CN107325163B - Astragalus seed protein and application thereof in anti-sports fatigue functional food - Google Patents

Astragalus seed protein and application thereof in anti-sports fatigue functional food Download PDF

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CN107325163B
CN107325163B CN201710580674.7A CN201710580674A CN107325163B CN 107325163 B CN107325163 B CN 107325163B CN 201710580674 A CN201710580674 A CN 201710580674A CN 107325163 B CN107325163 B CN 107325163B
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astragalus
saturation
seed protein
ammonium sulfate
astragalus seed
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索思卓
徐健
李续娥
徐晓阳
胡豪
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South China Normal University
University of Macau
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • 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

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Abstract

The invention discloses an astragalus seed protein and application thereof in anti-sports fatigue functional food, and the extraction and separation method of the protein comprises the following steps: pulverizing radix astragali seed, defatting with petroleum ether, and adding KH2PO4Stirring and leaching the NaOH buffer solution for 6 to 12 hours to obtain an extracting solution; adding powdered ammonium sulfate into the extract to reach 30% saturation, centrifuging, and collecting supernatant; adding powdered ammonium sulfate to 80% saturation, centrifuging, and collecting precipitate; dialyzing the precipitate until the dialyzate is colorless, and finally freeze-drying to obtain the astragalus seed protein. The astragalus seed protein is prepared by an ammonium sulfate saturation precipitation method, and the saturation of the ammonium sulfate is 30-80%. Can be used as nutritional supplement for resisting sports fatigue, and can be added into cookies, liquid beverage, and solid beverage, with convenient use, and can satisfy sports people who like different food types.

Description

Astragalus seed protein and application thereof in anti-sports fatigue functional food
Technical Field
The invention belongs to the field of natural products and functional research thereof, and particularly relates to astragalus seed protein and application thereof in an anti-sports fatigue functional food.
Background
When the training and competition loads of athletes exceed the body's ability to withstand, temporary physiological hypofunction can occur. The physical inadaptation, called exercise fatigue, is caused by the physiological process failing to maintain the function of the body at a specific level or a predetermined exercise intensity. The sports fatigue is relieved, and the sports injury caused by excessive sports and extreme sports can be avoided or reduced in the field of competitive sports; in the field of public body building, the body metabolism disorder caused by the excessive sports fatigue can be avoided, and the internal secretion and the injury of internal organs and tissues can be avoided.
The common methods for relieving exercise-induced fatigue mainly comprise: (1) the sporter is actively rested or trained to recover the therapy; (2) physical therapy using physical stimuli such as light, electricity, magnetism, heat, force, etc. to accelerate microcirculation, promote metabolism, and eliminate fatigue; (3) nutritional supplement therapy for supplementing specific functional foods (free of stimulant substances prohibited by International Olympic Commission) prepared from protein, amino acids, creatine, carnitine, vitamins, minerals, etc.; (4) using various Chinese herbal medicines such as ginseng, penis, pilose antler and the like and chemical medicines such as calcium gluconate, inosine, adenosine triphosphate and the like for medicine conditioning; (5) psychotherapy with cueing, relaxation, breathing regulation, recreation and recreational activities. Among them, nutritional supplement foods made using proteins and amino acids for alleviating exercise-induced fatigue have received much attention from researchers.
Radix astragali is dried root of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao) or Astragalus membranaceus (Fisch) Bge. of Leguminosae. Sweet in nature and slightly warm in taste. It enters lung and spleen meridians. Has the functions of invigorating qi, invigorating yang, consolidating superficial resistance, arresting sweating, inducing diuresis, relieving swelling, promoting fluid production, nourishing blood, activating stagnancy, relieving arthralgia, expelling toxin, expelling pus, healing sore and promoting granulation. Can be used for treating deficiency of vital energy, asthenia, anorexia, loose stool, collapse of middle-warmer energy, chronic diarrhea, rectocele, hematochezia, spontaneous perspiration due to exterior deficiency, edema due to qi deficiency, internal heat, diabetes, blood deficiency, hemiplegia, arthralgia, numbness, carbuncle, cellulitis, and intractable ulcer.
At present, the research on the astragalus extract is mainly focused on the astragalus medicinal material, namely the root of astragalus. Related researches on extracts derived from astragalus seeds are less reported, and researches on the anti-exercise fatigue efficacy of astragalus seed protein are not reported.
Disclosure of Invention
The invention aims at providing an extraction and separation method of astragalus seed protein, which is an ammonium sulfate saturation precipitation method, wherein the saturation degree of ammonium sulfate is 30-80%.
Another object of the present invention is to provide the seed protein of Astragalus membranaceus prepared by the above method.
The invention also aims to provide application of the astragalus membranaceus seed protein.
The purpose of the invention is realized by the following technical scheme:
an extraction and separation method of astragalus seed protein comprises the following steps:
(1) pulverizing radix astragali seed, defatting with petroleum ether, and adding KH2PO4Stirring and leaching the solution with NaOH buffer solution for 6 to 12 hours, centrifuging, and taking supernatant; repeating the above leaching operation for several times, and mixing the obtained supernatants to obtain extractive solution;
(2) slowly adding powdered ammonium sulfate into the extractive solution under stirring to 30% saturation, centrifuging, and collecting supernatant; slowly adding powdered ammonium sulfate into the supernatant under continuous stirring to reach 80% saturation, centrifuging, and collecting precipitate;
(3) transferring the precipitate into a dialysis bag, dialyzing in deionized water (changing water every 24h on average) until the dialysate is colorless, and freeze-drying the components in the dialysis bag to obtain astragalus seed protein;
the Astragalus seeds are dried seeds of Astragalus mongholicus (Fisch) Bge. var. mongholicus (Bge) Hsiao.) which is a leguminous plant (purchased from northern medicinal material seed company of Anguo city in Hebei province and identified by experts of Chinese academy of Guangdong pharmaceutical university);
the petroleum ether degreasing is to soak the astragalus seed powder for 2 to 4 hours in a sealed way by using petroleum ether, filtering, air-drying the astragalus seed powder, and recovering the filtrate by using a rotary evaporator;
the KH2PO4NaOH buffer, preferably at a concentration of 40mmol/L,the pH value is preferably 6.8-8.0;
the centrifugation is preferably 2000-3000r/min and 8-15 min;
the method is carried out in the environment of 4-10 ℃.
The astragalus seed protein prepared by the method has the function of resisting sports fatigue, can obviously prolong the swimming exhaustion time of a mouse, obviously reduces the blood lactic acid and blood urea nitrogen content of the mouse, and obviously increases the liver glycogen and muscle glycogen concentration of the mouse.
The astragalus seed protein is used for resisting sports fatigue of mice, the administration route is gastrointestinal tract route, the dosage is 0.1-5g/kg.d, preferably 0.5-2g/kg.d, and the administration lasts for 28 days. The dosage for human use can be determined by conversion.
Based on the above, the astragalus seed protein can be applied to anti-exercise fatigue functional food, and can be used as a nutritional supplement for exercise fatigue caused by overlarge exercise amount and extreme exercise in the field of competitive exercise and the field of public fitness;
the type of the anti-sports fatigue functional food comprises biscuits, liquid beverages, solid beverages and the like.
Compared with the prior art, the invention has the following advantages and effects:
1. the astragalus seed protein is prepared by an ammonium sulfate saturation precipitation method, and the saturation of the ammonium sulfate is 30-80%. Can be used as nutritional supplement for resisting sports fatigue, and can be added into cookies, liquid beverage, and solid beverage, with convenient use, and can satisfy sports people who like different food types.
2. The astragalus seed protein of the invention is unrelated to stimulant, and does not belong to or contain a compound forbidden by the international Olympic Commission.
3. The astragalus seeds have wide sources, sufficient medicinal materials are supplied, the protein content is rich, the extraction and separation method is mature, and the method is easy to popularize and apply industrially.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
An extraction and separation method of astragalus seed protein comprises the following steps:
(1) pulverizing radix astragali seed, defatting with petroleum ether, and adding KH2PO4Stirring and leaching the solution with NaOH buffer solution for 6 to 12 hours, centrifuging, and taking supernatant; repeating the above leaching operation for 2-3 times, and mixing the obtained supernatants to obtain extractive solution;
(2) slowly adding powdered ammonium sulfate into the extractive solution under stirring to 30% saturation, centrifuging, and collecting supernatant; slowly adding powdered ammonium sulfate into the supernatant under continuous stirring to reach 40% saturation, centrifuging, and collecting precipitate;
(3) transferring the precipitate into dialysis bag, dialyzing in deionized water (changing water every 24 hr on average) until the dialysate is colorless, and freeze drying the components in the dialysis bag to obtain radix astragali seed protein.
Example 2
A method for extracting and separating astragalus membranaceus seed protein adopts an ammonium sulfate saturation precipitation method, and also comprises 3 steps, wherein the steps (1) and (3) are the same as those in example 1, and the step (2) is as follows:
(2) slowly adding powdered ammonium sulfate into the extractive solution under stirring to reach 40% saturation, centrifuging, and collecting supernatant; and slowly adding powdery ammonium sulfate into the supernatant to reach 60% of saturation degree under continuous stirring, centrifuging, and collecting precipitate.
Example 3
A method for extracting and separating astragalus membranaceus seed protein adopts an ammonium sulfate saturation precipitation method, and also comprises 3 steps, wherein the steps (1) and (3) are the same as those in example 1, and the step (2) is as follows:
(2) slowly adding powdered ammonium sulfate into the extractive solution under stirring to reach saturation of 60%, centrifuging, and collecting supernatant; and slowly adding powdered ammonium sulfate into the supernatant to reach the saturation of 80% under continuous stirring, centrifuging, and collecting the precipitate.
Comparative example 1
An extraction and separation method of astragalus seed protein adopts an ammonium sulfate saturation precipitation method, and the saturation of ammonium sulfate fractional precipitation is less than 30%; the other steps are the same as in example 1.
Comparative example 2
An extraction and separation method of radix astragali seed protein adopts ammonium sulfate saturation precipitation method, wherein the saturation of ammonium sulfate fractional precipitation is more than 80%; the other steps are the same as in example 1.
Functional effect experiment:
healthy C57BL/6 mice were obtained, male, 8 weeks old, and 20-23g in body weight. A blank control group and an astragalus seed protein group are provided, and each group contains 30 mice. Raising at 23 + -2 deg.C and relative humidity of 55 + -5%, and setting light/dark at 12h/12h day and night cycle. The conventional feed is given every day, and the bedding materials are changed 2-3 times in 1 week after free feeding and water feeding. The astragalus membranaceus seed proteome is used for administering the proteins of each embodiment and each proportion, and the dosage is 0.5-2g/kg.d (see the table 1 in detail); the blank control group was given physiological saline for 28 days. Each group of mice underwent 3 passive swimming sessions for 15 minutes per week. And (4) killing after the last passive swimming experiment, and detecting related indexes. SPSS 17.0 statistical software is adopted to analyze experimental data, and results are obtained
Figure BDA0001352195990000051
Showing that the samples between groups are compared by adopting one-way analysis of variance.
Detection of swimming exhaustion time: 10 mice were taken out of each group for passive swimming experiments. The temperature of the swimming pool water is kept at 26 +/-1 ℃ and the water depth is 35 cm. A weight with the mass of 10 percent of the mouse body is added on the tail of each mouse. From the beginning of the experiment, the mice are judged to have exhausted movement when the coordinated movement disorder occurs and the water surface can not be exposed within 10s, and the swimming time of the mice is recorded. The results are shown in Table 2.
Detecting blood biochemical indexes: 10 mice are taken out from each group, the weight bearing (2 percent of the weight of the mice) is firstly carried out for 30min swimming movement, blood is collected after the rest for 60min, and the blood lactic acid content and the blood urea nitrogen content are measured by adopting a kit of Nanjing institute of bioengineering. The results are shown in Table 3.
Tissue glycogen analysis: the rest 10 mice in each group firstly do swimming exercise without load for 90min, after resting for 60min, the mice are killed, the liver and gastrocnemius are taken, and the concentration of hepatic glycogen and myoglycogen is detected by adopting a kit of Nanjing institute of bioengineering. The results are shown in Table 4.
The exhaustion time is the comprehensive manifestation of various functions of the body such as anti-stress capability, anti-fatigue capability and the like, and is an important direct index for measuring the motor capacity of the body. Swimming is forced whole body consumption exercise, a large amount of energy is consumed by violent exercise, and the swimming time is comprehensively reflected by the physiological activity of the organism, so the swimming exhaustion time is one of important indexes for measuring the sports fatigue.
Lactic acid is one of the most studied sports fatigue-causing substances at present, and during aerobic exercise, the organism produces little lactic acid, but under the condition of oxygen deficiency, glycogen is subjected to anaerobic decomposition to produce a large amount of lactic acid. With the increase of the exercise intensity, the yield of lactic acid in the organism is obviously increased. The increase in lactic acid can cause a decrease in exercise capacity through a variety of pathways: on the one hand, the lactic acid dissociation generates a large amount of H+The key enzyme of glycolysis is inhibited, so that the anaerobic oxidation energy supply of sugar is reduced, the synthesis of ATP during exercise is reduced, and the exercise capacity is reduced; on the other hand, H formed after dissociation of lactic acid+To decouple excitation-contraction, which results in the reduction of skeletal muscle contraction and reduced exercise capacity. The concentration of the blood lactic acid is increased during the sports fatigue, so the content change of the blood lactic acid after the astragalus seed protein is applied to the mice can reflect the anti-sports fatigue effect of the mice.
There are three main causes of elevated blood urea nitrogen caused by exercise: (1) the alanine-glucose circulation is enhanced, and the urea is increased; (2) the motor enhances the aging decomposition of the enzyme in the muscle, and the urea which is the final product of the catabolism is increased; (3) muscle is rapidly supplied with energy, ATP cannot be rapidly synthesized, AMP is generated, AMP is easy to deaminate in muscle to generate IMP, and ammonia is converted into urea. Research shows that blood urea nitrogen is an important index of sports fatigue, and the higher the content of the blood urea nitrogen is, the poorer the exercise endurance of the organism is.
Sugar is a main energy supply substance when a body moves, the existing mode in the body is blood sugar and glycogen, glycogen is a storage form of sugar, and 2 forms of liver glycogen and muscle glycogen are mainly used for maintaining the stability of blood sugar and meeting the energy requirement of skeletal muscle during movement, and is closely related to the occurrence of sports fatigue. Energy in exercise is derived from the breakdown of glycogen, muscle glycogen is first consumed in strenuous exercise, and then energy is obtained from glucose which is continuously released into the blood from liver glycogen. Therefore, the glycogen content of the body is a sensitive index for judging the sports fatigue.
As can be seen from the experimental results (Table 2, Table 3 and Table 4), administration of the astragalus seed protein of comparative example 1 had no effect on the swimming exhaustion time, blood lactic acid content, blood urea nitrogen content, liver glycogen and muscle glycogen concentrations of the mice, compared to the control group. Likewise, administration of the astragalus seed protein of comparative example 2 had no effect on the time to swimming exhaustion, blood lactate content, blood urea nitrogen content, liver glycogen and muscle glycogen concentrations in the mice. The prepared astragalus seed protein with the saturation of less than 30 percent and more than 80 percent of ammonium sulfate has no sports fatigue resistance effect and can not be used as a nutritional supplement for resisting sports fatigue.
However, administration of the astragalus seed proteins of examples 1-3 to each group of mice very significantly extended the time to swim exhaustion, very significantly reduced the blood lactate and blood urea nitrogen levels, and very significantly increased the liver glycogen and muscle glycogen concentrations, as compared to the control group. The obtained astragalus seed protein with 30-80% of saturation of ammonium sulfate has the effect of resisting sports fatigue and can be used as a nutritional supplement for resisting sports fatigue.
TABLE 1 saturation of ammonium sulfate fractional precipitation and experimental dosing of seed protein to Astragalus membranaceus
Figure BDA0001352195990000061
TABLE 2 Effect of Astragalus seed protein on mouse weight and swimming exhaustion time: (
Figure BDA0001352195990000071
n＝10)
Figure BDA0001352195990000072
P <0.01 compared to control group.
TABLE 3 Effect of Astragalus seed protein on blood lactate and blood urea Nitrogen in mice: (
Figure BDA0001352195990000073
n＝10)
Figure BDA0001352195990000074
P <0.01 compared to control group.
TABLE 4 Effect of Astragalus seed protein on liver glycogen and muscle glycogen in mice: (
Figure BDA0001352195990000081
n＝10)
Figure BDA0001352195990000082
P <0.01 compared to control group.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (5)

1. The application of the astragalus seed protein in the anti-sports fatigue functional food is characterized in that:
the method for extracting and separating the astragalus seed protein comprises the following steps:
(1) pulverizing radix astragali seed, defatting with petroleum ether, and adding KH2PO4Stirring and leaching the solution with NaOH buffer solution for 6 to 12 hours, centrifuging, and taking supernatant; repeating the above leaching operation for several times, and mixing the obtained supernatants to obtain extractive solution;
(2) adding powdered ammonium sulfate into the extractive solution under stirring to 30% saturation, centrifuging, and collecting supernatant; adding powdered ammonium sulfate into the supernatant under continuous stirring to reach 80% saturation, centrifuging, and collecting precipitate;
(3) transferring the precipitate into a dialysis bag, dialyzing in deionized water until the dialysate is colorless, and freeze-drying the components in the dialysis bag to obtain astragalus seed protein;
the operation is carried out in an environment of 4-10 ℃.
2. Use according to claim 1, characterized in that: the petroleum ether degreasing in the step (1) is to hermetically soak the astragalus seed powder for 2 to 4 hours by using petroleum ether, and air-dry the astragalus seed powder after filtering.
3. Use according to claim 1, characterized in that: KH in the step (1)2PO4NaOH buffer, concentration 40mmol/L, pH 6.8-8.0.
4. Use according to claim 1, characterized in that: the centrifugation in the step (2) is 2000-3000r/min and the centrifugation is 8-15 min.
5. Use according to claim 1, characterized in that: the functional food for resisting sports fatigue comprises biscuits, liquid beverages and solid beverages.
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