CN110663817B

CN110663817B - Application of lactococcus lactis KDLL2016-01 in preparation of protein feed by fermenting feathers

Info

Publication number: CN110663817B
Application number: CN201811442434.1A
Authority: CN
Inventors: 徐良梅; 陈志辉; 刘洋; 李岚雪; 郭建新
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2022-09-27
Anticipated expiration: 2038-11-29
Also published as: CN110663817A

Abstract

The invention discloses application of lactococcus lactis KDLL2016-01 in preparation of protein feed through feather fermentation, and belongs to the technical field of animal nutrition and feed. The application comprises pulverizing feather into feather powder, sterilizing, and adding into fermentation culture medium; inoculating lactococcus lactis KDLL2016-01 into a fermentation culture medium for fermentation culture; and drying the fermentation product after the fermentation is finished to obtain the protein feed. The lactococcus lactis KDLL2016-01 adopted in the application does not produce harmful metabolites in the culture process, products can be directly fed to livestock and poultry, detoxification and other treatment of fermented products are not needed, lactococcus peptides produced in the fermentation process can also have a killing effect on pathogenic bacteria possibly existing in the feed, and the feed quality is safer. The protein feed prepared by the method is suitable for feeding livestock and poultry.

Description

Application of lactococcus lactis KDLL2016-01 in preparation of protein feed by fermenting feathers

Technical Field

The invention relates to application of lactococcus lactis KDLL2016-01 in preparation of protein feed by fermentation of feathers, and belongs to the technical field of animal nutrition and feed.

Background

At present, the lack of protein feed is a worldwide problem, and the lack of good quality, sufficient protein feed, and the lack of good quality, sufficient meat products, has largely limited the rapid and healthy development of livestock and poultry farming. Feather resources in China are very abundant, but feather resources are not well utilized for a long time, which is mainly related to structural properties of the feather. The feather structure is very stable, so that the feather is stable in property and insoluble in water, dilute acid and dilute alkali, and before processing, the protease cannot hydrolyze the feather, so that animals cannot digest the feather and cannot utilize protein in the feather, and the feather contains high crude protein, mainly keratin which is difficult to digest by livestock and poultry. Therefore, the method is of great importance for destroying the stable structure of the feather, improving the water solubility of the feather and improving the digestibility of the feather so that the feather becomes easy to digest and absorb. The current methods commonly used to hydrolyze feathers are as follows:

the high-temperature high-pressure hydrolysis method is the most widely used treatment method at present, the color of the finished product can change along with the change of the raw materials, when the color of the raw materials is white, the finished product is dark yellow generally, when the color of the raw materials is black, the finished product is dark brown generally, and under general conditions, the finished product of the high-pressure hydrolyzed feather meal is light brown. The high-temperature high-pressure hydrolysis method has the disadvantages that the temperature and pressure are difficult to control, the quality of the produced product is not very stable, and the protein denaturation is caused by overhigh temperature, so that the digestibility of the feather meal is reduced.

The chemical hydrolysis method is a treatment method which is invented by the characteristic that the sensitivity of the feather to acid and alkali is greatly improved under the heating condition. However, the chemical hydrolysis method also has many defects, the production process of the chemical hydrolysis method is relatively complex, the produced hydrolyzed feather meal contains more salt, which can affect the palatability of the hydrolyzed feather meal, and more salt can easily absorb moisture, resulting in the deterioration of the hydrolyzed feather meal. The chemical hydrolysis method can produce a large amount of sewage in the production process and can cause serious pollution to the environment.

The principle of the puffing method is that feather powder is subjected to a great shearing effect when passing through the puffing channel, the external pressure disappears rapidly at the moment when the feathers are flushed out of the puffing outlet, the interior of the feather powder still keeps a high-pressure state, and the feathers expand rapidly due to the internal and external pressure difference. The firm space structure of the feather can be damaged during puffing, the structure of keratin becomes loose, the hydrophobicity is reduced to some extent, the hydrophilicity is improved, the feather is hydrolyzed more easily by protease, and the feather is digested more easily by animals and absorbs the required nutrition from the feather.

Microbial degradation means that certain microorganisms in nature can produce enzymes capable of hydrolyzing keratin during growth to degrade keratin into small peptides or amino acids. Compared with other treatment methods, the microbial degradation method has great advantages, mainly manifested in low cost, simple process, and less waste water and gas. Microorganisms having keratin degrading ability are mainly bacteria, fungi and actinomycetes. However, the secondary metabolites of conventional microorganisms may contain substances harmful to livestock and poultry, and thus usually require detoxification treatment.

The traditional acid-base treatment and puffing method can cause environmental pollution on one hand and damage to nutritional ingredients of feather keratin on the other hand.

In addition, the feather is used as protein feed, the cystine content of the feather is too high, the protein quality of the feather is poor compared with that of common fish meal, and amino acid imbalance is easily caused when the feed is prepared.

At present, domestic articles about degrading feather keratin by probiotics are rare, and the research on degrading feather keratin by lactococcus is not reported, and the method for feeding livestock and poultry by using fermentation products and the feeding effect are not reported.

Disclosure of Invention

Aiming at the problems that the feather is greatly damaged by nutritional ingredients and pollutes the surrounding environment when being processed by a chemical hydrolysis method and an expansion method to be used as a protein feed, and the problems that secondary metabolites possibly generate toxicity to livestock and poultry and the like in the microbial fermentation of the feather, lactococcus which is one of probiotics is adopted to ferment the feather to produce the feather protein feed. The method has little pollution to the surrounding environment, the amino acid of the fermented feather powder is more balanced, the nutritive value is improved to a certain extent, and secondary metabolites harmful to livestock and poultry are not generated, so the feather powder can be directly used for feeding livestock and poultry. The invention provides application of Lactococcus lactis KDLL2016-01 in preparation of protein feed by fermenting feathers, and discovers Lactococcus lactis KDLL2016-01 capable of degrading feather keratin and new application thereof for the first time, wherein the Lactococcus lactis can be used for preparing protein feed by fermenting and degrading feathersFermented protein feedThe amino acid of the protein feed product obtained by fermentation is more balanced, the content of cystine is reduced to a certain extent, and the protein feed product is rich in probiotics beneficial to the growth of livestock and poultry, and the specific scheme is as follows:

the invention provides an application of Lactococcus lactis (Lactococcus lactis) KDLL2016-01 in preparation of a protein feed by fermenting feathers, namely a method for preparing the protein feed by fermenting feathers by using Lactococcus lactis (Lactococcus lactis) KDLL2016-01, wherein the preservation number of the Lactococcus lactis (Lactococcus lactis) KDLL2016-01 is CGMCC No. 15450.

Preferably, said application comprises the steps of:

the method comprises the following steps: pulverizing feather into feather powder, sterilizing, and adding into fermentation culture medium;

step two: inoculating lactococcus lactis KDLL2016-01 into a fermentation medium for fermentation culture;

step three: and drying the fermentation product after the fermentation is finished to obtain the protein feed.

Preferably, the feather is pulverized into feather powder in the first step according to the following method: cleaning feather, mixing with water and agar, stirring uniformly, drying, pulverizing, placing on a nylon bag of 400 meshes, cleaning with flowing water to remove agar, drying, and sieving with a 60-mesh sieve; wherein: the weight ratio of the feather to the water is 10: 1; the weight ratio of the agar to the feather is 1: 200.

Preferably, the addition amount of the feather powder in the first step is as follows: the ratio of the weight of feather meal to the weight of fermentation medium was 1: 4.

Preferably, in the second step, the lactococcus lactis KDLL2016-01 is subjected to recovery culture before inoculation, then inoculated into a liquid seed culture medium for seed culture for 48 hours under the conditions of the temperature of 30 ℃, the pH value of 6.0 and the rotation speed of 200rpm to obtain a seed culture solution of the lactococcus lactis KDLL2016-01, and the obtained seed culture solution of the lactococcus lactis KDLL2016-01 is inoculated into a fermentation culture medium.

More preferably, the formula of the liquid seed culture medium is as follows: 8.0g of beef extract, 10.0g of peptone, 4.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 801.0g of tween, 0.05g of manganese sulfate and 1000mL of deionized water.

Preferably, the inoculation amount of the inoculation in the second step is 5.7% (v/v).

Preferably, the formula of the fermentation medium in the second step is as follows: 10g of beef extract, 10g of yeast extract, 100g of cane sugar, 2g of sodium chloride, 10g of dipotassium hydrogen phosphate and 0.5g of magnesium sulfate, and the pH value is 6.0.

Preferably, the conditions of the fermentation culture in the step two are as follows: aerobic, the initial pH value is 6.3, the fermentation temperature is 32 ℃, the rotating speed is 220rpm, and the fermentation time is 36 h.

Preferably, the drying of the fermentation product in the third step is to filter the fermentation product to obtain a fermentation liquid and a solid, spray-dry the obtained fermentation liquid, dry the obtained solid, and then combine the dried solid powder with the spray-dried solid powder to obtain the protein feed.

The invention also provides a protein feed prepared by any one of the applications.

The lactobacillus provided by the invention is a probiotic strain capable of degrading keratin, which is separated and screened from waste feathers accumulated in a chicken farm by using feather meal as a single nitrogen source, and the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 3 months and 14 days, and the preservation number is CGMCC No. 15450.

The invention has the beneficial effects that:

1. the invention discovers a lactococcus lactis KDLL2016-01 capable of degrading feather keratin for the first time, the preservation number of the lactococcus lactis KDLL2016-01 is CGMCC No.15450, the lactococcus lactis is a probiotic, harmful metabolites are not generated in the culture process, products can be directly fed to livestock and poultry, detoxification and other treatment on fermentation products are not needed, lactococcus peptides generated in the fermentation process can also kill pathogenic bacteria possibly existing in the feed, and the feed quality is safer.

2. The protein content in feather meal can reach more than 80%, but basically keratin with compact structure and difficult digestion is contained. After the feather is fermented by the method, the compact keratin structure contained in the feather is decomposed or destroyed by lactococcus and metabolites thereof, so that the digestion rate of livestock and poultry on feather protein feed is higher, the pH value of the feather fermented product is properly reduced, the palatability is better, and the feed intake of livestock and poultry can be improved.

3. The feather is used as protein feed, the cystine content of the feather is too high, the protein quality of the feather is poor compared with that of common fish meal, and the amino acid imbalance is easily caused when the feather is prepared into the feed. The content of the cystine in the fermented feather powder obtained by the method is reduced to a certain extent, and the proportion of the amino acid is more balanced.

3. The feather degradation rate can reach 35% by fermenting feathers with KDLL 2016-01.

4. The combined fermentation product obtained by the invention is used as a protein feed to replace fish meal to prepare a feed for feeding laying hens.

5. The invention adopts the feathers as the animal source protein feed, has low cost and is easy to obtain the feathers.

Drawings

FIG. 1 shows the degradation circle of the lactococcus strain.

FIG. 2 is a 16S rDNA phylogenetic tree.

FIG. 3 shows the apparent morphology of feather meal (left) and fermented feather meal (right).

Fig. 4 shows feather powder (left) and fermented feather powder (right) under a normal electron microscope.

FIG. 5 feather meal (left) and fermented feather meal (right) under scanning electron microscope.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

The "fermented feather meal" in the following examples is the "protein feed".

The first embodiment is as follows: separation, screening and identification of feather meal degrading bacteria

1. Experimental methods

(1) Collecting a sample: firstly, strain separation samples are collected from excrement of broiler chickens raised in animal raising bases of northeast agricultural university in 2016; secondly, 5g of chicken manure sample is put into a 250mL conical flask filled with 50mL of sterile physiological saline, the mixture is evenly stirred to prepare suspension, 1mL of suspension liquid is taken to be inoculated into a 250mL conical flask containing 100mL of enrichment medium, and enrichment culture is carried out at 30 ℃ and 220r/min for 48h to prepare enrichment culture liquid.

(2) Primary screening: diluting the enrichment culture solution according to gradient, respectively taking 0.1mL of the enrichment culture solution, coating the enrichment culture solution on a feather meal solid medium flat plate, carrying out inverted culture in a 30 ℃ constant temperature incubator for a plurality of days, observing, after the colonies grow out, selecting large and single colonies, scribing on an LB flat plate again, carrying out constant temperature culture at 30 ℃ for 3 days, and carrying out repeated scribing, separating and purifying to obtain single colonies.

(3) Re-screening: inoculating one ring of each strain obtained from the initial hoof into a seed culture medium, and culturing at 30 ℃ and 220r/min for 24 h. 1mL of each seed solution was inoculated into a 250mL flask containing 100mL of the initial liquid fermentation medium in parallel with three strains and cultured for 24 hours. During which the degradation of the feathers was closely observed. And (4) re-screening according to the feather degradation condition of each strain. The fermentation liquor is centrifuged for 15min in a centrifuge with 8000r/min at 4 ℃, and the supernatant is taken to determine the absorbance value and the content of soluble protein.

(4) And (3) soluble protein content determination: soluble protein content was determined by Coomassie Brilliant blue method.

(5) And (3) preservation of strains: the single bacterial strain obtained by separation and purification in the test is preserved for a long time by adopting a glycerol cryopreservation method, the bacterial strain is inoculated in a seed culture medium, shake culture is carried out for 24 hours at 30 ℃ and 220r/min, bacterial suspension and sterilized 30% glycerol-deionized water solution 1:1 are uniformly mixed, and the mixture is preserved at the low temperature of minus 20 ℃ for 1 year in validity period.

(6) And (3) identification of strains: the strain is coated on an LB solid medium plate and a feather meal medium plate, inverted culture is carried out at the temperature of 30 ℃, about 48 hours are carried out, the single colony is observed to be white and slightly convex in shape, the surface is smooth and moist, the edges are relatively regular, and 16SrDNA molecular identification and phylogenetic analysis are carried out.

The seed culture medium (MRS liquid culture medium) is composed of: 8.0g of beef extract, 10.0g of peptone, 4.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 801.0g of tween, 0.05g of manganese sulfate, 1000mL of deionized water and sterilization at 121 ℃ for 20 min.

2 results and conclusions of the experiment

The absorbance and soluble protein content of the supernatant of the rescreened strain are shown in table 1.

TABLE 1 absorbance of supernatant of re-screened strain and soluble protein content

Note: the data in the same column are marked with different letters to indicate significant difference (P <0.05), and the same letter or no letter to indicate insignificant difference (P > 0.05). The following table is the same.

And selecting a strain according to the re-screening result, respectively inoculating the strain to a nutrient agar culture medium coated with feather suspension on the surface layer, and observing the generation condition of a 'degradation ring' after 48 hours. As a result, as shown in FIG. 1, the colony showed a milky degradation circle.

DNA fragments generated by the strain through PCR amplification are single bands, and the PCR product is sequenced to obtain a gene sequence with the full length of 1379bp, which is consistent with the result of an electrophoretogram. The two end sequences are subjected to Blast comparison in NCBI, the 16S rDNA and most Lactococcus lactis are found to reach more than 95%, and a phylogenetic tree is constructed by utilizing MEGA 5.1 clustering analysis, as shown in figure 2. The strain can be further identified as lactococcus lactis from phylogenetic trees. Designated Lactococcus lactis KDLL2016-01, which is a probiotic.

The second embodiment: optimization of conditions for degrading feather meal by lactococcus fermentation

1 method of experiment

1.1 culture Medium

(1) Fermentation medium: 10g of beef extract, 10g of yeast extract, 100g of cane sugar, 2g of sodium chloride, 10g of dipotassium phosphate and 0.5g of magnesium sulfate, wherein the pH value is 6.0, and the beef extract is sterilized at 121 ℃ for 20 min.

(2) MRS liquid medium (seed medium): 8.0g of beef extract, 10.0g of peptone, 4.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium phosphate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 801.0g of tween, 0.05g of manganese sulfate, 1000mL of deionized water, 5.7 of pH value and 20min of sterilization at 121 ℃.

1.2 optimization of fermentation conditions

The factors influencing the galactococcus to ferment feather are researched by utilizing a single factor test: the influence of oxygen-free, time, rotating speed, temperature, inoculum size and pH value on feather degradation rate is found, and better levels are explored one by one.

(1) Marking nylon bags (6cm multiplied by 5cm) with 400 meshes by using a pencil, drying at 105 ℃ to constant weight, weighing the weight of each nylon bag, and marking as M1;

(2) washing feather, drying, crushing, and sieving with a 40-mesh sieve to obtain feather powder; weighing 5g of feather powder, placing the feather powder in a nylon bag, sealing the nylon bag, drying the feather powder at 105 ℃ to constant weight, and weighing the feather powder as M2;

(3) preparing MRS liquid culture medium, and culturing lactococcus for 24h under the conditions of anaerobism, 30 ℃, pH value of 6.0 and rotation speed of 180 rpm;

(4) preparing a fermentation culture medium, adding 50mL of the fermentation culture medium into a 250mL triangular flask, placing a sterilized nylon bag filled with feather in the culture medium, respectively under aerobic and anaerobic conditions, setting different gradients, keeping other factors unchanged, and setting 5 times of repetition while taking the inoculation amount of 4% (volume ratio), the pH value of 30.0, the rotation speed of 200rpm and the fermentation time of 30h as basic conditions, and researching factors influencing the degradation rate of the feather keratin, such as the existence of oxygen, the time (18h, 24h, 30h, 36h, 42h), the rotation speed (180rpm, 200rpm, 220rpm, 240rpm, 260rpm), the temperature (26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃), the inoculation amount (3%, 4%, 5%, 6%, 7%) and the pH value (5.0, 5.5, 6.0, 6.5, 7.0) and the like.

(5) After the fermentation, the nylon bag was fished out, the culture medium was washed away with tap water, and then repeatedly washed with distilled water three times, and then dried at 105 ℃ to a constant weight, and the total weight of the fermented nylon bag and the feathers inside the bag was weighed as M3. The calculation formula is as follows:

2 results of the experiment

2.1 Effect of oxygen and oxygen on degradation Rate of lactococcus feather powder

TABLE 2 Effect of oxygen and oxygen on feather meal degradation efficiency of lactococcus

Under the aerobic state, the degradation rate of lactococcus to the feather can reach 23.1 percent, which is higher than 13.7 percent of that under the anaerobic state, so the feather is degraded by aerobic fermentation in the test to prepare feather protein feed.

2.2 Effect of rotational speed on degradation efficiency of feather meal by lactococcus

TABLE 3 influence of rotational speed on the degradation efficiency of lactococcus feathers

The degradation rate of the hairs at different rotating speeds tends to increase firstly and then decrease, and when the rotating speed is 220rpm, the degradation rate of the lactococcus to the feathers is the highest and can reach 25.8 percent. Thus, a fermentation speed of 220rpm was selected for this test.

2.3 Effect of temperature on the efficiency of degradation of feather meal by lactococcus

TABLE 4 influence of temperature on feather degradation efficiency of lactococcus

The feather degradation rate is increased and then reduced at different temperatures, and the degradation rate of lactococcus to the feather is the highest and can reach 28.2% at the temperature of 30 ℃.

2.4 Effect of inoculum size on degradation efficiency of feather meal by lactococcus

TABLE 5 Effect of inoculum size on the feather degradation efficiency of lactococcus

The feather degradation rate is in the trend of increasing first and then decreasing in different inoculation amounts, and when the inoculation amount is 5%, the feather degradation rate of lactococcus is the highest and can reach 24.4%.

2.5 influence of pH on degradation efficiency of feather meal by lactococcus

TABLE 6 influence of pH on the degradation efficiency of lactococcus feathers

The feather degradation can reach 27.2% at a pH value of 6.5.

2.6 Effect of fermentation time on feather meal degradation efficiency by lactococcus

TABLE 7 influence of fermentation time on the degradation efficiency of lactococcus feathers

Under the aerobic condition, under the conditions of 220rpm of rotating speed, 30 ℃, 5% of inoculation amount and 6.5 of pH value, the degradation rate of the feathers is in a rapid growth state from 18-36 h, the degradation rate is increased from 17.4% in 18h to 35.3% in 36h, and the degradation rate is only increased by less than 0.5% in 42h, so the optimal fermentation time of the experiment is 36 h.

3 discussion and conclusions

The experiment adopts a method of weighing nylon bags, and factors influencing the degradation rate of feather keratin, such as oxygen-free property, rotating speed, temperature, inoculation amount, pH value, fermentation time and the like, are respectively subjected to optimization analysis on the conditions of lactococcus fermentation feathers. Since the dissolved oxygen in the medium increases with decreasing liquid loading, the effect on degradation rate is not significant, and the liquid loading was determined to be 20% according to literature during fermentation production in view of cost.

The result of the effect of anaerobic bacteria on feather powder degradation efficiency of lactococcus shows that the feather degradation rate of lactococcus can reach 23.08% in the aerobic state and is higher than 13.61% in the anaerobic state. Lactococcus is a facultative anaerobic microorganism and can produce acidic substances such as lactic acid and the like under anaerobic conditions. Research shows that under aerobic condition, oxygen can raise the growth rate of facultative anaerobe and the growth amount of the strain is relatively raised. Therefore, the reason for the improvement of the degradation rate of feather keratin can be analyzed because the number of lactococcus is increased, and the decomposition rate of feather is improved.

The high rotating speed can improve the oxygen dissolution of a liquid culture medium, but the contact probability of bacteria and a fermentation substrate can be reduced when the rotating speed is too high, researches show that the influence of 10 stirring blades on bacterial collagen fibers is discussed through experiments respectively, the results show that the growth and fermentation effects of thallus cells are synchronously coupled, the rotating speed is controlled in a step mode, the delay period of thallus growth is shortened, the logarithmic phase is advanced, the proliferation speed is accelerated, the highest bacteria concentration is improved to a certain extent, and compared with the rotating speed which is not controlled, the growth curve does not have an obvious decay period. Therefore, the rotating speed increases the fermentation rate of the thallus to a certain extent, and the maximum thallus concentration is increased along with the increase of the fermentation rate. In the test, the influence results of different rotating speeds on the feather degradation efficiency show that the feather degradation rate tends to increase and decrease along with the increase of the rotating speed when the rotating speed is 180-260 rpm, and the degradation rate is highest and can reach 25.78% when the rotating speed is 220 rpm. This shows that increasing the oxygen dissolution rate in the fermentation broth can indeed increase the growth rate of lactococcus, and thus the degradation rate of feather keratin, which is also identical to the previous research results with no oxygen. However, when the rotating speed is higher than 220rpm, the feather degradation rate is reduced, probably because the excessive rotating speed reduces the contact probability of lactococcus with the feather and reduces the attachment of lactococcus on the surface of the feather, thereby causing the feather degradation rate to be reduced.

In the process of fermenting feather meal by microorganisms, two factors mainly exist for influencing the degradation rate of the feather meal by the temperature. Firstly, the life activities of microorganisms are all formed by biochemistry and are extremely obviously influenced by temperature, and the temperature is an important factor influencing the growth and the propagation of the microorganisms; secondly, temperature can influence the enzymatic activity by influencing the speed of the enzymatic reaction or the protein structure of the active enzyme. Research shows that the maximum growth rate of lactococcus increases with the increase of temperature, the lag phase decreases with the increase of temperature, and when the optimal temperature is exceeded, the lag phase is obviously prolonged and the rate is reduced. In the test, the results of the influence of different temperatures on the feather degradation efficiency show that the feather degradation rate tends to increase and decrease at different temperatures, and the degradation rate of lactococcus to the feather is the highest at 30 ℃ and can reach 28.15%, which is consistent with the optimal growth temperature of a strain, and shows that the growth quantity of bacteria and the degradation rate have a certain proportional relation.

In the production practice, the inoculation amount is small, the fermentation process is prolonged, the fermentation time is prolonged, the energy consumption is increased, the fermentation cost is increased, the probability of mixed bacteria pollution is increased, the fermentation effect is reduced, if the inoculation amount of the seed liquid is enlarged, the fermentation period is shortened, the fermentation is facilitated, but the excessively large inoculation amount can cause that the microorganism concentration is higher at the beginning of the fermentation, a large amount of fermentation substrate is consumed, necessary nutrient substances in the degradation stage can not meet the growth requirement of the microorganism easily, and the bacteria can be promoted to be premature and autolyzed. In the test, the results of the influence of different inoculation amounts on the feather degradation efficiency show that the feather degradation rate tends to increase firstly and then decrease when the inoculation amounts are different, and when the inoculation amount is 5%, the degradation rate of lactococcus on the feather is the highest and can reach 24.36%.

The pH value in the environment has great influence on the life activity of microorganisms, and the main effects are as follows: causing a change in the cell membrane charge, thereby affecting the uptake of nutrients by the microorganisms; influence the activity of enzymes in metabolic processes; the availability of nutrient substances in the growing environment and the toxicity of harmful substances are changed. Research shows that pH value can affect the charge state of cell membrane, change the permeability of the membrane, affect the absorption of thallus to nutrients and the formation of metabolite, and affect the stability of the product. In the test, the results of the effect of different pH values on the feather degradation efficiency show that the feather degradation can reach 27.24% at a pH value of 6.5, and the feather degradation is within the pH range of the optimal growth condition and has a certain deviation from the optimal growth pH, which is probably because the lactococcus can accelerate the generation of lactic acid under the acidic condition to affect the feather degradation efficiency.

The selection of the fermentation time usually selects nodes which rapidly and slowly increase in the fermentation process, so that the cost is saved, and the efficiency can be improved, the test result shows that the degradation rate of the feather meal of lactococcus before 36h of fermentation is in an ascending trend, the feather meal slowly rises after 36h, and the feather meal has a leveling sign, so that the optimal fermentation time of the feather meal fermented by lactococcus in the test is 36 h.

Example three: analysis of physical and chemical properties of fermented feather meal

1 method of experiment

1.1 analysis of morphological Structure of fermented feather meal

Feather waste on the same day is collected from the market, washed by tap water and distilled water for 3 times, dried in a 65 ℃ oven to constant weight, crushed and sieved by a 40-mesh sieve to prepare feather powder. Washing the feather powder fermentation product and the nylon bag with tap water, washing with distilled water for 3 times, taking out the feather powder, sterilizing in a 120 ℃ oven for 15min, drying at 65 ℃ to constant weight, crushing and sieving with a 40-mesh sieve, and preparing the fermented feather powder (namely protein feed) according to the optimized conditions of the second embodiment by the following steps:

(1) cleaning feather, mixing with water and agar, stirring uniformly (the weight ratio of the feather to the water is 10: 1; the weight ratio of the agar to the feather is 1:200), drying, pulverizing, placing on 400-mesh nylon cloth, washing with flowing water to remove the agar, drying, and sieving with 60-mesh sieve; the addition amount of the feather powder after sterilization is as follows: adding feather powder into the fermentation medium at a ratio of 1: 4;

(2) resuscitating and culturing lactococcus lactis KDLL2016-01, inoculating the lactococcus lactis KDLL2016-01 into a liquid seed culture medium, performing seed culture for 48 hours under the conditions of the temperature of 30 ℃, the pH value of 6.0 and the rotation speed of 200rpm to obtain a seed culture solution of the lactococcus lactis KDLL2016-01, inoculating the obtained seed culture solution of the lactococcus lactis KDLL2016-01 into a fermentation culture medium according to the inoculation amount of 5.7% (v/v), and performing fermentation culture; wherein: the conditions of fermentation culture are as follows: aerobic, wherein the initial pH value is 6.3, the fermentation temperature is 32 ℃, the rotating speed is 220rpm, and the fermentation time is 36 h; the formula of the liquid seed culture medium is as follows: 8.0g of beef extract, 10.0g of peptone, 4.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 801.0g of tween, 0.05g of manganese sulfate and 1000mL of deionized water, and sterilizing at 121 ℃ for 20 min; the formula of the fermentation medium is as follows: 10g of beef extract, 10g of yeast extract, 100g of cane sugar, 2g of sodium chloride, 10g of dipotassium phosphate and 0.5g of magnesium sulfate, wherein the pH value is 6.0, and the beef extract is sterilized at 121 ℃ for 20 min.

(3) And after fermentation, drying the fermentation product, namely filtering the fermentation product to obtain fermentation liquor and solid, spray-drying the obtained fermentation liquor, drying the obtained solid, and then combining the dried solid powder with the spray-dried solid powder to obtain the protein feed.

The following experiments were performed:

(1) observing the apparent form of the feather powder before and after fermentation. Visual observation is carried out to directly observe the apparent properties of the feather powder and the fermented feather powder, such as apparent form, color and the like.

(2) Observing with optical microscope before and after fermentation. The pulverization degree, particle size and morphology of feather powder were observed by an optical micromirror (AMG Auto FL).

(3) Observing by scanning electron microscope before and after fermenting feather powder. The feather powder was put into a prepared mixture of ethyl ether and ethanol (95%) 1:1 mixing degreasing solution, carrying out ultrasonic cleaning for 2min, naturally drying in an operation table, placing a dried sample on a sample table by using a small forceps, slightly flattening, carrying out conductive treatment on the sample table by using an Eiko IB-3 ion sputtering instrument for 90S, and observing the microstructure change of feather powder before and after liquid fermentation by using a scanning electron microscope (HITACHI S-3400N).

1.2 analysis of conventional nutrient content and amino acid composition of fermented feather powder

(1) And (4) measuring the conventional nutrient components of the feather powder and the fermented feather powder. Measuring the content of the crude protein by adopting a Kjeldahl method; measuring the content of crude fat by using an ether extraction method; energy is measured by a Parr 6300 type oxygen bomb calorimeter; ca and P are respectively measured by a potassium permanganate method and a colorimetric method.

(2) Determining amino acid components of feather powder and fermented feather powder. The feather powder and the fermentation feather powder samples are firstly hydrolyzed by 6mol/L hydrochloric acid for 24h (110 ℃), and then are filtered by a 0.45 mu m filter membrane after transferring and fixing the volume, and the content of amino acid is measured on a Nigri L-8900 type amino acid automatic analyzer.

2 results of the experiment

2.1 analysis of the fermented feather meal Structure

In fig. 3, the left image is normal feather meal, and the right image is fermented feather meal (protein feed), as can be seen from fig. 3: compared with the normal feather powder, the fermented feather powder has the advantages of relatively compact texture, low feather shape retention degree, large crushing degree and slightly dark color.

In fig. 4, the left image is feather meal under a common electron microscope, and the right image is fermented feather meal (protein feed), as can be seen from fig. 4: after being crushed, the normal feathers are mostly strip-shaped short sections, the structure of the feather shaft is clear, and the feather small branch part is flaky; the particles of the fermented feather powder are smaller, and the crushing effect of the small sections of feather shafts and feather twigs is obvious.

In fig. 5, the left image is feather meal under a scanning electron microscope, and the right image is fermented feather meal (protein feed), as can be seen from fig. 5: although the crushing degree of the normal feather is changed after being crushed, all parts of the feather have complete structure, smooth surface and regular form, the myelin sheaths on the surfaces of the feather shaft and the feather twigs are not damaged, and the internal structure is not opened. The feather powder after fermentation culture has broken feather shaft, deepened irregular degree of section, decomposed myelin sheath, exposed internal structure and formed into bulk or scattered strip.

2.2 analysis of conventional nutrient content and amino acid composition of fermented feather meal

As can be seen from Table 8: the energy of the feather meal is reduced by 2% by the fermentation of lactococcus; the content of crude fat is increased by 0.3 percent; the content of crude protein is reduced by 6.95 percent; the content of coarse ash is increased by 78.26%; the calcium content is reduced by 4.44%; the phosphorus content is increased by 200%.

TABLE 8 conventional nutritional ingredients of fermented feather meal

As can be seen from Table 9: the content of feather meal methionine is increased by 2.44% by the fermentation of lactococcus; the tyrosine content is increased by 7.25%; the content of the other 15 amino acids is reduced, wherein the content of cysteine is reduced by 22.38 percent.

TABLE 9 fermented feather meal amino acid composition

3 discussion and conclusions

The change of the morphological structure of the feather meal by the lactococcus fermentation is studied from a macroscopic level to a microscopic level through visual observation, an optical microscope and a scanning electron microscope. The macroscopic observation results of the test show that compared with the normal feather powder, the fermented feather powder has relatively compact texture, low feather shape retention degree, large crushing degree and slightly dark color. The feather color may be the decomposition of feather surface substances in the course of fermenting feather by lactococcus so as to expose the darker inner color part, or the color of glucose in the fermentation medium becomes yellow after high-temperature and high-pressure sterilization, and the color of feather powder is changed along with the fermentation for a period of time.

The results of observing the structure of feather powder before and after degradation of lactococcus through an optical microscope show that the normal feather is mostly strip-shaped short sections after being crushed, the structure of a feather shaft is clear, the feather twigs are flaky and the particles of the fermented feather powder are smaller, the crushing effect of the small sections of feather shafts and the feather twigs is obvious, and further, the lactococcus is fermented and decomposed at different positions on the surface of the feather, so that the feather is broken, and the crushing degree is increased. According to the feather shaft crushing degree in the experimental electron microscope observation, the degradation effect of lactococcus on feather keratin in the research can be proved.

Compared with an optical microscope, the scanning electron microscope can directly observe the structure of the surface of a sample, the preparation process is simple, translation and rotation of a three-dimensional space can be performed in the observation process, the sample can be observed from various angles, the depth of field is hundreds of times greater than that of the optical microscope, the resolution ratio is higher, the image is rich in stereoscopic impression, the amplification range is wide, and the image can be amplified by dozens of times to hundreds of thousands of times. The observation result of the experimental scanning electron microscope shows that the structure of each part of the feather is changed into a bulk shape or a scattered strip shape from a complete shape with smooth surface. The decomposition of myelin sheaths on the surfaces of the feather axis and the feather twigs is obvious, and probably because disulfide bonds and chemical bonds in the feather structure are broken, the surface is rough and the structure is loose. The experimental electron microscope picture can also show that part of the feather structure is changed from the orderly arranged strips into loose blocks, and the feather powder has a structure similar to that of the puffed feather powder, thereby being beneficial to digestion and absorption of animals.

Example four application of fermented feather in egg-laying hens

1. Experimental methods

1.1 Experimental design and feed composition

(1) The test adopts a single-factor random test design, 150 feathers of 27-week-old healthy laying hens with similar body weight are selected and randomly divided into 3 groups, namely a control group (fish meal group), a 2% fermented feather powder group (50% substituted fish meal group) and a 4% fermented feather powder group (100% substituted fish meal group), each group is 5 repetitions, 10 feathers are repeated, the test is finished when the laying hens are 32 weeks old, the test period is 5 weeks, and the 'fermented feather powder' in the test is the fermented feather powder (protein feed) prepared by the method in the second embodiment.

(2) At the beginning of the test, the henhouse is cleaned thoroughly, the coop, the trough, the water tank, the dung pad and the ground are brushed, and a disinfection facility is arranged at the doorway. Potassium permanganate and formalin solution are mixed according to a certain proportion for closed fumigation and disinfection. Laying hens are fed in stacked metal cages, and each cage is provided with 1 laying hen. Dosing was done at 08: 00/day with free access to water. The temperature in the house is controlled to be about 20 ℃, the humidity is controlled to be about 60%, a mode of combining natural illumination and artificial illumination is adopted in the test period, the illumination period is gradually increased at a speed of 0.5h per week until 16h/d in the initial stage of egg laying, epidemic prevention and disinfection are carried out regularly, the water tank is cleaned every day, and the water supply and the cleanness are guaranteed. The conditions of the chicken flocks are observed every day, whether the drinking water and the food are normal and whether the feces are abnormal and the like are observed, and the daily record is made.

The basal diet nutrient content, with reference to NRC poultry nutritional requirements (1998 edition), met or exceeded this standard for the diet formula, which is shown in table 10 below.

TABLE 10 feed composition and nutritional level (air-dry basis) for laying hens in laying period

Note: the premix provides 12000 IU of vitamin A and D for one kilogram of feed ₂ 400IU, vitamin E30 IU, vitamin K ₃ 1.5mg, WeiShengsu B ₁₂ 0.012mg, vitamin B ₁ 2.0mg, biotin 0.20mg, folic acid 1.2mg, nicotinic acid 35mg, pantothenic acid 12mg, pyridoxine 4.5mg, riboflavin 9mg, copper 8mg, iodine 1.0mg, iron 80mg, manganese 100mg, selenium 0.30mg, zinc 80 mg.

1.2 measurement indices and methods

(1) Determination of apparent metabolic rate index of nutrient in feed

The metabolism test adopts a total dung collection method, and is started at the 29 th d of test laying hens at the age of 31 weeks. Accurately recording the feed intake of each group of test laying hens every day, accurately collecting excrement excreted by each group of test laying hens every day, removing hair shed from the collected excrement and impurities in the growth environment, and continuously collecting for 3 days. Weighing feces collected every day, adding a hydrochloric acid solution with the mass being 10% of the fresh weight of the feces, fully and uniformly mixing, storing at-20 ℃, fully and uniformly mixing the feces collected from the same group after all tests are finished, selecting about 100g of fresh feces samples in each group for air drying sample preparation, firstly drying the selected fresh feces samples in a 120 ℃ drying oven for 15min to inactivate enzyme, cooling to 65 ℃ for drying, fully dampening for 24h, weighing to constant weight, and crushing to prepare the sample to be tested. And (4) measuring the energy, crude protein, crude fat, Ca and P contents of the manure and the feed. Measuring the content of the crude protein by adopting a Kjeldahl method; measuring the content of crude fat by an ether extraction method; energy is measured by a Parr 6300 type oxygen bomb calorimeter; and respectively measuring Ca and P by a potassium permanganate method and a colorimetric method, and calculating the apparent nutrient metabolic rate.

(2) Determination of egg laying Performance indicators

And calculating the laying rate, the average egg weight, the feed conversion rate (feed-egg ratio) and the soft shell breaking rate according to the egg laying number, the egg weight and the feed consumption. The measurement method of each index is as follows:

1) laying rate: the number of eggs laid by each group at the same time (16:00) per day was recorded, and the average of the egg laying rate per repeat on the day and the daily egg laying rate of each group was calculated as follows:

2) average egg weight: eggs were collected (16:00) at the same time for three consecutive days per week (monday to wednesday), and the average egg weight was calculated by weighing each repeat on an electronic balance, as follows:

3) daily food intake: counting the feed consumption of each repeated laying hen in each group in the whole week every day, recovering the rest feed, and calculating the daily average feed intake;

4) soft shell breaking rate: the number of eggs broken in each replicate group was recorded at the same time each day (16: 00). And (3) calculating the soft shell breaking rate of each repetition and the average value of the soft shell breaking rates of all groups, wherein the calculation formula is as follows:

(5) feed conversion ratio (feed-egg ratio): the ratio is calculated according to daily feed intake and daily average egg weight.

(3) Determination of egg quality index

Randomly collecting 3 eggs in each group at the same time (12:00) every three days, 1 egg is reserved, and measuring the quality of the eggs on the same day by the following measuring sequence and method:

1) egg weight: accurately weighing the weight of each egg by an analytical balance to 0.0001 g;

2) egg shape index: the egg shape index was calculated by measuring the transverse diameter and longitudinal diameter of each egg using an egg shape index meter (NFN-385, fushihei industries, tokyo, japan) according to the following formula:

3) strength of eggshell: placing the whole egg on an eggshell strength tester (type II, Fushiping industries Co., Ltd., Tokyo, Japan), and selecting a corresponding procedure to test the eggshell strength;

4) half unit: the concentrated egg white height was measured using an egg white height measuring instrument (NFN-381, fushihei industries, tokyo, japan), which measured the egg white height at 2 or 3 points from the midpoint between the egg yolk edge and the concentrated egg white edge, averaged and calculated in hough units, as follows:

hough unit 100 × log (H-1.7 × W) ^0.37 +7.6)

H: concentrated protein height, unit: mm w: egg weight, unit: g;

5) yolk color: breaking eggs, separating egg yolk from egg white by using an egg yolk separator, carrying out color comparison with a Roche color comparison fan under a fluorescent lamp, respectively reading the same egg yolk by two persons in the test, and taking the average value;

6) thickness of eggshell: the eggshell thickness of the sharp end, blunt end and middle 3 portions of the egg was measured using an eggshell thickness meter (NFN-380, fushiping industries, tokyo, japan) and the average value thereof was taken;

7) yolk is relatively heavy: completely separating egg white and yolk by using a yolk separator, weighing the yolk by weight, and calculating according to the following formula:

2. results of the experiment

2.1 Effect of fermented feather meal on the metabolism of layer feed nutrients

As can be seen from Table 11: the apparent metabolic rate of dry matter, the apparent metabolic rate of fat, the apparent metabolic rate of protein, the apparent metabolic rate of metabolizable energy and the apparent metabolic rate of ash of the control group, the 2% fermented feather powder group and the 4% fermented feather powder group have no significant difference (P is more than 0.05); however, the apparent metabolic rate of calcium in the control group is obviously higher than that in the fermented feather group (P <0.05), and the apparent metabolic rate of calcium in the 2% fermented feather powder group is higher than that in the 4% fermented feather powder group, but the difference is not significant (P > 0.05). After the fermented feather meal is added, the apparent metabolic rate of phosphorus of the laying hens tends to be reduced, wherein 4% of the fermented feather meal groups are obviously lower than those of a control group (P < 0.05).

TABLE 11 influence of fermented feather meal on the nutrient metabolism rate of layers (%)

Note: the same row of data is shoulder marked with different letters to indicate significant difference (P <0.05), and the same letter or no letter to indicate insignificant difference (P > 0.05). (the same below)

2.2 Effect of fermented feather meal on egg laying Performance of laying hens

As can be seen from table 12: the laying rate, egg weight, daily feed intake, qualification rate, egg breakage rate and feed-egg ratio of the control group, the 2% fermented feather powder group and the 4% fermented feather powder group have no significant difference.

TABLE 12 influence of fermented feather meal on egg laying performance of layers

2.3 Effect of fermented feather meal on egg quality

As can be seen from table 13: the egg shape index, the eggshell strength, the yolk color, the egg white height, the Hough unit and the relative weight average of the yolk of the control group, the 2% fermented feather powder group and the 4% fermented feather powder group have no significant difference, and the eggshell thickness of the 4% fermented feather powder group is significantly higher than that of the control group and the 2% fermented feather powder group.

TABLE 13 influence of fermented feather meal on egg quality

3. Discussion and conclusions

Under the current landslide trend of poultry raising industry, how to reduce the feed cost under the condition of ensuring production becomes the current research hotspot, and the utilization of hydrolyzed feather meal, enzymolysis feather meal and the like to replace the protein feed with higher price in animal feed becomes a practical and effective solution. The apparent metabolic rate of nutrient substances is the most visual expression method for embodying the feeding effect of the feed, and the nutrient substances can be better absorbed and utilized by animal organisms to mainly improve the quality of the feed, so that the healthy growth and normal development of the organisms can be ensured. The apparent metabolic rate of nutrient substances is mainly influenced by various factors such as intestinal microorganisms, the composition of daily ration amino acid, the quality of feed raw materials and the like. Research and evaluation show that the energy apparent metabolic rates of the three feather powders on meat ducks are 81.51%, 73.07% and 76.85%, respectively, and the results are lower compared with the test result, which indicates that the lactococcus fermented feather powder in the test has higher feeding value and wide application prospect compared with other feather fermented feeds. In the test, the trace elements, particularly the phosphorus content in the fermented feather powder is obviously increased, and the apparent metabolic rate of phosphorus in the group of replacing fish meal by the fermented feather powder is also obviously higher than that in the group of replacing fish meal by the feather powder, which shows that the lactococcus fermented feather can improve the metabolism of laying hens by changing the trace elements. The fermented feather meal can obviously improve the apparent metabolic rate of each nutrient substance of the common feather meal instead of fish meal, and has no obvious influence on the feeding effect of fish meal feed besides the apparent digestibility of calcium is reduced, which shows that the hard 'shell' of feather keratin is really decomposed by the fermentation of lactococcus, so that the nutrient components are easier to digest and absorb, the digestive metabolism of laying hens is not adversely affected, meanwhile, the cost of feed can be reduced, the resource waste is reduced, and the environment-friendly development is promoted.

The egg laying performance is an important index for evaluating the value and the quality of the laying hens, the experiment shows that the fermented feather meal does not have obvious influence on the egg laying performance of the laying hens by replacing fish meal in daily ration with the lactococcus fermented feather meal at different levels to feed the laying hens. The 2% fermented feather meal group had an egg yield higher than the 4% fermented feather meal group, probably because the feather meal lacked Met, Lys, Trp, His, and other amino acids, and therefore these amino acid deficiencies were likely to be caused when the feather meal was used to formulate a ration. In the test formula, only the satisfaction of Lys and Met is considered, and the balance of amino acids such as His and Trp is not considered, so that when the addition amount of feather meal in daily ration of the laying hens is increased, His and Trp are more easily lacked, which may be the reason that the egg laying rate of the laying hens is reduced while the addition amount of feather meal is increased.

The yolk color is an important sensory index for measuring the egg quality, has no influence on the egg edible value, but gradually arouses the attention of consumers along with the improvement of the consumption level of people, directly influences the purchasing power of the consumers and has economic value. The color of egg yolk is mainly affected by genetic and coloring matter in feed. The protein height is mainly influenced by the energy and feed intake of the diet. The higher the Haff unit value, the thicker the protein, and the better the protein quality, which is mainly influenced by the content and composition of the nutrient components digested and absorbed. In addition, the yolk is rich in nutrition, and the relative weight of the yolk is also an important index for measuring the nutritional content of the eggs. The relatively large egg yolk indicates a high level of nutrition in the egg, and in addition to being influenced by genetic factors, the nutritional level of the diet is important. The test result shows that the fermented feather meal has no obvious influence on the strength of egg shells, the color of egg yolks, the height of egg white, Hafu units and the relative weight of egg yolks of laying hens instead of fish meal, and shows that the quality of the egg white of the fermented feather meal is almost not different from that of the fish meal.

The egg shape index is one of indexes of the germ plasm classification of the laying hens and is only related to packaging and transportation. Research shows that the egg shape index has certain correlation with the egg weight, and the egg shape index tends to decrease along with the increase of the egg weight. In the test, the fermented coarse powder instead of the fish meal has no obvious influence on the egg weight, and the egg shape index has no obvious difference.

The eggshell thickness is an important index for measuring the quality of eggs, directly influences the death rate of embryos and the hatching rate of hatching eggs, and has important economic value. The good eggshell strength and eggshell thickness can reduce the breakage rate of eggs and improve the economic benefit. The thickness and strength of the eggshell have certain influence on the freshness of the eggs. The test result shows that the thickness of the eggshell of the laying hen can be obviously improved by adding 4% of fermented feather powder into the feed. The analysis reason may be that the combined action of the higher protein content and the amino acid level in the fermented feather meal increases the protein deposition in the eggshells, increases the eggshell thickness and further improves the quality of the egg.

The test identifies a strain of probiotics (lactococcus) capable of degrading feather through screening, provides a new idea for the research of the feather degrading bacteria, and lays a foundation for further development and research by combining the characteristics of microorganisms and directly feeding test animals with the fermented feather powder of the inactivated lactococcus or adopting a complex enzyme fermentation method. In the research, the laying hen feeding test period is short, the purpose is to explore the preliminary feeding effect of the fermented feather powder, the degradation efficiency still needs to be further improved on the basis, and lactococcus fermented feathers are formed into products by combining various production means such as puffing and solid state fermentation, and the like, so that the method is applied to large-scale production verification and provides a theoretical basis for forming an industrial system.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of Lactococcus lactis KDLL2016-01 in preparing protein feed by fermenting feathers is disclosed, wherein the preservation number of the Lactococcus lactis KDLL2016-01 is CGMCC No. 15450.

2. Use according to claim 1, characterized in that it comprises the following steps:

step two: inoculating lactococcus lactis KDLL2016-01 into a fermentation culture medium for fermentation culture;

3. The use of claim 2, wherein the feather pulverization of the feathers into feather meal in step one is carried out according to the following method: cleaning feather, mixing with water and agar, stirring uniformly, drying, pulverizing, placing on a nylon bag of 400 meshes, cleaning with flowing water to remove agar, drying, and sieving with a 60-mesh sieve; wherein: the weight ratio of the feather to the water is 10: 1; the weight ratio of the agar to the feather is 1: 200.

4. The use of claim 2, wherein the feather meal of step one is added in an amount of: the ratio of the weight of feather meal to the weight of fermentation medium was 1: 4.

5. The application of claim 2, wherein in the second step, the lactococcus lactis KDLL2016-01 is subjected to recovery culture before inoculation, then inoculated into a liquid seed culture medium for seed culture for 48 hours under the conditions of 30 ℃ temperature, pH value of 6.0 and rotation speed of 200rpm to obtain a seed culture solution of the lactococcus lactis KDLL2016-01, and the obtained seed culture solution of the lactococcus lactis KDLL2016-01 is inoculated into a fermentation culture medium.

6. The use according to claim 5, wherein the liquid seed culture medium has a formulation of: 8.0g of beef extract, 10.0g of peptone, 4.0g of yeast extract, 20.0g of glucose, 2.0g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 801.0g of tween, 0.05g of manganese sulfate and 1000mL of deionized water.

7. The use of claim 2, wherein the fermentation medium of step one is formulated as: 10g of beef extract, 10g of yeast extract, 100g of cane sugar, 2g of sodium chloride, 10g of dipotassium hydrogen phosphate and 0.5g of magnesium sulfate, and the pH value is 6.0.

8. The use according to claim 2, wherein the conditions of the fermentation culture in step two are: the inoculation amount is 5.7% (v/v), the fermentation temperature is 32 ℃, the rotation speed is 220rpm, and the fermentation time is 36h, and the fermentation is aerobic, and the initial pH value is 6.3.

9. The use of claim 2, wherein the drying of the fermentation product in step three comprises filtering the fermentation product to obtain a fermentation broth and a solid, spray-drying the fermentation broth, drying the solid, and combining the dried solid powder with the spray-dried solid powder to obtain the protein feed.

10. A protein feed produced by the use of any one of claims 1 to 9.