Method for promoting production of cyclic dipeptides in porphyra haitanensis fermentation broth by using composite fermentation inoculant

Abstract

This invention discloses a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent, belonging to the field of microbial technology. This invention provides a compound fermentation agent comprising *Lactobacillus salivarius* HYD 1-1 and *Saccharomyces cerevisiae*, wherein the *Lactobacillus salivarius* HYD 1-1 is classified as... Ligilactobacillus salivarius HYD1-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252332, on October 24, 2025. Using this composite fermentation agent to ferment *Porphyra yezoensis*, 31 cyclic dipeptides were detected in the fermentation broth. The cyclic dipeptide content was 62% higher than that of *Lactobacillus salivarius* fermentation alone and 40% higher than that of *Saccharomyces cerevisiae* fermentation alone, indicating a significant synergistic effect between the two.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent. Background Technology

[0002] Cyclic dipeptides, also known as diketopiperazine compounds, are cyclic peptides composed of two amino acids linked by peptide bonds. They are the smallest and simplest known cyclic peptides. Compared to linear peptides, the six-membered ring structure of cyclic dipeptides increases their structural stability, and the presence of two hydrogen bond donors (NH) and two hydrogen bond acceptors (C=O) within the molecule allows them to specifically bind to various enzymes or proteins through a hydrogen bond network. Based on these structural characteristics, cyclic dipeptides have been shown to possess a variety of physiological functions beneficial to human health, including antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, immune-enhancing, and neurodegenerative disease prevention, thus arousing great interest in the scientific community. Currently, research on marine-derived cyclic dipeptides, both domestically and internationally, mainly focuses on marine microorganisms, with limited research on cyclic dipeptides in macroalgae. Cyclic dipeptides have only been found in a few algae such as *Ulva prolifera*, *Gracilaria*, *Gracilaria zebrina*, *Ulva spp.*, *Porphyra yezoensis*, and *Trichoderma urticata*.

[0003] Porphyra yezoensis, belonging to the red algae phylum, is widely cultivated in areas south of the Yangtze River and is an important economic algae crop along the South my country Sea coast. Porphyra yezoensis is not only rich in resources but also a vast nutritional treasure trove, possessing extremely high research and development value. However, research on cyclic dipeptides from Porphyra yezoensis is currently lacking. This is mainly because the content of cyclic dipeptides in natural Porphyra yezoensis is low, and they often coexist with complex matrices such as polysaccharides and proteins, making them difficult to extract and detect. Therefore, finding suitable methods to promote the production of cyclic dipeptides and increase their content in Porphyra yezoensis is of great significance for realizing its high-value utilization and developing novel Porphyra yezoensis health foods.

[0004] Microbial co-culture is an effective strategy for activating silent biosynthetic pathways and enhancing the production of latent secondary metabolites. This culture mode introduces interspecies interactions, which can stimulate the production of novel compounds not observed in monocultures, thereby significantly increasing metabolite diversity and yield. Furthermore, many microbial strains are widely used in the food processing industry due to their favorable fermentation properties. Currently, no research has been reported on the role of microbial fermentation in promoting the production of cyclic dipeptides in *Porphyra yezoensis* fermentation broth. Summary of the Invention

[0005] Based on the above problems, the purpose of this invention is to provide a strain of Lactobacillus salivarius HYD 1-1, a compound fermentation agent containing the strain and Saccharomyces cerevisiae, and a method for promoting the production of cyclic dipeptides in the fermentation broth of Porphyra yezoensis using the compound fermentation agent.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: First, the present invention provides a compound fermentation agent comprising *Lactobacillus salivarius* HYD 1-1 and *Saccharomyces cerevisiae*. Saccharomyces cerevisiae The classification name of the *Lactobacillus saliva-associated* HYD 1-1 is... Ligilactobacillus salivarius HYD1-1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252332, located at Wuhan University, Wuhan, China, on October 24, 2025; the volume ratio of *Lactobacillus salivarius* HYD1-1 to *Saccharomyces cerevisiae* is 1:0.5 to 1:2.

[0007] Preferably, the brewer's yeast was purchased from Beina Chuanglian Biotechnology Co., Ltd., and its Latin name is... Saccharomyces cerevisiae The depositary institution is the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, the accession number is BNCC336491, and the deposit date is April 25, 2018.

[0008] Preferably, the volume ratio is 2:1, 1:1, or 1:2.

[0009] Preferably, the total viable count of the *Lactobacillus saliva-associated* HYD 1-1 is ≥1×10⁻⁶. 8 CFU / mL; the total viable count of the *Saccharomyces cerevisiae* is ≥1×10⁻⁶. 8 CFU / mL.

[0010] Secondly, the present invention also provides a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent, comprising the following steps: (1) The hydatid lactobacillus HYD 1-1 and saccharomyces cerevisiae were activated separately and then mixed to obtain a compound fermentation agent; (2) Disperse the laver in distilled water, blend it into a slurry, and add glucose; (3) After sterilizing the laver pulp, inoculate it with the compound fermentation agent prepared in step (1); (4) Place the inoculated laver slurry at 28-32℃ for 5-10 days to ferment, and collect the supernatant by centrifugation to obtain laver fermentation liquid; (5) Extract cyclic dipeptides from the fermentation broth of Lavera rotundifolia.

[0011] Preferably, the activation method of Lactobacillus saliva-associated HYD 1-1 in step (1) includes: taking 100~500 μL of Lactobacillus saliva-associated bacterial suspension into 10~50 mL of MRS liquid culture medium, culturing at 30~37℃ and 180~200 rpm for 20~24 h, centrifuging to discard the supernatant, washing the bacterial precipitate twice with sterile water and then reconstitute for later use.

[0012] Preferably, the method for activating Saccharomyces cerevisiae in step (1) includes: taking 100-500 μL of Saccharomyces cerevisiae culture into 10-50 mL of YM liquid culture medium, culturing at 28-32℃ and 180-200 rpm for 20-24 h, centrifuging to discard the supernatant, washing the cell precipitate twice with sterile water and then reconstitute for later use.

[0013] Preferably, the volume ratio of Lactobacillus saliva-associated HYD 1-1 to Saccharomyces cerevisiae in step (1) is 1:0.5 to 1:2.

[0014] Preferably, in step (2), the ratio of laver to distilled water is 1 g: 30~80 mL, and the amount of glucose added is 0.5%~1.5% of the volume of laver slurry.

[0015] Preferably, the inoculation amount of the compound fermentation agent in step (3) is 2% to 10% of the volume of the seaweed slurry.

[0016] Preferably, the fermentation conditions in step (4) include a rotation speed of 180~200 rpm.

[0017] Preferably, the extraction method in step (5) is liquid-liquid extraction: take 50-100 mL of Porphyra yezoensis fermentation broth and 50-100 mL of dichloromethane / ethyl acetate (1:1, v / v), vortex at 1000-2000 rpm for 30-60 s, then sonicate on ice for 15-30 min, and then centrifuge at 5000-6000 rpm for 5-10 min, and take the lower organic phase; repeat the above operation once, and extract once more with 50-100 mL of ethyl acetate, combine the organic phases, filter to remove impurities, concentrate the liquid by rotary evaporation, and blow dry with nitrogen.

[0018] Third, the present invention also provides a fermented broth of laver prepared by the above method.

[0019] Fourth, the present invention also provides the application of the above-mentioned laver fermentation liquid in the preparation of anti-inflammatory drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a newly isolated Lactobacillus salivarius HYD 1-1 (CCTCC NO: M20252332), which enriches the resources of culturable microbial strains.

[0021] (2) The present invention uses a compound fermentation agent prepared by combining *Lactobacillus saliva-associated* HYD 1-1 with *Saccharomyces cerevisiae*, and there is a significant synergistic effect between the two. Using this compound fermentation agent (Examples 3-5), 31 kinds of cyclic dipeptides can be detected in the fermentation of laver. The content of cyclic dipeptides is 62% higher than that of *Lactobacillus saliva-associated* fermentation alone and 40% higher than that of *Saccharomyces cerevisiae* fermentation alone.

[0022] (3) The method provided by the present invention is simple, easy to implement, green and safe, and can improve the nutritional value of the fermented laver liquid, which helps to promote the development of healthy products made from fermented laver. Attached Figure Description

[0023] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 Microscopic image of Lactobacillus hyd 1-1 from salivary region; Figure 2 A schematic diagram of a phylogenetic tree constructed based on the 16S rRNA sequence for Lactobacillus salivarius HYD 1-1; Figure 3 The total ion chromatogram of cyclic dipeptides in the original solution of *Porphyra yezoensis* (blank control group) is shown. Figure 4 The total ion chromatogram of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* prepared using the combined fermentation agent of *Lactobacillus* and *Saccharomyces cerevisiae* in Example 3 of this invention is shown below. Figure 5 A bar chart showing the percentage content of different types of cyclic dipeptides in the Porphyra yezoensis fermentation broth prepared using the combined fermentation agent of Lactobacillus and Saccharomyces cerevisiae in saliva as described in Example 3 of this invention. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0027] In this invention, the source of the laver is not specifically required; commercially available laver is sufficient. In a specific embodiment of this invention, laver produced in Xiapu County, Ningde City, Fujian Province is selected.

[0028] Example 1 This example describes the isolation, purification, and identification of Lactobacillus salivarius HYD 1-1.

[0029] (1) Isolation and purification of Lactobacillus saliva Samples used for bacterial isolation and purification (collected from white-skinned fish intestines) were added to MRS liquid medium and incubated at 37°C for 48 h for bacterial enrichment. The enriched bacterial solution was then diluted 10-fold, vortexed to mix, and 100 μL of each solution was spread onto MRS agar medium and incubated at 37°C for 48 h. After incubation, representative single colonies were selected based on surface morphology, color, and degree of protrusion, and streaked onto MRS agar medium, incubated at 37°C for 48 h. After multiple subcultures, strains with uniform morphology observed visually were Gram-stained and examined microscopically. Strains with uniform microscopic examination were considered pure strains. Figure 1 As shown. The purified strain was stored at -80°C for later use.

[0030] (2) Identification of Lactobacillus saliva The purified strain was subjected to 16S rRNA sequencing, and the sequencing results are shown in SEQ ID NO:1. The sequencing results were then BLAST-aligned against the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 12.0 software, as shown below. Figure 2 As shown, the purified strain was identified as *Lactobacillus salivarius*, and named... Ligilactobacillus salivarius HYD1-1.

[0031] This strain was deposited on October 24, 2025, at the China Center for Type Culture Collection (CCTCCNO: M 20252332), located at Wuhan University, Wuhan, China. It is classified and named as follows: Ligilactobacillus salivarius HYD1-1.

[0032] Example 2 This embodiment describes a method for promoting the production of cyclic dipeptides in Porphyra yezoensis fermentation broth using saliva-based combined fermentation with Lactobacillus HYD 1-1, including the following steps: (1) Preparation of saliva-based Lactobacillus fermentation agent The *Lactobacillus saliva-associated* strain was activated. 100 μL of *Lactobacillus saliva-associated* bacterial culture was transferred to 10 mL of MRS liquid medium and incubated at 37°C and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the bacterial precipitate was washed twice with sterile water and reconstituted for later use.

[0033] (2) Preparation of Laver original solution and fermentation liquid (2-1) Prepare laver and distilled water at a ratio of 1 g: 50 mL. Disperse the laver in the distilled water and use a pulping machine to make it into a uniform slurry. Add 1% of the slurry volume of glucose as the carbon source required by the fermentation strain.

[0034] (2-2) Transfer the laver pulp into a high-pressure steam sterilizer and sterilize at 115℃ for 30 min. After the pulp cools to room temperature, divide it into two groups in a clean bench: one group is added with 5% of the volume of laver pulp in sterile water (blank control group), and the other group is added with 5% of the volume of laver pulp in saliva combined with Lactobacillus fermentation agent (fermentation group).

[0035] (2-3) Centrifuge the laver pulp of the blank control group at 8000 rpm for 10 min and collect the supernatant to obtain the unfermented laver original liquid.

[0036] (2-4) The slurry of the fermentation group of laver was placed in an incubator at 30℃ and 200 rpm for 7 days. After the fermentation was completed, it was centrifuged at 8000 rpm for 10 min and the supernatant was collected to obtain the laver fermentation liquid.

[0037] (3) Extraction and identification of cyclic dipeptides from the original liquid and fermentation broth of Porphyra yezoensis (3-1) Extraction of cyclic dipeptides from the original liquid and fermentation broth of *Porphyra yezoensis* Cyclic dipeptides were extracted from the original solution and fermentation broth of *Porphyra yezoensis* using liquid-liquid extraction. 50 mL of the original solution and fermentation broth were mixed with 50 mL of dichloromethane / ethyl acetate (1:1, v / v), vortexed at 2000 rpm for 30 s, followed by sonication in an ice bath for 30 min. The mixture was then centrifuged at 6000 rpm for 5 min, and the lower organic phase was collected. This process was repeated once, followed by extraction with 50 mL of ethyl acetate. The organic phases were combined, filtered to remove impurities, concentrated by rotary evaporation, and dried under nitrogen.

[0038] (3-2) Identification of cyclic dipeptides in the original liquid and fermentation broth of *Porphyra yezoensis* LC-HRMS was used to identify cyclic dipeptides in the original solution and fermentation broth of *Porphyra yezoensis*. Chromatographic conditions: A C18 column (2.1 mm × 100 mm, 1.7 μm) was used; the injection volume was 5 μL; the flow rate was 0.3 mL / min; the mobile phase was water (A) containing 0.1% formic acid / acetonitrile (B); the gradient elution program was 0 min 50% B; 20 min 90% B; 21 min 90% B; 22 min 50% B; and the column oven was set to 30℃. Mass spectrometry conditions: Electrospray ionization (ESI) in positive ion mode; the spray voltage was 3800 V; the ion source temperature was 500℃; ion source gas 1 (50 psi), ion source gas 2 (50 psi), and curtain gas (35 psi); the mass spectrometry scan range was m / z 50–1250; the maximum injection time was 100 ms; and the collision energy of the secondary mass spectrometer was 30 eV.

[0039] Example 3 This embodiment describes a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent, comprising the following steps: (1) Preparation of compound fermentation agent (1-1) The *Lactobacillus saliva* HYD 1-1, which had been isolated, purified, identified and preserved, was activated. 100 μL of *Lactobacillus saliva* culture was transferred to 10 mL of MRS liquid medium and cultured at 37℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the bacterial precipitate was washed twice with sterile water and then reconstituted for later use.

[0040] (1-2) Saccharomyces cerevisiae Saccharomyces cerevisiae BNCC336491 (purchased from Beina Chuanglian Biotechnology Co., Ltd.) was used for activation. 100 μL of Saccharomyces cerevisiae culture was added to 10 mL of YM liquid medium and cultured at 30℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the cell precipitate was washed twice with sterile water and then reconstituted for later use.

[0041] (1-3) A compound fermentation agent is obtained by mixing saliva-containing Lactobacillus bacterial solution and Saccharomyces cerevisiae bacterial solution in a volume ratio of 1:1.

[0042] (2) Preparation of Laver original solution and fermentation liquid (2-1) Prepare laver and distilled water at a ratio of 1 g: 50 mL. Disperse the laver in the distilled water and use a pulping machine to make it into a uniform slurry. Add 1% of the slurry volume of glucose as the carbon source required by the fermentation strain.

[0043] (2-2) Transfer the laver slurry into a high-pressure steam sterilizer and sterilize at 115℃ for 30 min. After the slurry cools to room temperature, divide it into two groups in a clean bench: one group is added with 5% of the volume of laver slurry in sterile water (blank control group), and the other group is added with 5% of the volume of laver slurry in compound fermentation agent (fermentation group).

[0044] (2-3) Centrifuge the laver pulp of the blank control group at 8000 rpm for 10 min and collect the supernatant to obtain the unfermented laver original liquid.

[0045] (2-4) The slurry of the fermentation group of laver was placed in an incubator at 30℃ and 200 rpm for 7 days. After the fermentation was completed, it was centrifuged at 8000 rpm for 10 min and the supernatant was collected to obtain the laver fermentation liquid.

[0046] (3) Extraction and identification of cyclic dipeptides from the original liquid and fermentation broth of Porphyra yezoensis (3-1) Extraction of cyclic dipeptides from the original liquid and fermentation broth of *Porphyra yezoensis* Cyclic dipeptides were extracted from the original solution and fermentation broth of *Porphyra yezoensis* using liquid-liquid extraction. 50 mL of the original solution and fermentation broth were mixed with 50 mL of dichloromethane / ethyl acetate (1:1, v / v), vortexed at 2000 rpm for 30 s, followed by sonication in an ice bath for 30 min. The mixture was then centrifuged at 6000 rpm for 5 min, and the lower organic phase was collected. This process was repeated once, followed by extraction with 50 mL of ethyl acetate. The organic phases were combined, filtered to remove impurities, concentrated by rotary evaporation, and dried under nitrogen.

[0047] (3-2) Identification of cyclic dipeptides in the original liquid and fermentation broth of *Porphyra yezoensis* Cyclic dipeptides in the original solution and fermentation broth of *Porphyra yezoensis* were identified using LC-HRMS. The chromatographic conditions were the same as in Example 2.

[0048] Test results as follows Figure 3 and Figure 4 As shown.

[0049] Example 4 This embodiment describes a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent, comprising the following steps: (1) Preparation of compound fermentation agent (1-1) The *Lactobacillus saliva* HYD 1-1, which had been isolated, purified, identified and preserved, was activated. 100 μL of *Lactobacillus saliva* culture was transferred to 10 mL of MRS liquid medium and cultured at 37℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the bacterial precipitate was washed twice with sterile water and then reconstituted for later use.

[0050] (1-2) Saccharomyces cerevisiae Saccharomyces cerevisiae BNCC336491 (purchased from Beina Chuanglian Biotechnology Co., Ltd.) was used for activation. 100 μL of Saccharomyces cerevisiae culture was added to 10 mL of YM liquid medium and cultured at 30℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the cell precipitate was washed twice with sterile water and then reconstituted for later use.

[0051] (1-3) A compound fermentation agent is obtained by mixing saliva-containing Lactobacillus bacterial solution and Saccharomyces cerevisiae bacterial solution at a volume ratio of 2:1.

[0052] (2) Preparation of Laver original solution and fermentation liquid Same as step (2) in Example 3.

[0053] (3) Extraction and identification of cyclic dipeptides from the original liquid and fermentation broth of Porphyra yezoensis Same as step (3) in Example 3.

[0054] The results showed that the types and distribution characteristics of cyclic dipeptides in the Porphyra yezoensis fermentation broth prepared in this embodiment were basically the same as those in Example 3, with 31 types of cyclic dipeptides detected in both.

[0055] Example 5 This embodiment describes a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using a compound fermentation agent, comprising the following steps: (1) Preparation of compound fermentation agent (1-1) The *Lactobacillus saliva* HYD 1-1, which had been isolated, purified, identified and preserved, was activated. 100 μL of *Lactobacillus saliva* culture was transferred to 10 mL of MRS liquid medium and cultured at 37℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the bacterial precipitate was washed twice with sterile water and then reconstituted for later use.

[0056] (1-2) Saccharomyces cerevisiae Saccharomyces cerevisiae BNCC336491 (purchased from Beina Chuanglian Biotechnology Co., Ltd.) was used for activation. 100 μL of Saccharomyces cerevisiae culture was added to 10 mL of YM liquid medium and cultured at 30℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the cell precipitate was washed twice with sterile water and then reconstituted for later use.

[0057] (1-3) A compound fermentation agent is obtained by mixing saliva-containing Lactobacillus bacterial solution and Saccharomyces cerevisiae bacterial solution at a volume ratio of 1:2.

[0058] (2) Preparation of Laver original solution and fermentation liquid Same as step (2) in Example 3.

[0059] (3) Extraction and identification of cyclic dipeptides from the original liquid and fermentation broth of Porphyra yezoensis Same as step (3) in Example 3.

[0060] The results showed that the types and distribution characteristics of cyclic dipeptides in the Porphyra yezoensis fermentation broth prepared in this embodiment were basically the same as those in Example 3, with 31 types of cyclic dipeptides detected in both.

[0061] Comparative Example 1 This comparative example describes a method for promoting the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis* using only *Saccharomyces cerevisiae*, including the following steps: (1) Preparation of Saccharomyces cerevisiae fermentation inoculum brewer's yeast Saccharomyces cerevisiae BNCC336491 (purchased from Beina Chuanglian Biotechnology Co., Ltd.) was used for activation. 100 μL of Saccharomyces cerevisiae culture was added to 10 mL of YM liquid medium and cultured at 30℃ and 200 rpm for 24 h. After centrifugation, the supernatant was discarded, and the cell precipitate was washed twice with sterile water and then reconstituted for later use.

[0062] (2) Preparation of Laver original solution and fermentation liquid (2-1) Prepare laver and distilled water at a ratio of 1 g: 50 mL. Disperse the laver in the distilled water and use a pulping machine to make it into a uniform slurry. Add 1% of the slurry volume of glucose as the carbon source required by the fermentation strain.

[0063] (2-2) Transfer the laver slurry into a high-pressure steam sterilizer and sterilize at 115℃ for 30 min. After the slurry cools to room temperature, divide it into two groups in a clean bench: one group is added with 5% of the volume of laver slurry in sterile water (blank control group), and the other group is added with 5% of the volume of laver slurry in yeast fermentation agent (fermentation group).

[0064] (2-3) Centrifuge the laver pulp of the blank control group at 8000 rpm for 10 min and collect the supernatant to obtain the unfermented laver original liquid.

[0065] (2-4) The slurry of the fermentation group of laver was placed in an incubator at 30℃ and 200 rpm for 7 days. After the fermentation was completed, it was centrifuged at 8000 rpm for 10 min and the supernatant was collected to obtain the laver fermentation liquid.

[0066] (3) Extraction and identification of cyclic dipeptides from the original liquid and fermentation broth of Porphyra yezoensis Same as step (3) in Example 3.

[0067] Results analysis: Analysis using UPLC-Q Exactive liquid chromatography-high-resolution mass spectrometry, based on multivariate data analysis including precise mass number, secondary mass spectrometry fragment ion matching, and retention time, revealed that no cyclic dipeptides were detected in the unfermented *Porphyra yezoensis* stock solution (blank control group). Figure 3 As shown. Thirty-one cyclic dipeptides were detected in the *Porphyra yezoensis* fermentation broth prepared according to Examples 3-5, and representative total ion chromatograms are shown below. Figure 4 As shown in the figure. Among them, six cyclic dipeptides—cyclic (phenylalanine-alanine), cyclic (proline-tyrosine), cyclic (proline-proline), cyclic (phenylalanine-proline), cyclic (proline-leucine), and cyclic (proline-alanine)—are highly abundant, and their percentage distribution is shown in the figure. Figure 5 As shown.

[0068] In Example 2 and Comparative Example 1, *Lactobacillus salivarius* HYD 1-1 and *Saccharomyces cerevisiae* BNCC336491 were used separately to ferment *Porphyra yezoensis*. The results showed that 5 and 12 cyclic dipeptides were detected in the *Porphyra yezoensis* fermentation broth prepared using the methods described in Example 2 and Comparative Example 1, respectively. Using peak area normalization, the total cyclic dipeptide content in the *Porphyra yezoensis* fermentation broth prepared using the method described in Example 2 (fermentation with *Lactobacillus salivarius* only) was 1.58 times that of the *Porphyra yezoensis* fermentation broth prepared using the method described in Comparative Example 1 (fermentation with *Saccharomyces cerevisiae* only) (i.e., a relative content of 158%, an increase of 58%). The total cyclic dipeptide content in the *Porphyra yezoensis* fermentation broth prepared using the method described in Example 3 (compound fermentation agent) was as high as 2.63 times the baseline (i.e., a relative content of 263%), an increase of 62% compared to Example 2 and 40% compared to Comparative Example 1.

[0069] The above results indicate a significant synergistic effect between *Lactobacillus salivarius* HYD 1-1 and *Saccharomyces cerevisiae*. The combination of these two strains significantly promotes the production of cyclic dipeptides in the fermentation broth of *Porphyra yezoensis*, with effects significantly superior to single-strain fermentation. The compound fermentation agent and its application method provided by this invention offer a new technical approach for the high-value utilization of *Porphyra yezoensis* and the green production of cyclic dipeptides.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound fermentation agent, characterized in that, A compound fermentation agent was obtained by mixing *Lactobacillus salivarius* culture and *Saccharomyces cerevisiae* culture at a volume ratio of 1:

1. The *Lactobacillus salivarius* culture was identified as *Lactobacillus salivarius* HYD 1-1. The classification of *Lactobacillus salivarius* HYD 1-1 was named... Ligilactobacillus salivarius HYD1-1, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252332, address: Wuhan University, Wuhan, China, deposited on October 24, 2025; the Latin name of the described Saccharomyces cerevisiae is... Saccharomyces cerevisiae The depositary institution is the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, the accession number is BNCC336491, and the deposit date is April 25, 2018; The total viable count of *Lactobacillus hydatidae* HYD 1-1 in the salivary sample is ≥1×10⁻⁶. 8 CFU / mL, the total viable count of the *Saccharomyces cerevisiae* is ≥1×10⁻⁶. 8 CFU / mL.

2. The application of the compound fermentation agent according to claim 1 in promoting the production of cyclic dipeptides in the fermentation broth of Lavera oleifera.

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