CN111528269B - A kind of protein fresh-keeping preservative for meat products and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of food preservation, and discloses a protein fresh-keeping preservative for meat products and a preparation method thereof. The preparation method comprises: (1) extracting protease inhibitors; (2) extracting lactoferrin, nisin, The enzyme and protease inhibitor are mixed with water, edetate disodium is added, and the concentrated solution is mixed with carboxymethyl cellulose sodium solution after nanofiltration and desalination to prepare meat product protein fresh-keeping preservative. The food antistaling agent of the present invention is mainly composed of protein, can inhibit the growth of microorganisms for a long time after being sprayed on the surface of meat, and does not change the color and flavor of food materials in a small amount, and is a relatively safe food antistaling agent.

Description

A kind of protein fresh-keeping preservative for meat products and preparation method thereof

technical field

The invention relates to the technical field of food preservation, in particular to a protein preservation preservative for meat products and a preparation method thereof.

Background technique

Traditional food preservation methods are divided into physical preservation methods and chemical preservation methods. Physical preservation methods, such as low-temperature freezing preservation and salt preservation, can only be used for short-term preservation, and will inevitably change the original flavor of aquatic products. The commonly used chemical preservation methods, such as adding chemical preservatives and antibiotics, are more and more rejected by consumers because of the addition of chemical components to varying degrees. Relatively speaking, biological preservatives are not only harmless to human health, but also do not change the flavor of food. Some biological preservatives are even more effective than chemical preservatives. Therefore, the development of new, safe and efficient biological preservatives has become the focus of food research workers. However, biological preservatives often have the problems of poor fresh-keeping effect and short duration. Therefore, it is necessary to develop a fresh-keeping preservative with good fresh-keeping effect, long-lasting fresh-keeping effect and harmless to human body.

Contents of the invention

In view of this, the object of the present invention is to provide a meat product protein fresh-keeping preservative and its preparation method, the meat product protein fresh-keeping preservative can inhibit the activity of microbial protease and amylase, and the color and luster and flavor of food materials are not changed in a small amount , is a safer food preservative.

In order to solve the above-mentioned technical problems, the invention provides a kind of preparation method of protein fresh-keeping preservative for meat products, comprising the following steps:

S1. Extraction of protease inhibitors: take the α ₂ M crude extract and concentrate it through a Sephacryl S-300 gel chromatography column, the eluent is 100mmol/L pH8.0 Tris-HCl buffer containing 0.5mol/L NaCl , detecting the effluent at the absorption peak of 280nm, collecting the first peak effluent and ultrafiltration, desalination and concentration to obtain protease inhibitors;

S2. mixing lactoferrin, nisin, lysozyme and the protease inhibitor with water, adding disodium edetate to obtain a mixed solution, and desalting the mixed solution to obtain a concentrated solution, The concentrated solution is mixed into sodium carboxymethyl cellulose solution to prepare meat product protein fresh-keeping preservative.

Preferably, in step S1, the molecular weight cut-off of the ultrafiltration membrane of the ultrafiltration is 300-500kDa.

Preferably, in step S1, the protein concentration of the protease inhibitor is 80-100 μg/L.

Preferably, in step S2, the dosage ratio of said lactoferrin, nisin, lysozyme, protease inhibitor, disodium edetate and water is (8-10)g:(3.5-4) g: (15-20) g: (3-5) mL: (8.4-16.8) g: 1000 mL.

Preferably, in step S2, the iron saturation of lactoferrin is 10%-15%, the specific activity of nisin is 1.0×10 ⁵ -1.0×10 ⁶ IU/mg, and the specific activity of lysozyme is 10000-20000IU/mg.

Preferably, in step S2, the volume of the concentrated solution is 1/8-1/5 of the volume of the mixed solution.

Preferably, in step S2, the dosing amount of the concentrated solution is 5-9 mL of the concentrated solution per 100 mL of sodium carboxymethyl cellulose solution.

Preferably, in step S2, the concentration of the sodium carboxymethyl cellulose solution is 10-30 g/L, and the molecular weight of the sodium carboxymethyl cellulose in the sodium carboxymethyl cellulose solution is 30-50 kDa.

The present invention also provides a protein fresh-keeping preservative for meat products prepared by the above preparation method.

Compared with the prior art, the present invention has the following beneficial effects:

1) The meat product protein fresh-keeping preservative of the present invention includes lactoferrin (LF), nisin (Nisin), lysozyme (LZ) and the protease inhibitor extracted by the method of the present invention. Wherein, the protease inhibitor extracted by the method of the present invention can inhibit the hydrolysis of protease to LF, LZ and Nisin, so that the three can exert long-acting antibacterial effect, LZ can destroy the bacterial cell wall, so that LF and Nisin can directly act on protoplasts, Through the synergistic antibacterial effect between several components, the purpose of food preservation is achieved.

2) In the preparation process of meat product protein fresh-keeping preservative of the present invention, disodium edetate is also added, disodium ethylenediaminetetraacetate can complex ferrous iron, and then remove by nanofiltration, just can reach LF The purpose of removing iron, thereby improving the antibacterial efficacy of LF.

3) Fresh meat is rich in iron. In the present invention, the concentrated solution after nanofiltration and desalination is mixed into the sodium carboxymethyl cellulose solution. The sodium carboxymethyl cellulose film can effectively isolate iron and LF, preventing LF from contacting The antibacterial effect caused by iron is ineffective.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

The invention provides a method for preparing meat protein fresh-keeping preservative, comprising the following steps:

S1. Extraction of protease inhibitors: take the α ₂ M crude extract and concentrate it through a Sephacryl S-300 gel chromatography column, the eluent is 100mmol/L pH8.0 Tris-HCl buffer containing 0.5mol/L NaCl , detecting the effluent at the absorption peak of 280nm, collecting the first peak effluent and ultrafiltration, desalination and concentration to obtain protease inhibitors;

S2. mixing lactoferrin, nisin, lysozyme and the protease inhibitor with water, adding disodium edetate to obtain a mixed solution, and desalting the mixed solution to obtain a concentrated solution, The concentrated solution is mixed into sodium carboxymethyl cellulose solution to prepare meat product protein fresh-keeping preservative.

Concretely, first get the α ₂ M crude extract to concentrate and pass through Sephacryl S-300 gel chromatography column, eluent is the 100mmol/L pH8.0 Tris-HCl buffer solution containing 0.5mol/L NaCl, at the absorption peak Detect the effluent at 280nm, collect the first peak effluent and ultra-filter, desalt and concentrate to obtain protease inhibitors; then mix lactoferrin, nisin, lysozyme and protease inhibitors with water, add ethylenediaminetetraacetic acid Disodium, to obtain a mixed solution, the mixed solution is nanofiltered and desalted to obtain a concentrated solution, the volume of the obtained concentrated solution is preferably 1/8-1/5 of the volume of the mixed solution; finally, the concentrated solution is mixed with sodium carboxymethyl cellulose solution to prepare protein fresh-keeping preservatives for meat products.

Fresh meat is rich in iron. In the present invention, the concentrated solution after nanofiltration and desalination is mixed into the sodium carboxymethyl cellulose solution. The sodium carboxymethyl cellulose membrane can effectively isolate iron and LF, preventing LF from being destroyed due to contact with iron. The antibacterial effect that causes loses effect, and wherein the incorporation amount of concentrated solution is preferably every 100mL sodium carboxymethyl cellulose solution is mixed with 5-9mL, and the molecular weight of sodium carboxymethyl cellulose is preferably 30-50kDa, sodium carboxymethyl cellulose solution The concentration is preferably 10-30g/L.

In the present invention, the molecular weight cut-off of the ultrafiltration membrane used for ultrafiltration and desalination is preferably 300-500 kDa, and the protein concentration of the protease inhibitor after ultrafiltration, desalination and concentration is preferably 80-100 μg/L. In the preparation process of meat protein fresh-keeping preservative of the present invention, the consumption ratio of lactoferrin, nisin, lysozyme, protease inhibitor, disodium edetate and water is preferably (8-10) g: (3.5-4)g:(15-20)g:(3-5)mL:(8.4-16.8)g:1000mL; Among them, the iron saturation of lactoferrin is preferably 10%-15%, lactis streptococcus The specific activity of lysozyme is preferably 1.0×10 ⁵ -1.0×10 ⁶ IU/mg, and the specific activity of lysozyme is preferably 10000-20000 IU/mg.

The present invention also provides a protein fresh-keeping preservative for meat products prepared by the above preparation method.

Lactoferrin (LF) adopted in the protein fresh-keeping preservative for meat products of the present invention is an important non-heme iron-binding glycoprotein in milk, a monomer glycoprotein with bactericidal activity in neutrophil granules, and the inhibitory effect of LF The bacterial effect is due to the fact that lactoferrin can highly bind iron, so that bacteria (except Lactobacillus) lose the basic element iron needed for growth and cannot grow. The antibacterial effect of LF is affected by many factors, among which the iron content of LF plays a decisive role in its antibacterial effect. When LF is saturated with iron, its antibacterial properties will disappear.

Nisin (Nisin), also known as nisin or transliterated as Nisin, used in the protein fresh-keeping preservative for meat products of the present invention, is a polypeptide substance produced by Streptococcus lactis, which can inhibit most Gram-positive bacteria , and has a strong inhibitory effect on Bacillus spores, so it is widely used in the food industry as a food preservative. After eating, it is quickly hydrolyzed into amino acids under the physiological pH conditions of the human body and the action of α-chymotrypsin, which will not change the normal flora in the human intestinal tract and cause resistance problems like other antibiotics, let alone interact with other antibiotics Cross-resistance appears, and it is a natural food preservative with high efficiency, non-toxicity, safety and no side effects.

The lysozyme (LZ) used in the protein fresh-keeping preservative for meat products of the present invention can effectively hydrolyze the peptidoglycan of the bacterial cell wall. For Gram-positive bacteria (G ⁺ ), such as Micrococcus luteus, Bacillus subtilis, or Micrococcus lyticus, the cell wall is almost entirely composed of peptidoglycan; for Gram-negative bacteria (G ^- ), such as Escherichia coli , Proteus, Shigella, Klebsiella pneumoniae, etc., the inner wall of the cell wall is peptidoglycan. Therefore, lysozyme can effectively destroy the bacterial cell wall, so that lactoferrin (LF) and nisin (Nisin) can directly act on the protoplast.

Through the synergistic antibacterial effect among several components, the protein fresh-keeping preservative for meat products of the present invention achieves the purpose of food safety and long-term fresh-keeping.

In order to further illustrate the present invention, a protein fresh-keeping preservative for meat products provided by the present invention and its preparation method are described in detail below in conjunction with examples.

The preparation method of α ₂ M crude extract:

Add 0.1% (w/v) soybean trypsin inhibitor to fresh porcine plasma at -5°C, and add EDTA (ethylenediaminetetraacetic acid) solution to make the final concentration to 10mmol/L. 18% Polyethylene Glycol 6000 fractional precipitation, after fully stirring for 60min, centrifuge at 10000r/min at 4°C for 30min, dissolve the precipitate in 50mmol/L pH7.4 Tris-HCl solution with 5 times the mass, and dissolve the precipitate in 50mmol/L pH7.4 Tris-HCl solution Fully dialyzed to obtain α ₂ M crude extract.

Example 1

1. Extraction of protease inhibitors: get the α ₂ M crude extract to concentrate and go through a Sephacryl S-300 gel chromatography column, the eluent is 100mmol/L pH8.0 Tris-HCl buffer containing 0.5mol/LNaCl, The effluent was detected at the absorption peak of 280nm, and the first peak effluent was collected and desalted and concentrated using an ultrafiltration membrane with a molecular weight cut-off of 400kDa to obtain a protease inhibitor. The protein concentration was determined to be 90 μg/L by a BCA protein quantification kit;

2. 9g of lactoferrin with an iron saturation of 12%, 3.7g of nisin with a specific activity of 5.0×10 ⁵ IU/mg, 17g of lysozyme with a specific activity of 15000IU/mg and 4mL of the product prepared in step 1 Mix protease inhibitors with 1000mL water evenly, add 12.4g disodium ethylenediaminetetraacetate, concentrate to 1/7 of the volume of the original solution after desalination by nanofiltration, mix 7mL concentrated solution into 100mL sodium carboxymethylcellulose with a molecular weight of 40kDa, Concentration is the sodium carboxymethyl cellulose solution of 20g/L, makes meat protein fresh-keeping preservative A.

Example 2

1. Extraction of protease inhibitors: the α ₂ M crude extract was concentrated and passed through a Sephacryl S-300 gel chromatography column, and the eluent was 100mmol/L pH8.0 Tris-HCl buffer containing 0.5mol/L NaCl , the effluent was detected at the absorption peak of 280nm, the first peak effluent was collected and concentrated by ultrafiltration and desalination with an ultrafiltration membrane with a molecular weight cut-off of 300kDa to obtain a protease inhibitor, and its protein concentration was determined to be 80 μg/L by a BCA protein quantification kit ;

2. 8g of lactoferrin with an iron saturation of 15%, 3.5g of nisin with a specific activity of 1.0×10 ⁵ IU/mg, 15g of lysozyme with a specific activity of 10000IU/mg and 3mL of the product prepared in step 1 Mix protease inhibitors with 1000mL water evenly, add 8.4g disodium ethylenediaminetetraacetate, concentrate to 1/8 of the volume of the original solution after desalination by nanofiltration, mix 9mL concentrated solution into 100mL sodium carboxymethylcellulose with a molecular weight of 50kDa, Concentration is 10g/L sodium carboxymethyl cellulose solution, makes meat product protein fresh-keeping preservative B.

Example 3

1. Extraction of protease inhibitors: the α ₂ M crude extract was concentrated and passed through a Sephacryl S-300 gel chromatography column, and the eluent was 100mmol/L pH8.0 Tris-HCl buffer containing 0.5mol/L NaCl , the effluent was detected at the absorption peak of 280nm, and the first peak effluent was collected and concentrated by ultrafiltration and desalination with an ultrafiltration membrane with a molecular weight cut-off of 500kDa to obtain a protease inhibitor, and its protein concentration was determined to be 100 μg/L by a BCA protein quantification kit ;

2. Mix 10g of lactoferrin with an iron saturation of 10%, 4g of nisin with a specific activity of 1.0× ¹⁰⁶ IU/mg, 20g of lysozyme with a specific activity of 20000IU/mg, and 5mL of the protease prepared in step 1 Mix the inhibitor with 1000mL water evenly, add 16.8g disodium ethylenediaminetetraacetate, concentrate to 1/5 of the volume of the stock solution after desalination by nanofiltration, mix 5mL concentrated solution into 100mL sodium carboxymethylcellulose with a molecular weight of 30kDa and a concentration of As a 30g/L sodium carboxymethylcellulose solution, meat protein fresh-keeping preservative C was prepared.

Experiment on Pork Preservation Effect

Meat sample processing: buy pork tenderloin in the supermarket, and cool it so that the center temperature drops to 0-4°C. Wipe the knives and chopping boards with 75% alcohol cotton balls in advance, and irradiate them with ultraviolet rays for 15 minutes. The surface layer of the meat piece was removed by aseptic operation to reduce the initial bacterial count on the surface, and the fascia and excess fat were removed, cut into meat pieces of about 100 g, and randomly divided into 6 groups, 5 pieces in each group.

Get 5 groups of meat pieces and spray 2-4mL of different protein fresh-keeping preservatives (meat product protein fresh-keeping preservatives A, B, C, 0.1wt% potassium sorbate solution and 0.1wt% sodium diacetate solution) respectively, use sterile distilled water Spray one group of meat pieces as the control group. The packaging method is plastic wrap tray packaging, and the storage and preservation temperature is 3±1°C. On the 0th, 7th, 14th, and 21d days, the total number of colonies was determined according to the method provided in GB4789.2-94 "Food Hygiene Microbiological Examination of the Total Number of Colonies", and the results were expressed in logarithmic values (LogCFU/g). Evaluation criteria: fresh meat is below 4LogCFU/g, sub-fresh meat is 4-6LogCFU/g, and spoiled meat is above 6LogCFU/g. Table 1 shows the logarithmic values of the total number of bacterial colonies in each group of meat pieces.

Table 1 The logarithmic result of the total number of bacterial colonies (LogCFU/g)

group 0d 7d 14d 21d control group 2.51±0.05 4.91±0.04 7.12±0.02 8.07±0.03 Example 1 2.43±0.04 2.75±0.03 3.24±0.05 3.77±0.04 Example 2 2.36±0.03 2.68±0.05 3.27±0.03 3.69±0.05 Example 3 2.61±0.02 2.91±0.02 3.33±0.04 3.95±0.02 0.1wt% potassium sorbate solution 2.48±0.05 3.51±0.03 4.65±0.03 5.87±0.03 0.1wt% sodium diacetate solution 2.39±0.03 3.78±0.04 4.82±0.02 5.79±0.03

It can be seen from Table 1 that with the prolongation of the storage period, the logarithmic value of the total number of colonies in each group showed an upward trend. After storage for 14 days, the logarithmic value of the total number of colonies in the control group is about 7.12, which has exceeded the national standard (6LogCFU/g), and it is spoiled meat with no fresh-keeping effect. Stored to 21 days, use 0.1wt% potassium sorbate solution and 0.1wt% sodium diacetate solution to process the logarithmic value of the total number of meat sample colonies not exceeding 6LogCFU/g, which is inferior fresh meat and has a certain fresh-keeping effect; while embodiment 1～ The logarithmic value of the total number of colonies of 3 does not exceed 4LogCFU/g, and still belongs to fresh meat, indicating that the protein fresh-keeping preservative for meat products prepared by the present invention can effectively inhibit bacterial growth and has a significant fresh-keeping effect.

The present invention provides a meat product protein fresh-keeping preservative and the idea and method of its preparation method. There are many methods and approaches for realizing the technical solution. The above description is only a preferred embodiment of the present invention. Those of ordinary skill in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components that are not specified in this embodiment can be realized by existing technologies.

Claims (6)

1. The preparation method of the meat product protein fresh-keeping preservative is characterized by comprising the following steps:

s1, extracting a protease inhibitor: take alpha ₂ Concentrating the M crude extract, passing through Sephacryl S-300 gel chromatography column, eluting with 100mmol/L Tris-HCl buffer solution containing 0.5mol/L NaCl and having pH of 8.0, detecting the eluate at absorption peak of 280nm, collecting the eluate of the first peak, ultrafiltering, desalting, and concentrating to obtain protease inhibitor; the cut-off molecular weight of the ultrafiltration membrane of the ultrafiltration is 300-500kDa; the protein concentration of the protease inhibitor is 80-100 mug/L;

s2, mixing lactoferrin, nisin, lysozyme, the protease inhibitor and water, adding ethylene diamine tetraacetic acid disodium to obtain a mixed solution, performing nanofiltration and desalination on the mixed solution to obtain a concentrated solution, and doping the concentrated solution into a sodium carboxymethylcellulose solution to obtain the meat product protein fresh-keeping preservative, wherein the consumption proportion of the lactoferrin, the nisin, the lysozyme, the protease inhibitor, the ethylene diamine tetraacetic acid disodium and the water is (8-10) g, (3.5-4) g, (15-20) g, (8.4-16.8) g and 1000mL.

2. The method for preparing the preservative for preserving the freshness of the protein of the meat products as claimed in claim 1, wherein in the step S2, the iron saturation of the lactoferrin is 10% -15%, and the specific activity of nisin is 1.0 x 10 ⁵ -1.0×10 ⁶ IU/mg, and the specific activity of the lysozyme is 10000-20000IU/mg.

3. The method for preparing the fresh-keeping preservative for protein of meat products as claimed in claim 1, wherein in step S2, the volume of the concentrated solution is 1/8-1/5 of the volume of the mixed solution.

4. The method for preparing the preservative for preserving meat product protein according to claim 1, wherein in step S2, the concentrated solution is added in an amount of 5-9mL per 100mL of the sodium carboxymethyl cellulose solution.

5. The method for preparing the fresh-keeping preservative for the protein of the meat products as claimed in claim 1, wherein in the step S2, the concentration of the sodium carboxymethyl cellulose solution is 10-30g/L, and the molecular weight of the sodium carboxymethyl cellulose in the sodium carboxymethyl cellulose solution is 30-50kDa.

6. The protein preservative for meat products prepared by the preparation method according to any one of claims 1 to 5.