95% ethanol is used for extracting the total flavonoids of the peanut meal, rutin is used as a standard sample for drawing a standard curve, the absorbance is determined, and the content of the total flavonoids is calculated. The DPPH radical scavenging ability was measured by referring to a method described in the DPPH radical scavenging ability assay kit-visible spectrophotometry (cat # BC4750, standard: 50T/24S).

Measurement of hydroxyl radical scavenging ability was carried out by referring to the method described in the hydroxyl radical scavenging ability test kit (cat. No. BC1320, specification: 50T/24S).

The experimental results are as follows:

(1) influence of fermentation process on functional components of peanut meal

Flavonoid (flavanones) generally refers to a series of compounds formed by connecting two benzene rings through three carbon atoms, has the effects of resisting free radicals and oxidation, can remarkably improve the production performance of animals, regulate lipid metabolism, reduce liver fat accumulation and improve the immune function, the influence of fermentation on the content of total Flavonoids in peanut meal is shown in figure 7, the content of the total Flavonoids is remarkably increased (P is less than 0.05) after fermentation, the change mainly occurs in a first fermentation stage and is not remarkable in a second fermentation stage, and under the process, the content of the total Flavonoids is increased from 1.15mg/g to 1.97mg/g and is increased by 71.30%.

(2) Influence of fermentation process on oxidation resistance of peanut meal

The influence of fermentation on the oxidation resistance of the peanut meal is shown in fig. 8, the oxidation resistance is gradually enhanced along with the extension of the fermentation time, the oxidation resistance tends to be stable after the two-stage fermentation process treatment, and the oxidation resistance is respectively improved by 95.63% and 102.02% by evaluating with two methods of DPPH (dehydroepiandrosterone) scavenging capacity and hydroxyl radical scavenging capacity.

Experiment four: influence of fermentation process on conventional nutrient components of peanut meal

The experimental method comprises the following steps: fermented peanut meal prepared by the final process identified in comparative example 2 and comparative example 3. Wherein,

moisture content: the method is implemented according to the method specified in GB/T6435-2014 (measurement of the water content in the feed);

crude protein: the method is implemented according to the method specified in GB/T6432-2018 (Kjeldahl method for measuring crude protein in the feed);

acid soluble protein: the method is implemented according to the method specified in NY/T3801-2020 (determination of acid soluble protein in feed raw materials);

crude fat: according to the method specified in GB/T6433-2006 (measurement of crude fat in feed);

energy value: by IKA-C2000-Basic type full-automatic oxygen bomb calorimeter (

-Werke GmbH&Staufen/germany) operation;

total acid: according to the method specified in GB 12456-2021 (determination of total acid in food);

coarse ash content: according to the method specified in GB/T6438-2007 (measurement of crude ash in feed);

SDS-PAGE: extracting fermented peanut meal protein by a denaturation and heating combined method, performing according to a method friendly to the whole process (microbial solid state fermentation of bean cypress and sensitization research thereof [ Master thesis ]: Nanchang university), and purchasing a separation gel and a concentrated gel pre-prepared solution from Bio-rad (USA), wherein the specific operation steps comprise gel preparation, sample loading, electrophoresis, dyeing and decoloration, and finally, the method is carried out until protein strips are clear;

amino acids: according to the method specified in GB/T18246-2019 (determination of amino acids in feed);

fatty acid: according to the method specified in GB/T21514-2008 (determination of fatty acid content in feed);

the experimental results are as follows:

(1) influence of fermentation process on conventional nutrient components of peanut meal

The influence of fermentation on the conventional nutrition of the peanut meal is shown in table 8, and indexes with significant changes comprise crude protein, acid-soluble protein and total acid content which are respectively improved by 9.88%, 559.87% and 418.84% compared with the content before fermentation.

TABLE 8 Effect of fermentation Process on conventional nutrient composition of peanut meal

Note: the absence of common superscripts in rows a-d indicates significant differences (P < 0.05);

(2) influence of fermentation process on molecular weight of peanut meal protein

The influence of fermentation on the molecular weight of peanut meal protein is shown in fig. 9, with the extension of fermentation time, macromolecular protein with a molecular weight of more than 35.0KD is obviously degraded, and after the fermentation is started for 24 hours, protein molecules with a molecular weight of about 17KD are also obviously degraded.

(3) Influence of fermentation process on amino acid spectrum of peanut meal

The influence of fermentation on amino acids of peanut meal is shown in fig. 10, and the amino acid composition changes greatly before and after fermentation. Free amino acids: the other amino acids except arginine and glutamic acid are maintained at a lower level before fermentation, and the content of arginine is reduced and the other amino acids are increased to different degrees after the fermentation by the process; and (3) hydrolysis of amino acid: lysine, histidine, phenylalanine, leucine, alanine, glycine and proline are all obviously increased to different degrees (P is less than 0.05), the content of arginine and serine is obviously reduced, and the ratio of arginine to lysine is adjusted to 1.52 from the initial 3.07, which is more close to the proportion of ideal amino acid.

(4) Influence of fermentation process on fatty acid spectrum of peanut meal

The influence of fermentation on fatty acids of peanut meal is shown in table 9, and the composition of fatty acids before and after fermentation is greatly changed. From classification, saturated fatty acid, monounsaturated fatty acid and polyunsaturated fatty acid are all improved remarkably to different degrees; from specific fatty acid type analysis, saturated fatty acid C14:0 (myristic acid) is remarkably reduced after fermentation, and excessive intake of the fatty acid can cause the increase of cholesterol in serum of a body, so that the health is not facilitated; the monounsaturated fatty acid C14:1 is obviously reduced, C16:1 and C18:1n9C are obviously increased, and the change can positively regulate the blood lipid metabolism, protect the vascular endothelium and reduce the high blood coagulation state; the content of C18:2n6C (linoleic acid) and C18:3n6 (linolenic acid) in the polyunsaturated fatty acid is obviously increased as the essential fatty acid for the body.

TABLE 9 influence of fermentation Process on fatty acid Profile of peanut meal

Note: "-" indicates that the content is too low or not detected; the absence of common superscripts in rows a-d indicates significant differences (P ≦ 0.05); SEM ═ standard error experiment five: fermentation process for reducing aflatoxin B of peanut meal ₁ (AFB ₁ ) And influence of phytic acid content

The experimental method comprises the following steps: fermented peanut meal prepared by the final process identified in comparative example 2 and comparative example 3. AFB ₁ : according to GB/T36858-2018 (aflatoxin B in feed) ₁ Measurement of high performance liquid chromatography) of the above-mentioned step; phytic acid: the method was carried out according to the method specified in the Phytic Acid (Total Phophorus) Assay Kit (Megazyme Company, Bray/Ireland).

The experimental results are as follows:

fermentation on AFB in peanut meal ₁ The effect of (2) is shown in fig. 11, the degradation of toxins mainly occurs in the first stage of fermentation, i.e. the strain bacillus beleisi LB-Y-1 plays a major role, and through the treatment process, AFB in peanut meal ₁ The initial concentration is reduced from 84.94 mu g/kg to 44.67 mu g/kg, and the degradation efficiency is 47.41 percent; the phytic acid is used as an anti-nutritional factor with higher content in the peanut meal raw material, the degradation effect mainly occurs in the second fermentation stage, and the degradation efficiency of the phytic acid is 45.49% by the process.

Experiment six: application of process-fermented peanut meal in improving meat quality of broiler chickens and reducing abdominal fat percentage

The experimental method comprises the following steps: fermented peanut meal prepared by the final process identified in comparative example 2 and comparative example 3. 180 healthy AA young cocks of 1 day age are selected and randomly divided into 3 treatment groups, each treatment group has 6 repetitions, each treatment group has 10 repetitions, and each group is respectively used for feeding corn-soybean meal type basic feed for a control group (CON), a peanut meal group (PNM, 10% additive amount), a fermented peanut meal group (FPNM, 10% additive amount), an antibiotic group (AGPs, with the addition of combined antibiotics aureomycin 75mg/kg, flavomycin 5mg/kg and kitasamycin 20mg/kg), which are all added according to the weight of the basic feed, and the test period is 42 d. The basic daily ration formula design refers to chicken feeding standard (NY/T33-2004), and the raw material composition and nutrition level are shown in Table 10.

TABLE 10 Experimental diet composition and Nutrition level (air-dried basis)

Note: the vitamin and trace element premix is provided for each kilogram of feed: vitamin A10000 IU, vitamin D ₃ 2000 IU, vitamin E10 IU, vitamin K ₃ 2.5mg, vitamin B ₁ 1mg of vitamin B ₂ 6mg of vitamin B ₃ 10mg of vitamin B ₅ 40mg of vitamin B ₆ 3mg of vitamin B ₁₁ 0.3mg, vitamin B ₁₂ 0.01mg, biotin 0.12mg, Cu (copper sulfate) 8mg, Fe (ferrous sulfate) 80mg, Mn (manganese sulfate) 60mg, Zn (zinc sulfate) 40mg, Se (sodium selenite) 0.15mg, I (potassium iodide) 0.35 mg; 2) the crude protein in the nutrition level is the measured value, the rest is the calculated value, and the amino acid is the total content.

Slaughter performance: at the age of 42 days, 1 repeated randomly is selected, the carcass weight is weighed after slaughtering after weighing live weight, and the carcass weight is weighed after full bore, half bore, leg muscle, pectoral muscle and abdominal fat weight are measured in dissection mode.

Calculating the formula:

slaughter (%) — carcass weight × 100;

the total open weight (%) -, total open weight/live weight × 100;

half bore ratio (%) ═ half bore weight/live weight × 100;

leg muscle ratio (%) — leg muscle weight/total bore weight × 100;

breast muscle rate (%) — breast muscle weight/total open weight × 100;

abdominal fat percentage (%). abdominal fat weight/(total dry weight + abdominal fat weight) × 100

Flavor characteristics of breast muscle:

the electronic nose is a low-cost and rapid detection instrument for simulating human smell. It consists essentially of a series of sensors sensitive to chemical composition, combined with multivariate statistical methods to measure volatile compounds in the headspace of a sample.

Sample pretreatment: the method comprises the steps of rapidly cutting a fresh chicken breast sample into meat particles with the side length of about 2mm at room temperature, weighing 10g of the meat particles in a 100mL conical flask, sealing the meat particles by using a 3-layer preservative film, carrying out water bath in a water bath kettle at 60 ℃ for 5min, taking out the meat particles, blowing headspace gas of the sample into an electronic nose system by using air filtered by activated carbon, and carrying out detection by using a PEN3 portable electronic nose and a W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2S, W2W and W3S sensor array (Airsense Company, Germany), wherein the specific sensor array and performance characteristics of the sensor array are shown in Table 11.

TABLE 11 electronic nose sensor array and Performance characteristics thereof

Breast muscle nutritional properties:

measuring the intramuscular water content: the method is carried out according to the method specified in GB 5009.3-2016 (measurement of moisture in food);

intramuscular fat content determination: the method was carried out according to the method prescribed in GB 5009.6-2016 (determination of fat in food);

intramuscular protein content determination: the method is carried out according to the method specified in GB 5009.5-2016 (determination of protein in food);

measuring the amino acid content of the breast muscle: the method is carried out according to the method specified in GB 5009.124-2016 (determination of amino acids in food);

measuring the content of fatty acid in the chest muscle: the method is carried out according to the method specified in GB 5009.168-2016 (determination of fatty acids in food); change of pH of thoracic muscle:

the PH of the breast muscle was measured at 45min and 24h post-slaughter using a calibrated PHS-25 digital acidimeter (r. mathhaus Company, PH-STAR, germany) with an accuracy of 0.01 and the difference Δ PH was calculated in parallel three times for each sample.

And (3) statistics and analysis: after the collected data are preliminarily arranged by Excel, one-way ANOVA one-factor analysis of variance method of SPSS 21.0 software is adopted for processing, Duncan's is selected for multiple comparison, the result is expressed by processing mean and mean Standard Error (SEM), and the difference probability value P <0.05 between different processing is determined as significant difference.

The experimental results are as follows:

(1) influence of fermented peanut meal on slaughtering performance of broiler chickens

The influence of the fermented peanut meal on the slaughtering performance of the broiler is shown in table 12, and the abdominal fat rate of the broiler is remarkably reduced (P is less than 0.05) by adding 10% of the fermented peanut meal into the feed.

TABLE 12 influence of fermented peanut meal on slaughter Performance of broiler chickens

Note: CON is basic daily ration group, PNM is peanut meal group, FPNM is fermented peanut meal group; the absence of common superscripts in rows a-d indicates significant differences (P < 0.05); SEM ═ standard error

(2) Influence of fermented peanut meal on flavor characteristics of breast muscle

Fig. 12 shows that the contribution rates of the 1 st principal component (PC1) and the 2 nd principal component (PC2) of the breast muscle are 88.52% and 9.24%, respectively, and the sum of the contribution rates is 97.76%, and the total contribution rate is higher than 85.00%, so that the characteristic flavor can be distinguished by using an electronic nose, and the analysis of the result after the distinction has certain significance. From the PCA results, the sensors W5C (sensitive to alkanes, aromatic compounds and weakly polar compounds), W1W (sensitive to inorganic sulfides), W2S (sensitive to alcohols, aldehydes, ethers and the like), W2W (sensitive to aromatic compounds and organic sulfides) and W3S (sensitive to alkanes) of the fermented peanut meal group are increased compared with the control group, which indicates that the content of the flavor substances corresponding to the sensors is increased, the flavor intensity of chicken is increased, and the flavor profile is improved.

(3) Influence of fermented peanut meal on breast muscle nutritional characteristics

The influence of the fermented peanut meal on the conventional composition of the breast muscle nutrition is shown in figure 13, the addition of the peanut meal in the daily ration obviously reduces the content of the breast muscle protein, and the addition after the fermentation of the process obviously increases the content of the breast muscle protein (P < 0.05); the moisture content and ash content in the meat have no obvious difference in each treatment group; the group of unfermented peanut meal with intramuscular fat content is obviously higher than that of the control group and the group of fermented peanut meal (P is less than 0.05), while in poultry, the excessively high intramuscular fat deposition easily causes lignified meat and white streaked meat, so that the appearance and the taste are greatly influenced, and the trend is relieved by fermentation treatment.

The influence of the fermented peanut meal on the amino acid composition of breast muscle of the broiler is shown in table 13, the total amount of amino acid in the breast muscle is obviously improved (P is less than 0.05) by replacing 10% of basic ration with the fermented peanut meal, and meanwhile, the content of essential amino acid such as arginine, isoleucine, methionine, phenylalanine and threonine in the breast muscle is obviously improved. The unfermented peanut meal group with the lysine content is obviously lower than the control group, but the content in the fermented peanut meal group is improved, and has no obvious difference with the control group. In the aspect of non-essential amino acids, the content of aspartic acid and proline in the fermented peanut meal group is obviously higher than that in the unfermented peanut meal group (P <0.05), and the content of glutamic acid in the fermented peanut meal group is obviously higher than that in the control group (P < 0.05).

TABLE 13 influence of fermented peanut meal on amino acid profile of breast muscle

Note: amino acid concentrations are expressed as percentage (%) of total identified amino acids, data are the average of 6 chickens per treatment; CON is basic daily ration group, PNM is peanut meal group, FPNM is fermented peanut meal group; the absence of common superscripts in rows a-d indicates significant differences (P < 0.05); SEM ═ standard error

The influence of the fermented peanut meal on the fatty acid spectrum of the breast muscle is shown in Table 14, compared with a control group, the content of monounsaturated fatty acid in the breast muscle is obviously improved (P is less than 0.05) by replacing basic ration with 10% of fermented peanut meal, the content of polyunsaturated fatty acid is obviously reduced (P is less than 0.05), the content of fatty acid C14:1 and C18:1n9 is obviously increased (P is less than 0.05), the content of C24:1 and C20:2, the content of C20:4n6 and the content of C22:6n3 are obviously reduced (P is less than 0.05), and the ratio of omega-6 and omega-6/omega-3 is obviously reduced (P is less than 0.05) in the 10% fermented peanut meal replacement group. Monounsaturated fatty acids help to reduce cholesterol, triglycerides and low density lipoprotein cholesterol (LDL-C) and do not have the lipid peroxidation problems that polyunsaturated fatty acids are prone to.

TABLE 14 Effect of fermented peanut meal on pectoral muscle fatty acid Profile

Note: fatty acid concentrations are expressed as a percentage (%) of total identified fatty acids, and data is the average of 6 chickens per treatment; SFA ═ saturated fatty acids, MUFA ═ monounsaturated fatty acids, PUFA ═ polyunsaturated fatty acids; CON is basic daily ration group, PNM is peanut meal group, FPNM is fermented peanut meal group; the absence of common superscripts in rows a-d indicates significant differences (P < 0.05); SEM ═ standard error

(4) Influence of fermented peanut meal on change of pH value of breast muscle

The change of the pH value of the muscle reflects the change of the quality of the meat and also influences the shelf life of the meat. If the pH of the pulp drops too quickly after slaughter, the meat becomes juicy, pale and poor in flavour and water binding (PSE meat). The influence of the fermented peanut meal on the quality of the breast muscle is shown in fig. 14, the pH value of the fermented peanut meal group is normally reduced and stabilized at about 5.80 after being refrigerated for 24 hours, the delta pH is obviously reduced compared with that of an unfermented peanut meal group and a control group (P is less than 0.05), and the fermented peanut meal group has a positive effect on prolonging the shelf life of the breast muscle.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (8)

1. A production method for stepwise fermenting peanut meal by using Bacillus belgii and Pediococcus acidilactici is characterized by comprising the following steps of: comprises the following steps of (a) carrying out,

(1) pretreatment of raw materials: pulverizing peanut meal, sieving, sterilizing with high pressure steam, and cooling to room temperature;

(2) stage one fermentation: preparing an activated starting strain fermentation inoculum to inoculate sterilized peanut meal, wherein the inoculum size is 2.0-4.0%, the water content is 36.0-42.0%, the inoculation amount is fully and uniformly mixed, the fermentation temperature is 36.0-39.0 ℃, the fermentation time is 48-60 h, an aerobic environment is kept, and the first-stage fermentation is carried out; the fermentation strain in the first stage is Bacillus belgii (Bacillus belgii)Bacillus velezensis) LB-Y-1, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC number 21344;

(3) adjusting discharging: uniformly mixing the fermented materials, and discharging;

(4) and (3) stage two fermentation: preparing an activated starting strain fermentation inoculum to be inoculated to the produced material, wherein the inoculum size is 1.8-3.3%, the water content is 39.0-46.0%, the mixture is fully and uniformly mixed, the fermentation temperature is 35.0-39.0 ℃, the fermentation time is 10-16 h, the mixture is subpackaged to a breathing bag, an anaerobic environment is kept, and the second-stage fermentation is carried out; the fermentation strain in the second stage is Pediococcus acidilacticiPediococcus acidilactici) LC-9-1, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC number 21345;

(5) adjusting discharging: and after the fermentation is finished, uniformly mixing, drying at a low temperature of 45-50 ℃ to ensure that the moisture of the final product is controlled to be 9.5-10.5%, crushing and sieving the materials, uniformly mixing, and discharging to obtain the final fermentation product.

2. The production process as claimed in claim 1, characterized in that: the pretreatment of the raw materials in the step (1): crushing and sieving the peanut meal, wherein the mesh number is 10-20 meshes, sterilizing for 15min by using high-pressure steam at 121 ℃, and cooling to room temperature after finishing sterilization; the step (5) of adjusting discharging: and after the fermentation is finished, uniformly mixing, drying at a low temperature, controlling the drying temperature to be 48 ℃, ensuring that the moisture of the final product is controlled to be 10.0%, crushing the materials, sieving the crushed materials by a 18-mesh sieve, uniformly mixing, and discharging to obtain the final fermentation product.

3. The production process as claimed in claim 1, characterized in that: the steps (2) and (4) are to clarify the process parameters of the inoculation amount, the water content, the fermentation temperature and the fermentation time through a single-factor experiment, a Plackett-Burman experiment and a Box-Behnken experiment; inoculating a fermentation inoculation liquid into sterilized peanut meal, taking the water content, the inoculation amount, the fermentation time and the fermentation temperature as independent variables, taking the TCA-N content in the first stage and the total acid content in the second stage as dependent variables, firstly, determining the independent variable selection range of the subsequent test and the extreme value of a Plackett-Burman test through a single-factor experiment, fully mixing according to various parameters, and fermenting under the aerobic condition of the first stage and the anaerobic condition of the second stage; and designing various parameters of Plackett-Burman according to the results of the single-factor test, performing the fermentation operation flow and the single-factor test, analyzing and judging according to a standard normal effect and a pareto chart, wherein independent variables which have obvious influence on the content of TCA-N or the content of total acid are only considered in the design of the Box-Behnken test.

4. The production process as claimed in claim 1, characterized in that: the stage I fermentation in the step (2): preparing an activated starting strain fermentation inoculation liquid to be inoculated into sterilized peanut meal, and the final process comprises the following steps: the inoculation amount is 2.0 percent, the water content is 37.38 percent, the materials are fully and uniformly mixed, the fermentation temperature is 38.18 ℃, the fermentation time is 54.41 hours, the stirring is carried out once every 4 hours, the aerobic environment is kept, and the first-stage fermentation is carried out; and (3) fermenting in the second stage of the step (4): preparing activated starting strain fermentation inoculation liquid to be inoculated to the produced material, and the final process comprises the following steps: inoculating 1.89% of the strain, adding water to 40.00%, mixing, fermenting at 37.11 deg.C for 12.42 hr, packaging into breathing bag, maintaining anaerobic environment, and fermenting at the second stage.

5. The production method according to claim 4, characterized in that: on the basis of the scheme, the activation method of the Bacillus belgii LB-Y-1 comprises the following steps: inoculating LB-Y-1 strain preserved in glycerol on an LB solid culture medium plate, carrying out inverted culture at 37 ℃ for 18h, picking out a single colony by using a disposable inoculating loop, inoculating the single colony in an LB liquid culture medium, carrying out culture at 37 ℃ and 160rmp/min for 24h to obtain a seed solution, continuously inoculating the seed solution into a new LB liquid culture medium according to the inoculation amount of 0.5 percent of the seed solution, carrying out amplification culture at 37 ℃ and 160rmp/min, and obtaining a liquid LB-Y-1 strain with the concentration of more than or equal to 3.0 x 10 ⁸ CFU/mL, namely the fermentation inoculation liquid of the first stage;

the method for activating the pediococcus acidilactici LC-9-1 comprises the following steps: inoculating the LC-9-1 strain preserved in glycerol on an MRS solid culture medium plate, performing inversion culture at 37 ℃ for 24h, picking out a single colony by using a disposable inoculating loop, inoculating the single colony in an MRS liquid culture medium, performing static culture at 37 ℃ for 24h to obtain a seed solution, continuously inoculating the seed solution to a new MRS liquid culture medium according to the inoculation amount of 1.0 percent of the seed solution, performing static culture at 37 ℃ for expansion culture, and obtaining liquid LC-9-1 strain with the concentration of more than or equal to 1.0 multiplied by 10 ⁸ CFU/mL is the second stage fermentation inoculation liquid;

the inoculation amounts of the Bacillus belgii LB-Y-1 and Pediococcus acidilactici LC-9-1 are w/w of the mass of the inoculated bacterial liquid and the inoculated culture medium.

6. A fermented peanut meal prepared by the method for producing a peanut meal by stepwise fermentation of Bacillus belgii and Pediococcus acidilactici according to claim 1.

7. The use of fermented peanut meal according to claim 6, wherein the use comprises:

1) the application of the method in improving the total flavone content, the antioxidant performance, the crude protein content, the acid soluble protein content, the total acid content and the essential amino acid content of the peanut meal and adjusting the relative proportion of arginine and lysine;

2) after degradation of peanut meal aflatoxin B ₁ Or in phytic acid;

3) the application of the method in improving the total unsaturated fatty acid content and the linoleic acid and linolenic acid content of the peanut meal;

4) the application of the composition in reducing the abdominal fat rate of the broiler chicken;

5) the application in improving the flavor characteristics and/or the meat quality of the breast muscle of the broiler;

6) the application of the compound in improving the content of monounsaturated fatty acid in the breast muscle of the broiler;

7) the application in improving the protein content of the breast muscle of the broiler chicken or prolonging the counter period of the breast muscle.

8. Use according to claim 7, characterized in that: the fermented peanut meal is added into the feed in a proportion of 5-15 g/100 g.

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