CN114376236A - Polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable enzyme - Google Patents

Polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable enzyme Download PDF

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CN114376236A
CN114376236A CN202210105257.8A CN202210105257A CN114376236A CN 114376236 A CN114376236 A CN 114376236A CN 202210105257 A CN202210105257 A CN 202210105257A CN 114376236 A CN114376236 A CN 114376236A
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selenium
fruit
polysaccharide
vegetable
nano
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魏启山
邓宇初
陈淑萍
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Bazhou Xindeyuan Food Co ltd
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Bazhou Xindeyuan Food Co ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/147—Helveticus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/249—Thermophilus

Abstract

The invention discloses polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable ferment, wherein the preparation method of the polysaccharide nano-selenium comprises the following steps: taking asparagus powder, extracting with alcohol, and filtering to obtain filtrate and filter residue; carrying out ultrasonic extraction on the filter residue by using water, and collecting supernatant; mixing the filtrate and the supernatant, concentrating, precipitating with ethanol, and drying to obtain crude polysaccharide; dissolving the crude polysaccharide in water, adding an extracting agent, dialyzing, and drying to obtain asparagus polysaccharide; mixing the dispersant aqueous solution and the sodium selenite aqueous solution uniformly, adding the asparagus polysaccharide aqueous solution, stirring, adding the ascorbic acid aqueous solution, stirring, dialyzing and drying to obtain the polysaccharide nano-selenium. The polysaccharide nano-selenium is applied to the selenium-rich fruit and vegetable enzyme, so that the fruit and vegetable enzyme is rich in nutrient fermentation active ingredients and also comprises the polysaccharide nano-selenium which is easy to absorb by a human body, the antioxidant activity is good, and the immunity of the organism is effectively enhanced.

Description

Polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable enzyme
Technical Field
The invention relates to the technical field of health-care food, in particular to polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable ferment.
Background
The fruit and vegetable enzyme is a product obtained by fermenting natural food materials such as fruits, vegetables and the like through microorganisms. It is rich in polyphenols, minerals, organic acids, dietary fiber, protein, enzyme, etc., and has good health promotion effect; for example: the antioxidant active ingredients in the ferment have the health-care effects of resisting oxidation and aging by inhibiting the damage of free radicals to the organism; the dietary fiber, the organic acid and other components in the ferment can promote intestinal tract movement, improve the microenvironment in vivo and achieve the health-care effect of relaxing bowel; polysaccharides, polyphenol, vitamins and other components in the ferment have certain antibacterial and anti-inflammatory effects; in addition, the active probiotics in the ferment can assist in removing the garbage such as toxin, blood fat and the like in the blood and improve the immunity of the organism.
Chinese patent CN106307512A discloses a preparation method of fruit and vegetable enzyme, which comprises the following steps: cleaning fruits and vegetables with a fruit and vegetable cleaning agent and sterile water, protecting color, pulping, and concentrating to obtain fruit and vegetable juice; pretreating fruit and vegetable juice by using a high-voltage pulse electric field; the fruit and vegetable ferment is prepared by adding concentrated fruit and vegetable juice, plant extract, konjac glucomannan, chitosan, amino acid, zinc sulfate, molasses and a leavening agent which are pretreated by a high-voltage pulse electric field into a fermentation tank for fermentation, so that crude fruit and vegetable ferment is obtained, and the fruit and vegetable ferment is obtained by ultrasonic treatment.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides polysaccharide nano-selenium and application thereof in selenium-rich fruit and vegetable ferment.
In order to solve the technical problems, the invention adopts the technical scheme that:
selenium is an essential trace element having important effects on human health, and the common existing modes in nature mainly comprise inorganic selenium and organic selenium, wherein the inorganic selenium and the organic selenium are combined with cysteine in organisms to be positioned in active centers of glutathione peroxidase, methionine sulfoxide reductase, iodothyronine deiodinase and the like, participate in the oxidation resistance and energy metabolism of organisms, have higher biological activity and important biological functions, such as effectively eliminating free radicals in the bodies, regulating the metabolism of the organisms, enhancing the immune regulation capability of the organisms, preventing cardiovascular and cerebrovascular diseases, resisting tumors, protecting the liver and the like.
Compared with the traditional inorganic selenium and organic selenium, the nano selenium has unique small size effect, quantum size effect, surface effect and macroscopic quantum tunneling effect, so that the nano selenium has lower toxicity, higher bioavailability and bioactivity and becomes a research hotspot of health food supplements.
The nano-selenium is prepared by the following method: uniformly mixing 5-15 parts by weight of 50-70mmol/L and 230 parts by weight of 170-230-.
The nano selenium has high specific surface energy and insufficient coordination of surface atoms, and is easy to aggregate and precipitate under the action of Brownian motion and Van der Waals attractive force to be converted into grey black elemental selenium, so that the biological activity and the bioavailability are reduced. The natural polysaccharide isolated from plants has a large number of hydrophilic groups, such as hydroxyl, carboxyl and amino groups, and proper molecular modification or structural reconstruction of the natural polysaccharide can lead the polysaccharide to generate new activity or further enhance certain activity. The polysaccharide has a highly complex branch structure and active hydroxyl groups, can well adsorb and wrap nano-selenium initially formed in a reduction reaction, control nucleation and growth of crystals, further prevent agglomeration and growth of nano-selenium particles, has good biological activity and biological adhesion, can prolong the retention time of the nano-selenium in organisms, and is an important regulator for preparing the nano-selenium. Therefore, the polysaccharide nano-selenium with good stability and high biological activity is prepared by taking natural polysaccharide as a stabilizer.
The preparation method of the polysaccharide nano-selenium comprises the following steps:
s1, drying the asparagus in a drying box at 50-70 ℃ for 5-8h, taking out, crushing, sieving with a 30-60-mesh sieve to obtain asparagus powder, putting 80-120 parts by weight of the asparagus powder into 500 parts by weight of anhydrous ethanol, heating to 65-75 ℃, refluxing for 2-5h, and filtering to obtain filtrate and filter residue; then adding the filter residue into 1800 parts by weight of 1200-1800 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at the rotation speed of 6000rpm at 65-75 deg.C for 15-30min and collecting supernatant, wherein the ultrasonic power is 150-; finally, combining the filtrate and the supernatant, carrying out vacuum concentration to one tenth of the original volume at 40-50 ℃ by using a rotary evaporator, adding absolute ethyl alcohol to enable the volume fraction of the ethyl alcohol to reach 75-85%, standing for 12-36h at 2-5 ℃, centrifuging for 15-30min at 6000-10000rpm after finishing, taking the precipitate, and freeze-drying to obtain the asparagus polysaccharide;
s2, mixing 10-30 parts by weight of 15-25mg/mL dispersant aqueous solution and 5-15 parts by weight of 50-70mmol/L sodium selenite aqueous solution uniformly, adding 160-200 parts by weight of 5-8mg/mL asparagus polysaccharide aqueous solution, stirring at the rotation speed of 600-1000rpm at room temperature for 40-80min, adding 5-15 parts by weight of 200-280mmol/L ascorbic acid aqueous solution, continuing stirring at 35-50 ℃ in the dark for 5-8h, then placing in a dialysis bag with the molecular weight cutoff of 3000-4000Da, dialyzing with water for 48-96h, wherein water is replaced once every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
Although the asparagus polysaccharide in the technical scheme is prepared by combining alcohol extraction and ultrasonic extraction, the asparagus polysaccharide still contains a lot of small molecular impurities such as proteins, pigments and some inorganic salts, so that the obtained asparagus polysaccharide has low utilization rate and low biological activity; therefore, the obtained crude polysaccharide of asparagus is further extracted and purified by adopting the organic solvent, so that the effective components in the crude polysaccharide of asparagus can be effectively and finely separated, the structure and the biological activity of the crude polysaccharide of asparagus can be better maintained, and the utilization rate of the crude polysaccharide of asparagus in the preparation of nano-selenium is improved.
Preferably, the preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 50-70 ℃ for 5-8h, taking out, crushing, sieving with a 30-60-mesh sieve to obtain asparagus powder, putting 80-120 parts by weight of the asparagus powder into 500 parts by weight of anhydrous ethanol, heating to 65-75 ℃, refluxing for 2-5h, and filtering to obtain filtrate and filter residue; then adding the filter residue into 1800 parts by weight of 1200-1800 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at the rotation speed of 6000rpm at 65-75 deg.C for 15-30min and collecting supernatant, wherein the ultrasonic power is 150-; finally, combining the filtrate and the supernatant, carrying out vacuum concentration to one tenth of the original volume at 40-50 ℃ by using a rotary evaporator, adding absolute ethyl alcohol to enable the volume fraction of the ethyl alcohol to reach 75-85%, standing for 12-36h at 2-5 ℃, centrifuging for 15-30min at 6000-10000rpm after finishing, taking the precipitate, and freeze-drying to obtain crude polysaccharide; dissolving crude polysaccharide in 30-50 parts by weight of water, adding 8-15 parts by weight of an extracting agent, stirring at the rotating speed of 300-; the extractant is formed by mixing n-butyl alcohol and chloroform according to the volume ratio of 1 (3-5);
s2, mixing 10-30 parts by weight of 15-25mg/mL dispersant aqueous solution and 5-15 parts by weight of 50-70mmol/L sodium selenite aqueous solution uniformly, adding 160-200 parts by weight of 5-8mg/mL asparagus polysaccharide aqueous solution, stirring at the rotation speed of 600-1000rpm at room temperature for 40-80min, adding 5-15 parts by weight of 200-280mmol/L ascorbic acid aqueous solution, continuing stirring at 35-50 ℃ in the dark for 5-8h, then placing in a dialysis bag with the molecular weight cutoff of 3000-4000Da, dialyzing with water for 48-96h, wherein water is replaced once every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
The dispersant is one or more of sodium dodecyl benzene sulfonate, cetyl trimethyl ammonium bromide, polyvinylpyrrolidone and tween-80; preferably, the dispersant is a mixture of Tween-80 and sodium dodecyl benzene sulfonate, wherein the mass ratio of the Tween-80 to the sodium dodecyl benzene sulfonate is (2-5) to (1-3).
Tween-80 is a nonionic surfactant, has remarkable water solubility, and can be used as a dispersant for a nano system. Tween-80 is adsorbed on the surface of the nano-selenium through strong interaction between hydroxyl and the nano-selenium, and has nonionic hydrophilic long chain-polyoxyethylene mutual repulsion, and the formed steric hindrance effect is utilized to prevent the aggregation of the nano-selenium. However, due to the fact that the polyoxyethylene chain structure of the Tween-80 is large, the Tween-80 is easily adsorbed on the surfaces of different nano selenium particles to generate a bridge effect, so that the nano selenium is flocculated, and a dispersion system is unstable. The sodium dodecyl benzene sulfonate is a linear surfactant, has a short molecular chain, is easy to penetrate into structural gaps of Tween-80, avoids the generation of a bridging effect, belongs to an anionic surfactant, and can be combined with polysaccharide hydroxyl on the surface of the nano-selenium by van der Waals force to form a stable adsorption coating system. According to the invention, Tween-80 and sodium dodecyl benzene sulfonate are compounded to be used as a dispersing agent, and the formed mixed system can reduce the interfacial tension, increase the adsorption capacity of the surface of the nano-selenium, provide more binding sites for polysaccharide, control the nucleation and growth of the nano-selenium and promote the formation of the polysaccharide nano-selenium with uniform size.
The forming process of the polysaccharide nano selenium is approximately as follows: in the reaction system, free selenite particles are firstly reduced into zero-valent selenium by ascorbic acid, and then when more zero-valent selenium is generated from the reduction reaction, the zero-valent selenium is taken as a core and gradually grows into nano selenium; and then, the polysaccharide and the dispersing agent are firmly adsorbed and coated on the surface of the nano-selenium by forming a C-O … Se bond or strong interaction between hydroxyl of the polysaccharide and the nano-selenium.
The polysaccharide nano-selenium is applied to selenium-rich fruit and vegetable ferment.
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into pieces with volume of 3-5cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
(2) adding pectinase with the mass of 0.01-0.03 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 40-50 ℃, the time is 60-120min, and the pH is 3-5;
(3) sterilizing the fruit and vegetable juice at the temperature of 115-125 ℃ for 8-12s, cooling to 35-38 ℃, inoculating mixed bacteria according to the inoculum size of 2-5 wt% under the aseptic condition, fermenting at the temperature of 35-38 ℃ for 50-80 days, filtering after the fermentation is finished, and removing filter residues to obtain a crude enzyme solution;
(4) mixing the crude enzyme solution, the polysaccharide nano selenium and honey according to a mass ratio of 100: (0.2-0.5): and (1-3) uniformly mixing, heating and sterilizing in a water bath at the temperature of 92-100 ℃ for 25-40min, and cooling to obtain the selenium-rich fruit and vegetable enzyme.
Preferably, the preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into pieces with volume of 3-5cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
(2) adding pectinase with the mass of 0.01-0.03 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 40-50 ℃, the time is 60-120min, and the pH is 3-5;
(3) sterilizing the fruit and vegetable juice at 125 deg.C under 115 deg.C for 8-12s, cooling to 35-38 deg.C, inoculating mixed bacteria at 2-5 wt% under aseptic condition, and fermenting at 35-38 deg.C for 40-50 days to obtain primary fermented product; adding a synergist accounting for 1-3 wt% of the fruit and vegetable juice, continuing to ferment for 10-30 days to obtain a secondary ferment, filtering, and removing filter residues to obtain a crude ferment liquid;
(4) mixing the crude enzyme solution, the polysaccharide nano selenium and honey according to a mass ratio of 100: (0.2-0.5): and (1-3) uniformly mixing, heating and sterilizing in a water bath at the temperature of 92-100 ℃ for 25-40min, and cooling to obtain the selenium-rich fruit and vegetable enzyme.
The preparation method of the synergist comprises the following steps:
pulverizing Curcumae rhizoma, sieving with 150-200 mesh sieve, adding into 85-95 wt% ethanol water solution according to the material-liquid ratio of 2kg (11-15) L, soaking for 5-10h, and performing ultrasonic extraction with the ultrasonic extraction parameters: the temperature is 70-80 ℃, the ultrasonic power is 200-300W, the ultrasonic frequency is 20-40kHz, and the ultrasonic time is 2-4 h; centrifuging at 8000rpm and 5000-; mixing the zedoary turmeric extract, ganoderma lucidum glucan and highland barley glucan uniformly according to the mass ratio of (0.2-0.5) to (0.5-1) to 2 to obtain the synergist.
The fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
The mixed bacteria are one or more of saccharomyces cerevisiae, lactobacillus plantarum, lactobacillus casei, lactobacillus helveticus, bacillus aceticus and streptococcus thermophilus; preferably, the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of (1-3): 1 are mixed to obtain the product.
The invention has the beneficial effects that: the invention provides polysaccharide nano-selenium which is applied to selenium-rich fruit and vegetable enzyme, so that the fruit and vegetable enzyme is rich in nutritional active ingredients generated by fruit and vegetable fermentation, also comprises the polysaccharide nano-selenium which is easy to absorb and utilize by a human body, can supplement essential trace elements for the organism in time, has good antioxidant activity, and can enhance the immunity of the organism. The invention separates and extracts natural plant polysaccharide with excellent biodegradability, stability and innocuity from natural plant asparagus, and the polysaccharide is combined with selenium which is an essential element participating in human body life activities by a chemical modification method due to the high complex branch structure and rich hydroxyl groups to prepare the polysaccharide nano selenium which has dual activities of selenium and polysaccharide and is easy to be absorbed and utilized by organisms, and has good stability and high bioactivity.
Detailed Description
The above summary of the present invention is described in further detail below with reference to specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples.
Introduction of some raw materials in this application:
pectinase, enzyme activity: 10 ten thousand U/g, supplied by Zhengzhou Yu and food additives Co., Ltd.
Saccharomyces cerevisiae, no: ACCC20034, purchased from China agricultural culture Collection for microorganisms.
Lactobacillus plantarum, No.: ACCC11016, purchased from China agricultural microbial culture Collection management center.
Ganoderma lucidum glucan, type: KZ-LZPJT, Kanzhou Biotech, Inc., Shaanxi.
Highland barley glucan, type: MH-QKPJT, Seaman grass Biotech Co., Ltd.
Gracilaria verrucosa belongs to Gracilaria genus plant of Gracilaria family, and is prepared by air drying naturally Gracilaria verrucosa.
Example 1
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 ℃ for 10s, cooling to 37 ℃, inoculating mixed bacteria according to 3 wt% of the inoculum size under aseptic condition, fermenting at 37 ℃ for 60 days, filtering after the fermentation is finished, and removing filter residue to obtain crude enzyme solution; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude ferment liquid, the nano selenium and the honey according to a mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The nano-selenium is prepared by the following method: uniformly mixing 10 parts by weight of 60mmol/L sodium selenite aqueous solution and 200 parts by weight of water, stirring at the room temperature for 60min at the rotating speed of 800rpm, then adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuously stirring for 6h under the dark condition of 40 ℃, then centrifuging at the rotating speed of 12000rpm for 30min, taking the bottom precipitate, and freeze-drying to obtain the nano-selenium.
Example 2
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 ℃ for 10s, cooling to 37 ℃, inoculating mixed bacteria according to 3 wt% of the inoculum size under aseptic condition, fermenting at 37 ℃ for 60 days, filtering after the fermentation is finished, and removing filter residue to obtain crude enzyme solution; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude enzyme solution, the polysaccharide nano-selenium and the honey according to the mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 60 ℃ for 6 hours, taking out the dried asparagus and crushing the dried asparagus and then sieving the dried asparagus with a 40-mesh sieve to obtain asparagus powder, then putting 100 parts by weight of the asparagus powder into 400 parts by weight of absolute ethyl alcohol, heating the mixture to 70 ℃ and refluxing the mixture for 3 hours, and filtering the mixture to obtain filtrate and filter residues; then adding the filter residue into 1500 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at 70 deg.C and ultrasonic power of 200W and ultrasonic frequency of 25kHz for 90min, and rotating at 5000rpm for 20min, and collecting supernatant; finally, mixing the filtrate and the supernatant, performing vacuum concentration to one tenth of the original volume at 45 ℃ by using a rotary evaporator, adding absolute ethyl alcohol to enable the volume fraction of the ethyl alcohol to reach 80%, standing for 24h at 4 ℃, centrifuging for 20min at 8000rpm after the completion, taking the precipitate, and performing freeze drying to obtain asparagus polysaccharide;
s2, uniformly mixing 20 parts by weight of 20mg/mL tween-80 aqueous solution and 10 parts by weight of 60mmol/L sodium selenite aqueous solution, adding 180 parts by weight of 6mg/mL asparagus polysaccharide aqueous solution, stirring at the room temperature at the rotating speed of 800rpm for 60min, adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuing stirring at 40 ℃ in the dark for 6h, placing the mixture in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing with water for 72h, wherein water is replaced every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
Example 3
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 ℃ for 10s, cooling to 37 ℃, inoculating mixed bacteria according to 3 wt% of the inoculum size under aseptic condition, fermenting at 37 ℃ for 60 days, filtering after the fermentation is finished, and removing filter residue to obtain crude enzyme solution; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude enzyme solution, the polysaccharide nano-selenium and the honey according to the mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 60 ℃ for 6 hours, taking out the dried asparagus and crushing the dried asparagus and then sieving the dried asparagus with a 40-mesh sieve to obtain asparagus powder, then putting 100 parts by weight of the asparagus powder into 400 parts by weight of absolute ethyl alcohol, heating the mixture to 70 ℃ and refluxing the mixture for 3 hours, and filtering the mixture to obtain filtrate and filter residues; then adding the filter residue into 1500 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at 70 deg.C and ultrasonic power of 200W and ultrasonic frequency of 25kHz for 90min, and rotating at 5000rpm for 20min, and collecting supernatant; finally, mixing the filtrate and the supernatant, concentrating the mixture in vacuum at 45 ℃ by using a rotary evaporator to one tenth of the original volume, adding absolute ethyl alcohol to ensure that the volume fraction of the ethyl alcohol reaches 80%, standing the mixture for 24 hours at 4 ℃, centrifuging the mixture for 20 minutes at 8000rpm after the completion, taking the precipitate, and freeze-drying the precipitate to obtain crude polysaccharide; dissolving the crude polysaccharide in 40 weight parts of water, adding 10 weight parts of extractant, stirring at 400rpm for 30min at room temperature, pouring into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with water for 48h, wherein water is replaced every 24h, collecting dialysate in the dialysis bag, and freeze-drying to obtain Gracilaria lemaneiformis polysaccharide; the extractant is formed by mixing n-butyl alcohol and chloroform according to the volume ratio of 1: 4.
S2, uniformly mixing 20 parts by weight of 20mg/mL tween-80 aqueous solution and 10 parts by weight of 60mmol/L sodium selenite aqueous solution, adding 180 parts by weight of 6mg/mL asparagus polysaccharide aqueous solution, stirring at the room temperature at the rotating speed of 800rpm for 60min, adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuing stirring at 40 ℃ in the dark for 6h, placing the mixture in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing with water for 72h, wherein water is replaced every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
Example 4
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 ℃ for 10s, cooling to 37 ℃, inoculating mixed bacteria according to 3 wt% of the inoculum size under aseptic condition, fermenting at 37 ℃ for 60 days, filtering after the fermentation is finished, and removing filter residue to obtain crude enzyme solution; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude enzyme solution, the polysaccharide nano-selenium and the honey according to the mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 60 ℃ for 6 hours, taking out the dried asparagus and crushing the dried asparagus and then sieving the dried asparagus with a 40-mesh sieve to obtain asparagus powder, then putting 100 parts by weight of the asparagus powder into 400 parts by weight of absolute ethyl alcohol, heating the mixture to 70 ℃ and refluxing the mixture for 3 hours, and filtering the mixture to obtain filtrate and filter residues; then adding the filter residue into 1500 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at 70 deg.C and ultrasonic power of 200W and ultrasonic frequency of 25kHz for 90min, and rotating at 5000rpm for 20min, and collecting supernatant; finally, mixing the filtrate and the supernatant, concentrating the mixture in vacuum at 45 ℃ by using a rotary evaporator to one tenth of the original volume, adding absolute ethyl alcohol to ensure that the volume fraction of the ethyl alcohol reaches 80%, standing the mixture for 24 hours at 4 ℃, centrifuging the mixture for 20 minutes at 8000rpm after the completion, taking the precipitate, and freeze-drying the precipitate to obtain crude polysaccharide; dissolving the crude polysaccharide in 40 weight parts of water, adding 10 weight parts of extractant, stirring at 400rpm for 30min at room temperature, pouring into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with water for 48h, wherein water is replaced every 24h, collecting dialysate in the dialysis bag, and freeze-drying to obtain Gracilaria lemaneiformis polysaccharide; the extractant is formed by mixing n-butyl alcohol and chloroform according to the volume ratio of 1: 4.
S2, uniformly mixing 20 parts by weight of 20mg/mL sodium dodecyl benzene sulfonate aqueous solution and 10 parts by weight of 60mmol/L sodium selenite aqueous solution, then adding 180 parts by weight of 6mg/mL asparagus polysaccharide aqueous solution, stirring at the room temperature at the rotating speed of 800rpm for 60min, then adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuing stirring for 6h under the dark condition of 40 ℃, then placing in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing with water for 72h, wherein water is replaced every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
Example 5
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 ℃ for 10s, cooling to 37 ℃, inoculating mixed bacteria according to 3 wt% of the inoculum size under aseptic condition, fermenting at 37 ℃ for 60 days, filtering after the fermentation is finished, and removing filter residue to obtain crude enzyme solution; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude enzyme solution, the polysaccharide nano-selenium and the honey according to the mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 60 ℃ for 6 hours, taking out the dried asparagus and crushing the dried asparagus and then sieving the dried asparagus with a 40-mesh sieve to obtain asparagus powder, then putting 100 parts by weight of the asparagus powder into 400 parts by weight of absolute ethyl alcohol, heating the mixture to 70 ℃ and refluxing the mixture for 3 hours, and filtering the mixture to obtain filtrate and filter residues; then adding the filter residue into 1500 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at 70 deg.C and ultrasonic power of 200W and ultrasonic frequency of 25kHz for 90min, and rotating at 5000rpm for 20min, and collecting supernatant; finally, mixing the filtrate and the supernatant, concentrating the mixture in vacuum at 45 ℃ by using a rotary evaporator to one tenth of the original volume, adding absolute ethyl alcohol to ensure that the volume fraction of the ethyl alcohol reaches 80%, standing the mixture for 24 hours at 4 ℃, centrifuging the mixture for 20 minutes at 8000rpm after the completion, taking the precipitate, and freeze-drying the precipitate to obtain crude polysaccharide; dissolving the crude polysaccharide in 40 weight parts of water, adding 10 weight parts of extractant, stirring at 400rpm for 30min at room temperature, pouring into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with water for 48h, wherein water is replaced every 24h, collecting dialysate in the dialysis bag, and freeze-drying to obtain Gracilaria lemaneiformis polysaccharide; the extractant is formed by mixing n-butyl alcohol and chloroform according to the volume ratio of 1: 4.
S2, uniformly mixing 20 parts by weight of 20mg/mL dispersant aqueous solution and 10 parts by weight of 60mmol/L sodium selenite aqueous solution, adding 180 parts by weight of 6mg/mL asparagus polysaccharide aqueous solution, stirring at the room temperature at the rotating speed of 800rpm for 60min, adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuing stirring for 6h under the dark condition of 40 ℃, placing the mixture in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing with water for 72h, wherein the water is replaced every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
The dispersing agent is a mixture of Tween-80 and sodium dodecyl benzene sulfonate, wherein the mass ratio of the Tween-80 to the sodium dodecyl benzene sulfonate is 3: 2.
Example 6
The preparation method of the selenium-rich fruit and vegetable ferment comprises the following steps:
(1) selecting fresh mature fruits and vegetables without mechanical damage, cleaning, peeling, removing core, and cutting into 4cm3Pulping the fruit and vegetable blocks to obtain fruit and vegetable pulp;
the fruit and vegetable is composed of apples, bananas, pears, white radishes, lettuce, ginger and carrots according to the mass ratio of 1:1:1:1:1:1: 1.
(2) Adding pectinase accounting for 0.02 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 45 ℃, the time is 90min, and the pH is 4;
(3) sterilizing the fruit and vegetable juice at 120 deg.C for 10s, cooling to 37 deg.C, inoculating mixed bacteria at 3 wt% under aseptic condition, and fermenting at 37 deg.C for 40 days to obtain primary fermented product; adding a synergist accounting for 2.5 wt% of the fruit and vegetable juice, continuing to ferment for 20 days to obtain a secondary ferment, filtering, and removing filter residues to obtain a crude ferment liquid; the mixed bacteria are prepared from saccharomyces cerevisiae and lactobacillus plantarum according to a mass ratio of 1:1, mixing to obtain;
(4) mixing the crude enzyme solution, the polysaccharide nano-selenium and the honey according to the mass ratio of 100: 0.3: 2, uniformly mixing, heating and sterilizing in a water bath at 95 ℃ for 30min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
The preparation method of the polysaccharide nano selenium comprises the following steps:
s1, drying the asparagus in a drying box at 60 ℃ for 6 hours, taking out the dried asparagus and crushing the dried asparagus and then sieving the dried asparagus with a 40-mesh sieve to obtain asparagus powder, then putting 100 parts by weight of the asparagus powder into 400 parts by weight of absolute ethyl alcohol, heating the mixture to 70 ℃ and refluxing the mixture for 3 hours, and filtering the mixture to obtain filtrate and filter residues; then adding the filter residue into 1500 parts by weight of water for ultrasonic extraction, wherein the ultrasonic extraction parameters are as follows: centrifuging at 70 deg.C and ultrasonic power of 200W and ultrasonic frequency of 25kHz for 90min, and rotating at 5000rpm for 20min, and collecting supernatant; finally, mixing the filtrate and the supernatant, concentrating the mixture in vacuum at 45 ℃ by using a rotary evaporator to one tenth of the original volume, adding absolute ethyl alcohol to ensure that the volume fraction of the ethyl alcohol reaches 80%, standing the mixture for 24 hours at 4 ℃, centrifuging the mixture for 20 minutes at 8000rpm after the completion, taking the precipitate, and freeze-drying the precipitate to obtain crude polysaccharide; dissolving the crude polysaccharide in 40 weight parts of water, adding 10 weight parts of extractant, stirring at 400rpm for 30min at room temperature, pouring into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with water for 48h, wherein water is replaced every 24h, collecting dialysate in the dialysis bag, and freeze-drying to obtain Gracilaria lemaneiformis polysaccharide; the extractant is formed by mixing n-butyl alcohol and chloroform according to the volume ratio of 1: 4.
S2, uniformly mixing 20 parts by weight of 20mg/mL dispersant aqueous solution and 10 parts by weight of 60mmol/L sodium selenite aqueous solution, adding 180 parts by weight of 6mg/mL asparagus polysaccharide aqueous solution, stirring at the room temperature at the rotating speed of 800rpm for 60min, adding 10 parts by weight of 240mmol/L ascorbic acid aqueous solution, continuing stirring for 6h under the dark condition of 40 ℃, placing the mixture in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing with water for 72h, wherein the water is replaced every 24h, finally collecting dialysate in the dialysis bag, and freeze-drying to obtain the polysaccharide nano-selenium.
The dispersing agent is a mixture of Tween-80 and sodium dodecyl benzene sulfonate, wherein the mass ratio of the Tween-80 to the sodium dodecyl benzene sulfonate is 3: 2.
The preparation method of the synergist comprises the following steps:
pulverizing rhizoma Curcumae, sieving with 200 mesh sieve, adding into 92 wt% ethanol water solution according to the material-liquid ratio of 2kg:13L, soaking for 8h, and performing ultrasonic extraction with the ultrasonic extraction parameters: the temperature is 75 ℃, the ultrasonic power is 300W, the ultrasonic frequency is 40kHz, and the ultrasonic time is 3 h; centrifuging at 6000rpm for 15min, collecting supernatant, vacuum concentrating at 45 deg.C with rotary evaporator to remove ethanol, and freeze drying to obtain Curcumae rhizoma extract; and uniformly mixing the curcuma zedoary extract, the ganoderma lucidum glucan and the highland barley glucan according to the mass ratio of 0.3:1:2 to obtain the synergist. The selenium-rich fruit and vegetable ferment of example 6 was evaluated according to the methods of test examples 1 and 2, and the superoxide anion removal rate was 80.1% and the lymphocyte proliferation rate was 31.95%.
Test example 1
Evaluation of antioxidant capacity in vitro: firstly, 2.4mL of 0.05mol/L Tris-HCl buffer solution with pH value of 8.2 is taken, then 1.0mL of selenium-rich fruit and vegetable ferment prepared in the embodiment is added, the mixture is kept warm in a water bath at 25 ℃ for 30min, taken out, 0.3mL of 7mmo1/L pyrogallol solution is added to start reaction, 0.5mL of 10mo1/L concentrated hydrochloric acid is added to stop the reaction at the 4 th min, and the absorbance at 325nm is measured by an ultraviolet spectrophotometer (A)i) In the control group, 1.0mL of distilled water was used to replace selenium-rich fruit and vegetable ferment (A)0). Each group of examples was tested in parallel for 5 groups and averaged. The superoxide anion radical clearance calculation formula is as follows:
superoxide anion scavenging ratio (%) - (A)0-Ai)/A0×100％
TABLE 1 in vitro antioxidant Capacity test results
Superoxide anion scavenging ratio (%)
Example 1 54.6
Example 2 65.8
Example 3 73.5
Example 4 73.2
Example 5 78.3
The above results show that the antioxidant capacity of the selenium-enriched fruit and vegetable ferment obtained in example 2 is significantly improved compared to that of example 1, which is probably because, compared to pure nano-selenium, the polysaccharide nano-selenium is used as a novel natural antioxidant, and combines the asparagus polysaccharide with natural antioxidant activity on the basis of the antioxidant activity of the original nano-selenium, so that more free radicals can be removed, cell membranes can be protected, the active oxygen damage resistance of the organism can be improved, and the antioxidant capacity of the prepared selenium-enriched fruit and vegetable ferment can be significantly improved. Example 3 based on example 2, the asparagus polysaccharide is further extracted and purified, so that not only can the effective components in the asparagus crude polysaccharide be effectively and finely separated, but also the structure and biological activity of the asparagus polysaccharide can be better maintained, and the utilization rate of the asparagus polysaccharide in the preparation of nano-selenium is improved, thereby promoting the improvement of the antioxidant capacity. Compared with the embodiments 3 and 4, the dispersant in the embodiment 5 adopts the compounding of tween-80 and sodium dodecyl benzene sulfonate, so that the synergistic effect is achieved, the antioxidant capacity of the selenium-rich fruit and vegetable ferment is further improved, probably because a mixed system formed by compounding the tween-80 and the sodium dodecyl benzene sulfonate can reduce the interfacial tension, increase the adsorption capacity of the surface of the nano selenium, provide more binding sites for the polysaccharide, control the nucleation and growth of the nano selenium, promote the formation of the polysaccharide nano selenium with uniform size, improve the binding rate of the polysaccharide, facilitate the absorption and utilization of the small molecular polysaccharide nano selenium by organisms, and facilitate the improvement of the antioxidant activity.
Test example 2
Evaluation of in vitro enhanced immune activity: taking heart of adult chicken for aseptic blood collection, performing heparin anticoagulation, adding equal volume of Hank's solution for dilution, adding into upper layer of lymphocyte separation solution, centrifuging at 2000rpm for 15min, sucking middle cloudy cell layer, washing with Hank's solution for 2 times, centrifuging at 1500rpm for 15min, counting viable cells more than 90%, and adjusting cell concentration to 2.5 × 10 with 10% calf serum-containing RPMI-1640 culture solution6each/mL of the selenium-enriched fruit and vegetable ferment is inoculated into a 96-well cell culture plate, each well is 100 mu L, then 100 mu L of the selenium-enriched fruit and vegetable ferment prepared in the embodiment is added, and each group of the embodiment is repeated for 4 wells; a4-well cell control group was also prepared, and only 100. mu.L of the cell culture medium was added. The 96-well cell culture plate is placed at 37.5 ℃ and 5% CO2Culturing for 44h under the condition, adding 30 μ L MTT into each well, culturing for 4h, centrifuging 96-well cell culture plate at 1500rpm for 10min, discarding supernatant, adding 100 μ L DMSO lysate into each well, placing the culture plate on a micro-oscillator, oscillating for 5min to dissolve precipitate completely, and detecting absorbance at 570nm wavelength (A) with enzyme linked immunosorbent assay detector570nm) And calculating the proliferation rate of the lymphocyte. The lymphocyte proliferation rate calculation formula is as follows:
lymphocyte proliferation rate (%) ═ aTest group-AControl group)/AControl group×100％
TABLE 2 in vitro potentiation of immune Activity test results
Lymphocyte proliferation rate (%)
Example 1 15.24
Example 2 23.17
Example 3 28.69
Example 4 28.65
Example 5 31.36
The polysaccharide nano-selenium prepared by the invention is an organic selenium compound bonded by selenium and natural plant polysaccharide, not only can play the dual functions of selenium and plant polysaccharide, but also can effectively improve the bioavailability of selenium, greatly reduces the toxicity and side effects, and greatly improves the biological activity.

Claims (9)

1. The preparation method of the polysaccharide nano selenium is characterized by comprising the following steps:
s1, drying and crushing asparagus, sieving to obtain asparagus powder, putting the asparagus powder into absolute ethyl alcohol, performing reflux extraction for 2-5h, and filtering to obtain filtrate and filter residue; then adding the filter residue into water for ultrasonic extraction, centrifuging after the ultrasonic extraction is finished, and collecting supernatant; mixing the above filtrate and supernatant, vacuum concentrating, adding anhydrous ethanol, standing for 12-36 hr, centrifuging, collecting precipitate, and freeze drying to obtain crude polysaccharide; dissolving crude polysaccharide in 30-50 weight parts of water, adding 8-15 weight parts of extractant, stirring at room temperature for 20-40min, dialyzing with water for 36-60h, collecting dialysate, and freeze drying to obtain thallus Gracilariae polysaccharide;
s2, uniformly mixing 10-30 parts by weight of 15-25mg/mL dispersant aqueous solution and 5-15 parts by weight of 50-70mmol/L sodium selenite aqueous solution, adding 160-200 parts by weight of 5-8mg/mL asparagus polysaccharide aqueous solution, stirring at room temperature for 40-80min, adding 5-15 parts by weight of 200-280mmol/L ascorbic acid aqueous solution, continuously stirring at 35-50 ℃ in the dark for 5-8h, dialyzing with water for 48-96h, collecting dialysate, and freeze-drying to obtain the polysaccharide nano-selenium.
2. The method for preparing polysaccharide nano-selenium as claimed in claim 1, wherein the extractant is formed by mixing n-butanol and chloroform in a volume ratio of 1 (3-5).
3. The method of claim 1, wherein the dispersant is one or more of sodium dodecylbenzene sulfonate, cetyltrimethylammonium bromide, polyvinylpyrrolidone, and tween-80.
4. Polysaccharide nanoseles, characterized in that it is prepared by a process as claimed in any one of claims 1 to 3.
5. The preparation method of the selenium-rich fruit and vegetable ferment is characterized by comprising the following steps:
(1) selecting fresh and mature fruits and vegetables without mechanical damage, cleaning, peeling, removing kernels, cutting into pieces, and pulping to obtain fruit and vegetable pulp;
(2) adding pectinase with the mass of 0.01-0.03 wt% of the fruit and vegetable pulp into the fruit and vegetable pulp for enzymolysis, and filtering to obtain fruit and vegetable juice; the enzymolysis conditions are as follows: the temperature is 40-50 ℃, the time is 60-120min, and the pH is 3-5;
(3) sterilizing the fruit and vegetable juice at 125 deg.C under 115 deg.C for 8-12s, cooling to 35-38 deg.C, inoculating mixed bacteria at 2-5 wt% under aseptic condition, and fermenting at 35-38 deg.C for 40-50 days to obtain primary fermented product; adding a synergist accounting for 1-3 wt% of the fruit and vegetable juice, continuing to ferment for 10-30 days to obtain a secondary ferment, filtering, and removing filter residues to obtain a crude ferment liquid;
(4) mixing crude ferment liquid, the polysaccharide nano selenium according to claim 4 and honey according to the mass ratio of 100: (0.2-0.5): (1-3) uniformly mixing, heating and sterilizing in a water bath at the temperature of 92-100 ℃ for 25-40min, and cooling to obtain the selenium-rich fruit and vegetable ferment.
6. The preparation method of the selenium-rich fruit and vegetable ferment as claimed in claim 5, wherein the preparation method of the synergist comprises the following steps:
pulverizing Curcumae rhizoma, sieving, adding into 85-95 wt% ethanol water solution according to a material-liquid ratio of 2kg (11-15) L, soaking for 5-10h, and performing ultrasonic extraction with ultrasonic extraction parameters: the temperature is 70-80 ℃, the ultrasonic power is 200-300W, the ultrasonic frequency is 20-40kHz, and the ultrasonic time is 2-4 h; centrifuging at 8000rpm and 5000-; mixing the zedoary turmeric extract, ganoderma lucidum glucan and highland barley glucan uniformly according to the mass ratio of (0.2-0.5) to (0.5-1) to 2 to obtain the synergist.
7. The method for preparing the selenium-rich fruit and vegetable enzyme according to claim 5, wherein the fruit and vegetable consists of apple, banana, pear, white radish, lettuce, ginger and carrot in a mass ratio of 1 (0.5-1): (0.5-1): (0.5-1): (0.5-1): 0.5-1).
8. The method for preparing the selenium-rich fruit and vegetable ferment as claimed in claim 5, wherein the mixed bacteria is one or more of Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Acetobacter aceti and Streptococcus thermophilus.
9. The selenium-rich fruit and vegetable ferment is characterized by being prepared by the preparation method of any one of claims 5 to 8.
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