Peptide sequence with high activity for promoting growth of lactic acid bacteria and application of peptide sequence

Abstract

The invention is applicable to the technical field of microbial fermentation, and provides a high-activity peptide sequence capable of promoting growth of lactic acid bacteria and application of the high-activity peptide sequence, a peptide sequence group is formed by a plurality of new peptides capable of promoting growth, and the peptide sequence group comprises an amino acid sequence as shown in SEQ ID NO: 1. The new peptide sequence has the activity of promoting the growth of lactic acid bacteria, effectively improves the growth efficiency of the lactic acid bacteria, increases the viable count of lactic acid bacteria culture, and greatly promotes commercialization of potential functional strains.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation technology improvement, and in particular relates to a peptide sequence with high activity for promoting the growth of lactic acid bacteria and an application thereof. Background Art

[0002] Lactic acid bacteria cultures and probiotic preparations, as key components of functional foods and health supplements, have garnered widespread attention worldwide in recent years. Lactic acid bacteria cultures are primarily used in dairy products and fermented foods, while probiotic preparations are widely used in a variety of industries, including food, health supplements, and pharmaceuticals. With rising consumer health awareness and advancements in scientific research and technology, both product categories have achieved significant growth in market size, technological innovation, and application areas. However, this rapid industry development has also exposed some common challenges that urgently need to be addressed. Within the probiotics industry, fastidious probiotics are often slow to grow, and their viable counts can decrease dramatically during processing, transportation, and storage. Consequently, poor fermentation performance and insufficient viable counts significantly hinder the commercialization of potentially functional strains. Exploring high-cell density culture strategies to ensure sufficient viable counts is one of the key challenges facing current probiotic products.

[0003] To solve this problem, researchers have been working to find effective methods to promote the growth of lactic acid bacteria. Traditional culture methods often fail to meet the probiotics industry's demand for high viable counts. Commercial MRS culture media can only activate lactic acid bacteria at a level of 10 7 -10 8 CFU / mL is around, which is far from enough. The culture medium needs to be optimized so that the number of viable bacteria is as high as possible. In previous studies, many scholars have selected different types of proliferation factors for culture tests on the proliferation of lactic acid bacteria. These proliferation factors are widely distributed. Oligosaccharides were used to study the proliferation of lactobacilli. The results showed that oligosaccharides can play a probiotic role in many aspects, such as enhancing the survival ability of probiotics in the digestive tract and promoting the growth of probiotics as a metabolic substrate for intestinal flora and lactic acid bacteria; the growth factors of different fruits and vegetables such as tomato juice, carrot juice, corn juice and oyster mushroom juice were studied to see how they affect the proliferation of Lactobacillus plantarum strains, and high-density fully automatic fermentation tanks were used to achieve high-density culture; some people have also comprehensively studied the effects of different carbon sources, nitrogen sources, trace elements and amino acids on the culture and proliferation of Lactobacillus plantarum NCU137, and the final number of viable bacteria reached 9.2×10 9 CFU / mL; All of the above studies can provide a certain reference for high-density cultivation of lactic acid bacteria. Among them, the simplest and most direct method for the proliferation of lactic acid bacteria is to add growth-promoting factors or prebiotics.

[0004] Research on prebiotics has primarily focused on indigestible oils and sugars, which can rapidly ferment and proliferate lactic acid bacteria. Therefore, the exploration of novel prebiotics is of great significance. Protein hydrolysates are mixtures of peptides and amino acids derived from protease degradation. Recent studies have shown that protein hydrolysates, peptides, or oligopeptides from various sources can promote the growth of probiotic bacteria with potential prebiotic functions. Proteins and peptides have also been extensively studied as rising potential prebiotics. Research on lactic acid bacteria growth-promoting peptides has primarily focused on the effects of proteins and their hydrolysates from different sources on their growth and metabolism. These include plant-based proteins such as soy protein hydrolysates and rubber protein hydrolyzed peptides, as well as animal proteins such as fish protein hydrolysates, meat protein and its hydrolysates, poultry egg protein hydrolysates, and casein macropeptides, which can promote the growth of lactic acid bacteria and bifidobacteria.

[0005] Therefore, it is particularly important to develop new prebiotics and their application methods. Summary of the Invention

[0006] The purpose of the present invention is to provide a peptide sequence with high activity in promoting the growth of lactic acid bacteria and its application, aiming to solve the technical problems of low growth rate and low number of viable bacteria of lactic acid bacteria.

[0007] The present invention is achieved by providing a peptide sequence, wherein the peptide sequence comprises a peptide sequence group consisting of a plurality of new growth-promoting peptides, and the peptide sequence group comprises the amino acid sequence shown in SEQ ID NO: 1 A further technical solution of the present invention is that the peptide sequence group also includes the amino acid sequence shown in SEQ ID NO:2.

[0008] A further technical solution of the present invention is that the peptide sequence group also includes the amino acid sequence shown in SEQ ID NO:4.

[0009] A further technical solution of the present invention is that the peptide sequence has the activity of promoting the growth of lactic acid bacteria.

[0010] A further technical solution of the present invention is that the peptide sequence is artificially synthesized.

[0011] Another object of the present invention is to provide an application of a peptide sequence having the activity of promoting the growth of lactic acid bacteria.

[0012] The beneficial effects of the present invention are: the new peptide sequence has the activity of promoting the growth of lactic acid bacteria, effectively improves the growth efficiency of lactic acid bacteria, increases the number of viable lactic acid bacteria cultured, and greatly promotes the commercialization of potential functional strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the promoting effect of adding different components on A51 provided by the embodiments of the present invention.

[0014] Figure 2 This is a schematic diagram showing the effect of the addition amount of the peptide component (<3 kDa) provided in the examples of the present invention on the growth of A51.

[0015] Figure 3 Schematic diagram of the effect of the synthetic peptide PAR11 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus A51.

[0016] Figure 4 This is a schematic diagram of the effect of the synthetic peptide PAR11 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus L1.

[0017] Figure 5 Schematic diagram of the effect of the synthetic peptide PAR11 provided in an embodiment of the present invention on the growth of Lactobacillus reuteri A115.

[0018] Figure 6 This is a schematic diagram of the effect of the synthetic peptide PAR11 provided in an embodiment of the present invention on the growth of Lactobacillus plantarum L3.

[0019] Figure 7 This is a schematic diagram of the effect of the synthetic peptide PAR11 provided in an embodiment of the present invention on the growth of Lactobacillus paracasei C125.

[0020] Figure 8 This is a schematic diagram of the effect of the synthetic peptide GAR12 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus A51.

[0021] Figure 9 This is a schematic diagram of the effect of the synthetic peptide GAR12 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus L1.

[0022] Figure 10 This is a schematic diagram of the effect of the synthetic peptide GAR12 provided in an embodiment of the present invention on the growth of Lactobacillus reuteri A115.

[0023] Figure 11 This is a schematic diagram of the effect of the synthetic peptide GAR12 provided in an embodiment of the present invention on the growth of Lactobacillus plantarum L3.

[0024] Figure 12 This is a schematic diagram of the effect of the synthetic peptide GAR12 provided in an embodiment of the present invention on the growth of Lactobacillus paracasei C125.

[0025] Figure 13 This is a schematic diagram of the effect of the synthetic peptide DGG14 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus A51.

[0026] Figure 14 This is a schematic diagram of the effect of the synthetic peptide DGG14 provided in an embodiment of the present invention on the growth of fermentative Lactobacillus mucilaginosus L1.

[0027] Figure 15Schematic diagram of the effect of the synthetic peptide DGG14 provided in an embodiment of the present invention on the growth of Lactobacillus reuteri A115.

[0028] Figure 16 This is a schematic diagram of the effect of the synthetic peptide DGG14 provided in an embodiment of the present invention on the growth of Lactobacillus plantarum L3.

[0029] Figure 17 This is a schematic diagram of the effect of the synthetic peptide DGG14 provided in an embodiment of the present invention on the growth of Lactobacillus paracasei C125. DETAILED DESCRIPTION

[0030] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0031] In the description of the present invention, it should be understood that the terms "length," "width," "up," "down," "front," "back," "left," "right," "vertical," "horizontal," "top," "bottom," "inside," "outside," and the like, indicating positions or location relationships, are based on the positions or location relationships shown in the accompanying drawings and are intended only to facilitate the description of the present invention and simplify the description. They do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting the present invention. Furthermore, in the description of the present invention, "plurality" means two or more, unless otherwise expressly and specifically defined.

[0032] The present invention provides a method for preparing a proteolytic peptide that promotes the proliferation of lactic acid bacteria and its application in high-density culture of lactic acid bacteria, aiming to increase the growth rate and number of viable lactic acid bacteria by optimizing fermentation conditions, thereby meeting the commercialization needs of probiotic products.

[0033] Preparation of peptide samples For casein peptide digestion, the protein was first defatted with n-hexane, filtered, and dried. The protein was dissolved in distilled water to a concentration of 10 g / L. The pH was adjusted to 2.0 with 36% hydrochloric acid, and pepsin was added at an enzyme-to-substrate ratio of 1:35 w / w. The mixture was stirred at 37°C for 2 hours. The pH was then adjusted to 5.3 with saturated sodium bicarbonate and 7.5 with 1 M sodium hydroxide. Pancreatin was added at an enzyme-to-substrate ratio of 1:25 w / w, and the mixture was stirred at 37°C for 2 hours. After protein hydrolysis, the reaction was terminated by heating to 95°C for 10 minutes. The precipitate was removed by centrifugation at 6500 rpm for 15 minutes. Large molecular weight impurities were filtered using 1.2 μm and 0.45 μm filter membranes, and peptides were separated and collected using 10,000 and 3000 Da ultrafiltration membranes. After pre-freezing for 4 hours, the mixture was freeze-dried in vacuo to obtain peptide samples of varying molecular weights.

[0034] A culture medium supplemented with active peptides was prepared, and A51 was inoculated therein to detect its growth curve and the real-time viable bacterial count at different time periods.

[0035] like Figure 1-2 As shown in the figure, the peptide component <3kDa has the best growth-promoting effect on A51 when added as a growth factor. Compared with MRS medium, it can also control the apoptosis of lactic acid bacteria in the late stage of culture. The addition amount shows that the addition amount of 0.3% to 0.4% has a better growth-promoting effect.

[0036] During the experiment, mixed peptides of different molecular weights of yak casein were prepared. It was found that the addition of peptide components with a size of less than 3kDa had a better growth-promoting effect. Subsequently, in order to determine which peptides were effective, peptideomics testing was carried out. Targeted screening of differential peptides was carried out. The peptides with the highest consumption were defined as those with activity that promoted the growth of lactic acid bacteria. Then, a search for novelty of the consumed peptides was conducted, and those that had been studied and had potential antibacterial properties were eliminated. Activity scores were performed, and those with high activity were selected for chemical synthesis, and their growth-promoting activity was verified. To explore the utilization of casein hydrolyzed peptides by A51 (Lactobacillus mucilaginosus A51), peptidomics analysis of the supernatant was performed at two key time points: the beginning of culture (0 h) and the end of culture (24 h).

[0037] Samples were removed from the frozen state and peptides were extracted. The peptide extracts were ultrafiltered and enriched using 3 kD ultrafiltration tubes, desalted using HLB, and dried using a vacuum concentrator. Peptides were fully dissolved in distilled water and quantified by UV spectrophotometry using a NANO DROP ONE (Thermo Scientific).

[0038] Peptide identification and analysis were performed using LC-MS / MS. Equal amounts of peptides were dissolved in mass spectrometry loading buffer and subsequently separated using a Vanquish Neo (Thermo) chromatograph. The analytical column was a uPAC High Throughput column (75 μm × 5.5 cm, Thermo, USA). Mobile phase A consisted of water (2% acetonitrile + 0.1% formic acid), and mobile phase B consisted of water (80% acetonitrile + 0.1% formic acid). The chromatographic gradient was set to 8 minutes, and the flow rate was 300 nL / min. Data acquisition software was ThermoXcalibur 4.7 (Thermo, USA). After HPLC separation, samples were analyzed by mass spectrometry using an Orbitrap Astral mass spectrometer (Thermo, USA). The Orbitrap Astral mass spectrometer (Thermo, USA) was operated in DDA mode, with positive ion detection and a source voltage of 1.5 kV. The mass spectrometer primary scan range was 380–980 m / z, and the secondary scan range was 150–2000 m / z. The raw data files were imported into the PEAKS Studio 8.5 software system for library search and analysis. The database used in this study is https: / / www.uniprot.org / . Parameter settings included: fixed modification: carbamidomethyl; variable modifications: oxidation and acetyl; protein digestion: trypsin; maximum tolerance for precursor mass error: ±10 ppm; and a false discovery rate (FDR) of ≤0.01 for peptide identification. Only peptides identified within a high confidence interval were selected for downstream protein identification analysis.

[0039] Peptidomics analysis of peptides before and after culture revealed 144 shared peptides between the two groups, of which 59 were upregulated, 49 were downregulated, and 39 showed no significant changes. Consumed peptides are defined as potentially nutritious peptides that promote the growth of Lactobacillus fermentum. Conversely, peptides that are not absorbed or newly produced are considered peptides not utilized by Lactobacillus fermentum. Thirty-two peptides with significant changes from the downregulated peptides were selected as potential nutritious peptides for Lactobacillus fermentum, and their sequences and functions were compared against databases. Mathematical statistics revealed that lactic acid bacteria prefer hydrophilic peptides with more than seven amino acids, including valine at the C-terminus and proline at the penultimate position (there was no significant difference in the N-terminal amino acid distribution), and peptides containing the characteristic PAG sequence. A novel search revealed 32 consumed peptides, including 20 previously unreported novel peptides, 12 previously studied functionally active peptides, and 6 with antimicrobial activity. Patent applications are anticipated for these 20 novel peptides.

[0040] Table 1 Prediction and research of physicochemical properties of peptide sequences The peptides used in the fermentation of Lactobacillus mucilaginosus A51 were screened for predicted activity scores greater than 0.6 and characteristic sequences containing PAGs, which were chemically synthesized and tested for lactic acid bacteria growth promoting activity.

[0041] Table 2 Prediction of active peptide activity The screened peptides were synthesized to verify the growth-promoting activity, and five strains were selected, including Lactobacillus mucilaginosus A51, Lactobacillus mucilaginosus L1, Lactobacillus mucilaginosus A115, Lactobacillus plantarum L3, and Lactobacillus paracasei C125.

[0042] The experimental method is to sterilize and cool the sterilized culture medium, inoculate 2% seed liquid, add different amounts of irradiated synthetic peptides, and perform growth curve measurement to judge the growth situation.

[0043] like Figure 3-7 As shown, Figure 3 The effect of different addition amounts of PAR11 on the growth of fermentative Lactobacillus mucilaginosus A51; Figure 4 The effect of different addition amounts of PAR11 on the growth of fermentative Lactobacillus mucilaginosus L1; Figure 5 The effect of different addition amounts of PAR11 on the growth of Lactobacillus reuteri A115; Figure 6 The effect of different addition amounts of PAR11 on the growth of Lactobacillus plantarum L3; Figure 7 Effects of different addition amounts of PAR11 on the growth of Lactobacillus paracasei C125.

[0044] From the growth curve, it can be seen that PAR11 has a significant promoting effect on Lactobacillus mucilaginosus lactic acid bacteria. The promoting effect of fermentation Lactobacillus mucilaginosus A51 is more obvious, with a large increase in biomass. The absorbance is about 0.3 higher than that of the basic MRS culture medium, and the growth promoting effect is significant (p<0.05); it has a promoting effect on fermentation Lactobacillus mucilaginosus L1, and the low-dose response is not obvious; Lactobacillus reuteri A115 has a significant growth-promoting response to this peptide, and the growth after adding more than 0.5 mg / mL and culturing for 12 hours is significantly different from that of the control group; it has no obvious effect on Lactobacillus plantarum L3; it shows a significant inhibitory effect on Lactobacillus paracasei C125, and the biomass is much lower than that of the control group in the later stage of culture.

[0045] like Figure 8-12 As shown, Figure 8 The effect of different addition amounts of GAR12 on the growth of fermentative Lactobacillus mucilaginosus A51; Figure 9 The effect of different addition amounts of GAR12 on the growth of fermentative Lactobacillus mucilaginosus L1; Figure 10 The effect of different addition amounts of GAR12 on the growth of Lactobacillus reuteri A115; Figure 11The effect of different addition amounts of GAR12 on the growth of Lactobacillus plantarum L3; Figure 12 Effects of different addition amounts of GAR12 on the growth of Lactobacillus paracasei C125.

[0046] GAR12 has a growth-promoting effect on the screened probiotics, such as Figure 8-12 As shown, the activity exhibited by PAR11 is similar to that of PAR11, with the best growth promotion effect on three Lactobacillus mucilaginosus strains, A51, L1, and A115. It has no significant effect on Lactobacillus plantarum, but still has a strong inhibitory effect on Lactobacillus paracasei. This may be because this type of active peptide is screened from Lactobacillus mucilaginosus and has a better growth promotion effect on strains with higher homology.

[0047] like Figure 13-17 As shown, Figure 13 The effect of different addition amounts of DGG14 on the growth of fermentative Lactobacillus mucilaginosus A51; Figure 14 The effect of different addition amounts of DGG14 on the growth of fermentative Lactobacillus mucilaginosus L1; Figure 15 The effect of different addition amounts of DGG14 on the growth of Lactobacillus reuteri A115; Figure 16 The effect of different addition amounts of DGG14 on the growth of Lactobacillus plantarum L3; Figure 17 Effects of different addition amounts of DGG14 on the growth of Lactobacillus paracasei C125.

[0048] Compared to peptides PAR11 and GAR12, DGG14 promoted the growth of all five strains screened, with a particularly significant effect after 12 hours. The effect of promoting biomass increase was also related to the amount of active peptide added. In addition to Lactobacillus reuteri, which had a better growth-promoting effect at 1 mg / mL than at 1.5 mg / mL, the other four strains, Lactobacillus mucilaginosus A51 and L1, Lactobacillus plantarum L3, and Lactobacillus paracasei C125, showed a positive correlation with the amount of active peptide added, demonstrating a certain biomass-increasing effect.

[0049] Comparative experiment on the growth-promoting effects of different peptide sequences on various lactic acid bacteria To further verify the differences in growth-promoting activity of different peptide sequences, three core peptides, PAR11 (SEQ ID NO: 1), GAR12 (SEQ ID NO: 2), and DGG14 (SEQ ID NO: 4), were selected, and comparative experiments were carried out using Lactobacillus plantarum NCU137 and Lactobacillus acidophilus LA-5 as test strains.

[0050] Experimental methods: Prepare basic MRS medium, add 0.3% target peptide sequence, sterilize and inoculate 2% seed solution, OD600=0.5.

[0051] The culture was carried out in a fully automatic fermenter (37°C, stirring speed 150 rpm) for 24 h. The temperature in the fermenter was around 37°C and the stirring speed was 150 rpm. Samples were taken every 2 h to determine the OD600 value and the number of viable bacteria (pour plate method).

[0052] Experimental results: The OD600 of the Lactobacillus plantarum NCU137:DGG14 group reached 1.82 at 12 h, and the number of viable bacteria was 9.6×10 9 CFU / mL, which was 42% higher than that of the MRS control group; the OD600 of the PAR11 group was 1.57, and the number of viable bacteria was 8.1×10 9 CFU / mL; there was no significant difference between the GAR12 group and the control group (p>0.05).

[0053] The viable bacterial count of Lactobacillus acidophilus LA-5:PAR11 group reached 1.2×10¹ at 16 hours. 0 CFU / mL increased by 38% compared with the control group; the DGG14 group increased by 27%, while the GAR12 group only increased by 15%.

[0054] Experimental Conclusion The growth-promoting effects of different peptide sequences on lactic acid bacteria are strain-specific. DGG14 has better growth-promoting activity on Lactobacillus plantarum, and PAR11 has a more significant effect on Lactobacillus acidophilus.

[0055] Stability studies of peptide sequences To evaluate the applicability of the peptide sequence in actual production, stability tests were performed under different conditions.

[0056] Experimental methods: Thermal stability: 1 mg / mL peptide solution was treated at 60°C, 80°C, and 100°C for 30 min, and the peptide content retention rate was determined by HPLC.

[0057] pH stability: The pH of the peptide solution was adjusted to 2.0, 4.5, 7.0, and 9.0, and the OD280 value was measured after standing at room temperature for 24 h.

[0058] Pepsin resistance: The peptide solution was incubated with pepsin at 37°C, pH 2.0 for 2 h, with an enzyme-substrate ratio of 1:100. The proportion of undegraded peptide was detected after ultrafiltration.

[0059] Experimental data: Thermal stability: After treatment at 80℃, the retention rates of PAR11 and DGG14 were 92.3% and 88.7% respectively; after treatment at 100℃, the retention rates of both were >75%.

[0060] pH stability: In the pH range of 2.0-9.0, the OD280 value of the peptide solution fluctuated by <5%, indicating good acid-base stability.

[0061] Enzyme resistance: After treatment with pepsin, the undegraded proportion of PAR11 was 68.5%, and that of DGG14 was 59.2%, which were significantly higher than those of commercially available prebiotic oligosaccharides (42.1%).

[0062] Experimental Conclusion Discussion on the mechanism of action: Preliminary transcriptomic analysis found that PAR11 can significantly upregulate the expression of ABC transporter genes (such as oppA and oppB) in lactic acid bacteria, promoting peptide absorption; DGG14 activates the activity of key enzymes in the glycolysis pathway (such as phosphofructokinase) and accelerates energy metabolism.

[0063] Industrial advantages: Compared with traditional prebiotics, artificially synthesized peptide sequences have the following characteristics: The activity is more stable and is not affected by degradation by intestinal flora; the effect efficiency is high, and the addition amount is only 1 / 5-1 / 10 of traditional prebiotics; it can be produced on a large scale through chemical synthesis, and the cost is controllable.

[0064] The peptide sequence group provided by the present invention (including SEQ ID NO: 1, NO: 2, NO: 4, etc.) achieves the goal of increasing the number of viable bacteria from 10 7 -10 8 CFU / mL increased to 10 10 This is a technological breakthrough, achieving a concentration of over 1000 CFU / mL. This peptide sequence has been validated in multiple scenarios to excel in promoting lactic acid bacteria growth and improving the quality and stability of fermented products, providing a novel solution for the commercialization of the probiotics industry. Further targeted modification of the peptide sequence, combined with protein engineering techniques, could be conducted to expand its applicable strain range and functional properties.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A peptide sequence, characterized in that The peptide sequence is a peptide sequence group consisting of a plurality of new growth-promoting peptides, and the peptide sequence group includes the amino acid sequence shown in SEQ ID NO:

1.

2. The peptide sequence according to claim 1, characterized in that The peptide sequence group also includes the amino acid sequence shown in SEQ ID NO:

2.

3. The peptide sequence according to claim 1 or 2, characterized in that The peptide sequence group also includes the amino acid sequence shown in SEQ ID NO:

4.

4. The peptide sequence according to claim 3, characterized in that The peptide sequence has the activity of promoting the growth of lactic acid bacteria.

5. The peptide sequence according to claim 4, characterized in that The peptide sequence is artificially synthesized.

6. Use of the peptide sequence according to any one of claims 1 to 5 in promoting the growth of lactic acid bacteria.

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