NL2026344A - Method for extracting type-i collagen through hydrochloric acid-pepsin combination - Google Patents

Abstract

The invention discloses a method for extracting a type-I collagen through hydrochloric acid-pepsin combination, which includes the following steps: step 1. 5 degreasing and decalcifying a bone raw material, and adjusting a pH value to 3 to 5, step 2. heating and boiling at 90°C to 95°C for 2 hours to 3 hours; step 3. cooling, and adding pepsin for enzymolysis, step 4. separating an enzymatic hydrolysate, and step 5. sequentially performing pepsin deactivation and sterilization, decolorization and desalination, concentration, sterilization and spray drying on the enzymatic 10 hydrolysate to obtain a type-I bone collagen powder. According to the invention, the type-I collagen is extracted, so that a utilization value of an edible bone is improved, and the preparation method of the invention is simple, and high in production efficiency, and a product is pure white in color and high in purity. 20

Description

METHOD FOR EXTRACTING TYPE-I COLLAGEN THROUGH HYDROCHLORIC ACID-PEPSIN COMBINATION

TECHNICAL FIELD The present invention belongs to the field of bone processing technologies, and more particularly, relates to a method for extracting a type-I collagen through hydrochloric acid-pepsin combination.

BACKGROUND China is rich in edible bone resources, and an annual output of livestock and poultry bones in China reaches up to 12 million tons at present, which accounts for one third of an output of the world, is equivalent to about two million tons of animal protein, and can meet annual protein demands of 75 million people. China will become an aging society immediately, and osteoporosis will become the biggest chronic threat to the old people. Under a condition of population pressure and limited bearing capacity of breeding environment, to strengthening the comprehensive development and utilization of the bone resources has important economic, social and environmental benefits. Studies have shown that a collagen is a main extracellular matrix component, which is mainly combined with glycoprotein, proteoglycan and the like to form insoluble biomacromolecules. The collagen may be roughly extracted through five methods such as an acid method, a pepsin method, hot-water extraction, an alkaline method and neutral salt extraction according to different extraction media. The acid extraction is to use swelling and dissolution of a collagen fiber caused by a low-concentration acidic condition to dissolve uncrosslinked collagen molecules and the collagen fiber containing an amide cross bond. The pepsin extraction is to enhance degradation of a telopeptide of an unspiralized region of the collagen by adding protease. The hot-water extraction, the alkaline extraction and the neutral salt extraction may destroy a natural triple helical structure of the collagen to varying degrees during extraction thereof. Since a peptide chain of the collagen is easy to break under an alkaline condition, which may cause racemization of amino acid with intensified degree of hydrolysis, thus greatly reducing nutrition and safety of products. The neutral salt extraction is to extract the collagen by using a solubility difference of the collagen in a salt solution of a certain concentration. However, a part of the salt 1 solution may reduce a molecular conformation stability of the collagen, so that the collagen with a natural structure cannot be completely extracted. The hot-water extraction is able to decompose intermolecular and intramolecular hydrogen bond and covalent crosslinking of the collagen, and hydrolyze a part of amide bonds in a S primary structure of collagen molecules. The triple helical structure of the collagen is destroyed and transformed into a random coil state, and an obtained Hot-water Extracted Collagen (HEC) has a high solubility, which may also be called Gelatin.

SUMMARY Aiming at the above technical problems, the present invention designs and develops a method for extracting a type-I collagen through hydrochloric acid-pepsin combination, which is simple in preparation and good in energy-saving effect, and a product is pure white in color and high in purity. The present invention provides the technical solutions as follows. A method for extracting a type-I collagen through hydrochloric acid-pepsin combination includes the following steps: step 1. crushing a bone raw material to a particle size of 10 mm to 20 mm, soaking the bone raw material in a sodium hydroxide solution with a concentration of

0.5 mol/L to 0.8 mol/L for 6 hours to 7 hours at a stirring speed of 12 rpm to 24 rpm to degrease the bone raw material, and then soaking the degreased bone raw material in a hydrochloric acid solution with a concentration of 0.01 mol/L to 0.02 mol/L for 10 hours to 12 hours at a stirring speed of 12 rpm to 24 rpm to decalcify the bone raw material, and adjusting a pH value to 3 to 5; step 2. heating and boiling the material obtained in the step 1 at 90°C to 95°C for 2 hours to 3 hours; step 3. cooling the material obtained in the step 2 to below 55°C, and adding pepsin for enzymolysis, wherein an addition amount of the pepsin is 3%o to 5%. of a mass of the material obtained in the step 2, an enzymolysis temperature is 50°C to 55°C, an enzymolysis time is 5 hours to 7 hours, and a pH value is maintained at 5 to 7 step 4. performing three-phase separation on the material obtained in the step 3, performing solid-liquid separation first, removing a solid-phase inorganic residue obtained by separation, and then separating an enzymatic hydrolysate and bone oil; and 2 step 5. sequentially performing deactivation and sterilization, decolorization and desalination, concentration, sterilization and spray drying on the enzymatic hydrolysate obtained in the step 4 to obtain a type-I bone collagen powder.

Preferably, according to the method for extracting the type-I collagen through 5S hydrochloric acid-pepsin combination, the pepsin is added for two times, an addition amount of the pepsin for the first time is 0.2%o to 0.5%e of the mass of the material obtained in the step 2, and an enzymolysis time is 20 minutes to 40 minutes, then the pepsin is added for the second time, and a total addition amount of the pepsin for the two times is 3%o to 5%. of the mass of the material obtained in the step 2, and a total enzymolysis time for the two times is 5 hours to 7 hours.

Preferably, according to the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the pepsin deactivation and sterilization is performed at 125°C to 130°C for 10 seconds to 15 seconds; and the decolorization and desalination includes a specific process of: decolorizing through an activated carbon column first, wherein a flow rate of the enzymatic hydrolysate subjected to the pepsin and sterilization is 4 times to 6 times a void volume, and then desalinating through an anion-cation resin column, wherein a flow rate of the enzymatic hydrolysate subjected to the decolorization is 6 times to 8 times the void volume.

Preferably, according to the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the concentration includes a specific process of: concentrating the decolorized and desalinated enzymatic hydrolysate to a concentration of Brix10° to 15° through a nanofiltration membrane, and then concentrating the decolorized and desalinated enzymatic hydrolysate to a concentration of Brix 40° to 45° through a double-effect falling membrane.

Preferably, according to the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the sterilization includes a specific process of: delivering the concentrated feed liquid into an insulation storage tank for pasteurization, with a sterilization temperature of 90°C to 95°C and a time of 10 minutes to 15 minutes.

Preferably, according to the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the spray drying includes a specific process of: delivering the sterilized feed liquid into a spray tower, with an air inlet temperature of 180°C to 185°C and an air outlet temperature of 80°C to 90°C, and drying until a 3 moisture content of a finished product is no more than 5.0%.

Hydrochloric acid-pepsin combination is used in the method of the present invention to extract the type-I collagen, thus greatly improving a comprehensive utilization value of an edible bone; the preparation method of the present invention is simple, 5S high in production efficiency and outstanding in economic benefit, membrane method and vacuum falling membrane cascade concentration are used in the process, which has an obvious energy-saving effect; a product obtained by the decolorization and desalination is pure white in color and high in purity., and has a high economic value; and the present invention is simple and convenient to operate, and scale production IO may be formed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process flow chart of an embodiment of a method for extracting a type-I collagen through hydrochloric acid-pepsin combination according to the present invention; FIG 2 is an ultraviolet spectrogram of a pepsin-soluble type-I collagen (PSC) extracted in a first embodiment of the present invention and an acid-soluble type-I collagen (ASC) extracted by an acid extraction process; FIG 3 is a Fourier infrared spectrogram of the pepsin-soluble type-I collagen (PSC) extracted in the first embodiment of the present invention and the acid-soluble type-I collagen (ASC) extracted by the acid extraction process; FIG 4(a) is an infrared spectrogram of the acid-soluble type-I collagen (ASC) extracted by the acid extraction process after deconvolution by curve fitting; and FIG 4(b) is an infrared spectrogram of the pepsin-soluble type-I collagen (PSC) extracted inthe first embodiment of the present invention after deconvolution by curve fitting; FIG 5 is a heat flow analysis diagram of the pepsin-soluble type-I collagen (PSC) extracted in the first embodiment of the present invention and the acid-soluble type-I collagen (ASC) extracted by the acid extraction process; FIG 6 shows electrophoresis results of the pepsin-soluble type-I collagen (PSC) extracted in the first embodiment of the present invention and the acid-soluble type-I collagen (ASC) extracted by the acid extraction process; and FIG. 7(a) is a scanning electron microscope photograph of the acid-soluble type-I collagen (ASC) extracted by the acid extraction process; and FIG 7(b) is a scanning electron microscope photograph of the pepsin-soluble type-I collagen (PSC) extracted 4 in the first embodiment of the present invention.

DETAILED DESCRIPTION The present invention is further described in detail with reference to the accompanying drawings, so that those skilled in the art can implement the present invention according to the description.

As shown in FIG 1, the present invention provides a method for extracting a type-I collagen through hydrochloric acid-pepsin combination, which includes the following steps: step 1: crushing a bone raw material to a particle size of 10 mm to 20 mm, soaking the bone raw material in a sodium hydroxide solution with a concentration of

0.5 mol/L to 0.8 mol/L for 6 hours to 7 hours at a stirring speed of 12 rpm to 24 rpm to degrease the bone raw material, and then soaking the degreased bone raw material in a hydrochloric acid solution with a concentration of 0.01 mol/L to 0.02 mol/L for 10 hours to 12 hours at a stirring speed of 12 rpm to 24 rpm to decalcify the bone raw material, and adjusting a pH value to 3 to 5; step 2. heating and boiling the material obtained in the step 1 at 90°C to 95°C for 2 hours to 3 hours.

step 3. cooling the material obtained in the step 2 to below 55°C, and adding pepsin for enzymolysis, wherein an addition amount of the pepsin is 3%o to 5% of a mass of the material obtained in the step 2, an enzymolysis temperature is 50°C to 55°C, an enzymolysis time is 5 hours to 7 hours, and a pH value is maintained at 5 to 7; added for two times added for two times step 4, performing three-phase separation on the material obtained in the step 3, wherein solid-liquid separation is performed first, a solid-phase inorganic residue obtained by separation is removed, and then an enzymatic hydrolysate and bone oil are separated; specifically, the solid-liquid separation is performed first during the three-phase separation, a horizontal screw decanter centrifuge needs to be used in solid-liquid separation for small materials such as poultry bones and fish bones, while solid-liquid separation for livestock bones may be performed by filtration; liquid-oil separation is performed by a disc centrifuge, and a heavy phase (enzymatic hydrolysate) and a light phase (bone oil) are separated by high-speed centrifugation; and 5 step 5. sequentially performing deactivation and sterilization, decolorization and desalination, concentration, sterilization and spray drying on the enzymatic hydrolysate obtained in the step 4 to obtain a type-I bone collagen powder.

A hydrochloric acid-pepsin combination method is used in the present invention to extract the type-I collagen, the extracted type-I collagen has a triple helical structure, a protein body of the triple helical structure is released, and an obtained product may be called a pepsin-soluble type-I collagen. Moreover, the prepared type-I collagen has a low antigenicity and can be used as a good biomedical material.

In a preferred embodiment, in the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the pepsin is added for two times, an addition amount of the pepsin for the first time is 0.2%0 to 0.5%. of the mass of the material obtained in the step 2, and an enzymolysis time is 20 minutes to 40 minutes, then the pepsin is added for the second time, and a total addition amount of the pepsin for the two times is 3%o to 5% of the mass of the material obtained in the step 2, and a total enzymolysis time for the two times is 5 hours to 7 hours. The pepsin is added for two times, the addition amount for the first time is small, with a short enzymolysis time, and the remaining pepsin is completely added for the second time, and is subjected to enzymolysis for a long time, so as to improve an enzymolysis efficiency and an extraction efficiency.

In a preferred embodiment, in the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the pepsin deactivation and sterilization is performed at 125°C to 130°C for 10 seconds to 15 seconds. The decolorization and desalination includes a specific process of: decolorizing through an activated carbon column first, wherein a flow rate of the enzymatic hydrolysate subjected to the pepsin and sterilization is 4 times to 6 times a void volume, and then desalinating through an anion-cation resin column, wherein a flow rate of the enzymatic hydrolysate subjected to the decolorization is 6 times to 8 times the void volume.

Preferably, the prepared enzymatic hydrolysate is heated at 125°C to 130°C for 10 seconds to 15 seconds in an ultra-high-temperature instantaneous sterilizer to sterilize the pepsin with inactivated materials when the pepsin and sterilization is performed on the enzymatic hydrolysate.

Preferably, the sterilized enzymatic hydrolysate flows through a filtering decolorizing column filled with activated carbon at a flow rate of 4 times to 6 times a void volume first, and then the decolorized liquid enters a resin column filled with anion-cation 6 resin at a flow rate of 6 times to 8 times the void volume in a series mode.

In a preferred embodiment, in the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the concentration includes a specific process of: concentrating the decolorized and desalinated enzymatic hydrolysate to a 5S concentration of Brix10° to Brix15° through a nanofiltration membrane, and then concentrating the decolorized and desalinated enzymatic hydrolysate to a concentration of Brix40° to Brix45° through a double-effect falling membrane.

Preferably, the decolorized and desalinated liquid enters a nanofiltration membrane concentration system, wherein a pore diameter of the nanofiltration membrane is 1 nm to 2 nm, and is concentrated to the concentration of Brix10° to Brix15°. The decolorized and desalinated liquid enters a double-effect falling membrane vacuum concentrator, and is concentrated to the concentration of Brix40° to Brix45°.

In a preferred embodiment, in the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the sterilization includes a specific process of: delivering the concentrated feed liquid into an insulation storage tank for pasteurization, with a sterilization temperature of 90°C to 95°C and a time of 10 minutes to 15 minutes. Preferably, the stirring is performed at 24 rpm to 36 rpm.

In a preferred embodiment, in the method for extracting the type-I collagen through hydrochloric acid-pepsin combination, the spray drying includes a specific process of: delivering the sterilized feed liquid into a spray tower, with an air inlet temperature of 180°C to 185°C and an air outlet temperature of 80°C to 90°C, and drying until a moisture content of a finished product is no more than 5.0%.

The sterilized material enters a centrifugal spray tower (or a pressure spray tower) after concentration, with the air inlet temperature of 180°C to 185°C and the air outlet temperature of 80°C to 90°C, and the moisture content of the finished product is no more than 5.0%.

The product is packed, and an inner layer is packed with a clean plastic bag by 5 Kg to 10 Kg per bag.

The technical solutions further describing the present invention are as follows, and the following comparative examples and embodiments are now provided.

Comparative examples A type-I beef bone collagen was extracted by an acid extraction process, and the specific process was as follows.

(1) 0.1 mol/L NaOH was added by a solid-liquid ratio of 1:10, and was shaken and 7 soaked at 4°C for 6 hours, and the NaOH was replaced every 2 hours.

(2) A bone sample was repeatedly washed to be neutral, and was shaken and degreased with 10% n-hexane by a solid-liquid ratio of 1:10 at 4°C for 3 days after draining, a solution was replaced every 8 hours, and if fat granules still obviously existed on a surface, a degreasing time was appropriately increased.

(3) The bone sample was repeatedly washed to be neutral, and was shaken and decalcified with 0.25 mol/L EDTA (pH 7.4) by a solid-liquid ratio of 1:10 at 4°C for 3 days after draining, and a solution was replaced every 8 hours.

(4) The bone sample was shaken and extracted with 0.5 mol/L acetic acid solution by a solid-liquid ratio of 1:10 for 24 hours to 5 days, a supernatant was collected, and a concentration of NaCl in the solution was adjusted to 0.9 mol/L. Tris (6 g/L extracting solution) was added, a pH was adjusted to 7.5, the mixture was stirred evenly and stood overnight, and then was centrifuged at 4°C at 10,000 rmp for 30 minutes, and freeze-drying was performed on a precipitate to obtain an ASC crude product.

(5) The crude product was dissolved with 0.5 mol/L acetic acid, then was put into a dialysis bag to be dialyzed with 0.1 mol/L acetic acid for 2 days first, and then was dialyzed with distilled water for 2 days, and a liquid outside the dialysis bag was replaced every 12 hours. Freeze-drying was performed on the precipitate after dialysis to obtain a relatively pure type-I beef bone collagen (ASC for short).

First embodiment Implementation of a method for extracting a type-I collagen through hydrochloric acid-pepsin combination (taking a beef bone as an example)

1. 10 kg of bone raw materials frozen at -18°C were selected, impurities in the bone raw materials were washed away with clear water first, and then the bone raw materials were crushed by a hard bone crusher, with a crushing particle size controlled to be 10 mm.

2. The crushed bones were washed with clear water to remove impurities and attachments.

3. The washed crushed bones were soaked by using a NaOH solution with a concentration of 0.5 mol/L for 6 hours at a stirring speed of 12 rpm during soaking for degreasing.

4. The degreased bones were soaked by using a HCI solution with a concentration of

0.01 mol/L for 10 hours at a stirring speed of 12 rpm during soaking for decalcifying, and a pH value was adjusted to be 4.

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5. The decalcified materials were boiled at 90°C for 2 hours at a stirring speed of 12 rpm during boiling.

6. The boiled materials were cooled to a temperature below 55°C, then pepsin was added with an addition amount of 3%. of a mass of the boiled material, an 5S enzymolysis temperature of 50°C, an enzymolysis time of 5 hours, and a pH value maintained at 5 and a stirring speed of at 12 rpm during enzymolysis.

7. Solid-liquid separation was performed on the above materials subjected to enzymolysis in a filtering mode first. A disc centrifuge was used in liquid-oil separation to process the materials, and a heavy phase (enzymatic hydrolysate) and a light phase (bone oil) were separated by high-speed centrifugation.

8. The prepared enzymatic hydrolysate was heated at 125°C for 10 seconds through an ultra-high-temperature instantaneous sterilizer to sterilize the pepsin with inactivated materials.

9. The sterilized enzymatic hydrolysate flowed through a filtering decolorizing column filled with activated carbon at a flow rate of 4 times a void volume first, and then the decolorized liquid entered a resin column filled with anion-cation resin at a flow rate of 6 times the void volume in a series mode.

10. The decolorized and desalinated liquid entered a nanofiltration membrane concentration system, wherein a pore diameter of a nanofiltration membrane was 1 nm, and was concentrated to a concentration of Brix10°; and then the decolorized and desalinated liquid entered a double-effect falling membrane vacuum concentrator, and was concentrated to a concentration of Brix40°.

11. The concentrated liquid entered an insulation storage tank, was pasteurized at 90°C for 10 minutes, and was stirred at 24 rpm.

12. The sterilized materials entered a centrifugal spray tower (or a pressure spray tower), with an air inlet temperature of 180°C and an air outlet temperature of 80°C, and a moisture content of a finished product was no more than 5.0%. An inner layer was packed with a clean plastic bag by 5 Kg per bag.

A yield of a pepsin-soluble type-I collagen (PSC) extracted in the embodiment is compared with a yield of the acid-soluble type-I collagen (ASC) extracted in the comparative example.

Yield (%) = mass of crude product of type-I collagen subjected to freeze-drying/mass of raw material bone for extraction x 100% The yield of the ASC in the comparative example and the yield of the PSC in the first 9 embodiment are 2.24% and 3.32% respectively.

Structures of the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment were analyzed, and a composition and a content of amino acid in the two type-I beef bone collagens were shown in Table 1. Table 1 Contents of Amino Acids in Type-I Beef bone Collagens of ASC in the Comparative Example and PSC in the First embodiment Amino acid ee It can be seen from Table 1 that a content of TAA in the ASC of the beef ribin the comparative example and a content of TAA in the PSC of the beef rib in the first embodiment are respectively 656.5 mg/g and 708.7 mg/g, and proportions of EAAs are 20.0% and 20.6%. Contents of Glys are the highest, accounting for 25.5% and

24.6% of the TAAs respectively, followed by Pro, Glu, Ala, Arg and Asp. Total amounts of characteristic amino acids (Gly, Lys, Pro) of the type-I collagens reach

277.9 mg/g and 297.5 mg/g. Trp is not included. Cys, Met and Tyr are three amino acids with the lowest content in the type-I beef bone collagen. Except Cys, Val and Met, contents of other amino acids in the PSC of the beef rib are higher than those in the ASC of the beef rib. Therefore, it is inferred that the ASC and PSC of the beef ribs have a typical composition characteristic of the type-I collagen, and a purity of the PSC is relatively high. Ultraviolet spectrum analysis was performed on the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. As shown in FIG 2, the beef bones ASC and PSC both reached maximum absorption at 234 nm. Infrared spectrum analysis was performed on the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. Peakfit 4.2 software was used to perform Gaussian fitting on an amide I-band in infrared spectrograms of the ASC and the PSC of the beef ribs (as shown in FIG 3) to obtain several sub-peaks, and peak positions were identified. 1616 cm’! to 1637 cm’! and 1681 cm™ to 1700 cm fell top-fold, 1638 cm’! to 1645 cm™ fell to random coil, 1646 cm"! to 1664 cm’! fell too-helix, 1665 cm’! to 1681 cm’! fell top-corner, and a relative percentage of a secondary structure was calculated. FIG 3 is a Fourier infrared spectrogram of the ASC and the PSC, and FIG. 4(a) and FIG. 4(b) are obtained after deconvolution by curve fitting. Analysis results of peak position identification show that a content of the a-helix (66.7%)} in the ASC of the beef rib is 2.0 times that in the PSC of the beef rib, the B-folds in the ASC and the PSC are 29.5% and 41.2% respectively, and the B-corners are 3.8% and 25.4% respectively. Neither the ASC nor the PSC contains the random coil, thus indicating that triple helical structures of the ASC and the PSC of the beef ribs are complete. DSC analysis was performed on the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. FIG 5 is a heat flow analysis diagram of the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. Heat shrinkage Temperatures (Ts) and Denaturation Enthalpies (DH) of the ASC and the PSC of the beef ribs were analyzed by differential scanning calorimetry. The type-I collagen had a superhelical helical structure, a hydrogen bond therein was broken due to heating, and a random coil was 11 formed due to denaturation, with a change in energy. Meanwhile, changes in a helical structure and a bioactivity of the type-I collagen were reflected. The Tss of the ASC and the PSC of the beef ribs are 40.12°C and 40.94°C respectively, and the DHs of the ASC and the PSC of the beef ribs are 0.25 J/g and 0.22 J/g respectively.

An electrophoresis experiment was performed on the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on the ASC and the PSC extracted from the beef ribs, as shown in FIG 6. The PSC and the ASC are trimers of o chains of the type-I collagens at a position above 250 kDa, ie. vy chains, and a B band exists at a position about 200 kDa. Two electrophoresis bands al and 02 exist at a position about 110 kDa. Therefore, the extracted type-I collagen is judged to basically have a complete triple helical structure.

Scanning electron microscope photographs of the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment were taken to observe micro-structures of the type-I beef bone collagens of the ASC in the comparative example and the PSC in the first embodiment. The ASC has a thin structure and relatively even distribution, thus indicating that a fiber structure thereof is basically not destroyed during extraction, while the PSC has many pore diameters which are unevenly distributed, thus indicating that the pepsin partially changes the structure of the type-I collagen, but a good network structure is still maintained. Second embodiment Implementation of a method for extracting a type-I collagen through hydrochloric acid-pepsin combination (taking a fish bone as an example)

1. Raw materials used were frozen at -18°C, and fish bones needed to be crushed by a crusher, with a crushing particle size controlled to be 20 mm.

2. The crushed bones were washed with clear water to remove impurities and attachments.

3. The washed crushed bones were soaked by using a NaOH solution with a concentration of 0.8 mol/L for 7 hours at a stirring speed of 24 rpm during soaking for degreasing.

4. The degreased bones were soaked by using a HCI solution with a concentration of

0.02 mol/L for 12 hours at a stirring speed of 24 during soaking for decalcifying.

5. The decalcified materials were boiled at 95°C for 3 hours at a stirring speed of 24 rpm during boiling.

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6. The boiled materials were cooled to a temperature below 55°C, then pepsin was added with an addition amount of 5%. of a mass of the boiled material, an enzymolysis temperature of 55°C, an enzymolysis time of 7 hours, and a pH value maintained at 7 and a stirring speed of at 24 rpm during enzymolysis.

S 7. Solid-liquid separation was performed on the above materials subjected to enzymolysis first, and a horizontal screw decanter centrifuge was used in solid-liquid separation for the fish bone in a solid-liquid separation mode. A disc centrifuge was used in liquid-oil separation to process the materials, and a heavy phase (enzymatic hydrolysate) and a light phase (bone oil) were separated by high-speed centrifugation.

8. The prepared enzymatic hydrolysate was heated at 130°C for 15 seconds through an ultra-high-temperature instantaneous sterilizer to sterilize the pepsin with inactivated materials.

9. The sterilized enzymatic hydrolysate flowed through a filtering decolorizing column filled with activated carbon at a flow rate of 6 times a void volume first, and then the decolorized liquid entered a resin column filled with anion-cation resin at a flow rate of 8 times the void volume in a series mode.

10. The decolorized and desalinated liquid entered a nanofiltration membrane concentration system, wherein a pore diameter of a nanofiltration membrane was 2 nm, and was concentrated to a concentration of Brix 15°; and then the decolorized and desalinated liquid entered a double-effect falling membrane vacuum concentrator, and was concentrated to a concentration of Brix45°.

11. The concentrated liquid entered an insulation storage tank, was pasteurized at 95°C for 15 minutes, and was stirred at 36 rpm.

12. The sterilized materials entered a centrifugal spray tower (or a pressure spray tower), with an air inlet temperature of 185°C and an air outlet temperature of 90°C, and a moisture content of a finished product was no more than 5.0%. An inner layer was packed with a clean plastic bag by 10 Kg per bag.

A yield of the pepsin-soluble type-I collagen (PSC) extracted in the second embodiment 1s 3.42%. Moreover, through analysis of the pepsin-soluble type-I collagen (PSC), the pepsin-soluble type-I collagen (PSC) has a complete triple helical structure, a good network structure is maintained, and a protein structure was not destroyed.

Third embodiment Implementation of a method for extracting a type-I collagen through hydrochloric 13 acid-pepsin combination (taking a beef bone as an example)

1. 10 kg of bone raw materials frozen at -18°C were selected, impurities in the bone raw materials were washed away with clear water first, and then the bone raw materials were crushed by a hard bone crusher, with a crushing particle size controlled tobe 10 mm.

2. The crushed bones were washed with clear water to remove impurities and attachments.

3. The washed crushed bones were soaked by using a NaOH solution with a concentration of 0.5 mol/L for 6 hours at a stirring speed of 12 rpm during soaking for degreasing.

4. The washed crushed bones were soaked by using a HCI solution with a concentration of 0.01 mol/L for 10 hours at a stirring speed of 12 rpm during soaking for decalcifying, and a pH value was adjusted to be 4.

5. The decalcified materials were boiled at 90°C for 2 hours at a stirring speed of 12 rpm during boiling.

6. The boiled materials were cooled to a temperature below 55°C, and then pepsin was added for two times, wherein an addition amount of the pepsin for the first time is

0.2%o of a mass of the boiled material at an enzymolysis temperature of 50°C, a pH value maintained at 5 and an enzymolysis time of 20 minutes; then the pepsin was added for the second time at an enzymolysis temperature of 50°C and a pH value maintained at 5, and a total addition amount of the pepsin for the two times was 3%o, a total enzymolysis time for the two times was 5 hours, and the stirring speed during enzymolysis was 12 rpm.

7. Solid-liquid separation was performed on the materials subjected to enzymolysis in afiltering mode first. A disc centrifuge was used in liquid-oil separation to process the materials, and a heavy phase (enzymatic hydrolysate) and a light phase (bone oil) were separated by high-speed centrifugation.

8. The prepared enzymatic hydrolysate was heated at 125°C for 10 seconds through an ultra-high-temperature instantaneous sterilizer to sterilize the pepsin with inactivated materials.

9. The sterilized enzymatic hydrolysate flowed through a filtering decolorizing column filled with activated carbon at a flow rate of 4 times a void volume first, and then the decolorized liquid entered a resin column filled with anion-cation resin at a flow rate of 6 times the void volume in a series mode.

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10. The decolorized and desalinated liquid entered a nanofiltration membrane concentration system, wherein a pore diameter of a nanofiltration membrane was 1 nm, and was concentrated to a concentration of Brix10°; and then the decolorized and desalinated liquid entered a double-effect falling membrane vacuum concentrator, and S was concentrated to a concentration of Brix40°.

11. The concentrated liquid entered an insulation storage tank, was pasteurized at 90°C for 10 minutes, and was stirred at 24 rpm.

12. The sterilized materials entered a centrifugal spray tower (or a pressure spray tower), with an air inlet temperature of 180°C and an air outlet temperature of 80°C, IO and a moisture content of a finished product was no more than 5.0%. An inner layer was packed with a clean plastic bag by 5 Kg per bag.

A yield of the pepsin-soluble type-I collagen (PSC) extracted in the third embodiment is 3.64%. Moreover, through analysis of the pepsin-soluble type-I collagen (PSC), the pepsin-soluble type-I collagen (PSC) has a complete triple helical structure, a good network structure is maintained, and a protein structure is not destroyed.

Fourth embodiment Implementation of a method for extracting a type-I collagen through hydrochloric acid-pepsin combination (taking a fish bone as an example)

1. Raw materials used were frozen at -18°C, and fish bones needed to be crushed by a crusher, with a crushing particle size controlled to be 20 mm.

2. The crushed bones were washed with clear water to remove impurities and attachments.

3. The washed crushed bones were soaked by using a NaOH solution with a concentration of 0.8 mol/L for 7 hours and at a stirring speed of 24 rpm during soaking for degreasing.

4. The degreased crushed bones were soaked by using a HCI solution with a concentration of 0.02 mol/L for 12 hours at a stirring speed of 24 rpm during soaking for decalcifying.

5. The decalcified materials were boiled at 95°C for 3 hours at a stirring speed of 24 rpm during boiling.

6. The boiled materials were cooled to a temperature below 55°C, and then pepsin was added for two times, wherein an addition amount of the pepsin for the first time is

0.5%o of a mass of the boiled material at an enzymolysis temperature of 55°C, a pH value maintained at 7 and an enzymolysis time of 40 minutes; then the pepsin was 15 added for the second time at an enzymolysis temperature of 55°C and a pH value maintained at 7, and a total addition amount of the pepsin for the two times was 5%o, a total enzymolysis time for the two times was 7 hours, and the stirring speed during enzymolysis was 24 rpm.

S 7. Solid-liquid separation was performed on the above materials subjected to enzymolysis first, and a horizontal screw decanter centrifuge was used in solid-liquid separation for the fish bone in a solid-liquid separation mode. A disc centrifuge was used in liquid-oil separation to process the materials, and a heavy phase (enzymatic hydrolysate) and a light phase (bone oil) were separated by high-speed centrifugation.

8. The prepared enzymatic hydrolysate was heated at 130°C for 15 seconds through an ultra-high-temperature instantaneous sterilizer to sterilize the pepsin with inactivated materials.

9. The sterilized enzymatic hydrolysate flowed through a filtering decolorizing column filled with activated carbon at a flow rate of 6 times a void volume first, and then the decolorized liquid entered a resin column filled with anion-cation resin at a flow rate of 8 times the void volume in a series mode.

10. The decolorized and desalinated liquid entered a nanofiltration membrane concentration system, wherein a pore diameter of a nanofiltration membrane was 2 nm, and was concentrated to a concentration of Brix15°; and then the decolorized and desalinated liquid entered a double-effect falling membrane vacuum concentrator, and was concentrated to a concentration of Brix45°.

11. The concentrated liquid entered an insulation storage tank, was pasteurized at 95°C for 15 minutes, and was stirred at 36 rpm.

12. The sterilized materials entered a centrifugal spray tower (or a pressure spray tower), with an air inlet temperature of 185°C and an air outlet temperature of 90°C, and a moisture content of a finished product was no more than 5.0%; and an inner layer was packed with a clean plastic bag by 10 Kg per bag.

A yield of the pepsin-soluble type-I collagen (PSC) extracted in the fourth embodiment 1s 3.69%. Moreover, through analysis of the pepsin-soluble type-I collagen (PSC), the pepsin-soluble type-I collagen (PSC) has a complete triple helical structure, a good network structure is maintained, and a protein structure is not destroyed.

Although the embodiments of the present invention have been disclosed above, the present invention is not limited to the applications listed in the specification and the 16 embodiments.

The present invention can be applied to various fields suitable for the present invention absolutely, and other modifications can be easily realized by those skilled in the art.

Therefore, the present invention is not limited to the specific details and the illustrations shown and described herein without departing from the general concepts defined by the claims and equivalent scopes. 17

Claims (6)

CONCLUSIES:CONCLUSIONS: 1. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie, omvattende de volgende stappen van: - stap 1. het vermalen van een botgrondstof tot een deeltjesgrootte van 10 mm tot 20 mm, het weken van de botgrondstof in een natriumhydroxideoplossing met een concentratie van 0,5 mol/l tot 0,8 mol/l gedurende 6 uur tot 7 uur bij een roersnelheid van 12 rpm tot 24 rpm om de botgrondstof te ontvetten, en vervolgens het weken van de ontvette botgrondstof in een zoutzuuroplossing met een concentratie van 0,01 mol/l tot 0,02 mol/l gedurende 10 uur tot 12 uur bij een roersnelheid van 12 rpm tot 24 rpm om de botgrondstof te ontkalken, en het instellen van een pH-waarde van 3 tot 5; - stap 2. het verhitten en koken van het in stap 1 verkregen materiaal bij 90 °C tot 95 °C gedurende 2 uur tot 3 uur; - stap 3. het afkoelen van het in stap 2 verkregen materiaal tot onder 55 °C, en het toevoegen van pepsine voor enzymolyse, waarbij een toevoegingshoeveelheid van de pepsine 3 %o tot 5 %o is van een massa van het in stap 2 verkregen materiaal, een enzymolysetemperatuur 50 °C tot 55 °C bedraagt, een enzymolysetijd 5 uur tot 7 uur is, en een pH-waarde wordt gehandhaafd op 5 tot 7; - stap 4. het uitvoeren van driefasige scheiding op het in stap 3 verkregen materiaal, het uitvoeren van scheiding van vaste stof en vloeistof eerst, het verwijderen van een door scheiding verkregen vastefasig anorganisch residu, en vervolgens het scheiden van een enzymatisch hydrolysaat en botolie; en - stap 5. het achtereenvolgens uitvoeren van pepsine-deactivering en sterilisatie, van het ontkleuren en ontzilten, van het concentreren, van het steriliseren en van het sproeidrogen op de in stap 4 verkregen enzymatische hydrolysaat om een type-I botcollageenpoeder te verkrijgen.1. A method for extracting a type I collagen by means of a hydrochloric acid-pepsin combination, comprising the following steps of: step 1: grinding a bone raw material to a particle size of 10 mm to 20 mm, the bone stock in a sodium hydroxide solution at a concentration of 0.5 mol / l to 0.8 mol / l for 6 hours to 7 hours at a stirring speed of 12 rpm to 24 rpm to degrease the bone stock, and then soak the degreased bone raw material in a hydrochloric acid solution at a concentration of 0.01 mol / l to 0.02 mol / l for 10 hours to 12 hours at a stirring speed of 12 rpm to 24 rpm to descale the bone raw material, and adjust a pH value from 3 to 5; - step 2. heating and boiling the material obtained in step 1 at 90 ° C to 95 ° C for 2 hours to 3 hours; - step 3, cooling the material obtained in step 2 to below 55 ° C, and adding pepsin for enzymolysis, wherein an addition amount of the pepsin is 3% o to 5% o of a mass of the obtained in step 2 material, an enzymolysis temperature is 50 ° C to 55 ° C, an enzymolysis time is 5 hours to 7 hours, and a pH value is maintained at 5 to 7; step 4. performing three-phase separation on the material obtained in step 3, performing separation of solid and liquid first, removing a solid phase inorganic residue obtained by separation, and then separating an enzymatic hydrolyzate and bone oil; and - step 5. performing pepsin deactivation and sterilization, decolorization and desalting, concentration, sterilization and spray drying sequentially on the enzymatic hydrolyzate obtained in step 4 to obtain a type I bone collagen powder. 2. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie volgens conclusie 1, waarbij de pepsine in twee keer wordt toegevoegd, een toevoegingshoeveelheid van de pepsine voor de eerste keer 0,2 %o tot 0,5 %o is van de massa van het in stap 2 verkregen materiaal, een enzymolysetijd 20 minuten tot 40 minuten is, vervolgens de pepsine voor de tweede keer wordt toegevoegd, een totale toevoegingshoeveelheid van de pepsine voor de twee keer 3 %o tot 5 %o is van de massa van het in stap 2 verkregen materiaal, en een totale enzymolysetijd voor de twee keer 5 uur tot 7 uur is. 18A method of extracting a type I collagen by means of a hydrochloric acid-pepsin combination according to claim 1, wherein the pepsin is added in two steps, an addition amount of the pepsin for the first time 0.2% o to 0 , 5% o of the mass of the material obtained in step 2, an enzymolysis time is 20 minutes to 40 minutes, then the pepsin is added a second time, a total addition amount of the pepsin for the two times 3% o to 5 % o is the mass of the material obtained in step 2, and a total enzymolysis time for the two times is 5 hours to 7 hours. 18 3. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie volgens conclusie 1, waarbij de pepsine-deactivering en sterilisatie wordt uitgevoerd bij 125 °C tot 130 °C gedurende 10 seconden tot 15 seconden; en het ontkleuren en ontzilten een specifiek proces omvat van: het ontkleuren door een actiefkoolkolom eerst, waarbij een stroomsnelheid van het enzymatische hydrolysaat dat aan de pepsine-deactivering en sterilisatie wordt onderworpen 4 maal tot 6 maal het interstitiële volume is, en vervolgens het ontzilten door een anion-kationharskolom, waarbij een stroomsnelheid van het enzymatische hydrolysaat dat aan het ontkleuren wordt onderworpen 6 maal tot 8 maal het IO interstitiéle volume is.A method of extracting a type I collagen by means of a hydrochloric acid-pepsin combination according to claim 1, wherein the pepsin deactivation and sterilization is performed at 125 ° C to 130 ° C for 10 seconds to 15 seconds; and the decolorization and desalination comprises a specific process of: decoloring through an activated carbon column first, wherein a flow rate of the enzymatic hydrolyzate subjected to the pepsin deactivation and sterilization is 4 times to 6 times the interstitial volume, and then desalting by an anion-cation resin column, where a flow rate of the enzymatic hydrolyzate subjected to decolorization is 6 times to 8 times the 10 interstitial volume. 4. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie volgens conclusie 1, waarbij het concentreren een specifiek proces omvat van: het concentreren van het ontkleurde en ontzilte enzymatische hydrolysaat tot een concentratie van Brix 10 ° tot 15 ° door een nanofiltratiemembraan, en vervolgens het concentreren van het ontkleurde en ontzilte enzymatische hydrolysaat tot een concentratie van Brix van 40 ° tot 45 ° door een vallend membraan met dubbel effect.A method of extracting a type I collagen by means of a hydrochloric acid-pepsin combination according to claim 1, wherein the concentrating comprises a specific process of: concentrating the decolorized and desalted enzymatic hydrolyzate to a concentration of Brix 10 °. up to 15 ° through a nanofiltration membrane, then concentrating the decolorized and desalted enzymatic hydrolyzate to a concentration of Brix from 40 ° to 45 ° through a dual effect falling membrane. 5. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie volgens conclusie 1, waarbij het steriliseren een specifiek proces omvat van: het afleveren van de geconcentreerde voedingsvloeistof in een isolatieopslagtank voor pasteurisatie, met een sterilisatietemperatuur van 90 °C tot 95 °C en een tijd van 10 minuten tot 15 minuten.A method of extracting a type I collagen by means of a hydrochloric acid-pepsin combination according to claim 1, wherein the sterilizing comprises a specific process of: delivering the concentrated nutrient liquid into an isolation storage tank for pasteurization, with a sterilization temperature of 90 ° C to 95 ° C and a time of 10 minutes to 15 minutes. 6. Werkwijze voor het extraheren van een type I-collageen door middel van een zoutzuur-pepsine-combinatie volgens conclusie 1, waarbij het sproeidrogen een specifiek proces omvat van: het afleveren van de gesteriliseerde voedingsvloeistof in een sproeitoren, met een luchtinlaattemperatuur van 180 °C tot 185 °C en een luchtuitlaattemperatuur van 80 °C tot 90 °C, en het drogen tot een vochtgehalte van een afgewerkt product niet meer dan 5,0% bedraagt. 19The method of extracting a type I collagen by means of a hydrochloric acid-pepsin combination according to claim 1, wherein the spray drying comprises a specific process of: delivering the sterilized nutrient fluid into a spray tower, with an air inlet temperature of 180 ° C to 185 ° C and an air outlet temperature of 80 ° C to 90 ° C, and drying to a moisture content of a finished product not exceeding 5.0%. 19