Method for preservation of flavor quality of aquatic products during refrigeration by ultrasonic synergistic low-temperature plasma active water

Abstract

This invention belongs to the field of non-thermal processing and preservation technology for aquatic products, specifically relating to a method for preserving aquatic products by regulating their flavor and quality under refrigeration using ultrasound-assisted low-temperature plasma activated water. The method involves immersing fresh aquatic products in low-temperature plasma activated water for ultrasound treatment, followed by continued immersion in the water after ultrasounding, and finally removing the fresh aquatic products for refrigeration. This preservation method is green, leaves no chemical residues, and effectively inhibits odors, providing a novel technical solution for high-quality sterilization and preservation of aquatic products.

Description

Technical Field

[0001] This invention belongs to the field of non-thermal processing and preservation technology of aquatic products, specifically involving a method for treating aquatic products, especially scallops, using ultrasound (US) in conjunction with low-temperature plasma activated water (PAW) to inhibit flavor deterioration and extend shelf life of scallops during cold storage from both microbial and metabolic pathway perspectives. Background Technology

[0002] Scallops (Patinopecten yessoensis) are an important marine fishery resource in my country. Their adductor muscles are rich in protein, flavor-enhancing free amino acids, and various essential trace elements, making them highly sought after in the global seafood market. However, scallop tissues have a high water content and are rich in nutrients, making them extremely susceptible to attack from endogenous enzyme activity and spoilage microorganisms during refrigeration, leading to rapid protein degradation and lipid oxidation. This quality deterioration not only manifests as softening of the muscle but also is accompanied by unpleasant odors (such as the foul sulfides produced by the metabolism of sulfur-containing amino acids and the rancid odor of fatty acids produced by lipid oxidation), which is a key factor limiting the shelf life and commercial value of scallops.

[0003] Traditional chemical preservatives (such as potassium sorbate and sodium benzoate) have a certain preservation effect, but they pose a risk of chemical residues, and consumer demand for "clean label" products is increasing. Non-thermal processing technologies have become a research hotspot due to their green, safe, and chemical-residue-free characteristics. Ultrasound, as a physical processing method, mainly disrupts the cell wall and cell membrane structure of microorganisms through the cavitation effect it generates; while plasma-activated water (PAW), rich in reactive oxygen species and reactive nitrogen species (ROS / RNS), exhibits excellent broad-spectrum bactericidal capabilities.

[0004] Currently, some studies have combined ultrasound with plasma-activated water for food preservation. CN116649400A discloses a green cleaning and sterilization method for shrimp using ultrasonic-coupled jet plasma activated water. The method involves selecting live shrimp as raw materials, soaking them in salt water to allow them to expel impurities from their gills and intestinal glands; placing the expelled shrimp in a cleaning tank and cleaning and sterilizing them using ultrasonic-coupled jet plasma activated water; draining the shrimp after cleaning, packing them into boxes, and then using modified atmosphere packaging with nitrogen and oxygen before storing the shrimp products at 0-4°C. This invention utilizes ultrasonic-coupled jet plasma activated water cleaning to effectively remove impurities from the surface and crevices of shrimp without the need for chemical cleaning agents, effectively improving shrimp quality, extending shelf life, and giving the product a strong market competitiveness. CN118266493A discloses a method for preserving fresh crayfish using ultrasonic coupling low-temperature plasma activation water combined with high-concentration carbon dioxide modified atmosphere packaging. The method involves selecting crayfish for sudden death treatment, followed by ultrasonic treatment, washing with low-temperature plasma activation water, and finally filling with high-concentration CO2 for modified atmosphere packaging and refrigeration. Compared with ultrasonic coupling low-temperature plasma activation water treatment alone or high-concentration CO2 modified atmosphere packaging alone, this invention can more effectively reduce the total bacterial count, volatile basic nitrogen, and thiobarbituric acid value of fresh crayfish during storage, significantly improve the sensory quality of fresh crayfish, and extend the shelf life of products stored at 4°C by 4 days. Simultaneously, the sterilization process does not produce chemical residues, significantly improving the safety of fresh crayfish products. CN119924441A discloses the application of ultrasonic combined with plasma activation water to reduce the sublethal damage rate of foodborne pathogens. The method discloses mixing the object to be treated with plasma activation water and then subjecting it to ultrasonic treatment. The ultrasonic treatment power is 200–600W, and the ultrasonic treatment time is 3–10 minutes. While significantly reducing foodborne pathogens, it can also significantly reduce the occurrence of sublethal damage from foodborne pathogens, improve the sterilization effect of foodborne pathogens, and thus be more conducive to solving food safety hazards. CN114711284B describes a method for extending the shelf life of scallops by using a composite preservative solution composed of bamboo leaf polyphenol extract, natural soybean trypsin inhibitor, and potassium sorbate combined with ultrasonic treatment. However, this method still relies on chemical preservatives and does not reveal the molecular mechanism of odor inhibition at the metabolic pathway level.

[0005] Therefore, developing a green, non-thermal preservation method that can inhibit key spoilage bacteria and their odor metabolic pathways in scallops is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing cold storage and preservation technologies for aquatic products (shellfish, especially scallops), such as chemical residues, unsatisfactory odor suppression, and short shelf life, this invention provides a preservation method for regulating the flavor and quality of cold-stored aquatic products using ultrasound-assisted plasma-activated water. This preservation method is not limited to macroscopic sterilization effects; it also intervenes in the metabolic pathways of cysteine ​​and methionine, as well as the oxidation pathways of glycerophospholipids and arachidonic acid, to block the source of characteristic spoilage odors (sulfide stench, rancid fatty acid odor, etc.) in aquatic products (shellfish, especially scallops), while maintaining a high level of umami amino acid retention.

[0007] The technical solution of the present invention is a method for preserving the flavor and quality of aquatic products by regulating the cold storage using ultrasound-assisted low-temperature plasma activated water. The steps include: immersing fresh aquatic products in low-temperature plasma activated water for ultrasound in a low-temperature environment; after ultrasound, continuing to immerse the fresh aquatic products in low-temperature plasma activated water; and after immersion, removing the fresh aquatic products from the low-temperature plasma activated water for cold storage.

[0008] After the ultrasound treatment, the fresh aquatic products are soaked in low-temperature plasma activated water to ensure that the active ingredients in the low-temperature plasma activated water come into full contact with the fresh aquatic products.

[0009] The mass-to-volume ratio of the fresh aquatic products to the low-temperature plasma activated water is 1:3-10, preferably 1:5-9, and more preferably 1:6-8.

[0010] The temperature of the low-temperature environment is controlled at 0-10℃, preferably at 4±0.5℃, such as by using an ice bath.

[0011] The preparation of the low-temperature plasma activated water includes: using a modulated pulsed plasma power supply and air as the working gas, immersing the plasma jet tip in sterile distilled water for discharge activation to obtain an aqueous solution rich in active oxygen and active nitrogen. The pressure of the working gas is 0.1–0.3 MPa, preferably 0.1–0.2 MPa; the gas flow rate is 10–30 L / min, preferably 15–20 L / min. The plasma jet tip is positioned 0.5–1.5 cm below the surface of the sterile distilled water, preferably 0.5–1 cm below the surface. The low-temperature plasma activation time for each 1 L of sterile distilled water is 5–15 min, preferably 10 min.

[0012] The fresh aquatic products are selected from fish, crustaceans, shellfish, etc., such as fresh fish bodies or fish meat, fresh shrimp or shrimp meat, fresh crab or crab meat, fresh shellfish or shellfish meat, etc. The shellfish are any one of bivalves, gastropods, and cephalopods. The shellfish meat includes, but is not limited to, adductor muscle, gonads, foot, foot, viscera, etc. As one embodiment, bivalves such as scallops, mussels, oysters, freshwater mussels, and hard clams are selected, and adductor muscles are further selected, and scallop adductor muscles are selected even further. The preparation of fresh shellfish adductor muscles includes: immediately extracting the adductor muscles from fresh live shellfish, rinsing the surface impurities with sterile physiological saline or sterile deionized water, and draining the surface water to obtain fresh shellfish adductor muscles.

[0013] The ultrasonic power is 200-400W, preferably 300-350W; the frequency is 20-50 kHz, preferably 40 kHz; and the ultrasonic time is 5-15 min, preferably 10 min.

[0014] The soaking time is 10–50 min, preferably 30 min; gentle stirring is performed during soaking. This ensures that the total contact time between the fresh aquatic product (scallop adductor muscle) and the low-temperature plasma activated water (PAW) during the ultrasonication and soaking process reaches 20–60 min, preferably 40 min.

[0015] The refrigeration temperature is 0–4°C.

[0016] This invention provides a refrigerated aquatic product that maintains its flavor and quality, prepared by the preservation method described above. The aquatic product is selected from fish, crustaceans, shellfish, etc., such as fresh fish bodies or fish meat, fresh shrimp or shrimp meat, fresh crab or crab meat, fresh shellfish or shellfish meat, etc. The shellfish are any one of bivalves, gastropods, and cephalopods. The shellfish meat includes, but is not limited to, adductor muscle, gonads, foot, foot, viscera, etc. As one embodiment, the shellfish are selected from bivalves, such as scallops, mussels, oysters, freshwater mussels, hard clams, etc., further selecting adductor muscles, and even further selecting scallop adductor muscles.

[0017] Based on a systematic analysis of the microbial dynamics, free amino acid profiles, and multidimensional flavor omics of scallops before and after US-PAW treatment, this invention elucidates for the first time the underlying mechanism by which US-PAW inhibits the deterioration of scallop flavor from the perspective of the entire chain of "microbe-metabolic pathway-flavor," and proposes a specific and efficient preservation method based on this mechanism.

[0018] This invention utilizes the synergistic effect of ultrasound and plasma-activated water to: (1) inhibit the proliferation of common spoilage bacteria in scallops, such as Shewanella and Pseudomonas; (2) significantly inhibit key metabolic reactions in the cysteine ​​and methionine metabolic pathways, blocking the generation of malodorous sulfides such as hydrogen sulfide, dimethyl disulfide, and dimethyl trisulfide; and (3) inhibit glycerophospholipid metabolism and arachidonic acid metabolism pathways, reducing the accumulation of lipid oxidation products (such as hexanal, nonanal, and 1-octen-3-ol, which are rancid fatty acid odor substances).

[0019] The shelf life of the refrigerated scallop adductor muscle of this invention is 30% to 50% longer than that of untreated scallops (directly refrigerated at 4°C). On the 10th day of refrigeration, the core umami amino acids (glycine and glutamic acid) still maintain more than 50% to 70% of their initial content. Attached Figure Description

[0020] Figure 1 This is a schematic flowchart of the ultrasonic-assisted plasma-activated water regulation method for preserving the flavor and quality of scallops during cold storage, as described in this invention.

[0021] Figure 2 The changes in volatile basic nitrogen (TVBN) content in scallops treated with different methods during refrigeration were shown. CK was the control group, US was the ultrasound group, PAW was the low-temperature plasma activated water group, and UP was the ultrasound-assisted low-temperature plasma activated water group, and so on.

[0022] Figure 3 The changes in thiobarbituric acid (TBA) content in scallops under different treatment methods during refrigeration.

[0023] Figure 4 The color changes of scallops under different processing methods during refrigeration.

[0024] Figure 5 Electronic nose radar images of scallops in different treatment groups during refrigeration (days 1, 4, and 7). Among them, W1C is sensitive to aromatic compounds, W5S is sensitive to nitrogen oxides, W3C is sensitive to ammonia and aromatic compounds, W6S is sensitive to hydrides, W5C is sensitive to short-chain alkanes and aromatic hydrocarbons, W1S is sensitive to methyl derivatives, W1W is sensitive to sulfides and terpenes, W2S is sensitive to alcohols, aldehydes, and ketones, W2W is sensitive to aromatic compounds and sulfides, and W3S is sensitive to long-chain alkanes. Figure 5 Image A is the electronic nose radar image for day 1. Figure 5 Image B is the electronic nose radar image on day 4. Figure 5 The image in C is the electronic nose radar image on day 7.

[0025] Figure 6The differences in the horizontal microbial community structure of scallops from different treatment groups during refrigeration were observed.

[0026] Figure 7 To investigate the differences in species-level microbial community structure of scallops from different treatment groups during refrigeration.

[0027] Figure 8 A statistical graph annotating the KEGG metabolic pathway, a differential metabolite, in scallops from different treatment groups during refrigeration.

[0028] Figure 9 Scatter plot of KEGG enrichment of differential metabolites in scallops on day 1 of refrigeration. Among them, Pancreatic secretion refers to pancreatic secretion, Saliva secretion refers to salivary secretion, Calcium signaling pathway refers to the calcium signaling pathway, Aldosterone-regulated sodium reabsorption refers to sodium reabsorption regulated by aldosterone, Oxytocin signaling pathway refers to the oxytocin signaling pathway, Biosynthesis of cofactors refers to cofactor biosynthesis, Thyroid hormone synthesis refers to thyroid hormone synthesis, Progesterone-mediated oocyte maturation refers to progesterone-mediated oocyte maturation, Oocyte meiosis refers to oocyte meiosis, Melanogenesis refers to melanin production, Long-term potentiation refers to long-term potentiation, Hormone signaling refers to hormone signaling pathways, GnRH signaling pathway refers to the GnRH signaling pathway, Glutamatergic synapse refers to glutamatergic synapse, Gastric acid secretion refers to gastric acid secretion, Dopaminergic synapse refers to dopaminergic synapse, Circadian entrainment refers to circadian rhythmic entrainment, Cholinergic Synapse refers to cholinergic synapse; Adrenergic signaling in cardiomyocytes refers to adrenergic signaling in cardiomyocytes; Ovarian steroidogenesis refers to ovarian steroid production; Glucagon signaling pathway refers to the glucagon signaling pathway; and Starch and sucrose metabolism refers to starch and sucrose metabolism.

[0029] Figure 10Scatter plot of KEGG enrichment of differential metabolites in scallops on day 4 of refrigeration. Among them, ABC transporters refer to ABC transport proteins, Starch and sucrose metabolism refers to starch and sucrose metabolism, Calcium signaling pathway refers to the calcium signaling pathway, Efferocytosis refers to endocytosis, Glutathione metabolism refers to glutathione metabolism, Metabolic pathways refer to metabolic pathways, One carbon pool by folate refers to the folate one-carbon pool, Thyroid hormone synthesis refers to thyroid hormone synthesis, Terpenoid backbone biosynthesis refers to terpenoid backbone biosynthesis, Pancreatic secretion refers to pancreatic secretion, Long-term potentiation refers to long-term potentiation, Glutamatergic synapse refers to glutamatergic synapse, Circadian entrainment refers to circadian rhythmic entrainment, Cholinergic synapse refers to cholinergic synapse, Arginine and proline metabolism refers to arginine and proline metabolism, Carbon metabolism refers to carbon metabolism, Oxytocin signaling pathway refers to the oxytocin signaling pathway, Fc epsilon RI signaling pathway refers to the FcεRI signaling pathway, Saliva secretion refers to salivary secretion, Gap Junction refers to a gap connection.

[0030] Figure 11Scatter plot of KEGG enrichment in scallops on day 7 of refrigeration. Among them, Calcium signaling pathway refers to the calcium signaling pathway, Efferocytosis refers to endocytosis, Thyroid hormone synthesis refers to thyroid hormone synthesis, Terpenoid backbone biosynthesis refers to terpenoid backbone biosynthesis, Pancreatic secretion refers to pancreatic secretion, Cholinergic synapse refers to cholinergic synapse, Oxytocin signaling pathway refers to the oxytocin signaling pathway, Oxidative phosphorylation refers to oxidative phosphorylation, Fc epsilon RI signaling pathway refers to the FcεRI signaling pathway, Neuroactive ligand-receptor interaction refers to neuroactive ligand-receptor interaction, Saliva secretion refers to salivary secretion, Olfactory transduction refers to olfactory transduction, Longevity regulating pathway refers to the longevity regulating pathway, Aldosterone-regulated sodium reabsorption refers to aldosterone-regulated sodium reabsorption, Serotonergic synapse refers to serotonergic synapse, ABC transporters refer to ABC transporters, Ether lipid metabolism refers to ether lipid metabolism, and cGMP-PKG signaling... pathway refers to the cGMP-PKG signaling pathway.

[0031] The CK control group refers to the 4℃ direct refrigeration group, US refers to the ultrasound group, PAW / P group refers to the low-temperature plasma activated water group, and UP refers to the ultrasound-assisted low-temperature plasma activated water group. Detailed Implementation

[0032] To make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0033] Example 1 A method for preserving the flavor and quality of scallops under cold storage by regulating water through ultrasound-assisted plasma activation, the specific steps of which are as follows: (1) PAW preparation: A modulated pulsed plasma power supply (CTP-2000KP, Nanjing Suman Plasma Technology Co., Ltd.) was used, with compressed air as the working gas (pressure 0.1 MPa, gas flow rate 15-20 L / min). The plasma jet tip was placed 0.5 cm below the surface of sterile deionized water. 1 L of sterile distilled water was taken and subjected to low-temperature plasma activation treatment for 10 min for later use.

[0034] (2) Scallop pretreatment: Take fresh scallops, remove the shells immediately and take the adductor muscle, rinse the surface impurities quickly with sterile deionized water, and drain for later use.

[0035] (3) US-PAW co-treatment: Scallop adductor muscles were immersed in 500 mL of PAW at a feed-to-liquid ratio of 1:8 (w / v) and placed in a 1000 mL beaker. The beaker was then placed in a 1 L beaker filled with ice (to maintain an ambient temperature of 4°C during the sonication and immersion process). The sonicator (single frequency, 40 kHz) was turned on and treated for 10 min at 300 W ultrasonic power. After the sonication, the scallops were gently stirred in the PAW for another 30 min, bringing the total PAW treatment time to 40 min.

[0036] (4) Drain and refrigerate: Remove the adductor muscle of the scallop, drain the surface moisture, and store it in a refrigerated room at 4°C.

[0037] Example 2 A method for preserving the flavor and quality of scallops under cold storage by regulating water through ultrasound-assisted plasma activation, the specific steps of which are as follows: (1) PAW preparation: Same as in Example 1.

[0038] (2) Scallop pretreatment: Same as in Example 1.

[0039] (3) US-PAW co-processing: Same as Example 1, except that the adductor muscle of scallops is immersed in PAW at a feed-to-liquid ratio of 1:6 (w / v) and the ultrasonic power is 350W.

[0040] (4) Drain and refrigerate: Same as in Example 1.

[0041] Example 3 This embodiment performs a single ultrasound treatment, as follows: (1) Scallop pretreatment: Same as in Example 1.

[0042] (2) Immerse the adductor muscle of the scallop in a 500 mL beaker of sterile deionized water at a feed-to-liquid ratio of 1:8 (w / v), and place it in a 1 L beaker filled with ice (maintaining the ambient temperature of 4℃ during the ultrasound process). The ultrasound frequency is 40 kHz, the ultrasound power is 300 W, and the ultrasound time is 10 min.

[0043] (3) Drain and refrigerate: Same as in Example 1.

[0044] Example 4 This embodiment performs single PAW processing, as follows: (1) Scallop pretreatment: Same as in Example 1.

[0045] (2) Soak the adductor muscle of the scallop in 500 mL PAW at a feed-to-liquid ratio of 1:8 (w / v) for 40 minutes, stirring gently to achieve uniform soaking. Place the soaking container in ice to maintain the ambient temperature at 4°C.

[0046] (3) Drain and refrigerate: Same as in Example 1.

[0047] Results analysis: (a) Detection of volatile basic nitrogen (TVBN) content in scallops under different treatment groups TVBN was determined according to the automatic Kjeldahl method in GB5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". 3 g of sample was mixed with 1 g of light magnesium oxide and distilled. The distillate was collected using methyl red-bromocresol green-boric acid indicator and then titrated with 0.01 M standard hydrochloric acid solution. The TVBN content was obtained as the milligram nitrogen content per 100 g of scallop (mg N / 100 g).

[0048] The results are as follows Figure 2 As shown, the TVBN of the CK group (directly refrigerated at 4℃) exceeded the normal value after day 4, reaching the spoilage standard, with a shelf life of only about 3-4 days. For the ultrasonic group, US treatment could delay the onset of spoilage to about day 6, but by day 7, spoilage was already obvious, and the preservation effect was limited. However, PAW treatment effectively inhibited microbial growth, keeping TVBN stable for the first 6 days, with a significant increase only on day 7, and a shelf life of more than 6 days. For the preferred UP group, ultrasonic treatment disrupted cell structure, causing some free amino acids or nitrogenous substances to dissolve, resulting in a slightly higher TVBN initially; synergistic treatment with PAW significantly suppressed the upward trend of TVBN on day 7 of refrigeration, maintaining it at 48.38±2.58 mg / 100g.

[0049] (II) Detection of thiobarbituric acid (TBA) content in scallops under different treatment groups Take 1 g of sample and add 10 mL of 7.5% trichloroacetic acid solution (containing 0.1% EDTA), homogenize for 1 min, let stand in an ice bath for 10 min, take 5 mL of the supernatant and add 5 mL of 0.02 mol / L TBA solution, boil in a water bath for 40 min, cool with ice water, and measure the absorbance at 532 nm. Use 1,1,3,3-tetraethoxypropane (TEP) as a standard curve. The TBABS content is expressed as mg MDA / kg.

[0050] The results are as follows Figure 3 As shown, the TBA value in the control group (CK, directly refrigerated at 4℃) increased rapidly from 0.31±0.01 mg / kg on day 1, reaching 1.86±0.03 mg / kg on day 4, and as high as 2.20±0.06 mg / kg on day 7. This indicates extremely severe lipid oxidation, producing a large amount of secondary oxidation products such as malondialdehyde. For the ultrasound group (US), the TBA value was significantly lower than the control for the first 5 days, approached the control from day 6, and was slightly lower than the control on day 7. The cavitation effect of ultrasound can break down lipid oxidases, but the inhibitory effect weakens in the later stages of storage. The TBA value in the PAW group was lower than that in the US group throughout the process, reaching 2.00±0.01 mg / kg on day 7. Reactive oxygen species / reactive nitrogen species in PAW can quench free radicals and delay the chain oxidation reaction, but high concentrations of reactive oxygen species may also promote initial oxidation. The optimal group was the UP group, with all TBA values ​​being relatively low, indicating that ultrasound and PAW have a synergistic effect. Ultrasound may promote the penetration of active ingredients in PAW into the deep tissue layers, while destroying lipase, thereby inhibiting lipid oxidation more persistently.

[0051] (III) Color changes of scallops in different treatment groups Measurements were performed using a colorimeter, calibrated with its built-in white plate. The sample completely covered the light-transmitting aperture, and the L*, a*, and b* values ​​were measured and recorded. L* represents luminance; a* represents red-green hue; and b* represents yellow-blue hue. Eight parallel samples were measured for each group.

[0052] Images of scallops from different treatment groups after a certain number of days are shown below. Figure 4 As shown. Typically, during refrigeration, the adductor muscle of scallops deteriorates due to microbial growth, lipid oxidation, and protein denaturation, resulting in decreased brightness (L value), yellowing (increased b value), and dark spots. In the control group, brightness decreased significantly from day 2, and significant yellowing and browning occurred after day 4, making it sensorily unacceptable. However, the ultrasound group maintained better color initially, and although it began to darken significantly after day 4, it was still better than the control. The PAW group maintained better color than the US group, possibly because PAW inhibited melanin-producing microorganisms (such as Pseudomonas) and enzymatic browning. The UP group had the best color. Synergistic treatment not only resulted in more thorough sterilization but also promoted uniform contact of PAW through ultrasound, delaying protein oxidation and pigment formation.

[0053] (iv) Electronic nose radar images of scallops in different treatment groups Accurately pipette 2 mL of sample into an electronic nose autosampler vial. Incubate at 60 ℃ for 40 min. Headspace generation parameters: generation time 120 s, agitation rate 500 r / min. Headspace injection parameters: injection volume 2400 μL; injection rate 2400 μL / s. Perform 5 replicates per sample group.

[0054] The results are as follows Figure 5 As shown, on day 1, the sensor response values ​​of all four groups were relatively low. However, on days 4 and 7, the sensor response values ​​of W1S (sensitive to alcohols), W1W (sensitive to sulfides), and W2S (sensitive to methyl compounds) in the CK group increased sharply. This indicates that the scallops produced a large amount of hydrogen sulfide, methanethiol, and lower alcohols and aldehydes during the later stages of cold storage. This is due to the putrid odor caused by the decomposition of proteins and oxidation of fats by microbial activity. Compared to the other three treatment groups, W1S and W1W showed a significant inhibition phenomenon. This may be because ultrasound disrupted the cell wall and cell membrane structure of the microbial membrane through the cavitation effect, while PAW, rich in ROS / RNS, effectively inhibited the growth of putrefactive bacteria that decompose proteins.

[0055] (v) Analysis of the species-level microbial community structure of scallops in different treatment groups Total genomic DNA was extracted from scallops using the CTAB method. The V3-V4 region of the bacterial 16S rRNA gene was selected and amplified by PCR using Q5 DNA high-fidelity polymerase with specific primers 338F (5'- barcode+ACTCCTACGGGAGGCAGCA-3') and 806R (5'- GGACTACHVGGGTWTCTAAT-3') (New England Biological Laboratory, Ipswich, Massachusetts, USA).

[0056] PCR products were quantified using the Quant-iT PicoGreen dsDNA Assay Kit on a microplate reader (BioTek, FLx800), and then pooled according to the required data volume for each sample. Library construction was then performed using the Illumina TruSeq Nano DNA LT Library Prep Kit (Illumina Inc., San Diego, CA, USA). Taxonomic analysis and data processing of the scallop microbiota were performed using QIIME2 2024.5 and DADA2.

[0057] like Figure 6 As shown, a total of 10 dominant bacterial genera were screened at the genus level, including the genus *Psychrophilus* (…). Psychrobacter Shewanella ( ) Shewanella ), Pseudomonas spp. Pseudomonas ), genus *Keratinobacterium* Cutibacterium Mycoplasma genus Mycoplasma ), Sphingosomalmonella ( Sphingomonas ), Borrelia burgdorferi ( Borreliella ), Pseudomonas ( Pseudolactococcus ), Vibrio genus ( Vibrio Larstonia spp. Ralstonia On day 1 of refrigeration, the initial microbial community structure of the scallops in each group was similar, indicating a relatively fresh state. In particular, common spoilage pathogens in aquatic products, such as Shewanella and Pseudomonas, were absent. However, as the refrigeration time increased, the microbial community underwent drastic changes. The levels of Shewanella and Pseudomonas increased in the control group (CK), while the levels of spoilage bacteria in the three treatment groups were significantly lower than in the control group. This may be because the ROS and RNS in the PAW exhibited a strong targeted killing or inhibitory effect on these bacteria. This is consistent with the results obtained from the electronic nose and free amino acids.

[0058] Species-level taxonomic units mainly include ten major taxonomic units, including Psychrobacter (psychrophilic bacillus) Shewanella putrefaciens (Shewanella putrefaction) Pseudomonas fragi (Pseudomonas freundii) Cutibacterium acnes (Propionibacterium acnes) Mycoplasma (Mycoplasma) Borreliella garinii (Garneri) Pseudolactococcus piscium (Pseudomonas stomatitis) Sphingomonas parapaucimobilis (Sphingosine parasitoids) Psychrobacter (Psychrophilic Bacillus) and Arcobacter (Vibrio) This is consistent with the results at the genus level (see) Figure 7 ).

[0059] (vi) Statistical chart of KEGG metabolic pathway annotation of differential metabolites in scallops from different treatment groups Scallop samples were subjected to untargeted metabolomics mass spectrometry (LC-MS) analysis, with three replicates per group. The samples were first ground with liquid nitrogen and placed in EP tubes, then 80% methanol aqueous solution was added. The mixture was then vortexed, incubated on ice for 5 min, centrifuged for 20 min (15000 g, 4℃), and a certain amount of the supernatant was diluted with mass spectrometry-grade water. This was followed by centrifugation for another 20 min (15000 g, 4℃), and the supernatant was collected for LC-MS analysis.

[0060] LC-MS analysis was performed using a Vanquish UHPLC system (Thermo Fisher) with a chromatograph (Vanquish UHPLC, Thermo Fisher, Germany) and a mass spectrometer (Q Exactive). TM The parameters for the HF (Thermo Fisher., Germany) column (Hypesil Gold column, Thermo Fisher., USA) were: column temperature 40℃, flow rate 0.2 mL / min; the parameters for the mass spectrometer were: scan range m / z 100-1500; and the ESI source settings were: Spray Voltage: 3.5 kV, Aux Gas flow rate: 10 L / min. The MS / MS secondary scan was set to data-dependent scans.

[0061] All data (.raw) files were imported into the Commod Discoverer 3.1 library for processing, enabling metabolite identification and relative quantification. These data results were then used to annotate the identified metabolites using the KEGG database (https: / / www.genome.jp / kegg / pathway.html), the HMDB database (https: / / hmdb.ca / metabolites), and the LIPIDMaps database (http: / / www.lipidmaps.org / ).

[0062] Data results showed that 1166 metabolites (651 positive ions and 515 negative ions) were annotated from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KEGG functional annotation was performed on the metabolites to explore their functional significance (see [link to KEGG database]). Figure 8In metabolomics mass spectrometry, two modes were used: positive ion mode (POS) and negative ion mode (NEG). POS was used to detect positively ionized compounds (such as amino acids and phospholipids), while NEG was used to detect negatively ionized compounds (such as nucleotides, fatty acids, and sugars). The metabolomics study of scallops primarily used the NEG mode. In the NEG mode, metabolites were classified into six KEGG groups, including 13 Metabolism groups, 10 Organismal Systems groups, 4 Cellular Processes, 4 Drug Development groups, 3 Environmental Information Processing groups, and 1 Genetic Information Processing group. Among the metabolites, amino acid metabolism accounted for the majority of the metabolites, including 61 metabolites; followed by lipid metabolism (52 ​​metabolites), carbohydrate metabolism (33 metabolites), and nucleotide metabolism (20 metabolites). The trend was similar in the POS mode, with 76 metabolites related to amino acid metabolism, 60 metabolites related to lipid metabolism, 13 metabolites related to nucleotide metabolism, and 7 metabolites related to carbohydrate metabolism. Therefore, with the extension of refrigeration time, the scallops in different treatment groups were mainly affected in terms of amino acid metabolism, lipid metabolism, carbohydrate metabolism and nucleotide metabolism.

[0063] like Figure 9-11As shown, the top 30 enrichment results were used for all enrichment. Functional annotation and pathway enrichment analysis revealed that on day 1 of refrigeration, the differentially enriched metabolites in the CK, US, P, and UP groups were Cysteine ​​and methionine metabolism, Folate biosynthesis, Phospholipase D signaling pathway, Phosphonate and phosphinate metabolism, Amino sugar and nucleotide sugar metabolism, Cholesterol metabolism, Pyrimidine metabolism, and Steroid biosynthesis. On day 4 of refrigeration, the differential metabolites among the four groups were mainly related to glutathione, arginine, proline, cysteine ​​and methionine metabolism, glycolysis / gluconeogenesis, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, amino acid biosynthesis, nucleotide metabolism, phospholipase D signaling pathway, phosphonate and phosphinate metabolism, pentose phosphate pathway, and amino sugar and nucleotide sugar metabolism.Finally, on day 7, the differential metabolites in the four groups were mainly involved in Cysteine ​​and methionine metabolism, Nicotinate and nicotinamide metabolism, Biosynthesis of nucleotide sugars, Ubiquinone and other terpenoids. The biosynthesis of ubiquinone and other terpenoids / quinones involves several pathways, including phospholipase D signaling, phosphonate and phosphinate metabolism, starch and sucrose metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, aldosterone synthesis and secretion, glutathione metabolism, and the pentose phosphate pathway. In summary, metabolites from amino acid metabolism, lipid metabolism, nucleotide metabolism, and the synthesis and secretion of aldehydes, ketones, and alcohols are the main contributors to volatile flavor compounds. Therefore, during 7 days of refrigeration after ultrasonic and plasma-activated water treatment, the microbial metabolites of scallops changed, affecting the quality of the scallops and the characteristics of their volatile flavor compounds.

[0064] (vii) Detection of free amino acids in scallops from different treatment groups Analysis was performed using an automated amino acid analyzer (L-8800, Hitachi Co., Ltd, Tokyo, Japan). The sample was homogenized with trichloroacetic acid solution (15% w / w) at a volume ratio of 1:5 for 1 min, allowed to stand for 2 h, centrifuged, and filtered. The supernatant was collected, the pH adjusted to 2.0 with NaOH solution, and then rapidly diluted to 10 mL with ultrapure water. The solution was then filtered through a 0.22 mm aqueous membrane before analysis. Each sample was analyzed in triplicate. The procedure was performed at temperatures below 4°C.

[0065] The results are shown in Table 1. On day 1 of storage, the UP group showed a significant advantage in core umami indicators (P < 0.05), with Gly (up to 383.47 mg / 100g) and Glu (37.91 mg / 100g) content almost twice that of the CK group. This fully demonstrates that the cavitation effect of ultrasound effectively disrupts the scallop muscle and myofibril protein structure or cell membrane, not only promoting the dissolution of free amino acids but also possibly moderately activating endogenous proteases, accelerating the degradation of proteins into flavor peptides and amino acids. As the refrigeration time progressed, the values ​​of each group generally declined, but the UP group remained at a high level (Gly 300.69 mg / 100g, Glu 33.07 mg / 100g), indicating that the combined treatment effectively delayed the decline of core flavor substances. The accumulation of bitter amino acids in the later stages usually indicates protein spoilage. On day 7, Val and Met surged in the CK and US groups, while bitter amino acids in the P and UP groups remained in a very low range. This directly proves that the ROS and RNS abundant in PAW inhibit spoilage microorganisms that decompose proteins to produce bitter peptides, thus cutting off the source of the bitterness and odor of scallops in the later stages of refrigeration.

[0066] Table 1. Content of free amino acids in scallops from different treatment groups during refrigeration (days 1, 4, and 7). comprehensive Figures 2 to 4 In the CK group, the TVBN and TBA values ​​exceeded normal levels after 4 days of refrigeration, reaching the spoilage standard. However, among the three treatment groups, the UP group, which was the best, did not begin to spoil until the 6th day, significantly extending the shelf life of the scallops. The color change was consistent with the TVBN and TBA values. The shelf life of the UP group was 30%-50% longer than that of the single ultrasonic group or the single PAW group.

[0067] comprehensive Figure 5 According to Table 1, on the 7th day of refrigeration, the core umami amino acids Gly and Glu in the UP group remained at 300.69 mg / 100g and 33.07 mg / 100g, respectively, which were significantly higher than those in the control group (Gly 191.19 mg / 100g and Glu 15.68 mg / 100g); the content of bitter amino acids (Val, Met, etc.) was significantly lower than that in the control group.

[0068] comprehensive Figures 6 to 11 The UP group could inhibit the proliferation of Shewanella putrefaciens and Pseudomonas fragi, the putrefactive bacteria of scallops, block the cysteine ​​and methionine metabolic pathways, and simultaneously inhibit the oxidation pathways of glycerophospholipids and arachidonic acid, reducing hexanal, nonanal, and 1 Octene 3 Accumulation of lipid oxidation products of rancid fatty acid odor substances such as alcohols.

[0069] In conclusion, the US-PAW treatment of this invention has significantly better preservation and odor suppression effects than the single treatment (P<0.05), and has obvious technological progress and substantial innovation compared with existing preservation technologies.

Claims

1. A preservation method for controlling the flavor quality of aquatic products during cold storage by ultrasonic synergistic low-temperature plasma active water, characterized in that, The steps include: In a low-temperature environment, fresh aquatic products are immersed in low-temperature plasma active water for ultrasonication. After ultrasonication, the fresh aquatic products continue to be immersed in low-temperature plasma active water. After immersion, the fresh aquatic products are removed from the low-temperature plasma active water and refrigerated.

2. The method of claim 1, wherein The mass-to-volume ratio of the fresh aquatic products to the low-temperature plasma activated water is 1:3-10.

3. The method of claim 1, wherein The temperature of the low-temperature environment is controlled between 0-10℃.

4. The method of claim 1, wherein The preparation of the low-temperature plasma activated water includes: using a modulated pulsed plasma power supply, with air as the working gas, immersing the plasma jet end in sterile distilled water for discharge activation, to obtain an aqueous solution rich in active oxygen and active nitrogen.

5. The method of claim 4, wherein The working gas pressure is 0.1–0.3 MPa, and the gas flow rate is 10–30 L / min; the plasma jet end is positioned 0.5–1.5 cm below the surface of sterile distilled water, and the low-temperature plasma activation time for each L of sterile distilled water is 5–15 min.

6. The method of claim 1, wherein The ultrasonic power is 200–400W, the frequency is 20–50 kHz, and the ultrasonic time is 5–15 min.

7. The method of claim 1, wherein The soaking time is 10 to 50 minutes.

8. The method of claim 1, wherein The fresh aquatic products are selected from any one of fish, crustaceans, and shellfish.

9. The method of claim 1, wherein The refrigeration temperature is 0–4°C.

10. Chilled fish products which retain flavour quality characterised in that, It is prepared by any one of the preservation methods according to claims 1-9.

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