A method for removing cadmium from squid viscera enzymatic hydrolysate

Abstract

The present invention provides a method for removing cadmium from a squid viscera enzymatic hydrolysate, and relates generally to the technical field of aquatic product processing. The method for removing cadmium from a squid viscera enzymatic hydrolysate provided by the present invention comprises the following steps: after crushing the fresh squid viscera, adding an enzyme preparation of 0.05%-0.5% by weight of the squid viscera for enzymolysis, sterilizing the squid viscera enzymatic hydrolysate in sequence, cooling, and centrifuging to obtain supernatant A, adding 2 times the volume of anhydrous ethanol to the supernatant A, collecting the supernatant B by centrifugation, and recovering the ethanol to obtain supernatant C, ultrasonically treating the supernatant C to obtain supernatant D, adding activated carbon to the supernatant D, filtering to obtain supernatant E, adding sodium phosphate to the supernatant E, and filtering to obtain supernatant F by chelation decadmium, and adsorbing and removing cadmium from the supernatant F to obtain decadmium-depleted squid viscera enzymatic hydrolysate G. The present invention provides a method for removing heavy metal cadmium from squid viscera, which has a high removal rate for heavy metal cadmium in squid viscera.

Description

Technical Field

[0001] The invention mainly relates to the technical field of aquatic product processing, and in particular to a method for removing cadmium from squid viscera enzymatic hydrolysate. Background Art

[0002] Squid is a globally highly produced marine bioresource. Squid viscera are the primary by-product of squid processing, accounting for approximately 15.0% of the total squid weight. Squid viscera are highly nutritious, comprising 15%-20% protein, 10%-15% lipids, and 2%-5% crude polysaccharides. Furthermore, squid viscera are rich in polysaccharides, taurine, flavor-forming amino acids, and other trace elements, offering broad development prospects.

[0003] Squid viscera are rich in protein and flavor-enhancing amino acids and can be processed into aquatic feeds such as squid paste and fish meal. They can also be made into natural condiments such as squid sauce through enzymatic fermentation. Reports indicate that squid sauce is already being produced and sold in Spain and Japan. While there are reports of squid sauce in domestic literature, actual production in China is still unavailable. This is largely due to excessive levels of the heavy metal cadmium in squid viscera. The market for feed-grade squid paste has been shrinking in recent years, largely due to the high levels of heavy metal cadmium in squid viscera. Cadmium can inhibit mitochondrial oxidative phosphorylation in liver cells, disrupting tissue metabolism and causing mutagenic effects in humans and animals. Cadmium-contaminated aquatic products enter the human body through the digestive tract and accumulate, potentially causing a variety of diseases. Similarly, heavy metal cadmium in feed can pose a significant threat to the livestock and poultry industry. Therefore, excessive levels of heavy metal cadmium in squid viscera have limited its use in food and feed applications, creating a technical bottleneck for the comprehensive processing and high-value development and utilization of squid viscera.

[0004] Prior art reports have used chitosan, chelating agents, and adsorption resins to remove cadmium from squid viscera and other aquatic products, but these methods only address the removal of free cadmium. Patent No. CN112273598 discloses a method for removing heavy metal cadmium from squid viscera, reporting the conversion of bound cadmium in squid viscera to free cadmium through enzymatic hydrolysis and the use of D401 resin for cadmium removal. The inventors believe that the claim that enzymatic hydrolysis can convert bound cadmium to free cadmium is inaccurate. The present invention provides a practical method for removing cadmium from squid enzymatic hydrolysates, offering a path to high-value, comprehensive processing of squid viscera. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides a method for removing cadmium from squid viscera enzymatic hydrolysate, which is achieved through the following technical solutions:

[0006] The present invention provides a method for removing cadmium from squid viscera enzymatic hydrolysate, comprising the following steps:

[0007] (1) Enzymatic hydrolysis: After crushing the fresh squid viscera, add 0.05%-0.5% of the weight of the squid viscera enzyme preparation for enzymatic hydrolysis. No exogenous acid or alkali is required to adjust the pH during the enzymatic hydrolysis process. After enzymatic hydrolysis, the squid viscera enzymatic hydrolysate is obtained.

[0008] (2) High-speed centrifugation: The squid viscera hydrolysate is sterilized, cooled, and centrifuged in sequence to obtain supernatant A, crude oil, and residue. The oil and residue are collected and processed separately, and the supernatant A enters the subsequent processing steps.

[0009] (3) Alcohol precipitation: Add 2 times the volume of anhydrous ethanol to the supernatant A, let it stand at 5-10°C for 12 hours, then centrifuge at 6000 rpm for 10 minutes, collect the supernatant B, and recover the ethanol to obtain supernatant C.

[0010] (4) Ultrasonic treatment: The supernatant C is ultrasonically treated at a frequency of 25-60 kHz and a temperature of 45-60°C for 30-60 minutes to obtain supernatant D.

[0011] (5) Decolorization by activated carbon: Add 0.3%-0.5% by weight of activated carbon to the supernatant D, decolorize the cadmium at 60-80°C for 30 minutes, and then filter with a plate and frame filter to obtain supernatant E.

[0012] (6) Sodium phosphate chelation precipitation: add 0.01-0.05% by weight of sodium phosphate to the supernatant E, chelate and remove cadmium at 50-60°C for 30 minutes, and then filter using a plate and frame filter to obtain supernatant F.

[0013] (7) Resin adsorption: The supernatant F was pumped into a D751 macroporous chelating resin column for adsorption and cadmium removal to obtain cadmium-depleted squid viscera hydrolysate G.

[0014] Preferably, the enzyme preparation is a composite protease obtained by compounding neutral protease, trypsin and flavor protease in a weight ratio of 2:1:2.

[0015] Preferably, the enzymolysis temperature is 50° C.-60° C., and the enzymolysis time is 6-8 hours.

[0016] Preferably, the sterilization is carried out at 100° C. for 5-10 minutes.

[0017] Preferably, the cooling is to cool the squid viscera hydrolysate after enzyme inactivation to below 55° C., and the centrifugal condition is high-speed centrifugation at 8000 rpm for 10 minutes.

[0018] Preferably, during the alcohol precipitation, the supernatant B is subjected to a vacuum degree of 0.06 MPa and a temperature of 60° C. to recover ethanol.

[0019] Preferably, during the chelating decadmium treatment, the mixture is first stirred continuously for 20 minutes and then allowed to stand for 10 minutes.

[0020] Preferably, after the resin adsorption is completed, the adsorption resin column is regenerated using distilled water, 1 mol / L HCl, and 1 mol / L NaOH.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. High-speed centrifugal separation process: 8000rpm high-speed centrifugal separation is used to achieve efficient separation of oil and fat in the enzymatic hydrolysate, and control the oil content in the supernatant A to be no higher than 0.5%, so as to avoid the contamination of the adsorption resin by oil in the later stage.

[0023] 2. Add an alcohol precipitation process to improve cadmium removal: Ethanol is added to the supernatant A of the squid viscera enzymatic hydrolysis solution to partially remove cadmium by removing the polysaccharide components in the supernatant through alcohol precipitation. Both the polysaccharides and proteins in the squid viscera form bound cadmium with cadmium. Enzymatic hydrolysis can convert and remove bound cadmium from proteins, but it cannot remove bound cadmium from crude polysaccharides. Therefore, it is necessary to add an alcohol precipitation process to remove the crude polysaccharides from the squid enzymatic hydrolysate and reduce the bound cadmium content in supernatant A. Furthermore, removing the crude polysaccharides through alcohol precipitation reduces the problem of adsorption resin clogging that can occur during adsorption cadmium removal, thereby improving the cadmium removal efficiency and regeneration rate of the adsorption column.

[0024] 3. Add ultrasonic treatment to promote the decomposition of cadmium from peptides and amino acids: Enzymatic hydrolysis technology degrades large protein molecules into small peptides and free amino acids, making it possible to convert bound cadmium into free cadmium. Ultrasonic treatment can disrupt the stability of cadmium binding to peptides and amino acids through high-frequency vibration and ultrasonic cavitation, achieving efficient decomposition of cadmium, converting bound cadmium into free cadmium, and facilitating its subsequent removal.

[0025] 4. Three-step synergistic cadmium removal technology improves the cadmium removal effect: first, activated carbon treatment is used to remove some free cadmium through adsorption. At the same time, activated carbon can also adsorb most of the pigments in the supernatant D to protect the adsorption resin; secondly, sodium phosphate can form a stable cadmium phosphate precipitate with the cadmium ions in the supernatant E and remove it by centrifugation; finally, D751 macroporous chelating resin is used to further adsorb and remove free cadmium ions in the supernatant F, thereby achieving efficient removal of bound cadmium and free cadmium in the squid enzymatic hydrolysate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for removing cadmium from squid viscera enzymatic hydrolysate, comprising the following steps:

[0028] (1) Enzymatic hydrolysis: After crushing the fresh squid viscera, add 0.05%-0.5% of the weight of the squid viscera enzyme preparation for enzymatic hydrolysis. No exogenous acid or alkali is required to adjust the pH during the enzymatic hydrolysis process. After enzymatic hydrolysis, the squid viscera enzymatic hydrolysate is obtained.

[0029] (2) High-speed centrifugation: The squid viscera hydrolysate is sterilized, cooled, and centrifuged in sequence to obtain supernatant A, crude oil, and residue. The oil and residue are collected and processed separately, and the supernatant A enters the subsequent processing steps.

[0030] (3) Alcohol precipitation: Add 2 times the volume of anhydrous ethanol to the supernatant A, let it stand at 5-10°C for 12 hours, then centrifuge at 6000 rpm for 10 minutes, collect the supernatant B, and recover the ethanol to obtain supernatant C.

[0031] (4) Ultrasonic treatment: The supernatant C is ultrasonically treated at a frequency of 25-60 kHz and a temperature of 45-60°C for 30-60 minutes to obtain supernatant D.

[0032] (5) Decolorization by activated carbon: Add 0.3%-0.5% by weight of activated carbon to the supernatant D, decolorize the cadmium at 60-80°C for 30 minutes, and then filter with a plate and frame filter to obtain supernatant E.

[0033] (6) Sodium phosphate chelation precipitation: add 0.01-0.05% by weight of sodium phosphate to the supernatant E, chelate and remove cadmium at 50-60°C for 30 minutes, and then filter using a plate and frame filter to obtain supernatant F.

[0034] (7) Resin adsorption: The supernatant F was pumped into a D751 macroporous chelating resin column for adsorption and cadmium removal to obtain cadmium-depleted squid viscera hydrolysate G.

[0035] The enzyme preparation is a composite protease prepared by compounding neutral protease, trypsin and flavor protease in a weight ratio of 2:1:2. The purpose of the enzymatic hydrolysis of the present invention is mainly to improve the enzymatic hydrolysis effect and increase the relative content of small molecular peptides and free amino acids in the enzymatic hydrolysis solution, so as to separate heavy metal cadmium ions from macromolecular proteins.

[0036] The enzymolysis temperature is 50° C.-60° C., and the enzymolysis time is 6-8 hours.

[0037] The sterilization is carried out at 100° C. for 5-10 minutes.

[0038] The cooling step is to cool the squid viscera hydrolysate after enzyme inactivation to below 55° C., and the centrifugal condition is high-speed centrifugation at 8000 rpm for 10 minutes.

[0039] During the alcohol precipitation, the supernatant B is precipitated at a vacuum degree of 0.06 MPa and a temperature of 60° C. to recover ethanol.

[0040] During the chelate decadmium treatment, the mixture was first stirred continuously for 20 minutes and then allowed to stand for 10 minutes.

[0041] After the resin adsorption is completed, the adsorption resin column is regenerated using distilled water, 1 mol / L HCl, and 1 mol / L NaOH.

[0042] Example 1: 2 kg of fresh squid viscera were taken, crushed with a beater, transferred to a triangular flask, heated to 95°C, maintained for 5 minutes for sterilization, then cooled to 60°C, 3 g of composite protease was added, and enzymatic hydrolysis was carried out at 60°C for 8 hours. The enzymatic hydrolyzate was heated to 95°C for 5 minutes to inactivate the enzyme, and then centrifuged at 8000 rpm for 10 minutes. Supernatant A, crude oil, and residue were collected separately to obtain 1600 g of supernatant A; supernatant A was cooled to below 25°C, and 3200 mL of anhydrous ethanol was added, and the mixture was allowed to stand at 5°C for 12 hours, and then centrifuged at 6000 rpm for 10 minutes to obtain supernatant B. 4750mL, the supernatant B was transferred to a rotary evaporator in batches, and the ethanol was recovered at a vacuum degree of 0.06MPa and a temperature of 60°C to obtain 1500g of supernatant C; the supernatant C was ultrasonically treated at 50°C and 40KHz for 40 minutes to obtain 1500g of supernatant D; 7.5g of activated carbon was added to the supernatant D, and the mixture was stirred at 70°C for 30 minutes, and then filtered with filter paper to obtain 1450g of supernatant E; adding 0.5 g of sodium phosphate to the supernatant E, maintaining stirring at 55° C. for 20 minutes, then allowing to stand for 10 minutes, and then filtering with filter paper to obtain 1400 g of supernatant F; slowly injecting the supernatant F into a D751 macroporous chelating resin column with a size of 30 mm×600 mm, controlling the flow rate to 0.5-1.0 L / h, and cyclically adsorbing for 3 times to finally obtain 1350 g of supernatant G, which is the squid viscera hydrolyzate after cadmium removal.

[0043] Example 2: 2 kg of fresh squid viscera were taken, crushed with a beater, transferred to a triangular flask, heated to 95°C, maintained for 5 minutes for sterilization, then cooled to 60°C, added with 5 g of composite protease, and enzymatically hydrolyzed at 55°C for 7 hours. The enzymatic hydrolyzate was heated to 95°C for 5 minutes to inactivate the enzyme, and then centrifuged at 8000 rpm for 10 minutes. Supernatant A, crude oil, and residue were collected separately to obtain 1620 g of supernatant A; supernatant A was cooled to below 25°C, and 3240 mL of anhydrous ethanol was added, and the mixture was allowed to stand at 10°C for 12 hours, and then centrifuged at 6000 rpm for 10 minutes to obtain supernatant B. 4850mL, supernatant B is moved in batches into the rotary evaporator, and under vacuum tightness 0.06MPa, temperature 60 ℃ reclaims ethanol to obtain 1550g of supernatant C; supernatant C is ultrasonically treated under 40 ℃, 50KHz conditions for 50 minutes to obtain 1550g of supernatant D; 5g of gac is added to the supernatant D, kept stirring at 80 ℃ for 30 minutes, then filtered with filter paper to obtain 1500g of supernatant E; 0.75g of sodium phosphate is added to the supernatant E, kept stirring at 55 ℃ for 20 minutes, then left standstill 10 minutes, then filtered with filter paper to obtain 1450g of supernatant F; supernatant F is slowly injected into the D751 macroporous chelating resin column with a size of 30mm * 600mm, and the controlled flow rate is 0.5-1.0L / h, and circulated adsorption is performed twice to obtain 1400g of decadmium supernatant G at last.

[0044] Example 3: 2000 kg of fresh squid viscera were pumped into an enzymolysis tank using a Roots pump, heated with a steam jacket, raised to 95°C, and maintained for 5 minutes for sterilization. The mixture was then cooled to 60°C using cold water in the jacket, 6 kg of composite protease was added, and enzymolysis was carried out at 60°C for 8 hours. The enzymolysis solution was heated to 95°C to inactivate the enzyme for 5 minutes, and then centrifuged at 8000 rpm using a disc centrifuge. Supernatant A, crude oil, and residue were collected to obtain 1600 kg of supernatant A. Supernatant A was cooled to 25°C, 3200 L of anhydrous ethanol was added, stirred evenly, and then allowed to stand at 8°C for 12 hours. The mixture was then centrifuged at 6000 rpm using a disc centrifuge to obtain supernatant B. 4650Kg, the supernatant B was pumped into the ethanol recovery tower, and the ethanol was recovered at a vacuum degree of 0.06MPa and a temperature of 60°C to obtain 1500Kg of supernatant C; the supernatant C was pumped into an ultrasonic tank and ultrasonically treated at 50°C and 50KHz for 60 minutes to obtain 1500Kg of supernatant D; 7.5Kg of activated carbon was added to the supernatant D, and the mixture was stirred at 80°C for 30 minutes, and then filtered with a plate and frame filter to obtain To 1460Kg of supernatant E; 700g of sodium phosphate was added to the supernatant E, the mixture was kept at 55°C and stirred for 20 minutes, then allowed to stand for 10 minutes, and then filtered with a plate and frame filter to obtain 1430Kg of supernatant F; the supernatant F was slowly injected into a D751 macroporous chelating resin column with a size of 50cm×300cm×6 groups, the flow rate was controlled to be 0.5-1m3 / h, and adsorbed once to finally obtain 1400Kg of cadmium-removed supernatant G.

[0045] Samples of supernatant A, supernatant C, supernatant E, supernatant F, and supernatant G during the above treatment were taken and tested for cadmium content. The specific results are shown in Table 1:

[0046] Table 1 Changes in cadmium content in squid viscera enzymatic hydrolysate

[0047]

[0048] It can be seen from the above examples that Example 1, Example 2 and Example 3 can all achieve efficient cadmium removal from squid viscera hydrolysate. Among them, the supernatant F in Example 1 was circulated and adsorbed three times by a D751 macroporous chelating resin column, and the cadmium content in the supernatant G already met the food hygiene requirements; the supernatant F in Example 2 and Example 3 was circulated and adsorbed 2 times and 1 time by a D751 macroporous chelating resin column, respectively, and the cadmium content in the supernatant G already met the standard requirements for feed and fertilizer. The cadmium removal experimental results show that the cadmium removal technology of the present invention can effectively remove heavy metal cadmium from squid viscera hydrolysate.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for removing cadmium from squid viscera enzymatic hydrolysate, characterized in that: The steps include: (1) Enzymatic hydrolysis: After crushing the fresh squid viscera, add 0.05%-0.5% of the weight of the squid viscera enzyme preparation for enzymatic hydrolysis. No exogenous acid or alkali is required to adjust the pH during the enzymatic hydrolysis process. After enzymatic hydrolysis, the squid viscera enzymatic hydrolysate is obtained; (2) High-speed centrifugation: The squid viscera hydrolysate is sterilized, cooled, and centrifuged in sequence to obtain supernatant A, crude oil, and residue. The oil and residue are collected and processed separately, and the supernatant A enters the subsequent processing steps; (3) Alcohol precipitation: add 2 volumes of anhydrous ethanol to the supernatant A, let it stand at 5-10°C for 12 hours, then centrifuge at 6000 rpm for 10 minutes, collect the supernatant B, and recover the ethanol to obtain supernatant C; (4) Ultrasonic treatment: Ultrasonic treatment of supernatant C at a frequency of 25-60 kHz and a temperature of 45-60°C for 30-60 minutes to obtain supernatant D; (5) Activated carbon decolorization: Add 0.3%-0.5% by weight of activated carbon to the supernatant D, decolorize the cadmium at 60-80°C for 30 minutes, and then filter with a plate and frame filter to obtain supernatant E; (6) Sodium phosphate chelation precipitation: add 0.01-0.05% by weight of sodium phosphate to the supernatant E, incubate at 50-60°C for 30 minutes to remove cadmium by chelation, and then filter using a plate and frame filter to obtain supernatant F; (7) Resin adsorption: The supernatant F was pumped into a D751 macroporous chelating resin column for adsorption and cadmium removal to obtain cadmium-depleted squid viscera enzymatic hydrolysate G; The enzyme preparation is a composite protease obtained by compounding neutral protease, trypsin and flavor protease in a weight ratio of 2:1:2; The enzymatic hydrolysis temperature is 50°C-60°C, and the enzymatic hydrolysis time is 6-8 hours; The sterilization is carried out at 100°C for 5-10 minutes; The cooling step is to cool the squid viscera hydrolysate after enzyme inactivation to below 55° C., and the centrifugal condition is high-speed centrifugation at 8000 rpm for 10 minutes.

2. The method for removing cadmium from a squid viscera enzymatic hydrolysate according to claim 1, wherein: During the alcohol precipitation, the supernatant B is precipitated at a vacuum degree of 0.06 MPa and a temperature of 60° C. to recover ethanol.

3. The method for removing cadmium from a squid viscera enzymatic hydrolysate according to claim 1, wherein: During the chelate decadmium treatment, the mixture was first stirred continuously for 20 minutes and then allowed to stand for 10 minutes.

4. The method for removing cadmium from a squid viscera enzymatic hydrolysate according to claim 1, wherein: After the resin adsorption is completed, the adsorption resin column is regenerated using distilled water, 1 mol / L HCl, and 1 mol / L NaOH.

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