A method for enriching vitamin a compounds from mussel viscera

Abstract

The application discloses a method for enriching vitamin A compounds from a mussel viscera group, and comprises the following steps: raw material pretreatment: after vacuum freeze-drying of mussel viscera and gonad, the mussel viscera and gonad are ground into powder as raw material; pectinase enzyme solution is added into the raw material for enzymolysis; after the enzymolysis is completed, inactivation is carried out, and then anhydrous ethanol is added for ultrasonic extraction; after filtration, suction filtration is carried out, supernatant is collected, and a crude extract is obtained; the crude extract is added into pretreated macroporous resin for adsorption treatment, and the macroporous resin after adsorption is collected; finally, an eluent is added for elution; the obtained eluent is freeze-dried to obtain a vitamin A compound product. The application is a method for efficiently, simply separating and purifying vitamin A compounds from mussel waste, the extract has good liver protection function, has important application potential in the food industry, and provides a new idea for diversified development of mussel resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of marine bioengineering, in particular to a method for extracting and purifying vitamin A compounds from the viscera of shellfish. BACKGROUND

[0002] Scallop belongs to Mollusca, and is an important shellfish breeding variety in China, mainly distributed in various sea areas of China. In recent years, the scallop breeding scale in China has gradually expanded, and the scallop processing amount has also increased. According to the China Fishery Statistical Yearbook, the scallop breeding yield in China in 2021 reached 1,829,924 tons. A large amount of low-value by-products are generated in the production and processing of scallop column products, which have not been effectively developed and utilized, causing resource waste and environmental pollution. Therefore, the high-value development of scallop resources has become an important research direction for the sustainable utilization of marine resources. Studies have shown that scallops are a good source of vitamin A compounds, but there are only sporadic research reports on the determination of vitamin A compounds in shellfish. In addition, scallop waste contains various nutrients such as fatty acids and taurine.

[0003] Therefore, it is of great significance to develop and utilize the nutritional elements in scallop and other shellfish waste. SUMMARY

[0004] The purpose of the present application is to provide a method for enriching vitamin A compounds from the viscera of shellfish, in order to make up for the shortcomings of the prior art.

[0005] To achieve the above purpose, the specific technical scheme adopted by the present application is as follows:

[0006] A method for enriching vitamin A compounds from the viscera of shellfish, comprising the following steps:

[0007] (1) Raw material pretreatment: the viscera and gonads of shellfish are vacuum freeze-dried and ground into powder as raw materials;

[0008] (2) Enzymatic hydrolysis of the above-mentioned raw materials by adding pectinase enzyme solution;

[0009] (3) After the enzymatic hydrolysis is completed, inactivation is carried out, and then anhydrous ethanol is added for ultrasonic extraction. After filtration, suction filtration is carried out, and the supernatant is collected to obtain a crude extract;

[0010] (4) Pre-treated macroporous resin is added to the crude extract for adsorption treatment, and the macroporous resin after adsorption is collected;

[0011] (5) Finally, an eluent is added for elution, and the obtained eluate is freeze-dried to obtain a vitamin A compound product.

[0012] Further, in the step (2), the ratio of the raw material to the enzyme liquid is 1:3-1:8; the enzyme liquid concentration of the pectinase is 20-50 U / mL; the enzymolysis time is 2-4 h; and the enzymolysis temperature is 45-55 DEG C.

[0013] Further, in the step (3), the inactivation temperature of the enzymolysis liquid is 75-85 DEG C, and the inactivation time is 5-10 min.

[0014] Further, in the step (3), the volume ratio of the total volume of the enzymolysis liquid to the ethanol extraction is 1:8-1:15; and the extraction is performed at least three times.

[0015] Further, in the step (3), the ultrasonic power is 80-120 W, the ultrasonic extraction temperature is 30-40 DEG C, the ultrasonic extraction time is 0.5 h; the ultrasonic extraction is performed three times; and the supernatant is collected by combining the filtration and the suction filtration.

[0016] Further, in the step (4), the macroporous resin is M0041 macroporous resin, the volume ratio of the macroporous resin to the adsorption solution is 1:4-1:6; and the oscillation rate of the macroporous resin in the adsorption process of the supernatant is 100-150 rpm, and the adsorption time is 4 h.

[0017] Further, in the step (4), the pretreatment of the macroporous resin is as follows: firstly, 95% ethanol solution is used for soaking for 24 h, the amount is about twice the amount of the macroporous resin to be treated, after being sufficiently swelled, the macroporous resin is washed with deionized water until the colorless solution is discharged; then, 5% hydrochloric acid solution and 5% sodium hydroxide solution are used for soaking for 12 h respectively, the macroporous resin is washed with deionized water, and the pH value is measured by using pH paper until the pH value approaches to neutral, and the macroporous resin is stored in deionized water.

[0018] Further, in the step (5), in the elution process, the macroporous resin is eluted by using 2 times the volume of anhydrous ethanol, 20% ethyl acetate-ethanol solution and 40% ethyl acetate-ethanol solution respectively, the filtrate is collected, and the freeze-drying is performed after the rotary evaporation.

[0019] The vitamin A compound includes retinol, retinol palmitate and beta-carotene.

[0020] The application of the vitamin A compound enriched in the mussel viscera group in the liver protection product.

[0021] The advantages and beneficial effects of the application are as follows:

[0022] The application takes the viscera and gonad of shellfish as raw materials, and uses enzymatic hydrolysis and macroporous resin to extract and purify vitamin A compounds, the retinol, retinol palmitate and beta-carotene yield is respectively 121.96 μg / 100 g, 280.15 μg / 100 g, 679.07 μg / 100 g; the extraction rate is respectively 85.14%, 80.02%, 96.60%; and the purification multiple is respectively 38.73 times, 30.48 times, 32.63 times.

[0023] The application is a kind of efficient, simple method for separating and purifying vitamin A compounds from shellfish waste, and the extract has good liver protection function, which has important application potential in the food industry, provides a new idea for the diversified development of shellfish resources, and is beneficial to promote the rapid development of green high-value development industry of shellfish resources. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a different macroporous resin for vitamin A compound standard desorption capacity comparison chart in embodiment 1 of the application.

[0025] Figure 2 It is a desorption capacity comparison chart of three kinds of macroporous resins screened in embodiment 2 of the application for comparing the sample of vitamin A compound.

[0026] Figure 3 It is a chart of the influence of different eluent volume fractions on desorption in embodiment 3 of the application.

[0027] Figure 4 It is a comparison chart of the content of vitamin A compounds obtained by directly using ethanol extraction and using enzymatic hydrolysis extraction in embodiment 4 of the application with scallop viscera and gonad as raw materials.

[0028] Figure 5 It is the liver protection activity of the product extracted in embodiment 4 of the application Figure 1 .

[0029] Figure 6 It is the liver protection activity of the product extracted in embodiment 4 of the application Figure 2 . IMPLEMENTATION

[0030] The application is further explained and described below through specific embodiments and in combination with the drawings. EMBODIMENT 1

[0031] Different macroporous resins desorption experiment of vitamin A compound standard:

[0032] (1) Preparation of standard solution, using anhydrous ethanol to prepare retinol, retinol acetate, retinol palmitate, all-trans-retinal, retinoic acid, β-carotene concentration of 50 μg / mL mixed standard solution.

[0033] (2) Macroporous resin pretreatment, select HP20, HPD750, M0041, M0042, M0073, M0069, M0032, M0058 macroporous resin, the same pretreatment method, first use 95% ethanol solution immersion 24 h, the amount is equal to the macroporous resin to be treated twice, fully swollen with deionized water to wash, is discharged for colorless for, then use 5% hydrochloric acid solution and 5% sodium hydroxide solution each immersion 12 h, respectively, macroporous resin with deionized water, use pH paper to measure its pH value until close to neutral, save in deionized water.

[0034] (3) each macroporous resin dosage 5.0 g, respectively, put in 15 mL of various standard 50 μg / mL mixed ethanol solution for adsorption experiment, 130 rpm speed under the shock 37 ℃ adsorption 4 h, after the completion of the adsorption experiment, filter, take out the macroporous resin added to 15 mL of ethyl acetate, in 130 rpm speed under the shock 37 ℃ desorption 4 h, after the completion of the desorption experiment, filter, measure the concentration of each compound in the desorption liquid (μg / mL).

[0035] Results analysis: as shown in Figure 1 M0041, M0042, M0073, three kinds of macroporous resin on vitamin A compound adsorption and desorption have a good effect. So choose these three kinds of resin for the detection of vitamin A compound in scallop viscera enrichment. Example 2

[0036] Select three kinds of macroporous resin (M0041, M0042, M0073) for desorption experiment of vitamin A compound in sample:

[0037] Sample preparation experiment: collect the inner organs and gonad of chlamys hastata, vacuum freeze-dried and ground into powder.

[0038] Take 5 g sample for extraction, use 100 mL ethanol ultrasonic extraction for three times, then use 5 g of M0041, M0042, M0073 macroporous resin into the extraction liquid for purification, after the completion of the adsorption process, use deionized water to wash the impurities, then use ethanol and ethyl acetate elution, respectively, collect the eluent, rotary evaporation to constant volume to 5 mL, liquid phase detection to calculate the content.

[0039] Results analysis: by Figure 2It can be seen that the effect of M0041 macroporous resin on the enrichment of vitamin A compounds in scallop viscera is the best, and the contents of retinol, retinol palmitate and beta-carotene are 74.43 μg / 100 g, 221.39 μg / 100 g and 457.46 μg / 100 g respectively. Therefore, M0041 macroporous resin is selected as the extraction material for subsequent experiments. Example 3

[0040] Effect of different eluent volume fractions on desorption experiment:

[0041] 5 g of sample was taken for extraction, and 100 mL of ethanol was used for ultrasonic extraction for three times. After filtration, suction filtration was performed, and then 5 g of M0041 macroporous resin was put into the extraction liquid for purification. After the adsorption process was completed, the impurities were washed with deionized water, and then different volume fractions of eluent were used for elution in turn. Each eluent was rotary evaporated to 5 mL, and the content was calculated by liquid phase detection.

[0042] Result analysis: from Figure 3 It can be seen that when different volume fractions of ethyl acetate-ethanol eluent are used in turn, the target compounds can be completely eluted from the macroporous resin when 40% ethyl acetate-ethanol eluent is used. Example 4

[0043] Using scallop viscera and gonad as raw materials, the content of vitamin A compounds obtained by using enzyme hydrolysis method on the raw materials and then combining with ethanol ultrasonic extraction method was explored:

[0044] 5 g of sample was taken, 25 mL of 30 U / mL pectinase enzyme solution was added, and then enzyme hydrolysis was carried out at 50℃ for 2.5 h. After that, the enzyme hydrolysate was inactivated at 80℃ for 5 min, and then anhydrous ethanol was added for ultrasonic extraction for three times. After each suction filtration, the supernatant was collected. M0041 macroporous resin was added to the crude extract for adsorption treatment. The macroporous resin adsorbed with vitamin A compounds was collected, packed into a column, and then washed with deionized water until the filtrate was colorless. Then, anhydrous ethanol, 20% ethyl acetate-ethanol eluent and 40% ethyl acetate-ethanol eluent were added in turn for elution. Finally, the obtained eluent was concentrated, freeze-dried, and the product was obtained.

[0045] According to the method of Example 4, the content of retinol in the extraction product was 4723.99 μg / 100 g, the content of retinol palmitate was 8537.93 μg / 100 g, and the content of beta-carotene was 22156.14 μg / 100 g.

[0046] Result analysis: from Figure 4As shown in Table 1, the results show that the contents of retinol, retinol palmitate and β-carotene extracted by the non-enzyme method are 70.32 μg / 100 g, 239.58 μg / 100 g and 527.62 μg / 100 g, respectively, and the contents of retinol, retinol palmitate and β-carotene in the sample raw material are 121.96 μg / 100 g, 280.15 μg / 100 g and 679.07 μg / 100 g, respectively, and the contents of retinol, retinol palmitate and β-carotene in the product obtained after enrichment by using the macroporous resin are 4723.99 μg / 100 g, 8537.93 μg / 100 g and 22156.14 μg / 100 g, respectively, and the enrichment fold of the purified product is 38.73, 30.48 and 32.63, respectively.

[0047] Table 1 Comparison of the extraction and enrichment contents of vitamin A group

[0048] Category Raw material content (pg / 100 g) Extracted content (pg / 100 g) Enriched content (pg / 100 g) Retinol 143.25 121.96 4723.99 Retinol palmitate 350.11 280.15 8537.93 Beta-carotene 702.96 679.07 22156.14 Example 5

[0049] Hepatoprotective activity test of the product:

[0050] The logarithmic growth phase HepG2 cells 1*10 4 single cell suspension was inoculated in a 96-well plate at 100 μL per well, and after 24 h of culture in the incubator, ethanol was added to prepare a culture medium with a concentration of 500 mM ethanol, and after 24 h of continuous culture, the culture medium was discarded. The product was prepared according to the method of Example 4, and the culture medium containing different concentrations of the product was added, and after 24 h of culture, MTT experiment was performed.

[0051] Results analysis: As Figure 5 shown in the results, the product has the effect of promoting the growth of HepG2 cells, and the concentration of 50 μg / ml has the maximum growth-promoting effect on the cells. The results show that the vitamin A compound product extracted from the scallop viscera and gonad has a significant growth-promoting effect on alcohol-induced liver injury cells. Figure 6 The results show that the product has a good intervention effect on alcohol-induced liver injury.

[0052] The above examples take the viscera and gonad by-product of Chlamys hirundo processing as raw material, and prepare retinol, retinol palmitate and beta-carotene through enzymolysis and macroporous resin purification processes. The present application can fully utilize the vitamin A compounds in the Chlamys hirundo and other shellfish wastes, and improve the added value of the shellfish products.

Claims

1. A method for enriching vitamin A group compounds from the viscera mass of shellfish, characterized in that, The method comprises the following steps: (1) raw material pretreatment: the viscera and gonad of shellfish are vacuum freeze-dried, ground into powder, and used as raw materials; (2) adding pectinase enzyme solution to the raw materials for enzymolysis; (3) after the enzymolysis is completed, inactivation is performed, and then anhydrous ethanol is added for ultrasonic extraction, filtration, and suction filtration, and the supernatant is collected to obtain a crude extract; (4) adding pretreated macroporous resin to the crude extract for adsorption treatment, and collecting the macroporous resin after adsorption; (5) finally, an eluent is added for elution, and the obtained eluate is freeze-dried to obtain a vitamin A compound product.

2. The method of claim 1, wherein, In step (2), the ratio of the raw materials to the enzyme solution is 1:3-1:8; the pectinase enzyme solution has a concentration of 20-50 U / mL; the enzymolysis time is 2-4 h; and the enzymolysis temperature is 45-55°C.

3. The method of claim 1, wherein, In step (3), the inactivation temperature of the enzyme solution is 75-85°C, and the inactivation time is 5-10 min.

4. The method of claim 1, wherein, In step (3), the total volume ratio of the enzyme solution to the ethanol extraction is 1:8-1:15; and the extraction is performed at least three times.

5. The method of claim 1, wherein, In step (3), the ultrasonic power is 80-120 W, the ultrasonic extraction temperature is 30-40°C, the ultrasonic extraction time is 0.5 h, and the ultrasonic extraction is performed three times; and the supernatant is collected by combining filtration and suction filtration.

6. The method of claim 1, wherein, In step (4), the macroporous resin is M0041 macroporous resin, the volume ratio of the macroporous resin to the adsorption solution is 1:4-1:6, and the oscillation rate of the macroporous resin during the adsorption process of the supernatant is 100-150 rpm, and the adsorption time is 4 h.

7. The method of claim 1, wherein, In step (4), the pretreatment of the macroporous resin is as follows: first, the macroporous resin is soaked in 95% ethanol solution for 24 h, and the amount of the ethanol solution is about twice the amount of the macroporous resin to be treated; after being fully swollen, the macroporous resin is washed with deionized water until the effluent is colorless; then, the macroporous resin is soaked in 5% hydrochloric acid solution and 5% sodium hydroxide solution for 12 h respectively, and then washed with deionized water; the pH value of the macroporous resin is measured by using pH paper until it approaches neutral; and finally, the macroporous resin is stored in deionized water.

8. The method of claim 1, wherein, In step (5), during the elution process, the macroporous resin is eluted with 2 times the volume of anhydrous ethanol, 20% ethyl acetate-ethanol solution, and 40% ethyl acetate-ethanol solution respectively, the filtrate is collected, and then rotary evaporation and freeze-drying are performed.

9. The use of the vitamin A compound-enriched viscera group of shellfish in claim 1 in the preparation of a liver protection product.

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