CN111374168B - Application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping - Google Patents
Application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping Download PDFInfo
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- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/42—Preservation of non-alcoholic beverages
- A23L2/44—Preservation of non-alcoholic beverages by adding preservatives
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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Abstract
The invention discloses an application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping. The natural antibacterial peptide As-CATH4 has broad-spectrum and efficient antibacterial action on common food spoilage bacteria, has quick bactericidal action, and has better synergistic action with the conventional preservative, namely butyl p-hydroxybenzoate. As-CATH4 is combined with butyl p-hydroxybenzoate to obviously inhibit the growth of spoilage bacteria, improve sensory evaluation score and prolong the storage time of food. In the storage experiments of salmon meat, cooked meat paste and fresh fruit juice, As-CATH4 obviously inhibits the growth of putrefying bacteria, improves the sensory evaluation score and prolongs the storage time of food. In an orange juice storage experiment, As-CATH4 is combined with butyl p-hydroxybenzoate, so that the growth of spoilage bacteria is obviously inhibited, the storage time of food is prolonged, and a good synergistic effect is presented. In addition, As-CATH4 can be completely degraded by trypsin, and has no toxic and side effects on human body. As-CATH4 has wide application prospect As a novel food preservative.
Description
Technical Field
The invention relates to an application of natural antibacterial peptide As-CATH4 in food preservation and freshness, belonging to the technical field of food chemistry.
Background
In the food industry, spoilage microorganisms are a significant hazard during the production and storage of food products, causing unnecessary waste and significant economic losses. Spoilage microorganisms can cause the physicochemical properties of the food to change, thereby causing the food to lose its original quality, nutritional value, and textural characteristics, ultimately resulting in non-compliance with food hygiene standards. Bacteria, mold and yeast can cause food spoilage, with bacteria and mold being the most common. To inhibit the growth of spoilage microorganisms, the food industry has adopted a variety of preservation methods, including chemical and physical preservation techniques. However, food spoilage is still a significant problem.
Chemical preservatives have been one of the most economical and effective methods widely used in the food industry over the past decades. Among the chemical preservatives that have been approved for use in food storage today are benzoic acid and its salts, sorbic acid and its salts, parabens, sulfur dioxide, sulfites and nitrites, and the like. However, the potential toxicity of chemical preservatives and consumer demand for natural healthy foods, there is an increasing urgent need to develop natural chemicals from plants, animals and microorganisms as novel food biological preservatives.
Antimicrobial peptides (AMPs) are a group of naturally occurring peptides, widely distributed in almost all types of multicellular organisms, are important substances of the host innate immune system, and play a key role in immune responses against microbial invasion. AMPs can act directly on almost all kinds of microorganisms (including bacteria, fungi, viruses, and even parasites) to exert antibacterial activity. Particularly has obvious effect on some microbial drug-resistant strains. In addition, many AMPs are also widely involved in enhancing and regulating the immune response of the host, enhancing the host's immunity to pathogenic microorganisms. Therefore, AMPs can be used as a substitute of conventional antibiotics, and have great potential application value in the field of treatment of bacterial infectious diseases. The antibacterial peptide as a food biological preservative has been reported at present, and a Nisin product, an antibacterial peptide product, has been applied to the market for many years. However, the antimicrobial peptide biological preservatives reported at present have two disadvantages: firstly, the antibacterial spectrum is narrow, and only part of food spoilage bacteria can be killed, for example, Nisin is only effective on gram-positive spoilage bacteria. Secondly, most of the antibacterial peptides have poor stability, are easily degraded by enzymes in food, and cannot exert the effect for a long time when being used as a preservative alone.
Disclosure of Invention
The invention aims to overcome the defects of the traditional chemical preservative and the existing antibacterial peptide, and provides the application of the natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping. As-CATH4 has broad-spectrum and high-efficiency antibacterial effect on common food spoilage bacteria, has quick bactericidal effect, and has better synergistic effect with the conventional preservative, namely butyl p-hydroxybenzoate. As-CATH4 is combined with butyl p-hydroxybenzoate to remarkably inhibit the growth of putrefying bacteria, improve sensory evaluation score and prolong the storage time of food. Has wide application prospect as a novel food preservative.
The first purpose of the invention is to provide an application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping.
Furthermore, the amino acid sequence of the natural antibacterial peptide As-CATH4 is shown in SEQ ID NO. 1.
Further, the natural antibacterial peptide As-CATH4 is used for inhibiting or killing putrefying bacteria in food.
Further, the putrefying bacteria is one or more of acinetobacter juni, acinetobacter johnsonii, psychrophilum coprophilum, proteus mirabilis, pseudomonas strawberrii, pseudomonas putida, serratia, sphingosine bacillus polyphagi, shewanella alga, shewanella putrefying, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus tequilensis, bacillus thuringiensis, bacillus belgii and roaming bacillus fluvialis.
Furthermore, the application also comprises the synergistic effect of the natural antibacterial peptide As-CATH4 and the butyl p-hydroxybenzoate, so that the putrefying bacteria in the food can be inhibited or killed.
The second purpose of the invention is to provide a food preservative containing the natural antibacterial peptide As-CATH 4.
Furthermore, the preservative also comprises butyl p-hydroxybenzoate.
Further, in the preservative, the mass ratio of the natural antibacterial peptide As-CATH4 to the butyl p-hydroxybenzoate is 1: 0.5-2.
The invention has the beneficial effects that:
the natural antibacterial peptide As-CATH4 has broad-spectrum and efficient antibacterial action on common food spoilage bacteria, has quick bactericidal action, and has better synergistic action with the conventional preservative, namely butyl p-hydroxybenzoate. As-CATH4 is combined with butyl p-hydroxybenzoate to remarkably inhibit the growth of putrefying bacteria, improve sensory evaluation score and prolong the storage time of food. In the storage experiments of salmon meat, cooked meat paste and fresh fruit juice, As-CATH4 obviously inhibits the growth of putrefying bacteria, improves the sensory evaluation score and prolongs the storage time of food. In an orange juice storage experiment, the As-CATH4 is combined with the butyl p-hydroxybenzoate, so that the growth of spoilage bacteria is obviously inhibited, the storage time of food is prolonged, and a good synergistic effect is presented. In addition, As-CATH4 can be completely degraded by trypsin, and has no toxic and side effects on human body. As-CATH4 has wide application prospect As a novel food preservative.
Drawings
FIG. 1 shows the bactericidal effect of As-CATH4 in sashimi;
FIG. 2 shows the bactericidal effect of As-CATH4 in cooked meat paste;
FIG. 3 shows the bactericidal effect of As-CATH4 in freshly squeezed orange juice;
FIG. 4 shows the results of pH measurement;
FIG. 5 is a graph showing the bactericidal effect in freshly squeezed orange juice when used in combination;
FIG. 6 shows the level of As-CATH4 degradation by trypsin.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1:
preparation of As-CATH4
(1) The chemical synthesis method of As-CATH4 comprises the following steps: the full sequence of As-CATH4 was synthesized using an automated peptide synthesizer (433A, Applied Biosystems) and purified by desalting by HPLC reversed-phase column chromatography.
(2) And the molecular weight is measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF).
(3) And the purity of the purified As-CATH4 is identified by a High Performance Liquid Chromatography (HPLC) method, the molecular weight is measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point is measured by isoelectric focusing electrophoresis, and the amino acid sequence structure is measured by an automatic amino acid sequencer.
The natural antibacterial peptide As-CATH4 consists of 38 amino acids, has a molecular weight of 4402.5Da and an isoelectric point of 12.78. The As-CATH4 amino acid sequence is As follows:
arg1Arg2Gly3Leu4Phe5Lys6Lys7Leu8Arg9Arg10Lys11Ile12Lys13Lys14Gly15Phe16Lys17Lys18Ile19Phe20Lys21Arg22Leu23Pro24Pro25Val26Gly27Val28Gly29Val30Ser31Ile32Pro33Leu34Ala35Gly36Arg37Arg38, wherein all amino acids are in L-form.
Example 2:
the antibacterial activity of As-CATH4 on common putrefying bacteria is detected:
and (4) measuring by using a minimum inhibitory concentration method. Putrefactive strains of fish are selected for MIC determination experiments. The test strains were inoculated into MH liquid medium (OXOID, UK), cultured with shaking at 37 ℃ until the logarithmic phase, and then the strain liquid cultured until the logarithmic phase was diluted to 2X 10 with fresh MH liquid medium 5 CFU/ml is ready for use.
100 mul LB liquid culture medium is added into each hole of sterile 96-hole plate in advance, then 100 mul As-CATH4 sample solution which is diluted to certain concentration by MH liquid culture medium and filtered by a filter membrane with 0.22 mu m holes is added into the first hole, 100 mul is added into the 2 nd hole after even mixing, and then dilution is carried out by multiple times (see table 1) in sequence, 100 mul is sucked out from the 9 th hole and discarded, and the 10 th hole is a control tube.
TABLE 1 dilution method
The 96-well plate was incubated at 37 ℃ for 18 hours with slow shaking, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible. The results are shown in Table 2.
As can be seen from Table 2, As-CATH4 exhibited stronger antibacterial activity against the isolated spoilage bacteria detected compared to p-hydroxybenzoic acid butyl ester preservative.
TABLE 2 As-CATH4 antibacterial Activity against putrefying bacteria
Example 3:
As-CATH4 sterilization rate measurement:
selecting four putrefying bacteria (Acinetobacter johnsonii, Shewanella alga, Proteus mirabilis, Bacillus subtilis) and culturing with MH liquid culture medium (OXOID, UK) at 37 deg.C for 12 hr, and diluting with fresh MH liquid culture medium to 10 6 CFU/ml bacterial suspension. A sample of As-CATH4 dissolved in sterile deionized water was added to the bacterial suspension to a final concentration of 5 × MIC. Placing the bacterial liquid added with the As-CATH4 sample in an incubator at 37 ℃ for shake culture, taking 10 mu l of bacterial liquid to dilute 1000 times at 0, 15, 30, 60, 90, 120 and 180 minutes respectively, then taking 50 mu l of diluted bacterial liquid to coat on an MH solid culture medium plate, and counting colonies after the culture in the incubator at 37 ℃ overnight. Butyl paraben was used as a positive control and sterilized deionized water as a negative control for this experiment.
As shown in tables 3-6, As-CATH4 showed extremely rapid sterilization of 4 spoilage bacteria strains, killing all bacteria within 15 minutes, while the positive control, butyl paraben, showed a slower sterilization rate.
TABLE 3 Sterilization rate of As-CATH4 against Acinetobacter johnsonii
TABLE 4 Sterilization rate of As-CATH4 against Shewanella alga
TABLE 5 Sterilization rates of As-CATH4 against Proteus mirabilis
TABLE 6 Sterilization rates of As-CATH4 against Bacillus subtilis
Example 4:
As-CATH4 and butyl p-hydroxybenzoate synergistic antibacterial effect determination:
the synergistic effect of the antibacterial peptide As-CATH4 and butyl paraben is determined by a chessboard microdilution method. Culturing Acinetobacter johnsonii, Shewanella alga, Proteus mirabilis and Bacillus subtilis in MH broth at 37 deg.C to exponential growth phase, and diluting with fresh MH to 2 × 10 6 CFU/ml。
As-CATH4 was serially diluted in MH medium in rows (50. mu.l) and butylparaben in MH medium in columns (50. mu.l), creating an 11X 7 matrix in 96-well plates. Mu.l of the bacterial culture was added to each well and incubated at 37 ℃ for 18 h. Each well was measured using a microplate reader (Bio-Tek ELX800, USA). The FIC of As-CATH4 was determined, where FICA is the MIC of antibacterial peptide A in combination with A alone (FICA + B/MICA) and FICB is the MIC of butylparaben in combination with B alone (FICB + MICA + B/MICB). The FIC index is calculated as FICindex ═ FICA + FICB. Synergy is defined as FICendex ≦ 0.5, no interaction is defined as 0.5< FICendex ≦ 4, and antagonism is defined as FICendex > 4.
As shown in Table 7, in addition to Proteus mirabilis, As-CATH4 and butylparaben showed strong synergistic effect on putrefying bacteria.
TABLE 7 synergistic antibacterial Effect of As-CATH4 with butyl p-hydroxybenzoate
Example 5:
use of As-CATH4 in food substrates:
(1) As-CATH4 bactericidal effect in sashimi
Fresh sashimi (4 ℃) were purchased from local supermarkets (Suzhou, Jiangsu, China) and immediately prepared for the experiment. Cutting salmon meat into small pieces of 1cm × 0.8cm × 0.2cm, soaking in 2mg/ml As-CATH4 for 20min, draining, placing into sterile centrifuge tube, and storing in refrigerator at 4 deg.C for 6 days. Fish samples were randomly selected, homogenized, spread on MH agar plates, and microbial colony assay was performed on days 0, 3, and 6, respectively. The positive control used was butyl paraben.
As a result, As-CATH4 did not provide effective preservation during the preservation of sashimi As shown in FIG. 1. The total viable count of the control and As-CATH4 was not significantly different throughout the storage period. The total bacterial count of the sashimi during storage was significantly reduced by butylparaben compared to the control group (P < 0.01).
(2) As-CATH4 has antibacterial effect in cooked meat paste
Sterilizing fresh salmon at 121 deg.C for 30min, adding sterile PBS solution, and homogenizing with tissue homogenizer to obtain meat paste. As-CATH4 value was added to the meat paste to a final concentration of 200. mu.g/mL. Acinetobacter junii was incubated in MH broth to logarithmic growth phase and washed three times with PBS buffer. Adding bacteria solution into meat paste to make final concentration 1 × 10 4 CFU/ml. Samples were stored at 25 ℃ for 5 days and sampled at the same time each day for dilution and MH solid plates were spread for colony counting. The positive control used was butyl paraben.
As shown in FIG. 2, As-CATH4 showed a certain bactericidal action in the fresh-keeping test of meat paste. The colony count of As-CATH4 group was significantly reduced within 4 days of initial storage (P <0.01) compared to the control group.
(3) As-CATH4 bactericidal effect in fresh orange juice
And extracting fresh orange juice by using a juice extractor. As-CATH4 was added to orange juice to give a final concentration of 200. mu.g/mL. The samples were stored at 25 ℃ for 5 days. And sampled and diluted at the same time every day, and MH solid plates were spread for colony counting. The positive control adopts potassium sorbate and sodium benzoate.
As shown in FIG. 3, the As-CATH4 showed the highest bactericidal activity in the fruit juice preservation test. As-CATH4 significantly reduced the total viable count (P <0.01) over the entire 5 day storage period compared to the control.
Example 6:
and (3) pH value measurement:
and extracting fresh orange juice by using a juice extractor. As-CATH4 was added to orange juice to a final concentration of 200. mu.g/mL. The samples were stored at 25 ℃ for 8 days. And the pH of the sample was measured at the same time each day using a digital pH meter. Butyl p-hydroxybenzoate, potassium sorbate and sodium benzoate were used as positive controls.
As shown in FIG. 4, the pH of As-CATH4 tended to decrease slightly when it was stored at 25 ℃ for 8 days. As-CATH4 was not statistically different from the conventional preservative treated groups (P > 0.05).
Example 7:
sensory evaluation analysis:
cutting fresh salmon into small pieces of 1cm × 0.8cm × 0.2cm, soaking in 2mg/ml As-CATH4 for 20min, draining, placing in sterile centrifuge tube, and storing at 25 deg.C for 5 days. Sensory evaluation was performed on the salmon at the same time every day. The method is based on a previously reported quality index method involving four important quality parameters including appearance, odor, hardness and overall acceptability. Seven experienced judges were randomly selected to score all samples according to the quality index method with a score range of 1-5, where 5 scores represent the best quality (table 2) and less than 3 scores are unacceptable.
Results as shown in table 8, all groups degraded in appearance, odor, hardness, and overall acceptability over extended storage times. However, the PBS group declined faster and the sensory score declined to less than 3 on day 2, which was unacceptable. In contrast, the As-CATH4 group and the conventional preservatives group still had a sensory score higher than 3 on day 2. The score of the antibacterial peptide group is very close to that of the traditional preservative groups of butyl p-hydroxybenzoate, potassium sorbate and sodium benzoate.
TABLE 8 sensory evaluation results
Example 8:
use of As-CATH4 in food matrices with a synergistic effect with butylparaben:
and extracting fresh orange juice by using a juice extractor. In normal experimental groups, As-CATH4 and butylparaben were added to orange juice to give a final concentration of 200. mu.g/mL, respectively. Synergistic experimental group in orange juice, As-CATH4 and butyl p-hydroxybenzoate were added in combination to give final concentrations of 100. mu.g/mL. The samples were stored at 25 ℃ for 5 days. And sampled and diluted at the same time every day, and MH solid plates were spread for colony counting.
As shown in FIG. 5, in the experiment of fruit juice preservation, As-CATH4 used in combination with P-hydroxybenzoic acid significantly reduced the total viable count (P <0.01) compared to the PBS group, and also significantly reduced the amount of preservative used.
Example 9:
protease degradation assay:
the degradation of As-CATH4 by trypsin was determined by reversed-phase high performance liquid chromatography. As-CATH4 and trypsin solution (Gibco, USA) were prepared in PBS buffer at a final molar ratio of 300:1 and incubated at 37 ℃ for 3h and 6h, respectively. Samples were extracted and analyzed by reverse phase high performance liquid chromatography (Thermo Scientific Syncronis C18 column, 250mm × 4.6mm) eluting with a linear gradient of acetonitrile/water (containing 0.1% TFA) mixed.
As shown in FIG. 6, As-CATH4 was gradually degraded by trypsin, gradually decreasing from 100% to 7.34% (3h), and finally to 0.87% (6 h). As-CATH4 is shown to be used As a novel preservative, can be completely degraded in intestinal tracts, and avoids the accumulation and toxicity of polypeptide in vivo.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> Suzhou university
<120> application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> PRT
<213> (Artificial sequence)
<400> 1
Arg Arg Gly Leu Phe Lys Lys Leu Arg Arg Lys Ile Lys Lys Gly Phe
1 5 10 15
Lys Lys Ile Phe Lys Arg Leu Pro Pro Val Gly Val Gly Val Ser Ile
20 25 30
Pro Leu Ala Gly Arg Arg
35
Claims (1)
1. A method for inhibiting or killing putrefying bacteria in food by using the synergistic effect of natural antibacterial peptide As-CATH4 and butyl p-hydroxybenzoate is characterized in that the amino acid sequence of the natural antibacterial peptide As-CATH4 is shown As SEQ ID NO. 1; the putrefying bacteria are one or more of acinetobacter junii, shiva alga and bacillus subtilis; the mass ratio of the natural antibacterial peptide As-CATH4 to the butyl p-hydroxybenzoate is 1: 0.5-2.
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| CN107261113A (en) * | 2017-05-26 | 2017-10-20 | 苏州大学 | Natural host defense peptide Alligatorin5 application |
| CN109160939A (en) * | 2018-09-21 | 2019-01-08 | 铜仁职业技术学院 | A kind of biologic antibiotic peptide and its application |
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