Compound essential oil yeast microcapsule based on quorum sensing and application thereof

Abstract

The invention discloses a composite essential oil yeast microcapsule based on quorum sensing and application thereof.The composite essential oil yeast microcapsule takes composite essential oil formed by compounding thyme and citral according to the volume ratio of 3: (1-3) as a core material, has a synergistic bacteriostatic effect on meat-derived specific putrefying bacteria pseudomonas fluorescens, takes yeast cell walls as a wall material, and has a synergistic bacteriostatic effect on meat-derived specific putrefying bacteria pseudomonas fluorescens; the lasting stability of the essential oil is ensured; the composite essential oil yeast microcapsule is prepared by mixing the composite essential oil microcapsule wall material and the yeast cell microcapsule core material according to the volume ratio of 1: (1-5), the decay-causing capacity of pseudomonas fluorescens is reduced under the sub-inhibitory concentration by regulating and controlling a quorum sensing system of target cells, and the composite essential oil yeast microcapsule can be used as a green, safe and efficient fresh-keeping agent. Spoilage of the fresh conditioned meat is slowed down, and the preservation time is prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of antiseptic and fresh-keeping, and in particular relates to a composite essential oil yeast microcapsule based on quorum sensing and an application thereof. Background Art

[0002] In recent years, with the development of social economy and the accelerated pace of life, fresh prepared meat has been favored by consumers due to its convenience and rich nutrition, and the demand has continued to rise. However, fresh prepared meat is not sterilized, and it is easy to deteriorate in quality, spoil, and have a short shelf life due to microbial contamination and lipid oxidation during production, circulation, and storage, which seriously restricts the development of the fresh prepared meat industry. Slowing down the spoilage of fresh prepared meat and extending its shelf life are the urgent needs of meat companies. The spoilage of fresh prepared meat is mainly caused by specific spoilage bacteria that decompose protein and fat. Pseudomonas fluorescens is the main spoilage bacteria of fresh prepared meat, and its putrefactive effect on fresh prepared meat is regulated by the quorum sensing system.

[0003] Quorum sensing (QS) is a mechanism for bacteria to communicate and transmit information. When the number of bacteria in a space reaches a certain density, they can spontaneously produce and release signal molecules. When the concentration of signal molecules reaches the threshold concentration, the bacteria will start the expression of related genes, thereby regulating certain biological behaviors. Many bacteria use this mechanism to regulate the expression of certain genes to achieve the effect of spoilage. Quorum sensing inhibitors (QSI) target the bacterial quorum sensing system without interfering with the normal life activities of bacteria, control the formation of bacterial biofilms and the expression of spoilage factors, thereby reducing the spoilage of spoilage bacteria, avoiding the generation of drug resistance, and achieving the purpose of preservation. At present, there are studies on the use of the action principle of quorum sensing inhibitors to achieve the effect of preservation and freshness preservation. CN111838295A discloses a method for preserving rainbow trout based on quorum sensing inhibitor cassia twig extract. By preparing a composite preservative, which includes 1%-3% cassia twig extract, 0.3%-0.5% ascorbic acid, 0.3%-0.5% water-soluble chitosan, 0.05%-0.15% phytic acid, 1%-3% salt, and the rest is water in a weight ratio, the quorum sensing phenomenon of specific spoilage bacteria is inhibited, the rainbow trout is preserved, and the preservation time is extended. Quorum sensing inhibitors provide a new strategy for controlling the formation of bacterial biofilms and the expression of spoilage factors. The development of more efficient quorum sensing inhibitors to achieve the purpose of preservation and freshness preservation is an important starting point for the development of the fresh prepared meat industry, but its research and development and application still face many challenges.

[0004] Plant essential oils are natural, widely available, safe and non-toxic. The active ingredients of some essential oils (alcohols, acetones, phenols, aldehydes and esters) have antibacterial and antioxidant effects and are an important source of bacterial QSI. However, the nature of essential oils is unstable, volatile, poorly water-soluble and has a pungent odor, which limits their use as food preservatives. Microencapsulation technology can reduce the impact of the external environment on the active ingredients in essential oils, improve their stability, and achieve a lasting antibacterial effect. In recent years, yeast has been found to be a new type of microcapsule biological wall material. The complete cell wall and cell membrane structure have certain strength and permeability, and no chemical reagents are required during the preparation process, and there is no solvent residue. It is very suitable as a microcapsule wall material in the fields of medicine, cosmetics and food. At present, there is no research on the preparation of microcapsule preservatives based on quorum sensing using plant essential oils as core materials and yeast cell walls as wall materials, and their application in the preservation of fresh prepared meat. Summary of the invention

[0005] The present invention discloses a composite essential oil yeast microcapsule based on quorum sensing and its application. The composite essential oil yeast microcapsule is prepared by using a mixture of thyme and citral essential oil as the core material and yeast cell wall as the wall material, which not only ensures the long-term stability of the essential oil, but also reduces the putrefaction ability of Pseudomonas fluorescens at a sub-inhibitory concentration by regulating the quorum sensing system of the target cells. The composite essential oil yeast microcapsule can be used as a green, safe and efficient preservative to slow down the spoilage of fresh prepared meat and extend the shelf life.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a quorum sensing-based composite essential oil yeast microcapsule, wherein the composite essential oil yeast microcapsule is made of a composite essential oil prepared by mixing thyme and citral in a volume ratio of 3:1-3 as a core material, and a yeast cell wall as a wall material.

[0008] The present invention provides a method for preparing composite essential oil yeast microcapsules based on quorum sensing, and the specific steps are as follows:

[0009] (1) Preparation of composite essential oil microcapsule core material: thyme and citral essential oils are mixed and dissolved in anhydrous ethanol to obtain thyme-citral composite essential oil microcapsule core material;

[0010] (2) Preparation of yeast cell microcapsule wall material:

[0011] ① Inactivate the active dry yeast at high temperature, dissolve it in warm water after cooling, and obtain a yeast solution;

[0012] ② After the yeast solution and the ethanol solution are mixed and allowed to stand, the precipitate is collected by centrifugation and washing to obtain the yeast cell microcapsule wall material;

[0013] (3) Preparation of composite essential oil yeast microcapsules:

[0014] The composite essential oil microcapsule core material obtained in step (1) and the yeast cell microcapsule wall material obtained in step (2) are mixed, ultrapure water is added for oscillation, the precipitate is collected by centrifugal washing, and the composite essential oil yeast microcapsule is obtained after freeze-drying.

[0015] Furthermore, in step (1), the thyme and citral essential oils are mixed in a volume ratio of 3:1-3; and the concentration of the composite essential oil microcapsule core material is 0.5-4 μL / mL.

[0016] Furthermore, in step ①, the inactivation temperature is 80-100° C., the time is 50-70 min; the temperature of the warm water is 35-42° C.; and the concentration of the yeast solution is 18-25%.

[0017] Furthermore, in step ②, the yeast solution and the ethanol solution are mixed in a volume ratio of 1:1-3, and the concentration of the ethanol solution is 70-85%; the standing temperature is 20-30°C, and the time is 1.5-2.5h; the centrifugal speed is 5000-6000r / min, and the time is 5-10min, and the ultrapure water is washed 2-4 times.

[0018] Furthermore, in step (3), the yeast cell wall material and the composite essential oil microcapsule core material are mixed in a volume ratio of 1:1-5; the amount of ultrapure water added is 2-5 times the volume of the core material and the wall material; the oscillation temperature is 20-50°C, the oscillation speed is 100-150r / min, and the oscillation time is 1-5h; the centrifugal speed is 5000-6000r / min, the time is 5-10min, and the anhydrous ethanol is washed 2-4 times; the freeze-drying conditions are a temperature of -20°C--50°C and a vacuum degree of 2-13Pa.

[0019] The present invention provides an application of the composite essential oil yeast microcapsule in bacterial quorum sensing inhibition.

[0020] The present invention also provides an application of composite essential oil yeast microcapsules based on quorum sensing in the preservation of fresh processed meat, and the specific method is as follows:

[0021] (1) Spreading the composite essential oil yeast microcapsules obtained above in a sterile culture dish and sterilizing them with ultraviolet light for later use;

[0022] (2) Cut the fresh cooked meat into slices, add salt, pepper and the composite essential oil yeast microcapsules obtained in step (1), stir evenly and store.

[0023] Furthermore, the thickness of the composite essential oil yeast microcapsules in step (1) is ≤0.5 cm; and the sterilization time is 15-30 min.

[0024] Furthermore, the fresh cooked meat in step (2) is cut to a thickness of 0.5-1 cm; the added amounts of salt, pepper and compound essential oil yeast microcapsules are 0.1-2%, 0.05-0.2% and 0.1%-4% of the mass of the fresh cooked meat, respectively; and the storage temperature is 0-4°C.

[0025] Beneficial effects:

[0026] 1. The composite essential oil prepared by compounding thyme and citral of the present invention has a synergistic antibacterial effect on the specific spoilage bacteria of meat source, Pseudomonas fluorescens. The minimum inhibitory concentration (MIC) of the composite essential oil is significantly lower than the MIC of the two essential oils used alone, which effectively reduces the usage of the essential oil.

[0027] 2. The composite essential oil prepared by the present invention has an inhibitory effect on the biofilm formation, clustering and swimming motility, extracellular protease activity and other spoilage factors of Pseudomonas fluorescens. By regulating the quorum sensing system of target cells, the spoilage ability of Pseudomonas fluorescens is reduced at sub-inhibitory concentrations, but does not affect the normal growth of bacteria, thus avoiding the generation of drug resistance. The composite essential oil is a new preservative based on the inhibitory effect of bacterial quorum sensing.

[0028] 3. The present invention uses yeast cell wall as wall material and composite essential oil as core material to prepare composite essential oil yeast microcapsules, which effectively solves the problems of poor water solubility, strong volatility, easy oxidation and other problems of plant essential oils that are not suitable for direct use. It can not only protect and slow-release the essential oils, but also reduce the influence of the essential oils on the odor of fresh prepared meat, thereby obtaining a microcapsule product with stable performance and effectively extending the shelf life of fresh prepared meat.

[0029] 4. The composite essential oil yeast microcapsules do not require chemical reagents and have no solvent residues during the preparation process. The obtained product has the advantages of uniform size, non-toxicity, and easy biodegradability. It can be used as a green and safe preservative for fresh processed meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effect of compound essential oil on the inhibition rate of purple pigment production and bacterial density of Bacillus violaceus CV026 at sub-inhibitory concentration;

[0031] Figure 2 The effect of compound essential oil on the inhibition rate and bacterial density of Pseudomonas fluorescens biofilm at sub-inhibitory concentration;

[0032] Figure 3 The effect of compound essential oil on the clustering, swimming and bacterial density of Pseudomonas fluorescens at sub-inhibitory concentrations;

[0033] Figure 4 The effect of compound essential oil on the extracellular protease activity of Pseudomonas fluorescens at sub-inhibitory concentration;

[0034] Figure 5 is the linear regression equation between the concentration of compound essential oil and absorbance value;

[0035] Figure 6 The effect of compound essential oil microcapsules on fresh processed meat TVB-N;

[0036] Figure 7 The effect of compound essential oil microcapsules on TBARS value of fresh prepared meat;

[0037] Figure 8 The effect of compound essential oil microcapsules on the juice loss rate of fresh prepared meat;

[0038] Fig. 9 To investigate the effect of composite essential oil microcapsules on the sensory quality of fresh processed meat. DETAILED DESCRIPTION

[0039] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Sources:

[0041] Thyme, citral, oregano, clove, Australian tea tree, and celery seed essential oils, with a purity of 95%, were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0042] Chromobacterium violaceum CV026 ATCC31532 was purchased from Biovector NTCC, and Pseudomonas fluorescens ACCC 01056 was purchased from China Agricultural Microbiological Culture Collection Center;

[0043] Peptone, tryptone, glucose, agar, sodium chloride, LB agar medium, LB broth medium, and PCA medium were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0044] Example 1 Determination of essential oil MIC

[0045] The activated Pseudomonas fluorescens and Bacillus violaceus CV026 were inoculated into LB broth and cultured overnight at 150 r / min. The concentration was adjusted to 10 6cfu / mL bacterial suspension. Thyme, citral, oregano, clove, Australian tea tree, and celery seed essential oils were selected as alternative essential oils, dissolved and diluted with anhydrous ethanol, filtered and sterilized with a sterile filter, and set aside. First, 100 μL of sterilized LB broth was added to each well of a 96-well cell culture plate, and then 100 μL of the essential oil solution was added. The two-fold dilution method was used to make the essential oil concentrations from left to right 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μL / mL. Finally, 100 μL of the diluted bacterial suspension was added to each well, and two groups A and B were set up. Group A was added with a suspension of Pseudomonas fluorescens, and group B was added with a suspension of Bacillus violaceus CV026. Negative and positive controls were set on the same culture plate. The blank broth group without bacterial suspension and essential oil was used as the negative control, and the group with bacterial suspension but without essential oil was used as the positive control. In order to eliminate the effect of anhydrous ethanol on bacteria, the positive control group added the same amount of anhydrous ethanol as the essential oil group. The culture plates of groups A and B were placed in a constant temperature incubator at 28°C and 37°C for 24 hours, respectively, and then taken out and placed in an ELISA instrument. The OD value was measured at a wavelength of 595nm. Compared with the negative control, the OD 595 The concentration with the smallest difference in values ​​was taken as the MIC of the essential oil. Each essential oil was repeated three times and the results were averaged.

[0046] The MIC results of thyme, citral, oregano, clove, Australian tea tree, and celery seed essential oils against Pseudomonas fluorescens and Bacillus violaceus CV026 are shown in Table 1 .

[0047] Table 1 MIC of essential oils against Pseudomonas fluorescens and Bacillus violaceus CV026

[0048]

[0049] Example 2 Determination of the synergistic antibacterial effect and optimal ratio of composite essential oils

[0050] According to the MIC results of the single essential oil measured in Example 1, the chessboard method was used to determine the combined MIC of the two essential oils to determine the synergistic antibacterial effect of the two essential oils. The dilution concentration of the essential oil was determined according to the MIC of the single essential oil for different strains. The maximum concentration was set to 4 times the MIC of the single essential oil. Seven rows were taken vertically and horizontally on the 96-well plate, with essential oil A in horizontal rows (concentration from high to low) and essential oil B in vertical columns (concentration from high to low). 100 μL of LB broth culture medium was added to each well, 100 μL of essential oils of different concentrations were added to the first column of wells, and the double dilution method was used to dilute them to 2, 1, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 MIC in sequence. The seventh well in the horizontal and vertical rows was a control tube without essential oil. Then 100 μL of the fluorescent Pseudomonas and purple Bacillus CV026 bacterial suspensions obtained in Example 1 were added to the corresponding wells, and cultured at 28°C and 37°C for 24 hours, respectively, and placed in an ELISA instrument to measure OD595 Value. Repeat 3 times and take the average value. The FIC index of the essential oil was used as the basis for judging the combined effect, and the calculation formula was as follows:

[0051]

[0052] When the FIC index ≤ 0.5, it was a synergistic effect; when 0.5 < FIC ≤ 1, it was an additive effect; when 1 < FIC ≤ 2, it was an irrelevant effect; when FIC > 2, it was an antagonistic effect. As shown in Table 2, the essential oil combinations with synergistic antibacterial effects were: thyme - citral, clove - thyme, thyme - celery seed. Among them, the synergistic effect of thyme - citral was the strongest. The MICs of the thyme - citral composite essential oil against Pseudomonas fluorescens and Chromobacterium violaceum CV026 were both 4.0 μL / mL, which were significantly lower than the MICs of the two used alone, reducing the dosage of the essential oil. The thyme - citral composite essential oil was selected for subsequent experiments.

[0053] Table 2 Determination results of the FIC index of essential oils

[0054]

[0055]

[0056] Taking the thyme - citral combination as an example, the following experiments were carried out. Using absolute ethanol as the solvent, the two were compounded in a volume ratio of 1:1, 1:3, 1:7, 1:9, 9:1, 7:1, 5:1, 3:1 to 2MIC (8 μL / mL) of the composite essential oil, and filtered and sterilized with a sterile filter for standby. First, add 0.2 mL of the Pseudomonas fluorescens bacterial suspension cultured overnight to a sterile petri dish, and then pour in PCA medium below 45 °C and mix well. Immerse a sterilized round filter paper with a diameter of 6 mm in the above - filtered and sterilized composite essential oil ethanol solution for 5 min, and use sterile forceps to pick up the filter paper and place it on the solidified medium. Each essential oil was repeated 4 times, and cultured at 28 °C for 24 h, and the diameter of the antibacterial circle was observed and measured. The results are shown in Table 3. When thyme:citral (v:v) = 3:1, the antibacterial circle was the largest and the antibacterial effect on Pseudomonas fluorescens was the best. The subsequent experiments were compounded according to thyme:citral (v:v) = 3:1.

[0057] Table 3 Influence of thyme - citral composite essential oil on the antibacterial circle of Pseudomonas fluorescens

[0058] Thyme: citral (v:v) Diameter of inhibition zone of Pseudomonas fluorescens (mm) 1:1 9.8 1:3 10.4 1:5 9.12 1:7 8.68 1:9 8 3:1 12.4 5:1 8.07 7:1 9.24 9:1 9.23

[0059] Example 3 Effect of the composite essential oil at sub - inhibitory concentration on the production of purple pigment by Chromobacterium violaceum CV026

[0060] The suspension of purple Bacillus CV026 activated overnight was inoculated into LB broth at 1%, and the filtered and sterilized composite essential oil was added to make the final concentration of the composite essential oil 0.5, 1.0, 2.0, and 3.0 μL / mL, respectively. The signal molecule C6-HSL was added to make the concentration 20 μg / mL, and the culture was shaken on a shaker until OD 595 300 μL of bacterial solution was taken into a 1.5 mL sterile centrifuge tube, 150 μL of 10% sodium dodecyl sulfate was added, vortexed for 5 seconds at room temperature, allowed to stand for 5 minutes, 600 μL of saturated n-butanol solution was added, vortexed for 8 seconds, centrifuged at 10000 r / min for 5 minutes, 200 μL of supernatant containing purple pigment was taken, added to a 96-well plate, and OD was measured. 595 LB broth without compound essential oil was used as blank control. The inhibition rate of purple pigment production in each compound essential oil treatment group was calculated:

[0061]

[0062] The function of Bacillus violaceus CV026 is to detect the quorum sensing inhibitory effect of the compound essential oil. Bacillus violaceus CV026 itself does not produce purple pigments, nor does it produce N-acyl homoserine lactone signal molecules (AHLs), but when exogenous signal molecules AHLs are present, it can sense their presence and produce purple pigments; the production of purple pigments by Bacillus violaceus CV026 is regulated by bacterial quorum sensing, and quorum sensing inhibitors will inhibit its production of purple pigments.

[0063] Effects of compound essential oils on the production of purple pigment by Bacillus violaceus CV026 Figure 1 As shown. The bacterial density values ​​of each treatment group are close, indicating that the sub-inhibitory concentration of the compound essential oil has no effect on the growth of purple Bacillus CV026. As the concentration of the compound essential oil increases, the inhibition rate of purple pigment gradually increases. When the concentration of the compound essential oil is 3.0μL / mL, the inhibition rate of purple pigment production reaches 64.8%. This shows that the compound essential oil has quorum sensing inhibitory activity on purple Bacillus CV026 without affecting the normal growth of the strain.

[0064] Example 4 Effect of composite essential oils on Pseudomonas fluorescens biofilm at sub-inhibitory concentrations

[0065] Incubate overnight until 10 6The fluorescent Pseudomonas bacterial solution with a cfu / mL was inoculated into LB broth at 1%, dispensed into sterile centrifuge tubes, and then filtered and sterilized composite essential oils were added to make the concentrations of the composite essential oils 0.5, 1.0, 2.0, and 3.0 μL / mL. The group without composite essential oils was used as the control, and the mixture was cultured at 28°C for 24 hours. The bacterial solution was poured out, washed with sterile water 3 times, and dried with sterile air for 30 minutes. 0.1% crystal violet solution was used for staining for 15 minutes, the stain was poured out, and washed with sterile water 5 times. 1 mL of 33% glacial acetic acid was added to fully dissolve, and 200 μL was taken in a 96-well plate, and the OD value at a wavelength of 595 nm was measured. The biofilm inhibition rate was calculated as follows:

[0066]

[0067] Under unfavorable external environmental conditions, bacteria adhere to each other and aggregate their own clones in their own secretions and disintegration products to form a film-like substance called biofilm, which is widely present on the surface of food processing equipment and is difficult to clean, which can easily cause food contamination. Figure 2 As shown in the figure, the compound essential oil can significantly inhibit the formation of fluorescent Pseudomonas biofilm, and the concentration is positively correlated with the inhibition rate. When the concentration of the compound essential oil is 3.0 μL / mL, the inhibition rate of the biofilm is 53.8%. There is no significant difference in the density of the bacterial solution in each treatment group, indicating that the compound essential oil has no effect on the growth of fluorescent Pseudomonas.

[0068] Example 5 Effect of compound essential oil on the clustering and swimming of Pseudomonas fluorescens at sub-inhibitory concentrations

[0069] After the composite essential oil was sterilized by filtration with a sterile filter, it was mixed with cluster culture medium (1% peptone, 0.5% sodium chloride, 0.5% glucose, 0.5% agar) and swimming culture medium (1% tryptone, 0.3% sodium chloride, 0.5% agar) and poured into plates to make the final concentrations of the composite essential oil 0.5, 1.0, 2.0, and 3.0 μL / mL, respectively. The culture medium without the composite essential oil was used as a blank control group. After the culture medium solidified, 5 μL of the overnight activated Pseudomonas fluorescens bacterial suspension was added to the center of the plate, dried with sterile air, and cultured at 28°C for 24 h. The migration of the strain was observed, and the clustering or swimming diameter of Pseudomonas fluorescens in the clustering and swimming plates was measured. The clustering and swimming inhibition rates were calculated based on the changes in diameter.

[0070]

[0071] The movement of bacteria contributes to the formation of the shape and structure of biofilms. Swarming and swimming are two ways of migration mediated by flagella. Fluorescent Pseudomonas has polar flagella, which have strong motility, allowing it to adhere to the surface of meat or invade muscle tissue more quickly. Therefore, motility can also reflect the spoilage ability of bacteria from a certain perspective. After 24 hours of cultivation on the surface of swarming and swimming plates, fluorescent Pseudomonas has a tendency to migrate outward. The control group has the strongest migration ability. As the concentration of the compound essential oil increases, the migration diffusion diameter gradually decreases. Figure 3 It can be seen that when the concentration of the compound essential oil is 3.0 μL / mL, the clustering and swimming inhibition rates are 83.13% and 77.43%, respectively. The differences in the bacterial density values ​​of each treatment group are not significant, indicating that the compound essential oil has no effect on the growth of Pseudomonas fluorescens. Studies have shown that the clustering and swimming characteristics of bacteria are regulated by QS. The compound essential oil inhibits the clustering and swimming ability of Pseudomonas fluorescens by inhibiting its QS, thereby reducing the spoilage characteristics of Pseudomonas fluorescens.

[0072] Example 6 Effect of compound essential oil on extracellular protease activity of Pseudomonas fluorescens at sub-inhibitory concentrations

[0073] Prepare skim milk plate, dissolve 15% skim milk powder in pure water and sterilize it separately (115℃, 30min), take 10mL skim milk and 90mL LB semi-solid nutrient medium containing 0.85% agar and mix them evenly, pour them into water agar plate, and set aside after solidification. Use a sterile filter paper with a diameter of 6mm, soak it in the fluorescent Pseudomonas solution containing different concentrations (0.5, 1.0, 2.0, 3.0μL / mL) of compound essential oil cultured overnight for 7min, take the filter paper flat on the surface of the solidified culture medium, and culture it at 28℃ for 24h, with the bacterial solution without compound essential oil as blank control. During the growth of skim milk plate, fluorescent Pseudomonas hydrolyzes casein in milk by secreting extracellular protease to form an obvious transparent circle. The level of protease hydrolysis activity can be judged by observing the size of the transparent circle. The larger the hydrolysis circle, the higher the activity of the extracellular protease.

[0074] Pseudomonas fluorescens can secrete extracellular proteases to hydrolyze proteins and free amino acids in meat, producing spoilage metabolites that cause meat to spoil. The activity of extracellular proteases in Pseudomonas fluorescens is regulated by the QS system, which intervenes in the expression of extracellular proteases by regulating the transcription of genes encoding extracellular proteases. Figure 4 It can be seen that the hydrolysis zone of the control group without adding compound essential oil was the largest, which was 28.2mm. The diameter of the protease hydrolysis zone of the group treated with 0.5μL / mL compound essential oil was 18mm. When the concentration of compound essential oil was further increased, no protease hydrolysis zone appeared, indicating that the sub-inhibitory concentration of compound essential oil inhibited the extracellular protease activity of Pseudomonas fluorescens.

[0075] Example 7 Preparation of composite essential oil yeast microcapsules

[0076] A composite essential oil yeast microcapsule based on quorum sensing, the preparation method is as follows:

[0077] (1) Preparation of composite essential oil microcapsule core material: thyme and citral essential oils were compounded in a volume ratio of 3:1, and the thyme-citral composite essential oil was dissolved in anhydrous ethanol to obtain a composite essential oil microcapsule core material with a concentration of 3 μL / mL;

[0078] (2) Preparation of yeast cell microcapsule wall material: Inactivate active dry yeast in an oven at 100°C for 1 hour, cool it, bottle it and store it in a desiccator for later use. Prepare a 20% solution of instant dry yeast in 40°C warm water, mix it with 80% ethanol solution in a volume ratio of 1:2, and let the mixture stand at 25°C for 2 hours. Then centrifuge the mixture at 5000r / min for 5 minutes, wash it with ultrapure water 3 times, collect the precipitate, and obtain the yeast cell microcapsule wall material for later use;

[0079] (3) Preparation of composite essential oil yeast microcapsules:

[0080] The composite essential oil microcapsule core material obtained in step (1) and the yeast cell microcapsule wall material obtained in step (2) were mixed in a volume ratio of 1:3, and ultrapure water 3 times the volume of the core material and the wall material was added. The mixture was oscillated at 140 r / min for 3 h in a 30° C. constant temperature oscillator to allow the essential oil to enter the yeast cells. The mixture was then centrifuged at 5000 r / min for 5 min, and washed with anhydrous ethanol 3 times to remove the surface essential oil. The precipitate was collected and freeze-dried at a temperature of -30° C. and a vacuum degree of 10 Pa to obtain composite essential oil yeast microcapsules for subsequent experiments.

[0081] Example 8 Determination of Encapsulation Efficiency of Composite Essential Oil Yeast Microcapsules

[0082] The composite essential oil was diluted to different concentrations with anhydrous ethanol, and a full wavelength scan was performed to determine the maximum absorption wavelength of the composite essential oil as its measurement wavelength. After scanning, the maximum absorption wavelength of the composite essential oil was 286nm. Accurately measure 500μL of essential oil into a 25mL volumetric flask, dilute to volume with anhydrous ethanol, and shake well to obtain a composite essential oil-ethanol standard dilution with a concentration of 20μL / mL. Then accurately measure 125, 250, 375, 500, and 625μL of solution from the volumetric flask into a 25mL volumetric flask, dilute to volume with anhydrous ethanol, and shake well to obtain composite essential oil-ethanol standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5μL / mL, respectively. Using anhydrous ethanol as a blank control, the absorbance value OD of essential oil solutions of different concentrations was measured at 286nm. 286 Repeat 3 times, take the average value, draw the standard curve, and obtain the composite essential oil concentration c and absorbance value OD 286The linear regression equation is as follows: Figure 5 shown.

[0083] The composite essential oil yeast microcapsules were dispersed in anhydrous ethanol at a volume ratio of 1:7 (w / w), and the nanocapsule wall material was broken by ultrasound for 15 minutes. The embedded essential oil was dissolved in anhydrous ethanol, centrifuged at 5000r / min for 5 minutes, 1mL of supernatant was drawn, and the volume was fixed to 10mL with anhydrous ethanol. The absorbance at a wavelength of 286nm was measured, and the total mass of the composite essential oil embedded in the microcapsules was calculated according to the composite essential oil standard curve equation. The encapsulation rate was calculated as follows:

[0084] Compound essential oil encapsulation efficiency (%) = total mass of compound essential oil embedded in microcapsules (g) / total mass of compound essential oil initially added to prepare microcapsules (g) × 100

[0085] The encapsulation rate of the composite essential oil yeast microcapsules obtained by the preparation method of the present invention is 82.9%, which is relatively high and meets the use requirements.

[0086] Example 9 Application of composite essential oil yeast microcapsules in the preservation of fresh processed meat

[0087] The application of compound essential oil yeast microcapsules in the preservation of fresh prepared meat is as follows:

[0088] The composite essential oil yeast microcapsules obtained in Example 7 were spread flat in a sterile culture dish with a thickness of ≤0.5 cm, and sterilized by ultraviolet irradiation for 20 min. The pork was cut into slices of 0.5-1 cm thick, 1% salt and 0.1% pepper were added, and the pork was randomly divided into 3 groups of equal amounts, and the experimental settings were: blank control group, 0.3% composite essential oil yeast microcapsule group, and 3% composite essential oil yeast microcapsule group. The composite essential oil yeast microcapsules were added according to the experimental settings and stirred evenly. The blank control group did not add composite essential oil yeast microcapsules. The processed pork was placed in a PP food-grade plastic tray and covered with plastic wrap and stored at 4°C. Samples were taken at 0, 2, 4, 6, 8, and 10 days, respectively, and the changes in volatile basic nitrogen (TVB-N), barbituric acid value (TBARS), juice loss rate and sensory quality in pork were determined.

[0089] Example 10 Effect of composite essential oil yeast microcapsules on fresh processed meat TVB-N

[0090] 10g of meat samples from the blank control group, 0.3% compound essential oil yeast microcapsule group, and 3% compound essential oil yeast microcapsule group at different treatment times in Example 9 were minced and placed in a conical flask, and 100ml of distilled water was added respectively. After shaking for 30 minutes, the samples were filtered, and the effects of each treatment group on the content of TVB-N in fresh prepared meat were determined with reference to GB5009.228-2016 "Determination of Volatile Basic Ammonia in Food". TVB-N is an alkaline nitrogen-containing substance such as ammonia and amines produced by the decomposition of protein in animal food during the corruption process due to the action of enzymes and bacteria. The higher its content, the more amino acids are destroyed, and the nutritional value will be greatly affected. It is the main indicator reflecting the freshness of meat.

[0091] Depend on Figure 6 It can be seen that the TVB-N content of each treatment group continued to increase during the cold storage of fresh prepared meat, among which the control group increased the fastest, and the TVB-N content was significantly higher than the two groups with compound essential oil yeast microcapsules, and the TVB-N of the 3% compound essential oil yeast microcapsules group was the lowest. On the 6th day, the TVB-N of the control group was 15.08 mg / 100g, reaching the critical point of meat corruption (fresh meat TVB-N ≤ 15 mg / 100g), and the 0.3% compound essential oil yeast microcapsules group and the 3% compound essential oil yeast microcapsules group reached the critical point of corruption at 8d and 10d of storage, respectively. During the storage of fresh prepared meat, the compound essential oil yeast microcapsules inhibited the activity of the extracellular protease of Pseudomonas fluorescens and reduced the decomposition of protein, so the content of alkaline substances such as ammonia and amines in the meat was low, which was manifested as a low TVB-N content.

[0092] Example 11 Effect of composite essential oil yeast microcapsules on TBARS value of fresh processed meat

[0093] Take 10g of meat samples from the blank control group, 0.3% composite essential oil yeast microcapsule group, and 3% composite essential oil yeast microcapsule group at different treatment times in Example 9, mince them, add 50mL of 5% trichloroacetic acid for homogenization, and filter. Take 5mL of supernatant, add 5mL of 0.02mol / L thiobarbituric acid solution. Heat the mixed solution in a constant temperature water bath at 80℃ for 40min to develop color, and after cooling to room temperature, measure the absorbance at a wavelength of 532nm. The TBARS value is the malondialdehyde content (mg / kg) in each kg of sample. The TBARS value is mainly used to evaluate the degree of lipid oxidation in food, which reflects the content of malondialdehyde compounds formed by lipid oxidation. The higher the TBARS value, the higher the degree of lipid oxidation in the food.

[0094] Depend on Figure 7It can be seen that the TBARS values ​​of each group showed an upward trend, rising slowly at the beginning of refrigeration and rising faster after the 4th day, indicating that the degree of lipid oxidation and peroxidation increased. There was no significant difference between the groups in the first 2 days of storage. From the 4th to the 10th day, the TBARS values ​​of the two groups with compound essential oil yeast microcapsules were significantly lower than those of the control group, among which the 3% compound essential oil yeast microcapsule group had the lowest TBARS value. The active ingredients such as thymol, pinepentin, citral, and limonene in the compound essential oil blocked the free radical chain reaction and reduced the degree of lipid oxidation and peroxidation in fresh processed meat.

[0095] Example 12 Effect of composite essential oil yeast microcapsules on the loss rate of juice from fresh prepared meat

[0096] Weigh 10 g of meat samples from the blank control group, 0.3% composite essential oil yeast microcapsule group, and 3% composite essential oil yeast microcapsule group in Example 9, record the initial weight of the meat sample W0 on day 0, take out the meat samples on days 2, 4, 6, 8, and 10, drain them, and weigh the meat sample weight W n The juice loss rate is the ratio of the juice loss mass to the initial mass of the meat sample, and its calculation formula is:

[0097]

[0098] During the storage of meat, the water holding capacity of the meat decreases due to the decomposition of proteins by extracellular proteases secreted by microorganisms, resulting in juice loss. The juice loss rate is one of the indicators for evaluating the freshness of meat. Figure 8 As shown in the figure, it gradually increased with time. After 2 days of storage, the juice loss rate of the compound essential oil microcapsule group was significantly lower than that of the control group, among which the juice loss rate of the 3% compound essential oil microcapsule group was the lowest. On the 10th day, the juice loss rates of the 0.3% compound essential oil microcapsule group and the 3% compound essential oil microcapsule group were 1.88 and 2.26 percentage points lower than those of the control group, respectively. The compound essential oil inhibited the activity of the Pseudomonas extracellular protease and reduced the decomposition of protein, thereby reducing the juice loss rate.

[0099] Example 13 Effect of composite essential oil yeast microcapsules on the sensory quality of fresh processed meat

[0100] A sensory evaluation panel consisting of 15 trained tasters evaluated the freshness of the meat samples of the blank control group, 0.3% compound essential oil yeast microcapsule group, and 3% compound essential oil yeast microcapsule group in Example 9 at different treatment times based on color, smell, elasticity, and tissue morphology. The evaluation criteria are shown in Table 1. The 5-point method was used for scoring, with 5 to 4 points for first-grade fresh meat; 4 to 3 points for second-grade fresh meat; 3 points for the acceptable limit, i.e., the sensory rejection point; and 1 to 3 points for spoiled meat. The sensory evaluation criteria are shown in Table 4.

[0101] Table 4. Sensory quality evaluation criteria for fresh processed meat

[0102]

[0103] Depend on Fig. 9 It can be seen that the sensory score of fresh prepared meat decreases with the extension of storage period. The sensory scores of each group change slowly in the first 4 days, and the sensory score decreases faster after 4 days. There is no significant difference between the groups on days 0 to 2, and the sensory score of the control group on days 4 to 10 is significantly lower than that of the composite essential oil yeast microcapsules. The sensory score of the control group dropped to 2.95 on the 6th day, which is lower than the sensory rejection point of 3.0, indicating that the meat sample has been corrupted and deteriorated. The meat samples of the 0.3% and 3% composite essential oil yeast microcapsules groups reached the corruption critical point on the 8th day and the 10th day, respectively. It can be seen that the composite essential oil yeast microcapsules prepared by the present invention effectively extend the shelf life of fresh prepared meat.

Claims

1. A composite essential oil yeast microcapsule based on quorum sensing, characterized in that: The composite essential oil yeast microcapsule uses composite essential oil prepared by mixing thyme and citral in a volume ratio of 3:1-3 as a core material and uses yeast cell walls as a wall material.

2. The composite essential oil yeast microcapsule according to claim 1, characterized in that: The wall material and the core material are mixed in a volume ratio of 1:1-5.

3. A method for preparing the composite essential oil yeast microcapsules based on quorum sensing according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of composite essential oil microcapsule core material: thyme and citral essential oils are mixed and dissolved in anhydrous ethanol to obtain thyme-citral composite essential oil microcapsule core material; (2) Preparation of yeast cell microcapsule wall material: ① Inactivate the active dry yeast at high temperature, dissolve it in warm water after cooling, and obtain a yeast solution; ② After the yeast solution and the ethanol solution are mixed and allowed to stand, the precipitate is collected by centrifugation and washing to obtain the yeast cell microcapsule wall material; (3) Preparation of composite essential oil yeast microcapsules: The composite essential oil microcapsule core material obtained in step (1) and the yeast cell microcapsule wall material obtained in step (2) are mixed, ultrapure water is added for oscillation, the precipitate is collected by centrifugal washing, and the composite essential oil yeast microcapsule is obtained after freeze-drying.

4. The preparation method according to claim 3, characterized in that: The concentration of the composite essential oil microcapsule core material in step (1) is 0.5-4 μL / mL.

5. The preparation method according to claim 3, characterized in that: In step ①, the inactivation temperature is 80-100° C. and the time is 50-70 minutes; the temperature of the warm water is 35-42° C. and the concentration of the yeast solution is 18-25%; in step ②, the yeast solution and the ethanol solution are mixed at a volume ratio of 1:1-3, and the concentration of the ethanol solution is 70-85%; the standing temperature is 20-30° C. and the time is 1.5-2.5 hours; the centrifugal speed is 5000-6000 r / min and the time is 5-10 minutes, and the ultrapure water is washed 2-4 times.

6. The preparation method according to claim 3, characterized in that: In step (3), the amount of ultrapure water added is 2-5 times the volume of the core material and the wall material; the oscillation temperature is 20-50°C, the oscillation speed is 100-150r / min, and the oscillation time is 1-5h; the centrifugal speed is 5000-6000r / min, the time is 5-10min, and the anhydrous ethanol is washed 2-4 times; the freeze-drying conditions are temperature -20°C--50°C, and vacuum degree 2-13Pa.

7. Use of the composite essential oil yeast microcapsule according to claim 1 in bacterial quorum sensing inhibition.

8. Use of the composite essential oil yeast microcapsule according to any one of claims 1-2 in preserving fresh processed meat.

9. A method for preserving fresh cooked meat using the composite essential oil yeast microcapsules according to claim 1, characterized in that: The method comprises the following steps: (1) Spread the composite essential oil yeast microcapsules in a sterile culture dish and sterilize them with ultraviolet light for later use; (2) Cut the fresh cooked meat into slices, add salt, pepper and the composite essential oil yeast microcapsules obtained in step (1), stir evenly and store.

10. The method according to claim 9, characterized in that: The flat thickness of the composite essential oil yeast microcapsules described in step (1) is ≤0.5 cm; the sterilization time is 15-30 min; the cutting thickness of the fresh prepared meat in step (2) is 0.5-1 cm; the added amounts of salt, pepper and composite essential oil yeast microcapsules are 0.1-2%, 0.05-0.2% and 0.1%-4% of the mass of the fresh prepared meat respectively in terms of mass ratio; and the storage temperature is 0-4°C.

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