Triticum aestivum antibacterial peptide and its application in low-temperature heat sterilized milk preservation

Abstract

This invention discloses a black wheat antimicrobial peptide and its application in the preservation of low-temperature heat-pasteurized milk. The invention involves separating and purifying fermented black wheat through solid-state fermentation or enzymatic hydrolysis to obtain the fermented black wheat antimicrobial peptide, which contains five novel peptides with the sequences: DVNES (Asp-Val-Asn-Glu-Ser), LPWF (Leu-Pro-Trp-Fhe), DHYE (Asp-His-Tyr-Glu), LLLCAF (Leu-Leu-Leu-Cys-Ala-Fhe), and ETPD (Glu-Thr-Pro-Asp). The black wheat antimicrobial peptides of this invention, used alone or in combination with other peptides, can effectively inhibit the rapid increase in total bacterial count during low-temperature heat-pasteurized milk storage, slow down quality deterioration, and extend the shelf life of the milk. The technical solution provided by this invention can solve the preservation application of low-temperature milk, and the novel black wheat antimicrobial peptides can be used in the development of new food products.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide from black wheat and its application in the preservation of milk subjected to low-temperature heat sterilization, belonging to the field of polypeptide applications. Background Technology

[0002] Fresh milk has a high water content and is rich in nutrients and bioactive substances. However, it also contains microorganisms such as bacteria, actinomycetes, yeasts, and molds that can cause spoilage. To better preserve the natural bioactive substances and flavor of fresh milk, low-temperature heat sterilization is currently the primary method. Although most bacteria and microorganisms are killed, some heat-resistant bacteria and spores remain. These can multiply rapidly at suitable temperatures during storage, transportation, and sales, leading to spoilage and shortening shelf life. Therefore, measures are needed to extend the shelf life of low-temperature milk.

[0003] Antimicrobial peptides are a collective term for a class of small molecule peptides with broad-spectrum antimicrobial activity. Antimicrobial peptides prepared from food proteins are highly desirable due to their safe origin, significant antimicrobial effects, good solubility, lack of unpleasant flavor, strong stability, and ability to be digested and broken down into non-toxic small peptides or amino acids in the human body. These advantages make them promising candidates for development as novel natural food ingredients, new food additives, or new food-related products, with excellent potential applications in various processed foods.

[0004] Currently, antimicrobial peptides from various sources have shown certain effects in inhibiting food spoilage bacteria and preserving fresh meat and fruit, and have been proven to be one of the good alternatives to chemical preservatives. For example, patent publication number CN111406872A discloses the preparation of a composite preservation film using the horseshoe crab antimicrobial peptide TacⅠ with chitosan and / or Nisin for the preservation of chilled fresh meat. Patent publication number CN111406872A discloses the inhibitory effect of the mud clam hemoglobin antimicrobial peptide on the food spoilage bacteria Listeria monocytogenes. Patent publication number CN111374168A discloses that the antibacterial effect of the natural antimicrobial peptide As-CATH4 is slightly weaker than that of the chemical substance butylparaben, but it can still extend the storage time of sashimi, cooked meat sauce, and freshly squeezed juice, and the effect is better when used in combination with butylparaben. Patent CN103404939B describes a 10% (w / w) antimicrobial peptide mixture applied evenly to tofu at a 20% spraying rate for preservation. Patent CN112094323B describes a combined treatment of *Lactobacillus plantarum* antimicrobial peptides and nisin to effectively delay spoilage of pork caused by microbial growth and reproduction. Currently reported antimicrobial peptides are mainly antimicrobial peptide complexes with unknown sequences or preservatives composed of antimicrobial peptides and nisin. These have narrow antimicrobial spectra, require high concentrations, and their stability is significantly affected by pH and temperature. Their effectiveness is not obvious when used alone, highlighting the urgent need to develop new antimicrobial peptides from food sources. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a black wheat antimicrobial peptide with a novel amino acid sequence, wherein the black wheat antimicrobial peptide is composed of one or more of the following polypeptides; the amino acid sequences of the polypeptides are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and the black wheat antimicrobial peptide is prepared from black wheat flour.

[0006] Another objective of this invention is the application of black wheat antimicrobial peptides in the preservation of milk through low-temperature heat sterilization.

[0007] When the black wheat antimicrobial peptide is composed of a mixture of multiple polypeptides, the ratio of the amounts of each polypeptide added is 1:1. The low-temperature heat sterilization described in this invention refers to heat sterilization at a temperature below 100°C.

[0008] The beneficial effects of this invention are as follows:

[0009] (1) This invention provides a novel amino acid sequence black wheat antimicrobial peptide, which is safer than chemical preservatives.

[0010] (2) The use of the black wheat antimicrobial peptides described in this invention in low-temperature heat-sterilized milk can effectively inhibit the increase in the total number of colonies during the refrigeration process, improve the sensory evaluation score, slow down the decrease in fat and protein content, delay the increase in lactic acidity, and prolong the storage time of milk.

[0011] (3) The black wheat antimicrobial peptide provided by the present invention can be developed into a new food product for the preservation of low-temperature heat sterilized milk. Attached Figure Description

[0012] Figure 1 This refers to the change in total bacterial count during the storage of low-temperature heat-sterilized milk;

[0013] Figure 2 This refers to the change in fat content of heat-sterilized milk during storage.

[0014] Figure 3 Changes in protein content during storage of low-temperature heat-sterilized milk;

[0015] Figure 4 Changes in acidity during the storage of low-temperature heat-sterilized milk. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: Preparation method of antimicrobial peptides from black wheat

[0018] In this embodiment, a polypeptide with the amino acid sequences Leu-Leu-Leu-Cys-Ala-Fhe (LLLCAF) and Glu-Thr-Pro-Asp (ETPD) was prepared. The specific preparation steps are as follows:

[0019] (1) Place 10g of black wheat flour in a fermentation bottle and sterilize it by high-pressure steam at 120℃ for 25min. After cooling, add sterile water at a material-to-liquid mass ratio of 1:8, add 1.2% ammonium sulfate and 0.3% potassium dihydrogen phosphate by mass, and inoculate with 2.0mL of Aspergillus oryzae (with a bacterial count of 1.0 x 10). 7 Fermentation was completed by culturing at 28°C and 90% humidity for 72 hours at natural pH (CFU / mL) until fermentation was complete.

[0020] (2) After fermentation, add 90 mL of sterile water to the solid fermentation product and extract by ultrasonication at room temperature for 40 min. After extraction, place it in a boiling water bath for 10 min, cool to room temperature, and then centrifuge at 4℃, 10000 r / min for 20-30 min to collect the supernatant.

[0021] (3) Use an ultrafiltration membrane with a molecular weight cutoff of 5000 Da to ultrafilter the supernatant in step (2) and collect the ultrafiltrate with a molecular weight of less than 5000 Da for later use.

[0022] (4) The ultrafiltrate from step (3) was separated and purified using a dextran G25 gel column with a loading volume of 0.5 ml, a flow rate of 1 mL / min, and water as the mobile phase. Each single peak was collected.

[0023] (5) Bacterial inhibition rate determination: Listeria monocytogenes, Salmonella, Shigella sonnei, Staphylococcus aureus, and Escherichia coli were cultured to the logarithmic growth phase, and the bacterial suspensions were resuspended at 1×10⁻⁶. 5 cfu / mL. The sample solutions of each peak in (4) were diluted to different concentrations using a two-fold dilution method. 100 μL of sample solutions of different concentrations and 100 μL of bacterial suspension were added to each well of a 96-well microplate and mixed. The plates were then incubated at 37°C for 24 h. The absorbance of each well was measured at 600 nm. The lowest concentration that completely inhibited bacterial growth was determined as the minimum inhibitory concentration (MIC) of the sample against that bacterium.

[0024] Fungal inhibition rate determination: *Candida albicans*, *Fusarium oxysporum*, *Aspergillus niger*, and *Trichoderma viride* were inoculated onto PDA solid medium and cultured at 28°C for 7 days. Fungal spores were washed away with sterile water. After counting with a hemocytometer, the spores were resuspended in fresh YPD liquid medium (2% glucose, 1% peptone, 0.5% yeast extract) at a concentration of 2 × 10⁻⁶. 4 CFU / mL. 50 μL of different single-peak samples were added to a 96-well microplate, followed by an equal volume of resuspended bacterial suspension. The plate was incubated at 30°C for 24 h. Then, 10 μL of MTT solution (5 mg MTT / mL, dissolved in PBS, pH 7.4) was added to each well, and the plate was incubated at 37°C for another 4 h. The absorbance at 570 nm was measured using a microplate reader. The lowest concentration that completely inhibited fungal growth was determined as the minimum inhibitory concentration (MIC) of the sample against that fungus, and the single-peak sample with the strongest antifungal activity was selected.

[0025] (6) The single-peak sample with the strongest antibacterial rate obtained from dextran gel column separation was further separated and purified using high-performance liquid chromatography (HPLC). The chromatographic column was Angilent Eclipse XDB-C18, the column temperature was 30–40℃, the sample loading volume was 5–15 μL, the flow rate was 0.5–1.2 mL / min, and the wavelength was 220 nm. The elution mobile phase A was acetonitrile (containing 0.1% trifluoroacetic acid), and the mobile phase B was ultrapure water (containing 0.1% trifluoroacetic acid). The elution conditions were: 0–5 min, mobile phase A with a volume percentage of 10%–20%, and mobile phase B with a volume percentage of 80%–90%; 5–30 min, mobile phase A with a volume percentage of 20%–45%, and mobile phase B with a volume percentage of 55%–80%. The single peak of the liquid phase was collected, freeze-dried, and stored for later use.

[0026] (7) The single peaks collected in step (6) were reconstituted and sequenced by LC-MS / MS. An Agilent Eclipse Plus C18 column was used, with a column temperature of 30℃, a wavelength of 220nm, a sample loading volume of 2μL, a flow rate of 0.2mL / min, and mobile phase A consisting of acetonitrile containing 0.1% formic acid and mobile phase B consisting of ultrapure water containing 0.1% formic acid. Elution conditions were as follows: 0-3 min, mobile phase A volume percentage from 5%, mobile phase B volume percentage from 95%-90%; 3-15 min, mobile phase A volume percentage from 5%-20%, mobile phase B volume percentage from 95%-80%; 15-25 min, mobile phase A volume percentage from 20%-40%, mobile phase B volume percentage from 80%-60%; 25-35 min, mobile phase A volume percentage from 40%-75%, mobile phase B volume percentage from 60%-25%; 35-40 min, mobile phase A volume percentage from 5%, mobile phase B volume percentage from 95%. Mass spectrometry conditions were: Full MS, dd-MS2, positive ion mode scan, resolution Full MS 35000, dd-MS2 17500, scan range 200-2000 m / z, collision energy 20 eV. The amino acid sequences were identified using PeaksStudio software. The obtained liquid chromatography-mass spectrometry fragment information was used to automatically identify peptide sequences and molecular weights using DeNovo™ software. Peptide sequences with an average local confidence level >85% were selected. Finally, the peptide powders with the amino acid sequences Leu-Leu-Leu-Cys-Ala-Fhe (LLLCAF) and Glu-Thr-Pro-Asp (ETPD) were selected.

[0027] Example 2: Preparation method of antimicrobial peptides from black wheat

[0028] In this embodiment, peptides with the amino acid sequences Asp-Val-Asn-Glu-Ser (DVNES), Leu-Pro-Trp-Fhe (LPWF), and Asp-His-Tyr-Glu (DHYE) were prepared. The specific preparation steps are as follows:

[0029] (1) Black wheat flour was dissolved in distilled water at a mass ratio of 1:15, stirred for 30 min, and then extracted by ultrasonication at 50℃ for 2 h. The pH was adjusted to 10.0 with 0.1M NaOH, and the mixture was extracted by stirring in a water bath at 35℃ for 2 h. The extract was centrifuged at 5000 r / min for 20 min, the supernatant was collected, and the pH was adjusted to 4.5 with 0.1M HCl. After standing for protein precipitation, the mixture was centrifuged at 5000 r / min for 20 min, the precipitate was collected, washed 3 times with pure water, the pH was adjusted to neutral, the precipitate was redissolved, and the mixture was freeze-dried to obtain crude black wheat protein.

[0030] (2) Weigh 10g of crude black wheat protein, add sterile water according to the material-to-liquid mass ratio of 1:12, stir, place in a constant temperature water bath at 35℃, and adjust the pH of the protein solution to 8.1 with NaOH solution.

[0031] (3) Add 1g of trypsin (5000U / g crude protein) for enzymatic hydrolysis. Maintain pH stability during hydrolysis. After 3.5h of hydrolysis, adjust the pH to neutral with HCl solution and incubate in a boiling water bath for 15min to inactivate the enzyme. After cooling, centrifuge at 10000r / min for 20min at 45℃ and collect the supernatant, which is black wheat peptide.

[0032] (4) Use an ultrafiltration membrane with a molecular weight cutoff of 5000 Da to ultrafilter the supernatant obtained in step (3) and collect the ultrafiltrate with a molecular weight of less than 5000 Da for later use.

[0033] (5) The ultrafiltrate obtained in step (4) was separated and purified using a dextran G25 gel column with a loading volume of 0.5 mL, a flow rate of 1 mL / min, and water as the mobile phase. Each single peak was collected.

[0034] (6) Bacterial inhibition rate determination: Listeria monocytogenes, Salmonella, Shigella sonnei, Staphylococcus aureus, and Escherichia coli were cultured to the logarithmic growth phase, and the bacterial suspensions were resuspended at 1×10⁻⁶. 5 cfu / mL. The single-peak sample solutions in (5) were diluted to different concentrations using a two-fold dilution method. 100 μL of each concentration of single-peak sample solution and 100 μL of bacterial suspension were added to each 96-well microplate and mixed. The plates were then incubated at 37°C for 24 h. The absorbance of each well was measured at 600 nm. The lowest concentration that completely inhibited bacterial growth was determined as the minimum inhibitory concentration (MIC) of the sample against that bacterium.

[0035] Fungal inhibition rate determination: *Candida albicans*, *Fusarium oxysporum*, *Aspergillus niger*, and *Trichoderma viride* were inoculated onto PDA solid medium and cultured at 28°C for 7 days. Fungal spores were washed away with sterile water. After counting with a hemocytometer, the spores were resuspended in fresh YPD liquid medium (2% glucose, 1% peptone, 0.5% yeast extract) at a concentration of 2 × 10⁻⁶. 4CFU / mL. 50 μL of single-peak sample solutions of different concentrations were added to each well of a 96-well microplate, followed by an equal volume of resuspended bacterial suspension. The plate was incubated at 30°C for 24 h. Then, 10 μL of MTT solution (5 mg MTT / mL, dissolved in PBS, pH 7.4) was added to each well, and the plate was incubated at 37°C for another 4 h. The absorbance at 570 nm was measured using a microplate reader. The lowest concentration that completely inhibited fungal growth was determined as the minimum inhibitory concentration (MIC) of the sample against that fungus. The single-peak sample with the highest inhibition rate was selected.

[0036] (7) The single peak with the strongest antibacterial rate obtained from the dextran gel column separation was further separated and purified using high performance liquid chromatography (HPLC). The chromatographic column was Angilent Eclipse XDB-C18, the column temperature was 30–40℃, the sample loading volume was 5–15 μL, the flow rate was 0.5–1.2 mL / min, and the wavelength was 220 nm. The elution mobile phase A was acetonitrile (containing 0.1% trifluoroacetic acid), and the mobile phase B was ultrapure water (containing 0.1% trifluoroacetic acid). The elution conditions were: 0–5 min, mobile phase A with a volume percentage of 10%–20%, and mobile phase B with a volume percentage of 80%–90%; 5–30 min, mobile phase A with a volume percentage of 20%–45%, and mobile phase B with a volume percentage of 55%–80%. The single peak was collected, freeze-dried, and stored for later use.

[0037] (8) The single peaks collected in step (7) were reconstituted and sequenced by LC-MS / MS. An Agilent Eclipse Plus C18 column was used, with a column temperature of 30℃, a wavelength of 220nm, a sample loading volume of 2μL, a flow rate of 0.2mL / min, and mobile phase A consisting of acetonitrile containing 0.1% formic acid and mobile phase B consisting of ultrapure water containing 0.1% formic acid. Elution conditions were as follows: 0-3 min, mobile phase A volume percentage from 5%, mobile phase B volume percentage from 95%-90%; 3-15 min, mobile phase A volume percentage from 5%-20%, mobile phase B volume percentage from 95%-80%; 15-25 min, mobile phase A volume percentage from 20%-40%, mobile phase B volume percentage from 80%-60%; 25-35 min, mobile phase A volume percentage from 40%-75%, mobile phase B volume percentage from 60%-25%; 35-40 min, mobile phase A volume percentage from 5%, mobile phase B volume percentage from 95%. Mass spectrometry conditions were: Full MS, dd-MS2, positive ion mode scan, resolution Full MS 35000, dd-MS2 17500, scan range 200-2000 m / z, collision energy 20 eV. The amino acid sequences were identified using PeaksStudio software. The obtained liquid chromatography-mass spectrometry fragment information was used to automatically identify peptide sequences and molecular weights using DeNovo™ software. Peptide sequences with an average local confidence level >85% were selected, and finally, peptide powders with the following amino acid sequences were screened out: Asp-Val-Asn-Glu-Ser (DVNES), Leu-Pro-Trp-Fhe (LPWF), and Asp-His-Tyr-Glu (DHYE).

[0038] Example 3: Application of black wheat polypeptides in the preservation of low-temperature heat-sterilized milk

[0039] The application of black wheat antimicrobial peptides in the preservation of low-temperature heat-sterilized milk described in this embodiment, the peptides used in this embodiment were prepared from Examples 1 and 2, specifically including the following steps:

[0040] (1) Experimental Groups: The experiment was set up with 9 groups: blank group, Nisin group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, experimental group 6, and experimental group 7, with 3 replicates per group. The amino acid sequence of the black wheat antimicrobial peptide added in experimental group 1 was Asp-Val-Asn-Glu-Ser (DVNES); the amino acid sequence of the black wheat antimicrobial peptide added in experimental group 2 was Leu-Pro-Trp-Fhe (LPWF); the amino acid sequence of the black wheat antimicrobial peptide added in experimental group 3 was Asp-His-Tyr-Glu (DHYE); and the amino acid sequence of the black wheat antimicrobial peptide added in experimental group 4 was... The amino acid sequence of the black wheat antimicrobial peptide is Leu-Leu-Leu-Cys-Ala-Fhe (LLLCAF). The amino acid sequence of the black wheat antimicrobial peptide added in experimental group 5 is Glu-Thr-Pro-Asp (ETPD). The amino acid sequences of the black wheat antimicrobial peptide added in experimental group 6 are Asp-Val-Asn-Glu-Ser and Leu-Pro-Trp-Fhe. The amino acid sequences of the black wheat antimicrobial peptide added in experimental group 7 are Asp-His-Tyr-Glu, Leu-Leu-Leu-Cys-Ala-Fhe and Glu-Thr-Pro-Asp.

[0041] (2) Purchase pasteurized milk produced on the day of the experiment with a shelf life of 7 days, and divide it into 27 portions of 100mL each under aseptic conditions.

[0042] (3) Add antimicrobial peptides. The final concentration of antimicrobial peptides in each pasteurized milk is 75 μg / mL. Store at 4℃ for 15 days.

[0043] The experimental samples were analyzed on days 0, 3, 6, 9, 12, and 15.

[0044] Sensory analysis

[0045] Take 20 mL of each sample and place it in a 50 mL beaker. Observe the color and texture under natural light and smell the odor. The evaluation was conducted by 10 members. The evaluation method was modified according to the "RHB101—2004 Sensory Quality Evaluation Rules for Pasteurized Milk". The scoring details are based on the sensory scoring criteria in Table 1.

[0046] Table 1 Sensory Evaluation Table

[0047]

[0048] The sensory evaluation results are shown in Table 2. The results indicate that the sensory quality of the control group did not change significantly in the early stages of storage. From day 9 onwards, the sensory scores of the control group samples decreased significantly, and obvious sedimentation appeared in the tissue. By day 12, the sensory quality had clearly deteriorated. The Nisin group samples and experimental groups 1 through 7 did not exhibit flocculent sedimentation throughout the entire 15-day experiment, effectively preserving the milky aroma of the low-temperature heat-sterilized milk. The tissue condition remained unchanged except for some adhesion to the walls, and the texture was uniform. The scores were higher than the control group at each storage day; among them, experimental groups 6 and 7 had the highest scores. The sensory evaluation results indicate that the black wheat antimicrobial peptides prepared in this invention can effectively maintain the sensory quality of low-temperature heat-sterilized milk during refrigeration.

[0049] Table 2 Sensory scores of low-temperature heat-sterilized milk during storage

[0050] 0 days 3 days 6 days 9 days 12 days 15 days Comparison 100 98.03 90.23 85.42 76.65 70.75 Experimental group 1 100 98.32 97.65 93.32 85.32 80.85 Experimental group 2 100 98.43 97.32 94.43 85.63 80.41 Experimental group 3 100 97.99 97.63 93.46 85.45 83.3 Experimental group 4 100 98.21 97.23 92.98 85.66 82.62 Experimental group 5 100 98.62 97.34 93.03 84.92 82.29 Experimental group 6 100 99.5 98.32 95.43 85.99 83.77 Experimental group 7 100 99.46 98.03 95.21 86.03 83.64 Nisin 100 98.12 97.72 94.33 84.13 80.87

[0051] Total bacterial count:

[0052] Following the method in GB 4789.2-2022, the plate count method was used to count the total bacterial count of heat-sterilized milk during storage at low temperatures. The specific steps are as follows:

[0053] (1) Take 25 mL of sample and dilute it with sterile physiological saline. Dilute it 3-6 times according to the actual situation.

[0054] (2) Spread 1.0 mL of the diluted solution onto a sterile petri dish, pour in 25 mL of plate counting agar medium, and mix well. Incubate the petri dish in a 37°C incubator for 48 h, and then count the colonies.

[0055] The results are as follows Figure 1 As shown: the total bacterial count of all treatment groups showed a continuous upward trend during storage, and the total bacterial count of the control group was significantly higher than that of the other groups, exceeding 10% after 9 days. 5 CFU / mL. The total bacterial count in the Nisin group was not significantly different from that in experimental groups 1-7 during the early stages of storage. From day 6 onwards, the total bacterial count was significantly higher than that in experimental groups 1-7. The increase in total bacterial count in treatment groups 6 and 7 was the slowest. The total bacterial count in experimental groups 1-7 met national standards within 15 days. The results indicate that the addition of antimicrobial peptides from black wheat enabled the low-temperature heat-sterilized milk to meet national hygiene standards over a longer storage period, demonstrating better stability.

[0056] Although it was not determined in this part of the experiment which specific bacteria the black wheat antimicrobial peptides of the present invention inhibited, it is sufficient to show that the antimicrobial peptides of the present invention play an antimicrobial role in the preservation of milk.

[0057] Fat content: The fat content was determined using the alkaline hydrolysis method, in accordance with the national standard GB 5009.6-2016. The specific steps are as follows:

[0058] (1) Weigh 10g of the sample into the liposuction bottle and add 10g of water to the blank test group. Add 2.0mL of ammonia water to each, mix thoroughly, and immediately place the liposuction bottle in a water bath at 65℃ for 20min, removing and shaking it from time to time.

[0059] (2) After the shaking ends, remove the product and cool it to room temperature, then let it stand for 30 seconds.

[0060] (3) Add 10 mL of ethanol and 25 mL of ether to two liposuction bottles and shake for 1 min.

[0061] (4) Add 25 mL of petroleum ether and shake for 30 seconds to mix.

[0062] (5) Centrifuge the stoppered liposuction bottle at 500 r / min for 5 min and take the supernatant.

[0063] (6) Repeat steps (3) to (5), using 15 ml of anhydrous diethyl ether and 15 ml of petroleum ether for the second and third extractions, and combine the supernatants.

[0064] (7) Place the fat collection bottle in an oven at 100℃ for 1 hour to dry. After removing it, place it in a desiccator to cool for 0.5 hours and weigh it. Repeat the above operation until a constant weight is achieved (until the difference between two weighings does not exceed 2 mg). Calculate the fat content according to the following formula:

[0065]

[0066] X: Fat content in the sample, in grams per 100 grams (g / 100g);

[0067] m1: The total mass of the fat collection bottle and fat after constant weight, in grams (g);

[0068] m2: Mass of the fat collection bottle, in grams (g);

[0069] m3: In the blank experiment, the total mass of the fat collection bottle and extract after constant weight, in grams (g);

[0070] m4: The mass of the fat collection bottle in the blank test, in grams (g);

[0071] m: Mass of the sample, in grams (g);

[0072] 100: Conversion factor.

[0073] Depend on Figure 2As shown, the fat content of all groups decreased during storage, with the control group showing the most significant decrease. Compared to the Nisin group, the fat content of experimental groups 1-7 decreased more slowly. After 15 days of storage, the fat content of the antimicrobial peptide groups (experimental groups 1-7) and the Nisin group remained higher than the national standard of 3.1 g / 100 g. This indicates that black wheat antimicrobial peptides can effectively slow down the decomposition of fat during the storage of low-temperature heat-sterilized milk, and the effect is better than that of Nisin.

[0074] Protein content detection:

[0075] The Kjeldahl method was used to determine the protein content of milk, in accordance with the national standard GB5009.5-2016. The specific steps are as follows:

[0076] (1) Weigh 10g of sample and add it to a nitrogen determination flask, along with 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid.

[0077] (2) Heat until completely carbonized and the foaming stops completely. Then increase the heat and keep the liquid in the bottle boiling gently until the liquid turns blue-green and becomes clear and transparent. Continue heating for another hour.

[0078] (3) After cooling, add 20 mL of water and transfer to a 100 mL volumetric flask for later use. The liquid in the volumetric flask is the sample processing solution.

[0079] (4) Add boric acid solution and 1 to 2 drops of mixed indicator solution (mix 2 parts methyl red ethanol solution and 1 part methylene blue ethanol solution immediately before use) to the bottle. Add 5 ml of sample processing solution to the reaction chamber, wash the small glass cup with 10 ml of water and let it flow into the reaction chamber, and then seal it with a rod-shaped glass stopper.

[0080] (5) Pour 10.0 mL of sodium hydroxide solution into a small glass cup, lift the glass stopper to let it slowly flow into the reaction chamber, and immediately tighten the glass stopper and water seal it.

[0081] (6) Clamp the screw clamp and begin distillation. After 10 minutes of distillation, move the receiving flask until the liquid level is above the bottom of the condenser, then distill for another 1 minute. Titrate to the endpoint as quickly as possible with a standard sulfuric acid or hydrochloric acid solution. Calculate the protein content using the following formula:

[0082]

[0083] X: Protein content in the sample, in grams per 100 grams (g / 100g);

[0084] V1: The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the test solution, in milliliters (mL);

[0085] V2: The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the reagent blank, in milliliters (mL);

[0086] c: Concentration of the standard titration solution of sulfuric acid or hydrochloric acid, in moles per liter (mol / L);

[0087] m: Mass of the sample, in grams (g);

[0088] V3: Volume of sample processing solution, in milliliters (mL).

[0089] like Figure 3 As shown, the protein content of all sample groups decreased slightly during storage. Although the protein content of the control group and all treatment groups still met the standard requirement of greater than 2.9 g / 100 g throughout the storage period, the protein content of experimental groups 1 to 7 decreased more slowly compared with the control group. This indicates that black wheat antimicrobial peptides can effectively delay the decomposition of proteins during the storage of low-temperature heat-sterilized milk.

[0090] Acidity testing:

[0091] Acidity was determined using the phenolphthalein indicator method, in accordance with the national standard GB5009.239-2016. The specific steps are as follows:

[0092] (1) Weigh 10g of sample and place it in a 150mL conical flask. Add 20mL of freshly boiled and cooled water to room temperature and mix well.

[0093] (2) Add 2.0 mL of phenolphthalein indicator solution, mix well, and titrate with sodium hydroxide standard solution while adding the solution and rotating the flask until the color is similar to that of the reference solution (3 g of cobalt sulfate heptahydrate dissolved in water and diluted to 100 mL) and does not fade within 5 seconds. The entire titration process should be completed within 45 seconds.

[0094] (3) Conduct a blank experiment using an equal volume of water. Calculate the acidity using the following formula:

[0095]

[0096] X2: Acidity of the sample, in degrees (°T)

[0097] c2: Molar concentration of sodium hydroxide standard solution, in moles per liter (mol / L);

[0098] V2: The volume of sodium hydroxide standard solution consumed during titration, in milliliters (mL);

[0099] V0: The volume of sodium hydroxide standard solution consumed in the blank experiment, in milliliters (mL);

[0100] m2: Mass of the sample, in grams (g);

[0101] 0.1: The acidity theory defines the molar concentration of sodium hydroxide, in units of moles per liter (mol / L).

[0102] The results are as follows Figure 4 As shown in the figure. The results showed that the acidity of all groups increased during storage. Within 0-6 days, the acidity of all samples did not exceed the national standard, while the blank group and Nisin group exceeded the national standard at 9 and 12 days, respectively. The experimental groups 3, 5, 6, and 7 did not exceed the national standard at 15 days. This indicates that black wheat antimicrobial peptides can effectively delay the increase in acidity during the storage of low-temperature heat-sterilized milk.

Claims

1. A black wheat antimicrobial peptide, characterized in that: The black wheat antimicrobial peptide is composed of one or more of the following polypeptides; the amino acid sequences of the polypeptides are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

2. The application of the black wheat antimicrobial peptide according to claim 1 in the preservation of milk by low-temperature heat sterilization.

3. The black wheat antimicrobial peptide according to claim 1, characterized in that: The black wheat antimicrobial peptides are prepared from black wheat flour.

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