Stenotrophomonas maltophilia and application of stenotrophomonas maltophilia in production of salty peptide

Abstract

The invention discloses a stenotrophomonas maltophilia AF15-5 strain, the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.32172, the preservation date is October 11, 2024, and the preservation unit is the China General Microbiological Culture Collection Center. The invention further discloses a preparation method of the stenotrophomonas maltophilia AF15-5 strain. The invention also discloses a screening method of the stenotrophomonas maltophilia AF15-5 strain and an application of the stenotrophomonas maltophilia AF15-5 strain in production of salty peptides. The stenotrophomonas maltophilia provided by the invention has relatively good safety and relatively good proteolysis capability, can produce salty peptides, enters a logarithmic phase of growth within 5-16 hours, is not resistant to high temperature (does not grow at the temperature of 51 DEG C or above), is alkali-resistant and is not acid-resistant, and the growth capability of the strain is strongly inhibited due to the salt concentration of 5% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Stenotrophomonas maltophilia and its application in producing salty peptides. Background Art

[0002] Fermented bean curd is one of the traditional Chinese special fermented foods. It is made from soybeans and fermented by multiple microorganisms synergistically, with its unique flavor and nutritional value. Its raw materials are rich in soy protein, which can be hydrolyzed by proteases produced by microorganisms into small molecule compounds such as polypeptides, amino acids, and nucleotides. These transformation products not only endow fermented bean curd with nutritional value, but also show a flavor-presenting or flavor-enhancing effect. As an essential condiment in the production of fermented bean curd, salt is crucial for maintaining the form of fermented bean curd and inhibiting harmful microorganisms. Insufficient salt will lead to a weakened flavor and a decline in quality, while excessive content may pose a threat to human health. Research indicates that the purpose of reducing salt without sacrificing saltiness can be achieved by optimizing the structure of salt or adding salt substitutes, such as non-sodium salts, salty peptides, and salty-enhancing peptides. Therefore, exploring and utilizing substances with salty characteristics has become a current research hotspot.

[0003] In the field of fermented foods, the excavation and research of salty substances are particularly crucial. Sensory evaluation and proteomics techniques have been used to reveal the core role of flavor amino acids and flavor-active peptides in flavor construction. The main methods for preparing such flavor-presenting substances include extraction, enzymatic hydrolysis, microbial fermentation, and synthesis. Microbial fermentation uses proteases produced by strains during their metabolic processes to hydrolyze active small molecule fragments from substrate proteins. The greatest advantage lies in its low cost. Zhou Xiao et al. used different Aspergillus flavus strains to enhance the fermentation of Liuyang fermented soybeans and found that the enhanced fermentation by strain A.flavus 5322 could promote the generation of umami peptides, and the umami score of this fermented soybean was the highest. Therefore, this study aims to excavate microorganisms in fermented bean curd that have high protease production and whose fermentation products have a strong ability to present saltiness, in order to provide reliable microbial resources for food "salt reduction" research. Summary of the Invention

[0004] In view of the above-mentioned deficiencies currently existing, the present invention provides a Stenotrophomonas maltophilia and its application in producing salty peptides. The Stenotrophomonas maltophilia AF15-5 strain of the present invention has good safety and good proteolytic ability. It enters the logarithmic growth phase at 5 - 16 h, is not heat-resistant (does not grow above 51 °C), is alkali-tolerant and acid-intolerant, and a salt concentration of more than 5% will cause a strong inhibitory effect on the growth ability of this strain.

[0005] To achieve the above object, the present invention provides a strain of Stenotrophomonas maltophilia AF15-5, with the preservation number of CGMCC NO.32172, the preservation date of October 11, 2024, and the preservation unit being the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

[0006] Through molecular biological identification, the above-mentioned strain of Stenotrophomonas maltophilia AF15-5 is round in light yellow color, with a smooth and shiny surface, slightly transparent edges, a raised center, and is easy to pick up; after Gram staining, its morphology is observed to be short rod-shaped, it is a Gram-negative bacterium, and has no spores. Based on the 16S rDNA gene sequence, a phylogenetic tree of strain AF15-5 is constructed, and AF15-5 has a 99% homology with Stenotrophomonas oligotrophica IAM 12423.

[0007] Based on the same inventive concept, the present invention also provides the application of the above-mentioned Stenotrophomonas maltophilia in the production of salty peptides.

[0008] According to one aspect of the present invention, the Stenotrophomonas maltophilia produces salty peptides in sufu; wherein, the salt concentration in the sufu is less than 5%.

[0009] Based on the same inventive concept, the present invention also provides a salty peptide, which is produced by the above-mentioned Stenotrophomonas maltophilia; the amino acid sequence of the salty peptide includes any one of LRK and YDR.

[0010] Advantages of the present invention:

[0011] The present invention obtains the Stenotrophomonas maltophilia AF15-5 that produces salty peptides through defined culture and screening. This strain has good safety (does not produce hemolysin, relatively good antibiotic sensitivity, and weak biofilm-producing ability), good proteolytic ability, enters the logarithmic growth phase at 5-16 h, is not heat-resistant (does not grow above 51 °C), is alkali-tolerant and acid-intolerant, and a salt concentration of more than 5% will cause a strong inhibitory effect on the growth ability of this strain. Through experimental identification, two novel salty peptides produced by the Stenotrophomonas maltophilia AF15-5 of the present invention include LRK and YDR. Description of the drawings

[0012] Figure 1 It is the protease activity diagram of 11 strains in Example 1 of the present invention under different acid-base conditions;

[0013] Figure 2 It is the taste evaluation of the decomposition and fermentation of tofu by 6 strains in Example 1 of the present invention, wherein, A is the sensory evaluation; B is the electronic tongue evaluation;

[0014] Figure 3 This shows the content of soluble proteins in the fermented bean curd of Example 1 of the present invention and the hydrolysates obtained after 6 strains decomposed proteins.

[0015] Figure 4 This shows the hemolytic properties of the 6 strains in Example 1 of the present invention; (a) Strain AF5-4; (b) Strain AF5-10; (c) Strain AF5-5; (d) Strain AF10-5; (e) Strain AF15-5; (f) Strain AF20-5;

[0016] Figure 5 This shows the biofilm-forming activities of the 6 strains in Example 1 of the present invention.

[0017] Figure 6 This shows the colony morphology and Gram staining morphology of Strain AF15-5 in Example 1 of the present invention; among them, A is the colony of the strain on nutrient agar; B is the Gram staining morphology magnified 1000 times.

[0018] Figure 7 This shows the phylogenetic tree of Strain AF15-5 in Example 1 of the present invention.

[0019] Figure 8 This shows the growth curve of Strain AF15-5 of the present invention.

[0020] Figure 9 This shows the effect of different temperatures on the growth of Strain AF15-5.

[0021] Figure 10 This shows the effect of different salt concentrations on the growth of Strain AF15-5.

[0022] Figure 11 This shows the effect of different pH values on the growth of Strain AF15-5.

[0023] Figure 12 This shows the salty taste score of the freeze-dried components in Example 3 of the present invention.

[0024] Figure 13 This shows the elution schematic diagram of the freeze-dried component S3 in Example 3 of the present invention.

[0025] Figure 14 This shows the length distribution of the screened polypeptides in Example 3 of the present invention.

[0026] Figure 15 This shows the interaction mode diagram of the ligand and TMC4 in Example 3 of the present invention; among them, A is LRK; B is DWR; C is WDR; D is YDR; E is YNLRE.

[0027] Figure 16Taste characteristics of 4 synthetic polypeptides screened for Example 3 of the present invention; among them, A is sensory evaluation; B is electronic tongue evaluation. Detailed implementation manners

[0028] To make the present invention more easily understandable, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as understood by those of ordinary skill in the art; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by well-known methods.

[0029] Example 1

[0030] A screening method for Stenotrophomonas maltophilia AF15-5 strain, comprising the following steps:

[0031] (1) Preparation process of fermented bean curd: soybean washing → soaking and grinding → filtering and boiling → coagulating and forming → draining and cutting into pieces → constant temperature fermentation at 18°C for 4-7 d → adding spices → packing into jars → post-fermentation at 20°C for 20 d.

[0032] (2) Primary screening: Samples of fermented bean curd were collected from tofu, the 3rd day of pre-fermentation, and the 5th, 10th, 15th, and 20th days of post-fermentation, and gradient dilution was carried out by 10-fold dilution method. The diluted solution was spread on casein medium (casein 8 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 0.36 g / L, disodium hydrogen phosphate 1 g / L, agar 20 g / L, pH 7.4 ± 0.1), and cultured at 37°C for 2 d. Strains with different morphologies and producing hydrolysis zones were picked and streaked on nutrient agar (peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar 15 g / L), and purified and cultured multiple times until single colonies were obtained. The single colonies were spot-inoculated into the casein medium for qualitative test of protease-producing ability, and 36 culturable strains were obtained. The results of the ratio of culturable strains to the diameter of the hydrolysis zone are shown in Table 1 below. The range of the diameter ratio (D / d) is 1.50-5.04. Strains with D / d greater than 4 (11 strains) were selected for the next experiment. Among them, the diameter of the hydrolysis zone is D; the diameter of the corresponding colony is d; the hydrolysis zone diameter ratio is D / d.

[0033] Table 1:

[0034]

[0035]

[0036] (3) Re-screening: The 11 strains obtained from the primary screening were activated and cultured with nutrient broth (peptone 10.0 g / L, sodium chloride 5.0 g / L, beef extract powder 3.0 g / L). The OD600 of the bacterial solution was adjusted to 0.8, and then inoculated into the fermentation medium (soybean powder 15 g / L, glucose 5 g / L, potassium dihydrogen phosphate 0.36 g / L, disodium hydrogen phosphate 1 g / L, pH 7.4 ± 0.1) at an inoculation amount of 2%. After culturing at 37°C and 120 r / min for 2 d, centrifugation was carried out at 4°C and 10,000 r / min for 10 min, and the supernatant was collected for standby.

[0037] (4) Screening:

[0038] Referring to the method in "GB / T 23527.1—2023 Part 1: Protease Preparations", the supernatant was inoculated into NB media with different pH values (4, 5, 6, 7, 8, 9, 10) respectively, and cultured at 37°C and 120 r / min for 2 d. The protease activity (OD600 value) was measured. As Figure 1 shown, since fermented bean curd is an alkaline fermented food, 6 strains (AF5-4, AF5-5, AF5-10, AF10-5, AF15-5, and AF20-5) that are suitable for growth in fermented bean curd and have strong protease activity were obtained.

[0039] Taste experiments on fermenting tofu with the above 6 strains: (1) Pretreatment: Adjust the OD600 of the bacterial solution (the seed solution obtained by inoculating the strain into NB medium and activating and culturing at 37°C and 120 r / min for 24 h) to 0.8, and inoculate it into sterilized tofu at an inoculation amount of 2% for fermentation at 30°C for 3 d. Another sterilized tofu inoculated with sterile water was used as a blank control (K), and cultured at 30°C for 3 d. 50 g of fermented tofu and 50 g of sterilized tofu fermented with sterile water were respectively added to 200 mL of deionized water, mixed evenly, cooled after boiling water bath for 30 min, centrifuged at 8000 r / min for 30 min, filtered, and the filtrate was fixed to 250 mL for standby. (2) Sensory evaluation: Refer to the method of Zhou Xiao et al. and make appropriate adjustments. 0.08% citric acid, 0.5% alum, 0.5% leucine, 0.7% sodium chloride, and 0.35% monosodium glutamate were used as references for the 5 flavors of sour, astringent, bitter, salty, and fresh respectively. The reference was recorded as 5 points, and the result adopted a 0-9 point system. (3) Electronic tongue evaluation: The AAE, CT0, CA0, C00, and AE1 sensors (corresponding to the 5 flavors of fresh, salty, sour, bitter, and astringent in turn) were used for adjustment and calibration. A reference solution prepared by mixing 0.03 mol / L potassium chloride and 0.0003 mol / L tartaric acid was used as the control for the sample. Each sample was tested 4 times, and the results of the last 3 measurements were taken for analysis. The results are as Figure 2As shown in the figure, from Figure 2 it can be seen that there are differences in different evaluation methods. The results of sensory evaluation show richer taste levels, which reflects the complexity and diversity of human taste perception. Although electronic tongue technology can provide accurate taste analysis, it still has certain limitations in simulating human taste interactions. The results of the two evaluation methods show that the extracts of strain-fermented tofu present obvious taste differences, indicating that microorganisms have the potential to improve food flavor. Compared with the blank group, saltiness and umami are the two more prominent tastes, and sourness is not obvious in both evaluation methods, probably because sourness is not the dominant taste in tofu. Among them, the saltiness of the AF15-5 group is the strongest, and the saltiness response value of the electronic tongue is 17.76, which is significantly higher than the saltiness response value of the blank group, -0.91.

[0040] Determination of the content of soluble proteins and their hydrolysates in tofu extract (tofu extract after taste experiment treatment): (1) Determination of soluble proteins: Take 2 mL of the sample solution and determine it by the Kjeldahl method referring to "GB5009.5—2016 Determination of proteins in foods". (2) Determination of soluble peptides: Take 1 mL of the sample solution, add 9 mL of 10% trichloroacetic acid solution, let it stand at 4 °C for 2 h, and centrifuge at 10,000 r / min for 10 min. Take the supernatant and determine it by referring to the lowry method. Using casein to establish a standard curve, the regression equation is y = 0.5797x + 0.0965, R 2 = 0.9954. Determination of amino acid nitrogen: Determine it by referring to "GB 5009.235—2016 Determination of amino acid nitrogen in foods". The results are as Figure 3 shown in the figure. From Figure 3 it can be seen that the contents of soluble peptides and amino acid nitrogen in the tofu samples treated with strains are significantly higher than those in the blank group (K), which indicates that the proteins in tofu are decomposed under the action of strains. Some strains show a relatively large diameter of the proteolytic zone and high enzyme activity, and there is no positive correlation with the content of their protein hydrolysates. Probably due to different substrates, proteases show different enzyme activity levels. Amino acid nitrogen can reflect the degree of fermentation, and its content is positively correlated with the degree of protein hydrolysis. AF15-5 shows the highest proteolytic ability, and its soluble peptide and amino acid nitrogen contents reach 3.48 g / 100 g and 0.67 g / 100 g respectively. The content of protein hydrolysates shows a consistent upward trend with the salty effect of tofu extract, and it is speculated that the increase in saltiness intensity is related to the content of soluble peptides and amino acids.

[0041] Safety evaluation of strains:

[0042] 1) Hemolytic test: Use an inoculation loop to pick up the bacterial solution and streak it in zones on a defatted fiber sheep blood plate, culture it at 37 °C for 2 d, and observe its hemolysis phenomenon. The results are as Figure 4As shown in the figure. From Figure 4 it can be seen that there are no clear zones or grass-green zones around the colonies, indicating that the six strains do not produce hemolysin and have good safety. The phenomenon that the medium around some colonies appears dark red may be caused by the agglutination of the growth metabolites of the strains.

[0043] 2) Drug sensitivity test: The disk diffusion method was used to determine the sensitivity of the strains to antibiotics. The results were judged according to the standards of the Clinical and Laboratory Standards Institute (CLSI) of the United States and the method of Lin Songquan. The results are as Figure 5 shown in the figure. From Figure 5 it can be seen that some microorganisms contain potential drug-resistant genes, which increases the difficulty of medical treatment in the human body. Therefore, evaluating the drug sensitivity of microorganisms is beneficial to reducing the risk of secondary infection of microorganisms. As shown in Table 2, 8 kinds of antibiotics show certain inhibitory effects on most strains, and inhibition zones are produced for both strains AF5-5 and AF15-5, and the diameters of the inhibition zones are greater than 20 mm, indicating that these two strains have good sensitivity to 8 kinds of antibiotics. Therefore, strains AF5-5 and AF15-5 have relatively good antibiotic sensitivity.

[0044] Table 2:

[0045]

[0046]

[0047] Note: "\ " indicates that it was not measured.

[0048] 3) Biofilm production activity test: Transfer 100 μL of the bacterial suspension to a 96-well plate containing 100 μL of sterile NB medium, and incubate the above six strains at 37 °C for 24 h. After incubation, discard the liquid in the wells, and rinse thoroughly with 0.1 mol / L sterile PBS buffer. Add anhydrous methanol, discard it after 15 min and let it dry. Stain with 1% crystal violet for 10 min, then wash with sterile water and let it dry. Add 33% glacial acetic acid, let it stand at 37 °C for 30 min, and measure the OD590 value. Use sterile NB medium as the negative control (K), and this value is twice the limit value (ODc). Result judgment: Strong biofilm formation ability: OD590 > 4ODc; Medium: 4ODc ≥ OD590 > 2ODc; Weak: 2ODc ≥ OD590 > ODc; No formation ability: ODc ≥ OD590. The results are as Figure 5 shown in the figure. From Figure 5 it can be seen that the measured OD590 values of the six strains are all less than 2ODc, indicating that the six strains have weak biofilm production activity.

[0049] Finally, the optimal strain AF15-5 was selected for molecular biological identification through the above experiments. As Figure 6 shown, the colony of strain AF15-5 was round, light yellow, with a smooth and shiny surface, slightly transparent edges, a raised center, and was easy to pick up. After Gram staining, it was observed that its morphology was short rod-shaped, it was a Gram-negative bacterium, and it had no spores. A phylogenetic tree of strain AF15-5 was constructed based on the 16S rDNA gene sequence, and the results were as Figure 7 shown. Strain AF15-5 had a 99% homology with Stenotrophomonas maltophilia IAM 12423. Finally, strain AF15-5 was identified as Stenotrophomonas maltophilia.

[0050] Example 2

[0051] Biological characteristics of strain AF15-5:

[0052] Strain AF15-5 was cultured according to the step (3) of Example 1, and its growth curve was as Figure 8 shown. As Figure 8 can be seen, when cultured for 0-5 h, the viable count of strain AF15-5 in the medium changed little, indicating that it was in the lag phase of growth. When the culture time was 5-16 h, the growth rate of the strain accelerated and it entered the logarithmic growth phase. After 16 h, the OD600 value of the strain was in a relatively stable range, indicating that the growth rate of the strain began to slow down at this stage, and the growth change tended to be stable, reaching the growth plateau phase.

[0053] Strain AF15-5 was cultured according to the step (3) of Example 1 to explore the effect of different temperatures on strain AF15-5, and the results were as Figure 9 shown. As Figure 9 can be seen, with the increase of temperature, the OD600 value of strain AF15-5 showed a trend of first increasing and then decreasing, reaching the maximum value at 37 °C, and the viable count in the culture medium was the largest at this temperature. When the strain was at 51 °C, the OD600 value was the smallest, and there was no visible change in the culture medium, while the culture media under other temperature conditions showed turbidity. It was speculated that the strain did not grow at 51 °C. Therefore, the growth of this strain was limited under high temperature conditions and it was not heat-tolerant.

[0054] Strain AF15-5 was cultured according to the step (3) of Example 1 to explore the effect of different salt concentrations on strain AF15-5, and the results were as Figure 10 shown. As Figure 10 can be seen, with the increase of salt concentration, the OD600 value of strain AF15-5 showed a downward trend, indicating that the growth of the strain was limited by the salt content. When the salt concentration was 5%-10%, the OD600 value of the strain decreased continuously. When the salt concentration was above 9%, the OD600 value had little difference and reached the minimum value. It can be inferred from this that the strain was not tolerant to high salt.

[0055] The strain AF15-5 was cultured according to the steps of Example 1(3) to explore the effects of different pH values on the strain AF15-5, and the results are as Figure 11 shown. As Figure 11 can be seen, with the increase of the pH value of the culture medium, the OD600 value of the strain AF15-5 first increased and then decreased. Under the neutral and alkaline conditions where the pH value of the culture medium was 6-9, the absorbance value was relatively high, indicating that the optimal growth pH value of the strain AF15-5 was 6-9, and it had good growth ability within this range. When the pH was too acidic or too alkaline, the OD600 value of this strain decreased. Therefore, this strain was alkali-tolerant but not acid-tolerant.

[0056] Example 3

[0057] Isolation and identification of salty peptides in fermented tofu by strain AF15-5:

[0058] (1) Preparation of fermented tofu extract: Adjust the OD600 of the bacterial liquid (the strain AF15-5 was inoculated into NB medium and activated and cultured at 37 °C and 120 r / min for 24 h to obtain the seed liquid) to 0.8, and inoculate it into sterilized tofu at an inoculation amount of 2% and culture it at 30 °C for 3 d for fermentation; another sterilized tofu inoculated with sterile water was used as a blank control (K) and cultured at 30 °C for 3 d. 50 g of fermented tofu and 50 g of sterilized tofu fermented with sterile water were respectively added to 200 mL of deionized water, mixed evenly, cooled after boiling water bath for 30 min, centrifuged at 8000 r / min for 30 min, filtered, and the filtrate was fixed to 250 mL for standby.

[0059] (2) Dialysis and ultrafiltration: The extract was filled into a dialysis bag with a molecular cut-off of 5 KD, and the liquid volume did not exceed 1 / 2 of the volume of the dialysis bag. Ultra-pure water was used as the dialysis solution, and the dialysis solution was changed every 4 h, and the replacement was repeated 4-6 times. The substances inside and outside the dialysis bag were respectively collected for concentration and freeze-drying. The dialysis solution was ultrafiltered and purified by an ultrafiltration centrifugal tube (3 KD), centrifuged at 3500 r / min for 5 min at room temperature, the precipitate and filtrate were collected and freeze-dried. A total of three freeze-dried components were obtained by the above treatment, namely S1 (>5 KD), S2 (3-5 KD) and S3 (<3 KD), and stored at -20 °C for standby. Each component was re-dissolved in ultra-pure water to 10 mg / mL, and sensory evaluation was carried out with reference to the method of taste experiment, and the results are as Figure 12 shown. As Figure 12 can be seen, the salty value of component S3 was the highest, which was 4.38±0.08, so it was selected for the next separation and purification.

[0060] (3) Sephadex G-15 gel chromatography: Weigh an appropriate amount of Sephadex G-15 gel dry powder, add deionized water with a volume 5 times that of the dry powder, soak it at room temperature for 2 days, and continuously stir during this period to make it fully swell. After complete swelling, remove the excess liquid on the upper layer and the non-settled gel impurities. Stir the Sephadex gel evenly and pour it into the chromatography column, and wait for it to settle naturally. Avoid phenomena such as dry column, bubbles, and faults during this process. Before loading the sample, use deionized water as the equilibration liquid, elute for 3 - 5 column volumes, then control the liquid level to be level with the gel surface, and close the lower valve. Redissolve the component S3 with the highest salty sensory score in pure water to make its concentration 50 mg / mL. After passing through a 0.45 μm aqueous filter membrane, slowly add it along the wall into the chromatography column. Open the lower valve to keep the sample liquid level with the gel surface, and load pure water into the chromatography column at a flow rate of 0.46 mL / min for sample elution. Collect one tube of components every 15 minutes for testing. The eluent and the equilibration liquid are pre-filtered through a 0.45 μm aqueous filter membrane to remove impurities and subjected to ultrasonic degassing treatment.

[0061] The elution schematic diagram of component S3 is as Figure 13 shown. As Figure 13 can be seen, four components can be obtained by separating and purifying with Sephadex G-15 column of Sephadex gel, which are named F1, F2, F3, and F4 respectively. The determination of the polypeptide content of the eluted components is carried out with reference to the lowry method. Draw an elution curve based on the absorbance value and time, merge the components of the same elution peak, and after freeze-drying treatment, redissolve each component to 5 mg / mL with ultrapure water for sensory evaluation. The results are shown in Table 3 below. As can be seen from Table 3, component F4 has no obvious salty taste. When components F1 and F2 present flavors, the interaction between the flavors is weak, which can enable sensory evaluators to make a better evaluation of a single flavor, while component F3 has a relatively strong taste, which may be related to various flavor components in the system. The bitterness of component F3 is prominent, which may be related to the hydrophobic amino acids contained.

[0062] Table 3:

[0063]

[0064]

[0065] (4) Identification by high-resolution liquid chromatography-mass spectrometry

[0066] Sample preparation: Add ultrapure water to dissolve the sample to make the sample concentration 2 mg / mL, and pass through a 0.22 μm organic filter membrane for LC-MS / MS instrument analysis and detection.

[0067] Detection method: Use an HSS T3 chromatographic column (2.1×100 mm, 1.8 μm, ) The peptide sequence was identified by an X500R LC-ESIQ-TOF liquid chromatography-mass spectrometry instrument. An aqueous solution of 0.1% (V / V) formic acid was used as mobile phase A, and acetonitrile was used as mobile phase B. Elution was carried out at a flow rate of 0.2 mL / min, with an injection volume of 1 μL and a column temperature of 40 °C. The elution method was as follows: 0 - 4 min, 5.0% B; 4.00 - 6.00 min, 5.0 - 10.0% B; 6.00 - 30.00 min, 10.0 - 40.0% B; 30.00 - 34.00 min, 40.0 - 90.0% B; 34.00 - 40.00 min, 90% B; 40.00 - 42.00 min, 90.0 - 5.0% B; 42.00 - 55.00 min, 5.0% B.

[0068] Collection method: Mass spectrometry was collected using the FullMS / DD-MS2 mode in the positive ion mode. The first-level resolution was set to 70000, the mass-to-charge ratio range was 100 - 1500 m / z, the TopN value was set to 4, the bombardment energy was stepped energy (15 - 27 - 40), the second-level resolution was set to 17500, and the dynamic mass-to-charge ratio collection range.

[0069] Analysis method: Proteo Wizard 3.0 was used to convert the mass spectrometry data from the wiff2 format to the Mgf format and the mzMXL format. Peptidomics identification and analysis were performed using PepOS 3.4 (Guangzhou Zhipeptide Biotechnology Co., Ltd.). The dual-engine setting was used, and the identification length was set to 2 - 30, where the infinite search engine length was 2 - 6, and the identification length for sequence library search was 7 - 30 (the sequence library was downloaded from Uniprot). For the first-level parent ions, the mass tolerance was 10 ppm, the error of the second-level daughter ions was 0.02 Da, the detection rate threshold for mixed ion clusters was 60%, the Bayesian scoring threshold was 50, the maximum drift time of the chromatographic peak was 1.0 min, and the number of parallel deconvolution cores was 60.

[0070] A total of 8 reported umami peptides and umami-enhancing peptides were identified by high-performance liquid chromatography-mass spectrometry, as shown in Table 4 specifically. As can be seen from Table 4, the lengths of the 8 polypeptides all contain 2 - 3 amino acids and are hydrophilic.

[0071] Table 4:

[0072]

[0073]

[0074] A total of 48 peptide sequences were screened for molecular docking based on peptide toxicity, hydrophilicity, and the amino acids near the C-terminus. Among them, the amino acid length distribution of the 48 polypeptides is shown in Figure 14 , and the mass spectrometry identification results are shown in Table 5. The results show that the 48 polypeptides screened are mainly 3 - 5 peptides, and their molecular weights are all below 1 KD.

[0075] Table 5:

[0076]

[0077]

[0078]

[0079] (5) Molecular docking screening

[0080] According to the identification results of high-performance liquid chromatography-mass spectrometry, the reported taste peptide sequences were removed by searching BIOPEP-UWM (http: / / www.uwm.edu.pl / biochemia). The toxicity of the peptide segments was predicted by ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ), and the polypeptide sequences with C-terminal residue amino acids R, K, E, and D were selected. PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) was used to search for the 3D structures of small molecule peptides. Transmembrane channel-like 4 (TMC4) was selected as the receptor for molecular docking, and the structure of Q7Z404·TMC4_HUMAN was obtained from the UniProtKB database. The TMC4 protein model was constructed by AlphaFold (99.6% of the amino acids in the TMC4 protein model are located in the allowed region, and only 0.3% of the amino acids are located in the disallowed region, indicating that the TMC4 protein model is reasonable). The Ramachandran plot of the receptor model was evaluated by the online tool PDBsum. CB-Dock2 (https: / / cadd.labshare.cn / cb-dock2 / php / index.php) and AutoDock Tools were used for virtual docking of the receptor protein and small molecule peptides. Finally, the visualization of the simulated docking results was analyzed by PyMOL. The results are as Figure 15 shown. It can be Figure 15 seen that the salty taste receptor TMC4 forms hydrogen bond interactions with five peptide segments (LRK, DWR, WDR, YDR, YNLRE) respectively and has two or more binding sites. According to the visualization map of the polypeptide ligand and the protein receptor, the binding sites and the strength of hydrogen bond interactions between the two can be obtained. As shown in Table 6, the main binding sites between the salty taste peptide and the salty taste receptor TMC4 are ILE-536, VAL-532, LEU-595, and SER-602.

[0081] Table 6:

[0082]

[0083]

[0084] In this study, the salty taste of 48 peptides was predicted by using the CB-dock2 online platform and Autodock tools software, and 5 peptide sequences were screened based on the binding energy less than -6 kcal / mol. As shown in Table 7, the binding sites of the four peptides LRK, YNLRE, YDR and DWR are consistent in center, which are (13, 6, 9), while the binding site of WDR is (-4, -11, 25).

[0085] Table 7:

[0086]

[0087] Except for YNLRE which was not analyzed due to its high risk of synthesis, the saltiness intensity of the other four synthetic peptides is as follows: Figure 16 As shown, WDR and DWR are similar in the five basic tastes, with no obvious outstanding taste; LRK has outstanding saltiness and umami, with sensory scores of 6.5 and 6.38 respectively. In terms of saltiness score, YDR (5.75) is similar to WDR (5.58) and DWR (5.25), which may be related to the same C-terminal amino acid of the three sequences. In the sensory evaluation results, the saltiness of the four synthetic peptides is higher than that of 0.40% NaCl aqueous solution.

[0088] In order to further understand the taste characteristics of the four synthetic peptides, electronic tongue technology was used for further evaluation. Figure 16 As shown in the figure, WDR and DWR are similar in the five basic tastes, which is consistent with the sensory evaluation results. In the electronic tongue evaluation results, YDR and LRK have similar taste characteristics, with obvious salty taste. Among them, LRK has the highest salty response value of 12.13; YDR is second, with a salty response value of 7.28. Based on the above results, LRK and YDR are preliminarily determined to be new salty peptides.

[0089] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A Stenotrophomonas maltophilia strain AF15-5, characterized in that, The preservation number of this strain is CGMCC NO. 32172, the preservation date is October 11, 2024, and the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Center.

2. Use of Stenotrophomonas maltophilia as described in claim 1 in the production of salty peptides.

3. The application according to claim 2, characterized in that, The Stenotrophomonas maltophilia produces salty peptides in sufu; wherein, the salt concentration in the sufu is less than 5%.

4. A salty peptide, characterized in that, The salty peptide is produced by the Stenotrophomonas maltophilia as described in claim 1; the amino acid sequence of the salty peptide includes any one of LRK and YDR.

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