A method for preserving triploid rainbow trout by combining plant extract with dielectric barrier discharge low temperature plasma technology

Abstract

The application provides a kind of plant extract combined dielectric barrier discharge low temperature plasma technology's triploid rainbow trout cold storage preservation method, belongs to the technical field of rainbow trout cold storage preservation, the application is fried after oil radish is extracted with water, adopt first with dielectric barrier discharge low temperature plasma technology processing 1 minute, then with oil-free fried radish water extract in 4 DEG C refrigerator soaking 10 minutes sequence.First, use dielectric barrier discharge to quickly kill the surface microorganism of triploid rainbow trout, then use the polyphenol in radish to remove the strong oxidizing substance generated by plasma treatment and form an antioxidant protective layer, solve the technical problem that low temperature plasma treatment accelerates the lipid oxidation of triploid rainbow trout, resulting in the decline of preservation effect.

Description

Technical Field

[0001] This invention belongs to the field of rainbow trout cold storage and preservation technology. Specifically, it relates to a method for cold storage and preservation of triploid rainbow trout using plant extracts combined with dielectric barrier discharge low-temperature plasma technology. Background Technology

[0002] Due to the characteristics of triploid rainbow trout meat, it is usually processed into sashimi to retain the original taste and nutrients to the greatest extent. The fat in sashimi is more easily oxidized. In addition, the high protein and high water content of aquatic products puts higher demands on their preservation during storage and transportation.

[0003] The dielectric barrier discharge (DBD) low-temperature plasma treatment process generates strong oxidizing substances such as reactive oxygen species and reactive nitrogen species, including ·OH, O3, H2O2, NO·, and ONOO. - This novel, green cold sterilization technology, which rapidly reduces the number of microorganisms by damaging their cell membranes, boasts advantages such as environmental friendliness, high efficiency, and no residual byproducts. It has been widely applied in aquatic product processing and preservation. However, the strong oxidizing agents also cause lipid oxidation and degradation in aquatic products, leading to a decline in quality characteristics such as deteriorated odor. Furthermore, DBD (Deep Dry Dispersion) only sterilizes the surface, and since samples are irregularly shaped, it suffers from uneven treatment, with some areas being overtreated and others undertreated, affecting preservation effectiveness. Therefore, it is urgent to combine it with other methods to overcome these weaknesses.

[0004] Plant extracts possess unparalleled advantages over chemical antioxidants due to their excellent antibacterial and antioxidant properties, as well as their natural and green characteristics. Radish seeds (Raphanus sativus L. seeds) are the dried, mature seeds of the radish, belonging to the Brassicaceae family. They are considered both food and medicine, and roasted radish seeds can be eaten directly. Summary of the Invention

[0005] In view of this, the present invention provides a method for cold storage and preservation of triploid rainbow trout using radish seed extract combined with dielectric barrier discharge low-temperature plasma technology, which can solve the technical problem in the prior art where low-temperature plasma treatment accelerates lipid oxidation in triploid rainbow trout, leading to a decrease in preservation effect.

[0006] This invention is achieved as follows: Roasted radish seeds are ground, and an appropriate amount of hexane is added to remove the radish seed oil. The mixture is then dried, soaked in water for extraction, and vacuum rotary evaporation is used to obtain a suitable concentration of roasted radish seed aqueous extract. Triploid rainbow trout are gutted and skinned, then cut into uniformly sized pieces and treated in a dielectric barrier discharge device. The sterilized roasted radish seed aqueous extract is then added according to the liquid-to-solid ratio for soaking. The treated triploid rainbow trout are then sealed in sterile homogenizing bags and stored in a refrigerated environment, with preservation indicators measured periodically. The strong oxidizing substances generated during plasma treatment are removed by using polyphenolic compounds in plant preservatives.

[0007] The plant extract is an aqueous extract of stir-fried radish seeds with de-oiled oil, determined by comparing the thiobarbituric acid value, total bacterial count, and volatile basic nitrogen value of stir-fried radish seed oil, stir-fried radish seed protein extract, stir-fried radish seed aqueous extract, and stir-fried radish seed aqueous extract with de-oiled oil on days 3 and 12.

[0008] In the preparation steps of the plant preservative, the ratio of roasted radish seed powder to n-hexane (g / mL) is 1:20, the soaking time is 1.5h, and the soaking is repeated 3 times. The content of residual radish seed oil in the n-hexane filtrate under different conditions is determined.

[0009] In the preparation step of the plant preservative, the fried radish seeds after oil removal are soaked in distilled water at a mass-to-volume ratio of 1:10 for 10 minutes and boiled for 10 minutes. The total phenol content in the extract under different conditions is determined.

[0010] In the preparation step of the plant preservative, the rotary evaporation concentration temperature is 50 to 60°C and the vacuum degree is 0.09 to 0.10 MPa, which is determined by measuring the retention rate of active ingredients and the concentration time under different temperature and vacuum conditions.

[0011] The concentration of the plant preservative is 1 g / mL, which is determined by comparing the thiobarbituric acid value and total bacterial count on day 0 and day 3 at different concentrations.

[0012] The liquid-to-material ratio of the plant preservative to the fish pieces is 5:1, which is determined by comparing the immersion of the fish pieces under different liquid-to-material ratios, the water absorption rate of the fish meat on day 0, and the total number of colonies on day 3.

[0013] In the aforementioned method for cold storage and preservation of triploid rainbow trout, the voltage of the dielectric barrier discharge device is 130kV, the frequency is 80Hz, the electrode spacing is 60mm, and the treatment time is 1 minute. The total number of colonies and the thiobarbituric acid value are determined by measuring different parameter combinations on the 3rd day after treatment.

[0014] The refrigerated environment temperature is 4°C, and the total bacterial count, volatile basic nitrogen value, and thiobarbituric acid value are measured every 3 days or every 2 days.

[0015] The measurement frequency is determined every 3 days or every 2 days by analyzing the changing trends of preservation indicators at different time points.

[0016] The preservation indicators include total bacterial count, volatile basic nitrogen (TVB-N) and thiobarbituric acid (TBA) values. Storage is stopped when the total bacterial count (TVC) exceeds 6 log CFU / g (first threshold), the volatile basic nitrogen (TVB-N) exceeds 20 mg / 100g (second threshold), or the thiobarbituric acid (TBA) value exceeds 2 mg / kg (third threshold).

[0017] This invention improves the preservation effect of stir-fried radish seeds by extracting the oil and water from them and preparing a plant-based preservative. The preservative's performance is optimized. After dielectric barrier discharge plasma treatment, triploid rainbow trout are soaked in the plant-based preservative. Polyphenolic compounds remove strong oxidizing substances such as hydroxyl radicals and hydrogen peroxide generated during plasma treatment, forming an antioxidant protective layer within the fish tissue. The polyphenolic compounds in the plant-based preservative have the ability to scavenge free radicals, and simultaneously, they protect proteins, reducing oxidative damage caused by dielectric barrier discharge. Furthermore, the plant extract itself has bactericidal properties. First, dielectric barrier discharge plasma treatment directly kills microorganisms on the surface of the fish. Then, soaking in the plant-based preservative neutralizes and removes residual strong oxidizing substances, compensating for unevenness in the plasma treatment area and achieving comprehensive sterilization. In summary, this invention solves the technical problem of accelerated lipid oxidation in triploid rainbow trout due to low-temperature plasma treatment, which leads to a decrease in preservation effect, by neutralizing the strong oxidizing substances generated during plasma treatment and forming an antioxidant protective layer. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method of the present invention.

[0019] Figure 2 This graph shows the changes in TVC in triploid rainbow trout during storage.

[0020] Figure 3 The graph shows the changes in TVB-N and TBA values ​​of triploid rainbow trout during storage.

[0021] Figure 4 This graph shows the changes in texture parameters of triploid rainbow trout during storage.

[0022] Figure 5 PCA plot and bar chart for the taste sensation of triploid rainbow trout during storage. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0024] like Figure 1 The diagram shown is a flowchart of a method for cold storage and preservation of triploid rainbow trout using plant extracts combined with low-temperature plasma technology, provided by this invention. This method includes the following steps:

[0025] S01. After grinding the roasted radish seeds and passing them through a 100-200 mesh sieve, add n-hexane at a material-to-liquid ratio of 1:20, soak at room temperature for 1.5 hours, filter, add the roasted radish seed powder to n-hexane again for soaking and filtration, and evaporate the filtrate by rotary evaporation. Measure the oil content in the filtrate. Stop soaking when the residual oil content in the radish seeds does not exceed 1% of the total weight. Soak and filter a total of 3 times.

[0026] S02. Add the degreased fried radish seeds to distilled water at a liquid-to-solid ratio of 1:10, soak for 10 minutes, then boil for 10 minutes, filter, concentrate the filtrate to 1 g / mL by rotary evaporation, autoclave and cool to room temperature to obtain the plant preservative.

[0027] S03. Place the processed triploid rainbow trout chunks into a dielectric barrier discharge device for processing. Set the voltage to 130kV, the frequency to 80Hz, the electrode spacing to 60mm, and the processing time to 1 minute.

[0028] S04. Add plant preservative to the plasma-treated triploid rainbow trout at a liquid-to-material ratio of 5:1 and soak for 10 minutes.

[0029] S05. After draining the processed fish meat, place it in a sterile homogenizing bag, seal it, and store it at 4°C. Measure the total bacterial count, volatile basic nitrogen, and thiobarbituric acid every 3 days. Stop storage when the total bacterial count exceeds the first threshold, the volatile basic nitrogen exceeds the second threshold, or the thiobarbituric acid exceeds the third threshold.

[0030] The dielectric barrier discharge plasma treatment and the plant preservative produced a synergistic preservation effect. The flavonoids and total polyphenols in the plant preservative inhibited microbial growth and scavenged free radicals generated by the plasma treatment, reducing the rate of lipid oxidation. The dielectric barrier discharge generated strong oxidizing substances that disrupted the microbial structure, while the antioxidant components of the plant preservative protected the fish tissue from oxidative damage. The combination of these two technologies achieved a dual effect of antibacterial and antioxidant properties. The total bacterial count of triploid rainbow trout treated with the combined technology remained below the first threshold on day 12 of storage; the volatile basic nitrogen value remained at a low level, below the second threshold; and the thiobarbituric acid value increased very slowly due to the presence of the plant preservative, remaining below 1.0 mg / kg on day 12, far below the third threshold, extending the shelf life to over 12 days.

[0031] Texture parameters, including hardness, cohesion, elasticity, and chewiness, reflect the mouthfeel quality of triploid rainbow trout. Fish treated with dielectric barrier discharge (DBD) exhibited higher hardness and cohesion but lower elasticity and chewiness. Fish treated with plant-based preservatives showed better elasticity and chewiness but slightly lower hardness. Fish treated with a combination of plant-based preservatives and DBD showed superior hardness, elasticity, and chewiness compared to either treatment alone. DBD treatment induced myofibril protein cross-linking, forming a denser network structure, thus increasing hardness. Polyphenols in radish seeds inhibited calpain activity, reducing muscle protein degradation and maintaining hardness. Radish seeds also contain antioxidants that protect cell membrane structures and maintain elasticity. Because degreased radish seeds removed the interference of fatty substances, some polyphenols and other substances could more easily contact the sample, resulting in better antioxidant and antibacterial effects.

[0032] Furthermore, flavor is a comprehensive sensory experience constructed by both taste and smell, including odor and taste characteristics, reflecting the flavor quality of triploid rainbow trout. Plasma treatment causes mild cross-linking of proteins, affecting odor and taste characteristics, while polyphenols in plant preservatives have a protective effect on proteins, reducing oxidative damage to proteins caused by dielectric barrier discharge. The combined treatment of these two technologies maintains the good flavor characteristics of the fish.

[0033] This method achieves multiple objectives—antibacterial activity, antioxidant effects, and maintenance of texture and flavor characteristics—by optimizing the processing sequence and parameter control. First, surface sterilization is performed using dielectric barrier discharge treatment, rapidly and precisely eliminating microorganisms on the fish surface. Then, the fish is immersed in a plant-based preservative, which immediately forms an antioxidant protective layer after plasma treatment, neutralizing residual strong oxidizing substances, preventing excessive oxidation of lipids and proteins, and comprehensively achieving sterilization of natural plant components. The results show that the synergistic effect of the two technologies significantly extends the cold storage shelf life of triploid rainbow trout, providing technical support for high-quality storage of aquatic products.

[0034] The specific implementation methods of the above steps are described in detail below.

[0035] In step S01, the fried radish seeds need to be de-oiled to improve their activity, based on the following experimental results. Equal amounts of fried radish seeds were weighed and extracted for oil and protein. Equal amounts of fried radish seeds and those after oil extraction were then subjected to water extraction. The protein, the water extract of fried radish seeds, and the de-oiled radish seed water extract were prepared into equal volumes of solution. All samples were autoclaved. Fish meat was soaked for 10 minutes in equal volumes of oil, protein, and the water extracts of radish seeds and de-oiled radish seeds, respectively. Distilled water was used as a control. The samples were stored at 4°C. On day 3, TVC, TVB-N, and TBA values ​​were measured in refrigerator C. The total bacterial count was 3.67 logCFU / g in the CK group, 2.99 logCFU / g in the oil group, 3.61 logCFU / g in the protein group, 2.93 logCFU / g in the water extract, and 2.56 logCFU / g in the defatted water extract. The TVB-N values ​​were 4.1 mg / 100g in the CK group, 3.8 mg / 100g in the water extract, 8.53 mg / 100g in the oil group, 17.87 mg / 100g in the protein group, and 5.03 mg / 100g in the defatted water extract. The TBA values ​​were 1.38 mg / kg in the CK group, 0.91 mg / kg in the oil group, 0.61 mg / kg in the protein group, 0.41 mg / kg in the water extract, and 0.25 mg / kg in the defatted water extract. On day 12, the total bacterial count was 7.31 log CFU / g in the CK group, 6.99 log CFU / g in the oil group, 6.8 log CFU / g in the protein group, 6.2 log CFU / g in the water extract, and 6.02 log CFU / g in the defatted water extract. The TVB-N values ​​were 17.03 mg / 100g in the CK group, 13.3 mg / 100g in the water extract, 17.73 mg / 100g in the oil group, 27.53 mg / 100g in the protein group, and 12.37 mg / 100g in the defatted water extract. The TBA values ​​were 1.38 mg / kg in the CK group, 0.91 mg / kg in the oil group, 0.61 mg / kg in the protein group, 0.41 mg / kg in the water extract, and 0.25 mg / kg in the defatted water extract. Experimental results showed that radish seed oil and protein had poor antibacterial, protein and lipid oxidation inhibition activities. The overall effect of the de-oiled radish seed aqueous extract was better than that of the radish seed aqueous extract. Considering that the pungent odor of radish seeds mostly comes from oil, the de-oiled radish seed aqueous extract was selected.

[0036] In step S01, with a material-to-liquid ratio of 1:20 for stir-fried radish seeds and 1.5 hours for hexane, the number of soaking cycles (3 times) was determined based on the following experimental results. With liquid-to-liquid ratios set to 1:5, 1:15, and 1:20, and an extraction time of 2 hours, each cycle was repeated 3 times. The extraction rates were found to be 26% for a liquid-to-liquid ratio of 1:5, 31% for 1:15, 34.5% for 1:20, and 34.5% for 1:25. Further, with a liquid-to-liquid ratio of 1:20 and extraction times of 0.5 hours, 1 hour, 1.5 hours, and 2.0 hours, each cycle was repeated 3 times. The extraction rates were 28% for 0.5 hours, 31.5% for 1 hour, 34.5% for 1.5 hours, and 34.5% for 2 hours. With a liquid-to-solid ratio of 1:20 and a soaking time of 1.5 hours, the radish seed oil obtained by evaporation from the filtrate after the first soaking accounted for 30% of the total weight of the radish seed powder. After the second soaking, the radish seed oil accounted for 3.5% of the total weight of the radish seed powder. After the third soaking, the radish seed oil accounted for 1% of the total weight of the radish seed powder. This shows that after three soaking extractions, the oil content in the radish seed residue did not exceed 1%. Therefore, the number of extractions was set at three. Based on the above results, to save materials and time, a liquid-to-solid ratio of 1:20, a soaking time of 1.5 hours, and three soakings were recommended.

[0037] The liquid-to-solid ratio of the de-oiled stir-fried radish seeds to distilled water in step S02 (1:10) and the boiling time (10 minutes) were determined through the following experiment. A certain amount of de-oiled stir-fried radish seed powder was taken and extracted with distilled water at mass-to-volume ratios of 1:5, 1:10, and 1:15, respectively. Each extraction was performed by soaking for 10 minutes, followed by boiling for 5 minutes, 10 minutes, and 15 minutes, respectively. The total phenol content in the extract was then measured. Experimental data showed that the total phenol content was 2.87 mg / mL at a mass-to-volume ratio of 1:5 after boiling for 10 minutes, 2.31 mg / mL at a mass-to-volume ratio of 1:10, and 1.54 mg / mL at a mass-to-volume ratio of 1:15. The total phenol content was 1.48 mg / mL at a mass-to-volume ratio of 1:10 after boiling for 5 minutes, and 2.45 mg / mL at a mass-to-volume ratio of 1:10 after boiling for 15 minutes. Considering extraction efficiency and subsequent concentration costs, a mass-to-volume ratio of 1:10 and boiling for 10 minutes is determined to be the optimal parameter, as it yields a high content of active ingredients while avoiding excessive energy consumption during the concentration process.

[0038] The rotary evaporation temperature (50-60℃) and vacuum level (0.09-0.10MPa) in step S02 were determined through the following experiments. Several portions of the extract obtained in step S01 were taken and concentrated by rotary evaporation under different temperatures and vacuum levels. The retention rate of active ingredients and the concentration time were measured after concentration. Experimental data showed that at 40℃ and 0.09MPa, the concentration time was 90 minutes with a total phenol retention rate of 90%; at 50℃ and 0.09MPa, the concentration time was 45 minutes with a total phenol retention rate of 92%; at 60℃ and 0.10MPa, the concentration time was 40 minutes with a total phenol retention rate of 89%; and at 70℃ and 0.10MPa, the concentration time was 35 minutes with a total phenol retention rate of 83%. The temperature range of 50-60℃ ensured both concentration efficiency and a high retention rate of active ingredients, while the vacuum level range of 0.09-0.10MPa provided a moderate evaporation rate; therefore, this range was determined to be the optimal parameter range.

[0039] The concentration of the plant preservative in step S02 was set at 1 g / mL, as determined by the following experiments. When the plant preservative concentration was 0.5 g / mL, the bacterial count on the fish meat on day 3 was 3.61 log CFU / g, and the thiobarbituric acid value was 0.35 mg / kg; when the plant preservative concentration was 1 g / mL, the bacterial count on day 3 was 3.41 log CFU / g, and the thiobarbituric acid value was 0.29 mg / kg; when the plant preservative concentration was 2 g / mL, the bacterial count on day 3 was 3.39 log CFU / g, and the thiobarbituric acid value was 0.28 mg / kg. Considering economic factors, a plant preservative concentration of 1 g / mL was chosen to treat triploid rainbow trout.

[0040] The voltage (130kV), frequency (80Hz), electrode spacing (60mm), and treatment time (1 minute) of the dielectric barrier discharge device in step S03 were determined through the following experiments. Triploid rainbow trout were treated with voltages of 70kV, 100kV, 130kV, and 150kV, frequencies of 60Hz, 80Hz, and 100Hz, electrode spacings of 60mm, 80mm, and 100mm, and treatment times of 0.5 minutes, 1 minute, and 2 minutes, respectively. The total bacterial count and thiobarbituric acid value were measured on the third day after treatment. Experimental data show that when treated for 1 minute at a voltage of 70kV, a frequency of 60Hz, and a spacing of 80mm, the total bacterial count was 4.15 logCFU / g and the thiobarbituric acid value was 1.21 mg / kg. At a voltage of 100kV, a frequency of 80Hz, and a spacing of 80mm, the total bacterial count was 4.06 logCFU / g and the thiobarbituric acid value was 1.27 mg / kg. At a voltage of 130kV, a frequency of 100Hz, and a spacing of 80mm, the total bacterial count was 3.98 logCFU / g and the thiobarbituric acid value was 1.32 mg / kg. Local overheating occurred at a voltage of 70kV, a frequency of 80Hz, and a spacing of 60mm. At a spacing of 100mm, the discharge efficiency decreased, and the total bacterial count was 4.12 logCFU / g. When treated for 1 minute at a voltage of 130kV, a frequency of 80Hz, and a plate spacing of 60mm, the total bacterial count was 3.75 logCFU / g and the thiobarbituric acid value was 1.37 mg / kg. After 2 minutes of treatment, the total bacterial count was 3.45 logCFU / g and the thiobarbituric acid value was 1.45 mg / kg. After 0.5 minutes of treatment, the total bacterial count was 3.95 logCFU / g and the thiobarbituric acid value was 1.24 mg / kg. Considering both the antibacterial effect and lipid oxidation control, the optimal parameter combination was 130kV, 80Hz, 60mm plate spacing, and 1 minute treatment time.

[0041] The liquid-to-solid ratio of 5:1 in step S04 was determined through the following experiment. With a fixed soaking time of 10 minutes using the plant preservative, soaking treatments were performed at liquid-to-solid ratios of 3:1, 5:1, and 7:1. The water absorption rate of the fish meat on the day of treatment and the total bacterial count on the third day were measured. Experimental data showed that at a liquid-to-solid ratio of 3:1, some areas of the fish pieces were not completely submerged, resulting in uneven treatment and a total bacterial count of 4.58 logCFU / g. At a liquid-to-solid ratio of 5:1, the fish pieces were completely submerged and treated evenly, with a total bacterial count of 3.42 logCFU / g and a water absorption rate of 6.2%. At a liquid-to-solid ratio of 7:1, the total bacterial count was 3.41 logCFU / g and the water absorption rate was 6.5%. Considering both treatment uniformity and solution volume, a liquid-to-solid ratio of 5:1 ensured complete submersion of the fish pieces while avoiding excessive waste of solution, and was therefore determined as the optimal parameter.

[0042] In step S05, the first threshold is 6 log CFU / g. A value exceeding 6 log CFU / g indicates severe microbial contamination, rendering the product unsuitable for consumption. The second threshold is derived from GB2733-2015, the National Food Safety Standard for Fresh and Frozen Freshwater Aquatic Products, where a volatile basic nitrogen value not exceeding 20 mg / 100g indicates compliance with national standards. The third threshold is from the national standard GB / T 35252-2017, "Determination of 2-Thiobarbituric Acid Value in Animal and Vegetable Oils: Direct Method," which provides a standardized method for this indicator. Generally, a TBA value of 2 mg / kg is considered an important warning value for flavor deterioration, indicating detectable lipid oxidation in the meat, but not yet reaching a level of severe spoilage.

[0043] The frequency of measuring preservation indicators every 3 days in step S05 was determined through the following experiment. Triploid rainbow trout samples that had undergone complete treatment were refrigerated at 4°C, and the total bacterial count, volatile basic nitrogen (VBN) value, and thiobarbituric acid (THBN) value were measured on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Experimental data showed that the total bacterial count increased little from 2.85 logCFU / g to 4.11 logCFU / g from days 0 to 3, rapidly increased to 6.15 logCFU / g from days 3 to 6, increased to 6.63 logCFU / g from days 6 to 9, and increased to 7.54 logCFU / g from days 9 to 12. The VBN value increased relatively slowly in the first 3 days, from 7.5 mg / 100g to 8.9 mg / 100g, and then increased steadily to 18 mg / 100g from days 3 to 12. The thiobarbituric acid (TBA) value increased relatively evenly from 0.92 mg / kg to 3.62 mg / kg throughout the storage period. The TBA value in samples treated with plant preservatives increased more gradually, from 0.15 mg / kg to 0.60 mg / kg. Analysis of the experimental data revealed that measuring every 3 days allowed for timely monitoring of quality changes while avoiding sample consumption and operational costs associated with frequent measurements; therefore, the measurement frequency was determined to be every 3 days.

[0044] In step S01, after the stir-fried radish seed powder is soaked in n-hexane, it is dried at 45°C for 3 hours to obtain dried, de-oiled radish seed powder. The de-oiled radish seeds contain more active ingredients per unit mass, which may be the reason why the de-oiled radish seeds have better activity. The preservative made after de-oiling is more hydrophilic and easier to combine with fish meat.

[0045] In step S02, the radish seeds that have been degreased are first soaked in distilled water for 10 minutes to allow the water to penetrate the radish seeds better, resulting in a more uniform state when boiled.

[0046] The dielectric barrier discharge treatment in step S03 is a method of generating non-equilibrium plasma by isolating electrodes with an insulating dielectric layer and applying high-frequency, high-voltage alternating current to ionize the gas. The hydroxyl radicals, hydrogen peroxide, reactive oxygen species, and reactive nitrogen species generated during the discharge process attack the nucleic acids and proteins of microorganisms, causing them to denature and become inactive, thus achieving a sterilization effect. Smaller electrode spacing results in better performance; excessive spacing reduces discharge efficiency. A treatment time of one minute represents a balance between antibacterial effect and avoiding excessive oxidation; longer treatment times accelerate the oxidative degradation of polyunsaturated fatty acids in triploid rainbow trout.

[0047] Step S04 employs a sequential treatment process: first, dielectric barrier discharge treatment, followed by immersion in a plant-based preservative. This utilizes the fluidity and antibacterial / antioxidant activity of the plant-based preservative to overcome the uneven treatment caused by the irregular surface of the sample when directly treated with dielectric barrier discharge. During immersion, the polyphenolic compounds in the plant-based preservative penetrate into the fish tissue, forming an antioxidant protective layer and providing antibacterial properties. A liquid-to-material ratio of 5:1 ensures the fish pieces are completely submerged. Insufficient immersion time affects the penetration of active substances, while excessive immersion time causes the fish to absorb water and swell, affecting its texture.

[0048] The total bacterial count in step S05 reflects the degree of microbial contamination, while the volatile basic nitrogen value reflects the degree of protein degradation. During refrigeration, the action of endogenous enzymes and microorganisms in triploid rainbow trout leads to protein degradation into alkaline nitrogenous substances such as ammonia and amines, and the volatile basic nitrogen value increases with prolonged storage time. A total bacterial count exceeding the first threshold indicates that the product is severely contaminated by microorganisms and is no longer suitable for consumption, while a volatile basic nitrogen value exceeding the second threshold indicates that a large amount of protein has decomposed. A thiobarbituric acid value exceeding the third threshold indicates that fat spoilage is already severe. Measure preservation indicators every 3 days to promptly monitor the quality changes of triploid rainbow trout. A refrigeration temperature of 4℃ inhibits microbial growth and enzyme activity, extending shelf life.

[0049] Specifically, the principle of this invention is as follows: Low-temperature plasma treatment sterilizes microorganisms by generating hydroxyl radicals, hydrogen peroxide, reactive oxygen species, and reactive nitrogen species that attack their nucleic acids and proteins. However, these strong oxidizing substances simultaneously attack the polyunsaturated fatty acids in triploid rainbow trout, triggering lipid peroxidation and generating oxidation products such as malondialdehyde, leading to increased thiobarbituric acid levels and off-flavors. This invention employs a strategy of immediately soaking the radish seeds in a plant preservative solution after plasma treatment. The sulforaphane in the stir-fried radish seeds has a destructive effect on microbial cell membranes, enhancing the antibacterial effect. The antioxidant properties of polyphenols neutralize the peroxidation reaction generated by the low-temperature plasma. Degreasing treatment makes the radish seeds more refreshing to use, with higher antioxidant and antibacterial activity. A 10-minute soaking time allows the active ingredients to fully penetrate into the fish meat tissue. A 1-minute dielectric barrier discharge treatment balances the antibacterial effect with the avoidance of excessive oxidation.

[0050] To better understand and implement this invention, the following is an embodiment 1 of a specific application scenario: Embodiment

[0051] In optimizing the cold storage and preservation technology for triploid rainbow trout, a technical team addressed the problems of short shelf life and rapid quality deterioration associated with traditional preservation methods by applying the preservation technology of this invention. The team purchased 15 kg of fresh triploid rainbow trout from a breeding farm; the average weight of the fish was 2.5 kg, totaling 6 fish. After transporting the triploid rainbow trout to the processing workshop, the internal organs and skin were immediately removed on a sterile operating table, and the fish were cut into pieces 80 mm long, 60 mm wide, and 12-13 mm thick, obtaining approximately 210 fish piece samples.

[0052] The technical team first prepared the plant preservative according to the method of this invention. 3 kg of roasted radish seeds were weighed, and half of them were directly extracted with water to prepare a radish seed aqueous extract: 1500 g of radish seeds were added to 15000 mL of distilled water and soaked at room temperature for 10 minutes. The extract was then transferred to a stainless steel pot and heated to boiling, continuing to boil for 10 minutes. The solid residue was removed by filtering through four layers of gauze. The filtrate was transferred to a rotary evaporator, with the water bath temperature set to 60℃ and the vacuum degree to 0.10 MPa. After rotary evaporation and concentration for 50 minutes, 500 mL of concentrated solution was obtained. The concentrated solution was then diluted to a 1500 mL volumetric flask to obtain a radish seed preservative (RS) with a concentration of 1 g / mL. The other half of the roasted radish seeds were soaked three times in an appropriate amount of n-hexane to reduce the oil content to below 1%. They were then dried in an oven at 45°C for 2 hours to completely remove the n-hexane. The de-oiled roasted radish seeds were weighed to be 1100g. They were then soaked in 11000mL of distilled water for 10 minutes, boiled for 10 minutes, and filtered through four layers of gauze to remove solid residue. The filtrate was transferred to a rotary evaporator, and the water bath temperature was set to 60°C and the vacuum degree to 0.10MPa. After rotary evaporation and concentration for 40 minutes, 500mL of concentrate was obtained. The concentrate was diluted to 1100mL in a volumetric flask to obtain a 1g / mL de-oiled radish seed (DRS) preservative. RS and DRS were transferred to an autoclave and sterilized at 121°C and 0.1MPa for 20 minutes. After sterilization, the mixture was cooled to room temperature for later use.

[0053] 210 triploid rainbow trout pieces were randomly divided into 6 groups of 35 pieces each. The technical team first treated them with DBD (Deep Booster Digestion), and then immediately added plant-based preservative solution at a feed-to-substrate ratio of 5:1 for soaking, i.e., 5000 mL of soaking solution for every 1 kg of fish pieces. The treatment plan is shown in Table 1.

[0054] Table 1 Treatment schemes for different treatment groups

[0055]

[0056] The technical team placed the soaked fish pieces in a dielectric barrier discharge device for processing. The samples were arranged in a single layer (layer thickness 12.22±0.84 mm) within a sealed polypropylene box (outer diameter 215(L)×125(W)×40(H) m). The box containing the rainbow trout samples was then placed at the center of a quartz plate above the grounding electrode. The distance between the box surface and the upper electrode was 20 mm to ensure uniform distance between the fish piece surface and the upper electrode. The voltage was set to 130 kV, the frequency to 80 Hz, and the electrode spacing to 60 mm. The dielectric barrier discharge treatment lasted for 1 minute. Uniform discharge was observed during the process, with no localized overheating or arcing.

[0057] All processed triploid rainbow trout fillets were placed into sterile homogenization bags, with 3 fillets (approximately 150g) per bag. The bags were vacuum-sealed and stored in a 4°C refrigerator. The technical team developed a detailed quality monitoring plan, taking samples on days 0, 3, 6, 9, and 12 of storage to determine the total bacterial count, volatile basic nitrogen, and thiobarbituric acid levels. Figure 2 As shown, the total bacterial count was determined using the plate count method. 10g of fish meat sample was added to 90mL of sterile 0.85% saline solution to make a homogenate. After serial dilution with 0.85% NaCl, 0.1mL of the appropriate dilution was spread on plate count agar medium and incubated at 37±1℃ for 48h before calculating the total bacterial count (TVC) of rainbow trout.

[0058] like Figure 3 As shown, the volatile basic nitrogen value was determined using the micro-diffusion method. 10g of fish sample was extracted with 75mL of water for 30 minutes. After filtration, 1mL of the extract was placed in the outer chamber of a diffusion dish. Boric acid absorbent was added to the inner chamber, and saturated potassium carbonate solution was added to the outer chamber. The dish was then sealed and diffused for 2 hours. The volatile basic nitrogen value was calculated by titration with a standard hydrochloric acid solution. The thiobarbituric acid value was determined using spectrophotometry. 10g of fish sample was extracted with trichloroacetic acid solution and filtered. After the filtrate reacted with thiobarbituric acid reagent, the absorbance was measured at 532nm. The thiobarbituric acid value was calculated based on the standard curve.

[0059] Depend on Figure 2It is evident that the TVC values ​​of each group gradually increased with increasing storage time. The initial TVC value of the CK group was 2.72 lg CFU / g, while the initial values ​​of the other groups were all lower than those of the CK group. The TVC values ​​of RS, DRS, DBD, DBD+RS, and DBD+DRS were 1.83 lg CFU / g, 1.77 lg CFU / g, 2.30 lg CFU / g, 1.43 lg CFU / g, and 1.33 lg CFU / g, respectively. On day 6 of storage, the CK group approached the limit of 5.95 lg CFU / g, while the TVC values ​​of the DBD+RS and DBD+DRS groups were just approaching the limit on day 12, at 6.09 lg CFU / g and 5.94 lg CFU / g, respectively. The TVC values ​​of RS, DRS, and DBD on day 12 were 6.40 lg CFU / g, 6.28 lg CFU / g, and 6.71 lg CFU / g, respectively.

[0060] Depend on Figure 3 (A) It can be seen that the TVB-N value of the CK group was 7.47 mg / 100g at the beginning of storage. The TVB-N content of the other treatment groups was slightly reduced, but the difference was not significant. The TVB-N content of all groups continued to increase during storage. The CK group reached 18.43 mg / 100g at the end of storage, while the TVB-N values ​​of RS, DRS, DBD, DBD+RS, and DBD+DRS were 15.16 mg / 100g, 14.93 mg / 100g, 16.57 mg / 100g, 14.23 mg / 100g, and 13.73 mg / 100g, respectively, all within the limit range. The changes in TBARS values ​​of rainbow trout samples after different treatments at 4℃ are shown in the figure. Figure 3As shown in (B), the CK group had a concentration of 0.39 mg / kg, the DBD group had a concentration of 0.43 mg / kg, and the remaining treatment groups all had concentrations below 0.2 mg / kg. This indicates that DBD treatment may cause slight lipid oxidation, but the sample quality remains good, which may be due to the high fat content of rainbow trout. All treatment groups containing RS and DRS showed good control over lipid oxidation. The reason why DBD treatment causes lipid oxidation may be that the reactive oxygen species it produces may promote lipid oxidation (especially in the early stage of treatment), resulting in higher TBA values ​​than other groups. On the 3rd day of storage, the TBARS values ​​of the CK group and the DBD group both exceeded 1 mg / kg, at 1.42 mg / kg and 1.38 mg / kg, respectively, while the remaining treatment groups were all below 0.35 mg / kg. By the 12-day mark of the final storage period, the TBARS values ​​of the CK group exceeded 3 mg / kg, and those of the DBD group exceeded 2 mg / kg. The values ​​of the remaining RS, DRS, DBD+RS, and DBD+DRS groups were all less than 1 mg / kg, specifically 0.76 mg / kg, 0.62 mg / kg, 0.48 mg / kg, and 0.40 mg / kg, respectively. The DBD+DRS group consistently exhibited low TBARS values ​​throughout the storage process, indicating that RS, DRS, and DBD treatments all alleviated lipid oxidation in rainbow trout during the later stages of cold storage, and the combined use of these treatments further enhanced this inhibitory effect. This may be because DBD treatment inhibits microbial growth in rainbow trout, thereby limiting lipid oxidation; and the polyphenols in radish seeds can scavenge free radicals (·OH, O2). - It blocks the oxidation chain reaction. Because defatted radish seeds remove lipid interference, antioxidant components (such as glucosinolate hydrolysates) can more easily come into contact with fish oil, thus increasing the efficiency of inhibiting lipid oxidation.

[0061] Based on a comprehensive analysis of the changing trends of various indicators, the technical team determined that the triploid rainbow trout after composite treatment can have a shelf life of more than 12 days under refrigeration conditions at 4℃.

[0062] The technical team measured the textural properties of the fish meat on days 0, 3, 6, 9, and 12 of storage. The measured indicators included hardness, elasticity, chewiness, and adhesiveness. The fish meat was cut into rectangular samples of 20mm × 20mm × 10mm and measured using a texture analyzer equipped with a P / 5N cylindrical probe. The test speed was 1mm / s, the deformation rate was 30%, and the trigger force was 0.15N. The test results are as follows: Figure 4 As shown, the firmness, elasticity, and chewiness of fish meat decreased with prolonged storage, while adhesiveness increased. The CK group showed the most severe decrease in firmness, elasticity, and chewiness, and the fastest increase in adhesiveness. RS, DRS, and DBD treatments delayed these changes (P<0.05). In contrast, the DBD+DRS treatment showed the best results during cold storage.

[0063] The technical team measured the color characteristics of the fish meat on days 0, 3, 6, 9, and 12 of storage, as shown in Table 2. After 12 days of storage, compared with the CK group, the DBD+DRS group had a lower L... * Value, higher a * Value, lower b * Value. Because RS is initially slightly yellow, it will slightly affect L. * The value decreases, b * The value increases, a * The value has little impact. The color of DRS will lighten after degreasing, which has little effect on L. * value, b * value and a * The value has a relatively small impact. Overall, it can be observed that L * The value shows a trend of first decreasing and then increasing, a * The value shows a downward trend, b * The value shows an upward trend. L * The changes in light scattering values ​​may be related to post-mortem rigor mortis and alterations in surface muscle light scattering. Myofibrils, the cytoskeleton, and interactions with other sarcoplasmic proteins can absorb and scatter light, influencing the whitening of fish muscle. In this study, structural changes in muscle proteins over time increased light scattering and brightening of the surface salmon fillets (L... * The color is opaque. These structural changes are more pronounced in the CK sample, which exhibits greater muscle protein hydrolysis (higher drip loss and lower stiffness), thus showing greater light scattering and a higher L* value at the end of storage. Simultaneously, the lipid oxidation product aldehyde can interact with the amino groups of the protein and result in a pale yellow color (higher b...). * (Value) Color. DBD+DRS treatment can inhibit microbial putrefaction, protein and lipid oxidation to a certain extent, and maintain hardness, so it can better maintain L. * value, b * value and a * Value, to maintain color stability.

[0064] Table 2. Changes in color parameters of triploid rainbow trout during refrigeration.

[0065]

[0066] The technical team measured the flavor characteristics of the fish meat on days 0, 3, 6, 9, and 12 of storage. The fish meat was cut into rectangular samples of 20mm × 20mm × 10mm, and the flavor characteristics were measured using an electronic tongue. The results are as follows: Figure 5 As shown.

[0067] The electronic tongue system has eight sensors: salty, sour, bitter, astringent, umami, bitter aftertaste, astringent aftertaste, and umami richness. Figure 5 (A) shows that PCA-1 and PCA-2 contributed 68.1% and 14.2% respectively, with a total contribution of 82.3%. This indicates that the two principal components can reflect the flavor changes of fish fillets under the two treatments at different storage times. On day 0, the values ​​of each group were similar, indicating that RS and DBD treatments had little effect on flavor. After 12 days, the positions of each group of samples in the PCA diagram shifted to the right, with the CK group shifting the most significantly to the right, indicating that it had the highest degree of spoilage and the greatest flavor change. The other RS, DBD+RS, DBD, DRS, and DBD+DRS groups were all to the left of the CK group, with the DBD+DRS group being the far left, which could maximally inhibit spoilage and enable the product to maintain a flavor characteristic close to fresh after 12 days of storage.

[0068] The technical team conducted further research on the taste response values ​​to clarify the taste characteristics of rainbow trout. The taste null point parameters for the reference solution were: sour -13, salty -6, and all other taste parameters were 0. This means that among the evaluated taste indices, only tastes with measured values ​​higher than the null point values ​​were considered tastes perceptible to rainbow trout. Figure 5 (B) The electronic tongue taste histogram shows that umami, saltiness, and a small amount of bitterness are the main taste components perceived by rainbow trout, and their scores are all higher than the tasteless point. The scores for sourness, astringency, and bitter aftertaste are all lower than the tasteless point, indicating that rainbow trout cannot be tasted for these flavors. Umami richness was not detected on day 0, but was detected in all treatment groups on day 12, with no significant difference between treatment groups. Bitterness decreased with each treatment, and the DBD+DRS group showed the largest decrease on both day 0 and day 12. The bitterness value on day 12 was slightly higher than on day 0. There was no significant difference in saltiness values ​​among treatment groups, but the value on day 12 was slightly higher than on day 0. There were no significant differences in umami values ​​among treatment groups on days 0 and 12, but the umami value on day 12 was lower than on day 0. Although umami richness was not detected on day 0, it was detected in the treatment groups on day 12, and the umami richness of the treatment groups was higher than that of the CK group. Overall, DBD+DRS treatment can preserve the original flavor characteristics of rainbow trout.

[0069] In conclusion, DBD+DRS can protect rainbow trout from deterioration in texture, color, and flavor characteristics during refrigeration to the greatest extent possible, and its effect is better than single technologies or DBD+RS.

[0070] The advancements of this invention compared to traditional single preservation technologies are reflected in the following aspects: First, dielectric barrier discharge treatment provides instantaneous surface sterilization under low-temperature, pollution-free conditions. Then, the polyphenolic compounds in the plant preservative utilize their free radical scavenging ability to neutralize the strong oxidizing substances generated by plasma treatment, preventing excessive oxidation of polyunsaturated fatty acids. The synergistic effect of these two technologies inhibits microbial growth while controlling lipid oxidation, solving the problem of accelerated lipid oxidation caused by single plasma treatment. The treatment sequence of dielectric barrier discharge followed by immersion in the plant preservative overcomes the uneven treatment caused by irregular sample surfaces when directly treated, reduces oxidative damage to proteins from dielectric barrier discharge, and maintains the good textural properties, flavor characteristics, and color characteristics of the fish meat, achieving multiple preservation goals: antibacterial, antioxidant, and maintenance of textural properties, flavor, and color characteristics.

[0071] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cold storage and preservation of triploid rainbow trout using plant extracts combined with dielectric barrier discharge low-temperature plasma technology, characterized in that, A plant preservative was prepared by extracting the oil from stir-fried radish seeds with water. Triploid rainbow trout were gutted and skinned, then cut into uniform pieces. They were first treated in a dielectric barrier discharge device, then soaked in the extract of the de-oiled stir-fried radish seeds according to the liquid-to-material ratio. Excess water was absorbed, and the treated triploid rainbow trout were sealed in sterile homogenizing bags and stored in a refrigerated environment with regular monitoring of preservation indicators. The polyphenolic compounds in the plant preservative were used to remove the strong oxidizing substances generated by the plasma treatment.

2. The method for cold storage and preservation of triploid rainbow trout according to claim 1, characterized in that, The plant material was an aqueous extract of fried radish seeds after oil removal. The composition was determined by comparing the thiobarbituric acid value, total bacterial count, and volatile basic nitrogen value of the fried radish seed oil, fried radish seed protein extract, radish seed aqueous extract, and oil-removed fried radish seed aqueous extract on days 3 and 12.

3. The method for cold storage and preservation of triploid rainbow trout according to claim 2, characterized in that, In the deoiling step of the plant preservative, the ratio of roasted radish seed powder to n-hexane (g / mL) is 1:20, the soaking time is 1.5h, and the soaking is repeated 3 times. The content of residual radish seed oil in the n-hexane filtrate under different conditions is determined.

4. The method for cold storage and preservation of triploid rainbow trout according to claim 3, characterized in that, The ratio of oil-removed stir-fried radish seeds to distilled water was 1:10 (mass to volume). The soaking time was 10 minutes, and the boiling time was 10 minutes. The total phenol content in the extract under different conditions was determined.

5. The method for cold storage and preservation of triploid rainbow trout according to claim 4, characterized in that, The temperature for rotary evaporation concentration during the preparation of plant preservatives is 50 to 60°C, and the vacuum degree is 0.09 to 0.10 MPa. The concentration time is determined by measuring the retention rate of active ingredients and the concentration time required under different temperature and vacuum conditions.

6. The method for cold storage and preservation of triploid rainbow trout according to claim 5, characterized in that, The concentration of the plant preservative was 1 g / mL, determined by comparing the thiobarbituric acid value and total bacterial count on day 0 and day 3 at different concentrations.

7. The method for cold storage and preservation of triploid rainbow trout according to claim 6, characterized in that, The liquid-to-material ratio of the plant preservative and the fish pieces was 5:1, which was determined by comparing the immersion of the fish pieces, the water absorption rate of the fish meat, and the total number of colonies on the third day of storage under different liquid-to-material ratio conditions.

8. The method for cold storage and preservation of triploid rainbow trout according to claim 7, characterized in that, The dielectric barrier discharge device has a voltage of 130kV, a frequency of 80Hz, an electrode spacing of 60mm, and a treatment time of 1 minute. The total number of colonies and the thiobarbituric acid value are determined by measuring different parameter combinations on the 3rd day after treatment.

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