Method for preparing camel milk polypeptide through response surface method microbial fermentation and application

Abstract

The invention relates to a method for preparing camel milk polypeptide through response surface method microbial fermentation and application, the method uses camel milk industrial sweeping tower powder as an exploration object, uses bacterial species, bacterial density, fermentation temperature and material-liquid ratio as independent variables, uses camel milk polypeptide yield as a response value, and utilizes response surface Box-Behnken experimental design to optimize the preparation method of camel milk polypeptide. By taking the yield of the camel milk polypeptide as a response value, the content of the camel milk polypeptide is measured to be 39.96%, and the content of the camel milk polypeptide is remarkably improved compared with the content of 5.8% before fermentation. Meanwhile, the recovery rate is 52.3%. The antioxidant activity of the camel milk polypeptide is evaluated, the antioxidant activity result shows that the camel milk polypeptide has OH and DPPH free radical scavenging capacity, and the IC50 value is 1.69 + / -0.27 mg / mL and 1.44 + / -0.14 mg / mL respectively. Meanwhile, the amino acid composition is determined, and the contents of histidine and lysine are high. The method for preparing the camel milk polypeptide is mature, the operation is simple and not tedious, and amplification and large-scale preparation are easy; the obtained camel milk polypeptide has good antioxidant activity and can be applied to the fields of medicine, health care and food.

Description

Technical Field

[0001] This invention relates to a response surface methodology method for preparing camel milk peptides by microbial fermentation and its application. Background Technology

[0002] Camel milk, as a traditional milk source, has attracted much attention due to its unique nutritional and bioactive components. Compared to cow's and goat's milk, camel milk is rich in lactoferrin, immunoglobulins, lysozyme, and various vitamins (such as vitamin C and vitamin D), while having lower milk fat and lactose content, making it more suitable for people with lactose intolerance. This invention uses camel milk powder, a solid byproduct generated during the deep processing of camel milk. It is rich in milk calcium, lactoferrin, and prebiotics, but due to its rough texture and poor solubility, it is often used as feed or disposed of as waste in traditional processes, resulting in low resource utilization. Therefore, there is an urgent need for a research method that can utilize this resource more efficiently, thereby extending the industrial chain and increasing added value.

[0003] Polypeptides are short-chain amino acid sequences produced from proteins through enzymatic hydrolysis or fermentation, combining the advantages of easy absorption as small molecules with the functionality of large molecules. In recent years, bioactive peptides have become a research hotspot due to their diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antihypertensive, and immunomodulatory effects. Summary of the Invention

[0004] The purpose of this invention is to provide a response surface methodology (RSM) method for preparing camel milk peptides through microbial fermentation and its application. This method uses camel milk as raw material and utilizes RSM optimization to determine the effects of factors such as the material-to-liquid ratio, fermentation temperature, fermentation time, and bacterial density on the yield and anti-inflammatory activity of camel milk peptides. Four types of bacteria—*Lactobacillus fermentatus*, *Lactobacillus paracasei*, *Lactobacillus casei*, and *Bifidobacterium thermophilum*—are used for fermentation to prepare peptides. The highest camel milk peptide yield can reach 39.96 ± 0.13% (n = 3). This method eliminates the need for harsh extraction methods such as high temperature and ultrasound, and also eliminates the use of organic solvents. Furthermore, it uses the camel milk peptide yield as a process evaluation index, achieving integrated control of the "strain-process-activity" of camel milk peptides. This method has advantages such as a mild preparation process, high yield, high activity, and low pollution.

[0005] The method for preparing camel milk peptides by microbial fermentation using response surface methodology according to this invention comprises the following steps: a. Take camel milk powder, use deionized water as solvent, and sterilize it at 85℃ for 5 hours. b. Add four types of bacteria, including Lactobacillus casei, to the sterilized camel milk powder obtained in step a at a material-to-liquid ratio of 15:1 to 35:1. L. case Lactobacillus fermentum L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum Lactobacillus paracasei L. paracaseiIn this process, the fermentation temperature is 33-41℃, the fermentation time is 2-18h, and the bacterial density is 5×10⁻⁶. 6 CFU / mL - 8 × 10 7 Fermentation at CFU / mL yields camel milk polypeptide extract; c. Centrifuge the camel milk polypeptide extract obtained in step b at 7000 rpm for 10 min at 4℃, take the supernatant, and dialyze it with deionized water as the exchange medium in a dialysis bag for 60 h. After that, freeze-dry the liquid in the dialysis bag, weigh it, and the camel milk polypeptide is obtained. Store it at -20℃.

[0006] The camel milk polypeptide obtained by the method is used in the preparation of food ingredients.

[0007] The camel milk polypeptide obtained by the method is used in the preparation of raw materials for health products.

[0008] This invention discloses a response surface methodology method for preparing camel milk peptides through microbial fermentation and its application. By employing multi-strain synergistic fermentation, it overcomes three major challenges in traditional camel milk peptide preparation: low activity, high cost, and long cycle time. Through the preparation of camel milk powder peptides using this invention, a closed-loop industrial chain is constructed: forming a resource recycling chain of camel milk, powder, peptides, and functional products. This reduces raw material costs, extends the industrial chain, brings more substantial economic benefits, increases added value, and continuously releases the industrial and social value of "desert gold." Its innovation lies in the fact that the response surface methodology optimizes the preparation method of camel milk peptides, resulting in a more applicable, milder, higher-yield, and less-contaminated preparation method. Attached Figure Description

[0009] Figure 1 This is a flowchart of the research process for this invention; Figure 2 This invention uses various time, temperature, liquid-to-material ratio, and bacterial density to measure the effects of CMP. Figure 3 The effects of different factors on CMP yield; Figure 4 This invention illustrates the change in the degree of hydrolysis of CMP at different fermentation times. Figure 5 This refers to the scavenging ability of 0-3 mg / mL CMP against •OH free radicals in this invention; Figure 6 This invention demonstrates the DPPH• free radical scavenging activity of 0-3 mg / mL CMP. Figure 7 The amino acid analysis curves of the standard and CMP in this invention are shown. Figure 8 This is an HPLC chromatogram of unfermented and fermented camel milk powder in this invention. Detailed Implementation

[0010] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual protection scope of the present invention, nor are they intended to limit the protection scope of the present invention to this. Example 1

[0011] a. Take camel milk powder, use deionized water as solvent, and sterilize it at 85℃ for 5 hours. b. Add four types of bacteria, including Lactobacillus casei, to the sterilized camel milk powder obtained in step a at a material-to-liquid ratio of 20:1. L. case Lactobacillus fermentum L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum Lactobacillus paracasei L. paracasei In the middle stage, the fermentation temperature was 36℃, the fermentation time was 10h, and the bacterial density was 5×10⁻⁶. 6 Fermentation at CFU / mL yields camel milk polypeptide extract; c. Centrifuge the camel milk polypeptide extract obtained in step b at 7000 rpm for 10 min at 4℃, take the supernatant, use deionized water as the exchange medium, dialyze and desalt it using a dialysis bag for 60 h, then freeze-dry the liquid in the dialysis bag, weigh it, and obtain the camel milk polypeptide, which can be stored at -20℃. Example 2

[0012] a. Take camel milk powder, use deionized water as solvent, and sterilize it at 85℃ for 5 hours. b. Add four types of bacteria, including Lactobacillus casei, to the sterilized camel milk powder obtained in step a at a material-to-liquid ratio of 15:1. L. case Lactobacillus fermentum L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum Lactobacillus paracasei L. paracasei In the middle stage, the fermentation temperature was 33℃, the fermentation time was 2 hours, and the bacterial density was 8×10⁻⁶. 7 Fermentation at CFU / mL yields camel milk polypeptide extract; c. Centrifuge the camel milk polypeptide extract obtained in step b at 7000 rpm for 10 min at 4℃, take the supernatant, use deionized water as the exchange medium, dialyze and desalt it using a dialysis bag for 60 h, then freeze-dry the liquid in the dialysis bag, weigh it, and obtain the camel milk polypeptide, which can be stored at -20℃. Example 3

[0013] a. Take camel milk powder, use deionized water as solvent, and sterilize it at 85℃ for 5 hours. b. Add four types of bacteria, including Lactobacillus casei, to the sterilized camel milk powder obtained in step a at a material-to-liquid ratio of 35:1. L. case Lactobacillus fermentum L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum Lactobacillus paracasei L. paracasei In the middle stage, the fermentation temperature was 41℃, the fermentation time was 18h, and the bacterial density was 8×10⁻⁶. 7 Fermentation at CFU / mL yields camel milk polypeptide extract; c. Centrifuge the camel milk polypeptide extract obtained in step b at 7000 rpm for 10 min at 4℃, take the supernatant, use deionized water as the exchange medium, dialyze and desalt it using a dialysis bag for 60 h, then freeze-dry the liquid in the dialysis bag, weigh it, and obtain the camel milk polypeptide, which can be stored at -20℃. Example 4

[0014] This invention uses temperature, time, liquid-to-solid ratio, and bacterial density as four factors. A Box-Behnken design was performed using Design Expert 13 software, with camel milk peptide yield as the response value. Response surface methodology was employed to optimize the peptide fermentation preparation method. Experimental data are the average of three parallel experiments, with P ≤ 0.05 considered statistically significant. A three-dimensional response surface plot was plotted based on the regression model. The results show that the optimal fermentation method is achieved with a liquid-to-solid ratio of 15:1–35:1 (v / w), a fermentation temperature of 33℃–41℃, a fermentation time of 2 h–18 h, and a bacterial density of 5 × 10⁻⁶. 6 CFU / mL - 8 × 10 7 Under the condition of CFU / mL, the predicted yield of camel milk peptides was 42.43%. Under these optimal conditions, a validation experiment was conducted three times, and the yield of camel milk peptides was 39.96 ± 0.13% (n=3), which was close to the predicted yield. This indicates that the response surface methodology is effective in optimizing peptide preparation. Figure 3 As shown; Regression analysis was performed on the response surface methodology results, and the regression equation was obtained after fitting: Y(%)=-1270.13-3.71*A+64.84*B+6.00*C+12.30*D+0.14*A*B+0.08*A*C+0.32*A*D-0.36*B*D +0.05*C*D-0.30*A 2 -0.96*B 2 -0.26*C 2 -0.34*D 2 ; The yield of camel milk peptides was the highest value. A three-dimensional response surface plot was plotted based on a regression model. The results showed that the optimal fermentation conditions were: fermentation temperature 35-39℃, fermentation time 8-10 h, liquid-to-solid ratio 25-35:1 (v / w), and bacterial density 4×10⁻⁶. 7 -8×10 7 Under the condition of CFU / mL, the predicted yield of camel milk peptides was 42.43%. Under this optimal method condition, a validation experiment was conducted 3 times, and the yield of camel milk peptides was 39.96±0.13% (n=3), which was close to the predicted yield, indicating that the response surface methodology is effective in optimizing peptide preparation. The yield of camel milk peptides was determined by the trichloroacetic acid (TCA) method. Table 1 Experimental design of mixed bacterial ratio

[0015] The peptide content is calculated using the formula: Camel milk peptide yield (%) = (soluble peptides (g) / total protein (g)) × 100% The yield of fermented camel milk peptides was 39.96±0.13% (n=3), while that of unfermented camel milk was 5.8±0.22% (n=3). Example 5

[0016] Determination of degree of hydrolysis: Preparation of digestion fluid samples: Mix the sample solution with fermentation time of 0-18h with an equal volume of 6.2% TCA (0.2mL + 0.2mL), shake well and let stand for about 30min, centrifuge at 10,000g for 30min at 4℃, and filter the supernatant through a 0.45 μm filter membrane for later use. Preparation of o-phthalaldehyde solution: Weigh 19.05 g of sodium tetraborate and 500 mg of sodium dodecyl sulfate, add 375 mL of deionized water and dissolve until completely transparent to obtain (a); 400 mg of phthalaldehyde was dissolved in 10 mL of anhydrous ethanol, and the resulting (a) was quantitatively transferred to the phthalaldehyde ethanol solution using deionized water to obtain (b); After complete dissolution, add 440 mg of dithiothreitol to the obtained (b), and dilute the solution to 500 mL with deionized water to obtain (c). Preparation of standard solution: Accurately weigh 50 mg of serine standard and dilute to 500 mL with deionized water; Determination procedure: Take 3 mL of o-phthalaldehyde solution and 400 μL of sample solution, mix well for 5 s, let stand for 2 min, and then measure the OD value at 340 nm using a spectrophotometer; set up blank group, standard group and sample group, where the sample solutions are serine standard solution, deionized water and digestion filtrate, respectively. The formula for calculating the degree of protein hydrolysis is as follows: DH(%) = h / h tot ×100; h=(Serine-NH2-β) / α; Serine-NH2=(OD sample -OD blank ) / (OD stand. -OD blank )×0.9516×X -1 ; Where: h, the number of peptide bonds cleaved per gram of protein, mmol / g; htot, the total number of peptide bonds in the protein, mmol / g; Serine-NH2, the amount of NH2 of serine in per gram of protein, meqv / g protein; α, β, constants, selected as 1.00 and 0.40 respectively for calculation of total amino acid content; 0.9516, milliequivalent of serine; X, the protein content in the sample solution, g / L; from Figure 4 It was learned that the degree of hydrolysis gradually increases with the increase of fermentation time. Example 6

[0017] • OH radical scavenging ability: Reagent: 6 mM FeSO4 solution: Weigh 166.8 mg of FeSO4, dissolve it in deionized water, and bring the volume to 100 mL; 6 mM salicylic acid: Weigh 138.12 mg of salicylic acid, dissolve it in ethanol, and bring the volume up to 100 mL; 0.1% hydrogen peroxide: Take 100 μL of 3% hydrogen peroxide and dilute it with deionized water to 3 mL; Sample preparation: Dissolve 6 mg of camel milk peptide (CMP) in 2 mL of deionized water, centrifuge and set aside, and dilute to equal concentrations. For activity assay, 300 μL of 6 mM salicylic acid, 300 μL of 6 mM FeSO4 solution, 500 μL of sample solution, and 150 μL of 0.1% hydrogen peroxide were added sequentially to a 4.5 mL EP tube and mixed thoroughly to form the sample group. A control group was prepared by replacing the sample solution with the sample solvent. A blank control group was prepared by replacing the hydrogen peroxide solution with deionized water. After adding reagents and mixing thoroughly, the mixture was incubated at 37 ℃ for 30 min and then transferred to a 96-well plate. The absorbance was measured at λ = 510 nm. VC was used as a positive control, and the assay method was the same as for the sample. Each experiment was repeated three times. •OH radical scavenging rate (%) = (A0 - A i -A)×100% / A0; In the formula, A0 represents the absorbance of the control group; Ai A represents the absorbance of the reagent blank control group; A represents the absorbance of the sample group. Depend on Figure 5 It can be seen that the •OH radical scavenging ability of CMP increases with increasing concentration, exhibiting a concentration-dependent effect, and the IC50 value is also high. 50 =1.69±0.27 mg / mL. Example 7

[0018] DPPH free radical scavenging activity: Sample preparation: Dissolve 6 mg of CMP in 2 mL of deionized water, centrifuge, and dilute to a series of concentrations for later use; Activity assay: 400 μL of samples of various concentrations were mixed with 400 μL of 0.02% DPPH•methanol solution in 1.5 mL EP tubes to form the sample group; 400 μL of deionized water was mixed with 400 μL of 0.02% DPPH•methanol solution to form the control group. A mixture of 400 μL of VC at various concentrations and 400 μL of 2 mM DPPH•methanol solution served as a positive control. After mixing thoroughly, the mixture was incubated at 37°C for 30 min, then transferred to a 96-well plate and the absorbance was measured at λ = 517 nm. DPPH free radical scavenging rate (%) = (A0 - AA) i )×100% / A0; A0 is the absorbance of the control group; A is the absorbance of the sample; A i Sample blank; the calculation method for the free radical scavenging rate of VC is the same as that for the sample; Depend on Figure 6 It is known that the DPPH• free radical scavenging ability of camel milk polypeptide (CMP) increases with increasing concentration, exhibiting a concentration-dependent effect, and the IC50 value is [not specified]. 50 =1.44±0.14 mg / mL. In the DPPH• free radical scavenging assay, CMP exhibited strong scavenging ability. Example 8

[0019] Amino acid composition analysis of camel milk peptides (CMP): Amino acid analysis was performed using pre-column derivatization with phenyl isothiocyanate (PITC). 8 mg of camel milk peptide (CMP) was dissolved in 6 mol / L hydrochloric acid (HCl) solution containing 0.1% phenol and reacted at 110℃ for 24 hours for acid hydrolysis. After hydrolysis, the sample was completely removed by rotary evaporation under reduced pressure at 70℃, and the dried sample was then dissolved in 12 mL of ultrapure water. 200 μL of the sample solution / amino acid standard mixture was mixed with 100 μL of 1 mol / L triethylamine acetonitrile solution and 100 μL of 0.2 mol / L acetonitrile solution. After derivatization at room temperature for 1 hour, 400 μL of n-hexane was added. The mixture was allowed to stand and separate into layers. 200 μL of the lower layer was mixed with 800 μL of water and filtered through a 0.22 μm filter. High-performance liquid chromatography (HPLC) was performed using a Diamonsil® AAA amino acid analysis column under the following conditions: Phase A: pH = 6.5 ± 0.05, sodium acetate solution 0.01. mol / L, Phase B: methanol, acetonitrile, and water volume ratio of 20:60:20, using a gradient elution program: 0-49 min, 5-48% B; 49-50 min, 48-100% B; 55-56 min, 100-5% B; 56-60 min, 5% B, elution flow rate of 0.6 mL / min, detection wavelength of 254 nm; according to Figure 8 The peak times of the sample and the peak times of different amino acids in the mixed standard were compared to determine the types of amino acids contained in the sample. The peak area ratio of each amino acid in the sample spectrum was compared to determine the proportion of amino acids contained in the sample, as shown in Table 2. The essential amino acid content of camel milk peptides is 34.14%, which meets the standard of high-quality peptides and can be absorbed and utilized as a nutritional supplement. Table 2. Relative molar ratio of amino acids in camel milk polypeptides

[0020] Note: "*" indicates essential amino acids, and "#" indicates hydrophobic amino acids.

[0021] The results showed that the histidine (His) content was significantly increased compared with that before fermentation. As an aromatic amino acid, His can provide H+. + It chelates metal ions and simultaneously enhances the stability of vitamin A. This suggests a possible connection to the antioxidant activity of camel milk peptides. Example 9

[0022] Molecular weight distribution of samples before and after fermentation Column: TSKgel 2000 SWXL, 300mm × 7.8mm; Mobile phase: acetonitrile / water / trifluoroacetic acid, volume ratio 45 / 55 / 0.1; Detection wavelength: 220 nm; Flow rate: 0.5 mL / min; Column temperature: 30℃; Sample preparation: Accurately weigh 100 mg of sample and place it in a 10 mL volumetric flask. Dilute quantitatively with the mobile phase and filter through a 0.45 μm microporous membrane. The unfermented and fermented (10 h) sample solutions were analyzed under the above chromatographic conditions. The data were processed using GPC software to obtain the peptide composition, paired molecular weight distribution and distribution range of the samples.

[0023] Table 3. Molecular weight distribution of camel milk peptides before and after fermentation

[0024] The table shows that the peptide content after fermentation is >99%, and the molecular weight distribution experiment shows that mixed-culture fermentation has successfully degraded large protein molecules into small peptides or amino acids, resulting in better biological stability. Example 10

[0025] LC-MS / MS mass spectrometry identification The prepared CMP was separated by capillary high-performance liquid chromatography and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (Thermo Fisher) for 60 min. Detection mode: positive ion. The mass-charge ratio of the peptide and its fragments was collected using the following method: 10 fragment spectra were acquired after each full scan (MS2scan).

[0026] The raw mass spectrometry test files were retrieved from the relevant database using MaxQuant 1.5.5.1 software, and finally the protein identification and quantification results were obtained.

[0027]

[0028] Experimental results: Experimental results showed that the final fermentation product was the most abundant in peptide components. This characteristic may stem from the fact that mixed-culture fermentation operates at highly specific enzyme cleavage sites (cleaving only the carboxyl-terminal peptide bonds of lysine or arginine), thereby producing more peptides with well-defined structures and controllable sequences.

[0029] This invention discloses a method for preparing camel milk polypeptide and its applications. The method uses camel milk industrial scavenging powder as the research object, with bacterial species, bacterial density, fermentation temperature, and solid-liquid ratio as independent variables and camel milk polypeptide yield as the response value. The preparation method of camel milk polypeptide was optimized using a Box-Behnken response surface methodology, yielding camel milk polypeptide (CMP). The content of CMP was determined to be 39.96%, a significant increase compared to 5.8% before fermentation. The recovery rate was 52.3%. Its antioxidant activity was evaluated, and the results showed that the camel milk polypeptide possesses •OH and DPPH• free radical scavenging capabilities, with an IC50 value of [missing value]. 50 The values ​​were 1.69±0.27 mg / mL and 1.44±0.14 mg / mL, respectively. Simultaneously, the amino acid composition was determined. This invention provides a more scientific method for preparing peptides, yielding peptides with strong antioxidant activity. The production process is simpler and more rational, providing a certain raw material basis for adjuvants in antioxidant health products or foods.

[0030] The above embodiments are preferred embodiments of the invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A response surface methodology method for preparing camel milk peptides by microbial fermentation, characterized in that... Follow these steps: a. Take camel milk powder, use deionized water as solvent, and sterilize it at 85℃ for 5 hours. b. Add four types of bacteria, including Lactobacillus casei, to the sterilized camel milk powder obtained in step a at a material-to-liquid ratio of 15:1 to 35:

1. L. case Lactobacillus fermentum L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum Lactobacillus paracasei L. paracasei In this process, the fermentation temperature is 33-41℃, the fermentation time is 2-18h, and the bacterial density is 5×10⁻⁶. 6 CFU / mL - 8 × 10 7 Fermentation at CFU / mL yields camel milk polypeptide extract; c. Centrifuge the camel milk polypeptide extract obtained in step b at 7000 rpm for 10 min at 4℃, take the supernatant, and dialyze it with deionized water as the exchange medium in a dialysis bag for 60 h. After that, freeze-dry the liquid in the dialysis bag, weigh it, and the camel milk polypeptide is obtained. Store it at -20℃.

2. The use of the camel milk polypeptide obtained by the method according to claim 1 in the preparation of food ingredients.

3. The use of the camel milk polypeptide obtained by the method according to claim 1 in the preparation of raw materials for health products.

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