Acipenser swimming bladder collagen as well as acid leaching extraction method and application thereof

Abstract

The invention provides sturgeon swimming bladder collagen as well as an acid leaching extraction method and application thereof, and relates to the technical field of protein extraction. The acid leaching extraction method provided by the invention comprises the following steps: performing alkali washing on sturgeon swimming bladders, degreasing, soaking in an acid solution, separating an extracting solution, and salting out to obtain collagen. The method provided by the invention can simply, conveniently and quickly obtain the sturgeon swimming bladder acid-soluble collagen with strong antioxidant activity, not only can effectively reduce the waste of sturgeon swimming bladders and sustainably utilize sturgeon resources, but also has higher extraction rate of the sturgeon swimming bladder collagen, and is beneficial to industrial production; meanwhile, the prepared acid-soluble collagen of the sturgeon swimming bladder can effectively inhibit and improve premature ovarian failure.

Description

Technical Field

[0001] The invention relates to the technical field of protein extraction, and in particular to sturgeon swim bladder collagen and an acid leaching extraction method and application thereof. Background Art

[0002] The ovaries are important female gonadal organs. Female growth, development, reproduction and other physiological activities are closely related to ovarian function. Premature ovarian failure refers to the phenomenon of amenorrhea in women before the age of 40 due to ovarian failure. It is characterized by primary or secondary amenorrhea accompanied by increased blood gonadotropin levels and decreased estrogen levels, leading to infertility, menopause and other serious complications, such as psychological stress, osteoporosis and autoimmune diseases.

[0003] In addition, the etiology and pathogenesis of premature ovarian failure are very complex. It is generally believed that this disease is related to genetics, autoimmunity, iatrogenic diseases and the environment. Researchers are aware that oxidative damage can directly damage the ovarian environment and have an adverse effect on follicle development, ovulation, oocyte maturation and granulosa cell function. At present, the treatment of premature ovarian failure mainly includes exogenous hormone replacement therapy, immunotherapy and stem cell therapy.

[0004] However, long-term use of exogenous hormones has significant side effects, such as vaginal bleeding, abdominal distension, breast cancer and endometrial cancer, while immunotherapy can cause serious adverse reactions and its long-term treatment effect is poor. In addition, most stem cell therapies are still in the animal testing stage, and their effectiveness and safety still require extensive research. Therefore, it is urgent to provide a solution to improve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a sturgeon maw collagen and an acid leaching extraction method and application thereof, which can simply and quickly obtain sturgeon maw acid-soluble collagen with strong antioxidant activity, which can not only effectively reduce the waste of sturgeon maw and sustainably utilize sturgeon resources, but also has a high extraction rate of sturgeon maw collagen, which is conducive to industrial production. At the same time, the obtained sturgeon maw acid-soluble collagen can effectively inhibit and improve premature ovarian failure.

[0006] In a first aspect, the present invention provides an acid leaching extraction method for sturgeon maw collagen, comprising: alkali washing the sturgeon maw and then defatting it, soaking it in an acidic solution, separating the extract, and salting out to obtain collagen.

[0007] Optionally, the sturgeon swim bladder is alkali washed and defatted and then soaked in an acidic solution at 20°C-30°C.

[0008] Optionally, the sturgeon swim bladder is alkaline washed and defatted and then soaked in an acidic solution for 36h-60h.

[0009] Optionally, after the sturgeon swim bladder is washed with alkali and defatted, the solid-liquid volume ratio is 1:(5-15) when it is immersed in an acidic solution.

[0010] Optionally, the pH of the acidic solution is 3.12-3.38.

[0011] Optionally, the acidic solute in the acidic solution includes one of acetic acid and citric acid.

[0012] Optionally, the concentration of the acidic solute in the acidic solution is 0.1 mol / L-5 mol / L.

[0013] Optionally, when the sturgeon swim bladder is washed with alkali and defatted, the volume of the sturgeon swim bladder is 1 cm 3 -2cm 3 .

[0014] Optionally, when the sturgeon maw is alkali-washed and defatted, the sturgeon maw is cut and crushed in advance.

[0015] Optionally, the sturgeon swim bladder includes frozen and thawed sturgeon swim bladder, and sturgeon swim bladder that has been isolated for 0h-24h.

[0016] Optionally, the sturgeon swim bladder is washed after being soaked in an alkaline solution.

[0017] Optionally, the alkaline solute in the alkaline solution includes one of sodium hydroxide and sodium bicarbonate.

[0018] Optionally, the concentration of the alkaline solute in the alkaline solution is 1 mol / L-1.5 mol / L.

[0019] Optionally, the sturgeon swim bladder is immersed in an alkaline solution at a solid-liquid ratio of 1 g: (5-15) mL.

[0020] Optionally, the sturgeon swim bladder is soaked in an alkaline solution for 12h-36h.

[0021] Optionally, the alkali-washed sturgeon maw is soaked in a defatting solution and then cleaned.

[0022] Optionally, the solute in the degreasing solution includes one of isopropanol and butanol.

[0023] Optionally, the mass fraction of the degreasing solution is 8%-12%.

[0024] Optionally, the solid-liquid ratio of the sturgeon maw after alkali washing to the defatting solution is 1 g: (5-15) mL.

[0025] Optionally, the alkali-washed sturgeon maw is soaked in a defatting solution for 12 h to 36 h.

[0026] Optionally, after separating the extract, an inorganic salt is added, stirred to dissolve, and then allowed to stand for 6 hours to 18 hours.

[0027] Optionally, the inorganic salt is sodium chloride.

[0028] Optionally, after adding inorganic salt to the extract and stirring to dissolve, the concentration of the inorganic salt is 0.5 mol / L-2 mol / L.

[0029] Optionally, the method comprises the following steps: alkali washing the sturgeon maw to obtain the non-collagen-free sturgeon maw; defatting the non-collagen-free sturgeon maw to obtain the defatted sturgeon maw; soaking the defatted sturgeon maw in an acidic solution and then separating the extract; salting out the extract to obtain crude collagen; and purifying the crude collagen to obtain sturgeon maw collagen.

[0030] In a second aspect, the present invention also provides a sturgeon maw collagen obtained by any of the above optional acid leaching extraction methods.

[0031] In a third aspect, the present invention further provides a use of sturgeon maw collagen in the preparation of a drug for preventing or treating premature ovarian failure, wherein the sturgeon maw collagen is an active ingredient in the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of collagen content in FP, AP, HP, and PP during property testing in the present invention;

[0033] Figure 2 This is a test diagram of polyacrylamide gel electrophoresis (SDS-PAGE) of FP, AP, HP, and PP during property detection in the present invention;

[0034] Figure 3 The ultraviolet spectra of FP, AP, HP and PP in the present invention when performing property detection;

[0035] Figure 4 The infrared spectra of FP, AP, HP and PP in the present invention when performing property detection;

[0036] Figure 5 The scanning electron microscope images of FP, AP, HP and PP at different magnifications when performing property detection in the present invention;

[0037] Figure 6 The thermogravimetric curves of FP, AP, HP and PP during property testing of the present invention are shown below;

[0038] Figure 7 This is a graph showing the in vitro antioxidant performance of FP, AP, HP, and PP during the property testing of the present invention;

[0039] Figure 8 This is a comparison diagram of ovarian pathological sections of mice with premature ovarian failure using FP, AP, HP, and PP during performance testing of the present invention;

[0040] Fig. 9 This is a graph showing the effect of FP, AP, HP, and PP on the serum of mice with premature ovarian failure during performance testing of the present invention;

[0041] Fig.10 This is a graph showing the uterine oxidative stress index of FP, AP, HP, and PP on premature ovarian failure mice during performance testing of the present invention;

[0042] Fig.11 The present invention provides a flowchart of an acid leaching extraction method for sturgeon swim bladder collagen. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0044] The invention provides an acid leaching extraction method for sturgeon swim bladder collagen, comprising: alkali washing the sturgeon swim bladder, degreasing it, soaking it in an acidic solution, separating the extract, and salting out to obtain the collagen.

[0045] In fact, the sturgeon maw can be cleaned by alkali washing, thereby removing non-collagenous substances in the sturgeon maw, and further removing lipids in the sturgeon maw after defatting, thereby obtaining a relatively pure sturgeon maw for acid leaching extraction.

[0046] In fact, the acid-soluble collagen in the sturgeon maw can be dissolved through acid leaching extraction, and the acid-soluble collagen can be precipitated after salting out treatment, thereby obtaining a relatively pure sturgeon maw collagen product.

[0047] In some embodiments, after the sturgeon swim bladder is washed with alkali and defatted, it is soaked in an acidic solution at 20°C-30°C, thereby promoting the dissolution of acid-soluble collagen in the sturgeon swim bladder. Specifically, the defatted sturgeon swim bladder can be soaked in the acidic solution at room temperature for 36h-60h.

[0048] In some embodiments, after the sturgeon maw is washed with alkali and defatted and then immersed in an acidic solution, the solid-liquid volume ratio is 1:(5-15). In fact, when the defatted sturgeon maw is mixed with the acidic solution, it is necessary to completely immerse the sturgeon maw in the acidic solution. In addition, during the acid leaching extraction process, physical methods such as external ultrasound can be used to improve the dissolution efficiency of acid-soluble collagen.

[0049] In some embodiments, the pH of the acidic solution used is 3.12-3.38, and the solute in the acidic solution includes one of acetic acid and citric acid, and the concentration of the acidic solute in the acidic solution is 0.1 mol / L-5 mol / L. In fact, by regulating the type and pH of the acidic solution, the efficiency of collagen extraction from defatted sturgeon swim bladder by the acidic solution can be effectively regulated.

[0050] In some embodiments, when the sturgeon maw is alkali-washed, the sturgeon maw is cut and crushed in advance, so as to effectively increase the specific surface area of ​​the sturgeon maw, which is beneficial for extracting collagen in the acidic solution. Specifically, the volume of the sturgeon maw during alkali washing is 1 cm 3 -2cm 3 In addition, the sturgeon maw used includes frozen and thawed sturgeon maw and sturgeon maw separated from the body for 0-24 hours.

[0051] In some embodiments, when alkali-washing the sturgeon maw, the sturgeon maw is soaked in an alkaline solution and then washed. In fact, soaking in an alkaline solution can improve the uniformity and thoroughness of alkali-washing the sturgeon maw, which is conducive to removing non-collagen components in the sturgeon maw to the maximum extent.

[0052] Specifically, the alkaline solute in the alkaline solution includes one of sodium hydroxide and sodium bicarbonate, and the concentration thereof is 1 mol / L-1.5 mol / L. In fact, the solid-liquid ratio of the sturgeon bladder when soaked in the alkaline solution is 1 g: (5-15) mL, and the sturgeon bladder is soaked in the alkaline solution for 12 h-36 h.

[0053] In some embodiments, the sturgeon swim bladder after alkali washing is washed with distilled water until the pH of the washing solution is neutral, thereby it can be considered that the alkaline solution adhering to the surface of the sturgeon swim bladder is completely washed away.

[0054] In some embodiments, the alkali-washed sturgeon maw is soaked in a defatting solution and then cleaned. In fact, soaking in a defatting solution can improve the uniformity and thoroughness of defatting the sturgeon maw, thereby improving the purity of collagen extracted by acid leaching of the sturgeon maw.

[0055] In some embodiments, the solute in the degreasing solution includes one of isopropanol and butanol, and the mass fraction of the degreasing solution is 8%-12%. In fact, when the sturgeon maw after alkali washing is degreased, the solid-liquid ratio of the sturgeon maw after alkali washing to the degreasing solution is 1g:(5-15)mL, and the sturgeon maw is soaked in the degreasing solution for 12h-36h.

[0056] In some embodiments, after extracting the sturgeon swim bladder in an acidic solution and separating the solid and liquid to obtain an extract, an inorganic salt is added, stirred and dissolved, and allowed to stand for 6 hours to 18 hours, thereby obtaining a collagen product after salting out. In fact, the inorganic salt used is sodium chloride, and the concentration of the inorganic salt after dissolution is 0.5 mol / L-2 mol / L.

[0057] In fact, see Fig.11 The acid leaching extraction method of sturgeon swim bladder collagen comprises the following steps:

[0058] S1. performing alkali washing on the sturgeon swim bladder to obtain the non-collagen-free sturgeon swim bladder;

[0059] S2, defatting the non-collagen-free sturgeon maw to obtain defatted sturgeon maw;

[0060] S3, soaking the defatted sturgeon swim bladder in an acidic solution and separating the extract;

[0061] S4, salting out the extract to obtain a crude collagen product;

[0062] S5. Purify the crude collagen to obtain sturgeon swim bladder collagen.

[0063] In fact, when executing step S5 to purify the crude collagen, common methods such as centrifugation and concentration are used.

[0064] Example 1

[0065] This embodiment 1 provides a method for acid leaching and extracting sturgeon swim bladder collagen, comprising the following steps:

[0066] S1. After thawing, cut the sturgeon swim bladder into 1 cm×1 cm swim bladder blocks, immerse the swim bladder blocks in a 1 mol / L sodium hydroxide aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, take out the swim bladder blocks after removing non-collagenous substances, and wash them with distilled water until the washing solution is neutral;

[0067] S2, soaking the washed swim bladder blocks in a 10% isopropanol aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, taking out the swim bladder blocks after removing the grease, and washing the isopropanol on the surface with distilled water;

[0068] S3, immersing the cleaned swim bladder pieces in an acetic acid solution with a concentration of 0.5 mol / L and a volume of 10 times the volume of the swim bladder pieces at a room temperature of 25°C for 48 hours, and then centrifuging and separating the supernatant to obtain an extract;

[0069] S4, adding sodium chloride (concentration after dissolution is 1 mol / L) to the extract, stirring to dissolve, standing at 25°C for salting out for 12 hours, separating and precipitating to obtain crude collagen;

[0070] S5. The crude collagen is centrifuged and concentrated to obtain an acid-soluble sturgeon swim bladder collagen product (AP).

[0071] Example 2

[0072] This embodiment 2 provides a hot water extraction method for sturgeon swim bladder collagen, comprising the following steps:

[0073] D1. After thawing, cut the sturgeon swim bladder into 1 cm × 1 cm swim bladder blocks, immerse the swim bladder blocks in a 1 mol / L sodium hydroxide aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, take out the swim bladder blocks after removing non-collagenous substances, and wash them with distilled water until the washing solution is neutral;

[0074] D2, soaking the washed swim bladder pieces in a 10% isopropanol aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, taking out the swim bladder pieces after removing the grease, and washing the isopropanol on the surface with distilled water;

[0075] D3, adding the washed swim bladder pieces into ultrapure water 10 times its own volume, heating in a 55°C water bath for 3 hours, and centrifuging the supernatant to obtain the extract;

[0076] D4. Add sodium chloride (concentration after dissolution is 1 mol / L) to the extract, stir and dissolve, let stand at 25°C for 12 hours to allow salting out, and separate the precipitate to obtain crude collagen;

[0077] D5. The crude collagen is centrifuged and concentrated to obtain an acid-soluble sturgeon maw collagen product (HP).

[0078] Example 3

[0079] This embodiment 3 provides a method for preparing sturgeon swim bladder collagen peptide, comprising the following steps:

[0080] F1. After thawing, cut the sturgeon swim bladder into 1 cm × 1 cm swim bladder blocks, immerse the swim bladder blocks in a 1 mol / L sodium hydroxide aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, take out the swim bladder blocks after removing non-collagenous substances, and wash them with distilled water until the washing solution is neutral;

[0081] F2, soak the washed swim bladder pieces in a 10% isopropanol aqueous solution at a solid-liquid ratio of 1:10 (w / v) for 24 hours, remove the grease from the swim bladder pieces and use distilled water to clean the isopropanol on the surface;

[0082] F3. After adding 10 times the volume of ultrapure water to the washed swim bladder, add 100 U / mL of alkaline protease and heat in a 55°C water bath for 4 hours, then inactivate the enzyme in a 100°C water bath for 10 minutes to obtain crude collagen peptide;

[0083] F4. The crude collagen peptide is centrifuged and concentrated to obtain an acid-soluble sturgeon maw collagen peptide product (PP).

[0084] Blank example 1

[0085] This blank example 1 is a dried sturgeon maw with a water content of less than 10%.

[0086] Property detection

[0087] (1) Determination of Hydroxyproline Content

[0088] 5 mg of FP, AP, HP, and PP were weighed and mixed with 2 mL of 6 mol / L hydrochloric acid, and digested in a 100°C water bath for 6 h until no lumps were visible. Then, the pH was adjusted to 6-8 with a 10 mol / L sodium hydroxide solution, and then distilled water was added to dilute the volume to 4 mL and centrifuged at 12000 rpm for 20 min to collect the supernatant. The absorbance of AP, HP, and PP at 560 nm was measured, and the collagen content was calculated according to the standard curve, where the collagen content = hydroxyproline content × 11.1 × 100%. The results are as follows: Figure 1 shown.

[0089] (2) Amino acid composition analysis

[0090] 30 mg of FP, AP, HP, and PP were weighed respectively and mixed with 10 mL of 6 mol hydrochloric acid and 1 g of phenol. After nitrogen blowing to remove air, the mixture was hydrolyzed in an oven at 110°C for 24 h, and then diluted to 50 mL with super-stored water. 1 mL of the fixed volume sample was taken and placed in a clean culture dish. After evaporation to dryness in a 60°C water bath, an appropriate amount of ultrapure water was added to dissolve the mixture, and the evaporation was repeated 1-2 times. After evaporation to dryness, 3 mL of sample diluent (pH 2.2) was added to dissolve the mixture again. After filtration through a 0.2 μm filter membrane, the mixture was analyzed using a Sykam S-433D amino acid analyzer. The analysis results are shown in Table 1 below.

[0091] Table 1 Amino acid composition analysis

[0092]

[0093] Note: Data are shown as SD±mean (n=3). Numbers followed by different letters are significantly different at p<0.05 according to Duncan test.

[0094] (3) Polyacrylamide gel electrophoresis (SDS-PAGE) determination

[0095] FP, AP, HP, and PP were heated in a water bath at 85°C for 1 hour and then centrifuged at 8500 rpm for 5 minutes. The samples were collected and mixed with 5× loading buffer at a volume ratio of 1:1. The samples were electrophoresed using SDS / PAGE technology. After the electrophoresis was completed, 0.05% Coomassie Brilliant Blue was used to stain in a mixture of 50% methanol and 7.5% (V / V) acetic acid for 30 minutes. The samples were then decolorized with a mixture of 5% (V / V) methanol and 7.5% (V / V) acetic acid for 1 hour. Figure 2 shown.

[0096] (4) Ultraviolet spectrum analysis

[0097] Using UV-Vis spectrophotometer, FP, AP, HP, and PP were analyzed in the wavelength range of 400nm-190nm, as shown in Figure 2. Figure 3 shown.

[0098] (5) Fourier transform infrared analysis

[0099] Fourier transform infrared spectroscopy was used at 400 cm -1 -4000cm -1 Within the spectral scanning range, FP, AP, HP, and PP are analyzed respectively with a resolution of 4 cm -1 , the scan is 32 times, and the results are as follows Figure 4 shown.

[0100] (6) Scanning electron microscopy analysis

[0101] Using a scanning electron microscope, FP, AP, HP, and PP were analyzed at 500x and 200x magnifications, respectively. The results are as follows: Figure 5 shown.

[0102] (7) Thermogravimetric analysis

[0103] The thermal properties of FP, AP, HP, and PP were analyzed using a thermogravimetric analyzer with an initial temperature of 30°C, an end temperature of 600°C, and a heating rate of 10°C / min. The results are as follows: Figure 6 shown.

[0104] (8) In vitro antioxidant test

[0105] Use microplate reader to detect ABTS of FP, AP, HP and PP respectively + Free Radicals and O2 - Free radical scavenging ability, the results are as follows Figure 7 shown.

[0106] Performance Testing

[0107] A cyclophosphamide-induced premature ovarian failure mouse model was pre-established, including: SPF female mice (25.0 g ± 2.0 g, 6-8 weeks old, purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., Animal Use License: SCXK (Zhejiang) 2019-0001) were adapted to growth at 22°C ± 2°C, 12 h light-dark cycle for 1 week, and then randomly divided into 7 experimental groups, and each experimental group had 6 mice. On days 8-21, except for the normal control group (N) mice injected with normal saline, the remaining mice were intraperitoneally injected with 10 mg / kg BW cyclophosphamide to establish an animal model of premature ovarian failure mice; subsequently, the positive control group (V) was based on 0.20 mg / kg BW estradiol valerate tablets, and the experimental groups were given 125 mg / kg FP (F), AP (A), HP (H), and PP (P) of BW. Meanwhile, the N group and the model control group (T) were given normal saline for 14 days, and 24 h after the last gavage, all mice were killed by cervical dislocation, and the uterus, bilateral ovaries, and serum were collected for analysis.

[0108] (1) H&E staining and follicle counting at different stages

[0109] One ovary was taken from each of the seven groups of mice and fixed with 10% formalin at room temperature of 25°C for 12 hours. The ovarian tissue after paraffin embedding was cut into 5 μm thick tissue sections. After H&E staining, images were collected using an LV1 pathological section scanner. Follicles of different stages in the ovarian images were classified and counted. The results are shown in Figure 2. Figure 8 shown.

[0110] (2) Serum hormone analysis

[0111] The serum levels of FSH, LH, and E2 in the seven groups of mice were measured using ELISA kits. The results are shown in Fig. 9 shown.

[0112] (3) Analysis of uterine oxidative stress

[0113] The uterine tissues of the seven groups of mice were mixed with pre-cooled PBS and ground, and then centrifuged at 10000rpm and 4℃ for 10min to collect the supernatant. The protein concentration in the supernatant was detected in advance using the BCA protein detection kit, and the activities of SOD and MDA in the supernatant were determined using SOD and MDA kits. The activities were expressed as the activities of SOD and MDA per mg of protein. The results are shown in Fig.10 shown.

[0114] Results Analysis

[0115] The biological activity of protein is closely related to the distribution of amino acids and the composition of R groups. As shown in Table 1, the basic amino acid unit of the triple helix region of sturgeon swim bladder collagen is glycine-XY. Among the amino acid compositions determined, glycine (Gly) is the most abundant, followed by glutamic acid (Glu), alanine (Ala), proline (Pro), arginine (Arg), aspartic acid (Asp), and serine (Ser). In addition, phenylalanine (Phe) and tyrosine (Tyr) are aromatic amino acids that can promote the antioxidant activity of protein (shown as PP>FP=AP>HP). However, hydrophobic amino acids such as valine (Val), leucine (Leu), isoleucine (ILe), alanine (Ala), methionine (Met), phenylalanine (Phe) and proline (Pro) are opposite to aromatic amino acids (shown as HP>AP>FP>PP).

[0116] At the same time, after identifying the essential amino acids in the collagen (peptide) products, it was found that the essential amino acid contents of FP, AP, HP and PP were 24.77%, 24.67%, 23.80% and 24.30%, respectively. This shows that the amino acid composition of collagen (peptide) obtained by different extraction methods is different, which may be due to different internal structural changes during the extraction process.

[0117] Hydroxyproline is a stable amino acid in collagen and plays an important role in collagen content. The conversion factor between fish bladder collagen and hydroxyproline is 11.1. Figure 1 It can be seen that the collagen content in the products obtained by different extraction methods is different, and the content of hydroxyproline has corresponding differences. After conversion, the collagen contents in FP, AP, HP and PP are 51.17±1.52%, 59.42±1.49%, 64.71±3.18% and 49.27±2.69%, respectively, among which the collagen content in HP is higher.

[0118] See also Figure 2Lane 2, lane 3, and lane 5 correspond to AP, HP, and FP, respectively. The three show similar results. α chains can be observed between 100 kDa and 130 kDa, including α1 or α3 chain (130 kDa) and α2 chain (110 kDa). The molecular weights of α1 and α3 are very similar. Figure 2 It can be seen that the molecular weight in AP is the most significant, which indicates that the collagen obtained by acid extraction retains a high-purity high-molecular structure, while HP and FP show weaker and mixed color bands. In addition, lane 4 representing PP cannot be analyzed because collagen peptides are mainly composed of small molecular polypeptides.

[0119] See also Figure 3 AP, HP, PP and FP all showed similar characteristic peaks of collagen, with absorption peaks of collagen at 220nm-230nm and 260nm-280nm respectively. The triple helix protein showed the maximum absorption peak at 220nm-230nm, which was consistent with the absorption characteristics of collagen. This was because of the n→π* electron transition of C=O, -COOH and CO-NH2 groups. In addition, the absorption peak at 260nm-280nm indicated that there were a small amount of aromatic amino acids in the product, with PP being the most significant and HP being the least significant, which was consistent with the amino acid composition analysis results in Table 1.

[0120] See also Figure 4 , AP, HP, PP, and FP all exhibit several characteristic peaks. This is because the NH group will migrate to a lower frequency direction after forming hydrogen bonds with other groups, thus -1 -3400cm -1 There is an absorption peak between 2930cm and 4960cm, and this range is called amide A band, which is caused by the stretching vibration of hydrogen bonds between NH groups and OH groups. This also shows that AP, HP, PP and FP all contain hydrogen bonds. In addition, at 2930cm -1 The observed peaks may be due to the stretching vibration of the CH bond and the =CH and NH3 + The asymmetric stretching vibration of -CH2 may be caused by the antisymmetric stretching vibration of -CH2, which indicates the presence of amide B.

[0121] Also, see Figure 4 , at 1600cm -1 -1660cm -1 The appearance of characteristic peaks indicates the presence of a triple helix structure. The appearance of characteristic peaks is related to the C=O bond, while the amide II band is at 1500 cm -1 -1600cm -1 The range is probably due to the bending vibration of NH and the stretching vibration of CN groups, which are characteristic peaks for predicting protein secondary structure.-1 -1550cm -1 The region represents the existence of antiparallel β structures and parallel β structures, both of which can indicate that AP, HP, PP and FP contain β structures.

[0122] See also Figure 5 The structure of FP is irregular, AP is a disordered petal-like layered structure with local pores, HP has a good polygonal structure, and PP has a finer fragment structure. This shows that the differences in collagen (peptide) products obtained by different extraction methods are very obvious. The acid leaching extraction method seriously damages the structure of collagen, while the hot water extraction method has little effect on the structure of collagen.

[0123] See also Figure 6 FP, AP and HP all showed two weight loss processes. The initial weight loss occurred at 30℃-160℃, and the temperatures were 92.08℃, 68.55℃ and 89.75℃ respectively. In addition, PP had the lowest initial weight loss rate, and the maximum weight loss rate of PP occurred at 47.58℃. In fact, the first weight loss process was mainly due to the evaporation of water, while the second weight loss process was due to the breakage of peptide bonds between collagen fibers. The temperatures at which FP, AP, HP and PP had the highest loss rates were 323.75℃, 320.14℃, 326.08℃ and 325.58℃ respectively. Therefore, it can be seen that HP has the best thermal stability, which may be because acid extraction and enzymes will affect collagen, resulting in a decrease in thermal stability.

[0124] See also Figure 7 , ABTS of AP, HP, PP and FP + The free radical scavenging ability gradually increased with the increase of concentration. At the highest concentration (5 mg / mL), ABTS of FP + The free radical scavenging rates were 28.61%, 33.58% for AP, 37.40% for HP and 46.00% for PP. + The free radical scavenging ability is the strongest. In addition, the superoxide anion scavenging rates of FP and AP increase with the increase of concentration. At the highest concentration (5 mg / mL), the superoxide anion scavenging rates of FP and AP are 61.82% and 42.88%, respectively. HP and PP reach the maximum superoxide anion scavenging rate at 4 mg / mL, and the increase of concentration no longer affects the superoxide anion scavenging rate, which are 37.86% and 34.99%, respectively.

[0125] See also Figure 8, the N group had the highest growth number and normal morphology. In contrast, the T group showed ovarian atrophy and irregular follicle morphology, characterized by a decrease in growing follicles and a significant increase in atretic follicles. After ingesting sturgeon swim bladder collagen (peptide), the follicle growth of the F, A, H and P groups increased, and the atretic follicles decreased. Compared with the F, A and P groups, the changes in the H group were more significant.

[0126] See also Fig. 9 Compared with group N, group T had higher levels of LH and FSH, while groups F, A, H, and P were able to significantly reduce these serum hormone levels. Group T had significantly reduced E2 levels, while groups F, A, H, and P had significantly increased E2 levels. In general, compared with collagen peptides, collagen had a more obvious effect, with group H having the best effect on regulating serum hormones.

[0127] See also Fig.10 , the oxidative stress level of the uterus of mice with premature ovarian failure was detected. Compared with the N group, the SOD level of the T group was significantly reduced, and the MDA level was significantly increased. This shows that cyclophosphamide disrupted the balance of redox reactions in the mouse uterus. After taking sturgeon bladder collagen (peptide), the SOD activity of the uterus increased and the MDA content decreased significantly, among which the H group had the most significant regulatory effect.

[0128] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A method for acid leaching and extracting sturgeon swim bladder collagen, characterized in that: include: The sturgeon swim bladder is washed with alkali and then defatted, and then soaked in an acidic solution to separate the extract, and the collagen is obtained by salting out.

2. The acid leaching extraction method according to claim 1, characterized in that: After washing and defatting the sturgeon maw with alkali, the sturgeon maw is soaked in an acidic solution at 20°C-30°C; and / or, after washing and defatting the sturgeon maw with alkali, the sturgeon maw is soaked in an acidic solution for 36h-60h; and / or, when the sturgeon maw is soaked in an acidic solution after washing and defatting the sturgeon maw with alkali, the solid-liquid volume ratio is 1:(5-15).

3. The acid leaching extraction method according to claim 1, characterized in that: The pH of the acidic solution is 3.12-3.38; and / or the acidic solute in the acidic solution includes one of acetic acid and citric acid; and / or the concentration of the acidic solute in the acidic solution is 0.1 mol / L-5 mol / L.

4. The acid leaching extraction method according to claim 1, characterized in that: When the sturgeon swim bladder is washed with alkali and defatted, the volume of the sturgeon swim bladder is 1 cm 3 -2cm 3 ; and / or, when the sturgeon maw is alkali-washed and defatted, the sturgeon maw is cut and crushed in advance; and / or, the sturgeon maw includes frozen and thawed sturgeon maw and sturgeon maw separated from the body for 0h-24h.

5. The acid leaching extraction method according to claim 1, characterized in that: The sturgeon maw is soaked in an alkaline solution and then washed; wherein: the alkaline solute in the alkaline solution includes one of sodium hydroxide and sodium bicarbonate; and / or the concentration of the alkaline solute in the alkaline solution is 1 mol / L-1.5 mol / L; and / or, when the sturgeon maw is soaked in the alkaline solution, the solid-liquid ratio is 1 g: (5-15) mL; and / or, the sturgeon maw is soaked in the alkaline solution for 12 h-36 h.

6. The acid leaching extraction method according to claim 1, characterized in that: The sturgeon maw after alkali washing is soaked in a degreasing solution and then cleaned; wherein: the solute in the degreasing solution includes one of isopropanol and butanol; and / or the mass fraction of the degreasing solution is 8%-12%; and / or the solid-liquid ratio of the sturgeon maw after alkali washing to the degreasing solution is 1g:(5-15)mL; and / or the sturgeon maw after alkali washing is soaked in the degreasing solution for 12h-36h.

7. The acid leaching extraction method according to claim 1, characterized in that: After separating the extract, adding inorganic salt, stirring and dissolving, and then standing for 6h-18h; wherein: the inorganic salt is sodium chloride; and / or, after adding inorganic salt to the extract and stirring and dissolving, the concentration of inorganic salt is 0.5mol / L-2mol / L.

8. The acid leaching extraction method according to any one of claims 1 to 7, characterized in that: The following steps are involved: The sturgeon maw is subjected to alkali washing to obtain a non-collagen-free sturgeon maw; the non-collagen-free sturgeon maw is defatted to obtain a defatted sturgeon maw; the defatted sturgeon maw is immersed in an acidic solution and then separated to obtain an extract; The extract is subjected to salt precipitation to obtain crude collagen; the crude collagen is purified to obtain sturgeon swim bladder collagen.

9. Sturgeon swim bladder collagen obtained by the acid leaching extraction method according to any one of claims 1 to 8.

10. Use of sturgeon bladder collagen obtained by the acid leaching extraction method according to any one of claims 1 to 8 or the sturgeon bladder collagen according to claim 9 in the preparation of a drug for preventing or treating premature ovarian failure, characterized in that: The sturgeon swim bladder collagen is an effective ingredient in medicine.

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