Pediococcus pentosaceus Ppe-YC39 capable of enhancing fish tea nutritional activity and flavor and inhibiting formation of biogenic amines and application thereof

Abstract

The application discloses a Pediococcus pentosaceus Ppe-YC39 capable of enhancing the nutritional activity and flavor of fish tea and inhibiting the formation of biological amines and an application thereof. The Pediococcus pentosaceus Ppe-YC39 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 66920. The Pediococcus pentosaceus Ppe-YC39 has the same or even better probiotic properties as a commercial strain of Lactobacillus plantarum LGG, and when applied to fish tea fermentation, the content of amino acids and fatty acids in the fermented fish tea is significantly increased, and the content of biological amines is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of food microbiology, and in particular to a strain of Pediococcus pentosacchariformis Ppe-YC39 that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines, and its applications. Background Technology

[0002] Fish tea is a traditional specialty food made from fish, rice, and salt through anaerobic fermentation, resulting in a unique sour, umami, and grain aroma. Fermented fish tea is rich in bioactive compounds and has significant nutritional value; however, its traditional production methods present numerous challenges, limiting industrialization and wider application. A key research gap lies in the uncontrolled fermentation process of fish tea, relying on natural microbial activity or continuous fermentation. While this contributes to rich microbial diversity and unique sensory characteristics, it also leads to unstable product quality, excessively long fermentation cycles, and potential safety risks such as pathogenic contamination or the accumulation of harmful biogenic amines. These issues hinder standardized production and commercialization, highlighting the necessity of optimizing fermentation strategies to ensure food safety and quality. Previous studies have emphasized the importance of microbial regulation in fermented foods, but systematic research on the challenges associated with tropical fermented products like fish tea is limited. Therefore, there is an urgent need to develop controllable fermentation methods that improve efficiency, stability, and safety while preserving the ideal sensory characteristics and nutritional value of these traditional foods. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide a strain of Pediococcus pentosacchariformis Ppe-YC39 that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines, and its application, aiming to solve the efficiency, stability and safety problems existing in the traditional fish tea fermentation process.

[0004] The technical solution of the present invention is as follows:

[0005] Firstly, a strain of Pediococcus pentosaceus is provided that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines. Pediococcus pentosaceus Pediococcus pentosaceus Ppe-YC39 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66920 and deposit date of September 5, 2025.

[0006] In a second aspect, a microbial agent is provided, said microbial agent comprising Pediococcus pentosaceus Ppe-YC39 as described in the first aspect.

[0007] Optionally, the microbial agent may also include food-acceptable additives.

[0008] Thirdly, a fermentation broth is provided, which is obtained by fermentation of Pediococcus pentosaceus Ppe-YC39 as described in the first aspect.

[0009] Fourthly, the application of Pediococcus pentosaceus Ppe-YC39 as described in the first aspect, the inoculant as described in the second aspect, or the fermentation broth as described in the third aspect in the preparation of fermented foods.

[0010] Optionally, the fermented food is fish tea.

[0011] Fifthly, a method for preparing fish tea is provided, including the following steps:

[0012] (1) Mix the fish meat with salt and marinate at 20-40℃ for 40-80 minutes to obtain marinated fish pieces;

[0013] (2) Steam the rice at 90-110℃ for 10-20 minutes to obtain steamed rice;

[0014] (3) Inoculate the Pediococcus pentosacchari Ppe-YC39 as described in the first aspect into MRS liquid medium, culture at 30-45℃ for 16-18h, centrifuge and discard the supernatant, wash, and then resuspend the precipitate in water to obtain Ppe-YC39 bacterial suspension.

[0015] (4) Mix the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension, and anaerobic ferment at 15-25℃ for 18-22 days to obtain fish tea.

[0016] Optionally, in step (1), the mass ratio of the surface-dehydrated fish meat to salt is 1:(0.07-0.11).

[0017] The method for preparing the water-free fish meat includes the following steps: washing and cutting fresh fish meat into pieces, draining the water until no water seeps out.

[0018] Optionally, in step (3), the specific conditions for centrifugation include: a rotation speed of 3500-4500 rpm, a temperature of 20-30℃, and a time of 10-20 min.

[0019] Optionally, in step (4), the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension are all mixed at 20-40°C;

[0020] The mass ratio of the marinated fish pieces to the steamed rice is 1:(1.5-2).

[0021] The volume-to-mass ratio of the Ppe-YC39 bacterial suspension and the total weight of the pickled fish pieces to the steamed rice is (0.02-0.08) mL:1g;

[0022] The bacterial concentration of the Ppe-YC39 bacterial suspension was 10. 3 -10 5 CFU / mL;

[0023] The specific conditions for the anaerobic fermentation include: oxygen volume ratio <0.5%.

[0024] Beneficial Effects: This invention provides a *Pediococcus pentosaceus* strain, Ppe-YC39, which enhances the nutritional activity and flavor of fish tea and inhibits the formation of biogenic amines. Compared to the existing commercial strain *Lactobacillus plantarum* LGG, *Pediococcus pentosaceus* strain Ppe-YC39 of this invention exhibits comparable to or even superior probiotic properties in most aspects. Furthermore, applying *Pediococcus pentosaceus* strain Ppe-YC39 of this invention to fish tea fermentation significantly increases the amino acid and fatty acid content of the fermented fish tea while significantly reducing the biogenic amine content. Attached Figure Description

[0025] Figure 1 This is the growth curve of Ppe-YC39.

[0026] Figure 2 This is a graph showing the results of the artificial gastric juice tolerance assessment for Ppe-YC39.

[0027] Figure 3 This is a graph showing the results of the artificial intestinal fluid tolerance assessment for Ppe-YC39.

[0028] Figure 4 This is a graph showing the evaluation results of the self-cohesion ability of Ppe-YC39.

[0029] Figure 5 This is a graph showing the evaluation results of the co-cohesion capacity of Ppe-YC39.

[0030] Figure 6 This is a graph showing the evaluation results of the hydrophobicity of Ppe-YC39.

[0031] Figure 7 This is a graph showing the results of the hemolytic activity assessment of Ppe-YC39. Detailed Implementation

[0032] This invention provides a strain of Pediococcus pentosacchariformis Ppe-YC39 that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines, and its application. To make the purpose, technical solution and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0033] Fish tea is a traditional fermented food. The quality and flavor of fermented foods are largely determined by key metabolites, including short-chain fatty acids, free amino acids, and biogenic amines.

[0034] Short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid) are primarily produced by microorganisms like lactic acid bacteria through the fermentation of carbohydrates. Studies have shown that higher levels of short-chain fatty acids are generally associated with superior flavor characteristics in fermented foods, as their pleasant acidity balances other tastes. Furthermore, their ability to lower the pH of the food matrix helps inhibit the growth of spoilage and pathogenic bacteria, thereby extending the product's shelf life. Beyond enhancing flavor and preservation, short-chain fatty acids are also associated with potential health benefits, such as maintaining gut microbiota balance and promoting gut health, which further enhances the nutritional value of fermented foods.

[0035] Free amino acids are another key class of metabolites, playing a dual role in nutrition and flavor. As the basic building blocks of proteins, free amino acids in food are essential for human health, contributing to various physiological functions such as promoting intestinal nutrient absorption, supporting energy metabolism, and maintaining nitrogen balance. In terms of flavor, different free amino acids possess unique taste characteristics—for example, glutamic acid and aspartic acid are key sources of umami, while glycine and alanine contribute to sweetness. Studies have shown that a balanced and abundant free amino acid composition is a hallmark of high nutritional value and a pleasant, complex taste in fermented foods, making it an important indicator for quality assessment.

[0036] In contrast, biogenic amines are a class of metabolites that require close monitoring due to their potential safety risks. Biogenic amines are primarily formed by the decarboxylation of amino acids by certain bacteria. Common biogenic amines in fermented foods include putrescine, histamine, and cadaverine. Furthermore, high concentrations of biogenic amines indicate potential spoilage or inadequate fermentation control, further compromising food quality and safety. Therefore, controlling biogenic amines such as putrescine at low levels is crucial for ensuring the safety of fermented products. Studies have shown that lactic acid bacteria can improve the metabolic profile by promoting the production of short-chain fatty acids and free amino acids and inhibiting the formation of biogenic amines, making them an ideal choice for inoculation fermentation. However, the application of lactic acid bacteria starter cultures in tropical fermented foods such as fish tea and shrimp paste still requires further investigation, particularly in optimizing fermentation conditions to achieve a balance between flavor, nutrition, and safety.

[0037] Based on this, embodiments of the present invention provide a strain of Pediococcus pentosaceus that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines. Pediococcus pentosaceus Pediococcus pentosaceus Ppe-YC39 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66920 and deposit date of September 5, 2025.

[0038] Based on the same inventive concept, embodiments of the present invention provide a microbial agent, the microbial agent comprising Pediococcus pentosaccharis Ppe-YC39 as described above.

[0039] In some embodiments, the microbial agent also includes food-acceptable additives.

[0040] Based on the same inventive concept, this embodiment of the invention provides a fermentation broth, which is obtained by fermentation of Pediococcus pentosacchari Ppe-YC39 as described above.

[0041] Based on the same inventive concept, embodiments of the present invention provide the application of Pediococcus pentosaceus Ppe-YC39, the bacterial agent, or the fermentation broth as described above in the preparation of fermented foods.

[0042] In some embodiments, the fermented food is fish tea.

[0043] Based on the same inventive concept, this invention provides a method for preparing fish tea, comprising the following steps:

[0044] (1) Mix the fish meat with salt and marinate at 20-40℃ for 40-80 minutes to obtain marinated fish pieces;

[0045] (2) Steam the rice at 90-110℃ for 10-20 minutes (that is, steam the rice until it is 60-80% cooked) to obtain steamed rice;

[0046] (3) Inoculate the Pediococcus pentosacchari Ppe-YC39 as described above into MRS liquid medium, culture at 30-45℃ for 16-18h, centrifuge and discard the supernatant, wash, and then resuspend the precipitate in water to obtain Ppe-YC39 bacterial suspension;

[0047] (4) Mix the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension, and anaerobic ferment at 15-25℃ for 18-22 days to obtain fish tea.

[0048] In some embodiments, in step (1), the mass ratio of the surface-dehydrated fish meat to salt is 1:(0.07-0.11).

[0049] The method for preparing the water-free fish meat includes the following steps: washing and cutting fresh fish meat into pieces, draining the water until no water seeps out.

[0050] In some implementations, the specific conditions for centrifugation in step (3) include: a rotation speed of 3500-4500 rpm, a temperature of 20-30℃, and a time of 10-20 min.

[0051] In some embodiments, in step (4), the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension are all mixed at 20-40°C;

[0052] The mass ratio of the marinated fish pieces to the steamed rice is 1:(1.5-2).

[0053] The volume-to-mass ratio of the Ppe-YC39 bacterial suspension and the total weight of the pickled fish pieces to the steamed rice is (0.02-0.08) mL:1g;

[0054] The bacterial concentration of the Ppe-YC39 bacterial suspension was 10. 3 -10 5 CFU / mL;

[0055] The specific conditions for the anaerobic fermentation include: oxygen volume ratio <0.5%.

[0056] The present invention will be further described below through specific embodiments.

[0057] The culture medium used in the examples was prepared with water and its components are as follows:

[0058] MRS liquid culture medium: peptone 10 g / L, Tween 80 1 mL / L, yeast extract 4 g / L, triammonium citrate 2 g / L, manganese sulfate 0.05 g / L, beef meal 5 g / L, dipotassium ammonium phosphate 2 g / L, glucose 20 g / L, magnesium sulfate 0.2 g / L, sodium acetate 5 g / L.

[0059] MRS plates: Add 15 g / L of agar to the MRS liquid medium.

[0060] LB agar plate: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar 15g / L.

[0061] Example 1

[0062] This embodiment provides a strain of Pediococcus pentosacchari, Ppe-YC39, which can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines, and its application, as detailed below:

[0063] 1. Screening and identification of strains

[0064] Fish tea samples were collected from Hainan, China, and lactic acid bacteria were isolated and purified in February 2024. The 16S rDNA region was amplified by PCR using forward primer 1492R and reverse primer 27F, and the isolated strains were identified. The DNA sequence of the 16S rRNA of strain Ppe-YC39 is shown in SEQ ID NO.1 (submitted to the GenBank database, accession number PX210472), and its Latin taxonomical name was determined by comparative analysis to be *Pediococcus pentosaceus*. Pediococcus pentosaceusPpe-YC39 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66920 and deposit date of September 5, 2025.

[0065] 2. Evaluation of Probiotics

[0066] 2.1 Evaluation of antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA)

[0067] The antibacterial activity of Ppe-YC39 against MRSA was evaluated using the agar well diffusion method. All isolated strains were cultured in MRS liquid medium at 37°C for 24 h to obtain fermentation broth. MRSA was inoculated onto LB agar plates at a final concentration of 1×10⁻⁶. 6 CFU / mL. After LB agar plates solidified, holes (8 mm in diameter) were punched in the agar, and 100 μL of fermentation broth was added. Vancomycin (50 μg / mL) was used as a positive control, and MRS as a negative control. The inhibition zone diameter was measured after incubation at 37°C overnight.

[0068] The results showed that the Ppe-YC39 fermentation broth had significant antibacterial activity (see Table 1).

[0069] 2.2 Assessment of biofilm formation capacity

[0070] Biofilm content was determined using crystal violet staining. Ppe-YC39 cells cultured overnight (1%, v / v) were inoculated into MRS plates and incubated at 37°C for 24 h in 96-well plates. After incubation, the bacterial culture was discarded and the cells were washed with physiological saline. The plates were then fixed at 65°C. Biofilms were stained with 0.1% (w / v) crystal violet for 5 min, and excess dye was washed away with PBS. The plates were dried at 37°C for 20 min, and the stained crystal violet was dissolved in 30% (v / v) acetic acid solution. The absorbance was measured at 570 nm. A commercial strain, *Lactobacillus rhamnosus* GG (LGG), isolated from the commercial product Cultulelle, was used as a positive control.

[0071] The results showed that Ppe-YC39 had a significantly higher biofilm formation capacity than the commercial strain LGG (see Table 1).

[0072] 2.3 Determination of growth curve and acid production capacity

[0073] The initial concentration was 1×10 6 Ppe-YC39 at CFU / mL was inoculated into MRS liquid medium and incubated at 37°C for 18 h. OD was measured every 2 h using a microplate reader. 600 The pH value was measured after 24 hours.

[0074] The results showed that Ppe-YC39 grew well and its acid production capacity was comparable to that of commercial LGG (see [link to study]). Figure 1 (and Table 1).

[0075] Table 1. Determination of antibacterial activity, acid production capacity, and biofilm formation ability of Ppe-YC39

[0076]

[0077] 2.4 Gastrointestinal Tolerance Assessment

[0078] To assess the strain's tolerance to the gastrointestinal environment, a suspension of Ppe-YC39 bacteria (bacterial concentration 10) was prepared. 9 CFU / mL (preparation method as described in Section 2.5 below) and LGG bacterial suspension (bacterial concentration 10). 9 CFU / mL, prepared as described in section 2.5 below, was added to simulated gastric and intestinal fluid environments at a volume ratio of 1:9. Simulated gastric fluid was prepared by adding 0.35% (w / v) pepsin and 0.2% (w / v) NaCl to MRS liquid medium at pH 2.0. Simulated intestinal fluid was prepared by adding 0.3% (w / v) ox bile salt to MRS liquid medium adjusted to pH 7.0. After incubation at 37°C for 3 hours, the viable bacterial count was determined using a method slightly modified from that described in Yang et al.'s work (DOI:10.1016 / j.scitotenv.2023.167587). Results are expressed as survival percentage (%).

[0079] Survival rate percentage (%) = Nt / N0 × 100;

[0080] N0 and Nt represent the number of viable bacteria at 0h and 3h, respectively.

[0081] The results showed that Ppe-YC39 exhibited significantly better gastrointestinal tolerance than the commercial strain LGG (see [link to study]). Figure 2 and Figure 3 ).

[0082] 2.5 Assessment of Self-Consolidation and Co-Consolidation Capabilities

[0083] The self-aggregation and co-aggregation of the isolated strains were evaluated using the method of Fan et al. (DOI:10.3390 / foods11213476). Ppe-YC39 was inoculated into MRS liquid medium and cultured at 37°C for 16–18 h. After centrifugation at 4000 rpm for 15 min, the supernatant was removed, and the suspension was resuspended in PBS to obtain the Ppe-YC39 bacterial suspension. The preparation method for the LGG bacterial suspension was the same as that for the Ppe-YC39 suspension, except that Ppe-YC39 was replaced with LGG. The self-aggregation ability of each strain was evaluated by measuring the absorbance at 600 nm at 0 h and 4 h. The percentage of self-aggregation was calculated as follows:

[0084] Self-aggregation (%) = [1 - (At / A0)] × 100;

[0085] Where A0 and At represent the OD at 0h and 4h, respectively. 600 .

[0086] To determine co-aggregation capacity, Ppe-YC39 and LGG bacterial suspensions were prepared as described above and mixed with MRSA bacterial suspensions of equal volume concentration, respectively. The mixtures were incubated at 37°C for 4 hours, and OD values ​​were measured at 0 h and 4 h, respectively. 600 The formula for calculating the co-aggregation rate is the same as the formula for calculating the self-aggregation rate.

[0087] The results showed that the self-aggregation and co-aggregation abilities of Ppe-YC39 were comparable to those of the commercial strain LGG (see [link to study]. Figure 4 and Figure 5 ).

[0088] 2.6 Hydrophobicity Assessment

[0089] Xylene was chosen as the hydrophobic organic solvent. Ppe-YC39 and LGG bacterial suspensions were prepared according to the method in Section 2.5, and then mixed with the organic solvent at a ratio of 3:1, and incubated at 37°C for 4 hours. OD was monitored. 600 The value is recorded as At. The formula for calculating the percentage of hydrophobicity is as follows:

[0090] Hydrophobicity (%) = [1 - (At / A0)] × 100;

[0091] Where A0 and At represent the OD at 0h and 4h respectively. 600 .

[0092] The results showed that Ppe-YC39 had significantly higher hydrophobicity than the commercial strain LGG (see [link to results]). Figure 6 ).

[0093] 3. Safety Evaluation

[0094] 3.1 Hemolytic assessment

[0095] To assess hemolytic activity, Ppe-YC39 was incubated at 37°C for 48 hours on Columbia agar plates (Guangdong Huankai Biotechnology, catalog number CP0160) containing 5% sheep blood (v / v). A positive hemolytic activity was considered when a distinct (β-hemolytic) hemolytic zone was observed around the colony. A negative activity was considered when a green or brown discoloration (α-hemolysis) or the absence of a hemolytic zone (γ-hemolysis) was observed in the culture medium.

[0096] MRSA is known to exhibit β-hemolysis, with a clear hemolytic zone surrounding the colony. However, experimental results showed that the Ppe-YC39 colony did not have a similar hemolytic zone, indicating a negative result and making it a safe strain (see...). Figure 7 ).

[0097] 4. Physicochemical and microbiological evaluation of fish tea fermented with lactic acid bacteria

[0098] 4.1 Preparation of Fermented Fish Tea

[0099] Fresh grass carp are scaled, headed, and gutted. The meat is washed, cut into 2cm x 2cm x 1cm pieces, and drained until no water seeps out. Salt is added (9% w / w relative to the fish weight), and the fish pieces are marinated at room temperature for 60 minutes. Rice is rinsed three times and then steamed for 15 minutes, until approximately 70% cooked. The steamed rice is cooled to room temperature. Ppe-YC39 is inoculated into MRS liquid medium and statically cultured at 37°C for 16-18 hours (bacterial concentration of 10). 9 CFU / mL). Centrifuge the culture medium at 4000 rpm and 25°C for 15 minutes. Discard the supernatant and wash the precipitate twice with sterile water. Then resuspend the cells in sterile water and mix thoroughly to obtain a Ppe-YC39 bacterial suspension. Mix the prepared fish pieces and rice at a ratio of 80:140 (w / w). Add the Ppe-YC39 bacterial suspension (bacterial concentration of 10). 4 The mixture was prepared at CFU / mL (5% v / w) relative to the fish and rice mixture and then compacted into 50 mL centrifuge tubes. Anaerobic fermentation was then carried out at 20°C for 20 days to obtain the final fish tea product.

[0100] The natural fermentation group (Control) follows the same steps as the control group, except that no inoculation is performed.

[0101] 4.2 Determination of pH value during fish tea fermentation

[0102] Fish tea samples from fermentation days 0, 7, and 20 were ground and thoroughly mixed with sterile water (10 mL / g fish tea sample). The pH of the mixture was measured.

[0103] The results showed that after inoculation with Ppe-YC39, the pH of the fish tea initially decreased rapidly and then remained stable, as shown in Table 2. The decrease in pH indicates enhanced antibacterial activity, which can inhibit spoilage microorganisms and extend shelf life, thereby improving food safety and product stability.

[0104] 4.3 Ppe-YC39 activity count during fish tea fermentation

[0105] Fish tea samples from fermentation days 0, 7, and 20 were ground and thoroughly mixed with sterile water (10 mL / g fish tea sample). Each mixed fish tea sample was serially diluted and plated onto MRS plates. The inoculated MRS plates were incubated at 37°C for 24 hours, and the viable bacterial count in the fish tea samples was calculated.

[0106] The results showed that in the Ppe-YC39 inoculated group, the lactic acid bacteria count increased rapidly in the first few days, and then remained stable, as shown in Table 2. This indicates that Ppe-YC39, as a starter culture for fish tea, can maintain relatively stable fermentation quality and state even after 20 days.

[0107] Table 2. Colony count and pH changes during fermentation.

[0108]

[0109] In Table 2, the numerical values ​​represent the mean ± standard error (SEM) of the HPLC analysis.

[0110] 4.4 Determination of Metabolite Content in Fermented Fish Tea

[0111] Fish tea samples were collected after 20 days of fermentation. The samples were ground to ensure thorough mixing of rice and fish. 50 mg of the fish tea sample was accurately weighed into a 1.5 mL centrifuge tube and sent to a sequencing company for subsequent LC-MS / MS analysis, including the content of short-chain fatty acids (SCFA), free amino acids (FAA), and biogenic amines (BA).

[0112] Short-chain fatty acids (SCFAs) are key determinants of the unique flavor characteristics of fish tea, and their type and concentration directly affect sensory attributes such as acidity and distinctive aroma. In the determination of SCFA content, the Ppe-YC39 inoculated group showed a significantly higher acetic acid content compared to the naturally fermented group (Control), and it was the most abundant SCFA (see Table 3). This indicates that Ppe-YC39 inoculation effectively enhances the acidity of fish tea. Besides sensory effects, acetic acid has functional properties such as regulating digestive tract pH, modulating appetite, and inhibiting pathogenic microorganisms. This suggests that these products may provide enhanced functional benefits related to digestive health, pathogen inhibition, and potential metabolic effects. In addition to acetic acid, the inoculated group showed lower levels of propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid, although these were not statistically significant. This trend may stem from competitive substrate utilization by lactic acid bacteria, mild inhibition of harmful microorganisms, and subtle changes in metabolic pathways, which may mitigate, to some extent, the formation of off-flavors typically associated with these acids.

[0113] Free amino acid (FAA) analysis provides important insights into the protein hydrolysis metabolism of the strain and the generation of bioactive and flavor-active compounds during fish tea fermentation. As shown in Table 4, after inoculation with Ppe-YC39, the content of each essential amino acid (EAAs) increased significantly relative to the natural fermentation group (Control). Specifically, L-lysine increased by 157%, L-phenylalanine by 72%, L-methionine by 79%, L-threonine by 207%, L-leucine by 89%, L-isoleucine by 110%, L-valine by 114%, and L-tryptophan by 151%. This indicates that microbial fermentation effectively promotes protein breakdown into bioavailable EAAs, thereby enhancing the nutritional value and amino acid balance of the product.

[0114] When analyzing flavor-related amino acids, Ppe-YC39 showed an increase in umami-related amino acids compared to the naturally fermented group (Control). Specifically, aspartic acid increased by 39%, L-glutamic acid by 182%, and L-glutamine by 164%, with the increases in L-glutamic acid and L-glutamine being significant. For sweetness-related amino acids, significant increases were observed in L-glycine (104%), L-serine (520%), L-threonine (207%), and L-methionine (79%). Additionally, L-proline increased by 44%. For bitter amino acids, the changes in the inoculated group were significant compared to the naturally fermented group, with significant increases in L-lysine (157%), L-histidine (174%), L-leucine (89%), L-isoleucine (110%), L-tyrosine (431%), L-valine (114%), L-phenylalanine (72%), and L-tryptophan (151%). Only the arginine content showed a significant decrease, reduced by 98%. Overall, the inoculation with lactic acid bacteria Ppe-YC39 diversified the flavor characteristics of the fish tea, significantly enhanced the umami and sweetness, and introduced a balanced bitterness.

[0115] Biogenic amines (BAs) are low-molecular-weight organic bases formed from the decarboxylation of amino acids by microorganisms, commonly found in fermented foods and biological tissues. They play a role in physiological processes, but excessive accumulation can lead to adverse reactions such as headaches or high blood pressure. As shown in Table 5, compared with the naturally fermented group (Control), the putrescine content in fish tea fermented with Ppe-YC39 was reduced by 64%, while the levels of spermine and spermidine fluctuated less significantly. The arginine content decreased by 81% compared with the naturally fermented group, while the ornithine content increased by 326%, showing a significant change. Therefore, this strain can be used as a fermentation agent to reduce biogenic amines and improve the safety and quality of fish tea.

[0116] Table 3. Short-chain fatty acid (SCFA) content in fish tea

[0117]

[0118] In Table 3, the numerical values ​​represent the mean ± standard error (SEM); * indicates a significant difference (p<0.05) compared to the natural fermentation group (Control), obtained through one-way ANOVA combined with the Turkey test.

[0119] Table 4. Free amino acid (FAAs) content in fish tea

[0120]

[0121] In Table 4, the numerical values ​​represent the mean ± standard error (SEM); * indicates a significant difference (p<0.05) compared to the naturally fermented group (Control), obtained through one-way ANOVA combined with the Turkey test. ND indicates that the sample concentration is below the detection limit of the analytical method used.

[0122] Table 5. Biogenic amines (BAs) content in fish tea

[0123]

[0124] In Table 5, the numerical values ​​represent the mean ± standard error (SEM); * indicates a significant difference (p<0.05) compared to the natural fermentation group (Control), obtained through one-way ANOVA combined with the Turkey test.

[0125] In summary, this invention employs an inoculation fermentation method to address the problems encountered during the fermentation process of fish tea. Lactic acid bacteria were isolated from fish tea grown in Hainan, and the strains were subsequently identified and screened based on criteria such as growth status, the final pH value of the fermentation medium, antibacterial activity, and biofilm formation ability. Next, the screened lactic acid bacteria strains were further evaluated for their probiotic properties, such as survival rate, aggregation ability, hydrophobicity, and safety in simulated gastrointestinal fluid. One strain of *Pediococcus pentosaceus* (Ppe-YC39) exhibiting good probiotic properties was selected as a single starter culture for fish tea production. pH changes, dynamic changes in the number of viable lactic acid bacteria, and the contents of short-chain fatty acids (SCFAs), free amino acids (FAAs), and biogenic amines (BAs) in different treatment groups were measured and compared. This invention contributes to a deeper understanding of the microbiology of tropical fermented foods and provides a practical strategy for meeting modern food safety and quality standards while preserving cultural heritage.

[0126] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A strain of Pediococcus pentosaceus that can enhance the nutritional activity and flavor of fish tea and inhibit the formation of biogenic amines ( Pediococcus pentosaceus Ppe-YC39, characterized in that, The Pediococcus pentosaceus Ppe-YC39 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66920 and deposit date of September 5, 2025.

2. A microbial agent, characterized in that, The bacterial agent includes Pediococcus pentosaceus Ppe-YC39 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent also includes food-acceptable additives.

4. A fermentation broth, characterized in that, The fermentation broth was obtained by fermentation of Pediococcus pentosaceus Ppe-YC39 as described in claim 1.

5. The application of Pediococcus pentosaceus Ppe-YC39 as described in claim 1, the inoculum as described in any one of claims 2-3, or the fermentation broth as described in claim 4 in the preparation of fermented foods.

6. The application according to claim 5, characterized in that, The fermented food is fish tea.

7. A method for preparing fish tea, characterized in that, Including the following steps: (1) Mix the fish meat with salt and marinate at 20-40℃ for 40-80 minutes to obtain marinated fish pieces; (2) Steam the rice at 90-110℃ for 10-20 minutes to obtain steamed rice; (3) Inoculate the Pediococcus pentosacchari Ppe-YC39 as described in claim 1 into MRS liquid medium, culture at 30-45℃ for 16-18h, centrifuge and discard the supernatant, wash, and then resuspend the precipitate in water to obtain Ppe-YC39 bacterial suspension; (4) Mix the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension, and anaerobic ferment at 15-25℃ for 18-22 days to obtain fish tea.

8. The method for preparing fish tea according to claim 7, characterized in that, In step (1), the mass ratio of the surface-dehydrated fish meat to salt is 1:(0.07-0.11). The method for preparing the water-free fish meat includes the following steps: washing and cutting fresh fish meat into pieces, draining the water until no water seeps out.

9. The method for preparing fish tea according to claim 7, characterized in that, In step (3), the specific conditions for centrifugation include: a rotation speed of 3500-4500 rpm, a temperature of 20-30℃, and a time of 10-20 min.

10. The method for preparing fish tea according to claim 7, characterized in that, In step (4), the marinated fish pieces, steamed rice and Ppe-YC39 bacterial suspension are all mixed at 20-40℃; The mass ratio of the marinated fish pieces to the steamed rice is 1:(1.5-2). The volume-to-mass ratio of the Ppe-YC39 bacterial suspension and the total weight of the pickled fish pieces to the steamed rice is (0.02-0.08) mL:1g; The bacterial concentration of the Ppe-YC39 bacterial suspension was 10 3 -10 5 CFU / mL; The specific conditions for the anaerobic fermentation include: oxygen volume ratio <0.5%.

Images