Process for extracting lycopene and polysaccharide from tomato pomace and membrane material prepared from lycopene and polysaccharide

Abstract

The invention relates to a process for extracting lycopene and polysaccharide from tomato pomace and a membrane material prepared from the same, and the process comprises the following steps: S1, uniformly spreading a certain mass of tomato pomace in a dryer for drying, crushing the tomato pomace dried to constant weight, and sieving with a 90-mesh standard sieve to prepare tomato pomace powder; s2, enzyme-assisted aqueous two-phase system extraction: adding the tomato peel residue powder into a disodium hydrogen phosphate-citric acid buffer solution, then adding neutral protease, uniformly mixing, and carrying out enzymolysis in a water bath at 45 DEG C for 60 minutes; and after the enzymolysis reaction is finished, purifying and drying to obtain lycopene and polysaccharide in the tomato pomace. The influence factors of the yield of the lycopene and the polysaccharide in the tomato pomace are explored through a single-factor test, the process conditions for extracting the yield of the lycopene and the polysaccharide are preliminarily obtained by means of an enzyme-assisted aqueous two-phase extraction method, and then the optimal process conditions are obtained through orthogonal test optimization. Finally, the optimal process is determined as follows: the mass fraction of ethyl acetate is 35%, the mass fraction of dipotassium phosphate is 14%, the enzyme activity is 2000U, and the pH value is 7.

Description

Technical Field

[0001] This invention belongs to the field of biological feed technology, and in particular to a process for extracting lycopene and polysaccharides from tomato peel and pomace. Background Technology

[0002] Lycopene possesses strong biological activities, including antioxidant effects, central nervous system protection, anti-cancer properties, and the ability to enhance immunity. Lycopene from tomato peels and pomace can be obtained through extraction methods such as organic solvent extraction, chromatographic separation, enzyme-assisted extraction, and microwave-assisted extraction. Existing solvent extraction methods suffer from low efficiency, long extraction times, large consumption of organic solvents, and potential safety concerns regarding the extraction solvents. Most current methods only extract lycopene from tomato peels and pomace, which also contain a significant amount of polysaccharides. Therefore, how to safely and efficiently extract these bioactive substances is a crucial issue.

[0003] Aqueous two-phase extraction has advantages such as low organic solvent consumption, low energy consumption, and high product yield, and has a wide range of applications in the extraction and purification of various active ingredients. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly efficient extraction process for lycopene and polysaccharides from tomato peel and pomace.

[0005] The technical problem solved by this invention is achieved through the following technical solution:

[0006] A process for extracting lycopene and polysaccharides from tomato peel and pomace, comprising the following steps:

[0007] S1. Take a certain mass of tomato peel and pomace and spread it evenly in a dryer for drying. Crush the dried tomato peel and pomace to a constant weight and pass it through a 90-mesh standard sieve to prepare tomato peel and pomace powder.

[0008] S2. Enzyme-assisted aqueous two-phase extraction: Tomato peel and pomace powder was added to disodium hydrogen phosphate-citric acid buffer solution, followed by neutral protease. The mixture was then incubated in a 45°C water bath for 60 min. After the enzymatic hydrolysis, dipotassium hydrogen phosphate solution was immediately added to dissolve the powder completely. Ethyl acetate was then added and mixed thoroughly. The mixture was allowed to stand for 1.5 h in the dark to obtain upper and lower phase solutions. The upper phase was the extract of lycopene and polysaccharides, while the lower phase was the polysaccharide extract. After purification and drying, a mixture of lycopene and polysaccharides from tomato peel and pomace, as well as tomato polysaccharides, were obtained.

[0009] Furthermore, the ethyl acetate has a mass fraction of 25% and the dipotassium hydrogen phosphate has a mass fraction of 14%.

[0010] Furthermore, the pH value of the disodium hydrogen phosphate-citric acid buffer solution is 7.

[0011] Moreover, the activity of the neutral protease is 1000-2000U.

[0012] A lycopene and polysaccharide-based membrane material component is prepared using the following steps:

[0013] ① Add chitosan, stearic acid, and lipase to tert-butanol solvent, mix and react at 50-60℃ for 24-48 hours, centrifuge or filter, wash with ethanol 2-3 times, and collect the modified product.

[0014] ② Take the above ethyl acetate solution of lycopene and polysaccharide, dissolve the modified product in DMSO, mix them according to the dry weight ratio of 1:1-3, vortex or sonicate to form a homogeneous transparent solution, and dry to obtain the membrane material composition.

[0015] Moreover, the mass ratio of chitosan to stearic acid is 1:5-10.

[0016] Moreover, the dry weight ratio of lycopene and polysaccharide to modified product is 1:1-3.

[0017] The advantages and positive effects of this invention are:

[0018] This invention simultaneously extracts lycopene and polysaccharides. A natural eutectic solvent is prepared, and then tomato peel and pomace are added for extraction. The mixture is then separated to extract and separate the economically valuable lycopene and polysaccharides. A cell anti-inflammatory experiment is designed to determine the physiological activity of lycopene.

[0019] This invention investigates the influencing factors on the yield of lycopene and polysaccharides from tomato pomace through single-factor experiments. The initial extraction conditions for lycopene and polysaccharides were obtained using enzyme-assisted aqueous two-phase extraction (A2PE). These conditions were then optimized through orthogonal experiments to determine the optimal process conditions. The final optimal process was determined to be: 35% ethyl acetate, 14% dipotassium hydrogen phosphate, 2000 U enzyme activity, and pH 7.

[0020] Under optimal processing conditions, the yield of lycopene in tomato pomace was 65 mg / 100g, and the yield of polysaccharides was 501 mg / g. Antioxidant evaluation using vitamin C as a positive control showed that lycopene reduced DPPH radicals, hydroxyl radicals, and ABTS. + The maximum free radical scavenging rates were 90.47%, 29.11%, and 91.27%, respectively. The polysaccharides scavenged DPPH, hydroxyl, and ABTS free radicals. + The maximum free radical scavenging rates were 84.01%, 72.01%, and 97.31%, respectively. Lycopene and polysaccharides exhibited activity against DPPH free radicals, hydroxyl radicals, and ABTS. +The free radicals were effectively scavenged. After lycopene treatment, the NO content decreased with increasing sample concentration, indicating that lycopene has certain anti-inflammatory activity, and the 10 μg / mL sample significantly reduced the NO content. This experiment improved the comprehensive utilization value of tomato pomace and provided a theoretical basis for the extraction and application of lycopene and polysaccharides from tomato pomace. Attached Figure Description

[0021] Figure 1 The effect of dipotassium hydrogen phosphate mass fraction on the extraction of lycopene and polysaccharides.

[0022] Figure 2 The effect of ethyl acetate mass fraction on the extraction of lycopene and polysaccharides.

[0023] Figure 3 The effect of enzyme activity on the extraction of lycopene and polysaccharides.

[0024] Figure 4 The effect of pH on the extraction of lycopene and polysaccharides.

[0025] Figure 5 This study aims to determine the DPPH free radical scavenging effect of the membrane material.

[0026] Figure 6 This study aims to determine the scavenging effect of membrane materials on hydroxyl radicals.

[0027] Figure 7 This study aims to determine the scavenging effect of membrane materials on ABTS+ free radicals.

[0028] Figure 8 The effect of different concentrations of membrane material on the survival rate of RAW 264.7 cells. Detailed Implementation

[0029] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0030] The tomato pomace used in this invention was sourced from Shandong Qicai Manor Vegetable Food Base Co., Ltd.; lycopene standard from Shanghai Aladdin Biochemical Technology Co., Ltd.; L(+)-ascorbic acid, glucose, and dipotassium hydrogen phosphate from Shanghai Guoyao Group Chemical Reagent Co., Ltd.; ethyl acetate from Jiangsu Taicang Hushi Reagent Co., Ltd.; neutral protease and Cell Counting Kit-8 (CCK-8) reagent from Beijing Solarbio Science & Technology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazine (DPPH) from TCI (Shanghai) Chemical Industry Development Co., Ltd.; 2,2-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) from Shandong Keyuan Biochemical Co., Ltd.; 30% hydrogen peroxide from Tianjin Damao Chemical Reagent Factory; and the mouse macrophage cell line RAW. 264.7, Hubei Boster Biological Technology Co., Ltd.; DMEM medium, penicillin-streptomycin antibiotics, fetal bovine serum (FBS), Gibco, USA; trypsin digestion solution, Wuhan Pronosai Life Science Technology Co., Ltd.; lipopolysaccharide (LPS), Sigma, USA.

[0031] A highly efficient extraction process for lycopene and polysaccharides from tomato peel and pomace includes the following steps:

[0032] I. Extraction Process

[0033] 1. Sample pretreatment

[0034] A certain amount of tomato peel and pomace is evenly spread in a dryer for drying. The dried tomato peel and pomace is then crushed and passed through a 90-mesh standard sieve to prepare tomato peel and pomace powder.

[0035] 2. Enzyme-assisted aqueous two-phase extraction

[0036] 0.1 g of tomato peel and pomace powder was mixed thoroughly with a certain volume concentration of disodium hydrogen phosphate-citric acid buffer solution and 1000 U of neutral protease. The mixture was then enzymatically hydrolyzed in a 45°C water bath for 60 min. After the enzymatic hydrolysis, a certain amount of dipotassium hydrogen phosphate solution was immediately added to ensure complete dissolution, followed by the addition of ethyl acetate and thorough mixing. The mixture was then allowed to stand for 1.5 h under light-protected conditions, and the volumes of the upper and lower phases were measured. The extracts of lycopene and polysaccharides from the upper phase and the polysaccharide extract from the lower phase were collected for later use. The yields of lycopene and polysaccharides were determined using the following formula:

[0037]

[0038] In the formula, Y A The yield of lycopene, mg / 100g; Y B The yield of polysaccharides is expressed in mg / g; C1 and C2 are the mass concentrations of the upper phase lycopene extract and the lower phase extract, respectively.b V1 represents the mass concentration of the upper phase polysaccharide extract, in mg / mL; V2 and V1 represent the volumes of the upper and lower phases, in mL, respectively; M represents the total mass of the tomato peel and pomace powder, in g.

[0039] 3. Determination of lycopene and polysaccharide content

[0040] The lycopene content in tomato pomace was determined using a spectrophotometer. The lycopene extract was diluted with ethyl acetate at a certain ratio, and the absorbance was measured at 503 nm. The linear regression equation for the lycopene standard solution was y = 318.8x + 0.0252, R² = 0.9946 (where y is the absorbance and x is the concentration of the lycopene standard solution, mg / mL).

[0041] The polysaccharide content in tomato peel and pomace was determined using the phenol-sulfuric acid colorimetric method. Prepared polysaccharide extracts were diluted 100-fold with buffer solution. 1 mL of each extract was first mixed with 0.5 mL of 6% phenol solution, then 2.5 mL of concentrated sulfuric acid was added. The mixture was heated in a 75℃ water bath for 30 min, and the absorbance was measured at 490 nm. The linear regression equation for the glucose standard solution was y = 14.74x + 0.1558, R0. 2 =0.9947 (where y is absorbance; x is the concentration of glucose standard solution, mg / mL).

[0042] 4. Optimization of the above-mentioned process conditions for lycopene and polysaccharides

[0043] Based on the single-factor experiments, orthogonal experiments were conducted according to Table 1.

[0044] Table 1. Factor Levels of Orthogonal Experiment

[0045]

[0046] Table 2. Results of the orthogonal experiment on lycopene and polysaccharides

[0047]

[0048]

[0049] The orthogonal experimental results for the extraction of lycopene and polysaccharides are shown in Table 2. Table 2 shows that the factors affecting lycopene extraction are D > C > A > B, with the optimal level being A1B1C3D2, i.e., ethyl acetate mass fraction of 25%, dipotassium hydrogen phosphate mass fraction of 14%, enzyme activity of 2000U, and pH of 7.5. Under these conditions, the yields of lycopene and polysaccharides were 72 mg / 100g and 495 mg / g, respectively.

[0050] Table 2 shows the orthogonal experimental results of polysaccharides. The order of influence on polysaccharide extraction is C > A > D > B. The optimal level is A3B3C3D1, which means ethyl acetate mass fraction of 35%, dipotassium hydrogen phosphate mass fraction of 18%, enzyme activity of 2000U, and pH value of 7. Under these conditions, the yields of lycopene and polysaccharides were calculated to be 65mg / 100g and 501mg / g, respectively.

[0051] Taking all factors into consideration, the optimal process conditions were determined to be 25% ethyl acetate, 14% dipotassium hydrogen phosphate, 2000 U enzyme activity, and pH 7. Repeated experiments were conducted under these conditions, and the yields of lycopene and polysaccharides were calculated to be 66 mg / 100g and 523 mg / g, respectively. Compared with the results of orthogonal experiments, the yields of both lycopene and polysaccharides were higher. The experimental results indicate that the optimization of the extraction process has certain reference value.

[0052] The present invention also provides a membrane material component based on lycopene and polysaccharides prepared by the above method, the steps of which are as follows:

[0053] ① Add chitosan, stearic acid, and lipase to tert-butanol solvent, mix and react at 50-60℃ for 24-48 hours. After centrifugation or filtration, wash with ethanol 2-3 times and collect the modified product. The mass ratio of chitosan to stearic acid is 1:5-10.

[0054] ② Take the above ethyl acetate solution of lycopene and polysaccharide, dissolve the modified product in DMSO, mix them according to the dry weight ratio of 1:1-3 of lycopene and polysaccharide: modified product, vortex or ultrasonically mix (avoid light exposure) to form a homogeneous transparent solution, and dry to obtain the membrane material composition.

[0055] To extend the duration of action of lycopene and polysaccharides, 0.1 wt% vitamin E is added during the preparation process. Vitamin E can protect the groups of lycopene, prevent oxidation, and prolong the antioxidant effect of the membrane material.

[0056] In this invention, the recommended ratio of lycopene to polysaccharide is 1-2:1 by weight.

[0057] This membrane material can be sprayed onto the surface of health products or cosmetics, such as capsules and cotton pads, to remove free radicals and reduce inflammation.

[0058] Antioxidant activity analysis of membrane materials

[0059] 1. DPPH free radical scavenging rate

[0060] 2.0 mL of sample solutions of different concentrations (test solutions after reconstitution of membrane material solution, all concentrations are based on lycopene concentration) and 2.0 mL of DPPH solution (0.2 mmol / L) were placed in test tubes respectively, mixed thoroughly, and then allowed to stand in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm as A1. The absorbance of 2 mL of sample solution and 2 mL of anhydrous ethanol was measured in the same way as A2, and the absorbance of 2 mL of DPPH solution with 2 mL of 70% ethanol was measured as A. At the same time, Vc of the same mass concentration as the sample solution was measured as a positive control. The DPPH free radical scavenging rate was calculated according to formula (3).

[0061]

[0062] Depend on Figure 5 It can be seen that the DPPH free radical scavenging rate increases with the increase of the membrane solution concentration. At a concentration of 10 μg / mL, the DPPH free radical scavenging rate is 90.47%, which is higher than the scavenging rate of vitamin C at the same concentration, indicating that lycopene in tomato peel and pomace has a good scavenging effect.

[0063] 5.2 Hydroxyl radical scavenging

[0064] Take 1 mL of sample solutions of different concentrations and add them to a test tube. Then add 1 mL of 8 mmol / L FeSO4 solution, 1 mL of 8 mmol / L salicylic acid-ethanol solution, and 1 mL of 8 mmol / L H2O2 in sequence. After mixing thoroughly, let stand for 1 h and measure the absorbance A2 at a wavelength of 510 nm. In the same way, use 70% ethanol instead of the sample to measure the absorbance A, and use purified water instead of H2O2 to measure the absorbance A1. At the same time, measure Vc with the same mass concentration as the sample solution as a positive control, and calculate the hydroxyl radical scavenging rate according to formula (4).

[0065]

[0066] Depend on Figure 6 It can be seen that the hydroxyl radical scavenging rate increases with the increase of lycopene concentration in the membrane material. At a lycopene concentration of 10 μg / mL, the hydroxyl radical scavenging rate of lycopene in tomato peel and pomace reached 29.11%, which is higher than the scavenging rate of vitamin C at the same concentration, indicating that lycopene in tomato peel and pomace has a good scavenging effect on hydroxyl radicals.

[0067] 5.3ABTS + Free radical scavenging

[0068] Mix 10 mL of 7 mmol / L ABTS solution with 10 mL of 2.45 mmol / L potassium persulfate solution until homogeneous, then let stand in the dark for 12 h. Before use, dilute the solution by a certain factor so that the absorbance at a wavelength of 734 nm is around 0.70. Take 0.5 mL of samples of different concentrations into test tubes, add 3 mL of the diluted solution to each, shake vigorously for 30 s, let stand in the dark for 10 min, and then measure the absorbance A1 at a wavelength of 734 nm. The blank group (A2) uses deionized water instead of the sample solution as a control, and the reference group (A) uses deionized water instead of the sample solution and anhydrous ethanol instead of the ABTS solution. At the same time, measure the Vc concentration of the same mass as the sample solution as a positive control. Calculate ABTS according to formula (5). + Free radical scavenging rate.

[0069]

[0070] Depend on Figure 7 It can be seen that the ABTS+ free radical scavenging rate increases with the increase of lycopene concentration in the membrane material. When the lycopene concentration is 10 μg / mL, the ABTS+ free radical scavenging rate of lycopene extracted from tomato peel pomace reaches 91.27%, which is higher than that of vitamin C at the same concentration for ABTS+ free radical scavenging. + The free radical scavenging rate indicates that the membrane material has a good scavenging effect on ABTS+ free radicals.

[0071] Part Two: Study on the Anti-inflammatory Activity of Membrane Materials

[0072] 1. Determination of cell viability

[0073] Samples were taken from cells in the logarithmic growth phase, and the cell density was adjusted to 5 × 10^6 cells. 4 100 μL of cell suspension was seeded into each well of a 96-well plate. The culture plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The treatment groups were as follows: the NC group (negative control group) was given an equal volume of basal medium, the model group was given 1.0 μg / mL LPS to induce an inflammatory response, and the drug groups were given different concentrations of lycopene (2, 5, 10, 20, 50, 100 μg / mL). 1.0 μg / mL LPS was added to each group at the same time. After 24 hours of culture, the supernatant was discarded, 10 μL of CCK-8 reagent was added to each well, and the reaction was continued in a 37°C and 5% CO2 incubator for 2 hours. After the reaction, the absorbance (OD value) was measured at 450 nm using an ELISA reader to reflect the cell viability. The cell viability was calculated using formula (6):

[0074]

[0075] Effects of membrane material on RAW 264.7 cell viability

[0076] Table 3. Effects of different membrane material solution concentrations (based on lycopene concentration) on RAW 264.7 cell viability.

[0077]

[0078] Macrophages are immune cells widely distributed in various tissues and organs of the body, playing a crucial role in anti-inflammatory progression and commonly used as a model for assessing anti-inflammatory activity. Table 3 shows that within the concentration range of 2.5-10 μg / mL membrane material solution (all concentrations calculated based on lycopene concentration), cell viability remained above 90%, and no cytotoxicity was observed. However, treatment with lycopene at concentrations exceeding 20 μg / mL significantly reduced cell viability, indicating that the samples at this concentration range exhibited some toxicity to RAW264.7 cells. Based on these results, membrane material solutions of 2.5, 5, and 10 μg / mL were selected for subsequent experiments.

[0079] 2. Effect of membrane material solution on LPS-induced NO production in RAW264.7 cells

[0080] Cells in the logarithmic growth phase were collected, and the cell density was adjusted to 5 × 10⁻⁶. 4 Cells were seeded per well, with 100 μL of cell suspension per well. The 96-well plates were incubated at 37°C and 5% CO2 for 24 hours. NC group (control group): An equal volume of basal culture medium was added; model group: 1.0 μg / mL LPS was added to induce an inflammatory response. Drug groups: Different concentrations of membrane material solution (equivalent to 2, 5, 10, 20, 50, and 100 μg / mL lycopene) were added, along with 1.0 μg / mL LPS in each group. After 24 hours of incubation, 100 μL of supernatant was collected from each well, and 100 μL of Griess' reagent was added. After reacting at room temperature for 10 minutes, the absorbance was measured at 540 nm. Based on the measured absorbance values, the relative amount of NO produced by the cells was calculated using a standard formula.

[0081]

[0082] Macrophages release large amounts of nitrogen (NO) upon activation by bacteria and other pathogens, thus NO is often considered a marker of macrophage activation. However, excessive NO production can trigger inflammation and inflammatory diseases. Therefore, reducing intracellular NO production is crucial for suppressing inflammation. Figure 8As shown, compared with the NC group, LPS stimulation significantly increased NO content (P<0.05), indicating that the LPS-induced RAW 264.7 cell inflammation model was successfully established. Compared with the model group, after lycopene treatment, NO content decreased with increasing sample concentration, indicating that the membrane material has certain anti-inflammatory activity, and the 10 μg / mL sample significantly reduced NO content.

[0083] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A process for extracting lycopene and polysaccharides from tomato peel and pomace, characterized in that: The steps are as follows: S1. Take a certain mass of tomato peel and pomace and spread it evenly in a dryer for drying. Crush the dried tomato peel and pomace to a constant weight and pass it through a 90-mesh standard sieve to prepare tomato peel and pomace powder. S2. Enzyme-assisted aqueous two-phase extraction: Tomato peel and pomace powder was added to disodium hydrogen phosphate-citric acid buffer solution, followed by neutral protease. The mixture was then incubated in a 45°C water bath for 60 min. After the enzymatic hydrolysis, dipotassium hydrogen phosphate solution was immediately added to dissolve the powder completely. Ethyl acetate was then added and mixed thoroughly. The mixture was allowed to stand for 1.5 h in the dark to obtain upper and lower phase solutions. The upper phase was the extract of lycopene and polysaccharides, while the lower phase was the polysaccharide extract. After purification and drying, a mixture of lycopene and polysaccharides from tomato peel and pomace, as well as tomato polysaccharides, were obtained.

2. The extraction process for lycopene and polysaccharides from tomato peel and pomace according to claim 1, characterized in that: The ethyl acetate has a mass fraction of 25%, and the dipotassium hydrogen phosphate has a mass fraction of 14%.

3. The extraction process for lycopene and polysaccharides from tomato peel and pomace according to claim 1, characterized in that: The pH value of the disodium hydrogen phosphate-citric acid buffer solution is 7.

4. The extraction process for lycopene and polysaccharides from tomato peel and pomace according to claim 1, characterized in that: The activity of the neutral protease is 1000-2000U.

5. A membrane material component based on lycopene and polysaccharides prepared by the process described in claim 1, characterized in that: The preparation steps are as follows: ① Add chitosan, stearic acid, and lipase to tert-butanol solvent, mix and react at 50-60℃ for 24-48 hours, centrifuge or filter, wash with ethanol 2-3 times, and collect the modified product. ② Take the above ethyl acetate solution of lycopene and polysaccharide, dissolve the modified product in DMSO, mix them according to the dry weight ratio of 1:1-3, vortex or sonicate to form a homogeneous transparent solution, and dry to obtain the membrane material composition.

6. The lycopene and polysaccharide-based membrane material composition according to claim 5, characterized in that: The mass ratio of chitosan to stearic acid is 1:5-10.

7. The lycopene and polysaccharide-based membrane material composition according to claim 5, characterized in that: The dry weight ratio of lycopene and polysaccharide to the modified product is 1:1-3.

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