AU2021106749A4 - Whey protein peptide with muscle-building effect and preparation method thereof, and compound protein powder - Google Patents

Whey protein peptide with muscle-building effect and preparation method thereof, and compound protein powder Download PDF

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AU2021106749A4
AU2021106749A4 AU2021106749A AU2021106749A AU2021106749A4 AU 2021106749 A4 AU2021106749 A4 AU 2021106749A4 AU 2021106749 A AU2021106749 A AU 2021106749A AU 2021106749 A AU2021106749 A AU 2021106749A AU 2021106749 A4 AU2021106749 A4 AU 2021106749A4
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Wei Wei
Jiuxun Zhang
Xiping Zhang
Xuejun Zhang
Zhao Zhang
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Zhongshiduqing Shandong Biotech Co Ltd
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Abstract

OF THE DISCLOSURE The present disclosure provides a whey protein peptide with a muscle-building effect and a preparation method thereof, and a compound protein powder, relating to the technical field of protein peptides. In the present disclosure, a whey protein is hydrolyzed to obtain a whey protein peptide using a whey protein isolate as a raw material, and through a selective enzymolysis and a nano-scale membrane treatment system. The muscle-building efficacy of the whey protein peptide is verified using animal experiments. It is found that the muscle-building effect of the whey protein peptide is significantly better than that of a whey protein powder.

Description

WHEY PROTEIN PEPTIDE WITH MUSCLE-BUILDING EFFECT AND PREPARATION METHOD THEREOF, AND COMPOUND PROTEIN POWDER TECHNICAL FIELD
[01] The present disclosure belongs to the technical field of protein peptides, and specifically relates to a whey protein peptide with a muscle-building effect and a preparation method thereof, and a compound protein powder.
BACKGROUNDART
[02] After 25 years of age, various functions of the human body will gradually decrease. The human body has the most muscle mass at 20-25 years of age, and then will gradually lose the muscle. The muscle mass will lose by 10% within about 10 years, leading to a decrease of the strength accordingly.
[03] A certain frequency of strength training is one of the effective means to maintain muscles. Exercise not only consumes a lot of energy, but also intensifies protein catabolism, and may even cause a negative balance of protein. Therefore, the protein intake should be increased, and especially the protein intake should be increased. Although an intake increase of the common protein powders in an appropriate amount, such as a whey protein, is important in supplementing the protein loss during exercise, increasing the muscle strength, promoting the hemoglobin synthesis, and eliminating the fatigue, etc. However, there are problems of difficult digestion and slightly insufficient muscle-building effect.
SUMMARY
[04] In view of this, the present disclosure provides a whey protein peptide with a muscle-building effect and a preparation method thereof, and a compound protein powder. The whey protein peptide is easier to be absorbed due to a small molecular weight, and has a significant muscle-building effect.
[05] In order to realize the above objective, the present disclosure provides the following technical solutions:
[06] The present disclosure provides a preparation method of a whey protein peptide, including the following steps: (1) preparing a whey protein powder into a whey protein aqueous solution, adjusting a pH of the whey protein aqueous solution to 8.5-8.8, and conducting heat preservation at 53-56°C for 25-30 min to obtain a homogenate; where the whey protein powder and water in the whey protein aqueous solution have a mass-to-volume ratio of 1 kg: (8-12) L; and
[07] (2) conducting enzymolysis on the homogenate using a papain and a flavor protease, decolorizing and deodorizing after enzyme deactivation, collecting a submembrane liquid of a 1 kDa organic membrane to obtain the whey protein peptide.
[08] Preferably, in step (1), a preparation method of the whey protein aqueous solution may include: dissolving 3 0 -5 0 % of the whey protein powder with 3 0 -5 0 % of water, stirring uniformly and adding the remaining water and whey protein powder.
[09] Preferably, in step (1), the pH may be adjusted to 8.5-8.8 using a NaOH solution with a concentration of 10 mol/L.
[10] Preferably, in step (2), the papain may be 0.8-1.2% the mass of the whey protein powder, and the flavor protease may be 0.4-0.6% the mass of the whey protein powder.
[11] Preferably, in step (2), the enzymolysis may include: mixing the homogenate with the papain for enzymolysis for 3 h, and then with the flavor protease for enzymolysis for 2 h.
[12] Preferably, in step (2), the decolorizing and deodorizing may be conducted with an activated carbon; and
[13] a decolorized and deodorized enzyme deactivation liquid may be passed through a ceramic membrane with a molecular weight cut-off of 30,000 Da and an organic membrane with a molecular weight cut-off of 1,000 Da sequentially, and the submembrane liquid may be collected.
[14] Preferably, the preparation method may further include: spray drying after collecting the submembrane liquid.
[15] The present disclosure further provides a whey protein peptide obtained by the preparation method, where a branched chain amino acid in the whey protein peptide has a mass percentage of more than 20%.
[16] The present disclosure further provides a compound protein powder with a muscle-building effect, including a whey protein, a collagen peptide, a food flavor, a sucralose and the whey protein peptide.
[17] Preferably, the compound protein powder includes the following components by mass percentage: the whey protein 60-70%, the whey protein peptide 25-36%, the collagen peptide 1-2%, the food flavor 1-2% and the sucralose 0.04-0.1%.
[18] The beneficial effects are as follows: in the present disclosure, a whey protein is hydrolyzed to obtain a whey protein peptide using a whey protein isolate as a raw material, and through a selective enzymolysis and a nano-scale membrane treatment system. The muscle-building efficacy of the whey protein peptide is verified using animal experiments. It is found that the muscle-building effect of the whey protein peptide is significantly better than that of a whey protein powder.
BRIEF DESCRIPTION OF THE DRAWINGS
[19] FIG. 1 is a whey protein peptide data report issued by Shandong Tianheng Inspection Co., Ltd.
[20] FIG. 2 is grip strength of forelimbs of different groups of mice.
[21] FIG. 3 is a testosterone content of different groups of mice.
[22] FIG. 4 is a content of insulin-like growth factor 1 (IGF-1) in serum of different groups of mice.
[23] FIG. 5 is an adenosine triphosphatase (ATPase) activity in skeletal muscle mitochondria of different groups of mice.
[24] FIG. 6 is a pea oligopeptide activating an insulin-like growth factor (IGF)/insulin-like growth factor 1 receptor (IGF-1R) signaling pathway.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[25] The present disclosure provides a preparation method of a whey protein peptide, including the following steps: (1) preparing a whey protein powder into a whey protein aqueous solution, adjusting a pH of the whey protein aqueous solution to 8.5-8.8, and conducting heat preservation at 53-56°C for 25-30 min to obtain a homogenate; where the whey protein powder and water in the whey protein aqueous solution have a mass-to-volume ratio of 1 kg: (8-12) L; and
[26] (2) conducting enzymolysis on the homogenate using a papain and a flavor protease, decolorizing and deodorizing after enzyme deactivation, collecting a submembrane liquid of a 1 kDa organic membrane to obtain the whey protein peptide.
[27] In the present disclosure, a whey protein powder is prepared into a whey protein aqueous solution, the pH of the whey protein aqueous solution is adjusted to 8.5-8.8, and heat preservation is conducted at 53-56°C for 25-30 min to obtain a homogenate; where the whey protein powder and water in the whey protein aqueous solution have a mass-to-volume ratio of 1 kg: (8-12) L. A preparation method of the whey protein aqueous solution preferably includes: dissolving 30-50% of the whey protein powder with 30-50% of water, stirring uniformly and adding the remaining water and whey protein powder. In an example, when 500 kg of the whey protein powder is used to prepare a solution, 2000 L of a deionized water is added, 200 kg of the whey protein powder is added to the water while stirring at a rate of 200 r/min; and 3000 L of the deionized water is added, and the remaining 300 kg of the whey protein powder is added while stirring at a rate of 200 r/min. There is no special limitation on the source of the whey protein powder, and a commercially-available whey protein isolate conventional in the art may be used.
[28] In the present disclosure, the pH of the whey protein aqueous solution is adjusted to 8.5-8.8 preferably using a NaOH solution with a concentration of 10 mol/L; and heat preservation is conducted at 53-56°C for 25-30 min.
[29] In the present disclosure, enzymolysis is conducted on the homogenate using a papain and a flavor protease, decolorizing and deodorizing are conducted after enzyme deactivation, and a submembrane liquid of a 1 kDa organic membrane is collected to obtain the whey protein peptide.
The enzymolysis preferably includes: mixing the homogenate with the papain for enzymolysis for 3 h, and then with the flavor protease for enzymolysis for 2 h. The papain is 0.8-1.2% the mass of the whey protein powder, more preferably 1%. The flavor protease is 0.4-0.6% the mass of the whey protein powder, more preferably 0.5%. In an example, preferably the papain is added to the homogenate twice within 30 min, where the two addition amounts are the same; and the enzymolysis is conducted for 3 h by starting timing after the second addition. The flavor protease is preferably added at one time for enzymolysis for 2 h. There is no special limitation on the sources of the papain and the flavor protease, and commercially-available papain and flavor protease conventional in the art may be used.
[30] In the present disclosure, the enzyme deactivation is preferably conducted by heating an enzymatic hydrolysate at 85-90°C for 5 min. The decolorizing and deodorizing of the enzymatic hydrolysate after enzyme deactivation preferably includes: cooling an enzyme deactivation solution to 50-53°C, adjusting the pH of the enzyme deactivation solution to 4.5-5.0 using a 1OM HCl, and adding an activated carbon to the enzyme deactivation solution and maintaining for 90-110 min. The activated carbon is preferably 4% the mass of the whey protein powder. The decolorized and deodorized enzyme deactivation solution is preferably passed through a ceramic membrane with a molecular weight cut-off of 30,000 Da and an organic membrane with a molecular weight cut-off of 1,000 Da sequentially, and the submembrane liquid is collected.
[31] In the present disclosure, the preparation method preferably further includes: spray drying with an inlet air temperature of 180°C, and an outlet air temperature of 140°C after collecting the submembrane liquid.
[32] The present disclosure further provides a whey protein peptide obtained by the preparation method, where a branched chain amino acid in the whey protein peptide has a mass percentage of more than 20%. The whey protein peptide has a protein content of more than 91%. In an example, the obtained whey protein peptides with a molecular weight of less than 1,000 Da account for more than 85%, which are easier to be absorbed by the human body; the whey protein peptide has a proportion of branched chain amino acids reaching 20.8%; and the finally obtained whey protein peptide has no lactose, which is more suitable for oriental physique.
[33] The present disclosure further provides a compound protein powder with a muscle-building effect, including a whey protein, a collagen peptide, a food flavor, a sucralose and the whey protein peptide.
[34] The compound protein powder preferably includes the following components by mass percentage: the whey protein 60- 7 0%, the whey protein peptide 25- 3 6 %, the collagen peptide 1- 2 %, the food flavor 1- 2 % and the sucralose 0.0 4 -0.1%; more preferably, the whey protein 6 6 .7 4 %, the whey protein peptide 30.2%, the collagen peptide 1.5%, the food flavor 1.5% and the sucralose 0.06%. There is no special limitation on a preparation method of the compound protein powder, and a preparation method conventional in the art, such as stirring, may be used.
[35] The whey protein peptide with a muscle-building effect and a preparation method thereof, and a compound protein powder provided in the present disclosure will be described in detail below with reference to examples, but these examples cannot be understood as limiting the claimed protection scope of the present disclosure.
[36] Example 1
[37] Production process of a whey protein peptide
[38] (1) 2,000 L of deionized water was added to an enzymolysis tank, and 200 kg of a whey protein powder was added to the enzymolysis tank while stirring at a rate of 200 r/min; and 3,000 L of the deionized water was added to the enzymolysis tank, and the remaining 300 kg of the whey protein powder was added to the enzymolysis tank while stirring at a rate of 200 r/min.
[39] (2) A pH of a homogenate obtained in step (1) was adjusted to 8.5-8.8 with a OM NaOH, and heated to 53-56°C for 25-30 min while maintaining the pH.
[40] (3) 5 kg of a papain was weighed and added to an adjusted slurry obtained in step (2) twice in the same amount within 30 min, and enzymolysis was conducted for 3 h to obtain an enzymatic hydrolysate; 2.5 kg of a flavor protease was weighed and added to the enzymatic hydrolysate at one time, and enzymolysis was continued for 2 h; and temperature was raised to 85-90°C for 5 min to inactivate the protease.
[41] (4) An enzyme deactivation solution was cooled to 50-53°C, the pH of the enzyme deactivation solution was adjusted to 4.5-5.0 using a 1OM HCl, and 20 kg of an activated carbon was weighed and added to the enzyme deactivation solution and maintained for 90-110 min.
[42] (5) The enzymatic hydrolysate treated by the activated carbon was passed through a ceramic membrane with a molecular weight cut-off of 30,000 Da and an organic with a molecular weight cut-off of 1,000 Da sequentially.
[43] (6) The enzymatic hydrolysate of a whey protein peptide obtained by the membrane treatment was spray-dried with an inlet air temperature of 180°C and an outlet air temperature of 140°C, to obtain the whey protein peptide.
[44] The physical and chemical indexes (m/m) of the obtained whey protein peptides were: protein content 91.3%, moisture 5.1%, and ash content 3 .6 %; the whey protein peptides with a molecular weight of less than 1,000 Da account for more than 85%, which are easier to be absorbed by the human body; the whey protein peptide has a proportion of branched chain amino acids reaching 20.8%; and the finally obtained whey protein peptide has no lactose, which is more suitable for oriental physique.
[45] Meanwhile, Shandong Tianheng Inspection Co., Ltd. was commissioned to determine the distribution and molecular weight of hydrolyzed amino acids in the whey protein peptide (Table 1 and FIG. 1). The molecular weight distribution results (peak area percentage, %,X220nm) were as follows: >3000 0.07%, 3000-2000 1.62%, 2000-1000 7.89%, 1000-500 23.90%, and <500 66.52%.
[46] Table 1 Amino acid content table Amino acid name Content (g/100 g) Amino acid name Content (g/100 g) Aspartic acid 9.4134 Threonine 6.1534 Serine 5.4371 Glutamic acid 11.8370 Glycine 1.2915 Alanine 5.2508 Cystine 1.0315 Valine 5.5380 Methionine 1.1595 Isoleucine 5.1048 Leucine 10.2868 Tyrosine 1.1821 Phenylalanine 2.4206 Lysine 7.7838 Histidine 1.2576 Arginine 2.0361 Proline 6.1206 Total amino acid 83.3046
[47] Example 2
[48] A preparation method of a compound protein powder was provided:
[49] The compound protein powder included the following components according to mass ratio: 66.74 g of a whey protein, 30.2 g of the whey protein peptide prepared in Example 1, 1.5 g of a collagen peptide, 1.5 g of a food flavor and 0.06 g of a sucralose; the components were subjected to rotary mixing and stirring to obtain the compound protein powder.
[50] The preparation method included:
[51] (1) pre-mixing: four components, the whey protein peptide, the collagen peptide, the food flavor and the sucralose were weighed in proportions separately, and passed through a 30-mesh sieve to obtain a primary mixture;
[52] (2) mixing: the primary mixture obtained in step (1) was slowly added to warm water (at pH 6.0) at 45°C in a weight ratio to water of 1:5, uniformly stirred at a rate of 70 rpm, and the whey protein was added for mixing evenly;
[53] (3) a product obtained in step (2) was frozen and vacuum-dried (using a YRDS-15010 Chinese-style freeze dryer for health products and foods) at a temperature of -40°C to -45°C and a vacuum degree of 220 Pa for 46 h; and
[54] (4) a product obtained in step 3) was pulverized through a 40-mesh sieve with a WN-500A pulverizer to obtain the compound protein powder.
[55] Example 3
[56] 1. Specified pathogen free-grade (SPF-grade) C57 mice were taken as model mice and randomly divide into 4 groups: a control group (group A), a testosterone group (group B), a compound protein powder group (the compound protein powder of Example 2, group C) and a whey protein peptide group (the whey protein peptide of Example 1, group D), and a gavage dose was 300 mg/kg. Each experimental group was subjected to resistance exercise with weight-bearing ladder crawling for 8 weeks. The grip strength of forelimbs before and after the experiment was ) detected. At the end of the experiment, the experimental mice were sacrificed, and the serum and rectus femoris were taken for later use.
[57] 2. Determination of grip strength
[58] The experimental mice were grasped and placed on a grip strength plate with the right hand, a tension plate was pushed forward with the left hand, and the right hand slid back to the mouse tail; pulled back the mice in time when the mice grasped the grip strength plate hard to measure the maximum grip strength of the mice.
[59] The results were shown in Table 2 and FIG. 2. The whey protein peptide can significantly improve the grip strength of the forelimbs of mice with an effect better than that of the compound protein powder group and the testosterone group. The grip strength of the mice in the compound protein powder group is significantly improved compared with that of the normal group. Table 2 Effect of proteins on the grip strength of the forelimbs of mice in different groups
Item Group A Group B Group C Group D
Initial grip strength (g) 136.6+8.9 139.3+10.1 141.9+12.3 137.1+9.5
Grip strength after 8 weeks 206.2+15.2 277.5±27.3 261.5+23.1 289.9+25.6
[60] 3. Determination of testosterone and IGF-1
[61] The experimental mouse serum was taken to detect the content of testosterone and IGF-1 separately according to operating instructions of an enzyme-linked immunosorbent assay (ELISA) kit.
[62] Testosterone is very closely-related to muscle growth, and high-level testosterone can significantly reduce protein degradation. Moreover, increasing the testosterone level can effectively reduce fat. The experimental results were shown in FIG. 3. Compared with the control group, taking whey protein peptide can significantly increase the testosterone level of mice, and the testosterone level is significantly higher than that of the mice in the normal group, the testosterone group and the compound protein powder group. The testosterone level of mice in the compound protein powder group is significantly higher than that in the normal group.
[63] IGF-1 is a key positive regulator in the skeletal muscle regeneration and is important in promoting the skeletal muscle growth. FIG. 4 shows that compared with the control group, taking the whey protein peptide can significantly increase the content of IGF-1 in the peripheral blood of mice, with an increase higher than that of the compound protein powder group and the testosterone group.
[64] 4. Determination of gastrocnemius cross-sectional area
[65] The gastrocnemius cross-sectional area ([m2 ) of the medial gastrocnemius of the mice in different groups was observed using a staining method.
[66] The increase in the gastrocnemius cross-sectional area is the most intuitive manifestation of muscle growth, and the increase of the muscle cross-sectional area is significantly positively-correlated with the increase of the muscle strength. Table 3 shows that compared with the control group, taking the whey protein peptide can significantly promote the growth of gastrocnemius with an increase higher than that of the compound protein powder group and the testosterone group.
[67] Table 3 Influence of different proteins on the gastrocnemius cross-sectional area
Item Group A Group B Group C Group D
Gastrocnemius 3092.3+265.3 6318.9+377.4 5873.2+346.1 7156.4+462.5 cross-sectional area (m 2 )
[68] 5. Determination of ATPase activity
[69] Ice physiological saline was added to a fresh muscle tissue, and liver tissues were cut up as soon as possible with small ophthalmic scissors. The chopped muscle tissue was poured into a glass homogenization tube, the fragments of tissue remaining in a beaker were rinsed using the remaining 1/3 ice physiological saline, and poured into the glass homogenization tube together for homogenization; the lower end of the glass homogenization tube was inserted by the left hand into a beaker containing an ice-water mixture, and a ramming rod was inserted vertically into a casing with the right hand; grinding was thoroughly conducted by rotating up and down for dozens of times (for 6-8 minutes) to homogenize the muscle tissue (10% tissue homogenate). Centrifugation was conducted to obtain a supernatant, and the ATPase activity in the mitochondria of rectus femoris tissue was determined according to the steps of an ATPase kit operating instruction.
[70] Mitochondria are the main place to synthesize ATP, and the ATP is the energy required for body movement. A high-energy phosphate bond may be hydrolyzed by an ATP hydrolase to generate a free ATP to provide energy for body activities. Compared with the control group and the compound protein powder group, the whey protein peptide can significantly increase the ATP hydrolase activity in skeletal muscle mitochondria (FIG. 5).
[71] 6. Effect of the whey protein peptide on insulin signaling pathways
[72] Insulin signaling pathway is important in the muscle growth. In this study, the effect of the whey protein peptide on the insulin signaling pathway was detected using the Western blot. The operation method specifically includes: triceps surae of an experimental rat was homogenized, cells were sonicated, a loading buffer was added, protein separation was conducted by a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electric rotation was conducted at
350 mA for 2.5 hours; a polyvinylidene fluoride (PVDF) membrane was taken out, and blocked with a 5% skimmed milk powder at room temperature for 1 hour; the membrane was washed with a Tris-buffered saline with Tween-20 (TBS-T) for 3 times, a primary antibody was added for incubation overnight at 4°C; the membrane was washed with TBS-T for 3 times, a secondary antibody was added for incubation at room temperature for 1 hour; the membrane was washed, and an electrochemical luminescence (ECL) luminescent agent was added to develop color.
[73] Insulin-like growth factor (IGFs) signaling pathway is important in muscle growth and damage repair. The results are shown in FIG. 6. The whey protein peptide can significantly increase the expression of IGF-1R, thereby promoting the binding of IGF-1 and IGF-1R, and further promoting the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). In addition, the up-regulated expression of cell cycle-related proteins cyclinD1 and CDK6 are induced, which eventually leads to cell proliferation.
[74] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims (5)

WHAT IS CLAIMED IS:
1. A preparation method of a whey protein peptide, comprising the following steps: (1) preparing a whey protein powder into a whey protein aqueous solution, adjusting a pH of the whey protein aqueous solution to 8.5-8.8, and conducting heat preservation at 53-56°C for 25-30 min to obtain a homogenate; wherein the whey protein powder and water in the whey protein aqueous solution have a mass-to-volume ratio of 1 kg: (8-12) L; and (2) conducting enzymolysis on the homogenate using a papain and a flavor protease, decolorizing and deodorizing after enzyme deactivation, collecting a submembrane liquid of a 1 kDa organic membrane to obtain the whey protein peptide.
2. The preparation method according to claim 1, wherein in step (1), a preparation method of the whey protein aqueous solution comprises: dissolving 30-50% of the whey protein powder with -50% water, stirring uniformly and adding the remaining water and whey protein powder; wherein in step (1), the pH is adjusted to 8.5-8.8 using a NaOH solution with a concentration of 10 mol/L; wherein in step (2), the papain is 0.8-1.2% the mass of the whey protein powder, and the flavor protease is 0.4-0.6% the mass of the whey protein powder; wherein in step (2), the enzymolysis comprises: mixing the homogenate with the papain for enzymolysis for 3 h, and then with the flavor protease for enzymolysis for 2 h; wherein in step (2), the decolorizing and deodorizing is conducted with an activated carbon; and a decolorized and deodorized enzyme deactivation liquid is passed through a ceramic membrane with a molecular weight cut-off of 30,000 Da and an organic membrane with a molecular weight cut-off of 1,000 Da sequentially, and the submembrane liquid is collected; further comprising: spray drying after collecting the submembrane liquid.
3. A whey protein peptide obtained by the preparation method according to any one of claims 1-2, wherein a branched chain amino acid in the whey protein peptide has a mass percentage of equal to or more than 20%.
4. A compound protein powder with a muscle-building effect, comprising a whey protein, a collagen peptide, a food flavor, a sucralose and the whey protein peptide according to claim 3.
5. The compound protein powder according to claim 4, comprising the following components by mass percentage: the whey protein 60-70%, the whey protein peptide 25-36%, the collagen peptide 1-2%, the food flavor 1-2% and the sucralose 0.04-0.1%.
－1/3－
DRAWINGS 24 Aug 2021
Shandong Tianheng Inspection Co., Ltd. Test Report Report No.: SP2021-0220006 Sample No.: N20210220006 1/1 Page Inspection department: Zhongshiduqing (Shandong) Biotech Co., Ltd. Sample name: Whey protein peptide 2021106749
Results of hydrolysis of amino acids:
Amino acid name Content (g/100 g) Amino acid name Content (g/100 g)
Aspartic acid 9.4134 Threonine 6.1534
Serine 5.4371 Glutamic acid 11.8370
Glycine 1.2915 Alanine 5.2508
Cystine 1.0315 Valine 5.5380
Methionine 1.1595 Isoleucine 5.1048
Leucine 10.2868 Tyrosine 1.1821
Phenylalanine 2.4206 Lysine 7.7838
Histidine 1.2576 Arginine 2.0361
Proline 6.1206 Total amino acid 83.3046 Molecular weight results: Peak area percentage (%, λ220 nm) >3000 0.07 2000-3000 1.62 1000-2000 7.89 500-1000 23.90 <500 66.52 Examination by Shandong Tianheng Inspection Co., Ltd. Checked by (sealed) Issued Date 2021-02-26 FIG. 1
－2/3－ 24 Aug 2021 2021106749
Initial grip After 8 weeks of strength resistance exercise Grip strength of mice (g)
Group A Group B Group C Group D
FIG. 2 Testosterone content of mice (control multiple)
Group A Group B Group C Group D
FIG. 3
－3/3－ 24 Aug 2021
IGF-1 content (pg/ml) 2021106749
Group A Group B Group C Group D
FIG. 4 ATPase activity
Group A Group B Group C Group D
FIG. 5
Group A Group B Group C Group D
FIG. 6
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