AU2020104369A4

AU2020104369A4 - Preparation Method of Active Peptide for Enhancing Selenium Absorption and Application Thereof

Info

Publication number: AU2020104369A4
Application number: AU2020104369A
Authority: AU
Inventors: Wenjuan Jiao; Weifeng Liu; Yinqing Qiu; Ying Shi; Yehui Zhang; Yousheng Zhang; Tiantian ZHAO
Original assignee: Sericultural & Agri Food Res Institute Guangdong Academy Of Agricultural Sciences
Current assignee: Sericultural & Agri Food Research Institute Guangdong Academy Of Agricultural Sciences
Priority date: 2020-12-07
Filing date: 2020-12-29
Publication date: 2021-03-18
Anticipated expiration: 2028-12-29
Also published as: CN112481341B; CN112481341A

Abstract

A b s t r a c t The present disclosure belongs to the technical field of food processing, and specifically relates to a preparation method of an active peptide for enhancing selenium absorption and application thereof. According to the present disclosure, a supernatant prepared from fish skin, whey protein powder, tapioca starch, carrot powder and selenium-enriched yeast powder are subjected to anaerobic fermentation to obtain a fermentation solution, and the fermentation solution is added with selenoprotein and subjected to ultrasonic treatment, then mixed with a solution consisting of fish skin mucus, mulberry leaf juice, peanut oil and seaweed polysaccharide, and finally subjected to high static pressure treatment to obtain the active peptide for enhancing selenium absorption; the active peptide for enhancing selenium absorption prepared by the present disclosure mainly includes 6-peptide compounds and 8-peptide compounds, which are easily absorbed by the small intestine and the duodenum; at the same time, the structure is stable, the tolerance in the stomach and intestines is high, and absorption of selenium by the human body can be effectively enhanced; in addition, the active peptide for enhancing selenium absorption in the present disclosure can be mutually matched with other nutrients or food ingredients to prepare selenium-enriched health-care foods, which have not only high selenium absorption efficiency but also unique flavors and are easily accepted by people. 1

Description

A b s tr ac t

The present disclosure belongs to the technical field of food

processing, and specifically relates to a preparation method of an active

peptide for enhancing selenium absorption and application thereof.

According to the present disclosure, a supernatant prepared from fish skin,

whey protein powder, tapioca starch, carrot powder and

selenium-enriched yeast powder are subjected to anaerobic fermentation

to obtain a fermentation solution, and the fermentation solution is added

with selenoprotein and subjected to ultrasonic treatment, then mixed with

a solution consisting of fish skin mucus, mulberry leaf juice, peanut oil

and seaweed polysaccharide, and finally subjected to high static pressure

treatment to obtain the active peptide for enhancing selenium absorption;

the active peptide for enhancing selenium absorption prepared by the

present disclosure mainly includes 6-peptide compounds and 8-peptide

compounds, which are easily absorbed by the small intestine and the

duodenum; at the same time, the structure is stable, the tolerance in the

stomach and intestines is high, and absorption of selenium by the human

body can be effectively enhanced; in addition, the active peptide for

enhancing selenium absorption in the present disclosure can be mutually

matched with other nutrients or food ingredients to prepare

selenium-enriched health-care foods, which have not only high selenium

absorption efficiency but also unique flavors and are easily accepted by

people.

D e s c r i p t io n

Preparation Method of Active Peptide for Enhancing Selenium

Absorption and Application Thereof

Technical Field

The present disclosure belongs to the technical field of food

processing, and specifically relates to a preparation method of an active

peptide for enhancing selenium absorption and application thereof.

Related Art

Selenium is a trace element necessary for the human body and

affects the health and development of the human body. It is recommended

by "Dietary Reference Intakes of Chinese Residents" that 50 micrograms

of selenium is needed every day. Selenium participates in synthesis of

various selenium-containing enzymes and selenoproteins in the human

body, can improve human immunity and promote proliferation of

lymphocytes and synthesis of antibodies and immunoglobulins, and has

obvious inhibition and protection effects on various cancers such as colon

cancer, skin cancer, liver cancer and breast cancer. Methyl selenol, as an

intermediate metabolite of selenium in the human body, has high

anti-cancer activity. Selenium, vitamin E, allicin, linoleic acid,

germanium, zinc and other nutrients have a synergistic antioxidant effect,

so that the antioxidant activity is improved. At the same time, selenium

D e s c r i p t io n

has the effect of reducing and alleviating toxicity of heavy metals. It is

proved by some studies that selenium can effectively inhibit tumor

growth, and an appropriate amount of selenium has a good auxiliary

improvement effect on patients after surgery, radiotherapy and

chemotherapy treatment. In addition, selenium is also the most important

anti-aging element discovered so far. Research by experts from the

Chinese Academy of Sciences on Bama, the home of longevity, show that

the content of selenium in soil and grain of Bama is about 10 times higher

than the national average level, and the content of selenium in the blood

of centenarians is 3 to 6 times higher than that of normal people.

China has a vast territory, and the content of selenium in different

soils varies greatly. In some selenium-deficient areas, the content of

selenium in crops is also low, and the content of selenium in food is not

enough to meet the needs of the human body. Therefore, various

selenium-enriched foods appear on the market. The selenium-enriched

foods, namely foods rich in trace element selenium, generally include

natural selenium-enriched foods (also called plant active selenium foods)

and exogenous selenium-enriched foods (also called artificial organic

selenium foods).

At present, although there are various kinds of foods with a selenium

supplement effect on the market, such as selenium-enriched peanuts and

selenium-enriched rice, most of the selenium supplement foods are

D e s c r i p t io n

expensive but have unclear selenium content. At the same time, the

selenium intake effect of the selenium supplement foods is not

satisfactory due to the influence of digestion and absorption by the human

body. Therefore, it is of great value to develop selenium-enriched

health-care foods capable of improving the selenium absorption

efficiency.

Summary

In order to overcome the shortcomings in the prior art, the present

disclosure provides a preparation method of an active peptide for

enhancing selenium absorption. The binding stability of protein peptide

and selenium can be improved by using biological fermentation and

physical enhancement methods, so that not only can the flavor of a

selenium-enriched peptide be improved, but also the effect of absorbing

selenium by the human body can be improved to achieve the purpose of

selenium supplementation.

In order to achieve the objective, the present disclosure adopted the

following technical scheme:

The present disclosure provides a preparation method of an active

peptide for enhancing selenium absorption, and the method includes the

following steps:

S. preparing fish skin into fish paste, addingpepsin for enzymolysi

D e s c r i p t io n

treatment and collecting the supernatant;

S2. adding whey protein powder, tapioca starch, carrot powder and

selenium-enriched yeast powder into the supernatant obtained in step S1

and then performing anaerobic fermentation;

S3. adding selenoprotein into a fermentation solution obtained in

step S2 and then performing ultrasonic treatment;

S4. uniformly mixing fish skin mucus with mulberry leaf juice,

standing for a period of time, adding peanut oil and seaweed

polysaccharide and then performing homogeneous stirring;

S5. adding a solution obtained in step S4 into a fermentation solution

obtained in step S3, performing vacuumizing and high static pressure

treatment and finally performing depressurization to obtain the active

peptide for enhancing selenium absorption.

Preferably, the fish skin and the fish skin mucus are both from

snakeheaded fish.

Selenium binds to protein to form selenoprotein, which can improve

the structural stability in complex processing environments and digestion

environments. This form of active selenium helps the human body absorb

selenium and improve the bioavailability. According to the present

disclosure, a green and safe selenium-enriched peptide with high

bioavailability is prepared by using the yeast directed fermentation

technology, the stable selenopeptide compound technology and the

D e s c r i p t io n

characteristic of high absorption of peptides. Stable crosslinking of fish

protein and organic selenium is formed under high pressure, a stable

structure is formed by using high static pressure transformation

technology, 6-peptide compounds and 8-peptide compounds which are

easily absorbed by the small intestine and the duodenum are obtained,

and the absorption of organic selenium by intestinal mucosal cells is

enhanced; at the same time, a micro-nano embedding substance can be

formed after a mixture of the body surface mucus of the snakeheaded fish

and the mulberry leaf juice is emulsified, so that the structure of a

selenopeptide compound is not likely to be affected by gastrointestinal

digestion, and the selenopeptide compound can smoothly reach the

absorption site of the small intestine.

Preferably, in order to achieve a better enzymolysis effect, in step Sl,

the added amount of pepsin is 20-40% of the weight of the fish paste.

Preferably, in order to improve the directional fermentation effect, in

step S2, the whey protein powder, the tapioca starch and the carrot

powder are firstly added into the supernatant and heated to 30°C, then the

selenium-enriched yeast powder is added, and finally the temperature is

raised to 37°C for anaerobic fermentation.

Preferably, in order to achieve a better fermentation effect, in step S2,

based on the mass of the supernatant, the added amount of the whey

protein powder is 0.5-1.5%, the added amount of the tapioca starch is

D e s c r i p t io n

2.5-2.8%, the added amount of the carrot powder is 0.3-0.5%, and the

added amount of the selenium-enriched yeast powder is 0.2-0.3%.

Preferably, in order to achieve a better emulsification effect, in step

S4, the volume ratio of the fish skin mucus to the mulberry leaf juice is

1:1.

Preferably, in step S5, the high static pressure treatment is performed

at 300-350 MPa for 10 minutes. The high static pressure treatment is

more conducive to the formation of a stable structure between fish protein

and organic selenium.

Preferably, pH is one of the important conditions affecting the

enzymolysis effect, and in step S1, the pH during the enzymolysis

treatment is 3.8-4.2. Further, the pH during the enzymolysis treatment is

4.0.

Preferably, in order to ensure a sufficient degree of enzymolysis, the

enzymolysis treatment is stirring enzymolysis, and that is to say, stirring

is performed at a rotation speed of 200-300 r/min for 2 hours for

enzymolysis.

Preferably, the temperature is raised to 80°C and maintained for 10

minutes after the enzymolysis treatment to achieve the effects of

sterilization and enzyme inactivation.

Preferably, anaerobic fermentation is terminated when the pH is 4.8.

D e s c r i p t i o n

Preferably, in step S3, the added amount of selenoprotein is

0.001%-0.003% of the total mass of the fermentation solution.

Preferably, in order to ensure that selenoprotein is fully dispersed in

the fermentation solution, in step S3, the powder of ultrasonic treatment

is 300-400 W, and the time is 15-20 minutes.

Preferably, in step S3, the acidity (pH 4.8-5.0) and temperature

(35°C-40°C) of a solution are controlled to be unchanged during

ultrasonic treatment.

Preferably, in step S4, the added amount of the peanut oil is 10-15%

of the total volume of the fish skin mucus and the mulberry leaf juice, and

the added amount of the seaweed polysaccharide is 0.5-0.8 wt% of the

total mass of the fish skin mucus and the mulberry leaf juice.

Preferably, in order to ensure a sufficient degree of stirring, in step

S4, homogeneous stirring is performed at 42°C-46°C for 30-40 minutes.

Preferably, in order to ensure that a selenopeptide compound is fully

emulsified to form a micro-nano emulsion, in step S5, the volume ratio of

a solution obtained in step S4 to a fermentation solution obtained in step

S3 is 1:(4-6). Further, the volume ratio of the solution obtained in step S4

to the fermentation solution obtained in step S3 is 1:5.

Preferably, in step S5, the depressurization is to reduce the pressure

to 80-120 MPa and maintained for 10 minutes.

D e s c r i p t io n

The present disclosure also provides the active peptide for enhancing

selenium absorption prepared by adopting the preparation method.

The present disclosure also provides application of the active peptide

for enhancing selenium absorption in preparation of selenium-enriched

health-care foods.

The active peptide for enhancing selenium absorption is mutually

matched with other nutrients or food ingredients to prepare various forms

of selenium-enriched health-care foods. Not only can healthy nutrients of

the active peptide be improved, various forms of selenium supplement

foods with good selenium absorption effect are formed, but also different

forms of foods are easily accepted by different groups of people due to

different flavors.

The present disclosure also provides a preparation method of a

selenium-enriched emulsion beverage for enhancing selenium absorption,

and the method specifically includes: 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 a perilla extract and

1.3-2.0 wt% of glucose into the active peptide for enhancing selenium

absorption, uniformly stirring and mixing the mixture, and performing

high-temperature sterilization to obtain the selenium-enriched emulsion

beverage for enhancing selenium absorption.

The present disclosure also provides a preparation method of

D e s c r i p t io n

selenium-enriched health-care powder for enhancing selenium absorption,

and the method specifically includes: drying the active peptide for

enhancing selenium absorption into powder, then 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, and performing uniform mixing and packaging to obtain the

selenium-enriched health-care powder for enhancing selenium

absorption.

Compared with the prior art, the beneficial effects of the present

disclosure are:

The present disclosure provides a preparation method of an active

peptide for enhancing selenium absorption. A supernatant prepared from

fish skin, whey protein powder, tapioca starch, carrot powder and

selenium-enriched yeast powder are subjected to anaerobic fermentation

to obtain a fermentation solution, and the fermentation solution is added

with selenoprotein and subjected to ultrasonic treatment, then mixed with

a solution consisting of fish skin mucus, mulberry leaf juice, peanut oil

and seaweed polysaccharide, and finally subjected to high static pressure

treatment to obtain the active peptide for enhancing selenium absorption.

The active peptide for enhancing selenium absorption prepared by the

present disclosure mainly includes 6-peptide compounds and 8-peptide

D e s c r i p t io n

compounds, which are easily absorbed by the small intestine and the

duodenum; at the same time, the structure is stable, the active peptide is

not likely to be affected by gastrointestinal digestion and can smoothly

reach the absorption site of the small intestine, the tolerance in the

stomach and intestines is high, and absorption of selenium by the human

body can be effectively enhanced. In addition, the active peptide for

enhancing selenium absorption in the present disclosure can be mutually

matched with other nutrients or food ingredients to prepare

selenium-enriched health-care foods, which have not only high selenium

absorption efficiency but also unique flavors and are easily accepted by

people.

Detailed Description

Specific embodiments of the present disclosure are further described

below. It should be noted that description of these embodiments is used to

help understand the present disclosure without limiting the present

disclosure. In addition, technical features involved in the embodiments of

the present disclosure described below can be combined with each other

as long as they do not conflict with each other.

Experimental methods in the following examples, unless otherwise

specified, are all conventional methods, and test materials used in the

D e s c r i p t i o n

following examples, unless otherwise specified, can be all purchased

through conventional commercial channels.

Example 1 Preparation method of an active peptide for enhancing

selenium absorption

(1) Fresh snakeheaded fish was used as a raw material and

slaughtered, and fish skin and fish skin mucus were collected separately;

(2) the collected fish skin was chopped and mashed, 4°C pure water

was added into the chopped fish skin at a material-liquid weight ratio of

1:3, the fish skin was prepared into fish paste by using the wet ultra-fine

pulverization technology, the pH of the fish paste was adjusted to 4.0,

then pepsin which was 30% of the weight of the fish paste was added,

mechanical stirring was performed at a rotation speed of 250 r/min for

enzymolysis for 2 hours, 5000 g of an obtained mixture was subjected to

centrifugal filtration, and the supernatant was kept and heated to 80°C

and maintained for 10 minutes to achieve the effects of sterilization and

enzyme inactivation.

(3) The supernatant was poured into a fermentation tank, based on

the mass of the fish paste (supernatant), 1.0% of whey protein powder,

2.65% of tapioca starch and 0.4% of carrot powder were added,

uniformly mixed and heated to 30°C, then 0.25% of selenium-enriched

yeast powder was added, uniformly mixed and continuously heated to

37 0 C, and anaerobic fermentation was performed in the sealed

D e s c r i p t io n

fermentation tank for 3.5 hours and terminated when the pH reached 4.8.

(4) According to the total mass of a fermentation solution, 0.002% of

selenoprotein was added into the fermentation solution, uniformly mixed

and subjected to ultrasonic treatment at the power of 350 W for 17.5

minutes, and the acidity (pH 4.9) and temperature (37.5°C) of the

fermentation solution were controlled to be unchanged in the ultrasonic

process.

(5) The fish skin mucus obtained by separation in step (1) was

uniformly mixed with mulberry leaf juice at a volume ratio of 1:1, an

obtained mixture was subjected to standing for 10 minutes, peanut oil

which was 13% of the total volume (volume percentage) and seaweed

polysaccharide which was 0.65 wt% of the total mass were added, and an

obtained mixture was subjected to homogeneous stirring at 44 0 C for 40

minutes for later use.

(6) A solution obtained in step (4) was slowly added into the

fermentation solution in step (3) at a volume ratio of 1:5, uniformly

stirred and packaged in a bag, the bag was vacuumized, sealed and

subjected to high static pressure treatment at 330 MPa for 10 minutes,

and then the pressure was reduced to 100 MPa and maintained for 10

minutes to obtain a selenium-rich active peptide solution for enhancing

selenium absorption.

According to a method in "GB/T 22492-2008 Soy Peptide Powder",

D e s c r i p t io n

it is measured that main protein components in the selenium-enriched

active peptide solution are 6-peptide and 8-peptide.

Example 2 Preparation method of an active peptide for enhancing

selenium absorption

(1) Fresh snakeheaded fish was used as a raw material and

slaughtered, and fish skin and fish skin mucus were collected separately;

(2) the collected fish skin was chopped and mashed, 4°C pure water

was added into the chopped fish skin at a material-liquid weight ratio of

1:3, the fish skin was prepared into fish paste by using the wet ultra-fine

pulverization technology, the pH of the fish paste was adjusted to 4.0,

then pepsin which was 20% of the weight of the fish paste was added,

mechanical stirring was performed at a rotation speed of 200 r/min for

enzymolysis for 2 hours, 5000 g of an obtained mixture was subjected to

centrifugal filtration, and the supernatant was kept and heated to 80°C

and maintained for 10 minutes to achieve the effects of sterilization and

enzyme inactivation.

(3) The supernatant was poured into a fermentation tank, based on

the mass of the fish paste (supernatant), 0.5% of whey protein powder,

2.5% of tapioca starch and 0.3% of carrot powder were added, uniformly

mixed and heated to 30°C, then 0.2% of selenium-enriched yeast powder

was added, uniformly mixed and continuously heated to 37°C, and

anaerobic fermentation was performed in the sealed fermentation tank for

D e s c r i p t io n

3.5 hours and terminated when the pH reached 4.8.

(4) According to the total mass of a fermentation solution, 0.001% of

selenoprotein was added into the fermentation solution, uniformly mixed

and subjected to ultrasonic treatment at the power of 300 W for 20

minutes, and the acidity (pH 4.8) and temperature (35°C) of the

fermentation solution were controlled to be unchanged in the ultrasonic

process.

(5) The fish skin mucus obtained by separation in step (1) was

uniformly mixed with mulberry leaf juice at a volume ratio of 1:1, an

obtained mixture was subjected to standing for 10 minutes, peanut oil

which was 10% of the total volume (volume percentage) and seaweed

polysaccharide which was 0.5 wt% of the total mass were added, and an

obtained mixture was subjected to homogeneous stirring at 42 0 C for 40

minutes for later use.

(6) A solution obtained in step (4) was slowly added into the

fermentation solution obtained in step (3) at a volume ratio of 1:4,

uniformly stirred and packaged in a bag, the bag was vacuumized, sealed

and subjected to high static pressure treatment at 300 MPa for 10 minutes,

and then the pressure was reduced to 80 MPa and maintained for 10

minutes to obtain a selenium-rich active peptide solution for enhancing

selenium absorption.

According to a method in "GB/T 22492-2008 Soy Peptide Powder",

D e s c r i p t io n

it is measured that main protein components in the selenium-enriched

active peptide solution are 6-peptide and 8-peptide.

Example 3 Preparation method of an active peptide for enhancing

selenium absorption

(1) Fresh snakeheaded fish was used as a raw material and

slaughtered, and fish skin and fish skin mucus were collected separately;

(2) the collected fish skin was chopped and mashed, 4°C pure water

was added into the chopped fish skin at a material-liquid weight ratio of

1:3, the fish skin was prepared into fish paste by using the wet ultra-fine

pulverization technology, the pH of the fish paste was adjusted to 4.0,

then pepsin which was 40% of the weight of the fish paste was added,

mechanical stirring was performed at a rotation speed of 300 r/min for

enzymolysis for 2 hours, 5000 g of an obtained mixture was subjected to

centrifugal filtration, and the supernatant was kept and heated to 80°C

and maintained for 10 minutes to achieve the effects of sterilization and

enzyme inactivation.

(3) The supernatant was poured into a fermentation tank, based on

the mass of the fish paste (supernatant), 1.5% of whey protein powder,

2.8% of tapioca starch and 0.5% of carrot powder were added, uniformly

mixed and heated to 30°C, then 0.3% of selenium-enriched yeast powder

was added, uniformly mixed and continuously heated to 37°C, and

anaerobic fermentation was performed in the sealed fermentation tank for

D e s c r i p t io n

3.5 hours and terminated when the pH reached 4.8.

(4) According to the total mass of a fermentation solution, 0.003% of

selenoprotein was added into the fermentation solution, uniformly mixed

and subjected to ultrasonic treatment at the power of 400 W for 15

minutes, and the acidity (pH 5.0) and temperature (40°C) of the

fermentation solution were controlled to be unchanged in the ultrasonic

process.

(5) The fish skin mucus obtained by separation in step (1) was

uniformly mixed with mulberry leaf juice at a volume ratio of 1:1, an

obtained mixture was subjected to standing for 10 minutes, peanut oil

which was 15% of the total volume (volume percentage) and seaweed

polysaccharide which was 0.8 wt% of the total mass were added, and an

obtained mixture was subjected to homogeneous stirring at 46 0 C for 30

minutes for later use.

(6) A solution obtained in step (4) was slowly added into the

fermentation solution obtained in step (3) at a volume ratio of 1:6,

uniformly stirred and packaged in a bag, the bag was vacuumized, sealed

and subjected to high static pressure treatment at 350 MPa for 10 minutes,

and then the pressure was reduced to 120 MPa and maintained for 10

minutes to obtain a selenium-rich active peptide solution for enhancing

selenium absorption.

According to a method in "GB/T 22492-2008 Soy Peptide Powder",

D e s c r i p t io n

it is measured that main protein components in the selenium-enriched

active peptide solution are 6-peptide and 8-peptide.

Example 4 Preparation method of a selenium-enriched emulsion beverage

for enhancing selenium absorption

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 a

perilla extract and 1.6 wt% of glucose were added into the

selenium-enriched active peptide solution prepared in Example 1,

uniformly stirred and mixed and subjected to high-temperature

sterilization to obtain the selenium-enriched emulsion beverage for

enhancing selenium absorption.

Example 5 Preparation method of a selenium-enriched emulsion beverage

for enhancing selenium absorption

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 a perilla

extract and 1.3 wt% of glucose were added into the selenium-enriched

active peptide solution prepared in Example 1, uniformly stirred and

mixed and subjected to high-temperature sterilization to obtain the

selenium-enriched emulsion beverage for enhancing selenium absorption.

Example 6 Preparation method of a selenium-enriched emulsion beverage

for enhancing selenium absorption

2 wt% of whey protein, 0.003 wt% of lactoferrin, 0.15 wt% of edible

D e s c r i p t io n

fungus superfine powder, 1.3 wt% of maltodextrin, 1.3 wt% of a perilla

extract and 1.3 wt% of glucose were added into the selenium-enriched

active peptide solution prepared in Example 1, uniformly stirred and

mixed and subjected to high-temperature sterilization to obtain the

selenium-enriched emulsion beverage for enhancing selenium absorption.

Example 7 Preparation method of selenium-enriched health-care powder

for enhancing selenium absorption

The selenium-enriched active peptide solution prepared in Example 1

was spray-dried into light yellow selenium-enriched 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 superfine

powder, 9 wt% of maltodextrin and 0.008 wt% of solid DHA powder

were added into the selenium-enriched active peptide powder, uniformly

mixed and packaged to obtain the selenium-enriched health-care powder

for enhancing selenium absorption.

Example 8 Preparation method of selenium-enriched health-care powder

for enhancing selenium absorption

The selenium-enriched active peptide solution prepared in Example 1

was spray-dried into light yellow selenium-enriched 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

D e s c r i p t io n

were added into the selenium-enriched active peptide powder, uniformly

mixed and packaged to obtain the selenium-enriched health-care powder

for enhancing selenium absorption.

Example 9 Preparation method of selenium-enriched health-care powder

for enhancing selenium absorption

The selenium-enriched active peptide solution prepared in Example 1

was spray-dried into light yellow selenium-enriched 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

were added into the selenium-enriched active peptide powder, uniformly

mixed and packaged to obtain the selenium-enriched health-care powder

for enhancing selenium absorption.

Experimental Example 1 In-vitro digestion and absorption experiment of

small intestinal mucosal cancer cells (Caco-2)

(1) Cytotoxicity test (survival rate):

Caco-2 cells were inoculated into a collagen-coated 96-well plate at a

density of 1x104 cells/well and incubated in a constant-temperatureCO 2

incubator at 37°C for 24 hours. After a culture medium was removed, 200

pL of samples of different concentrations were added into each well, and

8 to 10 parallel wells were prepared for each sample. After incubation in

D e s c r i p t io n

theCO 2 incubator at 37°C for 20 hours, 20 pL of MTT (detection reagent)

(5 mg/mL) was added into each well, and after constant-temperature

incubation for 4 hours, a precipitate was removed by centrifugation. Then,

150 pL of DMSO (dimethyl sulfoxide) was added into each well, and a

cell culture plate was shaken at a low speed for 10 minutes. The

absorbance was measured at 490 nm with a microplate reader, and finally

the cell survival rate (%) was calculated according to the following

formula:

Cell survival rate (%)=(B 1-Bo)*100/B 2-Bo.

In the formula: BO refers to the absorbance without cells; B1 refers to

the absorbance with addition of samples; B 2 refers to the absorbance

without addition of samples.

(2) Construction of an intestinal cell model in vitro

Caco-2 cells were inoculated into a 12-well plate at a density of

1x10' cells/well, a culture medium (Caco-2 cell culture medium with the

main component of DMEM and 20% FBS) was changed every other day

in the first week and changed every day in the second week, and after

culture for 21 days, an in-vitro intestinal cell model was obtained. The

Caco-2 cell culture medium was removed, the cells were washed twice

with a selenium-free buffer HBSS (cell culture solution) and preheated at

37°C for 10 minutes, the HBSS buffer was removed, different

concentrations (1 mg/mL, 5 mg/mL and 10 mg/mL) of samples (the

D e s c r i p t io n

selenium-enriched active peptide samples of Example 1, Example 4 and

Example 7) were added, the cells were cultured in an incubator for 2

hours, after reaction, the cells were washed twice with the HBSS buffer at

4°C to remove the residual extracting solution, 500 pL of 0.1% (v/v)

Triton X-100 cell lysate was added, repeat freezing and thawing were

carried out to lyse the cells, and then ultrasonic treatment was performed

for 10 minutes to obtain a cell suspension. The total protein mass

concentration (g/L) was measured with a BCA kit, another part was taken

from the cell suspension and centrifuged at a rotation speed of 15000

r/min for 10 minutes, the content of selenium ions in the solution was

determined by using an atomic absorption spectrophotometer, and the

AKP activity was determined by using an alkaline protease (AKP)

activity detection kit. During the experiment, a control group and a

sodium selenite group (commonly used selenium supplement products)

were set, wherein the control group included cells with addition of only

the culture medium, and the sodium selenite group included cells with

addition of a 0.02 mg/mL sodium selenite solution. At the same time, the

cell uptake rate was calculated according to the following formula:

Cell uptake rate (%)=C1/C2.

In the formula: C1 refers to the concentration of selenium in the cells;

C2 refers to the total protein concentration of the cells.

According to the survival rate of the small intestinal mucosal cancer

D e s c r i p t io n

cells (Caco-2) and the selenium uptake rate data in Table 1, it can be seen

that the survived rate of the Caco-2 cells treated with the samples of

Example 1, Example 4 and Example 7 is 94% or above and is slightly

lower than that of the control group (96.5%) and higher than that of the

sodium selenite group, which indicates that the samples of the present

disclosure are not toxic to Caco-2. At the same time, it can also be seen

that the selenium uptake rate of the Caco-2 cells in the samples of

Example 1, Example 4 and Example 7 is also significantly higher than

that of the sodium selenite group. Table 1 Survival rate and selenium uptake rate of small intestinal mucosal cancer cells (Caco-2)

Group Concentration of Cell survival Total retaining

samples rate amount of

(mg/mL) (%) selenium

(0%)

Control group 96.5±0.3

Sodium selenite 1 mg/mL 91.2±0.3 63.6±1.1

5 mg/mL 91.9+0.2 65.5+1.6

10 mg/nL 92.0+0.2 66.1+0.9

Example 1 mg/mL 94.5+0.5 76.1+0.6

5 mg/mL 94.7+0.4 77.8+1.0

10 mg/nL 95.0+0.3 77.4+0.8

D e s c r i p t io n

Example 4 1 mg/mL 94.1+0.3 75.9+0.7

5 mg/mL 94.5+0.3 76.0+1.3

10 mg/mL 94.6+0.2 77.8+1.2

Example 7 1 mg/mL 94.3+0.4 75.8+1.3

5 mg/mL 94.8+0.3 77.6+1.5

10 mg/mL 95.2+0.2 78.2+0.8

Experimental Example 2 Simulated gastrointestinal digestion test

(1) Simulated gastric digestion stage: the pH of simulated gastric

fluid (SGF) including 2 mg/mL of NaCl, 12 mmoL/L of HCl and 3.2

mg/mL of pepsin in deionized water was adjusted to 2.0. 5.0 mg/mL of

the samples (the selenium-enriched active peptide samples of Example 1,

Example 4 and Example 7) were mixed with SGF at a mass ratio of 1:1,

then the pH was adjusted to 2.0, an obtained mixture was continuously

stirred at a rotation speed of 100 r/min and at the constant temperature of

37°C for 2 hours to simulate gastric digestion, and a supernatant was

taken and measured with an atomic absorption spectrophotometer.

(2) Simulated small intestine digestion stage: simulated intestinal

fluid (SIF) containing a salt solution (100 mg of CaCl 2, 300 mg/mL of

NaCl, pH 7.0), a bile salt solution (375.0 mg of bile salt dissolved in 7.0

mL of deionized water, pH 7.0) and an enzyme solution (120.0 mg of

lipase dissolved in 5.0 mL of deionized water, pH 7.0) was prepared.

D e s c r i p t io n

After a gastric digestion stage was completed, the pH (60.0 mL) of a

previous solution (the solution of the simulated gastric digestion stage)

was raised to 7.0, and the prepared SIF was added into the previous

solution to make the final concentration of lipase and bile salt reach 1.6

mg/mL. The pH of an obtained mixture was adjusted to 7.0, then the

mixture was continuously subjected to shaking culture at a rotation speed

of 100 r/min at 37°C for 2 hours, collected, cooled in an ice-water bath

and then centrifuged at a rotation speed of 15000 r/min at 4°C for 30

minutes, and a supernatant was taken and measured with an atomic

absorption spectrophotometer.

During the experiment, a control group and a sodium selenite group

(commonly used selenium supplement products) were set, wherein the

control group included a mixture of the samples and deionized water, and

the sodium selenite group included a mixture of a 0.02 mg/mL sodium

selenite solution and the simulated gastric fluid.

Wherein, the retaining amount of selenium(%)= the content of

selenium with addition of digestive fluid/ the content of selenium without

addition of the digestive fluid*100%.

It can be seen from Table 2 that during the gastric digestion stage, the

content of selenium in the control group is not detected, and the retaining

amount of selenium in a sodium selenite sample is significantly lower

than that of the samples of Example 1, Example 4 and Example 7. After

D e s c r i p t i o n

gastric digestion, the previous gastric fluid is combined with the

simulated intestinal fluid for secondary digestion, the remaining amount

of selenium in the samples of Example 1, Example 4 and Example 7 in

the intestinal digestion stage is also higher than that of the sodium

selenite sample, but is further reduced, and the reason may be that the

ability of a compound to bind to selenium is reduced since the structure

of the compound is changed by trypsin. It is indicated that the structure of

the selenium-enriched active peptide sample of the present disclosure is

stable and is not likely to be affected by gastrointestinal digestion, the

selenium-enriched active peptide sample can smoothly reach the

absorption part of the small intestine, and the tolerance in the stomach

and intestines is high.

Table 2 Retaining amount of selenium in the gastric and intestinal stages

during in-vitro simulated digestion

Group Retaining amount of Retaining amount of

selenium in the selenium in the

simulated gastric simulated intestinal

digestion stage (%) digestion stage (%)

Control group

Sodium selenite 72.0+2.7 53.8+0.6

Example 1 81.3+4.1 71.0+0.5

Example 4 81.0+3.6 70.7+0.9

D e s c r i p t io n

Example 7 80.8+2.3 70.8±0.6

Experimental Example 3 Animal experiment of digestion and absorption

in mice

6-week-old clean mice were randomly divided into 3 groups with 12

mice in each group and fed with selenium-free feed to establish a

low-selenium model, the mice freely ate the feed and drank water, and the

room temperature was maintained to be (25+1)°C. The mice were fasted

6 hours before drug administration and freely drank water. The three

groups of mice were fed with the feed (the selenium-enriched active

peptide samples of Example 1, Example 4 and Example 7 and sodium

selenite) at the selenium content of 18 pg/kg • bw respectively by gavage,

600 pL of blood was collected from the tails before gavage and at 5, 10,

, 30, 40, 60, 80, 100 and 120 minutes after gavage separately, placed in

an EDTA anticoagulation tube and centrifuged at 3000 r/min for 10

minutes, and an upper layer of plasma was sucked into a centrifuge tube

and marked. The content of selenium in mouse plasma was detected by

using hydride generation-atomic fluorescence spectrometry (HG-AFS).

It can be seen from Table 3 that after gavage of the

selenium-enriched active peptide samples of Example 1, Example 4 and

Example 7 and the sodium selenite sample to the mice and after gavage

of the selenium-enriched active peptide samples of Example 1, Example

D e s c r i p t io n

4 and Example 7, the peak blood selenium value of the blood of the mice

is significantly higher than that of the sodium selenite sample, and the

peak appearance time of the peak blood selenium value is 10 minutes. It

is indicated that selenium in the selenium-enriched active peptide sample

of the present disclosure is more easily absorbed, the selenium

supplement effect is better than that of sodium selenite, and the reason

may be that the active peptide in the selenium-enriched active peptide

sample of the present disclosure mainly includes 6-peptide compounds

and 8-peptide compounds which are easily absorbed by the small

intestine and the duodenum.

Table 3 Digestion and absorption of selenium in mice after gavage

Group Total peak value of the Peak time of the blood

blood selenium value of the selenium value of the

blood of the mice (mg/L) blood of the mice (min)

Sodium 0.81 10

selenite

Example 1 1.23 10

Example 4 1.20 10

Example 7 1.99 10

The embodiments of the present disclosure are described in detail

above, but the present disclosure is not limited to the described

D e s c r i p t io n

embodiments. Various changes, modifications, substitutions and variants

can be made to these embodiments by those skilled in the art without

departing from the principle and spirit of the present disclosure, and the

changes, modifications, substitutions and variants are still within the

protection scope of the present disclosure.

Claims

1. A preparation method of an active peptide for enhancing selenium

absorption, comprising the following steps:

S1. preparing fish skin into fish paste, adding pepsin for enzymolysis

treatment and collecting the supernatant;

S2. adding whey protein powder, tapioca starch, carrot powder and

selenium-enriched yeast powder into the supernatant obtained in step S1

and then performing anaerobic fermentation;

S3. adding selenoprotein into a fermentation solution obtained in

step S2 and then performing ultrasonic treatment;

S4. uniformly mixing fish skin mucus with mulberry leaf juice,

standing for a period of time, adding peanut oil and seaweed

polysaccharide and then performing homogeneous stirring;

S5. adding a solution obtained in step S4 into a fermentation solution

obtained in step S3, performing vacuumizing and high static pressure

treatment and finally performing depressurization to obtain the active

peptide for enhancing selenium absorption.

2. The preparation method of the active peptide for enhancing

selenium absorption according to claim 1, wherein in step S2, the whey

protein powder, the tapioca starch and the carrot powder are firstly added

into the supernatant and heated to 30°C, then the selenium-enriched yeast

powder is added, and finally the temperature is raised to 37°C for

anaerobic fermentation.

Cl a i m s

3. The preparation method of the active peptide for enhancing

selenium absorption according to claim 2, wherein in step S2, based on

the mass of the supernatant, the added amount of the whey protein

powder is 0.5-1.5%, the added amount of the tapioca starch is 2.5-2.8%,

the added amount of the carrot powder is 0.3-0.5%, and the added amount

of the selenium-enriched yeast powder is 0.2-0.3%.

4. The preparation method of the active peptide for enhancing

selenium absorption according to claim 1, wherein in step S4, the volume

ratio of the fish skin mucus to the mulberry leaf juice is 1:1.

5. The preparation method of the active peptide for enhancing

selenium absorption according to claim 1, wherein in step S5, the high

static pressure treatment is performed at 300-350 MPa for 10 minutes.