Pumpkin peel comprehensive utilization method, functional pigment and dietary fiber

Abstract

The invention discloses a comprehensive utilization method of pumpkin peel, functional pigment and dietary fiber, which comprises the following steps: by taking pumpkin peel as a raw material, carrying out vacuum freeze drying and crushing treatment, carrying out ultrasonic-assisted pectinase pretreatment, and extracting the pigment of the pumpkin peel by combining a specific extracting agent with optimized ultrasonic extraction parameters. The pumpkin peel pigment extracted by the method disclosed by the invention has oxidative stress resistance and heat stress resistance, the survival rate of C.elegans under a 35 DEG C heat stress state can be remarkably prolonged, the average survival rate reaches 73%, the growth curve of C.elegans under hydrogen peroxide (H2O2) oxidative stress is remarkably prolonged to the right, and the Taver is prolonged to 169.3 min or above. The pumpkin peel pigment extracted by the method disclosed by the invention has a weight-losing function and anti-colitis activity; dietary fibers are free of peculiar smell, good in sensory property, free of post-treatment such as decoloration and the like, and combined with water power, expansibility and adsorption capacity are enhanced.

Description

(I) Technical field

[0001] The invention relates to a pumpkin peel comprehensive utilization method and pumpkin peel pigment and dietary fiber for weight loss and anti-colitis. (II) Background technology

[0002] Pumpkin is an annual vine herbaceous plant of the Cucurbitaceae family and the genus Cucurbita (Cucurbita moschata). Its cultivated species are often divided into three categories according to their characteristics: Chinese pumpkin (C.moschata Duch), Indian pumpkin (C.maxima Duch) and American pumpkin (C.pepo L.). Pumpkin is grown all over the world, with the largest cultivated area in Asia, followed by Europe and South America. According to the United Nations Food and Agriculture Organization Statistical Database (FAO-STAT), the global total pumpkin production in 2021 was 23.7839 million tons, and my country's pumpkin production was 7.3876 million tons, accounting for 31.06% of the world's total production that year, ranking first in the world, and the available resources are very abundant.

[0003] Pumpkin pulp is the main raw material for processing and consumption, while pumpkin skin, which accounts for 18-21% of the weight of pumpkin, is discarded as processing waste during processing because it is inedible, which not only wastes resources but also pollutes the environment. In order to promote the research and development of pumpkin skin residue, new green methods for comprehensive utilization of pumpkin skin residue are urgently needed. Pumpkin skin is rich in pectin, soluble sugar, cellulose, carotenoids and other nutrients. It is reported that 100g pumpkin skin powder contains 42.09g dietary fiber (DF), which is mainly crude fiber, and has poor physical and chemical properties such as water binding and swelling power. Traditional acid or alkali method of modifying dietary fiber often has disadvantages such as environmental pollution and complicated process, and green modification technology is urgently needed.

[0004] In addition, pumpkin peel also contains a variety of natural antioxidant carotenoids, including α-carotene, β-carotene, lutein, β-cryptoxanthin, etc., which can be used as V A The pumpkin peel is a precursor of pumpkin and has high nutritional value. Therefore, how to increase the dissolution amount of pumpkin peel pigment, optimize the extraction process of pumpkin peel pigment and explore its biological activity is an urgent problem to be solved. At present, there have been reports on the extraction and stability of pumpkin pulp pigment, and research on the separation, purification and antioxidant properties of pumpkin pulp pigment has been gradually carried out. However, pumpkin peel contains waxy components and insoluble crude fiber, and the extraction method suitable for pulp pigment may not be suitable for peel pigment. Recycling pumpkin peel, a by-product of pumpkin processing, not only solves the problems of pollution and waste, but also improves the comprehensive development and utilization value of pumpkin.

[0005] Inflammatory bowel disease (IBD) is a chronic, relapsing, nonspecific inflammatory disease, including ulcerative colitis and Crohn's disease. Ulcerative colitis is a chronic inflammatory disease with abdominal pain, bloody diarrhea, and bloody stool as its main symptoms. Nature's sub-journal reported that antioxidants can reduce oxidative DNA damage in IBD patients. C.elegans is a widely used model organism in the field of biomedicine. It has the advantages of short life cycle, strong reproductive ability, known whole genome, and simple feeding. It is internationally recognized as a model organism with advantages in the fields of anti-oxidation and anti-aging. ICR mice originated from Swiss albino mice. They are male and female albino non-inbred mice introduced from Lausanne by the Rockefeller Institute in 1926. The descendants of these mice are distributed all over the world. In 1948, the American Cancer Research Institute established the Ha / ICR strain. Because of its docile character, high reproductive performance, fast growth, strong adaptability to the environment, and good experimental repeatability, it is widely used in biomedical research and is an internationally used closed group mouse. (III) Summary of the invention

[0006] The invention aims to provide a method for comprehensive utilization of pumpkin peels and functional pigments and dietary fiber. The pumpkin peels are used as raw materials, and an ultrasound-assisted enzyme method is used to pretreat the pumpkin peels on the basis of vacuum freeze drying and pulverization. The functional pigments are prepared by an ultrasound-assisted natural acid-alcohol two-phase extraction process. The filter residues after the pigments are extracted are subjected to green modification treatment to obtain dietary fiber. The method not only improves the extraction rate of pumpkin peel pigments and solves the residue problem of conventional solvent extraction, but also has the activity of resisting oxidative stress and heat stress, and also has the ability of reducing weight and resisting mouse colitis. The physicochemical properties of the obtained dietary fiber, such as swelling power, binding water power, sodium cholate adsorption capacity, etc., are significantly improved, and the dietary fiber can be used to prepare baked foods, thereby realizing a new utilization of pumpkin peel processing.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides a method for comprehensive utilization of pumpkin peels, which comprises the following steps:

[0009] (1) Add pumpkin peel powder to deionized water to form a slurry, add pectinase, mix well, and perform ultrasound-assisted enzymolysis for 40 to 60 minutes at 40 to 60° C., pH 3.0 to 4.0 (1 M hydrochloric acid to adjust pH), 150 to 240 W, boil for 100 seconds, adjust pH to neutral after cooling (preferably with 1 M NaOH aqueous solution), and vacuum freeze-dry to obtain an enzymolysis mixture;

[0010] (2) adding a natural acid containing 20-80% ethanol by volume to the enzymatic hydrolysis mixture of step (1), performing ultrasonic-assisted extraction for 50-90 min at 40-60° C. and 180-300 W, centrifuging (8000 rpm, 30 min) to obtain a supernatant and a filter residue, and concentrating the supernatant to 3 / 5 of the original volume to obtain a pumpkin peel pigment; the natural acid is prepared by mixing n-octanoic acid and n-decanoic acid, and heating at 60° C. for 20-30 min (preferably 25 min), wherein the molar ratio of n-octanoic acid to n-decanoic acid is 2-3:1, preferably 2.6:1;

[0011] (3) The filter residue from step (2) is dried with hot air at 60° C., crushed and sieved through a 150-200 mesh sieve, and subjected to wall breaking and modification treatment for 180-720 s using hydrogen peroxide having a mass concentration of 0.5-2% as a green modifier by an ultrasonic wall breaking machine, and centrifuged (preferably at 8000 rpm for 15 min). The residue is rinsed three times with deionized water, dried with hot air at 60° C., crushed and sieved to obtain pumpkin peel dietary fiber.

[0012] Furthermore, the activity of the pectinase in step (1) is 30,000 U / g, which can be obtained commercially. The amount of pectinase used is 0.9%-1.5% (preferably 0.9%) based on the mass of the pumpkin peel powder.

[0013] Furthermore, the volume of deionized water in step (1) is 20-30 mL / g, preferably 25 mL / g, based on the weight of the pumpkin peel powder.

[0014] Furthermore, the ultrasonic-assisted enzymatic hydrolysis conditions in step (1) are: ultrasonic power 150 W, temperature 55° C., and ultrasonic treatment for 53 min.

[0015] Furthermore, the vacuum freeze-drying method in step (1) is as follows: the initial temperature is -30°C, heating is started when the vacuum degree is 80 Pa, and the temperature is kept at -20°C for 1 hour, and then the temperature is kept at 10°C for 1 hour each time the temperature is increased, and the temperature is raised to 20°C and kept for more than 24 hours until the water content of the sample is below 8%.

[0016] Furthermore, the pumpkin peel powder in step (1) is prepared according to the following steps: the yellow wolf pumpkin is washed and peeled, the pumpkin peel is shredded, and then placed in a vacuum freeze dryer with an initial temperature of -30°C. When the vacuum degree is 80 Pa, heating is started, and when it is heated to -20°C, it is kept for 1 hour, and then the temperature is increased by 10°C for 1 hour, and the temperature is increased to 20°C, and kept for 24 hours until the water content is below 8%, and then the pumpkin peel powder is obtained after being crushed and passed through a 200-mesh sieve.

[0017] Furthermore, in step (2), the volume amount of the natural acid containing ethanol with a volume concentration of 20-80% is 10-50 mL / g, preferably 30 mL / g, based on the mass of the enzymatic hydrolysis mixture.

[0018] Furthermore, the ultrasonic-assisted extraction conditions in step (2) are preferably: ultrasonic power 180 W, temperature 50° C., and ultrasonic time 64 min.

[0019] Furthermore, the volume amount of hydrogen peroxide in step (3) is 20-50 mL / g, preferably 30 mL / g, based on the mass of pumpkin peel powder.

[0020] Furthermore, in step (3), the concentration of hydrogen peroxide was 1.46%, and the modification treatment lasted for 368 seconds.

[0021] Furthermore, the crushing and screening in step (3) refers to sieving through 300 mesh, 200 mesh, 150 mesh and 100 mesh sieves to obtain pumpkin peel dietary fiber of different particle sizes of >300 mesh, 200-300 mesh, 150-200 mesh and 100-150 mesh, respectively.

[0022] The present invention also provides an application of the pumpkin peel pigment prepared by the method in preparing an antioxidant or heat stress preparation, wherein the preparation is an antioxidant or heat stress preparation for Caenorhabditis elegans.

[0023] The pumpkin peel pigment of the present invention, as a natural functional pigment, can be used to resist oxidative stress and heat stress of Caenorhabditis elegans (C.elegans); the oxidative stress refers to significantly prolonging the survival rate of C.elegans in a hydrogen peroxide (200 μL, 4 mM) stress environment, and the heat stress resistance refers to significantly prolonging the survival rate of C.elegans in a 35°C heat stress environment.

[0024] The present invention also provides an application of pumpkin peel pigment prepared by the method in preparing an anti-colitis preparation, wherein the preparation can significantly enhance the activity of the antioxidant enzyme SOD in mice with colitis induced by dextran sulfate sodium salt (DSS), and significantly reduce the levels of inflammatory factors TNF-α and IL-6 in the colon tissue of the mice.

[0025] The invention also provides application of pumpkin peel pigment prepared by a method in preparing weight-loss medicine.

[0026] The present invention also provides a functional pumpkin peel pigment prepared by the method, wherein the functionality refers to that the pumpkin peel pigment has antioxidant stress activation, anti-colitis activity, and the ability to reduce body weight.

[0027] The present invention also provides pumpkin peel dietary fiber prepared by the method, and the pumpkin dietary fiber can be used for preparing baked food.

[0028] The present invention uses pumpkin peel as raw material, after vacuum freeze drying and pulverization, ultrasound-assisted pectinase pretreatment, and then extracts pumpkin peel pigment through a specific extractant combined with optimized ultrasonic extraction parameters. The technical key points of the present invention are that the vacuum freeze drying treatment of the raw pumpkin peel can avoid the temperature damage to the pumpkin peel pigment during the high-temperature drying process and improve the biological activity; the pretreatment process of low-temperature ultrasound and pectinase can make the crude fiber and wax structure in the pumpkin peel loose, which is conducive to the dissolution of the pigment in the cell; the extraction process of ultrasound-assisted special extractants can make the pigment in the pumpkin peel fully and effectively dissolve, and fully maintain its functional activity, including anti-oxidative stress, heat stress ability, anti-colitis activity and weight loss activity.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] 1. Ultrasonic-assisted pectinase pretreatment helps to improve the extraction rate of pumpkin peel pigment. In view of the high insoluble fiber content and waxy surface structure of pumpkin peel, ultrasonic-assisted pectinase pretreatment can make the raw material simultaneously subject to the efficient enzymatic hydrolysis of enzymes and the cavitation and mechanical action of ultrasound, improve the tough cell wall structure of pumpkin peel, and make the pumpkin peel raw material form a loose and porous state, which is conducive to the effective dissolution of pigments in the next step of extraction, avoiding the destruction of pumpkin peel pigment structure and activity; at the same time, ultrasonic-assisted enzymatic treatment ensures that the subsequent products have no odor and good sensory properties.

[0031] 2. Ultrasound-assisted pectinase pretreatment and natural acid-alcohol two-phase system extraction process significantly improves the extraction rate of pumpkin peel pigment. After ultrasound-assisted pectinase pretreatment, the present invention adopts a natural acid-alcohol two-phase system combined with ultrasound extraction process, which has the advantages of being green, natural, low-cost, low-toxicity, easy to prepare, etc., will not destroy the activity of pumpkin peel pigment, can achieve efficient extraction of heat-labile pigments at a lower temperature, and can make the extraction rate of pumpkin peel pigment reach more than 4 mg / g. The extraction rate of pumpkin peel pigment is 20.94% higher than that of traditional hot air drying pretreatment, 45.97% higher than that of pumpkin peel pigment extracted from pumpkin peel powder, 32.71% higher than that of peel pigment obtained without ultrasound-assisted enzymatic pretreatment, 30.71% higher than that of peel pigment obtained without ultrasound-assisted pretreatment, and 36.90% higher than that of pumpkin peel pigment obtained without enzymatic pretreatment; and 14.45%, 52.10%, 65.65%, 110.49%, 101.39%, 47.75% and 98.81% higher than that of ethanol, acetone, methanol, n-butanol, isopropanol, ethyl acetate and n-hexane extraction, respectively. The process is mild, green and environmentally friendly, and meets the production requirements of health food.

[0032] 3. The pumpkin peel pigment extracted by the method of the present invention has the ability to resist oxidative stress and heat stress, and can significantly prolong the survival rate of C.elegans under 35°C heat stress, with an average survival rate of 73% (the negative control group was only 45%), an increase of 62.22% (p<0.05). It can significantly prolong the rightward shift of the growth curve of C.elegans under hydrogen peroxide (H2O2) oxidative stress, and T aver Extended to more than 169.3 minutes.

[0033] 4. The pumpkin peel pigment extracted by the method of the present invention has a weight-loss function. Compared with the model group, pumpkin peel pigment significantly reduced the weight of mice by 22.41% (p<0.05).

[0034] 5. The pumpkin peel pigment extracted by the method of the present invention has anti-colitis activity. Pumpkin peel pigment significantly enhanced the activity of the antioxidant enzyme SOD in the liver of DSS-induced colitis mice, and significantly reduced the levels of inflammatory factors TNF-α and IL-6 in the colon tissue of mice. It can enhance the activity of the antioxidant enzyme SOD in the liver of DSS-induced colitis mice by 114.81% (p<0.05), and significantly reduce the levels of inflammatory factors TNF-α and IL-6 in the colon tissue of mice by 116.29% and 121.2% respectively (p<0.05).

[0035] 6. The dietary fiber obtained by ultrasonic wall breaking assisted hydrogen peroxide green modified pumpkin peel residue of the present invention has no peculiar smell, good sensory properties, and does not require post-treatment such as decolorization. The cell wall of the modified dietary fiber forms a loose and porous state under a scanning electron microscope (SEM), which is conducive to the enhancement of binding water force, swelling force and adsorption capacity; the modification operation is carried out under neutral conditions without the introduction of chemical reagents such as acid and alkali. The concentration of hydrogen peroxide used in the modification operation is low, and it is easy to decompose into water and oxygen after the operation is completed, and no residue will be left in the entire unit operation process.

[0036] The expansion force and bound water capacity of the pumpkin peel DF obtained by the method of the present invention are respectively increased by 354.70% and 211.40% compared with the DF obtained from pumpkin peel coarse powder, respectively increased by 25.77% and 3.03% compared with the DF obtained without ultrasound-assisted enzymatic pretreatment, respectively increased by 16.16% and 17.36% compared with the DF obtained without ultrasound-assisted pretreatment, and respectively increased by 28.50% and 27.93% compared with the DF obtained without enzymatic pretreatment.

[0037] 7. The pumpkin peel pigment comprehensive utilization method of the present invention does not introduce impurities and is pollution-free during the entire operation process. It has low equipment requirements and low energy consumption. The process conditions are mild and it is green and environmentally friendly, and it meets the production requirements of health food. (IV) Description of the drawings

[0038] Figure 1, the effect of extractants on the extraction rate of pumpkin peel pigment; A represents different molar ratios of natural acid, n-octanoic acid and n-decanoic acid; B represents different ethanol contents in natural acid; different letters represent significant differences between groups, p<0.05; the same letters represent no significant differences between groups, p>0.05.

[0039] Figure 2 , enzyme dosage, enzymatic hydrolysis temperature, enzymatic hydrolysis time, pH and ultrasonic power on the extraction rate of pumpkin peel pigment; different letters represent significant differences among the groups, p<0.05; the same letters represent no significant differences among the groups, p>0.05.

[0040] Figure 3 , enzymatic hydrolysis temperature, enzymatic hydrolysis time, and response surface optimization diagram of system pH to absorbance value.

[0041] Figure 4 , ultrasonic temperature, time, solid-liquid ratio and ultrasonic power on the extraction rate of pumpkin peel pigment; different letters represent significant differences among the groups, p<0.05; the same letters represent no significant differences among the groups, p>0.05.

[0042] Figure 5 , ultrasonic temperature, ultrasonic time, and solid-liquid ratio on the response surface optimization plot of pumpkin peel pigment extraction yield.

[0043] Figure 6 , Effects of pumpkin peel pigments on the lifespan and average lifespan of nematodes under oxidative stress (n=60), repeated three independent experiments; different letters represent significant differences between groups, p<0.05; the same letters represent no significant differences between groups, p>0.05.

[0044] Figure 7 , Effect of pumpkin peel pigment on nematode survival rate under heat stress (n=60); different letters represent significant differences among groups, p<0.05; the same letters represent no significant differences among groups, p>0.05.

[0045] Figure 8 , bar graph of the average body weight of mice in each group at the end of the experiment (15th day); different letters represent significant differences between groups, p<0.05; the same letters represent no significant differences between groups, p>0.05.

[0046] Fig. 9 , Photos of the status of mice in the solvent group (a, b) and high-dose group (c, d) on the 15th day of the experiment.

[0047] Fig.10 , Effect of pumpkin peel pigment on serum SOD activity in mice (different letters indicate p<0.05).

[0048] Fig.11, Effects of pumpkin peel pigments on the levels of inflammatory factors TNF-α and IL-6 in mouse colon (different letters indicate p<0.05).

[0049] Fig.12 , Scanning electron microscopy image of pumpkin peel before modification (left: scale bar is 5 μm; right: scale bar is 1 μm).

[0050] Fig.13 , Scanning electron microscopy image of pumpkin peel before modification (left: scale bar is 5 μm; right: scale bar is 1 μm).

[0051] Fig.14 , the expansion force (left) and bound water force (right) of pumpkin peel DF before and after modification.

[0052] Fig.15 , the adsorption capacity of pumpkin peel DF for sodium cholate before and after modification. (V) Specific implementation methods

[0053] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0054] Example 1

[0055] 1 Materials and reagents

[0056] Fresh yellow wolf pumpkin was purchased from the market, free of pests and diseases and rot. Wild-type Caenorhabditis elegans N2 (C.elegans) and Escherichia coli OP50 were provided by the College of Animal Science, Zhejiang University; healthy male ICR mice (weight 28-32 g) were provided by Shanghai Slake Laboratory Animal Co., Ltd. [certificate number SCXK (Shanghai) 2017-0005]; superoxide dismutase (T-SOD, A001-1-1) kit was purchased from Nanjing Jiancheng Bioengineering Institute; TNF Alpha ELISA (EK0411) and IL-6 ELISA (EK0527) kits were purchased from Wuhan Boster Bioengineering Co., Ltd.; anhydrous ethanol, octanoic acid, decanoic acid, etc. were all analytically pure; β-carotene standard was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; pectinase (activity 10000U / g) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0057] Preparation of natural acid: Caprylic acid and decanoic acid are mixed in a molar ratio of 2.6:1, mixed thoroughly, and heated at 60°C for 25 minutes to obtain natural acid. Unless otherwise specified in the examples, natural acid refers to a mixture of caprylic acid and decanoic acid in a molar ratio of 2.6:1.

[0058] 2 Experimental instruments

[0059] Small high-speed pulverizer (model WK-400A, Beijing Guowei Hejiao Medical Equipment Co., Ltd.), AL04 electronic balance, SHA-B water bath shaker, DHG-9146A electric constant temperature blast drying oven, CT14R0 high-speed centrifuge, stirrer, BILON-10000FDA vacuum freeze dryer, UV1800PC ultraviolet-visible spectrophotometer, DS-5510DTH ultrasonic cleaner.

[0060] 3 Experimental methods

[0061] 3.1 Pumpkin peel pigment extraction method and extraction yield determination

[0062] (1) Pumpkin skin powder: Wash and peel the yellow wolf pumpkin, cut the pumpkin skin into strips, and place it in a vacuum freeze dryer. The initial temperature is -30°C. When the vacuum degree is 80Pa, start heating, heat to -20°C and keep it for 1 hour, then increase the temperature by 10°C and keep it for 1 hour, until the temperature reaches 20°C, and keep it for 24 hours until the water content is below 8%. After crushing, pass through a 200-mesh sieve to obtain pumpkin skin powder.

[0063] (2) Extraction method of pumpkin peel pigment: Take 5 g of pumpkin peel powder prepared in step (1), add 150 mL of natural acid as an extractant, and extract at an ultrasonic power of 150 W and 50° C. for 40 min.

[0064] (3) Extraction yield of pumpkin peel pigment: The extract in step (2) was centrifuged (8000 rpm, 20 min), the supernatant was collected, and the absorbance was measured by UV-visible spectrophotometry at a maximum absorption wavelength of 448 nm. The extraction yield of pumpkin peel pigment was calculated based on a standard curve of pigment concentration-absorbance.

[0065] (4) Pigment concentration-absorbance standard curve: β-carotene standard was prepared with ethanol at concentrations of 4, 8, 12, 16 and 20 μg / mL, and the absorbance was measured at a maximum absorption wavelength of 448 nm using UV-visible spectrophotometry. The standard curve was drawn with concentration as the ordinate and absorbance as the abscissa. The linear regression equation between concentration and absorbance was: y = 0.1329x - 0.0092 (R 2 =0.9882).

[0066] Pumpkin peel pigment extraction rate (mg / g) = C × V / M

[0067] C is the concentration of pumpkin peel pigment calculated according to the standard curve of pigment concentration-absorbance value (mg / mL); V is the volume of the diluted extract (mL), and the diluted volume ensures that the absorbance is below 0.2; M is the mass of pumpkin peel powder (g).

[0068] 3.2 Optimization of extraction agent

[0069] (1) Effect of the molar ratio of n-octanoic acid to n-decanoic acid in natural acid on the extraction yield of pumpkin peel pigment. The molar ratio of n-octanoic acid to n-decanoic acid in natural acid in step 3.1 was set to 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, and 3.0:1, respectively. The other operations were the same, and the extraction yield of pumpkin peel pigment was calculated.

[0070] (2) Effect of the ratio of natural acid (octanoic acid to decanoic acid at a molar ratio of 2.6:1) to ethanol on the extraction yield of pumpkin peel pigment. The extraction agent in step 3.1 was changed to natural acid containing ethanol at a volume concentration of 80%, 60%, 40%, and 20%. Other operations were the same and the extraction yield of pumpkin peel pigment was calculated.

[0071] 3.3 Optimization of enzymatic pretreatment conditions for pumpkin peel powder

[0072] (1) Single factor optimization of enzymatic pretreatment of pumpkin peel powder:

[0073] Amount of enzyme added:

[0074] 50g of pumpkin peel powder prepared by method 3.1 was placed in a brown bottle, deionized water was added in a ratio of 1g:25mL to form a slurry, pectinase was added (the amount of enzyme was 0.3%, 0.6%, 0.9%, 1.2% and 1.5% based on the weight of pumpkin peel powder), and after mixing, the pH of the system was adjusted to 3.5 with 1M hydrochloric acid. Then, the solution was ultrasonically treated at a temperature of 50°C and an ultrasonic power of 180W for 40min, and then centrifuged (8000rpm, 20min), the supernatant was taken, and the absorbance value was measured at a wavelength of 448nm by UV-visible spectrophotometry.

[0075] B Temperature: Based on A, the enzyme amount was selected to be 1.2%, and the temperature was changed to 20-60°C (20, 30, 40, 50, 60°C), and other operations were the same.

[0076] C Time: Based on A, the enzyme amount was selected to be 1.2%, and the time was changed to 20-60 min (20, 30, 40, 50, 60 min). Other operations were the same.

[0077] D pH: Based on A, the enzyme amount was selected to be 1.2%, and the pH was changed to 3.0-5.0 (3.0, 3.5, 4.0, 4.5, 5.0). Other operations were the same.

[0078] E Ultrasonic power: Based on A, the enzyme amount was selected to be 1.2%, and the power was changed to 120-240 W (120, 150, 180, 210, 240 W). Other operations were the same.

[0079] (2) Response surface optimization: Based on the single factor, the extraction rate of pumpkin peel pigment was taken as the response value. According to the principle of central composite experiment, the hydrolysis temperature, system pH and hydrolysis time were used as variables. The three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software to perform Box Benhnken Design response surface optimization.

[0080] (3) Optimized preparation process of pumpkin peel powder pretreated by enzyme method

[0081] 50g of pumpkin peel powder prepared by 3.1 method is placed in a brown bottle, deionized water is added to form a slurry with a solid-liquid ratio of 1g:25mL, 0.6g pectinase (added in an amount of 1.2% of the raw material mass) is added, and after mixing, the pH of the system is adjusted to 3.5 with 1M hydrochloric acid. Then, after ultrasonic treatment for 40min at a temperature of 50°C and an ultrasonic power of 180W, boil for 100s, and after cooling, adjust to neutrality with NaOH (1M) to obtain an enzymolysis mixture. The enzymolysis mixture is vacuum-freeze-dried (initial temperature is -30°C, heating is started when the vacuum degree is 80Pa, and heating is maintained for 1h when heated to -20°C, and then the temperature is maintained for 1h every 10°C increase, and the temperature is always raised to 20°C, and maintained for 24h until the water content is below 8%) to obtain an enzymolysis mixture of 48.6g, which is the enzymatic pretreatment pumpkin peel powder for subsequent pigment extraction.

[0082] 3.4 Pumpkin peel pigment extraction process

[0083] 3.4.1 Optimization of pumpkin peel pigment extraction process

[0084] (1) Single factor:

[0085] A. Ultrasonic power: 5 g of the enzymatically pretreated pumpkin peel powder prepared in step 3.3 was added with 150 mL of natural acid containing 60% by volume as an extractant, and the amount of the extractant added was 30 mL / g based on the weight of the enzymatically pretreated pumpkin peel powder. The powder was placed in an ultrasonic cleaner and extracted for 50 min at 40°C and an ultrasonic power of 180-300 W (180, 210, 240, 270, 300 W). The pumpkin peel pigment extraction rate was used as the screening index.

[0086] B Ultrasonic temperature: Based on A, the ultrasonic power was set to 180 W, and the temperature was changed to 30-70°C (30, 40, 50, 60, 70°C). Other operations were the same.

[0087] C Ultrasonic time: Based on A, the ultrasonic power was set to 180 W, and the time was changed to 50-90 min (50, 60, 70, 80, 90 min). Other operations were the same.

[0088] D. Material-liquid ratio: Based on A, the ultrasonic power was set to 180 W, and the amount of extractant added was changed to 10-50 mL / g (10, 20, 30, 40, 50 mL / g) based on the weight of the enzymatically pretreated pumpkin peel powder. Other operations were the same.

[0089] (2) Response surface optimization:

[0090] On the basis of single factor, taking pumpkin peel pigment extraction rate as response value, according to the principle of central composite test, with ultrasonic power, ultrasonic temperature and ultrasonic time as variables, a three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software, and Box Benhnken Design response surface analysis was performed.

[0091] 3.4.2 Optimal Process for Extracting Pumpkin Peel Pigment

[0092] After optimizing the optimal process parameters of the enzymatic pretreatment in 3.3 and the ultrasonic extraction in 3.4.1, the pumpkin peel powder obtained by the enzymatic method under the optimal pretreatment process parameters was further extracted by the ultrasonic method under the optimal extraction process parameters using a natural acid-alcohol two-phase system to obtain a high yield of pumpkin peel pigment, as follows:

[0093] 100g of enzymatically pretreated pumpkin peel powder was added to 2.5L of deionized water to form a slurry, and 0.9% of the weight of the pumpkin peel powder pectinase was added, mixed, and ultrasonic enzymolysis was performed for 53min at 55°C, pH 3.5 (1M hydrochloric acid to adjust pH), 150W, boiled for 100s, and adjusted to neutrality with NaOH (1M) after cooling to obtain an enzymolysis mixture. The enzymolysis mixture was vacuum freeze-dried (initial temperature was -30°C, heating was started when the vacuum degree was 80Pa, and it was kept for 1h when heated to -20°C, and then the temperature was kept for 1h every time it was increased by 10°C, and it was kept until it was heated to 20°C and kept for 24h until the water content was below 8%) to constant weight to obtain 95.2g of enzymolysis mixture. Take 10g of the enzymatic hydrolysis mixture, add 300mL of natural acid containing 40% ethanol by volume, ultrasonically extract for 64min at 50°C and 180W, centrifuge (8000rpm, 30min), obtain the filter residue and supernatant, and concentrate the supernatant to a pumpkin peel pigment concentrate with a pigment concentration of 15mg / mL for subsequent biological activity determination.

[0094] 3.5 Modification of pumpkin peel dietary fiber (DF)

[0095] Take 50g of the filter residue in 3.4.2, dry it with hot air at 60℃, and then crush it through a 150-mesh sieve to obtain pumpkin peel dietary fiber before modification. Use hydrogen peroxide with a mass concentration of 1.46% as a green modifier, and the liquid-to-solid ratio is 30mL / g. Use an ultrasonic wall-breaking machine for wall-breaking modification for 368s, centrifuge at 8000rpm for 15min, rinse the residue with deionized water three times, dry it with hot air at 60℃, crush it and sieve it, and prepare pumpkin peel dietary fiber with different particle sizes of >300 mesh, 200-300 mesh, 150-200 mesh and 100-150 mesh respectively.

[0096] 3.6 Determination of biological activity of pumpkin peel pigment

[0097] 3.6.1 Effect of pumpkin peel pigments on the survival rate of C.elegans under oxidative stress.

[0098] M9 buffer solution: Accurately weigh 15.0 g Na2HPO4·12H2O, 3.0 g KH2PO4, 5.0 g NaCl, and 0.12 g MgSO4, dissolve them in 800 mL of water and then make up to 1000 mL, mix well, and sterilize with high-pressure steam at 121°C for 30 min.

[0099] Sample solution: Take the pumpkin peel pigment concentrate prepared by the method in step 3.4.2, and use olive oil-pure milk (the two are prepared in a volume ratio of 1:1) as a solvent to prepare sample solutions with pumpkin peel pigment mass concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg / mL in sequence.

[0100] NGM medium (1L) was prepared by adding 2.5g peptone, 3g NaCl, 17g agar, 25mL 1mol / L PBS buffer (pH 6.0), 975mL distilled water, and after sterilization, adding 1mL 5mg / mL cholesterol solution (solvent is ethanol) sterilized by filter membrane, 1mL 1mol / L MgSO4 aqueous solution, and 1mL 1mol / L CaCl2 aqueous solution.

[0101] LB liquid culture medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjusted to neutral with 5 M NaOH, the solvent is water.

[0102] Lysis solution: 0.5 mL deionized water, 0.3 mL NaClO, 0.2 mL 10 mol / L NaOH aqueous solution, prepared and used immediately.

[0103] Nematode culture: 100 μL E. coli OP 50The bacterial solution was inoculated into 100 mL of LB liquid medium and cultured at 37°C in a shaking incubator for 12 h. When the medium became turbid, it was taken out and placed in a 4°C refrigerator for later use. 50 μL of E. coli OP 50 Bacterial solution (concentration of 10 9 CFU / mL) was spread on NGM plates and dried, and then wild-type Caenorhabditis elegans N2 was inoculated on the plates. After constant temperature culture at 20°C for 72 h, a large number of adults and larvae were visible on the culture medium.

[0104] Synchronization of nematodes: Select a plate with good nematode growth, pipette 1.5mL M9 buffer onto the plate, wash the plate several times until most of the worms are washed off, pipette 1mL of nematode suspension into a 1.5mL EP tube, centrifuge at 3000rpm for 1min, discard the supernatant, and re-dissolve the precipitate with 0.25mL deionized water. Then add 0.15mL lysis buffer, vortex for 5min, centrifuge at 4000rpm for 1min, discard the supernatant, and then add 1mL M9 buffer to re-dissolve, vortex and wash, centrifuge at 4000rpm for 1min, and repeat the washing until there is no smell of sodium hypochlorite. Place the lysed eggs on an uncoated E.coli OP tube. 50 The cells were cultured in a 20°C incubator for 12 to 18 hours on NGM medium to obtain L1 nematodes. The worms were washed with M9 buffer and placed on a plate coated with E. coli OP 50 The cells were cultured on NGM medium in a 20°C incubator for 48 h to obtain synchronized L4 nematodes for subsequent experiments.

[0105] C.elegans survival rate experiment: The experiment set up a negative control group (M9 buffer), a positive control group (Vc, 0.5 mg / mL, solvent sample solution) and a pumpkin peel pigment sample group (1, 0.5, 0.25, 0.125, 0.0625 mg / mL). Each group was set up in parallel. First, 150 μL of E. coli OP50 (concentration of 10 9 CFU / mL), then 1mL of M9 buffer was added to the negative control group, 1mL of Vc solution was added to the positive control group, and 1mL of sample solution of different concentrations was added to the pumpkin skin pigment sample group. Then, the L4 stage nematodes after synchronization were picked to each group of plates, 3 parallel groups of 60 C.elegans in total, and cultured at 20℃ for 48h. Then transferred to a 96-well plate, 200μL of 4mM hydrogen peroxide and 200μL of M9 buffer were added to each well, and cultured at 20℃. The number of C.elegans deaths was recorded every 40min, and the survival rate of C.elegans at different time points was calculated.

[0106] 3.6.2 Effect of pumpkin peel pigments on the survival rate of C.elegans under heat stress.

[0107] C.elegans survival rate experiment: The experiment set up a negative control group (M9 buffer) and pumpkin peel pigment sample groups (0.0625, 0.125, 0.25, 0.5, 1.0 mg / mL). Each group was set up in parallel. First, 150 μL of E. coli OP50 (concentration of 10 9 CFU / mL), then 1mL of M9 buffer was added to the negative control group, and 1mL of sample solution of different concentrations was added to the pumpkin skin pigment sample group. Then 20 L4 nematodes after synchronization were picked to each group of plates, 3 parallel groups of 60 C.elegans in total, and cultured at 20℃ for 48h. Then, each group of C.elegans was transferred to 35℃ for heat treatment for 5h, and the number of C.elegans surviving was recorded, and the survival rate was calculated according to the formula (surviving number / initial number)×100%.

[0108] 3.6.3 Determination of the weight loss effect of pumpkin peel pigment

[0109] (1) Grouping. ICR male mice (28-32g) were kept at 23℃±2℃, relative humidity 55-60%, and 12h light / 12h dark. The mice were fed adaptively for one week after purchase, during which they were free to eat. After one week, the mice were randomly divided into 4 groups, 6 mice in each group. There should be no difference in the weight of mice between the groups. They were normal control, natural acid solvent control group, pumpkin skin pigment low-dose group, and pumpkin skin pigment high-dose group.

[0110] (2) Administration. Mice were administered by gavage at a dosage of 0.3 mL / 30 g body weight. The normal control group was gavaged with physiological saline, the natural acid solvent control group was gavaged with a mixture of natural acid and olive oil at a volume ratio of 1:14, the pumpkin peel pigment low-dose group was gavaged with a mixture of the pumpkin peel pigment concentrate prepared in step 3.4.2 and olive oil at a volume ratio of 1:29 (equivalent to a daily gavage dose of 5 mg / kg body weight for pumpkin peel pigment), and the pumpkin peel pigment high-dose group was gavaged with a mixture of the pumpkin peel pigment concentrate prepared in step 3.4.2 and olive oil at a volume ratio of 2:28 (equivalent to a daily gavage dose of 10 mg / kg body weight for pumpkin peel pigment).

[0111] (3) Body fat percentage. The status of each group of mice was observed every day, and the weight of the mice was measured and recorded. The experimental period was 15 days. After 15 days, the mice were dissected, and the left and right epididymal fat was taken and weighed to calculate the body fat percentage. Body fat percentage = (left and right epididymal fat) / body weight × 100%.

[0112] 3.6.4 Determination of the ability of pumpkin peel pigment to resist colitis in mice

[0113] (1) Grouping. ICR male mice (28-32 g) were fed ad libitum with 12 h light / 12 h dark. After one week of adaptive feeding, they were randomly divided into five groups, each with six mice, namely, normal control group (CON), mesalamine (MES) positive drug control, dextran sulfate sodium (DSS) model group, low-dose pumpkin skin pigment group (d-PSP) and high-dose pumpkin skin pigment group (PSP (pumpkin skin pigment)).

[0114] (2) Administration. The mice were administered by gavage, with a dosage of 0.3 mL / 30 g body weight, and gavage was performed once a day. The mice were fed according to Table 1. They drank deionized water on days 1-14. The normal control group and the model group were gavaged with physiological saline, respectively. The positive drug control group was gavaged with mesalazine in physiological saline (mesalazine dosage was 100 mg / kg body weight). The low-dose group of pumpkin peel pigment was gavaged with a mixture of pumpkin peel pigment concentrate prepared by the method of step 3.4.2 and olive oil at a volume ratio of 1:14 (pumpkin peel pigment dosage was 10 mg / kg body weight). The high-dose group of pumpkin peel pigment was gavaged with a mixture of pumpkin peel pigment concentrate prepared by the method of step 3.4.2 and olive oil at a volume ratio of 1:7 (pumpkin peel pigment dosage was 20 mg / kg body weight). On days 15-21, except for the normal control group, the drinking water of mice in other groups was replaced with physiological saline containing a mass concentration of 2.5% DSS, and the administration of each group was the same as on days 1-14. The mice were observed every day, and their weights were measured and recorded. The experimental period was 21 days.

[0115] Table 1 Animal experiment design

[0116]

[0117] (3) Sample collection and related biochemical index detection. On the 21st day after the mouse model was established, the mouse blood was collected by orbital venous plexus sampling, and the supernatant was collected after standing at 4°C overnight and centrifuged (3000rpm, 15min). The T-SOD activity was determined according to the kit instructions. After blood collection, the mouse was dissected to obtain colon tissue, which was washed with saline and then quickly cooled with liquid nitrogen and stored at -80°C. During the test, the colon tissue was homogenized and the content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was determined according to the method in the kit instructions.

[0118] 3.7 Scanning electron microscopy (SEM) observation of pumpkin skin DF

[0119] 0.5 g pumpkin peel dietary fiber before modification and 0.5 g pumpkin peel DF (150-200 mesh) after modification were taken respectively, the samples were placed on MC1000 ion sputtering instrument (icn sputter) for coating, and observed in itachi SU8010 field emission scanning electron microscope (SEM).

[0120] 3.8 Determination of physicochemical properties of pumpkin peel DF

[0121] 3.8.1 Determination of the expansion force of pumpkin peel DF

[0122] Take 1.000g (mass is recorded as M0) of pumpkin peel DF samples of different mesh sizes and put them into a 10mL measuring cylinder, measure the volume of dry powder, and record it as V0. Then take 0.1g (mass is recorded as M1) of sample into the measuring cylinder, add water to 10mL, stir it with a glass rod to make it fully contact with water, let it stand at 37℃ for 24h (natural sedimentation), read its volume after expansion, and record it as V1. The expansion force is calculated according to the following formula:

[0123] Expansion force (mL / g) = (M0V1-M1V0) / M0M1

[0124] 3.8.2 Determination of binding hydraulic capacity of pumpkin peel DF

[0125] Take 1.000g pumpkin peel DF samples of different mesh sizes and place them in a 100mL beaker, add 50mL of distilled water, let stand at 37℃ for 2h, filter through a 0.75mm nylon mesh until no water drops, move the residue to a balance and weigh the wet weight of the sample, record it as M1, dry it at 110℃ to constant weight, and record the mass as M2. Calculate according to the following formula combined with hydraulic pressure:

[0126] Bound water power (g / g) = (M1-M2) / M2

[0127] 3.8.3 Determination of the adsorption capacity of pumpkin peel DF for sodium bile

[0128] Cholate standard curve: Prepare 0.3mmol / L sodium glycocholate solution with 0.1mol / L phosphate buffer (pH 6.3), take 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5mL in 25mL stoppered test tubes, add 0.1mol / L phosphate buffer (pH 6.3) to 2.5mL, add 7.5mL of 60% concentrated sulfuric acid aqueous solution, bathe in 70℃ water bath for 20min, then ice bath for 5min, measure absorbance at wavelength 387nm. Draw cholate standard curve with cholate content as horizontal axis and absorbance as vertical axis.

[0129] Bile salt binding capacity: 1.00 g of pumpkin peel DF samples of different mesh sizes were weighed into 50 mL conical flasks, and the pH value was adjusted to 6.3 with 0.1 mol / L NaOH aqueous solution, followed by adding 4 mL of trypsin solution (10 mg / mL) prepared with 0.1 mol / L phosphate buffer (pH 6.3), and then digested in a constant temperature water bath at 37°C for 1 h to simulate the intestinal environment. 4 mL of sodium glycocholate solution (0.3 mmol / L, the amount of sodium glycocholate added was recorded as C0) prepared with 0.1 mol / L phosphate buffer (pH 6.3) was added to each sample, and after shaking in a water bath at 37°C for 1 h, the samples were centrifuged at 3800 r / min for 20 min, 2.5 mL of the supernatant was taken, and 7.5 mL of 60% sulfuric acid aqueous solution was added, and the absorbance value was measured at a wavelength of 387 nm by colorimetry, and the sodium cholate content (C1) was obtained according to the bile salt standard curve. In addition to the above test groups, sample blank group and reagent blank group were also performed. Sample blank group means no pumpkin peel DF sample was added, and other operations were the same as the test group. Reagent blank group means no sodium glycocholate solution was added (the sodium glycocholate solution was replaced with an equal volume of 0.1 mol / L phosphate buffer), and other operations were the same as the test group.

[0130] The adsorption capacity of sodium cholate was calculated according to the following formula:

[0131] Sodium cholate adsorption rate (%) = (C0-C1) / C0×100

[0132] Wherein, C0 is the amount of sodium glycocholate added, μmol; C1 is the remaining amount of sodium glycocholate, μmol.

[0133] 3.9 Data Processing

[0134] The experimental data were expressed as Mean±SD, and the significance difference was analyzed by one-way analysis of variance (ANOVA) and LSD multiple comparison analysis in IBM SPSS Statistics 25 software. p<0.05 was considered significant.

[0135] 4 Experimental results

[0136] 4.1 Effect of extractants on the extraction rate of pumpkin peel pigment

[0137] (1) Natural acid (DES) composition screening

[0138] Depend on Figure 1 It can be seen from Figure A that when the molar ratio of caprylic acid to capric acid is 2.6:1, the dissolution rate of pumpkin peel pigment is the highest; the optimal natural acid formula for pumpkin peel pigment extraction is determined to be caprylic acid: capric acid = 2.6:1.

[0139] (2) Screening of the ratio of ethanol to natural acid

[0140] Depend on Figure 1 As can be seen from Figure B, adding 40% anhydrous ethanol (i.e., DES: anhydrous ethanol = 3:2) to the natural acid can further promote the dissolution of pumpkin peel pigment. Therefore, 40% anhydrous ethanol was prepared as the natural acid-alcohol two-phase extraction system for pumpkin peel pigment using caprylic acid and decanoic acid (molar ratio 2.6:1) in DES as the base solvent.

[0141] 4.2 Optimization of process parameters for enzymatic pretreatment of pumpkin peel powder

[0142] (1) Single factor experiment on the effect of enzyme dosage, enzymatic hydrolysis temperature, enzymatic hydrolysis time, pH and ultrasonic power on the dissolution rate of pumpkin peel pigment Figure 2 .

[0143] Depend on Figure 2 It can be seen that the absorbance of pumpkin peel pigment extract increases significantly with the increase of enzyme dosage, and no longer increases significantly after 0.9%. Considering the application cost, the subsequent enzyme dosage is determined to be 0.9%. The absorbance of pumpkin peel pigment extract shows a significant increasing trend with the increase of ultrasonic power. When the ultrasonic power increases to more than 150W, the absorbance no longer increases significantly, so the subsequent ultrasonic power is determined to be 150W. The effects of enzymatic hydrolysis time, enzymatic hydrolysis temperature and system pH on the absorbance of pumpkin peel pigment extract all have inflection points with the increase of levels, so the center points of the three factors are selected as 50min, 50℃ and pH3.5 respectively.

[0144] (2) The optimization of the enzymatic extraction process was based on a single factor experiment. The absorbance of the pumpkin peel pigment extract was used as the screening index. According to the principle of the central composite experiment, a three-factor three-level response surface analysis experiment was designed using Design-Expert 8.0.6 software. The factor levels of the response surface experiment are shown in Table 2, and the experimental design and results are shown in Table 3.

[0145] Table 2. Experimental design factor level table of enzymatic extraction of pumpkin peel pigment

[0146] Coding Level A: Enzymatic hydrolysis time (min) B: System pH C: Enzymatic hydrolysis temperature (℃) -1 40 3 40 0 50 3.5 50 1 60 4 60

[0147] Table 3 Experimental design and results of enzymatic extraction of pumpkin peel pigment

[0148]

[0149] The Design-Expert 8.0.6 software was used to perform multivariate fitting on the experimental data of the absorbance of the pumpkin peel pigment extract in Table 3, and the quadratic multivariate regression model of the absorbance of the pumpkin peel pigment extract (Y) and the enzymatic hydrolysis time (A), the system pH (B) and the enzymatic hydrolysis temperature (C) was obtained as follows:

[0150] Y=0.94+0.038*A+0.025*B+0.048*C-0.017*AB-0.043*AC-0.062*BC-0.032*A 2 -0.092*B 2 -0.042*

[0151] C 2

[0152] The significance analysis of the regression model is shown in Table 4, which shows that the influence of various factors on the extraction rate of pumpkin peel pigment is enzymatic hydrolysis temperature > enzymatic hydrolysis time > system pH. The linear relationship between the dependent variable and all independent variables is significant. In the regression equation, Y represents the predicted value of pumpkin peel pigment absorbance. R 2 (coefficient of determination) and R 2 Adj (adjusted determination coefficient) was 0.9639 and 0.9175, respectively, indicating that the predicted value and the observed value have a high correlation. At the same time, the low coefficient of variation (CV% 2.81) indicates a high degree of reliability and precision. p is used to detect the significance of each coefficient, among which the p of the model (0.0003) < 0.01, indicating that the model is significantly adaptable; the p of the lack of fit term (0.0788) > 0.05 is not significant, indicating that the experimental method is reliable and the model achieves a good fit.

[0153] Table 4 Regression model analysis of dissolution rate of pumpkin peel pigment extracted by cellulase

[0154] Source Sum of Squares df Mean Square F Value P-value Prob>F Model 0.11 9 0.012 20.76 0.0003 A-enzyme hydrolysis time 0.011 1 0.011 19.07 0.0033 B-System pH 5.000E-003 1 5.000E-003 8.47 0.0226 C-enzymatic hydrolysis temperature 0.018 1 0.018 30.59 0.0009 AB 1.225E-003 1 1.225E-003 2.08 0.1928 AC 7.225E-003 1 7.225E-003 12.25 0.0100 BC 0.016 1 0.016 26.48 0.0013 <![CDATA[A 2 ]]> 4.379E-003 1 4.379E-003 7.42 0.0296 <![CDATA[B 2 ]]> 0.036 1 0.036 60.73 0.0001 <![CDATA[C 2 ]]> 7.516E-003 1 7.516E-003 12.74 0.0091 Residual 4.130E-003 7 5.900E-004 Lack of fit 3.250E-003 3 1.083E-003 4.92 0.0788

[0155] Make a response surface diagram based on the experimental results ( Figure 3 ), that is, the three-dimensional space diagram composed of the response value Y and the corresponding factors A, B, and C, can intuitively reflect the influence of each factor on the response value Y. According to the response surface optimization diagram, the interaction of the influence of each factor on the absorbance of pumpkin peel pigment can be seen, so as to determine the appropriate process conditions.

[0156] The dissolution rate of pumpkin peel pigment was comprehensively analyzed by enzyme-assisted extraction, and the best response point was obtained: enzymatic hydrolysis time was 53.02min, system pH was 3.48, and enzymatic hydrolysis temperature was 54.43℃. Under this condition, the theoretical value of the absorbance of pumpkin peel pigment extract was predicted to be 0.9576. Considering the feasibility of actual operation, the optimal modification conditions were adjusted to: enzymatic hydrolysis time was 53min, system pH was 3.5, and enzymatic hydrolysis temperature was 55℃. In order to further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out under the optimized conditions. The average absorbance of pumpkin peel pigment extract was 0.94±0.03, which was basically consistent with the predicted value, indicating that the response surface analysis method is reliable and fits the actual situation well.

[0157] 4.3 Optimization of extraction process parameters of pumpkin peel pigment

[0158] The effects of ultrasonic temperature, time, material-liquid ratio and ultrasonic power on the dissolution rate of pumpkin peel pigment are shown in Figure 4 .

[0159] Depend on Figure 4 It can be seen that the extraction rate of pumpkin peel pigment increases significantly with the increase of ultrasonic power, and no longer increases significantly after 180W, so the subsequent ultrasonic power is determined to be 180W. The effects of temperature, liquid-to-solid ratio and time on the extraction rate of pumpkin peel pigment all have inflection points as the levels increase, so the center points of the three factors are selected as 50℃, 30mL / g and 70min respectively. According to the principle of central composite test, Design-Expert.V8.0.6 software was used, and the response surface experimental factor levels are shown in Table 5, and the experimental design and results are shown in Table 6.

[0160] Table 5 Factor level table of the experimental design for extracting pumpkin peel pigment

[0161] Coding Level A: Extraction temperature (℃) B: Ultrasonic time (min) C: Liquid-to-solid ratio (mL / g) -1 40 50 20 0 50 70 30 1 60 90 40

[0162] Table 6 Experimental design and experimental results of extracting pumpkin peel pigment

[0163]

[0164] Design-Expert 8.0.6 software was used to perform multivariate fitting on the experimental data of pumpkin peel pigment extraction yield in Table 6, and the quadratic multivariate regression model of pumpkin peel pigment extraction yield (Y) and extraction temperature (A), ultrasonic time (B), and liquid-to-solid ratio (C) was obtained as follows:

[0165] Y=4.43+0.038A+0.012B-0.011C-0.070AB-0.11AC+0.058BC-0.22A 2 -0.20B 2 -0.10C 2

[0166] According to the Box-Behnken Design model, variance analysis was performed on the experimental data in Table 6. The significance analysis of the regression model is shown in Table 7, which shows that the influence of various factors on the extraction rate of pumpkin peel pigment is in the order of ultrasonic temperature > ultrasonic time > solid-liquid ratio. The linear relationship between the dependent variable and all independent variables is significant. In the regression equation, Y represents the predicted value of pumpkin peel pigment dissolution. R 2 (coefficient of determination) and R 2The Adj (adjusted determination coefficient) was 0.9037 and 0.7799, respectively, indicating that the predicted value and the observed value have a high correlation. At the same time, the low coefficient of variation (CV% 2.23) indicates a high degree of reliability and precision. p is used to detect the significance of each coefficient, among which the p of the model (0.0079) < 0.01, indicating that the model is significantly adaptable; the p of the lack of fit term (0.1413) > 0.05 is not significant, indicating that the experimental method is reliable and the model achieves a good fit.

[0167] Table 7 Analysis of variance of model regression equation

[0168]

[0169] Make a response surface diagram based on the experimental results ( Figure 5 ), that is, the three-dimensional space diagram composed of the response value Y and the corresponding factors A, B, and C, can intuitively reflect the influence of each factor on the response value Y. According to the response surface optimization diagram, the interaction of each factor on the dissolution process of pumpkin peel pigment can be seen, so as to determine the appropriate process conditions.

[0170] The steepness of the response surface is positively correlated with the influence of the density of contour lines on the response value. Figure 5 The response surface of the BC interaction term is steep and the contour lines are denser, indicating that the BC interaction has a significant effect, which is consistent with the results of variance analysis. The model optimization conditions are: ultrasonic time 63.68min, solid-liquid ratio 1:30.25, and ultrasonic temperature 50.08℃. Under these conditions, the theoretical value of pumpkin peel pigment dissolution rate is predicted to be 4.4354mg / g. Considering the feasibility of actual operation, the optimal modification conditions are adjusted to: ultrasonic time 64min, solid-liquid ratio 1g:30mL, and ultrasonic temperature 50℃.

[0171] In order to further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out under the optimized conditions. The average pumpkin peel pigment yield was 4.499±0.094 mg / g, which was basically consistent with the predicted value, indicating that the response surface analysis method is reliable and fits the actual situation well.

[0172] 4.4 Effect of pumpkin peel pigment on the survival rate of C.elegans under oxidative stress and heat stress

[0173] (1) Oxidative stress

[0174] Hydrogen peroxide is easily metabolized to highly reactive hydroxyl radicals that attack organic molecules and is widely used as an effective oxidant that induces oxidative stress in vivo and in vitro. Figure 6 It can be seen that under hydrogen peroxide stress, the average survival time of C.elegans in the negative control group (T aver ) was only 130.57min, and the T of the positive control group C.elegansaver All concentrations (0.0625 mg / mL-1.0 mg / mL) of pumpkin peel pigment can shift the survival time curve of C. elegans to the right. The medium concentration (0.125 mg / mL) of pumpkin peel pigment can significantly prolong the T of C. elegans. aver (p<0.05) to 169.3min, with no difference compared with the positive control; while the high concentration (0.25-1.0mg / mL) pumpkin peel pigment had a weaker effect (p>0.05), which may be related to the increase in osmotic pressure; it may also be that the high concentration produces other physiological toxicity, affecting the normal physiological activities of C.elegans. In summary, all concentrations selected in the experiment can prolong the survival rate of C.elegans under stress, indicating that pumpkin peel pigment has a significant anti-oxidative stress effect.

[0175] (2) Heat stress

[0176] Heat stress can cause oxidative stress in cells, leading to an increase in oxidative metabolites in tissue cells, increased levels of oxygen free radicals and peroxides, and affecting the body’s normal physiological metabolism. Figure 7 As shown, after heat treatment at 35℃ for 5h, the survival rate of C.elegans in the negative group was only 45.0%. All concentrations of pumpkin peel pigments could increase the survival rate of C.elegans, but the increase was not significant at high concentration (1.0mg / mL) and low concentration (0.0625mg / mL). Medium concentration (0.125-0.5mg / mL) pumpkin peel pigments could significantly reduce the survival rate of C.elegans (p<0.05).

[0177] 4.5 Effect of pumpkin peel pigment on mouse body weight

[0178] The average weight of mice in each group during the experiment is shown in Table 8. The average weight of mice in each group at the end of the experiment is shown in the bar graph. Figure 8 . There was no significant difference in the weight of mice in each group at the beginning and after one week of adaptation, that is, on day 0. During the experiment, the weight of mice in the normal group increased steadily, indicating that the existing experimental conditions were suitable for mouse growth. After one week of adaptive feeding, the weight of mice in other groups increased significantly on the first day of the experiment; in the following 15 days, the weight of mice in each group showed a continuous downward trend, among which the weight of mice in the low-dose group decreased slowly. By the 15th day, the average weight had dropped to 33.52g, but there was no significant difference between them and the normal control group ( Figure 8 , p>0.05). The solvent control group decreased significantly, and on the 15th day, the average body weight dropped to 31.75g, which was significantly different from the normal control group ( Figure 8, p<0.05), which is due to the presence of a mixture of caprylic acid and capric acid in the extraction solvent. Caprylic acid and capric acid are medium-chain fatty acids that have a fat-reducing effect. The weight loss of mice in the high-dose group was the most obvious. By the 15th day, the average weight dropped to 30.47g, which was significantly different from the normal control group ( Figure 8 , p<0.05), and further reduced body weight compared with the solvent group, proving that pumpkin peel pigment can enhance the weight loss effect of the solvent.

[0179] Table 8 Dynamic effects of pumpkin peel pigment on mouse body weight

[0180]

[0181] Depend on Figure 8 It can be seen that although there is no significant difference in the weight of mice between the high-dose group and the solvent group, the average weight of the high-dose mice is lower than that of the solvent group. Most importantly, the condition of the mice in the high-dose group is significantly better than that in the solvent group. Fig. 9 In the later stage of the experiment, the mice in the solvent group not only lost weight, but also had rough, dull, loose and flaky fur, showing typical symptoms of stress response. However, the mice in the high-dose group did not have the stress response symptoms of the mice in the solvent group, and their fur was shiny and smooth.

[0182] The body fat of mice in different groups after the 15th day of the experiment is shown in Table 9. The normal group mice had the heaviest body fat, the low-dose group was close to the normal group, and the high-dose group was the lowest. The body fat percentage of mice in each group had the same pattern. In summary, high-dose pumpkin peel pigment (i.e., 10 mg / kg body weight) not only has the effect of reducing the weight of mice, but also can alleviate the stress response caused by natural acid solvents.

[0183] Table 9 Effects of pumpkin peel pigment on body fat and body fat degree in mice

[0184]

[0185] 4.6 Effects of pumpkin peel pigment on oxidative stress levels and colon inflammatory factors in mice

[0186] Oxidative damage and oxidative stress levels are important markers for evaluating colitis. The dextran sulfate sodium salt (DSS) colitis model is similar to humans and has become one of the most widely used colitis models. Superoxide dismutase (SOD), as one of the most important enzymes in the intracellular antioxidant system, is the first line of defense against aging and maintaining health. It catalyzes the dismutation of superoxide anion free radicals to generate hydrogen peroxide and oxygen. Fig.10There was no significant difference in T-SOD activity in the serum of mice between the CON group and the DSS group (p>0.05). Compared with the DSS group, both high and low doses of pumpkin peel pigment could significantly increase T-SOD activity in the serum of mice (p<0.05), and the effect was better than that of the positive drug MES group, indicating that pumpkin peel pigment can significantly enhance the anti-oxidative stress level of mice.

[0187] TNF-α and IL-6 are representative pro-inflammatory cytokines. TNF-α is involved in inflammatory response, immune regulation, etc., and is an important indicator for judging the body's immune function; IL-6 is a polypeptide substance that is mainly expressed in inflammatory response (released by inflammatory cells). Fig.11 , the serum TNF-α level of mice in the DSS group was significantly increased compared with that in the CON group (p<0.05), and the IL-6 level was also higher than that in the CON group, indicating that DSS increased the level of inflammation in mice. Compared with the DSS group, the levels of TNF-α and IL-6 in the serum of mice in the pumpkin peel pigment group were significantly reduced (p<0.05), and the IL-6 level was significantly lower than that in the MES positive drug group, and there was no difference in the TNF-α level between the MES group and the MES group. Low-dose pumpkin peel pigment can significantly increase the serum TNF-α level of mice (p<0.05), but the IL-6 level is no different from that in the DSS group. High-dose pumpkin peel pigment can significantly inhibit the increase in the level of serum inflammatory factors in mice induced by DSS.

[0188] 4.7 Analysis of the microstructure and physicochemical properties of pumpkin peel DF before and after modification

[0189] The scanning electron microscopy images of pumpkin peel before and after DF modification are shown in Fig.12 and Fig.13 .

[0190] Scanning electron microscopy technology has become an indispensable auxiliary means for the comprehensive analysis of macromolecular structural characteristics. Fig.12 and Fig.13 It can be seen that the cell wall of pumpkin peel dietary fiber was relatively intact before modification, with only slight ruptures. The cell wall of pumpkin peel dietary fiber after modification was quite different from that before modification. The cell wall surface had ridge peaks, forming a loose and porous state, and the holes and gaps were dark black, which was conducive to the enhancement of binding water, swelling power and adsorption capacity.

[0191] The hydration capacity (such as swelling power and water binding power) of pumpkin peel DF with different mesh sizes before and after modification is shown in Figure 2. Fig.14 .

[0192] The hydration properties of pumpkin peel DF are related to its chemical structure, fiber porosity, particle size, etc. Fig.14Under the same conditions, the particle size has a greater influence on the binding water capacity of pumpkin DF. The expansion force and binding water capacity of the modified pumpkin DF are greater than those of pumpkin peel powder with the same mesh size, and show a trend of first increasing and then decreasing as the particle size decreases. When the mesh size is 200-300 mesh, the expansion force reaches 53.23±0.45mL / g, and the binding water capacity reaches 14.21±0.081g / g, which are 1.62 times and 2.31 times before modification, respectively. The reason may be that as the particle size of pumpkin peel DF decreases, it expands and stretches after dissolving in water, producing a greater volume effect, which increases the hydration capacity of pumpkin peel DF. When the particle size of pumpkin peel DF is too small, the hydration capacity shows a decreasing trend. This is because its network structure is destroyed, causing it to stack on each other and cannot support a larger space.

[0193] The adsorption capacity of pumpkin peel DF with different mesh sizes before and after modification is as follows Fig.15 .

[0194] Bile salts refer to a class of amphipathic macromolecules with a steroidal core structure from the body's bile. DF promotes cholesterol metabolism by adsorbing bile salts and excreting them from the body, reducing the reabsorption of bile acid, cutting off the enterohepatic circulation of cholesterol, and significantly reducing the cholesterol level in the body. Therefore, understanding the ability of DF to adsorb bile salts can help understand its ability to regulate blood lipids. Fig.15 The adsorption capacity of modified pumpkin peel DF for sodium cholate was greater than that of pumpkin peel powder with the same mesh size, and it increased first and then decreased with the decrease of particle size. When the mesh size was 150-200, pumpkin peel DF had the highest adsorption capacity for sodium cholate, reaching 72.76±0.031 mg / g.

[0195] Example 2: Preparation of pumpkin peel pigment and pumpkin peel dietary fiber by optimal extraction process

[0196] According to the optimal process parameters optimized by ultrasound-assisted enzyme pretreatment and natural acid-alcohol ultrasonic extraction in Example 1, pumpkin peel powder was first prepared under low temperature conditions, and then the prepared pumpkin peel powder was pretreated by wall breaking according to the optimized process parameters, and then the pumpkin peel pigment was extracted using the optimized optimal extraction process parameters, and finally the anti-stress ability and anti-colitis activity of the extracted pumpkin peel pigment were measured. The details are as follows:

[0197] (1) 20 g of pumpkin peel powder (same as 3.1 in Example 1) was placed in a brown bottle, 500 mL of deionized water was added at a solid-liquid ratio of 1 g:25 mL to form a slurry, 0.9% of the mass of pectinase of the pumpkin peel powder was added, the mixture was mixed, and ultrasonic enzymolysis was performed at 55° C., pH 3.5 (1 M hydrochloric acid to adjust the pH), and 150 W for 53 min. The mixture was boiled for 100 s, cooled and adjusted to neutral with NaOH (1 M), and vacuum freeze-dried (the initial temperature was -30° C., heating was started when the vacuum degree was 80 Pa, and the mixture was heated to -20° C. and maintained for 1 h. The temperature was then increased by 10° C. and maintained for 1 h. The temperature was increased to 20° C. and maintained for more than 24 h until the sample was dry and the water content was below 8%) to obtain 19.4 g of an enzymolysis mixture, i.e., the pumpkin peel powder pretreated by the enzymatic method.

[0198] (2) Take 10g of the enzymatic hydrolysis mixture, add 300mL of natural acid (caprylic acid and capric acid molar ratio 2.6:1) containing 40% ethanol by volume, so that the material-liquid ratio is 1g:30mL. In an ultrasonic cleaner, ultrasonicate for 64min at an ultrasonic power of 180W and an ultrasonic temperature of 50°C. Centrifuge (8000rpm, 20min) to obtain a supernatant and a filter residue. The yield of pumpkin skin pigment in the supernatant is 4.499mg / g. The supernatant is concentrated to 3 / 5 of the original volume to obtain a crude pumpkin skin pigment. The method of Example 1 was used for detection. Pumpkin skin pigment can shift the growth curve of C.elegans to the right under oxidative stress, and T aver The survival rate of C.elegans under heat stress (5h at 35℃) was 73%. Pumpkin peel pigment can significantly inhibit the increase of inflammatory factors TNF-α and IL-6 in the serum of mice induced by DSS (p<0.05), and significantly enhance the activity of SOD in the serum of mice (p<0.05), increase the antioxidant stress level of colitis mice, and reduce the weight of mice. There is no stress reaction symptom of extraction solvent, and the fur is shiny and soft.

[0199] (3) The filter residue of step (2) was dried with hot air at 60° C., crushed and sieved through a 150-mesh sieve, and 1.46% hydrogen peroxide was used as a green modifier with a liquid-to-solid ratio of 30 mL / g. The filter residue was subjected to a wall breaking modification treatment using an ultrasonic wall breaking machine for 368 s, and centrifuged at 8000 r / min for 15 min. The filter residue was rinsed with deionized water three times, dried with hot air at 60° C., crushed and sieved, and pumpkin peel DF of different particle sizes of >300 mesh, 200-300 mesh, 150-200 mesh and 100-150 mesh were prepared respectively. The method of Example 1 was used for detection. When the mesh number of pumpkin peel DF was 200-300 mesh, the swelling force reached 53.2 mL / g, the bound water capacity reached 14.2 g / g, and the adsorption capacity for sodium cholate reached 55.1 mg / g; when the mesh number was 150-200 mesh, the swelling force reached 34.3 mL / g, the bound water capacity reached 4.3 g / g, and the adsorption capacity for sodium cholate reached 72.8 mg / g.

[0200] Comparative Example 1 Effect of pumpkin peel powder treatment process on pigment extraction rate and DF physical and chemical properties

[0201] Wash and peel the yellow wolf pumpkin, dry 20 g of pumpkin peel in a hot air dryer at 70° C., grind it, and pass it through a 200-mesh sieve to obtain 11.9 g of pumpkin peel powder.

[0202] The pumpkin peel powder described above was used to replace the pumpkin peel powder in step (1) of Example 2, and the pumpkin peel pigment was extracted and pumpkin peel DF powder with a mesh size of 200-300 was prepared using the method of Example 2. The test steps and operations for the average survival time of C.elegans under oxidative stress were the same as those of Example 1; the test steps and operations for the swelling power and binding water strength of pumpkin peel DF were the same as those of Example 1. The extraction rate of pumpkin peel pigment, its antioxidant stress resistance, and the swelling power and binding water strength indexes of pumpkin peel DF are shown in Table 10 below. The pumpkin peel pigment prepared in this comparative example had no obvious antioxidant stress effect on colitis mice and the effect of reducing the weight of mice.

[0203] Table 10 Pigment yield and antioxidant stress resistance of pumpkin peels from different processes, as well as DF expansion and binding water index of pumpkin peels

[0204]

[0205] As can be seen from Table 10, the processing technology of pumpkin peel has a certain influence on the pigment extraction rate and the antioxidant stress resistance. The vacuum freeze-drying treatment of pumpkin peel in Example 2 can improve the extraction rate of peel pigment and the average survival time of C.elegans under stress state, which is 20.94% and 11.53% higher than that of the peel pigment obtained by the traditional hot air drying treatment in Example 1.

[0206] Comparative Example 2 Effect of pumpkin peel powder treatment process on pigment extraction rate and DF physical and chemical properties

[0207] The yellow wolf pumpkin was washed and peeled, 20 g of pumpkin peel was freeze-dried at -30°C in a vacuum, and then crushed through a 40-mesh sieve to obtain 19.8 g of pumpkin peel powder.

[0208] The pumpkin skin powder was used to replace the pumpkin skin powder in step (1) of Example 2, and the pumpkin skin pigment was extracted and pumpkin skin DF powder with a mesh size of >40 was prepared in the same manner as in Example 2. The test steps and operations for the average survival time of C.elegans under oxidative stress were the same as in Example 1; the test steps and operations for the expansion force and binding water force of pumpkin skin DF were the same as in Example 1. The extraction rate of pumpkin skin pigment, the antioxidant stress capacity, and the expansion force and binding water force indexes of pumpkin skin DF are shown in Table 11 below.

[0209] Table 11 Pigment yield and antioxidant stress resistance of pumpkin peels from different processes, as well as DF expansion and binding water index of pumpkin peels

[0210]

[0211] As shown in Table 11, the processing technology of pumpkin peel powder has a certain influence on the extraction rate of pigment and the ability to resist oxidative stress. The extraction rate of pumpkin peel pigment extracted from pumpkin peel powder prepared by the method of Example 1 and the average survival time of C.elegans under stress state are improved, and the pumpkin peel pigment extracted from pumpkin peel coarse powder prepared by the method of Comparative Example 2 is increased by 45.97% and 9.91% respectively. The DF expansion force and binding water force of pumpkin peel prepared by the method of Example 2 are increased by 354.70% and 211.40% respectively compared with Comparative Example 2.

[0212] Comparative Example 3 Effect of Ultrasound-Assisted Enzymatic Pretreatment on Pumpkin Peel Pigment Extraction Yield and DF Physical and Chemical Properties

[0213] The enzymatic hydrolysis mixture in step (2) of Example 2 was replaced by pumpkin peel powder in step (1), and the experiment was carried out according to steps (2) and (3). The results are shown in Table 12.

[0214] Table 12 Pumpkin peel pigment yields from different processes and pumpkin peel DF expansion and combined hydraulic indexes

[0215]

[0216] As shown in Table 12, the enzymatic pretreatment process of pumpkin peel powder has a certain influence on the extraction rate of pigment. The ultrasonic-assisted enzymatic pretreatment of pumpkin peel powder in Example 2 can improve the extraction rate of peel pigment, which is 32.71% higher than the peel pigment obtained without ultrasonic-assisted enzymatic pretreatment in Comparative Example 3. The expansion force and binding water capacity of pumpkin peel DF are 25.77% and 3.03% higher than those obtained without ultrasonic-assisted enzymatic pretreatment, respectively.

[0217] Comparative Example 4 Effect of Ultrasonic-Assisted Pretreatment on Pumpkin Peel Pigment Extraction Yield and DF Physical and Chemical Properties

[0218] Take 20g of the pumpkin peel powder crushed through a 200-mesh sieve in Example 1 and place it in a brown bottle. Add 500mL of deionized water at a solid-liquid ratio of 1g:25mL, add 0.9% pectinase by weight of the pumpkin peel powder, mix well, pH 3.5 (1M hydrochloric acid to adjust pH), enzymatically treat in a 55°C water bath shaker for 53min, boil for 100s, cool and adjust to neutral with NaOH (1M) to obtain a slurry. Take 10g of the slurry to replace the enzymatic mixture in step (2) of Example 2, and perform other operations the same as steps (2) and (3) of Example 2. Use the method in Example 1 to detect the extraction rate of pumpkin peel pigment, pumpkin peel DF expansion force and binding water strength, see Table 13.

[0219] Table 13 Influence of ultrasound-assisted extraction on pumpkin peel pigment yield and pumpkin peel DF swelling power and binding water strength

[0220]

[0221] As shown in Table 13, the pumpkin peel processing technology has a certain influence on the extraction rate of pigment. The ultrasound-assisted enzymatic pretreatment of pumpkin peel powder in Example 2 can improve the extraction rate of peel pigment, which is 30.71% higher than the peel pigment obtained without ultrasound-assisted pretreatment in Comparative Example 4. The expansion force and binding water power of pumpkin peel DF in Example 2 are 16.16% and 17.36% higher than those obtained without ultrasound-assisted pretreatment in Comparative Example 4, respectively.

[0222] Comparative Example 5 Effect of Enzymatic Pretreatment on Pumpkin Peel Pigment Extraction Yield and DF Physical and Chemical Properties

[0223] 20 g of the pumpkin peel powder crushed through a 200-mesh sieve in Example 1 was placed in a brown bottle, and 500 mL of deionized water was added at a solid-liquid ratio of 1 g:25 mL. The mixture was treated at 55°C for 53 min under 150 W ultrasonic conditions to obtain a slurry. 10 g of the slurry was used to replace the enzymatic hydrolysis mixture in step (2) of Example 2. Other operations were the same as steps (2) and (3) of Example 2. The method of Example 1 was used to detect the extraction rate of pumpkin peel pigment, the DF expansion force and the bound water force of pumpkin peel, as shown in Table 14.

[0224] Table 14 Effect of enzyme pretreatment on pumpkin peel pigment yield and pumpkin peel DF swelling power and combined water strength

[0225]

[0226] As shown in Table 14, the pumpkin peel processing technology has a certain influence on the extraction rate of pigment. The ultrasound-assisted enzymatic pretreatment of pumpkin peel powder in Example 2 can improve the extraction rate of peel pigment, which is 36.90% higher than the pumpkin peel pigment obtained without enzymatic pretreatment in Comparative Example 5. The expansion force and binding water power of pumpkin peel DF in Example 2 are 28.50% and 27.93% higher than the DF obtained without enzymatic pretreatment in Comparative Example 5, respectively.

[0227] Comparative Example 6 Effect of Ultrasonic-Assisted Treatment on the Pigment Yield of Pumpkin Peel

[0228] The step (2) of Example 2, "ultrasound for 64 min at an ultrasonic power of 210 W and an ultrasonic temperature of 50° C.", was changed to extraction for 64 min at a temperature of 50° C. in a water bath shaker, and the other operations were the same as those of Example 2. The extraction rate of pumpkin peel pigment was 4.017 mg / g. The extraction rate of pumpkin peel pigment in Example 2 was 4.499 mg / g, which was 12.00% higher than that in Comparative Example 6.

[0229] It can be seen that the extraction process of pumpkin peel pigment has a certain influence on the pigment extraction rate, and ultrasound-assisted extraction can further improve the extraction rate of pumpkin peel pigment.

[0230] Comparative Example 7 Effect of Extraction Solvent on Extraction Yield of Pumpkin Peel Pigment

[0231] The natural acid containing 40% ethanol by volume in step (2) of Example 2 was replaced by ethanol, acetone, methanol, n-butanol, isopropanol, ethyl acetate, and n-hexane, respectively. The other operations were the same as those in Example 2. The extraction yield of pumpkin peel pigment is shown in Table 15.

[0232] Table 15 Effect of different extraction solvents on pumpkin peel pigment yield

[0233]

[0234] As shown in Table 15, the extraction solvent has a certain influence on the extraction rate of pumpkin peel pigment. Natural acid-alcohol extraction can improve the extraction rate of pumpkin peel pigment, which is 14.45%, 52.10%, 65.65%, 110.49%, 101.39%, 47.75% and 98.81% higher than that of ethanol, acetone, methanol, n-butanol, isopropanol, ethyl acetate and n-hexane extraction, respectively.

[0235] Example 3

[0236] (1) 10 g of pumpkin peel powder (200 mesh, same as step 3.1 of Example 1) was placed in a brown bottle, 250 mL of deionized water was added at a solid-liquid ratio of 1 g:25 mL to form a slurry, 1.5% of pectinase by weight of the pumpkin peel powder was added, the mixture was mixed, and ultrasonic enzymolysis was performed at 40° C., pH 4.0 (1 M hydrochloric acid to adjust the pH), and 120 W for 60 min. The mixture was boiled for 100 s, cooled and adjusted to neutral with NaOH (1 M), and vacuum freeze-dried (the initial temperature was -30° C., heating was started when the vacuum degree was 80 Pa, and the mixture was heated to -20° C. and maintained for 1 h. The temperature was then increased by 10° C. and maintained for 1 h. The temperature was raised to 20° C. and maintained for more than 24 h until the sample was dry and the water content was below 8%) to obtain 9.2 g of an enzymolysis mixture.

[0237] (2) 5 g of the enzymatic hydrolysis mixture was added with 100 mL of natural acid (octanoic acid to decanoic acid molar ratio 2.4:1) containing 40% ethanol by volume to make the material-liquid ratio 1 g:20 mL. Ultrasonication was performed in an ultrasonic cleaner at an ultrasonic power of 180 W and an ultrasonic temperature of 60 ° C for 50 min. Centrifugation (8000 rpm, 20 min) yielded 4.379 mg / g of pumpkin peel pigment in the supernatant. The supernatant was concentrated to 3 / 5 of the original volume to obtain a crude pumpkin peel pigment. Pumpkin peel pigment increases the T of C.elegans under oxidative stress aver Extended to 163.2 minutes, high-dose pumpkin peel pigment can significantly inhibit the increase in serum inflammatory factor levels in mice induced by DSS, and at the same time has the effect of reducing the weight of mice, without the symptoms of solvent stress response, and the fur is shiny and smooth.

[0238] (3) The filter residue was dried with hot air at 60°C and crushed through a 200-mesh sieve. A 1.6% mass concentration of hydrogen peroxide was used as a green modifier, and the liquid-to-solid ratio was 40 mL / g. The filter residue was treated with an ultrasonic wall-breaking machine for 180 s and centrifuged at 8000 rpm for 15 min. The filter residue was rinsed with deionized water three times and dried with hot air at 60°C, crushed and sieved to prepare pumpkin skin DF with a particle size of 200-300 mesh. The expansion force of pumpkin skin DF was 50.7 mL / g, and the binding water force was 13.9 g / g.

[0239] Example 4

[0240] (1) 10 g of pumpkin peel powder (200 mesh, the same as 3.1 in Example 1) was placed in a brown bottle, 250 mL of deionized water was added at a solid-liquid ratio of 1 g:25 mL to form a slurry, 1.2% of pectinase by weight of the pumpkin peel powder was added, and the mixture was mixed. The mixture was subjected to ultrasonic enzymolysis at 60° C., pH 3.0 (1 M hydrochloric acid to adjust the pH), and 180 W for 40 min, and then boiled for 100 s. After cooling, the mixture was adjusted to neutral with NaOH (1 M), and vacuum freeze-dried (the initial temperature was -30° C., and heating was started when the vacuum degree was 80 Pa. When heated to -20° C., the mixture was kept for 1 h, and then the temperature was kept for 1 h each time the temperature was increased by 10° C., and the temperature was raised to 20° C. and kept for more than 24 h until the sample was dry and the water content was below 8%) to obtain 9.1 g of an enzymolysis mixture.

[0241] (2) Add 200 mL of natural acid (octanoic acid to decanic acid molar ratio 2.8:1) containing 40% volume concentration to 5 g of the enzymatic hydrolysis mixture to make the material-liquid ratio 1 g:40 mL. Ultrasonication was performed in an ultrasonic cleaner at an ultrasonic power of 300 W and an ultrasonic temperature of 40°C for 90 min. Centrifugation (8000 rpm, 20 min) was performed, and the yield of pumpkin peel pigment in the supernatant was 4.395 mg / g. The supernatant was concentrated to 3 / 5 of the original volume to obtain a crude pumpkin peel pigment. Pumpkin peel pigment increases the T of C.elegans under oxidative stress aver Extended to 165.4 minutes, high-dose pumpkin peel pigment can significantly inhibit the increase in serum inflammatory factor levels in mice induced by DSS, and at the same time has the effect of reducing the weight of mice, without the symptoms of solvent stress response, and the fur is shiny and smooth.

[0242] (3) The filter residue was dried with hot air at 60°C and crushed through a 200-mesh sieve. Hydrogen peroxide with a mass concentration of 1.3% was used as a green modifier, and the liquid-to-solid ratio was 20 mL / g. The filter residue was treated with an ultrasonic wall-breaking machine for 720 s and centrifuged at 8000 rpm for 15 min. The filter residue was rinsed with deionized water three times and dried with hot air at 60°C, crushed and sieved to prepare pumpkin skin DF with a particle size of 200-300 mesh. The expansion force of pumpkin skin DF was 52.6 mL / g, and the binding water force was 14.1 g / g.

[0243] Example 5

[0244] (1) 10 g of pumpkin peel powder (200 mesh, the same as 3.1 in Example 1) was placed in a brown bottle, 300 mL of deionized water was added at a solid-liquid ratio of 1 g:30 mL to form a slurry, 0.9% of the mass of pectinase of the pumpkin peel powder was added, and the mixture was mixed. After ultrasonic enzymolysis treatment for 40 min at 50° C., pH 4.0 (1 M hydrochloric acid to adjust the pH), and 150 W, the mixture was boiled for 100 s, cooled and adjusted to neutral with NaOH (1 M), and vacuum freeze-dried (the initial temperature was -30° C., heating was started when the vacuum degree was 80 Pa, and the mixture was heated to -20° C. and maintained for 1 h, and then the temperature was increased by 10° C. and maintained for 1 h, and the temperature was increased to 20° C. and maintained for more than 24 h until the sample was dry and the water content was below 8%) to obtain 9.1 g of an enzymolysis mixture.

[0245] (2) 5 g of the enzymatic hydrolysis mixture was added with 150 mL of natural acid (octanoic acid to decanoic acid molar ratio of 2.6:1) containing 40% ethanol by volume to make the solid-liquid ratio 1 g:30 mL. Ultrasonication was performed in an ultrasonic cleaner at an ultrasonic power of 240 W and an ultrasonic temperature of 50 ° C for 70 min. Centrifugation (8000 rpm, 20 min) was performed, and the yield of pumpkin peel pigment in the supernatant was 4.417 mg / g. The supernatant was concentrated to 3 / 5 of the original volume to obtain a crude pumpkin peel pigment. Pumpkin peel pigment inhibits the T of C.elegans under oxidative stress aver Extended to 166.3 minutes, high-dose pumpkin peel pigment can significantly inhibit the increase in serum inflammatory factor levels in mice induced by DSS, and at the same time has the effect of reducing the weight of mice, without the symptoms of solvent stress response, and the fur is shiny and smooth.

[0246] (3) The filter residue was dried with hot air at 60°C and crushed to pass through a 200-mesh sieve. A 1.5% mass concentration of hydrogen peroxide was used as a green modifier, and the liquid-to-solid ratio was 30 mL / g. The filter residue was treated with an ultrasonic wall-breaking machine for 540 s and centrifuged at 8000 rpm for 15 min. The filter residue was rinsed with deionized water three times and dried with hot air at 60°C, crushed and sieved to prepare pumpkin skin DF with a particle size of 200-300 mesh. The expansion force of pumpkin skin DF was 51.8 mL / g, and the binding water force was 14.1 g / g.

[0247] Example 6

[0248] (1) 10 g of pumpkin peel powder (200 mesh, the same as 3.1 in Example 1) was placed in a brown bottle, 200 mL of deionized water was added at a solid-liquid ratio of 1 g:20 mL to form a slurry, 1.2% of pectinase by weight of the pumpkin peel powder was added, and the mixture was mixed. After ultrasonic enzymolysis for 50 min at 40° C., pH 3.5 (1 M hydrochloric acid to adjust pH), and 150 W, the mixture was boiled for 100 s, cooled, adjusted to neutral with NaOH (1 M), and vacuum freeze-dried (the initial temperature was -30° C., heating was started when the vacuum degree was 80 Pa, and the mixture was heated to -20° C. and maintained for 1 h, then the temperature was increased by 10° C. and maintained for 1 h, until the temperature reached 20° C. and maintained for more than 24 h until the sample was dry and the water content was below 8%) to obtain 9.2 g of an enzymolysis mixture.

[0249] (2) Add 200 mL of natural acid (octanoic acid to decanic acid molar ratio 2.8:1) containing 40% ethanol by volume to 5 g of the enzymatic hydrolysis mixture to make the solid-liquid ratio 1 g:40 mL. Ultrasonication was performed in an ultrasonic cleaner at an ultrasonic power of 210 W and an ultrasonic temperature of 50 ° C for 50 min. Centrifugation (8000 rpm, 20 min) yielded 4.307 mg / g of pumpkin peel pigment in the supernatant. The supernatant was concentrated to 3 / 5 of the original volume to obtain crude pumpkin peel pigment. Pumpkin peel pigment increases T of C.elegans under oxidative stress aver Extended to 160.4 minutes, high-dose pumpkin peel pigment can significantly inhibit the increase in serum inflammatory factor levels in mice induced by DSS, and at the same time has the effect of reducing the weight of mice, without the symptoms of solvent stress response, and the fur is shiny and smooth.

[0250] (3) The filter residue was dried with hot air at 60°C and crushed through a 200-mesh sieve. Hydrogen peroxide with a mass concentration of 1.4% was used as a green modifier, and the liquid-to-solid ratio was 30 mL / g. The filter residue was treated with an ultrasonic wall-breaking machine for 360 s and centrifuged at 8000 rpm for 15 min. The filter residue was rinsed with deionized water three times and dried with hot air at 60°C, crushed and sieved to prepare pumpkin skin DF with a particle size of 200-300 mesh sieve. The expansion force of pumpkin skin DF was 51.2 mL / g, and the binding water force was 13.8 g / g.

Claims

1. A method for comprehensive utilization of pumpkin peel, characterized in that: The method comprises the following steps: (1) adding pumpkin peel powder to deionized water to form a slurry, adding pectinase, mixing, treating with ultrasound-assisted enzymolysis for 40 to 60 minutes at 40 to 60° C., pH 3.0 to 4.0, and 150 to 240 W, boiling for 100 seconds, adjusting the pH to neutral after cooling, and vacuum freeze-drying to obtain an enzymolysis mixture; (2) adding a natural acid containing 20-80% ethanol by volume to the enzymatic hydrolysis mixture of step (1), performing ultrasonic-assisted extraction at 40-60° C. and 180-300 W for 50-90 min, centrifuging to obtain a supernatant and a filter residue, and concentrating the supernatant to 3 / 5 of the original volume to obtain a pumpkin peel pigment; the natural acid is prepared by mixing n-octanoic acid and n-decanoic acid, heating at 60° C. for 20-30 min, and the molar ratio of n-octanoic acid to n-decanoic acid is 2-3:1; (3) The filtrate from step (2) is dried with hot air at 60° C., crushed and sieved through a 150-200 mesh sieve, and subjected to a wall breaking and modification treatment for 180-720 s using hydrogen peroxide having a mass concentration of 0.5-2% as a green modifier by an ultrasonic wall breaking machine. The filtrate is centrifuged, and the filtrate is rinsed three times with deionized water, dried with hot air at 60° C., crushed and sieved to obtain pumpkin peel dietary fiber.

2. The method according to claim 1, characterized in that The activity of the pectinase in step (1) is 30,000 U / g, the amount of the pectinase used is 0.9%-1.5% based on the mass of the pumpkin peel powder; and the volume amount of deionized water used is 20-30 mL / g based on the weight of the pumpkin peel powder.

3. The method according to claim 1, characterized in that The vacuum freeze-drying method in step (1) is as follows: the initial temperature is -30°C, heating is started when the vacuum degree is 80 Pa, and the temperature is kept at -20°C for 1 hour, and then the temperature is kept at 10°C for 1 hour each time, until the temperature reaches 20°C, and kept for more than 24 hours until the water content of the sample is below 8%.

4. The method according to claim 1, characterized in that The volume amount of the natural acid containing ethanol with a volume concentration of 20-80% in step (2) is 10-50 mL / g based on the mass of the enzymatic hydrolysis mixture.

5. The method according to claim 1, characterized in that The volume amount of hydrogen peroxide in step (3) is 20-50 mL / g based on the mass of pumpkin peel powder.

6. Use of the pumpkin peel pigment prepared by the method of claim 1 in preparing an antioxidant or heat stress preparation.

7. Use of pumpkin peel pigment prepared by the method of claim 1 in preparing an anti-colitis preparation.

8. Use of pumpkin peel pigment prepared by the method of claim 1 in preparing weight loss drugs.

9. A functional pumpkin peel pigment prepared by the method of claim 1.

10. Pumpkin peel dietary fiber prepared by the method of claim 1.

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