A method for extracting collagen / collagen peptides from Bacillus laterosporus.

Abstract

This invention discloses a Bacillus laterosporus SCUT-BR-2 strain capable of producing collagenase and its application in the extraction of animal collagen / collagen peptides. Specifically, the method includes: screening collagenase-producing strains from meat products, and extracting collagen from animal skin using a crude extract of the strain's collagenase through morphological observation, physiological and biochemical tests, and determination; simultaneously, using this crude extract to decompose bovine bone collagen to obtain a bovine bone collagen peptide with good antioxidant properties and anti-wrinkle, firming, soothing, and moisturizing effects.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation culture and the use of microorganisms to hydrolyze collagen, specifically to a Bacillus laterosporus and a method for hydrolyzing collagen to obtain collagen peptides. Background Technology

[0002] *Bacillus laterosporus* is a rod-shaped, peritrichous-flagellated facultative anaerobic bacterium, commonly used as a probiotic in biocontrol. Its applications are extensive, including: in animal husbandry, it can be used as a microbial additive in animal feed; in agriculture, it can effectively improve soil fertility and promote crop growth as a microbial fertilizer; in fisheries, it can inhibit algal photosynthesis, making it difficult for algal cells to survive, and induce lipid peroxidation of algal cell membranes, leading to algal death, thus achieving water quality control and cyanobacterial control, which is beneficial for aquaculture; in the food industry, its antimicrobial peptides have broad-spectrum antibacterial activity, are heat-resistant, acid and alkali-resistant, and sensitive to proteolytic enzymes, thus applicable to food preservation.

[0003] Collagen is a triple-helix fibrous protein composed of three polypeptide chains. It belongs to the insoluble protein category of fibrous proteins and is the most abundant protein in many vertebrates and invertebrates. Collagen possesses excellent low-immunity, biocompatibility, and biodegradability, making it widely applicable in food, cosmetics, and medicine. Collagen peptides (also known as collagen polypeptides) are relatively small molecular weight products formed from the hydrolysis of collagen. Compared to collagen, collagen peptides have higher absorption and utilization rates, better effects, and can promote the absorption of other proteins in food. Collagen polypeptides are completely soluble in water (even cold water) and possess certain antihypertensive and angiotensin (ACE) inhibitory activities. They also improve skin and promote wound healing, have antibacterial and antioxidant activities, and promote bone synthesis. Therefore, collagen polypeptides play an irreplaceable role in the food (including health products), medical, and skincare industries, and the demand is increasing daily.

[0004] Currently, collagen extraction primarily utilizes animal skin, employing methods including alkaline extraction, acid extraction, hot water extraction, enzymatic extraction, and combined methods. Alkaline extraction alone, or a combination of enzyme and acid methods, easily damages protein structure, while alkaline extraction causes protein racemization. Hot water extraction leads to incomplete collagen breakdown and low collagen yield. Enzymatic extraction, compared to other methods, is gentler, more efficient, and preserves the product's activity better. Existing technologies, such as the patent application with authorization number CN113943770B, employ an acid + enzyme combination + salting-out method to extract fish skin collagen; this process is complex, uses commercially available enzymes, and is costly. In the patent application with publication number CN110922475A, collagen is extracted from pig skin using two or more combined enzymes, including "pepsin treatment at pH 2-3 and temperature 30-40℃; papain treatment at pH 6-7 and temperature 50-60℃; and trypsin treatment at pH 7-8 and temperature 40-50℃". In existing methods, commercially available enzymes are used for enzyme selection. 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 expensive and complicated.

[0005] There are currently two main methods for obtaining collagen peptides from collagen through further hydrolysis: synthesis and degradation. Some researchers have used synthesis to produce bovine bone collagen peptides, but this method requires determining the bovine bone collagen peptide sequence, which is a high barrier to entry. Degradation is relatively easier. Degradation commonly uses enzymatic hydrolysis, including pepsin, alkaline protease, neutral protease, trypsin, animal complex protease, flavor protease, and papain. Similarly, in the degradation of lower molecular weight collagen peptides, the current mainstream method directly uses commercially available enzymes. As mentioned earlier, these mature commercial enzymes are not only very expensive, but also require stringent degradation conditions (degradation needs to be carried out under optimal enzyme activity conditions to achieve the best results and avoid enzyme waste; therefore, there are many requirements regarding temperature, substrate concentration, etc.), and the large quantities used undoubtedly increase the production costs for enterprises.

[0006] Therefore, developing a strain that can naturally produce proteolytic enzymes to replace the aforementioned expensive and high-volume commercial enzymes, and directly combining a simple fermentation process with self-produced enzymes to extract collagen and / or obtain collagen peptides, would be crucial for large-scale production and widespread application of collagen / collagen peptides. This would reduce extraction costs, simplify the extraction process, lower equipment requirements, and minimize environmental pollution. It also represents a new method for achieving higher resource utilization of fermentation products, providing new insights and ideas for environmental protection and product processing in fermentation enterprises. Summary of the Invention

[0007] To address the practical technical problems encountered in the production processes of numerous enterprises, this invention develops a novel *Bacillus laterosporus* species, thereby providing a method for directly extracting collagen / obtaining collagen peptides using the fermentation products of this *Bacillus laterosporus*. The specific technical solution is as follows:

[0008] A species of Brevibacillus laterosporus, named Brevibacillus laterosporus SCUT-BR-2, was deposited on October 15, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 65271.

[0009] As a further limitation of the above scheme, the *Bacillus laterosporus* is obtained by isolating, culturing and purifying it from meat products.

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

[0011] As a further limitation of the above scheme, the formulation of culture medium A is as follows: 0.8% collagen powder, 0.1% beef extract, 0.1% glucose, 0.05% KH2PO4, 0.01% MgSO4·7H2O, 2% agar, and the pH of culture medium A is adjusted to 7.0.

[0012] As a further limitation of the above scheme, the steps for isolating, culturing, and purifying *Bacillus laterosporus* from meat products include: grinding the meat products, weighing 1g and adding it to an Erlenmeyer flask containing 49mL of sterile physiological saline, shaking thoroughly for 30min, allowing it to stand, taking the middle layer supernatant as the bacterial suspension, and performing serial dilutions. Using the plate dilution method, the serially diluted bacterial suspensions are spread onto LB agar solid medium and cultured in a constant temperature incubator at 30-40℃ until single colonies grow. Single colonies are selected and transferred to screening medium A plates, and incubated upside down at 30-40℃ for 1-2 days. Based on screening characteristics, single colonies with normal growth and significant differences in colony morphology are selected and repeatedly streaked on beef extract peptone medium for pure culture, numbered, and stored for later use.

[0013] This invention also applies for protection of a method for enzymatic hydrolysis of bovine bone collagen using the aforementioned *Brevibacillus laterosporus* SCUT-BR-2, comprising: preparing a 12.5% ​​bovine bone collagen solution, stirring and dissolving it at 50°C, adding 0.3% (based on bovine bone collagen protein content) of crude collagenase extract, reacting at 50°C for 8-24 hours, then adding 0.5%-3% activated charcoal powder (based on bovine bone collagen protein content) to the reaction solution for adsorption and deodorization for 1 hour, filtering once with double-ring qualitative rapid filter paper, and then filtering once with a 0.45μm microporous membrane to obtain a clear and transparent filtrate; heating the filtrate at 70°C for 5 minutes to inactivate the enzyme, collecting the filtrate and spray drying it to obtain bovine bone collagen peptides.

[0014] As a further limitation of the above scheme, the obtained bovine bone collagen peptides must satisfy at least one of the following properties:

[0015] (1) The scavenging rate of bovine bone collagen peptides against 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 bone collagen peptides 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 lateralis SCUT-BR-2 colonies are picked from beef extract peptone medium plates, 1.5 times the weight of the bacterial cells are added to sterile water, shaken well, and centrifuged at 8000 rpm for 10 min at 4℃. The resulting supernatant is the crude collagenase extract used in this invention.

[0020] As a further limitation of the above scheme, the yield of bovine bone collagen peptides was 42.48%.

[0021] This application also claims the use of the above-mentioned Brevibacillus laterosporus SCUT-BR-2 in the extraction of collagen from fish skin, pig skin, cow skin, and donkey skin.

[0022] Beneficial technical effects

[0023] The *Bacillus laterosporus* SCUT-BR-2 provided by this invention exhibits excellent efficacy in the extraction of collagen from animal skins such as fish skin. Furthermore, when using bovine bone collagen as a raw material, it significantly decomposes collagen into small molecule peptides. Specifically, the obtained bovine bone collagen peptides show a DPPH free radical scavenging rate of 10.53±0.47%, an elastase inhibition rate of 14.39±0.52%, and a hyaluronidase inhibition rate of 47.02±2.49%, indicating that bovine bone collagen peptides possess certain antioxidant, anti-wrinkle, firming, soothing, and moisturizing effects, making them well-suited for use in various skincare products and beauty serums. The enzyme extracted from Bacillus laterosporus SCUT-BR-2 provided by this invention is highly suitable for collagen extraction and processing under neutral conditions, avoiding the need for additional acid-base adjustments required by commercially available enzymes such as pepsin or alkaline protease, which can easily introduce inorganic ionic impurities. Furthermore, this invention directly uses the crude fermentation extract of Bacillus laterosporus SCUT-BR-2, i.e., the crude collagen extract used in this invention, for collagen extraction and processing of bovine bone collagen peptides, which not only… This invention omits steps such as enzyme purification and comprehensively utilizes other beneficial components in the crude extract besides the extracellular collagenase produced by *Bacillus laterosporus* SCUT-BR-2 itself, such as extracellular secretions produced by *Bacillus laterosporus* SCUT-BR-2 during fermentation. The presence of these components provides the collagenase in the crude extract with certain activity and nutrients, promoting enzyme activity and making the enzymatic hydrolysis more efficient. The promotion of extracellular enzyme activity by extracellular secretions is mainly reflected in: enhancing enzyme stability. Some extracellular secretions can provide enzymes with a relatively stable and familiar working environment, thereby improving enzyme stability and activity. Secondly, the extracellular secretions in the crude extract themselves provide a familiar substrate for maintaining enzyme activity, promoting the enzyme reaction rate. The applicant has discovered that the *Bacillus laterosporus* SCUT-BR-2 provided in this invention can improve the degradation efficiency of its extracellular enzymes on substrates by secreting specific polysaccharides or proteins. Furthermore, different components in the extracellular secretions of the bacteria in the crude extract can interact to enhance the catalytic effect of the enzyme. In this invention, small molecules secreted by Bacillus laterosporus SCUT-BR-2 can activate enzyme activity or increase the accessibility of its substrate, thereby accelerating the enzymatic hydrolysis reaction rate.From a cost perspective, the culture conditions for *Bacillus laterosporus* SCUT-BR-2 provided by this invention are simpler and easier to preserve than those for commercially available enzymes. Furthermore, it is lower in cost and easier to industrialize, demonstrating promising development and application prospects. The crude enzyme extract eliminates a series of cumbersome purification and separation steps, simplifying the entire process and comprehensively utilizing all components in the crude extract. This significantly reduces production costs, conserves resources, and minimizes environmental pollution from wastewater during production, as well as the associated processes and costs of wastewater treatment. Simultaneously, *Bacillus laterosporus* SCUT-BR-2 and its crude extract provided by this invention also have significant application potential in addressing environmental pollution and resource waste issues. By applying it to extract collagen from animal skin, animal skin can be comprehensively utilized to obtain high-value products, providing a sustainable development solution for biotechnology applications and promoting the green development of biotechnology. Attached Figure Description

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

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

[0026] Figure 3 : 16S rDNA sequence diagram 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 collagen (unground);

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

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

[0032] Figure 9 Infrared spectrum of collagen;

[0033] Figure 10 CD diagram of collagen;

[0034] Figure 11 : Bovine bone collagen peptides after spray drying. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below in conjunction with various specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials, unless otherwise specified, can be obtained through ordinary commercial channels.

[0036] This invention obtained a novel *Brevibacillus laterosporus* SCUT-BR-2 strain through culture medium cultivation and subsequent screening and isolation. This strain was deposited on October 15, 2024, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 65271. The *Brevibacillus laterosporus* strain was isolated and purified from meat products, specifically pork products.

[0037] The screening steps for *Brevibacillus laterosporus* SCUT-BR-2 used the following culture media: LB medium, medium A, and beef extract peptone medium. Medium A was formulated as follows: 0.8% collagen powder, 0.1% beef extract, 0.1% glucose, 0.05% KH₂PO₄, 0.01% MgSO₄·7H₂O, and 2% agar, with the pH adjusted to 7.0. This medium was obtained by adding the above-mentioned components in the specified proportions to 1 liter of sterile or purified water and adjusting the pH to 7.0.

[0038] The method for isolating, culturing, and purifying Bacillus laterosporus from meat products includes:

[0039] Ground pork products were weighed, and 1g was added to an Erlenmeyer flask containing 49mL of sterile physiological saline. The mixture was shaken thoroughly for 30 minutes, allowed to stand, and the supernatant was taken as the bacterial suspension and serially diluted. The serially diluted bacterial suspensions were spread onto LB agar solid medium using the plate dilution method and cultured in a constant temperature incubator at 30-40℃ until single colonies grew. Single colonies were selected and placed on screening medium plates and incubated upside down at 30-40℃ for 1-2 days. Based on screening characteristics, such as single colony size, surface structure, texture, gloss, and color, single colonies with normal growth and obvious differences in colony morphology were selected and repeatedly streaked on beef extract peptone medium for pure culture. After numbering, they were stored for later use.

[0040] The preferred temperature of the constant temperature incubator is 35°C, and the preferred temperature for the inverted culture is also 35°C. The inventors were pleasantly surprised to find that the culture results were even better under these preferred conditions.

[0041] Bacterial testing

[0042] (1) Morphological identification

[0043] The experimental methods were followed according to the "Handbook of Systematic Identification of Common Bacteria", and the colony morphology, staining characteristics and optical microscope morphological characteristics were recorded.

[0044] (2) Physiological and biochemical identification

[0045] Physiological and biochemical indicators were determined for each strain in accordance with the "Handbook for Systematic Identification of Common Bacteria". These indicators included catalase, VP reaction, methyl red reaction, and nitrate reduction test.

[0046] (3) 16S rDNA sequence analysis

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

[0048] Morphological identification of Bacillus lateralis

[0049] The colony morphology of strain SCUT-BR-2 is as follows Figure 1 As shown, on beef extract peptone agar, its colonies appear milky white, nearly round, with smooth, regular edges, a raised center, and are translucent. Colonies picked from the plate were Gram-stained, and the results are as follows... Figure 2 As shown, some are Gram-negative and some are Gram-positive, but the vast majority are Gram-negative. This is because the Gram staining reaction of Bacillus laterosporus is variable: Gram-positive in the logarithmic phase, and Gram-negative in the late stationary phase and the death phase.

[0050] Physiological and biochemical identification of Bacillus laterosporus

[0051] The physiological and biochemical characteristics of strain SCUT-BR-2 are shown in Table 1. Based on its morphological characteristics and in accordance with the "Manual of Systematic Identification of Common Bacteria" and "Berge's Manual of Systematic Identification of Bacteria", the strain can be preliminarily identified as Brevibacillus.

[0052] Table 1. Results of physiological and biochemical tests on strain SCUT-BR-2

[0053]

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

[0055] 16S rDNA sequence analysis of Bacillus laterosporus

[0056] Using the bacterial genome as a template, the PCR product of the isolated strain SCUT-BR-2, after PCR amplification, showed a fluorescent band upon detection by agarose gel electrophoresis. Further sequencing of the PCR amplification product revealed the 16S rDNA fragment sequence as follows: Figure 3 As shown. Based on this sequence, a phylogenetic tree was constructed using MEGA 11.0 software, and the results are as follows. Figure 4 As shown. 16S rDNA sequence analysis showed the highest homology with the known strain *Brevibacillus laterosporus* (GenBank accession number NR 112727.1), with a score of 2543, coverage of 99%, and homology of 99.71%. Based on the colony morphology, individual morphology, physiological and biochemical characteristics, and 16S rDNA identification results, strain SCUT-BR-2 was identified as *Brevibacillus laterosporus*. The preservation information for this strain, *Brevibacillus laterosporus* SCUT-BR-2, is as described above.

[0057] Collagen was extracted from animal skin using the crude collagenase extract produced by Bacillus laterosporus SCUT-BR-2 as proposed in this invention. The crude collagenase extract was obtained by: picking a certain amount of Bacillus laterosporus SCUT-BR-2 colonies from a beef extract peptone medium plate, adding 1.5 times the weight of the bacterial cells to sterile water, shaking well, and centrifuging at 8000 rpm for 10 min at 4°C. The resulting supernatant was the crude collagenase extract used in this invention.

[0058] The specific extraction examples are as follows:

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

[0060] (1) Preprocessing

[0061] Weigh dried fish skin, cut it into 3×3cm pieces, and soak it in 0.1mol NaOH solution (20 times the weight of the fish skin) for 6 hours to remove protein and color. After soaking in the alkali solution, wash the fish skin and soak it in 20% ethanol solution (20 times the dry weight of the fish skin) to remove pigments, fats, and other impurities. After soaking for 6 hours, wash it clean. Then add 15 times the weight of the fish skin in purified water, boil it, boil for 10 minutes, let it stand and cool, and adjust the pH to neutral (7.0).

[0062] (2) Enzyme extraction reaction

[0063] Add the crude collagenase extract produced by Bacillus laterosporus SCUT-BR-2 of this invention to the fish skin solution at a dosage of 1% of the dry weight of the fish skin, and place it on a shaker for 18-36 hours. Specifically, the time can be 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 hours, etc., but 24 hours is preferred. Experimental comparison has shown that this preferred time can best balance efficiency, reaction quality, and the effective amount of enzyme in the extract.

[0064] (3) Separation

[0065] The reaction solution was filtered through double-loop qualitative filter paper, then through a 0.45μm microporous membrane. The filtrate was then heated at 70℃ for 5 minutes to inactivate the enzyme. The liquid was then collected and frozen for storage.

[0066] (4) Freeze-drying preservation

[0067] The extracted fish skin collagen was freeze-dried, weighed, collected, and stored.

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

[0069] (1) Preprocessing

[0070] Fresh cowhide, pighide, and donkey hide are scraped to remove hair and subcutaneous fat, then cut into 1cm x 1cm cubes for later use. Weigh out the prepared hides and add 1.5 times their weight of 0.1mol NaOH solution to a shaker for soaking to remove fat. After 6 hours, drain the liquid, wash the hides, and then add 1.5 times their weight of 10% (w / w) NaCl solution to soak again to remove salt-soluble proteins. After soaking in the brine for 24 hours, wash with water to remove residual sodium chloride. Then add 1.5 times their weight of purified water, boil, and boil for 10 minutes. Let stand and cool, adjust the pH to neutral, and set aside for use. All hide weights mentioned above refer to the wet hide weight of the prepared hides, and the same applies below.

[0071] (2) Enzyme extraction reaction

[0072] Add the crude collagenase extract of Bacillus retroflexus SCUT-BR-2 (of the present invention) at a dosage of 1% of the skin weight to the animal skin solution, and place it 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 has shown that this preferred method can best balance efficiency, reaction quality, and the effective amount of enzyme in the extract.

[0073] (3) Separation

[0074] The reaction solution is mixed with 0.5-3% (by weight of the obtained wet skin) of activated carbon powder. After 3-5 hours, the mixture is centrifuged at 5500 rpm for 5 minutes. The supernatant is then filtered through rapid qualitative filter paper, followed by filtration through a 0.45 μm microporous membrane. The filtrate is then heated at 70°C for 5 minutes to inactivate the enzyme. The liquid is then collected and frozen for storage. The 0.5-3% activated carbon powder can preferably be 1%-2% to ensure more thorough and clean adsorption of color and odor. However, considering the ease of removal of the adsorbed activated carbon, the amount of activated carbon powder added is more preferably 1%.

[0075] (4) Freeze-drying preservation

[0076] The extracted cowhide, pigskin, and donkey skin collagen was freeze-dried, weighed, collected, and stored.

[0077] For a rough description of the morphology of the extracted samples, please refer to the appendix. Figure 5 , 6 As shown in Figures 7 and 8.

[0078] Tests on the collagen extraction properties or physicochemical parameters of each raw material in Examples 1 and 2

[0079] Relative molecular weight detection

[0080] The extracted collagen samples were mixed with water to prepare test solutions with a concentration of 1 mg / mL. The solutions were vortexed and allowed to stand for 24 hours to dissolve. The solutions were then filtered through a 0.22 μm aqueous syringe filter using a disposable sterile syringe. The chromatographic conditions for detection were: detector temperature 25℃, Agilent 1260 Infinity II gel permeation column, flow rate 1 mL / min, and injection volume 10 μL.

[0081] Infrared spectroscopy detection

[0082] The extracted collagen sample coarse powder was ground evenly with potassium bromide at a mass ratio of 1:100, then compressed into tablets. The secondary structure of the samples was determined using Fourier transform infrared spectroscopy (FTIR) with a Shimadzu IRTracer 100 instrument (Japan), at a range of 4000-400 cm⁻¹. -1 The spectrum was obtained within the range. The automatic signal was measured at 2 cm⁻¹. -1 A 32-frequency spectral scan was performed at a resolution of [resolution value missing].

[0083] Circular two-column chromatography detection

[0084] The lyophilized 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 190–240 nm using a cuvette with a path length of 0.1 cm⁻¹.

[0085] Test results

[0086] (1) Collagen yield

[0087] Collagen yield is calculated according to Formula 1.

[0088]

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

[0090] Table 2 Collagen yield from the skin of four animal species

[0091]

[0092] (2) Relative molecular weight of collagen

[0093] The relative molecular weights of various collagen varieties are shown in Table 3 below.

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

[0095]

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

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

[0098] Figure 9 These are the infrared spectra of four extracted collagen samples obtained by FTIR detection. All four samples exhibit characteristic absorption peaks of collagen, including amide A, amide I, amide II, and amide III bands. The amide A band is the strongest absorption band among amide bonds and is related to the stretching vibration of the NH bond; the free NH stretching vibration occurs in the 3300-3500 cm⁻¹ range. -1 Within the specified range, the amide A bands of dried fish skin collagen, bovine skin collagen, porcine skin collagen, and donkey skin collagen were located at 3329.6 cm⁻¹. -1 3316cm -1 3338.8cm -1 and 3302.3cm -1The amide B band, located in the 1680-1780 cm⁻¹ range, is primarily generated by the combined C=O stretching and NH bending vibrations, representing the asymmetric extension of the CH₂ functional group, a characteristic group of tertiary structure. The amide I band originates from C=O stretching vibrations, NH bending vibrations, CN stretching, and C-CN deformation, and is typically used to reflect the secondary structure of proteins. Previous studies have shown that the following secondary structure regions in amide I are located at: β-turn, 1640–1660 cm⁻¹. -1 α-helix, 1650–1660 cm -1 Irregular curl, 1640–1650 cm -1 ; and β-fold, 1600–1640 cm -1 The amide I bands of dried fish skin collagen, bovine skin collagen, porcine skin collagen, and donkey skin collagen were located at 1660.6 cm⁻¹. -1 1658.3cm -1 1658.3cm -1 and 1660.6cm -1 Amide II band (1510–1580 cm) -1 The stretching vibrations of α-hydroxyl groups correspond to the stretching vibrations of CH and NH bonds. The amide II bands of dried fish skin collagen, bovine skin collagen, porcine skin collagen, and donkey skin collagen are located at 1544.3 cm⁻¹. -1 1551.1cm -1 1548.9cm -1 and 1544.3cm -1 The amide III band is located at 1200–1335 cm⁻¹. -1 The absorption, which reflects the vibration peak of the CH2 group in the glycine backbone and proline side chain, is related to the integrity of the collagen triple helix structure.

[0099] (4) Results of circular dichroism spectroscopy for collagen detection

[0100] Figure 10 The CD spectrum of collagen extracted from the skins of four different animals is shown. Figure 10 It is known that collagen has characteristic absorption peaks at 198 and 225 nm. The negative absorption peak at 198 nm is a typical feature of the random coil structure in the collagen molecule conformation, while the positive absorption peak at 225 nm is a typical feature of the L-polyproline (P-II) peptide chain conformation. The characteristic peak of collagen at 225 nm can reflect the triple helix level of collagen. The small positive absorption peak at 225 nm of these four collagen proteins indicates that there are relatively few higher-order triple helix structures within collagen.

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

[0102] Table 4. Results of secondary structure analysis of collagen

[0103]

[0104] Therefore, collagen was extracted from donkey skin, pig skin, cow skin, and fish skin using the crude extract of collagenase produced by Bacillus laterosporus SCUT-BR-2 isolated in this application. Infrared spectroscopy analysis revealed that the extracts all exhibited amide-specific absorption peaks. Circular dichroism spectroscopy showed fewer high-order triple helical structures but more irregular coil structures (a typical feature in collagen molecule conformation), all of which prove that the extracts are collagen. This also demonstrates that it is feasible to directly use the crude extract of collagenase produced by Bacillus laterosporus SCUT-BR-2 to extract collagen from animal skin, providing a feasible low-cost alternative to currently expensive and complex commercial enzyme products for collagen extraction.

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

[0106] (1) Preparation of bovine bone collagen peptides

[0107] Prepare a 12.5% ​​bovine bone collagen solution (dissolved by stirring at 50℃), add 0.3% (based on bovine bone collagen protein content) of crude collagenase extract, and react at 50℃ for 8-24 hours, specifically 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, but preferably 12 hours, as this optimal time results in the best degradation rate and overall experimental efficiency of bovine bone collagen. Then add 0.5%-3% activated charcoal powder (based on bovine bone collagen protein content) to the reaction solution for adsorption and deodorization for 1 hour. Filter once with double-ring qualitative rapid filter paper and once with a 0.45μm microporous membrane to obtain a clear and transparent filtrate. Heat the filtrate at 70℃ for 5 minutes to inactivate the enzyme, collect the liquid, and spray dry. The resulting dried solid is shown in the attached figure. Figure 11 As shown. The 0.5-3% activated carbon powder here can preferably be 1%-2% to ensure more thorough and clean adsorption of color and odor. However, considering the ease of removal of activated carbon after adsorption, the amount of activated carbon powder added is more preferably 1%.

[0108] The specific injection rate for spray drying is 40-110 L / h, and the inlet air temperature is 120-240℃. Preferably, the injection rate is 60-90 L / h, and the inlet air temperature is 150-200℃. For example, the injection rate can be 60, 65, 70, 75, 80, 85, or 90 L / h, and the inlet air temperature can be 150, 160, 170, 180, 190, or 200℃. Under the optimal injection rate of 70 L / h and inlet air temperature of 180℃, the spray drying effect is the best, characterized by high spray drying efficiency, minimal particle adhesion to the inner wall of the equipment, high yield, optimal process control, and better particle uniformity of the bovine bone collagen peptides with specific properties and molecular weight obtained by the enzymatic hydrolysis reaction of this application.

[0109] Relative molecular weight determination of bovine bone collagen peptides

[0110] Bovine bone collagen peptide samples were mixed with mobile phase solutions to prepare test solutions with a concentration of 1 mg / mL. The solutions were shaken using a vortex mixer, allowed to stand for 24 hours to dissolve, and then filtered through a 0.22 μm aqueous syringe for later use.

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

[0112] Activity assay of bovine bone collagen peptides

[0113] DPPH free radical scavenging rate

[0114] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption around 517 nm. In the presence of a free radical scavenger, the light absorption of the DPPH ethanol solution decreases due to single electron pairing. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of a test sample to scavenge free radicals.

[0115] Weigh a quantitative sample and dilute it with water to prepare multiple concentrations. Mix thoroughly by shaking / vacuuming before reaction and allow to stand. Dissolve the DPPH reagent in 95% ethanol to prepare a 0.12 mg / mL working solution, and prepare it in the dark. For Trolox, prepare a 0.08 mg / mL stock solution using 95% ethanol, and dilute it 1 / 2 times to create a series of concentration gradients to validate the experimental system. The concentration settings should include 0.01, 0.02, 0.04, and 0.08 mg / mL as positive controls. Refer to the sample addition grouping list and conduct the experiment using a 96-well microplate. Establish a sample group (T), a sample background group (T0), a negative control group (C), and a solvent background group (C0), with the positive control group treated the same as the sample group. Prepare three replicates for each group. Add reagents sequentially using a multi-channel pipette to ensure consistent reaction times.

[0116] Table 5. List of sample addition groups for antioxidant test

[0117]

[0118] After reacting in the dark for 5 minutes, immediately use a microplate reader for detection. The parameters are set as follows: slow shaking for 10 seconds, absorbance measured 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 crucial role in maintaining youthful skin, and its loss is a major cause of skin aging. Inhibiting elastase activity can effectively slow down the breakdown of elastin and maintain skin elasticity. Elastase catalyzes the hydrolysis of hydroxyl groups in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, a colored substance is produced, the absorbance of which can be measured at 410 nm using a spectrophotometer. Based on the change in absorbance, the inhibitory effect of the test substance on elastase activity can be calculated, and the firming and anti-wrinkle effects of the test substance can be evaluated.

[0123] Referring to the sample addition grouping list, experiments were conducted using 96-well microplates, establishing a sample group (T), a sample background group (T0), an enzyme reaction group (C), and a solvent background group (C0). The positive control group was treated the same as the sample group. Each group had three replicates.

[0124] Table 6. List of Liquid Addition Groups for Firming and Anti-wrinkle Test Samples

[0125]

[0126] Immediately use an ELISA reader for detection, with the parameters set as follows: slow oscillation for 10 seconds, absorbance measured at OD410nm. Export the file after the instrument completes the reading.

[0127] The elastase activity inhibition rate is calculated using the following formula:

[0128]

[0129] Hyaluronic acid inhibition

[0130] Hyaluronic acid, as an important natural moisturizing substance, plays a vital role in maintaining the volume of the extracellular matrix and normal skin physiological functions. However, hyaluronic acid can be specifically decomposed by hyaluronidase, and its products promote skin inflammation, leading to increased histamine production in the body. Furthermore, hyaluronidase expression is also associated with most IgE-mediated type I and T-cell-mediated type IV hypersensitivity reactions. Inhibiting hyaluronidase activity can, to some extent, help suppress the development of skin inflammation and allergic reactions, and improve the irritated state of the skin caused by inflammation and allergic reactions. Therefore, inhibiting hyaluronidase activity is often used as an important indicator for evaluating the soothing and moisturizing effects of products. 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, the absorbance can be measured at 540 nm to calculate the hyaluronidase activity.

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

[0132] Refer to the sample addition grouping list and conduct the experiment using 2mL centrifuge tubes. Each group should have 3 replicates.

[0133] Table 7. List of Samples for Soothing and Moisturizing Tests (Grouped by Solution)

[0134]

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

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

[0137]

[0138] Yield of bovine bone collagen peptides

[0139] Bovine bone collagen peptides obtained after spray drying, such as Figure 11 As shown. From the yield formula, the yield of bovine bone collagen peptides is...

[0140]

[0141] Relative molecular weight determination results of bovine bone collagen peptides

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

[0143] Table 8. GPC Detection Results of Bovine Bone Collagen Peptides

[0144]

[0145] Activity assay results of bovine bone collagen peptides

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

[0147] Table 9. Results of bovine bone collagen peptide activity assay

[0148]

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

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

[0151] The above tests demonstrate that the *Bacillus laterosporus* SCUT-BR-2 provided by this invention significantly decomposes bovine bone collagen into small molecule peptides. Furthermore, the obtained bovine bone collagen peptides exhibited a DPPH free radical scavenging rate of 10.53±0.47%, an elastase inhibition rate of 14.39±0.52%, and a hyaluronidase inhibition rate of 47.02±2.49%, indicating that the bovine bone collagen peptides possess certain antioxidant, anti-wrinkle, firming, soothing, and moisturizing effects. Therefore, using these bovine bone collagen peptides in skincare products, such as creams, lotions, serums, toners, and masks, can effectively combat free radicals and provide hydration and soothing effects, which is entirely feasible.

[0152] The embodiments described above only illustrate some implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A type of Bacillus retroflexus, characterized in that: The specimen, named *Brevibacillus laterosporus* SCUT-BR-2, was deposited on October 15, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 65271. This *Brevibacillus laterosporus* was isolated, cultured, and purified from meat products. The culture medium used for the *Bacillus laterosporus* includes: LB medium, medium A, and beef extract peptone medium; the formula for medium A is: 0.8% collagen powder, 0.1% beef extract, 0.1% glucose, 0.05% KH2PO4, and MgSO4·7H2O. 0.01%, 2% agar, adjust the pH of medium A to 7.0; the steps for isolating, culturing and purifying *Bacillus laterosporus* from meat products include: grinding the meat products, weighing 1g and adding it to a 49mL sterile physiological saline in an Erlenmeyer flask, shaking thoroughly for 30min and letting it stand, taking the middle supernatant as the bacterial suspension and performing serial dilutions; using the plate dilution separation method, spreading the serially diluted bacterial suspensions onto LB agar solid medium, culturing in a constant temperature incubator at 30-40℃ until single colonies grow, selecting single colonies and picking them onto screening medium A plates, and incubating them upside down at 30-40℃ for 1-2 days; according to the screening characteristics, selecting single colonies that grow normally and have obvious differences in colony morphology, repeatedly streaking them on beef extract peptone medium for pure culture, numbering them and storing them for later use.

2. A method for enzymatically hydrolyzing bovine collagen using *Bacillus laterosporus* as described in claim 1, characterized in that, include: Prepare a 12.5% ​​bovine bone collagen protein solution and stir to dissolve it at 50℃. Add 0.3% crude collagenase extract (calculated based on bovine bone collagen protein content) and react at 50℃ for 8-24 hours. Then add 0.5%-3% activated carbon powder (calculated based on bovine bone collagen protein content) to the reaction solution for adsorption and deodorization for 1 hour. Filter once with double-ring qualitative rapid filter paper and then once with a 0.45μm microporous membrane to obtain a clear and transparent filtrate. Heat the filtrate at 70℃ for 5 minutes to inactivate the enzyme. Collect the filtrate and spray dry it to obtain bovine bone collagen peptides.

3. The method according to claim 2, characterized in that: The bovine bone collagen peptides described herein possess at least one of the following properties: (1) The scavenging rate of bovine bone collagen peptides against 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 bone collagen peptide is 1870 g / mol.

4. The method according to claim 3, characterized in that: The crude collagenase extract was obtained by the following method: a certain amount of Bacillus lateralis SCUT-BR-2 colonies were picked from beef extract peptone agar plates, 1.5 times the weight of the bacterial cells were added to sterile water, shaken well, and centrifuged at 4℃ and 8000 rpm for 10 min. The resulting supernatant was the crude collagenase extract used.

5. The method according to claim 4, characterized in that: The yield of bovine bone collagen peptides obtained was 42.48%.

6. The application of bovine bone collagen peptides prepared according to any one of claims 2-5 in skin care products.

7. The application of a crude extract of collagenase produced by Bacillus retroflexus as described in any one of claims 1-4 in the extraction of collagen from fish skin, pig skin, cow skin, and donkey skin.

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