CN110915995A - Method for preparing pet feed additive from poultry liver protein hydrolysate - Google Patents
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Abstract
The invention relates to a method for preparing a pet feed additive from poultry liver protein hydrolysate, which comprises the steps of firstly taking poultry livers, carrying out enzyme deactivation treatment on the poultry livers, then carrying out liver protein hydrolysis by adopting 1-2% protease to prepare hydrolysate with the hydrolysis degree of 15-25%, and after the hydrolysis is finished, carrying out centrifugal separation to obtain supernatant and carrying out freeze drying; preparing a hydrolysate with the mass percent of 4-6% from a freeze-dried sample, performing ultrasonic treatment for 10-15min, sequentially adding a reducing sugar solution with the mass percent of 4-6% of the sample, VB6 with the mass percent of 0.05-0.15% of the sample and amino acid with the mass percent of 0.05-0.15% of the sample into the solution, uniformly mixing, placing the mixture into a microwave for preheating, adjusting the pH value of the mixed solution to 7.0-9.0, reacting at 90-130 ℃ for 1.0-2.0 h, immediately performing ice bath after the reaction is finished, and finishing the reaction to obtain a Maillard product of the liver protein hydrolysate. The preparation method is simple, the steps are easy to operate, the poultry liver protein hydrolysate is subjected to Maillard reaction to obtain the pet feed additive which is safe to eat and has strong aromatic odor, and the comprehensive utilization of poultry by-products is improved.
Description
Technical Field
The invention relates to a method for preparing a pet feed additive from poultry liver protein hydrolysate, belonging to the technical field of food processing.
Background
The poultry by-products include, in addition to hair, blood, manure, egg shells, internal organs, etc. The waste pollutes the environment, infects the disease source and harms the poultry industry, but can be comprehensively utilized, changed into valuables, reduced the pollution, improved the environment and increased the comprehensive economic benefit of the poultry industry after being properly processed and treated. At present, the utilization degree of poultry livers is single, how to play the role of byproducts more effectively, and the realization of the comprehensive utilization is the key point of research.
Maillard reaction (Maillard reaction), also known as carbonamido reaction (Amino-carbonyl reaction), refers to the reaction of compounds containing hemiacetal hydroxyl group (aldehyde, reducing sugar) and compounds containing Amino group (Amino acid, protein, amine, peptide) through condensation and polymerization reaction to generate high molecular weight polymer, which is discovered at the earliest by French chemist Maillard in 1912 when the mixed solution of glycine and glucose is heated together.
Disclosure of Invention
The invention aims to fully utilize poultry by-products and realize comprehensive utilization, and provides a method for preparing a pet feed additive from poultry liver protein hydrolysate.
The invention adopts the following technical scheme: a method for preparing pet feed additive from poultry liver protein hydrolysate comprises the following steps:
(1) pretreatment: removing connective tissues from poultry livers, processing the poultry livers into minced liver (6mm 10mm 12 mm-12 mm 16mm 20mm) by a meat grinder, adding 2-4 times of volume of phosphate buffer solution with pH7.0-7.4, processing the mixture in a high-speed homogenizer at 5000 plus 10000rpm for 2-4min, working time 3-5 s and dwell time 3-5 s, mixing the mixture at high speed, crushing the mixture, performing enzyme deactivation, adding 10-20 times of volume of isopropanol and 5-10 times of volume of 75% ethanol, stirring the mixture uniformly, performing centrifugal separation to remove supernatant, and flatly spreading and volatilizing separated precipitates until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 4-8, adding distilled water into the precipitate according to a material-liquid ratio, stirring for 20-40 s at 3000-6000 r/min, heating to 45-50 ℃, adjusting the pH of the solution to 7.5-8.5, adding protease accounting for 1-2% of the mass of the raw material, and hydrolyzing to prepare hydrolysate with the hydrolysis degree of 15-25%;
(3) preparation of liver protein hydrolysate: immediately putting the mixture into a boiling water bath after hydrolysis is finished to inactivate enzyme for 10-30 min, cooling to room temperature, adjusting the pH to 7.0-8.0 by using 0.8-1.2M hydrochloric acid, centrifuging after hydrolysis is finished, taking supernatant, and freeze-drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing a hydrolysate with the mass percent of 4-6% from a freeze-dried sample, performing ultrasonic treatment for 10-15min, sequentially adding a reducing sugar solution with the mass percent of 4-6% of the sample, VB6 with the mass percent of 0.05-0.15% of the sample and amino acid with the mass percent of 0.05-0.15% of the sample into the solution, uniformly mixing, placing the mixture into a microwave for preheating, adjusting the pH value of the mixed solution to 7.0-9.0, reacting at 90-130 ℃ for 1.0-2.0 h, immediately performing ice bath after the reaction is finished, and finishing the reaction to obtain a Maillard product of the liver protein hydrolysate;
(5) the pet feed additive comprises: uniformly mixing the Maillard product and the green tea extract according to the mass ratio of 100-10:1 to obtain the pet feed additive.
Further, the power of the ultrasound in the step (4) is 150-250W, and the ultrasound is closed for 3s after 2 s.
Further, the frequency of the microwave in the step (4) is 600-900MHz, and the preheating time is 8-12 min.
Further, the centrifugal rotating speed in the step (1) and the step (3) is 10000-15000 g, and the centrifugal time is 10-20 min.
Further, the hydrolysis time in the step (2) is 30-60 min.
Further, the reducing sugar in the step (4) is one or more of glucose, maltose, xylose, galactose, lactose and fructose.
Further, the green tea extract is an aqueous extract of green tea.
VB6 and amino acid are added in the Maillard reaction process, the amino acid is one or more of alanine, cystine, L-lysine, L-histidine or L-arginine, so that the Maillard reaction is more complete and the obtained product has stronger fragrance.
The structure of the protein after ultrasonic treatment is changed, the functional characteristics of the protein are changed, the ultrasonic treatment method and the intermittent ultrasonic treatment method are adopted, the intermittent ultrasonic treatment is carried out for 3 seconds after every 2 seconds of ultrasonic treatment, the steps are repeated, the ultrasonic power is 150-.
The invention has the beneficial effects that: (1) the preparation method is simple, the steps are easy to operate, the poultry liver protein hydrolysate is subjected to Maillard reaction to obtain the pet feed additive which is safe in edibility and has strong aromatic odor, and the comprehensive utilization of poultry by-products is improved;
(2) the addition of VB6 and amino acid during the Maillard reaction process can realize that the Maillard product generates stronger fragrance.
Drawings
FIG. 1 is a graph showing the effect of the degree of hydrolysis on the progress of the Maillard reaction in accordance with the present invention.
FIG. 2 is a graph showing the effect of reaction time on the progress of the Maillard reaction in accordance with the present invention.
FIG. 3 is a graph showing the effect of pH on the progress of the Maillard reaction in accordance with the present invention.
FIG. 4 is a graph showing the effect of temperature on the progress of the Maillard reaction in accordance with the present invention.
FIG. 5 is a graph showing the effect of example of the present invention on the growth of E.coli.
FIG. 6 is a graph showing the effect on the growth of Staphylococcus saprophyticus in the example of the present invention.
FIG. 7 is a graph showing the results of the degree of grafting of example two, comparative example two, and comparative example three in the present invention.
FIG. 8 is a graph showing the results of measurement of intrinsic fluorescence of examples two and comparative examples of the present invention.
FIG. 9 is an XRD result pattern of example two and comparative examples of the present invention.
FIG. 10 is an XRD result pattern of xylose in the present invention.
Detailed Description
The invention will be further explained and illustrated with reference to specific embodiments.
Example 1
A method for preparing pet feed additive from poultry liver protein hydrolysate comprises:
(1) pretreatment: taking 100g of chicken liver in a beaker, removing connective tissues, processing the chicken liver into minced liver shape 6mm x 10mm x 12mm by using a meat grinder, adding 2 times of volume of phosphate buffer solution with pH7.0, processing the mixture in a high-speed homogenizer at 10000rpm for 2-4min, working for 3-5 s, stopping for 3-5 s, inactivating enzyme at 100 ℃ for 20min, adding 20 times of volume of 10% isopropanol and 5 times of volume of 75% ethanol, stirring the mixture on a magnetic stirrer for 12h, centrifuging the mixture for 20min by 10000g, removing supernatant, putting the precipitate in a tray, and volatilizing the precipitate until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 4, adding distilled water, stirring at 3000r/min for 20s, adjusting the solution to pH 7.5 with 0.4mol/L sodium hydroxide at 50 ℃, adding 1% pancreatin for hydrolysis to prepare hydrolysate with 15% hydrolysis Degree (DH),
(3) preparation of liver protein hydrolysate: immediately after hydrolysis, the mixture was put into a boiling water bath to inactivate the enzyme for 30min, cooled to room temperature, and the pH was adjusted to 7.0 with 0.8M hydrochloric acid. Centrifuging the solution after hydrolysis at 4000r/min for 10min, and freeze-drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass fraction of 6% from a freeze-dried sample, carrying out ultrasonic treatment for 10min, wherein the ultrasonic power is 150W, the ultrasonic time is 2s, then closing the ultrasonic treatment for 3s, and then adding reducing sugar solution with the mass fraction of 6% of the sample; adding VB6 accounting for 0.05 percent of the mass of the sample and amino acid accounting for 0.05 to 0.15 percent of the mass of the sample, uniformly mixing, placing in a microwave for preheating, wherein the frequency of the microwave is 600MHz, the preheating time is 12min, adjusting the pH value of the mixed solution to 7.0, reacting at 90 ℃ for 1.0h, immediately carrying out ice bath after the reaction is finished, and finishing the reaction by taking the browning degree and the antioxidant activity as indexes, wherein the reducing sugar is one or more of glucose, maltose, xylose, galactose, lactose and fructose;
(5) preparing the pet feed additive: mixing the Maillard product and the green tea extract according to the mass ratio of 100: 1.
example 2
A method for preparing pet feed additive from poultry liver protein hydrolysate comprises:
(1) pretreatment: taking 100g of chicken liver in a beaker, taking 100g of chicken liver in the beaker, removing connective tissues, processing the chicken liver into minced liver shape with a meat grinder, namely 6mm 10mm 12mm, adding 2 times of volume of phosphate buffer solution with pH7.0, processing the mixture in a high-speed homogenizer at 10000rpm for 4min, working time 3s, dwell time 3s, inactivating enzyme at 100 ℃ for 20min, adding 15 times of volume of 12% isopropanol and 8 times of volume of 75% ethanol, stirring the mixture on a magnetic stirrer for 15h, centrifuging the mixture for 15min at 12000g, removing supernatant, putting sediment in a tray and volatilizing the sediment until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 5, adding distilled water, stirring at 4000r/min for 30s, adjusting the pH of the solution to 8.0 with 0.5mol/L sodium hydroxide at 45 ℃, adding 1.5% pancreatin for hydrolysis, preparing hydrolysate with 20% hydrolysis Degree (DH),
(3) preparation of liver protein hydrolysate: immediately putting into boiling water bath to inactivate enzyme for 15min after hydrolysis, cooling to room temperature, adjusting pH to 7.0 with 1.0M hydrochloric acid, centrifuging the solution at 5000r/min for 15min, and freeze drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass fraction of 5% from a freeze-dried sample, carrying out ultrasonic treatment for 12min, wherein the ultrasonic power is 200W, the ultrasonic time is 2s, then closing the ultrasonic treatment for 3s, and then adding reducing sugar solution with the mass fraction of 5% of the sample; adding VB6 accounting for 0.10 percent of the mass of the sample and amino acid accounting for 0.1 percent of the mass of the sample, uniformly mixing, placing in a microwave for preheating, wherein the frequency of the microwave is 800MHz, the preheating time is 10min, adjusting the pH value of the mixed solution to 8.0, reacting for 1.5h at 130 ℃, immediately carrying out ice bath to finish the reaction after the reaction is finished, and taking the browning degree and the antioxidant activity as indexes, wherein the reducing sugar is one or more of glucose, maltose, xylose, galactose, lactose and fructose, and the amino acid is a mixture of alanine, cystine, L-lysine, L-histidine and L-arginine;
(5) the pet feed additive comprises: mixing the Maillard product and the green tea extract according to the mass ratio of 80: 1.
example 3
A method for preparing pet feed additive from poultry liver protein hydrolysate comprises:
(1) pretreatment: taking 100g of chicken liver in a beaker, taking 100g of chicken liver in the beaker, removing connective tissues, processing the chicken liver into liver paste 6mm 10mm 12mm by a meat grinder, adding 2 times of volume of phosphate buffer solution with pH7.0, processing in a high-speed homogenizer at 10000rpm for 4min, working time 4s, dwell time 3s, enzyme deactivation at 100 ℃ for 20min, adding 10 times of volume of 15% isopropanol and 10 times of volume of 75% ethanol, stirring on a magnetic stirrer for 24h, centrifuging at 15000g for 10min, removing supernatant, putting precipitate in a tray, and volatilizing until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 8, adding distilled water, stirring at 6000r/min for 40s, adjusting the solution to pH 8.5 with 0.6mol/L sodium hydroxide at 45 ℃, adding 2% neutral protease for hydrolysis to prepare hydrolysate with hydrolysis Degree (DH) of 25%,
(3) preparation of liver protein hydrolysate: immediately after hydrolysis, the mixture was put into a boiling water bath to inactivate the enzyme for 10min, cooled to room temperature, and the pH was adjusted to 7.0 with 1.2M hydrochloric acid. Centrifuging the solution after hydrolysis at 6000r/min for 20min, and freeze drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass fraction of 4% from a freeze-dried sample, carrying out ultrasonic treatment for 15min, wherein the ultrasonic power is 250W, the ultrasonic time is 2s, then closing the ultrasonic treatment for 3s, and then adding reducing sugar solution with the mass fraction of 4% of the sample; adding VB6 accounting for 0.15 percent of the mass of the sample and amino acid accounting for 0.15 percent of the mass of the sample, uniformly mixing, placing in a microwave for preheating, wherein the frequency of the microwave is 900MHz, the preheating time is 12min, adjusting the pH of the mixed solution to 9.0, reacting at 100 ℃ for 2.0h, immediately carrying out ice bath after the reaction is finished, finishing the reaction by taking the browning degree and the antioxidant activity as indexes, and taking reducing sugar as one or more of glucose, maltose, xylose, galactose, lactose and fructose;
(5) the pet feed additive comprises: mixing the Maillard product and the green tea extract according to the mass ratio of 10: 1.
comparative example one:
a method for preparing pet feed additive from poultry liver protein hydrolysate comprises:
(1) pretreatment: taking 100g of chicken liver in a beaker, taking 100g of chicken liver in the beaker, removing connective tissues, processing the chicken liver into minced liver shape with a meat grinder, namely 6mm by 10mm by 12mm, adding 2 times of volume of phosphate buffer with pH7.0, treating the chicken liver in a high-speed homogenizer at 10000rpm for 2-4min, working time of 3-5 s, stopping time of 3-5 s, inactivating enzyme at 100 ℃ for 20min, adding 15 times of volume of 10% isopropanol, stirring the mixture on a magnetic stirrer for 20h, centrifuging the mixture for 15min at 12000g, removing supernatant, putting precipitate in a tray, and volatilizing the precipitate until no alcohol smell exists;
(2) mixing the raw materials in a ratio of 1: 8, adding distilled water, stirring at 6000r/min for 40s, adjusting the solution to pH 8.5 with 0.6mol/L sodium hydroxide at 45 ℃, adding 2% pancreatin for hydrolysis, preparing hydrolysate with hydrolysis Degree (DH) of 25%,
(3) preparation of liver protein hydrolysate: immediately after hydrolysis, the mixture was put into a boiling water bath to inactivate the enzyme for 10min, cooled to room temperature, and the pH was adjusted to 7.0 with 1.2M hydrochloric acid. Centrifuging the solution after hydrolysis at 6000r/min for 20min, and freeze drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass fraction of 4% from a freeze-dried sample, carrying out ultrasonic treatment for 10-15min, wherein the ultrasonic power is 250W, the ultrasonic time is 2s, then closing for 3s, and then adding reducing sugar solution with the mass fraction of 4% of the sample; preheating in microwave, adjusting pH of the mixed solution to 9.0, reacting at 130 deg.C for 2.0h, immediately performing ice bath to complete the reaction, and taking browning degree and antioxidant activity as indexes, wherein the reducing sugar is one or more of glucose, maltose, xylose, galactose, lactose and fructose;
(5) the pet feed additive comprises: mixing the Maillard product and the green tea extract according to the mass ratio of 10:1 and mixing.
Comparative example two: non-sonicated chicken liver protein Maillard product panel
(1) Pretreatment: taking 100g of chicken liver in a beaker, taking 100g of chicken liver in the beaker, removing connective tissues, processing the chicken liver into minced liver shape with a meat grinder, namely 6mm by 10mm by 12mm, adding 2 times of volume of phosphate buffer with pH7.0, processing the mixture in a high-speed homogenizer at 10000rpm for 2-4min, working time being 3-5 s, stopping time being 3-5 s, inactivating enzyme at 100 ℃ for 20min, adding 15 times of volume of 10% isopropanol, stirring the mixture on a magnetic stirrer for 20h, centrifuging the mixture for 15min at 12000g, removing supernatant, putting precipitate in a tray, and volatilizing the precipitate until no alcohol smell exists to obtain chicken liver protein;
(2) taking the chicken liver protein in the step (1) as a sample to prepare hydrolysate with the mass fraction of 4%, then adding reducing sugar solution with the mass of 4% of the sample, adding VB6 with the mass of 0.10% of the sample and amino acid with the mass of 0.1% of the sample, and uniformly mixing; preheating in microwave, adjusting pH of the mixed solution to 9.0, reacting at 130 deg.C for 2.0h, immediately performing ice bath to complete the reaction, and taking browning degree and antioxidant activity as indexes, wherein the reducing sugar is one or more of glucose, maltose, xylose, galactose, lactose and fructose;
(3) the pet feed additive comprises: mixing the Maillard product and the green tea extract according to the mass ratio of 10:1 and mixing.
Comparative example three: maillard reaction group of chicken liver protein hydrolysate without ultrasonic treatment
A method for preparing pet feed additive from poultry liver protein hydrolysate comprises:
(1) pretreatment: taking 100g of chicken liver in a beaker, taking 100g of chicken liver in the beaker, removing connective tissues, processing the chicken liver into minced liver shape with a meat grinder, namely 6mm 10mm 12mm, adding 2 times of volume of phosphate buffer solution with pH7.0, processing the mixture in a high-speed homogenizer at 10000rpm for 4min, working time 3s, dwell time 3s, inactivating enzyme at 100 ℃ for 20min, adding 15 times of volume of 12% isopropanol and 8 times of volume of 75% ethanol, stirring the mixture on a magnetic stirrer for 15h, centrifuging the mixture for 15min at 12000g, removing supernatant, putting sediment in a tray and volatilizing the sediment until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 5, adding distilled water, stirring at 4000r/min for 30s, adjusting the pH of the solution to 8.0 with 0.5mol/L sodium hydroxide at 45 ℃, adding 1.5% pancreatin for hydrolysis, preparing hydrolysate with 20% hydrolysis Degree (DH),
(3) preparation of liver protein hydrolysate: immediately putting into boiling water bath to inactivate enzyme for 15min after hydrolysis, cooling to room temperature, adjusting pH to 7.0 with 1.0M hydrochloric acid, centrifuging the solution at 5000r/min for 15min, and freeze drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass fraction of 5% from a freeze-dried sample, and then adding reducing sugar solution with the mass fraction of 5% of the sample; adding VB6 accounting for 0.10 percent of the mass of the sample and amino acid accounting for 0.1 percent of the mass of the sample, uniformly mixing, placing in a microwave for preheating, wherein the frequency of the microwave is 800MHz, the preheating time is 10min, adjusting the pH value of the mixed solution to 8.0, reacting for 1.5h at 130 ℃, immediately carrying out ice bath to finish the reaction after the reaction is finished, and taking the browning degree and the antioxidant activity as indexes, wherein the reducing sugar is one or more of glucose, maltose, xylose, galactose, lactose and fructose, and the amino acid is a mixture of alanine, cystine, L-lysine, L-histidine and L-arginine;
(5) the pet feed additive comprises: mixing the Maillard product and the green tea extract according to the mass ratio of 80: 1.
detecting the browning degree of the Maillard reaction:
after the Maillard reaction under different conditions is finished, a sample cooled in ice bath is immediately centrifuged for 10min under the condition of 3000r/min, and the final product of the Maillard reaction is evaluated by adopting the absorption value under the wavelength of 420nm (diluted by 150 times).
The Maillard reaction is divided into three stages, namely a primary stage, a middle stage and a high stage, and browning phenomena are usually accompanied with the progress of the reaction, mainly because the high stage can generate a class of nitrogenous brown polymers which are called as melanoidins, the color of a reaction system can become darker and darker along with the continuous progress of the Maillard reaction, and therefore, the glycosylation degree of the protein can be reflected by the change of the color of the reaction system. Generally, the color change can be reflected by measuring the absorbance value of the reaction system at 420nm, so that the browning degree of the Maillard reaction system can be obtained, which is one of the methods for detecting the Maillard reaction rate, and the increase of the browning usually indicates that the grafting reaction is accelerated. Therefore, in the research, the action effect and the reaction process of the Maillard reaction between the liver protein and the reducing sugar after the ultrasonic treatment are analyzed by measuring the browning index of the reaction system.
The results are shown in FIGS. 1 to 4.
As can be seen from FIGS. 1 to 4, the optimum conditions for the Maillard reaction of poultry liver protein hydrolysate were 20% hydrolysis degree, 130 ℃ reaction temperature, 90min reaction time and 8.0 reaction pH, i.e., the conditions in example two of the present invention were the optimum conditions for the Maillard reaction.
Secondly, the antioxidant performance of the antioxidant obtained in the second example and the antioxidant obtained in the first comparative example are detected:
1. determination of reducing power: putting 1.0mL of sample solutions with different concentrations into a glass test tube, adding 1.0mL of 0.2mol/L phosphate buffer solution (pH6.6) and 2mL of 1% potassium ferricyanide solution, uniformly mixing, putting the mixture into a water bath at 50 ℃, reacting for 20min, adding 2mL of 10% trichloroacetic acid solution, uniformly mixing, centrifuging for 15min at 5000g, taking 1.5mL of supernate, adding 1.0mL of distilled water and 0.2mL of 0.1% ferric trichloride solution, mixing, reacting for 10min at room temperature, and measuring the absorbance at 700 nm; VC was also used as a positive control. The blank group is chicken liver protein hydrolysate which has not undergone Maillard reaction.
The results are shown in table 1:
| serial number | Group of | Reducing power A700nm |
| 1 | Blank group | 0.231-0.256 |
| 2 | Comparative example 1 | 2.157-2.224 |
| 3 | Example two | 2.543-2.567 |
As can be seen from the results in Table 1, the Maillard product of chicken liver protein hydrolysate with VB6 and amino acids added has better reducing power.
2. Chelating Fe2+Determination of the Capacity:
mu.L of sample solution is mixed with 5. mu.L of ferrous chloride (FeCl 2; 2mM) solution, then 10. mu.L of phenazine (5mM) is added and thoroughly mixed, the reaction is carried out for 20min at room temperature, and the absorbance value is measured at 562 nm. Deionized water was used as a blank in place of the sample solution. Carnosine solution served as a positive control. The percentage (%) of the chelating effect was calculated by the following formula:
chelating Fe2+Capacity (%) - (A)B-As)/AB]×100
In the formula: AB — absorbance of blank; absorbance value of AS-sample
The results are shown in table 2:
| serial number | Group of | Fe2+Chelating ability |
| 1 | Blank group | 8.5％-10.2％ |
| 2 | Comparative example 1 | 45.7％-48.1％ |
| 3 | Example two | 76.5％-80.2％ |
As can be seen from Table 2: at 1.0mg/mL, the Maillard product of chicken liver protein hydrolysate added with VB6 and amino acid has better Fe content2+Chelating ability.
3. Measurement of hydroxyl radical scavenging ability: the tubes were filled with 50. mu.L of 10mmol/L salicylic acid-ethanol solution (salicylic acid is insoluble in water and therefore formulated in absolute ethanol), 50. mu.L of 1.0mM FeSO4 solution in 50. mu.L of sample solution, and 100. mu.L of 0.5M H2O2 (the existing 30% H2O2 molar concentration was about 10M, diluted in deionized water). After mixing, the mixture is reacted for 10min at room temperature. The absorbance A1 was measured at 510 nm. 50 mu L of distilled water is taken to replace the 1.0mM FeSO4 solution, and the absorbance A2 is measured; absorbance A3 was measured by replacing the protein solution with 50. mu.L of distilled water. The hydroxyl radical clearance is calculated according to a formula.
Clearance (%) [1- (a1-a2)/A3] × 100
The results are shown in Table 3:
| serial number | Group of | Hydroxyl radical scavenging ability |
| 1 | Blank group | 88.5％-90.2％ |
| 2 | Comparative example 1 | 95.2％-97.6％ |
| 3 | Example two | 96.7％-98.8％ |
As can be seen from Table 3, the Maillard product of chicken liver protein hydrolysate supplemented with VB6 and amino acids has better hydroxyl radical scavenging ability.
Third, bacteriostasis test
1. Four commonly used microorganisms were selected: escherichia coli (gram negative bacteria) and Staphylococcus saprophyticus (gram positive bacteria) are used as test strains for determining the Maillard antibacterial property of poultry liver protein hydrolysate.
2. The components of the fungus culture medium are as follows: potato culture medium: peeled potato 300g, glucose 20g, water 1000mL, 12l C sterilization for 15 min.
3. The bacterial culture medium comprises the following components: LB medium, NA medium.
4. Activating strains: escherichia coli was inoculated to LB medium and Staphylococcus saprophyticus was inoculated to NA medium, and activated at 37 ℃ for 24 hours.
5. Preparation of bacterial suspension: and respectively transferring a proper amount of activated bacterial suspension into a new sterile culture medium, and culturing the bacteria at 37 ℃ for 24h to prepare initial bacterial suspension with the bacterial content of about 106-108 CFU/mL.
6. Method for measuring bacterial bacteriostasis effect by turbidimetry
A0.1-1.0 g Maillard product (dry weight) sample is added into 200mL of bacterial suspension, and then the bacterial suspension is placed at 37 ℃ for culturing for 8 h. Starting from 0h, the OD600 values of the bacterial suspensions were determined every 2 h. To exclude interference from membrane lysis, the OD600 value of the actual bacterial suspension was equal to the OD600 value of the membrane-added bacterial suspension minus the OD600 value of the membrane-added medium. The experiment was controlled with an un-filmed original bacterial suspension.
The bacteria culture solution generally has the maximum light absorption value at about 600nm, so the number of bacteria can be judged according to the OD600 value of the bacteria suspension. As can be seen from FIGS. 5 and 6, the growth of the control group of Escherichia coli and Staphylococcus saprophyticus was normal, the 2-12 h stage was the logarithmic growth phase of the bacteria, the growth of the number of bacteria at this stage was rapid, the growth was slow after 12h, and the cells entered the stationary phase. Compared with the control group, the growth rate of the Maillard reaction product group in the second embodiment is obviously reduced after 2-12 h, and the bacterial quantity of the Maillard reaction product group in the second embodiment is also obviously smaller than that of the control group after the Maillard reaction product group enters the stable period (12 h). The results show that the Maillard reaction product of example two is treated to obviously inhibit the growth of Escherichia coli and Staphylococcus saprophyticus.
And fourthly, further researching the structure and the property of the product.
1. The results of the graft degree measurements of the maillard reaction products of example two, comparative example two, and comparative example three are shown in fig. 7.
The degree of grafting is an indication of the degree of maillard reaction. In the maillard reaction, the reaction rate is faster with the ammonia carbonyl reacting, consuming free amino groups. As is clear from FIG. 7, the degree of grafting was the greatest in example two, because the degree of grafting was increased as the number of free amino groups on the peptide chain of the protein molecule increased after hydrolysis sonication, and the decrease in the number of free amino groups by binding to xylose was observed as an increase in the degree of grafting.
2. The Maillard reaction products of example two, comparative example two, and comparative example three were subjected to particle size measurements, and the results are shown in Table 1, using non-sonicated chicken liver protein, sonicated chicken liver protein hydrolysate, and non-sonicated chicken liver protein hydrolysate as controls.
TABLE 1
As can be seen from Table 1, the particle size of the hydrolyzed chicken liver protein is reduced because the protein is hydrolyzed into small molecular substances, and the ultrasonic treatment accelerates the hydrolysis of the protein, so that the particle size of the protein is smaller; the average particle size of the protein is increased by the maillard reaction, which is due to the access of xylose molecules.
3. Determination of endogenous fluorescence
The Maillard reaction products of example two, comparative example two, and comparative example three were measured for intrinsic fluorescence using sonicated chicken liver protein hydrolysate, non-sonicated chicken liver protein hydrolysate as controls, and the results are shown in FIG. 8.
As can be seen from FIG. 8, the fluorescence intensity of the sonicated chicken liver protein hydrolysate was the highest, followed by the non-sonicated chicken liver protein hydrolysate, and the fluorescence intensity of the product after the Maillard reaction was decreased. This is probably because the molecular weight of the protein is reduced by enzymolysis, the spatial structure of the protein is opened, and the amino acid residue with fluorescent residue in the protein is exposed, so that the fluorescence intensity is increased; and the products after Maillard reaction, xylose access protein or small molecular peptide cause steric hindrance effect, and the fluorescence signal of amino acid is shielded to a great extent, so that the fluorescence of the copolymer is reduced.
Measurement of XRD
XRD measurements were performed on the Maillard reaction products of example two, comparative example two, and comparative example three, and the control was unasonified chicken liver protein, sonicated chicken liver protein hydrolysate, unasonified chicken liver protein hydrolysate, and xylose. The results are shown in FIGS. 9 and 10.
As can be seen from fig. 9 and 10, the non-sonicated chicken liver protein, the sonicated chicken liver protein hydrolysate, and the non-sonicated chicken liver protein hydrolysate were all amorphous structures, and xylose was crystalline structure. After Maillard reaction, the non-sonicated chicken liver protein Maillard product of comparative example two, the sonicated chicken liver protein hydrolysate-Maillard product of example two, and the non-sonicated chicken liver protein hydrolysate-Maillard product of comparative example three were also amorphous, indicating that the non-sonicated chicken liver protein, the sonicated chicken liver protein hydrolysate, the non-sonicated chicken liver protein hydrolysate and xylose interacted, and the highly disordered carbon atom in the product resulted in a broad peak at 20.32 °. As can be seen from fig. 10, the diffraction angle of the product after the maillard reaction is shifted to the left (19.12 °), which is probably because the xylose-inserted protein or protein hydrolysate facilitates the chain movement of the protein, reduces the crystalline structure by interfering with the protein chain alignment, and reduces the crystallinity of the reactant after the reaction.
Claims (8)
1. A method for preparing pet feed additive by poultry liver protein hydrolysate is characterized in that: the method comprises the following steps:
(1) pretreatment: processing poultry livers into minced liver, adding 2-4 times of volume of phosphate buffer solution to a high-speed homogenizer for 2-4min for uniformly mixing and crushing, then performing enzyme deactivation, adding 10-20 times of volume of isopropanol and 5-10 times of volume of ethanol, uniformly stirring, performing centrifugal separation to remove supernatant, and flatly spreading and volatilizing separated precipitates until no alcohol smell exists;
(2) poultry liver proteolysis: mixing the raw materials in a ratio of 1: 4-8, adding distilled water into the precipitate according to a material-liquid ratio, stirring for 20-40 s at 3000-6000 r/min, heating to 45-50 ℃, adjusting the pH of the solution to 7.5-8.5, adding protease accounting for 1-2% of the mass of the raw material, and hydrolyzing to prepare hydrolysate with the hydrolysis degree of 15-25%;
(3) preparation of liver protein hydrolysate: immediately putting the mixture into a boiling water bath after hydrolysis is finished to inactivate enzyme for 10-30 min, cooling to room temperature, adjusting the pH to 7.0-8.0 by using 0.8-1.2M hydrochloric acid, centrifuging after hydrolysis is finished, taking supernatant, and freeze-drying the supernatant;
(4) maillard reaction of liver protein hydrolysate: preparing hydrolysate with the mass percent of 4-6% from a freeze-dried sample, carrying out ultrasonic treatment for 10-15min, and sequentially adding a reducing sugar solution with the mass of 4-6% and VB with the mass of 0.05-0.15% into the solution6And 0.05-0.15% of amino acid by mass of the sample, uniformly mixing, placing in a microwave for preheating, adjusting the pH value of the mixed solution to 7.0-9.0, reacting at 90-130 ℃ for 1.0-2.0 h, immediately carrying out ice bath after the reaction is finished, and finishing the reaction to obtain a Maillard product of the liver protein hydrolysate;
(5) preparing the pet feed additive: uniformly mixing the Maillard product and the green tea extract according to the mass ratio of 100-10:1 to obtain the pet feed additive.
2. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: and (4) in the step (4), the power of the ultrasound is 150-250W, and the ultrasound is closed for 3s after 2 s.
3. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: the frequency of the microwave in the step (4) is 600-900MHz, and the preheating time is 8-12 min.
4. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: and (3) the centrifugal rotating speed in the step (1) and the step (3) is 10000-15000 g, and the centrifugal time is 10-20 min.
5. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: the hydrolysis time in the step (2) is 30-60 min.
6. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: the reducing sugar in the step (4) is one or a mixture of glucose, maltose, xylose, galactose, lactose and fructose.
7. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: the green tea extract is an aqueous extract of green tea.
8. The method for preparing an antioxidant from poultry liver protein hydrolysate of claim 1, wherein: the protease is pancreatin, neutral protease or alkaline protease.
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