CN108486087B

CN108486087B - Complex enzyme preparation for enzymolysis of chicken liver and application thereof

Info

Publication number: CN108486087B
Application number: CN201810316703.3A
Authority: CN
Inventors: 吴广兵; 龚俊勇
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-04-10
Filing date: 2018-04-10
Publication date: 2020-11-10
Anticipated expiration: 2038-04-10
Also published as: CN108486087A

Abstract

The invention discloses a compound enzyme preparation for enzymolysis of chicken liver and application thereof, belonging to the field of compound enzyme preparations. The compound enzyme preparation comprises the following components in parts by mass: 1-3 parts of neutral protease, 1-6 parts of alkaline protease, 1-3 parts of lipase, 0.1-0.3 part of visceral enzyme, 0.1-0.4 part of flavor enzyme and 3-5 parts of carrier. According to the invention, neutral protease, alkaline protease, lipase, visceral enzyme, flavor enzyme and a carrier are compounded into a complex enzyme preparation according to different enzyme activities, proportions and dosages, the chicken liver is subjected to enzymolysis, the product proteolysis degree and nitrogen recovery rate are higher, the amino acid molecular weight and bitter amino acid are less, the palatability is strong, and the nutritional value is very high. The enzymolysis product is further prepared into chicken liver protein powder through precipitation and freeze drying, the protein content is high, partial fish meal is replaced to feed animals, the growth performance of the animals can be improved, the breeding benefit is improved, and the breeding cost is saved.

Description

Complex enzyme preparation for enzymolysis of chicken liver and application thereof

Technical Field

The invention belongs to the field of complex enzyme preparations, and particularly relates to a complex enzyme preparation for enzymolysis of chicken livers and application thereof.

Background

In recent years, the chicken yield in China is steadily increased, the total yield is stabilized in 2013 and reaches the 2 nd position in the world, the chicken becomes the second big meat product with the average human consumption only second to pork, the side product chicken liver accompanying the chicken accounts for 2-2.5% of each chicken, and the yield reaches more than twenty thousand tons every year. The chicken liver has high nutritive value, contains protein, fat, carbohydrate, vitamin A, vitamin D, phosphorus and other components, has rich iron content, and has the effect of enriching the blood. In addition, the vitamin A content in the chicken liver is far higher than that of conventional foods such as milk, eggs, meat, fish and the like, the chicken liver has the effects of maintaining normal growth and reproduction, protecting eyes and maintaining normal eyesight, and the chicken liver is popularized and applied to the aspects of feed cultivation, food, medicine and the like to a certain extent. In the existing chicken liver treatment process, chicken meat powder is prepared by simple crushing and drying and is used in feed cultivation production. However, the simple process easily causes nutrition loss, the product has heavy fishy smell and high cholesterol content, the palatability of animals is poor, the digestibility is low, the market consumption is not large, and waste is caused. Therefore, research, development and use of a new process for improving the degree of hydrolysis and the utilization rate of chicken liver protein become one of the hot problems of people's attention.

The complex enzyme preparation enzymolysis method for extracting the protein in the chicken liver becomes one of effective ways for solving the problem of comprehensive utilization of the chicken liver. Research reports that a proper amount of enzyme preparation is added into chicken livers, the chicken livers are subjected to enzymolysis under the conditions of proper temperature, pH and reaction time, the degree of proteolysis is taken as a measurement index, and hydrolysis products mainly comprise amino acid, small molecular polypeptide, flavor nucleotide and the like, so that the chicken livers have the characteristics of high protein content and high nutritional value, can be further processed into high-protein feed raw materials, are added into livestock and poultry feed, are recycled, and improve the added value of the chicken livers. At present, the research of the enzymolysis process method of the chicken liver is more focused on, such as additive amount, temperature, pH, extraction time and the like, but the enzyme activity, the optimal proportion and the content of bitter amino acid of the enzyme preparation cannot be quantized, the aim of hydrolyzing the chicken liver on a large scale is achieved by adopting which enzyme preparation, which enzyme activity and the optimal proportion are adopted for enzymolysis of the chicken liver, meanwhile, the proteolysis degree and the nitrogen recovery rate can be improved, and the generation of flavor amino acid can be reduced. The chicken liver after enzymolysis can be further processed into protein feed to be fed to animals, so that the growth performance of the animals, such as daily gain and daily feed intake, can be improved, the feed-weight ratio can be reduced, the breeding benefit can be improved, and the breeding cost can be reduced. Therefore, the research and development of the complex enzyme preparation capable of improving the utilization rate of the chicken liver have important significance.

Disclosure of Invention

In order to overcome the defects that the utilization rate of chicken liver is not high, the content of bitter amino acid in an enzymolysis product is high, the fishy smell is heavy, and the large-scale utilization cannot be realized in the prior art, the invention mainly aims to provide a compound enzyme preparation for enzymolysis of chicken liver.

The invention also aims to provide the application of the compound enzyme preparation for enzymolysis of chicken livers.

Another object of the present invention is to provide a high protein feed.

Still another object of the present invention is to provide a method for preparing the above high protein feed.

The purpose of the invention is realized by the following technical scheme:

a complex enzyme preparation for enzymolysis of chicken liver comprises the following components in parts by mass:

1-3 parts of neutral protease, 1-6 parts of alkaline protease, 1-3 parts of lipase, 0.1-0.3 part of visceral enzyme, 0.1-0.4 part of flavor enzyme and 3-5 parts of carrier.

Preferably, the composition comprises the following components in parts by mass:

1-3 parts of neutral protease, 2-6 parts of alkaline protease, 1-3 parts of lipase, 0.1-0.3 part of visceral enzyme, 0.1-0.25 part of flavor enzyme and 3-5 parts of carrier.

Further preferably, the coating comprises the following components in parts by mass:

1 part of neutral protease, 2-6 parts of alkaline protease, 1 part of lipase, 0.1 part of visceral enzyme, 0.1-0.25 part of flavor enzyme and 4 parts of carrier.

Still more preferably, the composition comprises the following components in parts by mass:

1 part of neutral protease, 4 parts of alkaline protease, 1 part of lipase, 0.1 part of visceral enzyme, 0.15-0.25 part of flavor enzyme and 4 parts of carrier.

Most preferably, the composition comprises the following components in parts by mass:

1 part of neutral protease, 4 parts of alkaline protease, 1 part of lipase, 0.1 part of visceral enzyme, 0.2 part of flavor enzyme and 4 parts of carrier.

In the compound enzyme preparation, the enzyme activity of neutral protease is less than or equal to 5 ten thousand U/g and less than or equal to 10 ten thousand U/g; the enzyme activity of the alkaline protease is less than or equal to 20U/g and more than or equal to 10 ten thousand U/g; the enzyme activity of the lipase is less than or equal to 5 ten thousand U/g and less than or equal to 10U/g; the enzyme activity of the visceral enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g; the enzyme activity of the flavor enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g.

The compound enzyme preparation has the particle size: the 80-mesh passing rate is more than or equal to 99.0 percent.

The carrier comprises: one or more of corn starch, rice bran, talcum powder, wheat bran and wheat coarse powder.

The compound enzyme preparation for enzymolysis of chicken liver is applied to preparing high-protein feed after enzymolysis of chicken liver.

A high protein feed is prepared by adding the above complex enzyme preparation into chicken liver, performing enzymolysis, and freeze drying.

The preparation method of the high-protein feed comprises the following steps:

(1) taking a proper amount of chicken liver, then stirring into a paste shape, and uniformly mixing;

(2) weighing the complex enzyme preparation for enzymolysis of the chicken liver according to the addition amount of 1.00-2.50% of the dry weight of the chicken liver by mass percent, dissolving the complex enzyme preparation in water, and stirring until the complex enzyme preparation is completely dissolved;

(3) then adding the complex enzyme preparation solution into chicken livers preheated to 45-55 ℃, mixing and stirring for reaction to obtain an enzymatic hydrolysate; wherein, in the reaction conditions, the pH is 7.5-8.5, the enzymolysis temperature is 45-55 ℃, and the raw materials are as follows: and (3) adding water to the mixture (1:1) - (6:1), and performing enzymolysis for 1.5-2.5 hours.

(4) Precipitating the enzymolysis liquid obtained in the step (3), and freeze-drying to prepare chicken liver protein powder; namely: a high protein feed.

Preferably, the addition amount of the complex enzyme preparation for chicken liver enzymolysis in the step (2) is 1.50-2.50%; more preferably 1.50-2.00%; more preferably 1.50%.

Preferably, the reaction conditions in step (4) are: the pH is 8.0, the enzymolysis temperature is 50 ℃, and the raw materials are as follows: water-4: 1, the enzymolysis time is 2.0 hours.

The preparation method increases the degree of proteolysis of the product and the recovery rate of nitrogen.

The preparation method reduces the amino acid in the taste of the enzymolysis product.

The high protein feed is applied to feeding animals instead of part of fish meal.

The high-protein feed replaces part of fish meal to feed animals, can improve the growth performance of the animals, improve the quality of chicken livers and the utilization rate of the chicken livers in actual production, provide a high-quality protein source for feed cultivation, and promote food safety.

Compared with the prior art, the invention has the following advantages and effects:

according to the invention, neutral protease, alkaline protease, lipase, visceral enzyme, flavor enzyme and a carrier are compounded into a complex enzyme preparation according to different enzyme activities, proportions and dosages, the chicken liver is subjected to enzymolysis, the product proteolysis degree and nitrogen recovery rate are higher, the amino acid molecular weight and bitter amino acid are less, the palatability is strong, and the nutritional value is very high. The enzymolysis product is further prepared into chicken liver protein powder through precipitation and freeze drying, the protein content is high, partial fish meal is replaced to feed animals, the growth performance of the animals can be improved, the breeding benefit is improved, and the breeding cost is saved.

Drawings

FIG. 1 is a graph showing the effect of different complex enzyme preparations on the degree of proteolysis of chicken liver in example 1.

FIG. 2 is a graph showing the effect of different complex enzyme preparations on chicken liver nitrogen recovery in example 1.

FIG. 3 is the effect of different complex enzyme preparations in example 2 on the degree of chicken liver proteolysis under specific conditions.

FIG. 4 shows the results of detecting the molecular weight of proteins in the enzymatic hydrolysates obtained after the enzymatic hydrolysis of different complex enzyme preparations in example 2.

FIG. 5 is the effect of different doses of Complex enzyme preparation 3 on the degree of proteolysis of chicken liver in example 3.

FIG. 6 is a graph showing the effect of different doses of Complex enzyme preparation 3 on the recovery of nitrogen from chicken liver in example 3.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The neutral protease, the alkaline protease and the lipase used in the examples are all produced by Shenzhen Luweikang animal nutrition Co., Ltd, and are universal and have models of ZD500, JD1000 and F800 respectively. Visceral enzymes are derived from animal viscera and are purchased from bioscience, Henghuadao, Nanning, Inc. under model number NZ 100. Flavourzyme was purchased from south-ning-pointbo bioengineering ltd under model FW 100.

The carrier is purchased from Chinese food biochemical energy (elm) company Limited.

Example 1

1. The enzyme activity in the compound enzyme preparation needs to meet the following requirements:

the enzyme activity of the neutral protease is less than or equal to 5 ten thousand U/g and less than or equal to 10 ten thousand U/g; the enzyme activity of the alkaline protease is less than or equal to 20U/g and more than or equal to 10 ten thousand U/g; the enzyme activity of the lipase is less than or equal to 5 ten thousand U/g and less than or equal to 10U/g; the enzyme activity of the visceral enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g; the enzyme activity of the flavor enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g.

2. The particle size of the compound enzyme preparation is as follows: the 80-mesh passing rate is more than or equal to 99.0 percent.

3. The carrier includes: one or more of corn starch, rice bran, talcum powder, wheat bran and wheat coarse powder.

4. The lipase, the visceral enzyme, the flavor enzyme and the carrier are fixed in parts in the compound enzyme preparation, and the neutral protease and the alkaline protease are mixed into the compound enzyme preparation according to different proportions.

(1) Complex enzyme preparation 1:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 2: 1: 0.1: 0.2: 4;

(2) and (3) complex enzyme preparation 2:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 3: 1: 0.1: 0.2: 4;

(3) compound enzyme preparation 3:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.1: 0.2: 4;

(4) and (3) complex enzyme preparation 4:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 5: 1: 0.1: 0.2: 4;

(5) and (3) complex enzyme preparation 5:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 6: 1: 0.1: 0.2: 4.

5. taking a plurality of frozen chicken livers, unfreezing, then stirring into a paste shape, and uniformly mixing.

(1) Taking 3 conical flasks of 250mL, respectively weighing 200g of chicken liver paste, placing the chicken liver paste in the conical flasks, placing the conical flasks in an HZQ-F160Q full-temperature shaking incubator at 55 ℃, shaking at a speed of 220r/min, and preheating for 20 min.

(2) Weighing different complex enzyme preparations 1, 2, 3, 4 and 5 according to the addition amount of 1.50%, placing in a 50mL beaker, adding 20mL of water, placing in a HJ-4 magnetic heating stirrer, and stirring for 10min to completely dissolve the enzyme in the water.

(3) Respectively adding the 5 groups of complex enzyme preparation solutions into a preheated conical flask filled with chicken livers, and mixing and stirring. Continuously placing the HZQ-F160Q in a full-temperature shaking incubator at 55 ℃, shaking at the speed of 220r/min, and reacting for 2.5 hours.

(4) And after the reaction is finished, placing the mixture in a water bath kettle at 100 ℃ for 5-10 minutes for inactivation.

(5) After inactivation, the reaction solution is centrifuged for 15 minutes at 5000r/min by a desktop centrifuge.

(6) The volume of the supernatant of 3 groups was measured using a measuring cylinder measuring 100 mL.

(7) Measuring the nitrogen content in the supernatant and the chicken liver material by a Kjeldahl method.

(8) And measuring the content of the amino nitrogen in the supernatant of the 3 groups of the supernatant by using a formaldehyde potentiometric titration method, and multiplying the volume of the supernatant by the content of the amino nitrogen in the supernatant to obtain the mass of the amino nitrogen.

(9) The molecular weight distribution of the protein in the supernatant was determined by gel electrophoresis.

6. The compound enzyme preparation is used for enzymolysis of proteins in chicken livers, the additive amount is 1.00-2.00%, the pH is 7.5-8.5, the enzymolysis temperature is 45-55 ℃, and the raw materials are as follows: water (1:1) - (6:1), enzymolysis time is 1.5-2.5 hours, the obtained additive amount is 1.5%, the most suitable pH value is 8.0, the enzymolysis temperature is 50 ℃, and raw materials: water-4: 1, the enzymolysis time is 2 hours.

7. Measurement indexes are as follows: amino nitrogen, degree of proteolysis, and nitrogen recovery rate.

8. Measurement indexes are as follows:

the determination of the content of the amino nitrogen refers to a formaldehyde potentiometric titration method;

the total nitrogen amount is determined by a Kjeldahl method;

the method for measuring the proteolysis degree in the enzymolysis liquid comprises the following steps:

degree of proteolysis (DH,%) × 100% (mass of amino nitrogen in supernatant/mass of total nitrogen in sample);

the nitrogen recovery rate (NR,%) is (ammonia basic nitrogen content in the supernatant/total nitrogen content in the raw material) × 100%.

9. Enzymolysis effect of different complex enzyme preparations on chicken liver

The enzymolysis results of the complex enzyme preparations 1-5 on chicken livers under specific conditions are shown in table 1 and fig. 1 and 2.

TABLE 1 enzymolysis results of different complex enzyme preparations on chicken livers under specific conditions

Different enzyme preparations	Degree of proteolysis DH (%)	Nitrogen recovery DR (%)
			Complex enzyme preparation 1	28.20	94.83
Complex enzyme preparation 2	29.29	97.18
			Complex enzyme preparation 3	31.55	99.71
Complex enzyme preparation 4	30.03	96.81
			Complex enzyme preparation 5	28.92	95.26

As can be seen from table 1, fig. 1 and fig. 2, when the additive amount is 1.5%, the pH is 8.0, the enzymolysis temperature is 50 ℃, the raw materials: water-4: 1, the enzymolysis time is 2 hours, and the enzymolysis effect of the compound enzyme preparation 3 is the best, namely neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.10: 4, the chicken liver protein has higher degree of proteolysis and high nitrogen recovery rate.

Analyzing possible reasons: when the neutral protease: alkaline protease: lipase the following 1: 4: under the condition that the pH is 8.0 according to the proportion of 1, the compound enzyme preparation is in the most suitable pH state, the decomposition rate of the compound enzyme preparation can be exerted to the maximum extent, and the protein in the chicken liver is decomposed into amino acid and micromolecular polypeptide, so that the compound enzyme preparation 3 has the best enzymolysis effect on the chicken liver, and the proteolysis degree and the nitrogen recovery rate are highest.

Example 2

1. The neutral protease, the alkaline protease, the lipase, the visceral enzyme and the carrier are unchanged in parts, the flavor enzyme parts are changed, and the enzymolysis effect, the protein molecular weight and the flavor amino acid content of the chicken liver are measured.

(1) Compound enzyme preparation 6:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.05: 4;

(2) and (3) complex enzyme preparation 7:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.10: 4;

(3) and (3) complex enzyme preparation 8:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.15: 4;

(4) compound enzyme preparation 9:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.20: 4;

(5) compound enzyme preparation 10:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.10: 0.25: 4.

2. the compound enzyme preparation is used for enzymolysis of chicken livers, the additive amount is 1.0-2.0%, the pH value is 7.5-8.5, the enzymolysis temperature is 45-55 ℃, and the raw materials are as follows: water (1:1) - (6:1), enzymolysis time is 1.5-2.5 hours, the additive amount is 1.50%, the optimum pH value is 8.0, the enzymolysis temperature is 50 ℃, and raw materials: water-4: 1, enzymolysis time is 2 hours.

3. Measurement indexes are as follows: total nitrogen content, amino nitrogen, degree of proteolysis, protein molecular weight, and free amino acid composition content.

4. The determination method comprises the following steps:

the total nitrogen amount is determined by a Kjeldahl method;

protein molecular weight determination protein relative molecular mass determination was determined by reference to SDS-PAGE electrophoresis.

Determination of the content of free amino acid composition: the composition and content of 18 free amino acids were analyzed using an amino acid autoanalyzer. Accurately taking 3mL of sample, adding 0.5mL of 42% sulfosalicylic acid solution into a 5mL plastic centrifuge tube, shaking and shaking uniformly. Standing in a refrigerator for 12 hr, centrifuging at 13000r/min for 2min, filtering with 0.22m filter membrane, and analyzing on a computer.

Each sample analysis cycle was 53min, with 2 columns for analysis, separation column: 4.6mm multiplied by 60mm, eluent flow rate of 0.4mL/min, column temperature of 700 ℃ and column pressure of 11.627 MPa; reaction column: the flow rate of the ninhydrin and ninhydrin buffer solution is 0.35mL/min, the column temperature is 135 deg.C, and the column pressure is 1.078 MPa.

E/T value-essential amino acid/total amount of amino acids (essential amino acids: lysine (Lys), phenylalanine (Phe), valine (Val), leucine (Leu), threonine (Thr), isoleucine (Ile), methionine (Met), tryptophan (Trp))

The TAV value (taste activity value) is the ratio of the concentration of an amino acid component to its threshold value.

5. Influence of different complex enzyme preparations on degree of proteolysis of chicken liver

The influence of the complex enzyme preparations 6-10 on the degree of hydrolysis of chicken liver protein under specific conditions is shown in table 2 and fig. 3.

TABLE 2 Effect of different Complex enzyme preparations on the degree of proteolysis of Chicken liver under specific conditions

Different complex enzyme preparations	Degree of proteolysis DH (%)
		Complex enzyme preparation 6	29.72
Complex enzyme preparation 7	30.08
		Complex enzyme preparation 8	30.63
Complex enzyme preparation 9	31.55
		Complex enzyme preparation 10	30.96

As can be seen from Table 2 and FIG. 3, the complex enzyme preparation 9 has the highest degree of proteolysis and the best hydrolysis effect on chicken liver. The possible reasons for the analysis are: complex enzyme preparation 9 neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier ratio is 1: 4: 1: 0.10: 0.20: 4, the optimum proportion and the optimum pH value are kept, and the enzymatic action of various enzyme preparations in the compound enzyme preparation achieves the optimum decomposition effect.

6. Detection of protein molecular weight after enzymolysis of chicken livers by different complex enzyme preparations

The detection result of the molecular weight of the protein in the enzymatic hydrolysate after 6-10 enzymolysis of the complex enzyme preparation is shown in figure 4.

FIG. 4 shows that the supernatant of chicken liver after enzymolysis by different complex enzyme preparations 6, 7, 8, 9, 10 is diluted by 10 times, and the protein molecular weight in the supernatant is determined by SDS-PAGE electrophoresis. As can be seen from FIG. 4, the main products of the 5 kinds of complex enzyme preparations after enzymolysis are small peptides with the molecular weights of 14.4-20 KD and 20-27 KD, but a small amount of small peptides with the molecular weights of 9.5-14.4 KD exist in the complex enzyme preparation 9 product, which shows that the chicken liver is more thorough after enzymolysis by the complex enzyme preparation 9, and the effect is more obvious.

7. The free amino acid composition content of enzyme liquid after enzymolysis of chicken liver by different complex enzyme preparations

6-10 parts of the complex enzyme preparation is used for hydrolyzing chicken livers, and the composition content (g/dL) of free amino acids in an enzyme solution is shown in a table 3.

TABLE 3 free amino acid composition content (g/dL) in enzyme solution after enzymolysis of chicken liver by different complex enzyme preparations

As can be seen from Table 3, after the chicken liver is subjected to enzymolysis by different complex enzyme preparations 6, 7, 8, 9 and 10, the proportion (E/T) of essential amino acids (Lys, Phe, Val, Leu, Trp, Thr, Ile and Met) in the enzyme solution to free amino acids is respectively 49.37%, 49.88%, 49.25%, 49.07% and 49.70%, and the difference is small, and the proportion is higher than the WHO/FAO standard (35.38%). The essential amino acid is beneficial to the digestion and absorption of livestock and poultry, the higher the content and the higher the nutritional value, and the easier the additive is added into the feed to be absorbed by animal bodies.

8. The content of flavor amino acids in free amino acids after enzymolysis of chicken liver by different complex enzyme preparations

The content of flavor-developing amino acids in free amino acids after the chicken livers are subjected to enzymolysis by the complex enzyme preparation 6-10 is shown in table 4.

TABLE 4 proportion of flavor-developing amino acids in free amino acids

As can be seen from Table 4, after the chicken livers are subjected to enzymolysis by different complex enzyme preparations 6, 7, 8, 9 and 10, the sum of the contents of 3 umami amino acids, namely aspartic acid (Asp), glutamic acid (Glu) and proline (Pro), in the product is in an ascending trend, wherein after the chicken livers are subjected to enzymolysis by the complex enzyme preparation 9, the content of the umami amino acids in the product is the highest, and the effect is the most obvious. The sum of the 4 sweet amino acids threonine (Thr), serine (Ser), glycine (Gly) and alanine (Ala) is not very different. The content of the sum of the sweet/bitter amino acids valine (Val), methionine (Met), lysine (Lys) and arginine (Arg) is gradually reduced; the content of bitter amino acids is gradually reduced, wherein the content of the complex enzyme preparation 9 is the lowest, and the effect is the best.

After the chicken liver is subjected to enzymolysis by different complex enzyme preparations, the content difference of the enzymolysis products is not large according to the content of 4 flavor amino acids in the table 4, but the complex enzyme preparation 9 has relatively good effect.

Example 3

1. Compound enzyme preparation 3:

neutral protease: alkaline protease: lipase: visceral enzymes: flavor enzyme: the carrier is 1: 4: 1: 0.1: 0.2: 4.

2. additive amount, 5 gradients: 0.50%, 1.00%, 1.50%, 2.00%, 2.50%;

3. taking chicken liver as a raw material, and performing enzymolysis on the compound enzyme preparation 3 at the optimum pH of 8.0 and the enzymolysis temperature of 50 ℃, wherein the raw material comprises the following components: water-4: 1, enzymolysis time is 2 hours.

4. Measurement indexes are as follows: total protein nitrogen, amino nitrogen, degree of proteolysis, nitrogen recovery.

5. The determination method comprises the following steps:

total protein nitrogen: kjeldahl method;

amino nitrogen: potentiometric titration of formaldehyde;

6. Enzymolysis effect of different gradient complex enzyme preparation 3 on chicken liver

The results of enzymolysis of chicken livers by the complex enzyme preparation 3 with different dosages under specific conditions are shown in table 5 and fig. 5 and 6.

TABLE 5 enzymolysis results of different dosages of Complex enzyme preparation 3 on chicken liver under specific conditions

Additive amount (%)	Degree of proteolysis DH (%)	Nitrogen recovery DR (%)
			0.50	26.63	93.67
1.00	29.06	95.83
			1.50	31.55	99.71
2.00	30.74	98.64
			2.50	30.07	98.02

As can be seen from table 5, fig. 5, and fig. 6, when the additive amount was 1.50%, the complex enzyme preparation 3 exhibited the highest degree of proteolysis on chicken livers and the highest nitrogen recovery rate. At the beginning of the reaction, along with the gradual increase of the addition amount, the contact density of the enzyme and the substrate is increased, the enzymatic reaction rate is accelerated, the decomposition effect is good, and the proteolysis degree and the nitrogen recovery rate are high. When the adding amount reaches 1.50%, the degree of proteolysis and the nitrogen recovery rate reach the highest, and if the adding is continued, the degree of proteolysis and the nitrogen recovery rate are not changed too much.

Example 4

The enzymatic hydrolysate of the complex enzyme preparations 6, 7, 8, 9 and 10 is precipitated and freeze-dried to prepare protein feeds 1, 2, 3, 4 and 5 respectively, and the protein feeds are used for feeding piglets by replacing 2 percent of fish meal respectively.

(1) 288 weaned piglets (grown up) with similar body weight and good growth condition at 21 days are selected in the test and randomly divided into 6 groups, each group has 4 repetitions, each repetition has 12 pigs, the 1 st group is a positive control group, and the 2 nd, 3 rd, 4 th, 5 th and 6 th groups are respectively protein feeds 1, 2 nd, 3 rd, 4 th and 5 th groups which respectively replace 2% of fish meal.

(2) Feed formula and nutritional level

The experimental ration formula was designed according to the nutritional requirements of us NRC (2012) pigs using a corn-soybean meal type basal ration, see table 6.

TABLE 6 basic diet formula and nutritional level

Raw materials	Proportioning	Trophic level	Content (wt.)
				Corn (%)	49.87	Digestion energy (MJ/kg)	14.23
Soybean meal (%)	34.16	Crude protein (%)	23.70
				Puffed soybean (%)	4.91	Crude fat (%)	3.51
Whey powder (%)	2.68	Calcium (%)	0.80
				Concentrated soy protein (%)	2.17	Total phosphorus (%)	0.65
Fish meal (%)	2.17	Available phosphorus (%)	0.41
				Soya oil (%)	1.07	L-lysine (%)	1.36
Calcium hydrogen phosphate (%)	0.84	Egg + cystine (%)	0.76
				Stone powder (%)	0.83
Salt (%)	0.30
				Additive premix (%)	1.00
Total up to	100.00

Note: the premix provides vitamin A8-15(KIU), vitamin D3(KIU)1.5-3.0, vitamin E (mg) is not less than 32, vitamin K3(mg) is not less than 2.5, iron (mg) is not less than 175, copper (mg)125-200, zinc (mg)200-2250 and manganese (mg) is not less than 75 for each kilogram of diet; nutritional levels are calculated, dry matter content 88%.

(3) Feeding management

The test field is a pig farm in the flower east town of Guangzhou Huadu area, and the test period is 28 days. The pigsty is a semi-closed type high bed slotted floor. Piglets in the experimental period eat freely, and immunity and other management are operated according to the normal production management program of a pig farm.

(4) Index and method of measurement

1. Average Daily Gain (ADG):

average Daily Gain (ADG) ═ end of test-initial test weight/days of test

2. Average Daily Feed Intake (ADFI):

average Daily Feed Intake (ADFI) — (total feed intake-total residue intake)/test period

3. Feed consumption weight ratio (FCR):

feed consumption weight ratio (ADFI) Average Daily Feed Intake (ADFI)/Average Daily Gain (ADG)

4. Diarrhea Rate (DR):

the diarrhea rate is the number of diarrhea piglets in the test period/(number of test piglets) times 100%

(5) Measurement results

TABLE 7 Effect of different protein feed groups instead of 2% Fish meal on piglet growth Performance

Compared with a control group, the daily gain of the protein feed groups 2, 3, 4 and 5 is respectively increased by 7.4%, 10.4%, 12.5% and 10.2%, and the average difference is significant (P is less than 0.05); in the aspect of daily feed intake, the protein feed 1, 2, 3, 4 and 5 groups are respectively increased by 1.6%, 4.8%, 5.4%, 6.1% and 5.3%, and the average difference is not significant (P is more than 0.05); in the aspect of the feed consumption weight increase ratio, the protein feed 3, 4 and 5 groups are respectively reduced by 4.8%, 5.9% and 4.3%, and the average difference is obvious (P is less than 0.05). In the aspect of diarrhea rate, the protein feed 1, 2, 3, 4 and 5 groups are respectively reduced by 20.1%, 29.2%, 39.6%, 36.1% and 33.3%, and the difference is significant (P is less than 0.05).

From table 7, it can be seen that 2% of fish meal is replaced by 3, 4 and 5 groups of protein feeds, so that the daily gain of piglets can be effectively increased, the feed-weight ratio can be reduced, the growth performance can be improved, and the diarrhea rate can be obviously reduced.

Example 5

(1) Test animal grouping and handling

48 day-old, approximately 15kg (insignificant weight difference), good-growing-condition castrated ducrex (long x big) nursery pigs were selected, randomly divided into 6 groups, control group, protein feed 1, 2, 3, 4, 5 groups, each group was 4 replicates, and 2 pigs were replicated each.

(2) The diet formulation and nutritional levels were the same as in example 4.

(3) Feeding management

The test is carried out on Vietnam building of animal science institute of university of south China agricultural, a single-cage digestion test device is adopted for feeding, the temperature of a pigsty is controlled to be 25-30 ℃, natural illumination is carried out, and feeding management is carried out according to conventional operation rules.

(4) Test method

The digestion test adopts a full manure collection method. 8 in the morning of each day: 00 o and 6 o later: and feeding at a point 00 in a fixed amount, withdrawing the trough after one hour, weighing the rest material in the trough, and replacing the trough with a water trough. The specific operation is as follows:

in the adaptation period (day 1-7), feeding complete feed, feeding freely, and recording the daily feed intake of each pig.

In the pre-feeding period (8-12 days), test materials are fed according to 85% of the feed intake of each pig.

In a positive feeding period (day 13-16), feeding test materials according to 85% of the free feed intake of each pig, starting to collect dung 1 hour after feeding in the morning of day 1 in the positive feeding period, and continuously feeding and collecting the dung for 4 days, namely stopping collecting the dung 1 hour after early feeding in day 17; spraying a small amount of 10% hydrochloric acid after collecting the feces, and simultaneously recording the daily feed amount and the excess amount of each pig, wherein the feed intake is obtained by subtracting the excess amount from the total feed amount in the positive feeding period.

Taking repetition as a unit, placing a feces sample collected in a forward feeding period of 4 days in a 65 ℃ oven to be dried to constant weight, dampening for 24 hours at room temperature, weighing, then crushing and sieving with a 40-mesh sieve to prepare an air dried sample, and storing the air dried sample in a 4 ℃ refrigerator for later use.

(5) Index and method for determining digestion test

Determining Dry Matters (DM) in daily ration and excrement samples according to the method of GB/T6435-2006; pretreating Crude Protein (CP) according to a method of GB/T6432-1994, and then determining by using a FOSS Kjeltec-2300 full-automatic Kjeltec instrument; crude fat (EE) was measured by using a SZC-C type fat measuring instrument for Shanghai fiber test, with reference to GB/T6433-2006; total energy (GE) was measured using an IKAC2000 calorimeter.

(6) Nutrient digestibility calculation method

(7) Serum biochemical index determination and method

Serum samples were collected after the digestion test was completed. Feed and free feed began on day 1 after the digestion test was completed. On day 4 morning 8: before 00 feeds, 20mL of blood is collected from an anterior vena cava of each pig, the pig is obliquely placed in a cool place at room temperature, when serum is separated out, the pig is centrifuged at 3000rpm for 10 minutes, upper serum is taken out for subpackaging, the pig is placed in an ice box for precooling and then is stored at minus 80 ℃ for testing, a kit (purchased from Nanjing) is adopted for testing serum high-density lipoprotein (HDL), low-density lipoprotein (LDL), Total Cholesterol (TC) and free fatty acid (NEFA), and the ratio of the high-density lipoprotein to the low-density lipoprotein is calculated.

(8) Test results

TABLE 8 Effect of different protein feed groups replacing 2% Fish meal on digestibility of nutritional ingredients of piglets (%)

As can be seen from Table 8, the dry matter, protein, crude fat and total apparent digestibility were improved to some extent in the protein feeds 1, 2, 3, 4 and 5 compared to the control group, but the differences were not significant (P > 0.05). Compared with the control group, the protein digestibility of the protein feed groups 2, 3, 4 and 5 is respectively improved by 3.5 percent, 3.9 percent, 4.3 percent and 3.6 percent, and the difference is obvious (P is less than 0.05).

TABLE 9 Effect of protein feed group substituting 2% Fish meal on serum Biochemical index of piglets

As can be seen from table 9, the free fatty acids were reduced by 12.9%, 15.0%, 25.8% and 19.1% in the protein feeds 2, 3, 4 and 5, respectively, and the differences were significant (P < 0.05); in the aspect of total cholesterol, the protein 3, 4 and 5 groups are respectively reduced by 22.2%, 27.8% and 24.1%, and the average difference is obvious (P is less than 0.05); in the aspect of high-density lipoprotein, compared with a control group, the protein feed 3, 4 and 5 groups are respectively increased by 18.4%, 25.0% and 21.1%, and the average difference is obvious (P is less than 0.05); in the aspect of low-density lipoprotein, compared with a control group, the protein feed 4 and the protein feed 5 are respectively reduced by 33.0 percent and 27.1 percent, and the difference between the two groups is obvious (P is less than 0.05); the HDL/LDL ratio was increased by 48.3%, 87.6%, 66.3% in the groups 3, 4, and 5, and the differences were significant (P < 0.05).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A complex enzyme preparation for enzymolysis of chicken liver is characterized by comprising the following components in parts by mass:

1 part of neutral protease, 4 parts of alkaline protease, 1 part of lipase, 0.1 part of visceral enzyme, 0.2 part of flavor enzyme and 4 parts of carrier;

in the compound enzyme preparation, the enzyme activity of neutral protease is less than or equal to 5 ten thousand U/g and less than or equal to 10 ten thousand U/g; the enzyme activity of the alkaline protease is less than or equal to 20 ten thousand U/g and less than or equal to 10 ten thousand U/g; the enzyme activity of the lipase is less than or equal to 5 ten thousand U/g and less than or equal to 10 ten thousand U/g; the enzyme activity of the visceral enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g; the enzyme activity of the flavor enzyme is less than or equal to 5000U/g and less than or equal to 1 ten thousand U/g;

2. The complex enzyme preparation for enzymolysis of chicken livers as claimed in claim 1, which is characterized in that:

3. The use of the complex enzyme preparation for enzymolysis of chicken liver as claimed in any one of claims 1-2 in the preparation of high protein feed after enzymolysis of chicken liver.

4. A high protein feed characterized by: the compound enzyme preparation for enzymolysis of chicken liver as claimed in any one of claims 1-2 is added into chicken liver for enzymolysis, and then the mixture is frozen and dried to obtain the compound enzyme preparation.

5. The process for preparing a high protein feed according to claim 4, characterized in that it comprises the following steps:

(3) then adding the complex enzyme preparation solution into chicken livers preheated to 45-55 ℃, mixing and stirring for reaction to obtain an enzymatic hydrolysate; wherein, in the reaction condition, the pH is 8.0, the enzymolysis temperature is 50 ℃, and the raw materials are as follows: water = 4: 1, the enzymolysis time is 2.0 hours;

6. The method for preparing a high protein feed according to claim 5, wherein:

the addition amount of the complex enzyme preparation for chicken liver enzymolysis in the step (2) is 1.50-2.50%.

7. Use of the high protein feed of claim 4 to replace fish meal for feeding animals.