CN112481341A - Preparation method and application of selenium absorption enhancing active peptide - Google Patents

Preparation method and application of selenium absorption enhancing active peptide Download PDF

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CN112481341A
CN112481341A CN202011414163.6A CN202011414163A CN112481341A CN 112481341 A CN112481341 A CN 112481341A CN 202011414163 A CN202011414163 A CN 202011414163A CN 112481341 A CN112481341 A CN 112481341A
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selenium
powder
active peptide
absorption
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CN112481341B (en
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张业辉
张友胜
焦文娟
赵甜甜
刘伟峰
施英
丘银清
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Sericulture and Agri Food Research Institute GAAS
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Abstract

The invention belongs to the technical field of food processing, and particularly relates to a preparation method and application of selenium absorption-enhancing active peptide, wherein the preparation method comprises the steps of carrying out anaerobic fermentation on supernatant liquid prepared from fish skin, whey protein powder, cassava starch, carrot powder and selenium-enriched yeast powder to obtain fermentation liquid, adding selenium protein to carry out ultrasonic treatment, mixing the fermentation liquid with solution consisting of fish skin mucus, mulberry leaf juice, peanut oil and algal polysaccharide, and finally carrying out high static pressure treatment to obtain the selenium absorption-enhancing active peptide; the selenium absorption-enhanced active peptide prepared by the invention is mainly a 6 peptide and 8 peptide compound and can be easily absorbed by the small intestine and the duodenum; meanwhile, the selenium-rich milk powder has a stable structure and good tolerance in the stomach and intestine, and can effectively enhance the absorption of the selenium by a human body; in addition, the selenium absorption enhancing active peptide can be matched with other nutrient components or food ingredients to prepare a selenium-rich health food form, so that the selenium absorption enhancing active peptide has good selenium absorption efficiency and unique flavor and is easily accepted by people.

Description

Preparation method and application of selenium absorption enhancing active peptide
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a preparation method and application of selenium absorption-enhanced active peptide.
Background
Selenium is a trace element necessary for human body, which affects the health and development of human body, and the reference intake of dietary nutrients of Chinese residents recommends that 50 micrograms of selenium is needed every day. Selenium participates in the synthesis of various selenase and selenoprotein in a human body, the selenium can improve the immunity of the human body, promote the proliferation of lymphocytes and the synthesis of antibodies and immune globulin, the selenium has obvious inhibiting and protecting effects on colon cancer, skin cancer, liver cancer, breast cancer and other cancers, an intermediate metabolite methyl selenol in the body has strong anticancer activity, the selenium and vitamin E, allicin, linoleic acid, germanium, zinc and other nutrients have the synergistic antioxidant effect, the antioxidant activity is increased, and meanwhile, the selenium has the effects of reducing and relieving the heavy metal toxicity. Some researches prove that the selenium can effectively inhibit the growth of tumors, and a proper amount of selenium has good auxiliary improvement effect on patients after operation and radiotherapy and chemotherapy treatment. In addition, selenium is the most important anti-aging element discovered so far, and the research of experts of Chinese academy of sciences on the rural Bama with long life shows that: the selenium content in the barma soil and grains is about 10 times higher than the average level in China, and the selenium content in the blood of the centenarian is 3-6 times higher than that of normal people.
China is wide in territory, the content of selenium element in different soils is greatly different, and in some selenium-poor areas, the selenium content in crops is low, and the selenium content in food is not enough to supplement the needs of human bodies. Therefore, various selenium-rich foods are derived on the market, namely foods rich in trace element selenium; generally, the selenium-enriched food is divided into natural selenium-enriched food (also called plant active selenium food) and exogenous selenium-enriched food (also called artificial organic selenium food).
At present, various foods with selenium supplementing efficacy, such as selenium-enriched peanuts, selenium-enriched rice and the like, appear in the market, but most selenium supplementing foods are expensive, the selenium content of the selenium supplementing foods is uncertain, and the selenium in the selenium supplementing foods is influenced by the digestion and absorption of a human body, so that the selenium intake effect is not good. Therefore, the development of the selenium-rich health food for improving the selenium absorption efficiency has important value.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of selenium absorption-enhanced active peptide, which utilizes biological fermentation and physical enhancement methods to improve the stability of combination of protein peptide and selenium, so that the flavor of selenium-enriched peptide can be enhanced, the selenium absorption effect of a human body can be improved, and the purpose of selenium supplement is achieved.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of active peptide for enhancing selenium absorption, which comprises the following steps:
s1, preparing fish skin into fish paste, adding pepsin for enzymolysis, and collecting supernatant;
s2, adding whey protein powder, cassava starch, carrot powder and selenium-enriched yeast powder into the supernatant obtained in the step S1, and then carrying out anaerobic fermentation;
s3, adding selenoprotein into the fermentation liquor obtained in the step S2, and then carrying out ultrasonic treatment;
s4, uniformly mixing the fish skin mucus and the mulberry leaf juice, standing for a period of time, adding peanut oil and algal polysaccharide, and then homogenizing and stirring;
s5, adding the solution obtained in the step S4 into the fermentation liquid obtained in the step S3, vacuumizing, performing high static pressure treatment, and finally reducing the pressure to obtain the selenium absorption enhancing active peptide.
Preferably, the fish skin and the fish skin mucus are both from snakeheads.
Selenium and protein are combined to form selenoprotein, so that the structural stability in a complex processing environment and a digestion environment can be enhanced, and the active selenium form is beneficial to the absorption of the selenium by a body and improves the bioavailability. The invention utilizes the yeast directional fermentation technology and the selenium peptide stable compound technology, utilizes the characteristic of strong absorptivity of the peptide to prepare the selenium-rich peptide with green safety and high bioavailability, the fish protein and the organic selenium form stable cross connection through high pressure, and then form a stable structure through the high static pressure variable pressure technology, so as to obtain 6 peptide and 8 peptide compounds which are easy to be absorbed by small intestine and duodenum, and enhance the absorption of intestinal mucosa cells to the organic selenium; meanwhile, the mixture of the body surface mucus of the snakeheads and the mulberry leaf juice can form micro-nano embedding after emulsification, so that the structure of the selenium peptide compound is not easily affected by gastrointestinal digestion, and the selenium peptide compound can smoothly reach the absorption part of the small intestine.
Preferably, in step S1, the amount of pepsin added is 20-40% of the weight of the fish paste, in order to obtain better enzymolysis effect.
Preferably, in order to enhance the effect of the directional fermentation, in step S2, whey protein powder, tapioca starch and carrot powder are added into the supernatant, the mixture is heated to 30 ℃, then selenium-enriched yeast powder is added, and finally the mixture is heated to 37 ℃ for anaerobic fermentation.
Preferably, in order to obtain a better fermentation effect, in step S2, according to the mass of the supernatant, the addition amount of the whey protein powder is 0.5-1.5%, the addition amount of the tapioca starch is 2.5-2.8%, the addition amount of the carrot powder is 0.3-0.5%, and the addition amount of the selenium-enriched yeast powder is 0.2-0.3%.
Preferably, in order to obtain a better emulsification effect, in step S4, the volume ratio of the fish skin mucus to mulberry leaf juice is 1: 1.
Preferably, in step S5, the high static pressure treatment is a high static pressure treatment at 300-350MPa for 10 minutes. Under the high static pressure treatment, the stable structure formed by the fish protein and the organic selenium is more favorably formed.
Preferably, the pH is one of the important conditions affecting the enzymolysis effect, and in step S1, the pH of the enzymolysis treatment is 3.8-4.2. Further, the pH of the enzymatic treatment is 4.0.
Preferably, to ensure the sufficient degree of enzymolysis, the enzymolysis treatment is stirring enzymolysis, i.e. stirring enzymolysis at a rotation speed of 200-.
Preferably, the temperature is raised to 80 ℃ for 10 minutes after the enzymolysis treatment, so as to achieve the effects of sterilization and enzyme inactivation.
Preferably, the fermentation is terminated when the anaerobic fermentation reaches a pH of 4.8.
Preferably, in step S3, the amount of selenoprotein added is 0.001% -0.003% of the total mass of the fermentation broth.
Preferably, to ensure adequate distribution of the selenoprotein into the fermentation broth, the power of the sonication is 300- "400W for 15-20 minutes in step S3.
Preferably, the acidity (pH4.8-5.0) and temperature (35 deg.C-40 deg.C) of the solution are controlled to be constant during the sonication in step S3.
Preferably, in step S4, the addition amount of the peanut oil is 10-15% of the total volume of the skin mucus and the mulberry leaf juice, and the addition amount of the algal polysaccharides is (0.5-0.8) wt% of the total mass of the skin mucus and the mulberry leaf juice.
Preferably, in order to ensure sufficient degree of stirring, the homogeneous stirring is performed at 42 ℃ to 46 ℃ for 30 to 40 minutes in step S4.
Preferably, to ensure that the selenopeptide compound is sufficiently emulsified to form micro-emulsion nano-particles, the volume ratio of the solution of step S4 to the fermentation broth of step S3 in step S5 is 1 (4-6). Further, the volume ratio of the solution of the step S4 to the fermentation liquid of the step S3 is 1: 5.
Preferably, in step S5, the pressure reduction is to (80-120) MPa and maintained for 10 minutes.
The invention also provides the selenium absorption enhancing active peptide prepared by the preparation method.
The invention also provides application of the selenium absorption enhancing active peptide in preparation of selenium-rich health food.
The selenium-enriched health food with various forms is prepared by matching the selenium-enriched absorption active peptide with other nutrient components or food ingredients, so that the healthy nutrient components of the active peptide can be improved, and selenium-enriched functional foods with various forms and good selenium absorption effects are formed.
The invention also provides a preparation method of the selenium-enriched emulsion beverage for enhancing selenium absorption, which comprises the following steps: adding 1.8-2 wt% of whey protein, 0.002-0.003 wt% of lactoferrin, 0.1-0.15 wt% of edible fungus superfine powder, 1.3-2.0 wt% of maltodextrin, 1.3-2.0 wt% of perilla extract and 1.3-2.0 wt% of glucose into the selenium-enriched active peptide, stirring, mixing uniformly, and sterilizing at high temperature to obtain the selenium-enriched active peptide.
The invention also provides a preparation method of the selenium-rich health powder for enhancing selenium absorption, which comprises the following steps: drying the selenium-enriched absorption active peptide into powder, adding 5-6 wt% of whey protein powder, 0.6-1.2 wt% of walnut powder, 0.001-0.0015 wt% of lactoferrin, 0.8-1.0 wt% of edible fungus superfine powder, 8-10 wt% of maltodextrin and 0.005-0.01 wt% of solid DHA (docosahexaenoic acid) powder, uniformly mixing and packaging to obtain the selenium-enriched absorption active peptide.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a preparation method of selenium absorption enhancing active peptide, which comprises the steps of carrying out anaerobic fermentation on supernatant liquid prepared from fish skin, whey protein powder, cassava starch, carrot powder and selenium-enriched yeast powder to obtain fermentation liquid, adding selenoprotein for ultrasonic treatment, mixing the fermentation liquid with solution consisting of fish skin mucus, mulberry leaf juice, peanut oil and algal polysaccharides, and finally carrying out high static pressure treatment to obtain the selenium absorption enhancing active peptide. The selenium absorption-enhanced active peptide prepared by the invention is mainly a 6 peptide and 8 peptide compound and can be easily absorbed by the small intestine and the duodenum; meanwhile, the selenium-enriched milk powder has a stable structure, is not easily influenced by gastrointestinal digestion, can smoothly reach the absorption part of the small intestine, has good tolerance in the stomach and intestine, and can effectively enhance the absorption of the selenium by the human body. In addition, the selenium absorption enhancing active peptide can be matched with other nutrient components or food ingredients to prepare a selenium-rich health food form, so that the selenium absorption enhancing active peptide has good selenium absorption efficiency and unique flavor and is easily accepted by people.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1A method for preparing an active peptide with enhanced selenium absorption
(1) Taking fresh snakeheads as raw materials, slaughtering, and respectively collecting fish skin and fish skin mucus;
(2) cutting the collected fish skin, pulping, adding pure water at 4 ℃ into the crushed fish skin according to the feed liquid weight ratio of 1:3, preparing the fish skin into fish paste by a wet superfine grinding technology, adjusting the pH value of the fish paste to 4.0, then adding pepsin according to 30% of the weight of the fish paste, mechanically stirring and performing enzymolysis for 2 hours at the rotating speed of 250 revolutions per minute, then centrifugally filtering 5000g to retain supernatant, and finally heating the supernatant to the temperature of 80 ℃, keeping the temperature for 10 minutes to achieve the effects of sterilization and enzyme inactivation.
(2) Pouring the supernatant into a fermentation tank, adding 1.0% of whey protein powder, 2.65% of cassava starch and 0.4% of carrot powder according to the mass of fish paste (supernatant), uniformly mixing, heating to 30 ℃, then adding 0.25% of selenium-rich yeast powder, uniformly mixing, continuously heating to 37 ℃, then sealing the fermentation tank for anaerobic fermentation for 3.5 hours, and stopping fermentation when the pH reaches 4.8.
(3) Adding 0.002% selenoprotein into the fermentation liquor according to the total mass of the fermentation liquor, uniformly mixing, and then acting in ultrasonic waves with the power of 350W for 17.5 minutes, wherein the acidity (pH4.9) and the temperature (37.5 ℃) of the fermentation liquor are controlled to be constant in the ultrasonic process.
(4) Uniformly mixing the fish skin mucus obtained by separation in the step (1) and the mulberry leaf juice according to the volume ratio of 1:1, standing for 10 minutes, adding peanut oil (volume percentage) with the total volume of 13 percent and algal polysaccharide with the total mass of 0.65wt percent, and homogenizing and stirring at 44 ℃ for 40 minutes for later use.
(5) Slowly adding the solution in the step (4) into the fermentation liquor in the step (3) according to the volume ratio of 1:5, uniformly stirring, then placing in a bag, vacuumizing, sealing, treating for 10 minutes at high static pressure of 330MPa, then reducing the pressure to 100MPa, and maintaining for 10 minutes to obtain the selenium-enriched active peptide solution for enhancing selenium absorption.
The main protein components in the selenium-rich active peptide solution are measured by referring to the method in GB/T22492-2008 soybean peptide powder, namely 6 peptides and 8 peptides.
Example 2A method for preparing active peptide with enhanced selenium absorption
(1) Taking fresh snakeheads as raw materials, slaughtering, and respectively collecting fish skin and fish skin mucus;
(2) cutting the collected fish skin, pulping, adding pure water at 4 ℃ into the crushed fish skin according to the feed liquid weight ratio of 1:3, preparing the fish skin into fish paste by a wet superfine grinding technology, adjusting the pH value of the fish paste to 4.0, then adding pepsin according to 20% of the weight of the fish paste, mechanically stirring and performing enzymolysis for 2 hours at the rotating speed of 200 r/min, then centrifugally filtering 5000g to retain supernatant, and finally heating the supernatant to the temperature of 80 ℃ for 10 minutes to achieve the effects of sterilization and enzyme inactivation.
(2) Pouring the supernatant into a fermentation tank, adding 0.5% of whey protein powder, 2.5% of cassava starch and 0.3% of carrot powder according to the mass of fish paste (supernatant), uniformly mixing, heating to 30 ℃, adding 0.2% of selenium-rich yeast powder, uniformly mixing, continuously heating to 37 ℃, then sealing the fermentation tank for anaerobic fermentation for 3.5 hours, and stopping fermentation when the pH value reaches 4.8.
(3) Adding 0.001% selenoprotein into the fermentation liquor according to the total mass of the fermentation liquor, uniformly mixing, and acting in ultrasonic waves with the power of 300W for 20 minutes, wherein the acidity (pH4.8) and the temperature (35 ℃) of the fermentation liquor are controlled to be constant in the ultrasonic process.
(4) Uniformly mixing the fish skin mucus obtained by separation in the step (1) and the mulberry leaf juice according to the volume ratio of 1:1, standing for 10 minutes, adding peanut oil (volume percentage) with the total volume of 10 percent and algal polysaccharide with the total mass of 0.5wt percent, and homogenizing and stirring at 42 ℃ for 40 minutes for later use.
(5) Slowly adding the solution in the step (4) into the fermentation liquor in the step (3) according to the volume ratio of 1:4, uniformly stirring, then placing in a bag, vacuumizing, sealing, treating for 10 minutes at a high static pressure of 300MPa, then reducing the pressure to 80MPa, and maintaining for 10 minutes to obtain the selenium-enriched active peptide solution for enhancing selenium absorption.
The main protein components in the selenium-rich active peptide solution are measured by referring to the method in GB/T22492-2008 soybean peptide powder, namely 6 peptides and 8 peptides.
Example 3A method for preparing active peptide with enhanced selenium absorption
(1) Taking fresh snakeheads as raw materials, slaughtering, and respectively collecting fish skin and fish skin mucus;
(2) cutting the collected fish skin, pulping, adding pure water at 4 ℃ into the crushed fish skin according to the feed liquid weight ratio of 1:3, preparing the fish skin into fish paste by a wet superfine grinding technology, adjusting the pH value of the fish paste to 4.0, then adding pepsin according to 40% of the weight of the fish paste, mechanically stirring and performing enzymolysis for 2 hours at the rotating speed of 300 r/min, then centrifugally filtering 5000g to retain supernatant, and finally heating the supernatant to the temperature of 80 ℃ for 10 minutes to achieve the effects of sterilization and enzyme inactivation.
(2) Pouring the supernatant into a fermentation tank, adding 1.5% of whey protein powder, 2.8% of cassava starch and 0.5% of carrot powder according to the mass of fish paste (supernatant), uniformly mixing, heating to 30 ℃, then adding 0.3% of selenium-rich yeast powder, uniformly mixing, continuously heating to 37 ℃, then sealing the fermentation tank for anaerobic fermentation for 3.5 hours, and stopping fermentation when the pH reaches 4.8.
(3) Adding 0.003% selenoprotein into the fermentation liquor according to the total mass of the fermentation liquor, uniformly mixing, and then acting in ultrasonic waves with the power of 400W for 15 minutes, wherein the acidity (pH5.0) and the temperature (40 ℃) of the fermentation liquor are controlled to be constant in the ultrasonic process.
(4) Uniformly mixing the fish skin mucus obtained by separation in the step (1) and the mulberry leaf juice according to the volume ratio of 1:1, standing for 10 minutes, adding peanut oil (volume percentage) with the total volume of 15 percent and algal polysaccharide with the total mass of 0.8wt percent, and homogenizing and stirring at 46 ℃ for 30 minutes for later use.
(5) Slowly adding the solution in the step (4) into the fermentation liquor in the step (3) according to the volume ratio of 1:6, uniformly stirring, then placing in a bag, vacuumizing, sealing, treating for 10 minutes at high static pressure of 350MPa, then reducing the pressure to 120MPa, and maintaining for 10 minutes to obtain the selenium-enriched active peptide solution for enhancing selenium absorption.
The main protein components in the selenium-rich active peptide solution are measured by referring to the method in GB/T22492-2008 soybean peptide powder, namely 6 peptides and 8 peptides.
Embodiment 4 a method for preparing selenium-enriched emulsion beverage for enhancing selenium absorption
Adding 1.9 wt% of whey protein, 0.0025 wt% of lactoferrin, 0.125 wt% of edible fungus superfine powder, 1.6 wt% of maltodextrin, 1.6 wt% of perilla extract and 1.6 wt% of glucose into the selenium-rich active peptide solution prepared in the example 1, stirring and uniformly mixing, and then sterilizing at high temperature to obtain the selenium-rich active peptide.
Example 5A method for preparing a selenium enriched emulsion beverage for enhanced selenium absorption
Adding 1.8 wt% of whey protein, 0.002 wt% of lactoferrin, 0.1 wt% of edible fungus superfine powder, 1.3 wt% of maltodextrin, 1.3 wt% of perilla extract and 1.3 wt% of glucose into the selenium-rich active peptide solution prepared in the example 1, stirring and uniformly mixing, and then sterilizing at high temperature to obtain the selenium-rich active peptide.
Embodiment 6A method for preparing a selenium-enriched emulsion beverage for enhancing selenium absorption
Adding 2 wt% of whey protein, 0.003 wt% of lactoferrin, 0.15 wt% of edible fungus superfine powder, 1.3 wt% of maltodextrin, 1.3 wt% of perilla extract and 1.3 wt% of glucose into the selenium-rich active peptide solution prepared in the example 1, stirring and uniformly mixing, and then sterilizing at high temperature to obtain the selenium-rich active peptide.
Embodiment 7 a method for preparing selenium-rich health powder for enhancing selenium absorption
The selenium-rich active peptide solution prepared in the embodiment 1 is spray-dried into light yellow selenium-rich active peptide powder, and then 5.5 wt% of whey protein powder, 0.9 wt% of walnut powder, 0.0013 wt% of lactoferrin, 0.9 wt% of edible fungus ultrafine powder, 9 wt% of maltodextrin and 0.008 wt% of solid DHA powder are added into the selenium-rich active peptide powder, and the selenium-rich active peptide solution is obtained after uniform mixing and packaging.
Embodiment 8 a method for preparing selenium-rich health powder for enhancing selenium absorption
The selenium-rich active peptide solution prepared in the embodiment 1 is spray-dried into light yellow selenium-rich active peptide powder, and then 5 wt% of whey protein powder, 0.6 wt% of walnut powder, 0.001 wt% of lactoferrin, 0.8 wt% of edible fungus superfine powder, 8 wt% of maltodextrin and 0.005 wt% of solid DHA powder are added into the selenium-rich active peptide powder, and the selenium-rich active peptide solution is obtained after uniform mixing and packaging.
Embodiment 9 a method for preparing selenium-rich health powder for enhancing selenium absorption
The selenium-rich active peptide solution prepared in the embodiment 1 is spray-dried into light yellow selenium-rich active peptide powder, and then 6 wt% of whey protein powder, 1.2 wt% of walnut powder, 0.0015 wt% of lactoferrin, 1.0 wt% of edible fungus superfine powder, 10 wt% of maltodextrin and 0.01 wt% of solid DHA powder are added into the selenium-rich active peptide powder, and the selenium-rich active peptide solution is obtained after uniform mixing and packaging.
Experimental example 1 in vitro digestion and absorption experiment of Small intestinal mucosal cancer cells (Caco-2)
(1) Cytotoxicity assay (survival):
caco-2 cells were cultured at 1X 104The density of individual cells/well was plated in collagen-coated 96-well plates, which were incubated at 37 ℃ with CO2Incubate in incubator for 24 h. After removing the medium, 200. mu.L of samples of different concentrations were added to each well, and 8-10 parallel wells were prepared for each sample. CO at 37 deg.C2Culture boxAfter 20 hours of incubation, 20. mu.L of MTT (detection reagent) (5mg/mL) was added to each well, and after 4 hours of incubation, the precipitate was removed by centrifugation. Then, 150 μ L DMSO (dimethyl sulfoxide) was added to each well, and the cell culture plate was shaken at low speed for 10 min. The absorbance was measured at 490nm with a microplate reader, and finally the cell survival rate (%) was calculated according to the following formula:
cell survival rate (%) ═ B1-B0)*100/B2-B0。
In the formula: b is0Absorbance without cells; b is1Is the absorbance of the added sample; b is2Absorbance without sample.
(2) In vitro construction of intestinal cell model
Caco-2 cells were cultured at 1X 105The density of individual cells/well was seeded in 12-well plates, and the culture medium (Caco-2 cell culture medium, DMEM + 20% FBS as the main component) was changed every other day in the first week, and the culture medium was changed every day in the second week, and after 21 days of culture, an in vitro intestinal cell model was obtained. Removing the Caco-2 cell culture medium, washing twice with selenium-free buffer HBSS (cell culture solution), preheating at 37 ℃ for 10min, removing the HBSS buffer solution, adding samples (selenium-rich active peptide samples of examples 1, 4 and 7) with different concentrations (1mg/mL, 5mg/mL and 10mg/mL), culturing in an incubator for 2 hours, after reaction, washing twice with HBSS buffer solution at 4 ℃ to remove residual extract, adding 500. mu.L of 0.1% (v/v) Triton X-100 cell lysate, repeatedly freezing and thawing to lyse cells, and then performing ultrasonic treatment for 10min to obtain a cell suspension. The total protein mass concentration (g/L) was measured with BCA kit, another portion was removed from the cell suspension, centrifuged at 15000r/min for 10min, and the selenium ion content of the solution and alkaline protease (AKP) activity was determined by atomic absorption spectrophotometer using the AKP activity assay kit. A control group and a sodium selenite group (common selenium supplement products) are arranged in the experimental process, wherein the control group is cells added with culture medium only, and the sodium selenite group is cells added with 0.02mg/mL sodium selenite solution. Meanwhile, the cellular uptake rate was calculated according to the following formula:
the cellular uptake rate (%) ═ C1/C2.
In the formula: c1 is intracellular selenium concentration; c2 is the total cell protein concentration.
As can be seen from the survival rate and selenium uptake data of the small intestinal mucosal cancer cells (Caco-2) in Table 1, the survival rate of the Caco-2 cells treated by the samples of example 1, example 4 and example 7 was above 94%, slightly lower than that of the control group (96.5%) and higher than that of the sodium selenite group, indicating that the samples of the present invention were not toxic to Caco-2. It can also be seen that the uptake rate of selenium by Caco-2 cells was also significantly higher in the samples of examples 1, 4 and 7 than in the sodium selenite group.
TABLE 1 survival of small intestinal mucosal cancer cells (Caco-2) and their uptake of selenium
Figure BDA0002819639720000081
Experimental example 2 simulated gastrointestinal digestion test
(1) Simulating the gastric digestion stage: simulated Gastric Fluid (SGF) in deionized water was adjusted to pH 2.0, which included 2mg/mL NaCl, 12mmoL/L HCl and 3.2mg/mL pepsin. 5.0mg/mL of the sample (selenium-rich active peptide sample of example 1, example 4, example 7) was mixed with SGF at a mass ratio of 1:1, then adjusted to pH 2.0 and stirred continuously at a constant temperature of 37 ℃ at a rotation speed of 100r/min for 2h to simulate digestion of the stomach, and the supernatant was taken and measured with an atomic absorption spectrophotometer.
(2) Simulating the small intestine digestion stage: preparation of Simulated Intestinal Fluid (SIF): containing salt solution (100mg CaCl)2And 300mg/mL NaCl, pH 7.0), a bile salt solution (375.0mg bile salt dissolved in 7.0mL deionized water, pH 7.0), and an enzyme solution (120.0mg lipase dissolved in 5.0mL deionized water, pH 7.0). After the end of the gastric phase digestion phase, the pH (60.0mL) of the previous solution (the solution mimicking the gastric digestion phase) was raised to 7.0 and the prepared SIF was added to the previous solution to a final concentration of 1.6mg/mL lipase and bile salts. Adjusting pH of the mixture to 7.0, continuously shaking and culturing at 37 deg.C at 100r/min for 2h, collecting, and cold-cooling in ice-water bathHowever, it was then centrifuged at 15000r/min at 4 ℃ for 30min, and the supernatant was taken and measured with an atomic absorption spectrophotometer.
A control group and a sodium selenite group (common selenium supplement products) are arranged in the experimental process, wherein the control group is formed by mixing a sample with deionized water, and the sodium selenite group is formed by mixing a 0.02mg/mL sodium selenite solution with simulated gastric fluid.
Wherein the remaining amount (%) of selenium is 100% of the selenium content of the added digestive juice/the selenium content of the non-added digestive juice.
As can be seen from table 2, no selenium content was detected in the control group during the gastric digestion stage, and the selenium retention of the sodium selenite samples was significantly lower than that of the samples of examples 1, 4 and 7. After gastric digestion, the samples of examples 1, 4 and 7 were also retained higher than the sodium selenite sample during the intestinal digestion phase by performing a second digestion with a combination of the previous gastric juice and simulated intestinal fluid, but the retained amount of selenium was further reduced, probably due to the structural change of the compound caused by trypsin, thereby reducing the binding capacity to selenium. The results show that the selenium-rich active peptide sample has stable structure, is not easily influenced by gastrointestinal digestion, can successfully reach the absorption part of the small intestine and has good tolerance in the stomach and intestine.
TABLE 2 in vitro simulation of selenium retention in the gastric and intestinal stages of digestion
Figure BDA0002819639720000091
Experimental example 3 animal experiment of digestion and absorption of mice
The 6-week-old clean mice are randomly divided into 3 groups of 12 mice, and the mice are fed with selenium-free feed to establish a low-selenium model, and are fed with drinking water freely and kept at the room temperature of (25 +/-1) ° C. Mice were fasted 6h prior to dosing and had free access to water. Three groups of mice are respectively fed with gastric lavage feed (selenium-rich active peptide samples and sodium selenite in examples 1, 4 and 7) with the selenium content of 18 mug/kg · bw, and 600 mul of blood is taken from tails before and after gastric lavage 5, 10, 20, 30, 40, 60, 80, 100, 120min, and is placed in an EDTA anticoagulant tube, centrifuged for 10min at 3000r/min, and upper plasma is sucked into a centrifuge tube and marked. And (3) detecting the selenium content in the plasma of the mouse by using hydride generation-atomic fluorescence spectrometry (HG-AFS).
As can be seen from table 3, after the selenium-rich active peptide samples of example 1, example 4 and example 7 and the sodium selenite samples were perfused into the gavage mice, the peak values of the blood selenium values in the blood of the mice were all significantly higher than those of the sodium selenite samples, and the peak appearance times of the blood selenium values were all 10 minutes. The reason why the selenium in the selenium-rich active peptide sample is easier to be absorbed and the effect of supplementing selenium is better than that of sodium selenite is probably because the active peptides in the selenium-rich active peptide sample are mainly 6 peptide and 8 peptide compounds and are easy to be absorbed by small intestine and duodenum.
TABLE 3 digestive absorption of selenium after gastric lavage of mice
Group of Peak Total blood selenium value (mg/L) in mouse blood Peak time of blood selenium value in mouse blood
Sodium selenite 0.81 10
Example 1 1.23 10
Example 4 1.20 10
Example 7 1.99 10
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (10)

1. A preparation method of active peptide for enhancing selenium absorption is characterized by comprising the following steps:
s1, preparing fish skin into fish paste, adding pepsin for enzymolysis, and collecting supernatant;
s2, adding whey protein powder, cassava starch, carrot powder and selenium-enriched yeast powder into the supernatant obtained in the step S1, and then carrying out anaerobic fermentation;
s3, adding selenoprotein into the fermentation liquor obtained in the step S2, and then carrying out ultrasonic treatment;
s4, uniformly mixing the fish skin mucus and the mulberry leaf juice, standing for a period of time, adding peanut oil and algal polysaccharide, and then homogenizing and stirring;
s5, adding the solution obtained in the step S4 into the fermentation liquid obtained in the step S3, vacuumizing, performing high static pressure treatment, and finally reducing the pressure to obtain the selenium absorption enhancing active peptide.
2. The method of claim 1, wherein the fish skin and the fish skin mucus are from snakeheads.
3. The method of claim 1, wherein in step S2, whey protein powder, tapioca starch and carrot powder are added to the supernatant, and after heating to 30 ℃, selenium-rich yeast powder is added, and finally the temperature is raised to 37 ℃ for anaerobic fermentation.
4. The method of claim 3, wherein in step S2, the whey protein powder is added in an amount of 0.5-1.5%, the tapioca starch is added in an amount of 2.5-2.8%, the carrot powder is added in an amount of 0.3-0.5%, and the selenium-enriched yeast powder is added in an amount of 0.2-0.3% by mass of the supernatant.
5. The method of claim 1, wherein in step S4, the volume ratio of the fish skin mucus to mulberry leaf juice is 1: 1.
6. The method of claim 1, wherein the high static pressure treatment is 300-350MPa for 10 minutes in step S5.
7. The selenium absorption-enhanced active peptide prepared by the preparation method of any one of claims 1 to 6.
8. Use of the selenium absorption-enhancing active peptide of claim 7 for the preparation of selenium-enriched health food.
9. A preparation method of a selenium-enriched emulsion beverage for enhancing selenium absorption is characterized in that 1.8-2 wt% of whey protein, (0.002-0.003) wt% of lactoferrin, (0.1-0.15) wt% of edible fungus superfine powder, (1.3-2.0) wt% of maltodextrin, (1.3-2.0) wt% of perilla extract and (1.3-2.0) wt% of glucose are added into the selenium absorption enhancing active peptide of claim 7, and the selenium absorption enhancing active peptide is obtained by stirring, uniformly mixing and then sterilizing at high temperature.
10. A preparation method of selenium-rich health powder for enhancing selenium absorption is characterized in that the selenium-absorption enhancing active peptide of claim 7 is dried into powder, then added with (5-6) wt% whey protein powder, (0.6-1.2) wt% walnut powder, (0.001-0.0015) wt% lactoferrin, (0.8-1.0) wt% edible fungus superfine powder, (8-10) wt% maltodextrin and (0.005-0.01) wt% solid DHA powder, and the selenium-rich health powder is obtained after uniform mixing and packaging.
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