Process for extracting giant salamander protein peptide based on solid state fermentation

Abstract

The invention discloses a process for extracting giant salamander protein peptide based on solid state fermentation. The process comprises the following steps: pretreating fresh giant salamander meat; and carrying out solid state fermentation on the pretreated giant salamander meat. The invention further provides application of the bacillus licheniformis to extraction of the giant salamander protein peptide. The process disclosed by the invention has the advantages of energy conservation, low cost, high extraction rate, high safety, stable product quality, strong controllability, simple and easy-to-operate equipment, environmental friendliness and the like.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically designs a process for extracting giant salamander protein peptides based on solid-state fermentation. Background Art

[0002] The giant salamander is the world's largest amphibian and possesses unique biological characteristics. It belongs to the family Cryptobranchidae and is primarily found in Asia. It includes two species: the Chinese giant salamander and the Japanese giant salamander. The Chinese giant salamander is often called the "baby fish" because of its cry, which resembles a baby's cry.

[0003] Giant salamanders have a strong regenerative ability and can regenerate short limbs after amputation. The regeneration process can be subdivided into the following stages: wound healing, blastema formation, and final cell differentiation to replace all lost cell types. During the wound healing stage, cells migrate from the distal epithelial cells (commonly called the wound epithelium) to close the wound. During this process, these cells regulate gene expression and may begin to secrete specific proteins to activate surrounding cells to form blastema. Earlier studies have shown that covering uninjured skin on the wound epithelium inhibits the regeneration process, which further emphasizes the importance of changes in gene expression in response to injury in scarless regeneration.

[0004] Extracting relevant peptides from giant salamander tissue and developing their tissue regeneration function has enormous research value. When it comes to the preparation of giant salamander collagen peptides, scientists are more concerned about how to obtain collagen peptides with smaller molecular weight and easier to be absorbed by the body. For example, patent application CN105018555A discloses a method for preparing giant salamander skin collagen peptides. This method uses giant salamander skin as raw material and undergoes a series of processing steps. The final giant salamander skin collagen peptide powder obtained has a molecular weight of less than 1000 Daltons. However, the specific functional activity of the collagen peptides prepared by this method is still unclear.

[0005] There are currently three main methods for extracting protein peptides from meat: acid extraction, alkaline extraction, and enzymatic extraction. Acid extraction mainly uses low ion concentration acidic conditions to immerse the raw materials, thereby destroying the salt bonds and Schiff bases between molecules, causing fiber expansion and dissolution. Acid extraction has the following disadvantages: (1) Amino acid destruction: Acid hydrolysis may cause the destruction of some amino acids, especially tryptophan (Trp), which will be completely destroyed during the hydrolysis process; (2) Racemization: Acid hydrolysis may cause racemization during protein hydrolysis, destroying the three-dimensional structure of the protein; (3) Corrosiveness: The acid used in acid hydrolysis is corrosive and has high requirements for processing equipment, requiring the use of corrosion-resistant equipment; (4) Reaction condition control: The conditions of the acid hydrolysis reaction need to be strictly controlled, otherwise it may affect the extraction effect; (5) Purity problem: Acid hydrolysis may cause some impurities in the protein hydrolyzate, such as ash and salt, which affect the purity of the protein; (6) It is easy to cause environmental pollution.

[0006] Although alkaline extraction can easily cause hydrolysis of peptide bonds and the resulting hydrolysis products have relatively low molecular weights, its disadvantages are that it causes severe environmental pollution, has violent reactions, can easily destroy the nutritional components of the product, and contains impurities.

[0007] Enzymatic extraction is the most widely used extraction method on the market. It uses proteases to dissolve collagen. The hydrolysis conditions are mild and the reaction speed is fast. The extracted collagen still has a complete triple helix structure, high protein purity, stable physical and chemical properties, and no environmental pollution. However, enzymatic hydrolysis also has many disadvantages: (1) Extraction efficiency needs to be improved: Although enzymatic hydrolysis usually has a high extraction efficiency, it may sometimes take a long time to achieve the desired extraction effect; (2) Protein denaturation: Some enzymes may denature proteins, thereby affecting their functions and properties; (3) Impurity removal: During the extraction process, some impurities such as pigments and polysaccharides may be produced, which may be difficult to remove or require special treatment methods; (4) High cost: The enzymes required for enzymatic hydrolysis are usually expensive, which may increase the extraction cost; (5) Substrate specificity: The specificity of the enzyme for the substrate may limit its scope of application, because not all proteins can be effectively extracted by enzymatic hydrolysis; (6) Reaction condition control: The conditions of the enzymatic hydrolysis reaction need to be strictly controlled, otherwise it may affect the extraction effect; (7) Activity loss: In some cases, the enzymatic hydrolysis process may cause the loss of protein activity, which may limit its use in certain applications; (8) Safety issues: For some applications, such as food and medicine, the safety of the enzymes used needs to be considered. For protein peptides extracted by enzymatic hydrolysis, trypsin, pepsin, and papain are mainly used. Pepsin is the most widely used of these enzymes because it can specifically remove the telopeptides located in the non-helical region of collagen without affecting the helical region. Therefore, the protein peptides can still maintain a relatively complete triple helical structure after being extracted. In general, although acid hydrolysis and enzymatic hydrolysis have many advantages in protein extraction, their disadvantages and limitations still need to be comprehensively considered to determine whether they are suitable for specific applications.

[0008] Although there are many different extraction methods, scientists are still exploring more environmentally friendly and efficient extraction methods. Summary of the Invention

[0009] The purpose of the present invention is to provide a giant salamander protein peptide extraction process with simple operation, high extraction efficiency and stable product quality in response to the above technical problems to be solved.

[0010] In order to achieve the above object of the invention, the present invention provides a process for extracting giant salamander protein peptides based on solid-state fermentation, which comprises the following steps:

[0011] Step 1. Pre-processing fresh giant salamander meat;

[0012] Step 2. Perform solid-state fermentation on the pretreated giant salamander meat.

[0013] Preferably, in step 1, the pretreatment method is: removing the skin and fat tissue of fresh giant salamander meat and then mincing it into minced meat.

[0014] Preferably, in step 2, Bacillus licheniformis seed liquid is added to the giant salamander meat for solid-state fermentation.

[0015] More preferably, the ratio of giant salamander meat to Bacillus licheniformis seed solution is 1 gram of giant salamander meat mixed with 10 milliliters of Bacillus licheniformis seed solution.

[0016] On the other hand, the present invention also provides the use of Bacillus licheniformis in extracting protein peptides from giant salamanders.

[0017] Compared with the liquid fermentation treatment mostly carried out by microorganisms such as Bacillus subtilis and Pichia pastoris in domestic research, the present invention adopts the cheaper Bacillus licheniformis for solid-state fermentation, which can further reduce the cost of protein peptide preparation.

[0018] The advantages of solid-state fermentation for extracting protein peptides mainly include:

[0019] (1) Energy saving: Solid-state fermentation does not require the use of large amounts of water, nor does it require the evaporation of water, thus effectively saving energy.

[0020] (2) High extraction rate: The extraction rate of solid-state fermentation is higher because no additional nutrients need to be added during the fermentation process and the fermentation time is also shorter.

[0021] (3) Low cost: Solid-state fermentation is cheaper than enzymatic hydrolysis in extracting protein peptides. When using solid-state fermentation, it is only necessary to ensure a constant and suitable temperature, a sealed environment, and an appropriate stirring speed to carry out the fermentation step. However, the enzymatic hydrolysis method requires the purchase of expensive proteases, and the temperature and pH value of the reaction system need to be adjusted multiple times during the implementation process to provide a survival environment for the protease, which increases the complexity of the operation and the production cost. The equipment and process required for solid-state fermentation are relatively simple, so the production cost is relatively low.

[0022] (4) High safety: The solid-state fermentation process does not require the use of large amounts of chemicals, so the protein peptides produced are relatively safe.

[0023] (5) Wide range of applications: Solid-state fermentation is applicable to a variety of substrates and microorganisms, and can therefore be used to produce a variety of different protein peptides.

[0024] (6) Stable product quality: During the solid-state fermentation process, since there is no churning and stirring in the liquid fermentation process, the quality of the product is relatively stable and is not easily affected by the external environment.

[0025] (7) Strong controllability: During solid-state fermentation, the fermentation process can be controlled by controlling temperature, humidity, oxygen and other conditions, thereby obtaining a product that better meets the requirements.

[0026] (8) The equipment is simple and easy to operate: The equipment required for solid-state fermentation is relatively simple, easy to operate, and has low environmental requirements.

[0027] (9) Safety: Compared with acid-base extraction and enzymatic hydrolysis, solid-state fermentation does not involve any chemical reagents that are harmful to the human body. Therefore, this method is safer when handling samples, has no safety risks, and is more environmentally friendly.

[0028] In summary, solid-state fermentation has many advantages in extracting giant salamander protein peptides, including energy conservation, high extraction rate, low cost, high safety, wide application range, stable product quality, strong controllability, simple and easy equipment operation, etc. These advantages make solid-state fermentation-based extraction of giant salamander protein peptides an efficient, environmentally friendly and economical method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a growth curve of Bacillus licheniformis.

[0030] Figure 2 This is the growth curve of Bacillus subtilis.

[0031] Figure 3 This is a standard curve graph of hydroxyproline. DETAILED DESCRIPTION

[0032] The following is a further detailed description of the technical solution of the present invention in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] 1. Activation and expansion of bacterial strains

[0034] Prepare 1200-1500 mL of culture medium (formula: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, pH 7.2-7.4) and autoclave all necessary glassware. This sterilized medium will be used to activate the deposited strains Bacillus licheniformis (China Industrial Microbiological Culture Collection, Accession No. CICC 10092) and Bacillus subtilis (Shanghai Yubo Biotechnology Co., Ltd., Catalog No. YB00590). 20 mL of each culture will be used for subculture.

[0035] Take the third generation of bacteria for fermentation: add the activated bacteria into 10 250mL conical flasks for subculture (according to the subculture system of 2mL bacterial solution + 100mL culture medium), and take out the seed liquid according to the logarithmic phase time period for fermentation experiment. The logarithmic phase of Bacillus licheniformis is 18-24 hours, and the logarithmic phase of Bacillus subtilis is 10-14 hours. In addition, set up two control groups without meat samples and culture them on a shaking table at the same time. The culture conditions are: temperature 36.9℃~37℃, carbon dioxide concentration 400-450PPM, incubator speed 180r / min.

[0036] Figure 1 A graph of the growth curve of Bacillus licheniformis is shown. Figure 2 A graph of the Bacillus subtilis growth curve is shown.

[0037] Bacillus subtilis and Bacillus licheniformis are aerobic bacteria, with an optimal growth temperature of 37°C. Therefore, to ensure proper fermentation, the fermenter must be maintained at a constant temperature, with sterile air flow and agitation. The entire fermentation process is cost-effective and easy to implement, thanks to the low cost of the bacteria and related equipment, low energy consumption, and simple operation.

[0038] To better control contaminants, a method of continuous expansion of the bacterial strain was employed in advance. This ensured that a dominant bacterial strain was already present in the fermentation broth before the sample was added. Once the sample was added to the fermentation broth, interspecies competition suppressed the physiological activity of most contaminants. Therefore, from a macroscopic perspective, the solid-state fermentation method achieved a more impressive contaminant control rate than the enzymatic hydrolysis method used in the open experiment.

[0039] To determine the optimal cell age, we observed and studied the growth cycle of the Bacillus licheniformis strain used in the experiment. After multiple experiments, we found that solid-state fermentation achieved the best results when using a bacterial seed generation that had been cultured for at least the third generation and using a culture solution that had been incubated for 18-24 hours as the seed solution. This cell age is selected based on the growth status and vitality of the strain. By controlling the growth stage and incubation time of the strain, the solid-state fermentation process can be optimized and the extraction efficiency of protein peptides can be improved.

[0040] 2. Pretreatment of Giant Salamander Meat Samples

[0041] After slaughtering the live artificially bred giant salamander, the internal organs and skin were removed, and most of the fat tissue was scraped off. The remaining muscles were put into a meat grinder and ground into minced meat. The fresh minced meat samples were then placed in the refrigerator for later use.

[0042] 3. Solid-state fermentation of giant salamander minced meat samples

[0043] 10 g of minced giant salamander meat was added to the seed liquid (100 mL) of each growth system in the logarithmic growth phase for mixing and fermentation. The fermentation conditions were the same as those of the control group (temperature: 36.9°C~37°C), and the culture was shaken for 12 hours, 24 hours, 36 hours, 48 ​​hours, and 60 hours at a speed of 180 r / min.

[0044] During solid-state fermentation, fermentation time is a key factor influencing extraction efficiency. Experimental studies have shown that the optimal collagen peptide extraction rate occurs when meat samples are fermented in seed liquid for 36-48 hours. During this timeframe, solid-state fermentation is fully underway, effectively breaking down and converting the collagen in the meat sample, thereby increasing the collagen peptide extraction rate.

[0045] To simulate the fermentation process of industrial production, the experiment employed a shaking culture method for solid-state fermentation. By adjusting the incubator's rotational speed, the agitation environment of the fermentation system could be controlled. Experimental comparisons revealed that the optimal extraction rate for fermentation samples was achieved when the incubator's rotational speed reached 180 r / min. At this speed, oxygen supply and material exchange during solid-state fermentation were effectively guaranteed, thereby promoting microbial growth and metabolism and increasing the yield and quality of protein peptides.

[0046] 4. Determination of results

[0047] After the fermentation is completed, 0.2 mL of sample is taken from each conical flask and diluted 10 times for sample determination. The determination method is as follows:

[0048] (1) Use a pipette to transfer a certain volume (V) of sample into a 250 mL volumetric flask. After the volume is fixed, the concentration of hydroxyproline is between 0.5 μg / mL and 2 μg / mL.

[0049] (2) Pipette 4.00 mL of the above solution into a colorimetric tube, add 2.00 mL of chloramine T reagent, mix, and let stand at room temperature for 20 minutes.

[0050] (3) Add 2.00 mL of color developer (10.0 g of p-dimethylaminobenzaldehyde was weighed and dissolved in 35 mL of perchloric acid solution [60% (mass fraction)], and then slowly added 65 mL of isopropyl alcohol. Prepare immediately before use. The specific contents are all based on the national standard document GB / T9695.23-2008 Determination of Hydroxyproline Content in Meat and Meat Products) and mix thoroughly in a colorimetric tube. Seal the colorimetric tube with aluminum or plastic film.

[0051] (4) Place the colorimetric tube quickly in a 60°C water bath and heat for 20 minutes.

[0052] (5) Take out the colorimetric tube, cool it with running water for at least 3 minutes, and place it at room temperature for 30 minutes.

[0053] (6) Using water as a reference, measure the absorbance at 558 nm using a spectrophotometer or photoelectric colorimeter.

[0054] (7) Subtract the absorption of the blank solution and obtain the acid content in the hydrolysis product from the hydroxyproline standard curve.

[0055] (8) Blank test: Replace the diluted solution with distilled water and repeat the steps (1) to (7).

[0056] 5. Drawing of standard curve

[0057] Take 10 mg of L-hydroxyproline standard, dissolve it in 0.1 mol / L hydrochloric acid solution, dilute to 100 mL with distilled water as the standard stock solution, and store at 4°C.

[0058] Take 0.5, 1, 1.5, 2, 2.5, and 3 mL of hydroxyproline stock solution respectively in a 100 mL volumetric flask and dilute to 100 mL as the working solution.

[0059] Pipette 2 mL each of hydroxyproline working solution and distilled water (blank group) into a test tube, add 1 mL of chloramine T oxidant to each test tube, shake well and let it stand for 20 minutes, then add 1 mL of p-dimethylbenzaldehyde colorant, stopper and shake well, heat in a 60℃ water bath for 20 minutes, cool to room temperature with running water, and measure the absorbance at 560 nm.

[0060] The absorbance results are as follows:

[0061] Table 1: Solid-state fermentation absorbance results of giant salamander muscle tissue (unit: A)

[0062] Fermentation time 12 hours 24 hours 36 hours 48 hours 60 hours Control group 1 0.015 0 0 0.005 0.013 Control group 2 0.009 0.027 0.026 0.026 0.028 Sample 1 0.094 0.143 0.694 0.555 0.472 Sample 2 0.089 0.115 0.7 0.553 0.5

[0063] In Table 1, sample 1 was fermented by Bacillus licheniformis, sample 2 was fermented by Bacillus subtilis, the data of control group 1 without adding meat sample were the data of Bacillus licheniformis group, and the data of control group 2 without adding meat sample were the data of Bacillus subtilis group.

[0064] The standard curve of hydroxyproline was drawn with the mass concentration of hydroxyproline as the horizontal axis and the absorbance value of the working solution after deducting the blank as the vertical axis. Figure 3 shown.

[0065] 5. Calculation of extraction rate

[0066] According to the relationship between absorbance (A) and hydroxyproline concentration (c): A = 0.094c + 0.009, the hydroxyproline concentration in the diluted sample is obtained as: c = (A-0.009) / 0.094.

[0067] After obtaining the hydroxyproline concentration c of the diluted sample, convert μg to g and multiply the dilution factor by 10. The hydroxyproline content in the 100mL system in each conical flask is: m = c*10*100*10 -6 .

[0068] During the experiment, every 100 mL of seed liquid contained 10 g of fish meat. According to the reference document (national standard document [1] GB / T9695.23-2008), every 100 g of fish meat contains 0.3489 g of hydroxyproline. Theoretically, 10 g of fish meat in 100 mL of seed liquid contains M = 0.0348 g of hydroxyproline, and the extraction rate n = 100%*(m / M) is obtained.

[0069] Table 2: Hydroxyproline extraction rate from solid-state fermentation of giant salamander muscle tissue

[0070] Fermentation time 12 hours 24 hours 36 hours 48 hours 60 hours Control group 1 0.18341％ -0.27512％ -0.27512％ -0.12227％ 0.12227％ Control group 2 0％ 0.55025％ 0.51968％ 0.51968％ 0.58082％ Sample 1 2.59836％ 4.09635％ 20.94032％ 16.69112％ 14.15383％ Sample 2 2.44552％ 3.24040％ 21.12375％ 16.62998％ 15.00978％

[0071] In Table 2, sample 1 was fermented by Bacillus licheniformis, sample 2 was fermented by Bacillus subtilis, the data of control group 1 without adding meat sample were the data of Bacillus licheniformis group, and the data of control group 2 without adding meat sample were the data of Bacillus subtilis group.

[0072] As shown in Table 2, compared with the control group, the hydroxyproline extraction rates of Samples 1 and 2 were significantly improved, ranging from 14% to 21% within 36 to 60 hours of fermentation. This method is low-cost, has good extraction effects, and has high feasibility and practical application value.

[0073] Compared to Bacillus subtilis, Bacillus licheniformis offers similar extraction results, lower fermentation costs, greater stress resistance, and improved ecological adaptability. It has a strong tolerance to environmental stresses such as drought and high temperature, adapts to specific ecosystems, and can grow and function better in specific environments. This is the primary advantage of Bacillus licheniformis fermentation. Furthermore, the growth and fermentation conditions for Bacillus subtilis are complex, requiring the presence of aerobic conditions and the absence of other bacteria as much as possible. Consequently, production costs are higher, including equipment and consumables, which limits its widespread application. In comparison, fermentation of Bacillus licheniformis is simpler and production is more efficient.

Claims

1. A process for extracting giant salamander protein peptides based on solid-state fermentation, comprising the following steps: Step 1. Pre-processing fresh giant salamander meat; Step 2. Perform solid-state fermentation on the pretreated giant salamander meat.

2. The process according to claim 1, characterized in that: In step 1, the pretreatment method is: removing the skin and fat tissue of fresh giant salamander meat and then mincing it into minced meat.

3. The process according to claim 1 or 2, characterized in that: In the step 2, Bacillus licheniformis seed liquid is added to the giant salamander meat for solid-state fermentation.

4. The process according to claim 3, characterized in that: The ratio of giant salamander meat to Bacillus licheniformis seed liquid is 1 gram of giant salamander meat mixed with 10 milliliters of Bacillus licheniformis seed liquid.

5. Application of Bacillus licheniformis in extracting giant salamander protein peptides.

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