阅读说明：本技术 一种基于co或co释放试剂的水产品保鲜剂及其应用 (Aquatic product preservative based on CO or CO release reagent and application thereof ) 是由 吴根福 于 2020-11-18 设计创作，主要内容包括：本发明公开了一种基于CO或CO释放试剂的水产品保鲜剂及其应用,所述的CO释放试剂为过渡金属羰基化合物。本发明还公开了利用所述水产品保鲜剂的保鲜方法,具体包括如下步骤：在水产品冷藏、冷冻前或者在冷藏、冷冻的同时,用所述的保鲜剂处理水产品。本发明所述保鲜剂的主要有效成分为一氧化碳,作用于水产品后,可抑制细菌活性,达到延长货架时间的效果。(The invention discloses an aquatic product preservative based on a CO or CO release reagent and application thereof, wherein the CO release reagent is a transition metal carbonyl compound. The invention also discloses a preservation method using the aquatic product preservative, which specifically comprises the following steps: before or during the refrigeration and freezing of the aquatic products, the aquatic products are treated by the preservative. The main effective component of the preservative is carbon monoxide, and after the preservative acts on aquatic products, the activity of bacteria can be inhibited, so that the effect of prolonging the shelf life is achieved.)

1. An aquatic product preservative is characterized by consisting of a CO or CO release reagent.

2. The aquatic product preservative according to claim 1, wherein the aquatic product is a freshwater product or a seawater product.

3. The aquatic product preservative according to claim 1, wherein the freshwater product is a carp, a silver carp, a shrimp or a crayfish; the seawater product is hairtail, tuna, prawn or cuttlefish.

4. The aquatic product preservative according to claim 1, wherein the CO release reagent is a transition metal carbonyl compound formed by combining a transition metal, CO and an auxiliary ligand.

5. The aquatic product preservative according to claim 4, wherein the transition metal carbonyl compound is CORM-2, CORM-3, CORM-F, CORM-A1, ALF021 or ALF 062.

6.A preservation method of aquatic products is characterized by comprising the following steps: before or while refrigerating and freezing aquatic products, treating the aquatic products with the preservative according to any one of claims 1 to 5.

7. The method for preserving aquatic products according to claim 6, wherein the time for treating the aquatic products with the preservative is 10 minutes or more.

8. The aquatic product preservation method according to claim 6, wherein the aquatic product treated by the preservative specifically comprises: putting the aquatic product into a closed container or a freshness protection package, and filling the aquatic product freshness protection agent into the closed container or the freshness protection package to enable the volume percentage of CO in the closed container or the freshness protection package to be 0.1-10%; the aquatic product preservative is a gaseous preservative.

9. The aquatic product preservation method according to claim 6, wherein the aquatic product treated by the preservative specifically comprises: putting the aquatic product into a water tank, and filling the water tank with an aquatic product preservative to ensure that the effective concentration of CO in the water body is 0.01-1 mmol/L; the aquatic product preservative is a gaseous preservative, a liquid preservative or a solid preservative.

10. The application of the aquatic product preservative in preparation of the bacteriostatic agent according to any one of claims 1-5, wherein the bacteriostatic agent is a Shewanella inhibitor.

Technical Field

The invention relates to the field of food, in particular to an aquatic product preservative based on a CO or CO release reagent and application thereof.

Background

The aquatic product has rich nutrition and high water content, and is easy to decay and deteriorate in the storage and transportation processes. Statistics shows that aquatic products losing economic value due to spoilage account for about 30% of China each year. The spoilage of aquatic products is mainly due to three reasons: microbial action, self-enzymatic action and environmental oxidation, among which the microbial action is the main cause (Ghaly AE et al. Fish sponge mechanisms and preservation technologies [ J ]. American Journal of Applied Sciences,2010,7(7): 859-. The growth and reproduction of microorganisms not only cause the decay and deterioration of aquatic products, but also generate putrescine, cadaverine, aldehydes, sulfides, etc. during the metabolic process, and also emit malodorous gases (Dalgaard P et al. biogenic amine formation and microbial spore in solid garfish: effect of modified and immobilized packing and previous from storage [ J ]. Journal of Applied Microbiology,2006,101(1): 80-95).

It is generally accepted that spoilage bacteria in fresh water products, particularly in seawater products, are primarily Shewanella sp. Shewanella is widely distributed in water body, especially in ocean, and has optimal growth temperature of 30 deg.c and slow growth at low temperature. Can be metabolized by aerobic respiration, and can also be subjected to anaerobic respiration by utilizing various inorganic or organic electron acceptors. Especially, organic or inorganic sulfides such as sulfite, thiosulfate and dimethyl sulfoxide can be utilized to carry out metabolism, hydrogen sulfide or organic sulfur is released, unpleasant odor is emitted, and the quality of aquatic products is seriously influenced (Wu GF et al.

In order to prolong the shelf life of aquatic products, people adopt various methods to keep the aquatic products fresh. The fresh-keeping of aquatic products is mainly realized by controlling the growth and the propagation of microorganisms, particularly controlling the quantity of putrefying bacteria. The method is characterized by comprising the following common means of low-temperature fresh-keeping, chemical fresh-keeping, modified atmosphere fresh-keeping, irradiation fresh-keeping and the like (Xiancei, fresh food fresh-keeping technical research progress [ J ]. Chinese food academy of academic, 2010,10(3): 1-12).

Low temperature is the most common and widely used means for preservation. According to the difference of temperature, the method can be divided into refrigeration preservation (0-4 ℃), ice temperature preservation (0-2 ℃), micro-freezing preservation (-2-4 ℃), freezing preservation (-18-40 ℃) (OlafDOTRER G et al evaluation of shelf life of super code files and the filling of temperature structures of refrigerating storage on biological and chemical quality indicators [ J ]. Journal of Food Science,2010,71(2): S97-S109). Quick freezing preservation is an effective preservation means, but large-scale refrigeration equipment is needed, so that the energy consumption is high, the cost is high, and the quick freezing preservation is difficult to popularize in small and medium-sized enterprises. The refrigeration and preservation are the most easily realized means, but for aquatic products with a slightly larger volume, the cooling process needs more than 2 hours, some spoilage bacteria grow and propagate in the cooling process, and in addition, cold-resistant spoilage bacteria such as Shewanella and the like can slowly grow and metabolize under non-icing conditions, so that the quality guarantee time of the refrigeration and preservation is relatively short.

The chemical fresh-keeping method is a fresh-keeping method for prolonging the quality guarantee period of aquatic products by means of the sterilization and oxidation resistance of chemicals, and has the characteristics of low cost, simple and convenient operation and the like. Chemical preservatives can be divided into a plurality of categories, including preservatives (bacteriostats), bactericides and antioxidants. Among them, nitrite, sorbate, sodium benzoate, sodium diacetate, etc. (Yi S et al. effect of tea polysaccharides on microbiology and biochemical quality of colloidal fish ball J. Journal of the Science of Food & Agriculture 2011,91(9): 1591) 1597) are widely used. Along with the improvement of environmental protection and health consciousness of people, the requirements on chemical preservation are more and more strict, and preservation means of chemicals such as antibiotics and preservatives are forbidden by regulatory departments gradually.

Irradiation preservation technique refers to utilization⁶⁰Co or¹³⁷The gamma ray or high-energy electron beam generated by Cs can sterilize food, so that it can delay the growth and reproduction of microbe. Irradiation preservation requires special equipment, has strict treatment requirements (radiation has strong side effect on human body), and has certain damage to nutrient components (Molins RA. food irradation: principles and applications [ J ]].Food Irradiation Principles&Applications,2001,18(3):219), are less used in practice.

Modified atmosphere preservation refers to a preservation technology which is used for creating an environment which is not beneficial to the growth of microorganisms by changing the components or the concentration of gas in food packages, thereby inhibiting the propagation of the microorganisms and slowing down the decay of food. Often convert CO₂、N₂And filling the gas into a closed bag containing aquatic products for preservation. The method has better inhibition effect on aerobic bacteria, but the preservation effect on anaerobic bacteria and facultative anaerobic bacteria is not very good (Yi Lei, Xie Jing. aquatic product modified atmosphere preservation technical research progress [ J]Guangdong agricultural science, 2015, 42 (5): 92-97). In view of the fact that aquatic spoilage bacteria such as Shewanella and the like are facultative anaerobic bacteria, metabolic products such as sulfide and the like are generated through sulfite respiration or thiosulfate respiration under anaerobic conditions, and the application of the method is limited.

The Chinese patent application with publication number CN107114461A discloses a fish cold fresh preservation method, which comprises the following steps: (1) nondestructive freezing, (2) coating a chemical preservative, (3) modified atmosphere packaging and sealing, and (4) refrigerating and storing. The method comprises the steps of killing and cutting fresh fish, slowly freezing to 20 ℃ below zero, quickly thawing, coating the surface of the fish with a chemical preservative, and performing controlled atmosphere cold storage for preservation, wherein the shelf life can reach 7 days. The chemical preservative used in the method consists of 2% of lysozyme, 2% of calcium propionate, 1% of chitosan, 5% of vitamin C, 5% of glycine and 5% of glycerol, and the gas for modified atmosphere preservation consists of nitrogen: oxygen: the ratio of CO is 4: 1: 1 are mixed.

The invention has the following problems: (1) can only be used for the fish after cleaning, killing and cutting; (2) the steps are complicated, various processing procedures of chemical fresh-keeping, modified atmosphere fresh-keeping and freezing fresh-keeping are mixed, and the labor intensity is high; (3) the price is high, lysozyme, vitamin C and glycine are expensive chemicals, a large amount of electric energy is consumed for vacuumizing before modified atmosphere preservation, a large amount of electric energy is consumed for freezing and thawing, and the cell tissues of the fish are easily damaged due to the destructive effect of ice crystals in the thawing process; (4) when the modified atmosphere storage is carried out, the CO concentration is too high and reaches 16.7 percent, a large amount of CO is combined with heme molecules in fish protein, although the color of the fish is improved, the health of a human body is adversely affected by excessive ingestion of carboxyhemoglobin.

Therefore, it is necessary to explore a new preservation method to solve the above problems.

Disclosure of Invention

The invention provides an aquatic product preservative based on a CO or CO release reagent and application thereof, and the aquatic product preservative is simple and convenient to operate, wide in application range, green, environment-friendly, free of residue, free of damage to refrigerated food and free of edible safety problem.

The technical scheme provided by the invention for solving the technical problem is as follows:

an aquatic product preservative, which consists of a CO or CO release reagent.

CO is a gaseous substance and can freely penetrate through a cell membrane to enter cells, so that intracellular NADH dehydrogenase is inactivated; CO also acts on cytochrome transport proteins and terminal oxidases in the respiratory chain to inhibit the progression of aerobic and anaerobic respiration. The research shows that the CO of 10-40 mu M can obviously inhibit the growth and the reproduction of bacteria, and the concentration required for inhibiting eukaryotic cells is more than 100 mu M.

The aquatic product is a fresh water product or a seawater product.

The freshwater product is carp, silver carp, river shrimp or crayfish; the seawater product is hairtail, tuna, prawn or cuttlefish.

The CO release reagent is a transition metal carbonyl compound formed by combining transition metal, CO and an auxiliary ligand.

The transition metal element is ruthenium, manganese or molybdenum.

The main effective component of the aquatic product preservative is CO which can be at least one of gaseous, liquid and solid preparations.

The transition metal carbonyl compound is tricarbonyldichlororuthenium (II) dimer, CORM-2, tricarbonylchlororuthenium (II) (tricarbonylchloride (glycine) ruthenium (II)), CORM-3, iron-containing tricarbonyl compound (iron-binding carbonyls, CORM-F), sodium carbonyl boron compound (sodium boranocarbonate, CORM-a1), bromine (pentacarbonyl) manganese compound (bromine (pentacarbonyl) manganene, ALF 021), and tetraethylammonium molybdenum bromide (tetra ethyl ammonium bromide, ALF 062).

The invention also provides a preservation method of aquatic products, which comprises the following steps: before or during the refrigeration or freezing of aquatic products, the aquatic products are treated by the preservative.

The invention discovers that CO has a strong inhibiting effect on putrefying bacteria, particularly Shewanella, aquatic products are treated by using a reagent (preservative) containing CO or capable of releasing CO in advance, and then traditional preservation measures are adopted, or low-temperature preservation is carried out while treatment is carried out, so that the shelf life of the aquatic products can be obviously prolonged.

The specific method for treating the aquatic products by the preservative comprises the following steps: putting the aquatic product into a closed container or a freshness protection package, and filling the aquatic product freshness protection package into the closed container or the freshness protection package to ensure that the volume percentage of CO in the closed container or the freshness protection package is 0.1-10% (v/v); the aquatic product preservative is a gaseous preservative. Preferably, the percentage by volume of CO is 1-2% (v/v).

As CO is a gaseous compound, can freely penetrate cell membranes to enter cells, has high affinity with second-order iron in cytochrome, and is easy to combine into a carboxin molecule, thereby inhibiting the activity of various enzymes. As putrefying bacteria are mainly distributed on the surface of a fish body, and the pronucleus (bacteria) has higher sensitivity to CO than the eukaryote (fish body cells), CO in a closed container or a freshness protection package is preferentially combined with heme molecules in the bacteria, so that the aim of inhibiting or killing the bacteria is fulfilled. The concentration of CO is preferably 1-2%, if the concentration of CO is too high, not only the material is wasted, but also the redundant CO can be combined with the heme molecules of fish body cells to denature fish protein.

The specific method for treating the aquatic products by the preservative comprises the following steps: putting the aquatic product into a water tank, and filling the water tank with an aquatic product preservative to ensure that the effective concentration of CO in the water body is 0.01-1 mmol/L; the aquatic product preservative is a gaseous preservative, a liquid preservative or a solid preservative. Preferably, the effective concentration of CO is 0.1-0.2 mmol/L.

According to the figure 1, CO dissolved in water has a remarkable inhibiting effect on Shewanella putrefaciens, and the death rate can reach 99.9 percent after 20 mu M (0.02mmol/L) CORM-2 is treated for 30 minutes. Considering that the preservation environment is complex when aquatic products are preserved, the water body contains 0.1-0.2 mmol/L of CO which is more appropriate when the aquatic products are actually used, so that the effect of inhibiting putrefying bacteria can be achieved, and the quality of the aquatic products cannot be influenced.

The time for treating the aquatic product by the preservative is at least 10 minutes. Preferably, the time for treating the aquatic products by the preservative is 30-60 minutes.

According to fig. 1 and 2, the higher the treatment concentration of CO, the longer the treatment time, and the better the bacteriostatic effect. In order to save cost, it is relatively reasonable to treat at low concentration for a long time. However, the concentration of CO in the antistaling agent cannot be too low, and when the concentration of CO is 0.01mmol/L, if the treatment time is less than 10 minutes, the bacteriostatic effect is poor, so the time for treating aquatic products by the antistaling agent is at least 10 minutes.

The invention also provides application of the aquatic product preservative in preparation of a bacteriostatic agent, wherein the bacteriostatic agent is a Shewanella inhibitor.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention explores new application of the known compound CO and develops a new application field.

(2) The aquatic product preservative adopts CO or a CO release preparation as a main or key component, and the released CO can be rapidly combined with heme molecules of bacterial cells, so that the activity of various enzymes such as NADH dehydrogenase, cytochrome oxidase and the like is inhibited, bacteria are effectively inhibited or killed, and the shelf life of aquatic products is prolonged.

(3) The aquatic product preservative is suitable for whole fish, even live fish. The fish can be cut without killing in advance, and a chemical preservative (preservative) is not coated on the surface of the fish. Since the concentration of CO required for inhibiting bacteria is much lower than that for inhibiting eukaryotic cells (fish somatic cells), treatment with CO at said concentration is effective in inhibiting growth and reproduction of putrefying bacteria with less effect on live fish.

(4) The CO concentration in the modified atmosphere fresh-keeping of the invention is not more than 10% (v/v), preferably 1-2% (v/v). Since putrefying bacteria are generally located on the surface of fish bodies, they preferentially bind CO. Because the treated fish is whole fish, the remaining small amount of CO does not cause obvious chemical change of the fish meat, and the safety is high.

(5) The fresh-keeping method provided by the invention has the advantages of good effect, low labor intensity and low cost.

(6) The preservative provided by the invention can be industrially produced, the use amount of antibiotics and preservatives is reduced, and the environmental pollution and the possible side effect on human bodies are reduced.

Drawings

FIG. 1 is a graph showing the inhibition effect of CO on Shewanella in example 1, wherein A is the growth status in shake flask culture after treatment with CORM-2 at various concentrations; b is the survival rate measured by a viable count method after the treatment of CORM-2 with different concentrations, and C is a picture of a dibbling test; d is the growth of the bacteria treated with 10. mu.M CORM-2 for various periods of time.

FIG. 2 is a graph showing the effect of CO on Shewanella biofilm formation in example 2, wherein A is the biomass of suspension cells measured by Shewanella putrefaciens treated with CORM-3 at various concentrations and cultured for 16 hours at room temperature under standing; b is a plan view photograph of a biofilm formation test of Shewanella putrefaciens treated with CORM-3 at different concentrations, and the left and right are repeated tests.

Detailed Description

The invention is further described with reference to the accompanying drawings and the embodiments.

Examples 1 to 2 were processed as follows:

the Shewanella putrefaciens is inoculated into an LB culture set, cultured at 30 ℃ and 200rpm to logarithmic phase, centrifuged to remove supernatant, cells are resuspended in buffer solution, treated with CO release reagent with certain concentration for certain time, and the survival rate of the Shewanella putrefaciens is measured, so that the inhibition or killing effect of CO on Shewanella putrefaciens can be judged.

Example 1 inhibition or killing of Shewanella putrefaciens by CORM-2

Shewanella putrefaciens was cultured in LB medium to logarithmic growth phase (OD)₆₀₀0.4), 1mL was pipetted into a centrifuge tube, the supernatant was centrifuged, the cell was washed 1 time with phosphate buffer, the cell was resuspended in 1mL of phosphate buffer, CORM-2 was added at a constant concentration for 30 minutes, then 0.2mL of the treated solution was pipetted into 2.8mL of LB medium (OD tube), the cell was shake-cultured at 30 ℃, and the amount of cell growth was measured every 1 hour, and the results are shown in fig. 1A.

The cells treated with different CORM-2 were diluted and plated to calculate the mortality, and the results are shown in FIG. 1B.

2.5. mu.l of the treated cells diluted 10 times were spotted on LB plates and the survival rate was observed after the culture, and the results are shown in FIG. 1C.

Experiments also found that the duration of the CO action also had a large effect on the growth of the bacteria, with the survival rate decreasing with the treatment time within 0-30 minutes, as shown in FIG. 1D.

As can be seen from FIGS. 1A to 1D, CO has a significant inhibitory effect on Shewanella putrefaciens, and the mortality rate reaches 99.9% after 20 μ M CORM-2 treatment for 30 minutes. Indicating that CORM-2 is a potential antibacterial bactericide.

Example 2 inhibition of stationary growth of Shewanella putrefaciens by CORM-3

Shewanella putrefaciens was cultured in LB medium to logarithmic phase (OD)₆₀₀0.4), 1mL of each was pipetted into a centrifuge tube, the supernatant was centrifuged, after washing 1 time with phosphate buffer, the cells were resuspended in 1mL of phosphate buffer, and a certain concentration of CORM-3 was added for 30 minutes, then 0.2mL of the treatment solution was pipetted into 2.8mL of LB medium, and the cells were cultured by standing at room temperature (25 ℃) for 16 hours, and the growth amount and biofilm formation amount of the suspended cells were measured, and the results are shown in FIG. 2A. Biofilm formation (top view, cultured on 24-well plates) is shown in FIG. 2B. As is apparent from FIG. 2, CORM-3 significantly affected the amount of suspended cells and biofilm biomass in Shewanella static culture.

Examples 3 to 5 the following procedure was followed:

fresh aquatic products are put into a plastic bag in duplicate (identical in size and weight), one part of the fresh aquatic products is filled with CO gas, the other part of the fresh aquatic products is not processed, after the fresh aquatic products are stored for a certain time under the same conditions, sterile normal saline with the same volume is added, the fresh aquatic products are shaken in a shaking table at 150rpm for 10min, bacteria in the aquatic products are washed into the normal saline, the washed bacteria are immediately coated on an LB flat plate by a dilution coating method, and the bacteria are counted after being cultured for 48 hours at 30 ℃.

Example 3 Effect of gaseous CO on carp shelf-Life

Putting fresh Cyprinus Carpio into sealed plastic bag (fresh-keeping bag), charging CO into the bag at an amount of 10% of the bag volume, and charging no gas (original air) into the other group, sealing, storing in a 4 deg.C refrigerator, taking out one part after 2 days, 7 days and 15 days, respectively, injecting 0.2V (bag volume) of physiological saline into the bag, shaking for 10min in a shaking table at 150rpm, and measuring the total bacterial count in the physiological saline, the results are shown in Table 1. As can be seen, the shelf life can be significantly extended after CO treatment.

Table 1 influence of shelf life (viable count) of CO gas carp (. times.10)⁶cfu/mL)

Example 4 Effect of gaseous CO on the shelf-Life of hairtails

Fresh hairtails are put into a closed plastic bag (freshness protection bag), one group is filled with CO with the filling amount being 0.1% of the volume of the bag, the other group is not filled with any gas (original air), then the bag is sealed, and the bag is stored at room temperature (25 ℃) for 1 day, 3 days and 7 days, then one part is respectively taken, 0.2V-volume physiological saline is injected, the bag is shaken in a shaking table at 150rpm for 10min, the total bacterial count in the physiological saline is measured, and the result is shown in Table 2. As can be seen, the shelf life was extended after CO treatment.

Table 2 influence of shelf life (viable count) of CO gas hairtail (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	1 day	3 days	7 days
					CO treatment	----	1.8±0.3	15.7±0.8	89±7.7
Control	0.15±0.06	3.7±0.4	38±4.5	243±26

Example 5 Effect of gaseous CO on the shelf-Life of prawn

Fresh prawns are put into a sealed plastic bag (freshness protection bag), the experimental group fills CO into the bag, the filling amount is 0.5 percent, 1 percent, 2 percent and 5 percent of the volume of the bag, the control group does not fill any gas (original air), then the bag is sealed, the prawns are taken out and put into another clean freshness protection bag after being treated for 1 hour at room temperature (25 ℃), a refrigerator at 4 ℃ is used for storage, after 1 day, 3 days and 7 days, one part of the prawns is respectively taken out, physiological saline with the volume of 0.2V is injected, the prawns are shaken in a shaking table at 150rpm for 10min, the total bacterial count in the physiological saline is measured, and the result is. As can be seen from the table, the shelf life can be remarkably prolonged after the CO treatment, 1-2% is more appropriate, and the effect of inhibiting bacteria by further increasing the CO concentration is certain. But increases the cost and may affect the quality of the fish meat (CO reacts with heme in fish meat cells to produce carboxyheme).

TABLE 3 Effect of CO gas prawn shelf life (viable count) (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	1 day	3 days	7 days
					Control	0.12±0.08	3.2±0.5	18.1±3.7	73.5±8.8
0.5% CO treatment	----	2.1±0.6	14.5±0.6	58.1±8.3
					1% CO treatment	----	0.9±0.2	5.5±0.6	24.0±4.9
2% CO treatment	----	0.3±0.3	1.3±1.0	8.7±3.8
					5% CO treatment	----	0.2±0.1	0.9±0.4	5.8±1.3

Examples 6 to 10 were processed as follows:

fresh aquatic products are put in plastic boxes in duplicate (identical in size and weight), sterile normal saline is added to enable the aquatic products to be just immersed in water, CO or a CO release reagent is added to one part of the aquatic products, the other part of the aquatic products is not processed, after the aquatic products are stored for a certain time under the same condition, bacteria in the normal saline are coated on an LB plate through a dilution plate coating method, and the bacteria are counted after the aquatic products are cultured for 48 hours at the temperature of 30 ℃.

Example 6 Effect of CO Release formulation CORM-2 on the shelf-Life of shrimp

Fresh shrimp are placed in a plastic box and sterile normal saline is added to just immerse the shrimp in water. One group was filled with CORM-2 in a plastic box to make the concentration of CO in water 20. mu.M.the other group was not filled, and then the cover was closed, and the mixture was stored at room temperature, and after 1 day, 3 days, and 7 days, the total number of bacteria in the water was measured by sampling, respectively, and the results are shown in Table 4. As can be seen, the shelf life of the shrimp can be prolonged obviously after the CO treatment.

TABLE 4 Effect of CORM-2 shrimp shelf life (viable count) (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	1 day	3 days	7 days
					CO treatment	-----	0.4±0.1	1.3±0.5	8.6±1.2
Control	0.2±0.2	7.1±0.6	53±4.5	297±31

Example 7 Effect of CORM-3 on the shelf-life of prawn

Fresh penaeus vannamei boone is put into a plastic box, and sterile normal saline is added to immerse the penaeus vannamei boone in water. One group was filled with CORM-3 in a plastic box to make the CO concentration in the water 1mM., the other group was not filled, and then the cover was closed, and the water was stored in a refrigerator at 4 ℃ and after 2 days, 7 days, and 15 days, the total number of bacteria in the water was measured by sampling, respectively, and the results are shown in Table 5. As can be seen from the table, the shelf life of the prawns can be obviously prolonged after the CO treatment.

TABLE 5 Effect of CORM-3 on the shelf life (viable count) of Penaeus vannamei (x 10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	2 days	7 days	15 days
					CO treatment	----	0.2±0.1	1.2±0.8	7.3±0.7
Control	0.15±0.2	3.5±0.4	19±3.1	98±12

Example 8 influence of ALF062 and ALF021 on shelf life of tuna

Fresh tuna is put into a plastic box, and sterile normal saline is added to ensure that the tuna is just immersed in water. One group of 2 with ALF062 and ALF021 added to the plastic boxes: 1 the formulations were mixed so that the CO concentration in the water was 0.01mM., and the other was not added, and then the lid was closed, and the mixture was stored in a refrigerator at 4 ℃ for 2 days, 7 days, and 15 days, after which time the total number of bacteria in the water was measured by sampling, respectively, and the results are shown in Table 6. As can be seen, the shelf life of tuna can be remarkably prolonged after CO treatment.

TABLE 6 influence of ALF062 and ALF021 on tuna shelf life (viable count) (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	2 days	7 days	15 days
					CO treatment	-----	1.5±0.3	13±2.5	101±18
Control	0.08±0.12	2.4±0.8	28±4.3	756±88

Example 9 Effect of CORM-F3 and CORM-A1 on the shelf life of silver carp

Putting fresh silver carp into plastic box, adding sterile normal saline to make silver carp just immerged in water. Experimental groups a plastic box was charged with 1: 1 the preparations were mixed so that the CO concentration in water was 0.05mM, 0.1mM., 0.2mM, 0.5mM, and the other group was stored in a 4 ℃ refrigerator without control, and the total number of bacteria in physiological saline was measured after 1 day, 3 days, 7 days, and the results are shown in Table 7. As can be seen from the table, after the CO treatment, the growth and the propagation of bacteria can be obviously inhibited, the concentration is more suitable to be 0.1-0.2mM, and the further improvement of the CO concentration has certain effect on bacteriostasis, not only can the cost be increased, but also the fish quality can be influenced (CO can react with heme in fish cells to generate carbo-oxy heme).

TABLE 7 Effect of CORM-F3 and CORM-A1 shelf life (viable count) of silver carp (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	1 day	3 days	7 days
					Control	0.15±0.3	2.3±0.7	37±2.5	189±23
0.05mM. CO treatment	-----	2.1±0.6	26±2.9	120±11
					0.1mM. CO treatment	-----	1.3±0.5	9.8±2.7	50±5.8
0.2mM. CO treatment	-----	0.4±0.2	1.5±0.6	7.8±1.4
					0.5mM. CO treatment	-----	0.3±0.2	1.3±0.4	4.5±0.5

Example 10 Effect of gaseous CO on the shelf-Life of Cray

Fresh crayfish is placed in a plastic box and sterile normal saline is added to just immerse the crayfish in water. One set was filled with CO gas to saturation (saturation concentration of CO in water at 25 ℃ C. was 1mM), the other set was not added, and then the cover was closed, and the mixture was stored in a refrigerator at 4 ℃ for 2 days, 7 days, and 15 days, and then the total number of bacteria in the water was measured by sampling, respectively, and the results are shown in Table 8. As can be seen, the shelf life of the crayfish can be prolonged remarkably after the CO treatment.

TABLE 8 Effect of CO gas Cray shelf life (viable count) (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	2 days	7 days	15 days
					CO treatment	-----	6.5±0.7	18±2.2	52±4.0
Control	3.7±0.6	23±0.8	189±32	765±64

Examples 11 to 12 were processed as follows:

fresh aquatic products are put in plastic boxes in duplicate (identical in size and equal in weight), sterile normal saline is added to immerse the aquatic products in water, one part of the aquatic products is filled with CO or a CO release reagent, the other part of the aquatic products is not treated, the aquatic products are taken out after being treated for a certain time under the same conditions, the aquatic products are put in a freshness protection bag and stored by a conventional method, after the aquatic products are stored for a certain time, 0.2V (volume of the freshness protection bag) of normal saline is injected, the aquatic products are shaken for 10min in a shaking table at 150rpm, bacteria in the normal saline are coated on an LB flat plate by a dilution coating method, and the bacteria are counted after being cultured for 48 hours.

Example 11 Effect of ALF021 on the shelf life of prawn

Fresh prawns are placed in a plastic box, and sterile normal saline is added to immerse the prawns in water. One group was filled with the CO release formulation ALF021 in a plastic tank to a CO concentration of 1mM. in water, and the other group was not filled. After 10 minutes of treatment, the prawns were fished out, placed in a freshness protection package, stored in a refrigerator at 4 ℃, and after 2 days, 7 days, and 15 days, one portion was taken out, and 0.2V volume (volume of the freshness protection package) of physiological saline was injected thereto, shaken in a shaker at 150rpm for 10 minutes, and the total number of bacteria in the physiological saline was measured, and the results are shown in table 9.

TABLE 9 Effect of ALF021 on the shelf life (viable count) of prawn (× 10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	2 days	7 days	15 days
					CO treatment	-----	0.9±0.4	4.0±1.5	16±2.2
Control	0.2±0.1	1.7±0.5	27±3.2	106±15

Example 12 Effect of gaseous CO on shelf-Life of Loligo chinensis Gray

Putting fresh Loligo chinensis Gray into plastic box, adding sterile normal saline, introducing gas CO into the plastic box to make CO concentration in water 0.2mM., and adding no other group. Soaking the squids in water respectively, treating for 60 minutes, taking out the squids in the two groups, putting the squids in freshness protection bags respectively, hermetically storing the squids in a refrigerator at 4 ℃, taking one part after 2 days, 7 days and 15 days, injecting 0.2V (volume of the freshness protection bag) of normal saline, shaking in a shaking table at 150rpm for 10min, and determining the total number of bacteria in the normal saline, wherein the results are shown in Table 10.

TABLE 10 influence of CO gas on shelf life (viable count) of Loligo chinensis Gray (. times.10)⁶cfu/mL)

Retention time	Day 0 (before treatment)	2 days	7 days	15 days
					CO treatment	------	0.7±0.2	6.6±2.1	47±9.0
Control	0.15±0.2	2.5±0.8	38±3.1	157±38