Brevibacillus laterosporus and method for extracting collagen / collagen peptide from brevibacillus laterosporus

Abstract

The invention discloses brevibacillus laterosporus SCUT-BR-2 capable of producing collagenase and a method for applying the brevibacillus laterosporus SCUT-BR-2 to extraction of animal collagen / collagen peptide. The method specifically comprises the following steps: screening a bacterial strain capable of producing collagenase from a meat product, carrying out morphological observation, physiological and biochemical tests and determination, extracting collagen in animal skin by adopting a collagenase crude extract of the bacterial strain, and detecting the relative molecular weight and the like of the collagen; meanwhile, bovine bone collagen is decomposed by adopting the crude extract, and the bovine bone collagen peptide which is good in oxidation resistance and has anti-wrinkle, firming, soothing and moisturizing effects is obtained.

Description

Technical Field

[0001] The present application relates to the technical field of microbial fermentation and culture and the hydrolysis of collagen using microorganisms, and specifically to a Brevibacillus laterosporus and a method for hydrolyzing collagen to obtain collagen peptides. Background Art

[0002] Bacillus laterosporus is a type of facultative anaerobic bacteria with rod-shaped bodies and peritrichous flagella. It is also a probiotic commonly used for biological control. Bacillus laterosporus has a wide range of uses, including: in animal husbandry, it can be used as a microbial additive in animal feed. In agriculture, it can also effectively improve soil fertility and promote crop growth as a microbial fertilizer. In fisheries, Bacillus laterosporus can inhibit the photosynthesis of algae cells, making it difficult for them to survive, and cause lipid peroxidation in the algae cell membrane, causing the algae to die, thereby achieving the effect of water quality regulation and cyanobacteria control, which is beneficial to aquaculture. In the field of food, the antimicrobial peptides it produces have a broad spectrum of antibacterial activity, are resistant to high temperatures, acids and alkalis, and are sensitive to proteolytic enzymes, and can therefore be used in food preservation.

[0003] Collagen is a triple-helix fibrous protein twisted into three peptide chains. It is an insoluble protein among fibrous proteins and is the most abundant protein in many vertebrates and invertebrates. Collagen has good low immunity, biocompatibility and degradability, and can be widely used in food, cosmetics and medicine. Collagen peptides (also known as collagen polypeptides) are products with relatively small molecular weights formed by the hydrolysis of collagen. Compared with collagen, collagen peptides have higher absorption and utilization rates, better effects, and can promote the absorption of other proteins in food. Collagen polypeptides can be completely dissolved in water (cold water can also dissolve), and also have certain antihypertensive and angiotensin (ACE) inhibitory activities, improve skin and heal wounds, inhibit bacteria, antioxidant activity, promote bone synthesis, etc. Therefore, collagen polypeptides have an irreplaceable and important role in food (including health products), medical, beauty and skin care industries, and the demand is increasing.

[0004] At present, the extraction of collagen mainly adopts animal skin extraction, and the method includes alkali extraction, acid extraction, hot water extraction, enzymatic extraction and composite method. The extraction of acid method alone or enzyme-acid combined method is easy to destroy the protein structure, and the alkali extraction will cause the protein to racemize. The method of extracting collagen by hot water extraction method will lead to incomplete decomposition of collagen tissue, and the yield of collagen is low. Enzymatic extraction method is relatively mild and more efficient than other methods, and the activity retention degree of the product is better. In the prior art, as in the patent application with the authorization number CN113943770B, it adopts the method of acid + enzyme combination + salting out to extract fish skin collagen, which is complicated in process and uses commercial enzymes, and the cost is high. In the patent application with publication number CN110922475A, more than two combined enzymes are used to extract collagen from pig skin, including "pepsin treatment, pH 2-3, temperature 30-40°C; papain treatment, pH 6-7, temperature 50-60°C; trypsin treatment, pH 7-8, temperature 40-50°C". In the existing methods, commercial enzymes are used for the selection of enzymes. However, commercial enzymes are expensive, and the reaction conditions are based on the optimal conditions for enzyme activity. Therefore, the use of commercial enzymes makes the entire collagen extraction process costly and complicated.

[0005] There are currently two main methods for obtaining collagen peptides by further hydrolyzing collagen: synthesis and decomposition. Some scholars have used the synthesis method to make bovine collagen peptides, but this method requires the determination of the bovine collagen peptide sequence first, which has a high threshold and a relatively easy decomposition method. Enzymatic hydrolysis is commonly used in the decomposition method, including pepsin, alkaline protease, neutral protease, trypsin, animal compound protease, flavor protease and papain. Similarly, in the process of degrading low-molecular collagen peptides, the current mainstream method is to directly use commercial enzymes. As mentioned above, these mature commercial enzymes are not only very expensive, but also have harsh degradation conditions (degradation needs to be carried out under the reaction conditions with the best enzyme activity to achieve the best effect without wasting enzymes, so there are more requirements for temperature, substrate concentration, etc.), and the dosage is large, which undoubtedly increases the production cost of the enterprise.

[0006] Therefore, how to develop a bacterium that can naturally produce proteolytic enzymes to replace the above-mentioned expensive and large-volume commercial enzymes, and directly combine them through a simple fermentation + self-produced enzyme process, and use this process to extract collagen and / or obtain collagen peptides, reduce extraction costs, simplify the extraction process, reduce equipment requirements, and reduce environmental pollution. This is crucial for enterprises to carry out large-scale preparation and wide application of collagen / collagen peptides, and is also a new method to achieve higher resource utilization of fermentation process products, providing new reference and ideas for fermentation enterprises in environmental protection, product processing, etc. Summary of the invention

[0007] In order to solve the practical technical problems in the production process of the above-mentioned enterprises, the present invention has developed a new Brevibacillus laterosporus, and then obtained a method for directly extracting collagen / obtaining collagen peptides using the fermentation product of Brevibacillus laterosporus. The specific technical scheme is as follows:

[0008] A Brevibacillus laterosporus, whose name is Brevibacillus laterosporus SCUT-BR-2, was deposited in Guangdong Microbiological Culture Collection Center on October 15, 2024, and the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the storage number is: GDMCC No.65271.

[0009] As a further limitation of the above scheme, the Brevibacillus laterosporus is isolated, cultured and purified from meat products.

[0010] As a further limitation of the above scheme, the culture medium used for Brevibacillus laterosporus includes: LB culture medium, culture medium A, and beef extract peptone culture medium.

[0011] As a further limitation of the above scheme, the formula of the culture medium A is: 0.8% collagen powder, 0.1% beef extract, 0.1% glucose, KH 2 PO 4 0.05%,MgSO 4 7H 2 O 0.01%, agar 2%, and adjust the pH of medium A to 7.0.

[0012] As a further limitation of the above scheme, the steps of isolating, culturing and purifying Brevibacillus laterosporus from meat products include: grinding the meat products, weighing 1g and adding it to a triangular flask containing 49mL of sterile physiological saline, shaking it for 30 minutes, letting it stand, taking the middle layer of clear water as the bacterial suspension and performing gradient dilution. The gradient diluted bacterial suspension is spread on LB agar solid culture medium by plate dilution separation method, cultured in a constant temperature incubator at 30-40°C until a single colony grows, and the single colony is selected and picked on the screening culture medium A plate, and inverted and cultured at 30-40°C for 1 to 2 days; according to the screening characteristics, single colonies with normal growth and obvious differences in colony morphology are selected, and they are repeatedly streaked on beef extract peptone culture medium for pure culture, and stored for later use after numbering.

[0013] The present invention also claims a method for enzymatic hydrolysis of bovine collagen by using the Brevibacillus laterosporus SCUT-BR-2, comprising: preparing a 12.5% ​​bovine collagen solution, stirring and dissolving it at 50°C, adding 0.3% (based on the quality of bovine collagen) of a crude collagenase extract, reacting at 50°C for 8-24h, then adding 0.5%-3% activated carbon powder (based on the quality of bovine collagen) to the reaction solution, adsorbing and removing odor for 1h, first filtering once with a double-circle qualitative rapid filter paper, and then filtering once with a 0.45μm microporous filter membrane to obtain a clear and transparent filtrate; heating the filtrate at 70°C for 5min to inactivate the enzyme, collecting the filtrate for spray drying, and obtaining bovine collagen peptides.

[0014] As a further limitation of the above scheme, the obtained bovine collagen peptide satisfies at least one of the following properties:

[0015] (1) The scavenging rate of bovine collagen peptide on DPPH free radicals was 10.53±0.47%;

[0016] (2) The inhibition rate of elastase was 14.39±0.52%;

[0017] (3) The inhibition rate of hyaluronidase was 47.02±2.49%;

[0018] (4) The weight average molecular weight of bovine collagen peptide is 1870 g / mol.

[0019] As a further limitation of the above scheme, the crude collagenase extract is obtained by the following method: a certain amount of Bacillus laterosporus SCUT-BR-2 colonies are picked from a beef extract peptone culture medium plate, 1.5 times the mass of the bacteria is added with sterile water, shaken well, and centrifuged at 4°C and 8000 rpm for 10 minutes. The obtained supernatant is the crude collagenase extract used in the present invention.

[0020] As a further limitation of the above scheme, the yield of the obtained bovine collagen peptide is 42.48%.

[0021] The present application also claims to protect a use of the above-mentioned Brevibacillus laterosporus SCUT-BR-2 in extracting collagen from fish skin, pig skin, cow skin and donkey skin.

[0022] Beneficial technical effects

[0023] The Brevibacillus laterosporus (B. laterosporus) SCUT-BR-2 provided by the present invention has a good effect in the collagen extraction process of animal skins such as fish skin. At the same time, when bovine collagen is used as a raw material, its decomposition of collagen into small molecule peptides is remarkable; specifically, the obtained bovine collagen peptide has a scavenging rate of 10.53±0.47% for DPPH free radicals, an inhibition rate of 14.39±0.52% for elastase, and an inhibition rate of 47.02±2.49% for hyaluronidase, respectively indicating that the bovine collagen peptide has a certain antioxidant effect, anti-wrinkle and firming effect, and soothing and moisturizing effect, and can be well applied to various skin care products, beauty essences, etc. The enzyme extracted from Brevibacillus laterosporus SCUT-BR-2 provided by the present invention is very suitable for extracting and processing collagen under neutral conditions, avoiding the need for additional acid-base reagents to be added for acid and base adjustment of commercial enzymes such as pepsin or alkaline protease, which easily introduces inorganic ion impurities; in addition, the present invention directly uses the crude fermentation extract of Brevibacillus laterosporus (B. laterosporus) SCUT-BR-2, that is, the crude collagen extract used by the present invention, to extract collagen and process bovine bone collagen peptides, not only The steps of enzyme purification are omitted, and other beneficial components that promote enzymolysis in addition to the extracellular collagenase produced by Bacillus laterosporus SCUT-BR-2 itself are comprehensively utilized, such as the extracellular secretions produced by Bacillus laterosporus SCUT-BR-2 during the fermentation process. The presence of these components can provide certain vitality and nutrients to the collagenase in the crude extract, promote the activity of the enzyme, and make the enzymolysis of the enzyme more efficient. The promotion of extracellular secretions to the activity of extracellular enzymes is mainly reflected in: enhancing the stability of the enzyme. Certain extracellular secretions can provide a relatively stable and familiar living and working environment for the enzyme, thereby improving the stability and activity of the enzyme. Secondly, the extracellular secretions in the crude liquid itself provide a familiar substrate for the activity of the enzyme, promoting the reaction rate of the enzyme. The applicant found that the Bacillus laterosporus SCUT-BR-2 provided in the present invention can improve the degradation efficiency of its extracellular enzyme to the substrate by secreting specific polysaccharides or proteins. In addition, different components in the extracellular secretions of bacteria in the crude extract will interact with each other to enhance the catalytic effect of the enzyme. In the present invention, the small molecules secreted by Brevibacillus laterosporus SCUT-BR-2 can activate the activity of the enzyme or increase the accessibility of its substrate, thereby accelerating the reaction rate of enzymatic hydrolysis.From the perspective of processing cost, the culture conditions of the Brevibacillus laterosporus SCUT-BR-2 provided by the present invention are simpler than those of commercial enzymes on the market, easy to preserve, and low in cost, easy to industrialize, and have good development and application prospects; and the crude extract of the enzyme omits a series of cumbersome steps such as purification and separation, that is, simplifies the entire process flow, and comprehensively utilizes the various components in the crude extract, greatly reducing production costs, and saving resources, reducing environmental pollution caused by wastewater in the production process or the process and cost of treating wastewater. At the same time, the Brevibacillus laterosporus SCUT-BR-2 and its crude extract provided by the present invention also have important application potential in solving environmental pollution problems and resource waste problems. By using it to extract collagen from animal skins, animal skins can be comprehensively utilized as resources and high-value products can be obtained, providing a sustainable development solution for the application of biotechnology and promoting the green development of biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Colony morphology of SCUT-BR-2;

[0025] Figure 2 : Gram staining of SCUT-BR-2;

[0026] Figure 3 :16S rDNA sequence map of SCUT-BR-2;

[0027] Figure 4 : Phylogenetic tree of SCUT-BR-2;

[0028] Figure 5 : Freeze-dried fish skin collagen (unground);

[0029] Figure 6 : Freeze-dried bovine skin collagen (unground);

[0030] Figure 7 : Freeze-dried pig skin collagen (unground);

[0031] Figure 8 : Freeze-dried donkey skin collagen (unground);

[0032] Fig. 9 : Infrared spectrum of collagen;

[0033] Fig.10 : CD image of collagen;

[0034] Fig.11 : Spray dried bovine collagen peptide. DETAILED DESCRIPTION

[0035] The technical solutions of the specific embodiments of the present invention will be clearly and completely described below in combination with various specific embodiments. Obviously, the described embodiments are only 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. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained through ordinary commercial channels.

[0036] The present invention obtains a new Brevibacillus laterosporus SCUT-BR-2 through culture medium cultivation and subsequent screening and separation. The strain was deposited in Guangdong Microbiological Culture Collection Center on October 15, 2024. The deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, and the deposit number is: GDMCC No.65271; the Brevibacillus laterosporus is separated and purified from meat products. The meat products are pork products.

[0037] In the screening step of Brevibacillus laterosporus SCUT-BR-2, the culture medium used includes: LB culture medium, culture medium A, beef extract peptone culture medium. The formula of culture medium A is: collagen powder 0.8%, beef extract 0.1%, glucose 0.1%, KH 2 PO 4 0.05%,MgSO 4 7H 2 O 0.01%, agar 2%, adjust the pH of medium A to 7.0; the medium is obtained based on 1 liter of sterile water or purified water, and the substances in the above formula are added thereto and the pH is adjusted to 7.0.

[0038] The method for separating, culturing and purifying Brevibacillus laterosporus from meat products comprises:

[0039] Grind the pork product, weigh 1 g and add it into a conical flask containing 49 mL of sterile saline, shake it thoroughly for 30 minutes, let it stand, take the middle layer of the clear water as the bacterial suspension and perform gradient dilution; use the plate dilution separation method to spread the gradient diluted bacterial suspension on the LB agar solid culture medium, culture it in a constant temperature incubator at 30-40°C until a single colony grows, select the single colony and pick it on the screening culture medium plate, and culture it upside down at 30-40°C for 1 to 2 days; according to the screening characteristics, such as single colony size, surface structure, texture, gloss and color, pick the single colony with normal growth and obvious differences in colony morphology, repeatedly streak it on the beef extract peptone culture medium for pure culture, and store it for later use after numbering.

[0040] The temperature of the constant temperature incubator is preferably 35° C., and the temperature of the inverted culture is preferably 35° C. The inventors surprisingly found that the culture effect is better under the preferred conditions.

[0041] Bacteria detection

[0042] (1) Morphological identification

[0043] Refer to the experimental methods in the Manual of Common Bacterial System Identification and record the colony morphology, staining characteristics and optical microscope morphological characteristics.

[0044] (2) Physiological and biochemical identification

[0045] Referring to the physiological and biochemical indices in the Manual of Identification of Common Bacterial Systems, the physiological and biochemical indices such as catalase, VP reaction, methyl red reaction, and nitrate reduction test were measured for each strain.

[0046] (3) 16S rDNA sequence analysis

[0047] The isolated bacterial test tube slant (beef extract peptone medium) was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results of the strain 16S rDNA were compared with the NCBI database, and a phylogenetic tree was constructed.

[0048] Morphological identification of Brevibacillus laterosporus

[0049] The colony morphology of strain SCUT-BR-2 is as follows Figure 1 As shown in the figure, the colonies on the beef extract peptone medium are milky white, approximately round, with smooth and neat edges, a raised center, and translucent. The colonies on the plate were selected for Gram staining, and the results were as follows: Figure 2 As shown in the figure, some are Gram-negative and some are Gram-positive, but Gram-negative accounts for the vast majority. This is because the Gram staining reaction of Brevibacillus laterosporus is uncertain: Gram-positive in the logarithmic phase, Gram-negative in the late stable phase and the decay phase.

[0050] Physiological and biochemical identification of Brevibacillus laterosporus

[0051] Some physiological and biochemical characteristics of strain SCUT-BR-2 are shown in Table 1. Combined with the morphological characteristics, according to the Manual of Systematic Identification of Common Bacteria and Bergey's Manual of Systematic Identification of Bacteria, the strain can be preliminarily identified as Brevibacillus.

[0052] Table 1 Physiological and biochemical test results of strain SCUT-BR-2

[0053]

[0054] Note: + indicates a positive reaction, - indicates a negative reaction.

[0055] Analysis of 16S rDNA Sequence of Brevibacillus laterosporus

[0056] Using the bacterial genome as a template, after PCR amplification, the PCR product of the isolated strain SCUT-BR-2 showed fluorescent bands after agarose gel electrophoresis. Further sequencing results of the PCR amplification product showed that the 16S rDNA fragment sequence was as follows Figure 3 Based on this sequence, MEGA11.0 software was used to construct a phylogenetic tree. Figure 4 As shown. The results of 16S rDNA sequence analysis showed that it had the highest homology with the known strain Brevibacillus laterosporus (GenBank accession number NR 112727.1), with a score of 2543, a coverage rate of 99%, and a homology of 99.71%. Combined with the strain colony morphology, individual morphology, physiological and biochemical characteristics and 16S rDNA identification results, the strain SCUT-BR-2 is Brevibacillus laterosporus. The strain Brevibacillus laterosporus SCUT-BR-2, the preservation information is as recorded in the above content.

[0057] The crude collagenase extract produced by Brevibacillus laterosporus SCUT-BR-2 proposed by the present invention is used to extract collagen from animal skin, wherein the crude collagenase extract is obtained by picking a certain amount of Brevibacillus laterosporus SCUT-BR-2 colonies from a beef extract peptone culture medium plate, adding sterile water 1.5 times the mass of the colonies, shaking well, centrifuging at 4°C and 8000 rpm for 10 minutes, and the obtained supernatant is the crude collagenase extract used by the present invention.

[0058] Specific extraction examples are as follows:

[0059] Example 1: The specific extraction method of extracting collagen from fish skin using Brevibacillus laterosporus SCUT-BR-2 is as follows:

[0060] (1) Pretreatment

[0061] Weigh the dried fish skin, cut it into small pieces of 3×3 cm, add 0.1 mol NaOH solution of 20 times the weight of the fish skin, soak for 6 hours to remove protein and decolorize. After alkali soaking, wash it, add 20% ethanol solution (20 times the dry weight of the fish skin) to soak the fish skin to remove impurities such as pigment and fat from the fish skin, soak for 6 hours and wash it. Then add 15 times the weight of the fish skin, boil it, boil it for 10 minutes, let it stand and cool, adjust the pH to neutral (7.0), and set it aside.

[0062] (2) Enzyme extraction reaction

[0063] The crude extract of collagenase produced by Brevibacillus laterosporus SCUT-BR-2 of the present invention is added to the fish skin solution in an amount of 1% of the dry weight of the fish skin, and the reaction is placed on a shaker for 18-36 hours. Specifically, it can be 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 hours, etc., but preferably 24 hours. Experimental comparison shows that this preference can best take into account efficiency and reaction quality, the effective amount of enzyme in the extract, etc.

[0064] (3) Separation

[0065] The reaction solution was filtered through a double-circle qualitative filter paper and then filtered through a 0.45 μm microporous filter membrane. The filtrate was then heated at 70° C. for 5 min to inactivate the enzyme, and the liquid was collected and stored in a freezer.

[0066] (4) Freeze-dried storage

[0067] The fish skin collagen extracted above is freeze-dried by freeze-drying, weighed, collected and stored.

[0068] Example 2: Collagen was extracted from cowhide, pig skin and donkey skin using Brevibacillus laterosporus SCUT-BR-2. The specific extraction scheme is as follows:

[0069] (1) Pretreatment

[0070] Shave fresh cowhide, pig skin and donkey skin, remove subcutaneous fat with a knife, and cut into 1cm×1cm cubes for later use. Weigh the spare skin, add 0.1mol NaOH solution 1.5 times the weight of the skin, and soak on a shaker to degrease. Pour out the liquid after 6 hours, wash the skin, and then soak in 10% (w / w) NaCl solution 1.5 times the weight of the skin to remove salt-soluble proteins. After soaking in salt water for 24 hours, wash with water to remove residual sodium chloride. Then add 1.5 times the weight of the skin, pure water, boil, boil for 10 minutes, let stand and cool, adjust the pH to neutral, and set aside. The above skin weights are all wet skin weights of spare skins, the same below.

[0071] (2) Enzyme extraction reaction

[0072] The crude collagenase extract produced by Brevibacillus laterosporus SCUT-BR-2 of the present invention is added in an amount of 1% of the skin mass, added to the animal skin solution, and placed on a shaker for shaking reaction for 18-36 hours, specifically 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 hours, etc., but preferably 24 hours. Experimental comparison found that this preference can best take into account efficiency and reaction quality, the effective amount of enzyme in the extract, etc.

[0073] (3) Separation

[0074] The reaction liquid is mixed with 0.5-3% of the obtained wet leather mass of activated carbon powder, and after 3-5 hours, centrifuged at 5500r / min for 5 minutes, and then the supernatant is filtered with a rapid qualitative filter paper, and then filtered with a 0.45μm microporous filter membrane, and then the filtrate is heated at 70°C for 5 minutes to inactivate the enzyme, and then the liquid is collected and frozen for storage. The 0.5-3% activated carbon powder here can be preferably 1%-2% to ensure that the adsorption of color and odor is more thorough and clean, but at the same time, considering the difficulty of removing the activated carbon after adsorption, the addition amount of the activated carbon powder is more preferably 1%.

[0075] (4) Freeze-dried storage

[0076] The extracted cowhide, pig skin and donkey skin collagen is freeze-dried by freeze-drying, weighed, collected and stored.

[0077] The rough appearance of the samples extracted above is shown in the attached Figure 5 , 6 , 7, and 8.

[0078] Detection of collagen properties or physicochemical parameters extracted from various raw materials in Examples 1 and 2

[0079] Relative molecular weight detection

[0080] The extracted collagen samples were mixed with water to prepare a test solution with a concentration of 1 mg / mL, shaken with a vortex mixer, left to dissolve for 24 hours, and filtered with a disposable sterile syringe through a 0.22 μm water system needle filter for use. The chromatographic conditions for the test were: the detector temperature was 25°C, the chromatographic column was an Agilent 1260 InfinityII gel chromatography column, the flow rate was 1 mL / min, and the injection volume was 10 μL.

[0081] Infrared spectrum detection

[0082] The extracted collagen sample powder was ground with potassium bromide at a mass ratio of 1:100 and then pressed into tablets. The secondary structure of the sample was determined by Fourier transform infrared spectroscopy (FTIR). The instrument Shimadzu IRTracer100 was used at 4000-400 cm -1 The spectrum is acquired within the range of 2 cm. -1 The spectral scan was performed 32 times with a resolution of 1.

[0083] Circular dichromatographic detection

[0084] The freeze-dried collagen was dissolved in 0.1 mol / L acetic acid to a mass concentration of 0.5 mg / mL, and the CD curve of the extract was recorded in the range of 190 to 240 nm using a cuvette with a path length of 0.1 cm-1.

[0085] Test results

[0086] (1) Collagen yield

[0087] The yield of collagen is calculated according to formula 1

[0088]

[0089] The collagen yields of the four animal skins can be obtained, as shown in Table 2.

[0090] Table 2 Collagen yield of four animal skins

[0091]

[0092] (2) Relative molecular weight of collagen

[0093] The relative molecular weight of each type of collagen is shown in Table 3 below.

[0094] Table 3 GPC results of collagen from four animal skins

[0095]

[0096] (3) Infrared spectrum detection results of collagen

[0097] The infrared spectrum of collagen can obtain amide A band, B band, I band, II band and III band.

[0098] Fig. 9 The following are infrared spectra of four extracted collagens obtained by FTIR detection. All four samples have characteristic absorption peaks of collagen, including amide A band, amide I band, amide II band and amide III band. Amide A band is the strongest absorption band in the amide bond and is related to the stretching vibration of the NH bond. The free NH stretching vibration occurs at 3300-3500cm -1 The amide A bands of dried fish skin collagen, cow skin collagen, pig skin collagen and donkey skin collagen are at 3329.6 cm -1 、3316cm -1 、3338.8cm -1 and 3302.3cm -1 The amide B band is located in the range of 1680-1780 cm-1, which is mainly produced by the stretching vibration of C=O and the bending vibration of NH, representing CH 2 Asymmetric extension of the functional group, CH 2 It is a characteristic group of the tertiary structure. The amide I band originates from the stretching vibration of the C=O bond, the bending vibration of the NH bond, the stretching of the CN bond, and the deformation of the C-CN bond, and is usually used to reflect the secondary structure of proteins. Previous studies have shown that the positions of the following secondary structure regions in amide I are: β-turn, 1640-1660cm -1 ; α-helix, 1650~1660cm -1 ; Irregular curl, 1640~1650cm -1 ; and β-folding, 1600-1640cm -1 The amide I bands of dried fish skin collagen, cow skin collagen, pig skin collagen and donkey skin collagen are at 1660.6 cm -1 、1658.3cm -1 、1658.3cm -1 and 1660.6cm -1 . Amide II band (1510~1580cm -1 The stretching vibration of ) corresponds to the stretching vibration of CH and NH bonds. The amide II bands of dried fish skin collagen, cow skin collagen, pig skin collagen and donkey skin collagen are at 1544.3 cm -1 、1551.1cm -1 、1548.9cm -1 and 1544.3cm -1 The amide III band (Amide III) is located at 1200-1335 cm -1, reflecting the CH 2 The absorption caused by the group vibration peak is related to the integrity of the triple helix structure of collagen.

[0099] (4) Circular dichroism test results of collagen

[0100] Fig.10 The CD spectra of collagen extracted from four animal skins are shown. Fig.10 It can be seen that collagen has characteristic absorption peaks at 198 and 225nm. The negative absorption peak at a wavelength of 198nm is a typical feature of the irregular coil structure in the conformation of collagen molecules, and the positive absorption peak at 225nm is a typical feature of the L-polyproline (P-II) configuration peptide chain. The characteristic peak of collagen at 225nm can reflect the level of collagen triple helix. These four collagens have very small positive absorption peaks at 225nm, indicating that there are fewer advanced triple helical structures in collagen.

[0101] The results of the secondary structure analysis of collagen are shown in Table 4. Random coils account for the vast majority of the four conformations, while α-helices account for the smallest proportion, indicating that the collagenase crude extract hydrolyzes collagen while extracting it.

[0102] Table 4 Secondary structure analysis results of collagen

[0103]

[0104] Therefore, by using the crude extract of collagenase produced by Brevibacillus laterosporus SCUT-BR-2 screened and isolated in the present application, collagen was extracted from donkey skin, pig skin, cattle skin and fish skin, and infrared spectroscopy was performed on them. It was found that the extracts all had amide-specific absorption peaks, and the circular dichroism spectrum found that they had fewer high-order triple helical structures but more irregular coil structures (typical features in the conformation of collagen molecules), which proved that the extract was collagen. It also proved that it is feasible to directly use the crude extract of collagenase produced by Brevibacillus laterosporus SCUT-BR-2 in the present application to extract collagen from animal skins, which is a feasible solution for extracting collagen at low cost to replace the current commercial enzyme products with high prices and complex acquisition processes.

[0105] Example 3: The technical scheme for obtaining collagen peptides by decomposing bovine collagen using Brevibacillus laterosporus SCUT-BR-2 is as follows:

[0106] (1) Preparation of bovine collagen peptide

[0107] Prepare 12.5% ​​bovine collagen solution (stir and dissolve at 50°C), add 0.3% (based on the quality of bovine collagen) crude collagenase extract, and react at 50°C for 8-24 hours, specifically 8, 10, 12, 14, 16, 18, 20, 22, 24 hours, etc., but more preferably react for 12 hours, because at this preferred time, the degradation rate of bovine collagen and the comprehensive experimental efficiency are optimal. Then add 0.5%-3% activated carbon powder (based on the quality of bovine collagen) to the reaction solution, adsorb and deodorize for 1 hour, then filter once with double-circle qualitative rapid filter paper, and filter once with a 0.45μm microporous filter membrane to obtain a clear and transparent filtrate. Heat the filtrate at 70°C for 5 minutes to inactivate the enzyme, collect the liquid and spray dry it, and the resulting dry solid is as shown in the attached Fig.11 The 0.5-3% activated carbon powder here can be preferably 1%-2% to ensure that the adsorption of color and odor is more thorough and clean, but at the same time considering the difficulty of removing the activated carbon after adsorption, the addition amount of the activated carbon powder is more preferably 1%.

[0108] The specific injection volume of spray drying is 40-110L / h, and the inlet air temperature is 120-240°C. Preferably, the injection volume is 60-90L / h, and the inlet air temperature is 150-200°C. For example, the injection volume can be 60, 65, 70, 75, 80, 85, 90L / h, etc., and the inlet air temperature can be 150, 160, 170, 180, 190, 200°C, etc. Under the most preferred injection volume of 70L / h and inlet air temperature of 180°C, the spray drying effect is optimal, which is manifested in high spray drying efficiency, the least amount of particles adhering to the inner wall of the equipment, high yield, optimal process control process, and better uniformity of the product particles of bovine collagen peptides with certain properties and molecular weight obtained under the enzymatic hydrolysis reaction of the present application.

[0109] Relative molecular weight detection of bovine collagen peptide

[0110] The bovine collagen peptide samples were mixed with the mobile phase solution to prepare a test solution with a concentration of 1 mg / mL, shaken with a vortex mixer, left to dissolve for 24 hours, and filtered with a disposable sterile syringe through a 0.22 μm water system needle filter for use.

[0111] The chromatographic conditions were as follows: the detector temperature was 25°C, the chromatographic column was an Agilent 1260 InfinityII gel chromatography column, the flow rate was 1 mL / min, and the injection volume was 40 μL.

[0112] Activity detection of bovine collagen peptide

[0113] DPPH free radical scavenging rate

[0114] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH for short) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to pairing with a single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals.

[0115] Weigh the quantitative sample and dilute it with water to a multi-level concentration sample. During the reaction, shake / pump thoroughly to mix and then let it stand. Use 95% ethanol to dissolve the DPPH reagent and prepare it to 0.12 mg / mL. Prepare the DPPH working solution in the dark. Trolox is prepared into a 0.08 mg / mL stock solution using 95% ethanol, and half-diluted into a series of concentration gradients to verify the test system. The concentration setting should include 0.01, 0.02, 0.04, and 0.08 mg / mL as positive controls. Refer to the sample liquid addition grouping list, use 96 microplates for the test, set up a sample group (T), a sample background group (T0), a negative control group (C), and a solvent background group (C0), where the positive control group is treated the same as the sample group. Set up 3 parallels for each group. Use a spray gun to add reagents in sequence to ensure consistency in reaction time.

[0116] Table 5 Antioxidant test sample liquid addition grouping list

[0117]

[0118] After 5 minutes of reaction in the dark, use an ELISA reader to detect immediately. The parameters are set as follows: slow oscillation for 10 seconds and absorbance at OD517nm. Export the file after the instrument completes the reading.

[0119] The formula for calculating the DPPH free radical scavenging rate is as follows:

[0120]

[0121] Elastase inhibition rate

[0122] Elastin plays a key role in the youthfulness of the skin, and the lack of elastin is the main cause of skin aging. Inhibiting the activity of elastase can effectively slow down the decomposition of elastin and maintain the elasticity of the skin. Elastase can catalyze the hydrolysis of hydroxyl groups contained in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, it will produce colored substances, and its absorbance can be measured at 410nm in a spectrophotometer. According to the change in absorbance, the inhibitory effect of the test substance on the activity of elastase is calculated, and whether the test substance has firming and anti-wrinkle effects can be evaluated.

[0123] According to the sample liquid addition grouping list, the test was carried out using a 96-well microplate to set up a sample group (T), a sample background group (T0), an enzyme reaction group (C) and a solvent background group (C0), wherein the positive control group was treated in the same manner as the sample group. Three parallels were set up for each group.

[0124] Table 6 List of groups of samples for firming and anti-wrinkle test

[0125]

[0126] Immediately use the microplate reader to detect, set the parameters: slow oscillation for 10 seconds, OD410nm absorbance measurement. Export the file after the instrument completes the reading.

[0127] The method for calculating the inhibition rate of elastase activity is as follows:

[0128]

[0129] Hyaluronic acid inhibition

[0130] As an important natural moisturizing substance, hyaluronic acid plays an important role in maintaining the volume of extracellular matrix and normal physiological function of skin. However, hyaluronic acid can be specifically decomposed by hyaluronidase, and its products promote skin inflammation, leading to increased histamine production in the body. In addition, hyaluronidase expression is also related to most IgE-mediated type I and T-cell-mediated type IV allergic reactions. Inhibiting the activity of hyaluronidase is beneficial to inhibit the development of skin inflammation and allergic reactions to a certain extent, and improve the irritation state of the skin caused by inflammation and allergic reactions. Therefore, inhibiting the activity of hyaluronidase is usually used as an important indicator for product soothing and moisturizing evaluation. Hyaluronidase breaks the glycosidic bond between N-acetylglucosamine and D-glucuronic acid in the hyaluronic acid HA sugar chain. The resulting reducing sugar can reduce DNS to an amino compound under alkaline conditions. After a boiling water bath, it can be fully colored and its absorbance can be measured at 540nm to calculate the hyaluronidase activity.

[0131] Reagents and materials: hyaluronidase, hyaluronic acid, positive control: dexamethasone, DNS reagent.

[0132] Refer to the sample addition grouping table and use 2mL centrifuge tubes for the test. Set up 3 parallel tubes for each group.

[0133] Table 7 Soothing and moisturizing test sample liquid addition grouping list

[0134]

[0135] After the test tube has cooled, shake it well, take 150 μL of the reaction solution and add it to a 96-well plate, and use an ELISA reader to measure the absorbance at OD540 nm.

[0136] The hyaluronidase activity inhibition rate is calculated according to the following formula:

[0137]

[0138] Yield of bovine collagen peptides

[0139] The bovine collagen peptide obtained after spray drying is as follows Fig.11 As shown in the figure. According to the yield formula, the yield of bovine collagen peptide is

[0140]

[0141] Results of relative molecular weight test of bovine collagen peptide

[0142] The weight average molecular weight of the bovine collagen peptide is 1870 g / mol, and the dispersion coefficient is relatively close to 1, indicating that the bovine collagen peptide is a product obtained by decomposing bovine collagen with the collagenase of Brevibacillus laterosporus SCUT-BR-2, and the molecular weight distribution of the bovine collagen peptide is relatively uniform.

[0143] Table 8 GPC test results of bovine collagen peptides

[0144]

[0145] Activity test results of bovine collagen peptide

[0146] The results of the activity test of collagen peptides are shown in Table 9. 1% (W / V) of the test collagen peptide has a scavenging effect on DPPH free radicals, with a scavenging rate of 10.53±0.47%, and the data have significant differences (P=0.0003). This shows that the test results can be used as evidence to support the efficacy evaluation of the test collagen peptide by scavenging DPPH free radicals. 2% (W / V) of the test collagen peptide has an inhibitory effect on the decomposition of AAAPAN catalyzed by elastase, with an inhibition rate of 14.39±0.52%, and the data have significant differences (p=0.0280). This shows that the test results can be used as evidence to support that the test collagen peptide achieves firming and anti-wrinkle effects by inhibiting the activity of elastase. 2% (V / V) of the test collagen peptide has an inhibitory effect on the decomposition of hyaluronic acid by hyaluronidase, with an inhibition rate of 47.02±2.49%, and the data have significant differences (p=0.0025). This indicates that the test results can serve as evidence to support that the test collagen peptide achieves soothing and moisturizing effects by inhibiting the activity of hyaluronidase.

[0147] Table 9 Activity test results of bovine collagen peptides

[0148]

[0149] Compared with blank: *, p<0.05; **, p<0.01; ***, p<0.0001.

[0150] Among them, the positive control used for DPPH free radical scavenging rate was 40 μg / mL vitamin E, and the sample was 1% (W / V) bovine collagen peptide; the positive control used for elastase activity inhibition rate was 200 μg / mL EGCG, and the sample was 2% (W / V) bovine collagen peptide; the positive control used for hyaluronidase activity inhibition rate was 0.5 mg / mL dexamethasone sodium phosphate, and the sample was 2% (V / V) bovine collagen peptide.

[0151] The above-mentioned test shows that the SCUT-BR-2 provided by the present invention decomposes bovine collagen into small molecule peptides with remarkable effect. And the obtained bovine collagen peptide has a scavenging rate of 10.53±0.47% for DPPH free radicals, an inhibition rate of 14.39±0.52% for elastase, and an inhibition rate of 47.02±2.49% for hyaluronidase, respectively indicating that the bovine collagen peptide has certain antioxidant effects, anti-wrinkle and firming effects, and soothing and moisturizing effects. Therefore, it is completely feasible to use the bovine collagen peptide in skin care products, such as creams, lotions, essences, skin care water, facial masks, etc., which can play the effects of resisting free radicals, replenishing water, soothing and moisturizing.

[0152] The above-mentioned embodiments only express some implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A Brevibacillus laterosporus, characterized in that: The name is: Brevibacillus laterosporus SCUT-BR-2, and it was deposited in Guangdong Microbial Culture Collection Center on October 15, 2024. The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the storage number is: GDMCC No.65271.

2. The Brevibacillus laterosporus according to claim 1, characterized in that: The Brevibacillus laterosporus is separated, cultured and purified from meat products.

3. The Brevibacillus laterosporus according to claim 1, characterized in that: The culture medium used for Brevibacillus laterosporus includes: LB culture medium, culture medium A, and beef extract peptone culture medium.

4. The Brevibacillus laterosporus according to claim 1, characterized in that: The formula of the culture medium A is: 0.8% collagen powder, 0.1% beef extract, 0.1% glucose, 0.05% KH2PO4, 0.01% MgSO4·7H2O, and 2% agar, and the pH of the culture medium A is adjusted to 7.

0.

5. The Brevibacillus laterosporus according to claim 1, characterized in that: The steps of separating, culturing and purifying Brevibacillus laterosporus from meat products include: grinding the meat products, weighing 1g of the meat products, adding them into a triangular flask containing 49mL of sterile physiological saline, shaking them fully for 30 minutes and then standing them, taking the middle layer clear liquid as a bacterial suspension and performing gradient dilution; using a plate dilution separation method to spread the gradient diluted bacterial suspensions onto LB agar solid culture medium, culturing them in a constant temperature incubator at 30-40°C until single colonies grow, selecting single colonies and placing them on screening culture medium A plates, and inverting and culturing them at 30-40°C for 1-2 days; selecting single colonies with normal growth and obvious differences in colony morphology according to screening characteristics, repeatedly streaking them on beef extract peptone culture medium for pure culture, and storing them for later use after numbering.

6. A method for enzymatically hydrolyzing bovine collagen using the Brevibacillus laterosporus according to any one of claims 1 to 5, characterized in that: include: Prepare 12.5% ​​bovine collagen solution, stir and dissolve at 50°C, add 0.3% collagenase crude extract calculated based on the mass of bovine collagen, react at 50°C for 8-24h, then add 0.5%-3% activated carbon powder calculated based on the mass of bovine collagen to the reaction solution, adsorb and deodorize for 1h, filter once with double-circle qualitative rapid filter paper, and then filter once with a 0.45μm microporous filter membrane to obtain a clear and transparent filtrate; heat the filtrate at 70°C for 5min to inactivate the enzyme, collect the filtrate and spray dry it to obtain bovine collagen peptide.

7. The method according to claim 6, characterized in that: The bovine collagen peptide has at least one of the following properties: (1) The scavenging rate of bovine collagen peptide on DPPH free radicals was 10.53±0.47%; (2) The inhibition rate of elastase was 14.39±0.52%; (3) The inhibition rate of hyaluronidase was 47.02±2.49%; (4) The weight average molecular weight of bovine collagen peptide is 1870 g / mol.

8. The method according to claim 6, characterized in that: The collagenase crude extract is obtained by the following method: a certain amount of Brevibacillus laterosporus SCUT-BR-2 colonies are picked from a beef extract peptone culture medium plate, sterile water 1.5 times the mass of the colonies is added, shaken well, and centrifuged at 4°C and 8000rmp for 10 minutes. The obtained supernatant is the collagenase crude extract used in the present invention.

9. The method according to claim 6, characterized in that: The yield of bovine collagen peptide was 42.48%.

10. Use of the crude extract of collagenase produced by Brevibacillus laterosporus according to any one of claims 1 to 5 in extracting collagen from fish skin, pig skin, cow skin and donkey skin.

Images