Strain for efficient synthesis of gamma-glutamyl peptide and application thereof

Abstract

The application discloses a strain for efficiently synthesizing gamma-glutamyl peptides and application thereof, and has the characteristics that the strain is Rhodotorula mucilaginosa EIODSF019, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.32529; the application of the Rhodotorula mucilaginosa EIODSF019 in producing extracellular protease, producing gamma-glutamyl transpeptidase and synthesizing gamma-glutamyl peptides, and in fermented dry-cured ham is also provided; and the Rhodotorula mucilaginosa EIODSF019 has high protease activity and high gamma-glutamyl transpeptidase activity, and can significantly improve umami and rich flavor when applied to fermented meat products.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial screening and application technology, and in particular relates to a strain for efficient synthesis of γ-glutamyl peptide and its application. Background Technology

[0002] Gamma-glutamyl peptides (GGPs) are short peptides linked by gamma-glutamyl bonds. They can activate calcium-sensitive receptors, significantly enhancing the umami and rich flavor of food, and are important substances that produce the "Kokumi" flavor in traditional fermented meat products. GGPs such as γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Cys, γ-Glu-Ala, and γ-Glu-Lys are typical examples of rich-flavor peptides. During the processing of fermented meat products, the formation of these GGPs mainly depends on two stages: In the first stage, muscle proteins, under the action of proteolytic enzymes, convert structural proteins such as myosin, actin, and troponin into small peptides and free amino acids; in the second stage, small peptides and free amino acids, under the action of gamma-glutamyl transferase (GGT), form GGPs, which are formed by the γ-COOH of glutamate or glutamine reacting with the NH4+ of gamma-glutamyl receptors (mainly small peptides or free amino acids). 3+ Dehydration condensation forms a γ-peptide bond, thereby forming γ-glutamyl peptide.

[0003] In the natural fermentation process of traditional dry-cured ham (such as Jinhua ham), its formation depends on protein degradation and the catalysis of gamma-glutamyl transferase (GGT). However, the endogenous GGT activity in meat tissue is low, the fermentation cycle is long, and the conditions are harsh, making it difficult to achieve efficient synthesis of gamma-glutamyl peptides. Currently, although there are technologies such as enzymatic synthesis and microbial fermentation, the direct addition of enzyme preparations is easily inhibited by the high salt and low water activity of the ham processing environment, leading to enzyme activity loss. Therefore, its applicability to the actual production of dry-cured ham is limited.

[0004] Yeast is one of the dominant microbial groups in the fermentation and maturation process of dry-cured ham. Although it has been reported that Rhodotorula glutinis exists on the surface of ham and plays an important role in maintaining the quality stability of dry-cured ham, different strains have significant differences in physiological metabolic mechanisms, environmental adaptability and expression levels of key enzyme systems. Therefore, screening a salt-tolerant yeast strain with both high protease activity and high GGT activity is of great significance for the targeted regulation of γ-glutamyl peptide synthesis in dry-cured ham, shortening the fermentation cycle and improving product quality. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a strain with high protease activity and high γ-glutamyl transpeptidase activity for efficient synthesis of γ-glutamyl peptide and its application, which can significantly improve the umami and rich flavor when applied to fermented meat products.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: One of the objectives of this invention is to provide a strain for the efficient synthesis of γ-glutamyl peptide, wherein the strain is classified and named Rhodotorula glutinis (…). Rhodotorula mucilaginosa The strain EIODSF019, with accession number CGMCC No. 32529, was deposited on November 7, 2024. The depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0007] A second objective of this invention is to provide the application of the aforementioned Rhodotorula glutinis EIODSF019 in the production of extracellular proteases.

[0008] A third objective of this invention is to provide the application of the aforementioned Rhodotorula glutinis EIODSF019 in the production of γ-glutamyl transpeptidase (GGT).

[0009] Furthermore, the optimal reaction temperature for the γ-glutamyl transpeptidase produced by the Rhodotorula glutinis EIODSF019 is 45~50 °C, and the optimal pH value is 7.5~8.0.

[0010] The fourth objective of this invention is to provide the application of the aforementioned Rhodotorula glutinis EIODSF019 in the synthesis of γ-glutamyl peptide.

[0011] Furthermore, the γ-glutamyl peptide includes γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Cys, γ-Glu-Ala, and γ-Glu-Lys.

[0012] The fifth objective of this invention is the application of EIODSF019, a type of red yeast, in fermented and dried ham.

[0013] Furthermore, Rhodotorula glutinis EIODSF019 was inoculated at a density of 10. 5 ~10 7 The ham samples were inoculated with a concentration of CFU / mL after curing and fermented under the following temperature and humidity conditions: 25-35 days at 20-25 ℃ and 70-85% humidity, 25-35 days at 25-30 ℃ and 65-75% humidity, and 15-25 days at 20-25 ℃ and 60-80% humidity.

[0014] Compared with the prior art, the advantages of the present invention are as follows:

[0015] 1. Strong environmental adaptability: This strain can grow well under conditions of 24-40 ℃, 2-10% NaCl concentration, and 0-150 mg / kg sodium nitrite. In particular, its growth performance is significantly better than the control strain under 10% high salinity conditions. P. kudriavzevii XS-5 is an ideal fermentation environment for dry-cured ham.

[0016] 2. Excellent enzyme activity characteristics: This strain exhibits high protease activity and high γ-glutamyl transferase (GGT) activity. The optimal pH for its GGT enzyme is 8.0, and the optimal temperature is 50℃. After isolation and purification, the γ-glutamyl transferase activity produced was 552.90 U / g, and the protease activity was 15.92 U / mL, which are 2.63 times and 3.31 times that of the control strain, respectively, significantly higher than the control strain.

[0017] 3. High substrate affinity: Kinetic studies showed that the Michaelis constant (Km) of this strain GGT was only 4.31 nmol / mL, which was significantly lower than that of the control strain (10.88 nmol / mL), indicating a higher affinity for the substrate and the ability to catalyze transpeptide reactions more efficiently.

[0018] 4. High product accumulation rate: In the fermentation application of Jinhua ham, inoculation with this strain can increase the total content of γ-glutamyl peptide in the final product to 518.18 μg / g, which is 3.8 times that of the uninoculated control group (136.40 μg / g) and 2.86 times that of the control strain group (181.04 μg / g).

[0019] 5. Significantly improved flavor: Inoculation with this strain significantly enhanced the umami, aftertaste, and richness of the ham, with the aftertaste intensity increasing by 1.58 times compared to the control group, effectively improving the sensory quality of the product.

[0020] In summary, this invention provides a highly efficient Rhodotorula glutinis strain for synthesizing γ-glutamyl peptide isolated from Jinhua ham. This strain exhibits excellent salt and nitrite tolerance, high γ-glutamyl transferase (GGT) activity, and can significantly enrich γ-glutamyl peptide. When this strain is inoculated until the fermentation of the ham surface reaches maturity, the total γ-glutamyl peptide content in the product reaches 518.18 μg / g, which is 2.86 times that of the control strain. This significantly enhances the umami, aftertaste, and rich flavor (kokumi flavor) of the dry-cured ham, and has broad application prospects in fermented foods.

[0021] The above-mentioned red yeast is classified and named red yeast ( ). Rhodotorula mucilaginosaThe strain EIODSF019, with accession number CGMCC No.32529, was deposited on November 7, 2024. The deposit address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the depositary institution is the China General Microbiological Culture Collection Center. Attached Figure Description

[0022] Figure 1 (A) shows the colony morphology and hydrolysis zone of Rhodotorula glutinis EIODSF019 on YPD plates, and (B) shows the protease activity of Rhodotorula glutinis EIODSF019. Different letters indicate significant differences between samples in the groups (p < 0.05).

[0023] Figure 2 Phylogenetic tree of Rhodotorula glutinis EIODSF019;

[0024] Figure 3 Tolerance analysis of Rhodotorula glutinis EIODSF019 and control strain, where (A) represents temperature tolerance, (B) represents salt tolerance, and (C) represents nitrite tolerance;

[0025] Figure 4 (A) represents the effect of temperature on GGT enzyme activity, and (B) represents the effect of pH on GGT enzyme activity. Different letters indicate significant differences between samples in the groups (p < 0.05).

[0026] Figure 5 The enzyme kinetics of Rhodotorula glutinis EIODSF019 and the control strain GGT were compared and analyzed. (A) is the control strain and (B) is Rhodotorula glutinis EIODSF019.

[0027] Figure 6 The bar chart shows the comparison of purified enzyme activities between Rhodotorula glutinis EIODSF019 and the control strain. Different letters indicate significant differences between the samples in the groups (p < 0.05).

[0028] Figure 7 To analyze the content of γ-glutamyl peptide synthesized by GGT enzyme in vitro under different temperatures and NaCl concentrations, (A) shows the effect of different temperatures on the content of different γ-glutamyl peptides, (B) shows the effect of different temperatures on the total content of γ-glutamyl peptides, (C) shows the effect of different NaCl concentrations on the content of different γ-glutamyl peptides, and (D) shows the effect of different NaCl concentrations on the total content of γ-glutamyl peptides. Different letters indicate significant differences between samples in different groups (p < 0.05).

[0029] Figure 8 The cumulative amount of γ-glutamyl peptide in fermented ham inoculated with Rhodotorula glutinis EIODSF019 and control strains and uninoculated yeast was represented by different letters, indicating significant differences between samples in the groups (p < 0.05).

[0030] Figure 9 To illustrate the changes in the flavor characteristics of ham after inoculation with Rhodotorula glutinis EIODSF019 and the control strain versus no yeast inoculation, different letters indicate significant differences between the samples in the groups (p < 0.05). Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] Example 1: Isolation, screening and identification of strains.

[0033] Take 10 g of naturally matured Jinhua ham sample, add 90 mL of 0.9% sterile physiological saline, and homogenize using a homogenizer for 2 min. After serially diluting the suspension, spread it on YPD agar medium (0.01% chloramphenicol) containing 2 wt% skim milk powder and incubate at 28℃ for 48 h. Observe the colony morphology and select single colonies with obvious transparent hydrolysis zones.

[0034] The strains obtained from the initial screening were inoculated into YPD liquid medium and cultured at 28℃ and 140 rpm for 48 h with shaking. The supernatant was collected by centrifugation (8000 g, 20 min, 4℃). The protease activity in the supernatant was determined using the Folin-Ciocalteu method: 1 mL of sample supernatant was mixed with 1 mL of 2 wt% casein solution, reacted at 40℃ for 10 min, the reaction was terminated by adding trichloroacetic acid, and the absorbance at 680 nm was measured. A strain with high fermentation performance, deposited at the China Industrial Microbial Culture Collection Center, was used. Pichia kudriavzevii XS-5 (denoted as PK) was used as a control to screen for the strain with the highest extracellular protease activity. The results are as follows: Figure 1 As shown in (A), a strain EIODSF019 was isolated through initial plate screening. This strain produced a distinct transparent hydrolysis zone on a plate containing skim milk powder. Morphological observation revealed that the colonies of this strain were red, spherical, and smooth with a moist surface, indicating its ability to secrete extracellular proteases. Further testing of the protease activity in the secondary screening fermentation broth yielded the following results: Figure 1 As shown in (B), the protease activity of EIODSF019 was as high as 15.92 U / mL, which was significantly higher than that of the control strain PK (4.81 U / mL).

[0035] Genomic DNA was extracted from this strain, and the 26S rDNA sequence was amplified using primers NL1 and NL4 and sequenced. Its nucleotide sequence is shown in SEQ ID NO.1.

[0036] The sequences were compared with the NCBI database, and a phylogenetic tree was constructed to determine their taxonomic position. The phylogenetic tree is as follows: Figure 2 As shown. 26S rDNA sequencing and phylogenetic tree analysis indicated that strain EIODSF019 is related to... Rhodotorula mucilaginosa They clustered together, with a homology greater than 99%, and were identified as Rhodotorula glutinis.

[0037] This strain is currently deposited at the China General Microbiological Culture Collection Center and is classified as *Rhodotorula glutinis*. Rhodotorula mucilaginosa The strain EIODSF019, with accession number CGMCCNo.32529, was deposited on November 7, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the depository is the China General Microbiological Culture Collection Center.

[0038] Example 2: Analysis of strain tolerance.

[0039] To assess the adaptability of the strain to the ham fermentation environment, Rhodotorula glutinis EIODSF019(RE) and the control strain were compared. P. kudriavzevii A comparative test was conducted on the XS-5 (PK).

[0040] 1. Temperature tolerance: The activated EIODSF019 and control strain PK were inoculated onto YPD plates and cultured at different temperatures (24, 28, 32, 36, 40 ℃) for 3 days. The changes in colony diameter were observed.

[0041] 2. Salt tolerance: The strain was inoculated onto YPD plates containing different concentrations of NaCl (2%, 4%, 6%, 8%, 10%) and cultured at 28 °C for 3 days. The growth was then observed.

[0042] 3. Nitrite tolerance: The strain was inoculated onto YPD plates containing different concentrations of NaNO2 (0, 50, 100, 150 mg / kg), and cultured at 28 ℃ for 3 days. The growth was then observed.

[0043] like Figure 3 As shown in (A), EIODSF019 can grow in the range of 24-40 ℃, with the optimal growth temperature being 32 ℃. Figure 3 As shown in (B), regarding salt tolerance, when the NaCl concentration reached 10%, the growth of the control strain PK was significantly inhibited, resulting in small colonies; while strain EIODSF019 maintained good growth, with full colonies. Figure 3 As shown in (C), regarding nitrite tolerance, the growth of EIODSF019 was not inhibited at NaNO2 concentrations of 0-150 mg / kg, and the addition of appropriate amounts of NaNO2 had a certain promoting effect on its growth. This indicates that EIODSF019 is highly adaptable to the high-salt, high-osmotic-pressure environment in the later stages of dry-cured ham fermentation.

[0044] Example 3: Enzymatic properties and kinetic analysis of γ-glutamyl transpeptidase (GGT).

[0045] 1. Purification of γ-glutamyl transferase (GGT): EIODSF019 was cultured at 28 ℃, 140 rpm, for 48 h, and the supernatant was collected by centrifugation. Ammonium sulfate (80% saturation) was added for salting out precipitation. The precipitate was reconstituted and filtered through a 0.45 μm filter membrane. The filtrate was loaded onto a Sephadex G-75 gel filtration chromatography column and eluted with 0.02 M PBS buffer (pH 7.0). Different fractions were collected based on peak appearance and lyophilized, then stored at -20 ℃. The lyophilized fractions were dissolved in PBS to obtain EIODSF019 GGT enzyme solution, which was used for GGT enzyme activity determination.

[0046] 2. Enzymatic property analysis

[0047] (1) Effects of pH and temperature on GGT enzyme activity: Different pH systems were constructed using 0.1 M sodium citrate buffer (pH 3-5), 0.1 M phosphate buffer (pH 6-8), and 0.1 M glycine-NaOH buffer (pH 9-11). The optimal pH of GGT enzyme was evaluated using γ-glutamyl-p-nitroaniline (γ-GpNA) as a substrate. After thoroughly mixing the enzyme solution with the substrate solutions at each pH, ​​the mixture was incubated at 37 °C for 30 min.

[0048] GGT activity assay: 450 μL of 10 mM γ-glutamyl-p-nitroaniline (γ-GpNA), 450 μL of 40 mM Gly-Gly, and 100 μL of enzyme solution were mixed and incubated at 37 °C for 30 min. The reaction was then terminated by adding 450 μL of 0.1 M HCl. The absorbance of the mixture was measured at 410 nm using a 96-well plate. The control group was treated the same way as the experimental group, but the enzyme solution was preheated at 90 °C for 10 min to inactivate the enzyme. One unit of enzyme activity (U / g) was defined as 1 μmol of p-nitroaniline released from γ-GpNA per minute. Results are expressed as relative enzyme activity (maximum enzyme activity was set at 100%). The effect of temperature on γ-glutamyl transpeptidase activity was evaluated using a temperature gradient experiment from 20–70 °C. After mixing the GGT enzyme solution with the substrate solution, the mixture was incubated at different temperatures for 10 min, and the relative enzyme activity was calculated as described above. Results are shown below. Figure 4 (A) and Figure 4 As shown in (B), the enzymatic property test results indicate that the optimal reaction pH for GGT production by EIODSF019 is 8.0 and the optimal temperature is 50 °C.

[0049] (2) Determination of GGT enzyme kinetic parameters: Using different concentrations (2.5-40 mg / mL) of γ-GpNA as substrates, the Michaelis-Menten equation was fitted by the initial rate method, and the maximum reaction rate (Vmax) and Michaelis constant (Km) of γ-glutamyl transpeptidase were calculated by the double reciprocal plotting method (Lineweaver-Burk method).

[0050] The results are as follows Figure 5 (A) and Figure 5 As shown in (B), kinetic analysis of the purified enzyme revealed that the GGT Michaelis constant (Km) of EIODSF019 was 4.31 nmol / mL, significantly lower than that of the control strain PK (10.88 nmol / mL). A lower Km value indicates higher enzyme affinity for the substrate, meaning that in practical fermentation systems with limited substrate concentrations, the GGT of EIODSF019 can bind to the substrate more efficiently and catalyze the reaction. Furthermore, as... Figure 6 As shown, after purification, the GGT specific activity of EIODSF019 was as high as 552.90 U / g, which is 2.63 times that of the PK strain (210.48 U / g).

[0051] Example 4: Construction of an in vitro synthesis system for γ-glutamyl peptide and analysis of the product

[0052] A mixed substrate solution system containing Glu, Gln, Cys, Ala, and Lys was constructed (each substrate concentration was 20 mM, PBS buffer pH 8.0). The purified EIODSF019 GGT enzyme solution from Example 3 was added, and different reaction temperatures (30, 40, 50 °C) and different NaCl concentrations (0, 0.6, 1.2 M) were set. After reacting for 10 h, the enzyme was inactivated by heating at 90 °C for 5 min. The reaction product was filtered through a 0.45 μm filter and then lyophilized for concentration. The lyophilized and concentrated sample was reconstituted using 600 μL of 0.15 M K2HPO4 / NaH2PO4 buffer (pH 7.4, containing 50% D2O and 0.1% sodium 3-(trimethylsilyl)propane-1-sulfonate (DSS)). After centrifugation at 4 °C and 10000 rpm for 10 min, the supernatant was collected. Take 500 μL of supernatant and transfer it to an NMR tube with an outer diameter of 5 mm. Then, analyze the NMR signal by nuclear magnetic resonance (NMR). 1 The content of each γ-glutamyl peptide (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Cys, γ-Glu-Ala and γ-Glu-Lys) was quantitatively analyzed by ¹H NMR.

[0053] like Figure 7 (A) and Figure 7As shown in Figure (B), the in vitro synthesis experiments revealed that increased temperature favored peptide synthesis. Compared to 30 °C, the total content of γ-glutamyl peptide increased by 19.20% and 10.34% at 40 °C and 50 °C, respectively. This indicates that 40 °C is the optimal temperature for the enzyme to catalyze the transpeptidation reaction. Notably, at high temperature (50 °C), the enzyme not only maintained high synthesis levels of γ-Glu-Cys and γ-Glu-Gln, but also exhibited excellent stability in the synthesis of γ-Glu-Lys and γ-Glu-Glu. This demonstrates that the GGT enzyme possesses excellent thermostability and good affinity for various amino acid substrates, including Lys and Glu, which is beneficial for the continuous synthesis of various flavor peptides during the high-temperature fermentation stage.

[0054] like Figure 7 (C) and Figure 7 As shown in Figure (D), regarding the effect of salt concentration, in the NaCl concentration range of 0-1.2 M, although the total peptide content decreased with increasing salt concentration (9.52% and 20.31% lower than 0 M under 0.6 M and 1.2 M NaCl conditions, respectively), it is noteworthy that under the high-salt environment of 1.2 M, in addition to γ-Glu-Cys and γ-Glu-Gln, γ-Glu-Lys and γ-Glu-Glu also maintained high levels. This indicates that under high salt stress, the GGT enzyme of EIODSF019 exhibits high selective affinity not only for Cys and Gln but also for Lys and Glu substrates, which is beneficial for synthesizing richer key flavor peptides in the high-salt environment of ham.

[0055] Example 5: Analysis of the cumulative amount of γ-glutamyl peptide in ham.

[0056] 1. Preparation of bacterial culture: EIODSF019 Rhodotorula glutinis was inoculated into YPD liquid medium for activation, the cells were collected by centrifugation, washed with sterile physiological saline and resuspended, and the concentration of the bacterial suspension was adjusted to 10. 6 CFU / mL. The control strain PK was treated in the same way.

[0057] 2. Ham Production and Inoculation: Using pig hind legs as raw material, the shaped raw legs were placed on a steel frame and left to stand for 48 hours in a cold storage room at a temperature of 0-4 ℃ and a relative humidity of 85-90%. The pre-cooled pig legs were then cured with salt at a ratio of 0.15 g KNO3, 0.15 g NaNO2, and 60 g NaCl per kilogram of leg meat, applied in five batches. When no visible salt particles remained on the ham surface, all hams were further cured for approximately 40 days at 0-4 ℃ and a relative humidity of 75-85% to achieve salt balance. After salt balance, the ham surface was washed and air-dried to obtain ham samples. The ham samples were randomly divided into three groups: a natural fermentation group (CK) sprayed with an equal volume of sterile physiological saline; and a control strain group (PK) sprayed with PK bacterial solution at an inoculation amount of 10 g / kg. 6 CFU / kg. The experimental group (RE) was sprayed with bacterial suspension of the present invention strain EIODSF019, with an inoculum size of 10... 6 CFU / kg.

[0058] 3. Fermentation and maturation: Place the inoculated ham in a temperature- and humidity-controlled fermentation room and ferment according to the following procedure: ferment for 30 days at 20-25℃ and 70-85% humidity; ferment for 30 days at 25-30℃ and 65-75% humidity; and mature for 20 days at 20-25℃ and 60-80% humidity.

[0059] 4. Index Determination: After fermentation and maturation, 10 g of minced biceps femoris muscle was taken, and 15 mL of methanol / water (volume ratio 2:1) was added. The mixture was homogenized at 12000 rpm on ice for 2 min (homogenization 30 s, rest 30 s), then centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was collected, and the process was repeated twice. Finally, the supernatants were combined, and methanol was removed by rotary evaporation. The extract was then freeze-dried. The freeze-dried sample was reconstituted in 600 μL of 0.15 M K2HPO4 / NaH2PO4 buffer (pH 7.4, containing 50% D2O and 0.1% sodium 3-(trimethylsilyl)propane-1-sulfonate). After centrifugation at 4 ℃ and 12000 rpm for 10 min, 500 μL of the supernatant was transferred to a 5 mm outer diameter NMR tube for NMR analysis. The flavor quality of ham was evaluated using an electronic tongue and a sensory evaluation panel (10 people). The electronic tongue testing method was as follows: ham samples were thawed at 4 ℃, connective tissue and fat were removed, 20 g of ham biceps femoris muscle sample was weighed and minced, 120 mL of distilled water was added, and the mixture was extracted for 20 min under electromagnetic stirring. After centrifugation at 8000 g for 20 min, the mixture was filtered. 80 mL of filtrate from each sample was used for electronic tongue testing, with a sampling time of 120 s and a washing time of 10 s. Each sample was washed after testing, and the intensity of bitterness, aftertaste, umami, and richness was recorded. The sensory evaluation method involved a sensory evaluation panel consisting of 5 women and 5 men aged 25-50 years with evaluation experience. Slices approximately 0.5 cm thick were tasted at room temperature. The panel members rated the flavor intensity and overall acceptability of each sample on a scale of 0–10 (1–3: weak; 4–6: medium; 7–10: strong) based on their senses.

[0060] 5. Analysis of the cumulative amount of γ-glutamyl peptide in ham

[0061] At the end of fermentation and maturation, the total content of γ-glutamyl peptide in each group of hams was determined as follows: Figure 8 As shown, the total content of the CK group (natural fermentation) was 136.40 μg / g; the total content of the PK group (control strain) was 181.04 μg / g; while the total content of the RE group inoculated with the strain of this invention was as high as 518.18 μg / g.

[0062] The total γ-glutamyl peptide content in the RE group was 3.8 times that of the CK group and 2.86 times that of the PK group. Specific component analysis showed that γ-Glu-Lys, γ-Glu-Gln, and γ-Glu-Glu accumulated most significantly in the RE group, at 8.34 times, 8.27 times, and 7.17 times that of the control group, respectively. This confirms that EIODSF019 can efficiently exert its GGT enzyme activity in actual fermentation systems, significantly promoting the synthesis of target active peptides.

[0063] 6. Sensory and electronic tongue analysis of ham

[0064] Electronic tongue and sensory evaluation results, such as Figure 9 As shown in the figure, compared with the CK and PK groups, the RE group ham inoculated with EIODSF019 showed significant improvements in umami, aftertaste, and richness. Specifically, compared with the CK group, the RE group ham exhibited a 0.65-fold increase in umami intensity, a 1.58-fold increase in aftertaste intensity, and a 0.52-fold increase in richness. Simultaneously, the bitterness intensity was significantly reduced. This indicates that strain EIODSF019 effectively enhances the richness and overall flavor quality of dry-cured ham by promoting the production of γ-glutamyl peptide.

[0065] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A strain for efficient synthesis of γ-glutamyl peptides, characterized in that The strain is Rhodotorula mucilaginosa (R. mucilaginosa) Rhodotorula mucilaginosa ) EIODSF019, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 32529.

2. The use of Rhodotorula mucilaginosa EIO D SF019 in the production of extracellular protease according to claim 1.

3. The use of Rhodotorula mucilaginosa EIO D SF019 in the production of γ-glutamyl transpeptidase according to claim 1.

4. The use of the Rhodotorula mucilaginosa EIODSF019 according to claim 3 for the production of gamma-glutamyltranspeptidase, characterized by: The γ-glutamyl transpeptidase produced by the Rhodotorula mucilaginosa EIO D SF019 has a reaction temperature of 45 ~ 50℃ and a pH value of 7.5 ~ 8.

5. The use of Rhodotorula mucilaginosa EIO D SF019 in the synthesis of γ-glutamyl peptide according to claim 1.

6. The use of the Rhodotorula mucilaginosa EIODSF019 according to claim 5 for the synthesis of gamma-glutamyl peptides, characterized in that: The γ-glutamyl peptide includes γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Cys, γ-Glu-Ala and γ-Glu-Lys.

7. The use of Rhodotorula mucilaginosa EIO D SF019 in the fermentation of dry-cured ham according to claim 1.

8. The use of Rhodotorula mucilaginosa EIODSF019 according to claim 7 in the fermentation of dry-cured hams, characterized in that: The Rhodotorula mucilaginosa EIODSF019 was inoculated to the surface of the cured ham sample at a concentration of 10 5 ~10 7 CFU / mL, and fermented and matured under the following temperature and humidity conditions: 20-25 ℃ and 70-85% humidity for 25-35 days, 25-30 ℃ and 65-75% humidity for 25-35 days, and 20-25 ℃ and 60-80% humidity for 15-25 days.

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