Aeromonas media HHR-1-2 and application thereof in degrading carotenoids and preparing essence

Abstract

This invention discloses an *Aeromonas media* strain HHR-1-2 and its application in degrading carotenoids and preparing flavorings, belonging to the field of microbial fermentation technology. The *Aeromonas media* strain HHR-1-2 of this invention has the accession number CCTCC NO:M 20252744 and was deposited at the China Center for Type Culture Collection on December 1, 2025. *Aeromonas media* HHR-1-2 can degrade carotenoids with a degradation rate of 85%–95%, and its degradation products are mainly aldehyde compounds. Furthermore, the fermentation broth of *Aeromonas media* HHR-1-2 and carotenoids can be used for flavoring preparation; therefore, *Aeromonas media* HHR-1-2 has application value in flavoring preparation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and specifically relates to an intermediate Aeromonas hydrophila HHR-1-2 and its application in degrading carotenoids and preparing flavorings. Background Technology

[0002] Carotenoids are a class of fat-soluble pigments widely found in nature, with over 700 species discovered, mainly including β-carotene, lutein, and zeaxanthin. In vivo, carotenoids are degraded by specific enzymes such as carotenoid cleavage dioxygenases (CCDs) to produce flavorings, including β-ionone, dihydroactinolone, and β-damascone. These flavorings have low aroma thresholds and are widely used in the food, daily chemical, and other flavoring industries.

[0003] Biodegradation is a highly efficient method for degrading carotenoids that has emerged in recent years, attracting widespread attention due to its high efficiency and environmental friendliness. However, current technologies in the field of carotenoid biodegradation rely on strains of Bacillus and Escherichia, primarily utilizing CCD enzyme pathways for degradation. Research on degradation products is highly focused on β-ionone and dihydroactinolone. Therefore, screening for non-traditional, highly efficient degrading strains and exploring non-CCD enzyme-mediated degradation pathways are of great significance for enriching the diversity of carotenoid degradation products. Summary of the Invention

[0004] The main objective of this invention is to propose an intermediate Aeromonas hydrophila HHR-1-2 and its application in the degradation of carotenoids and the preparation of flavorings, aiming to solve the problem of insufficient diversity of carotenoid biodegrading strains in the prior art.

[0005] To achieve the above objectives, the present invention proposes an Aeromonas media HHR-1-2, characterized in that the accession number of the Aeromonas media HHR-1-2 is CCTCC NO:M 20252744, and the Aeromonas media HHR-1-2 was deposited at the China Center for Type Culture Collection on December 1, 2025.

[0006] In one embodiment, the 16S rDNA gene sequence of Aeromonas intermedius HHR-1-2 is shown in SEQ ID NO.1.

[0007] The present invention also provides an application of the aforementioned Aeromonas intermedius HHR-1-2 in the degradation of carotenoids.

[0008] In one embodiment, the carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, the degradation rate of carotenoids by Aeromonas intermedius HHR-1-2 is 85% to 95%.

[0009] In one embodiment, the products of *Aeromonas intermedius* HHR-1-2 that degrade carotenoids include aldehydes, ketones, alcohols, and esters: the aldehydes include at least one of hexanal, heptanal, trans-2-hexenal, octanal, trans-2-heptanal, nonanal, trans-2-octenal, trans-2-nonenal, ethyl-cyclocitral, trans-2-decenal, and trans-2-undecenal; the ketones include at least one of 2,2,6-trimethylcyclohexanone, isophorone, ethyl-ionone, and 5,6-epoxy-β-ionone; the alcohols include at least one of hexanol, heptanol, octanol, and nonanol; and the esters include dihydroactinol.

[0010] In one embodiment, in the product of the degradation of carotenoids by Aeromonas intermedius HHR-1-2: the peak area of ​​the aldehyde compound accounts for 65% to 75% of the peak area of ​​the product; the total peak area of ​​β-ionone, 5,6-epoxy-β-ionone and dihydroactinolone accounts for less than 2% of the peak area of ​​the product.

[0011] The present invention also provides an application of the aforementioned Aeromonas intermedius HHR-1-2 in the preparation of fragrances.

[0012] In one embodiment, the application includes the following steps:

[0013] A culture medium is provided, wherein the carbon source in the culture medium includes carotenoids; The Aeromonas intermedius strain HHR-1-2 was cultured in the culture medium to obtain a fermentation broth; The fermentation broth is used to prepare a flavoring.

[0014] In one embodiment, the step of preparing the fermentation broth into a flavoring includes: preparing a fermentation concentrate from the fermentation broth; and mixing the fermentation concentrate with mango ester, pineapple ester, ethyl butyrate, vanillin, and a solvent to obtain the flavoring.

[0015] In one embodiment, the fermentation concentrate has a mass percentage of 0.90-1.10%, the pineapple ester has a mass percentage of 6-10%, the ethyl butyrate has a mass percentage of 8-12%, and the vanillin has a mass percentage of 1-3%; and / or, the solvent includes propylene glycol, glycerol, and ethanol.

[0016] The technical solution of this invention provides an intermediate Aeromonas ( AeromonasThe *Aeromonas intermedius* HHR-1-2, with accession number CCTCC NO:M 20252744, was deposited on December 1, 2025, at the China Center for Type Culture Collection (CCCCC), Wuhan University, Wuhan, China. *Aeromonas intermedius* HHR-1-2 can degrade carotenoids with a degradation rate of 85%–95%. The peak area of ​​aldehydes in the degraded carotenoid products accounts for 65%–75% of the peak area of ​​the products; the total peak area of ​​β-ionone, 5,6-epoxy-β-ionone, and dihydroactinolone accounts for less than 2% of the peak area of ​​the products. Its carotenoid degradation products differ from those of the traditional CCD degradation pathway, indicating that *Aeromonas intermedius* HHR-1-2 uses a different degradation pathway than the traditional CCD pathway for β-carotene degradation. In addition, the fermentation broth of Aeromonas intermedius HHR-1-2 and carotenoids can be used for flavor preparation, so Aeromonas intermedius HHR-1-2 has application value in flavor preparation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 This is a growth diagram of HHR-1-2 and HHR-1-3 in Example 1 of the present invention on a medium with β-carotene as the sole carbon source; Figure 2 This is the standard curve of β-carotene in Example 2 of the present invention; Figure 3 The graph shows the degradation rate of β-carotene by HHR-1-2 and HHR-1-3 in Example 2 of this invention. Figure 4 These are photographs of the control group, HHR-1-2, and HHR-1-3 fermentation broths in Example 2 of this invention. Figure 5 This is a colony diagram of HHR-1-2 after streaking culture in LB medium for 24 hours in Example 3 of the present invention; Figure 6 The image shown is an HS-GC-MS image of the fermentation broth of Aeromonas intermedius HHR-1-2 in Example 4 of this invention.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Carotenoids are a class of fat-soluble pigments widely found in nature, with over 700 species identified, mainly including β-carotene, lutein, and zeaxanthin. Carotenoids can be degraded by specific enzymes such as carotenoid cleavage dioxygenases (CCDs) to produce flavoring substances such as β-ionone, dihydroactinolone, and β-damascone. These flavorings possess a significant advantage of low aroma threshold and are widely used in the food, cosmetics, and other flavoring industries.

[0022] Biodegradation is a highly efficient method for degrading carotenoids that has emerged in recent years and has attracted widespread attention due to its high efficiency and environmental friendliness. However, current technologies in the field of carotenoid biodegradation rely on strains of Bacillus and Escherichia, and primarily utilize CCD enzyme pathways for degradation. Research on degradation products is highly focused on substances such as β-ionone and dihydroactinolone. Therefore, screening for non-traditional, highly efficient degrading strains and exploring non-CCD enzyme-mediated degradation pathways are of great significance for enriching the diversity of carotenoid degradation products.

[0023] In view of this, the present invention provides an Aeromonas media HHR-1-2, the accession number of which is CCTCC NO:M 20252744, and the Aeromonas media HHR-1-2 was deposited at the China Center for Type Culture Collection on December 1, 2025.

[0024] In the technical solution of this invention, Aeromonas intermedius HHR-1-2 can degrade carotenoids with a degradation rate of 85% to 95%. Its degradation products include aldehydes, ketones, alcohols, and esters. In addition, the fermentation broth of Aeromonas intermedius HHR-1-2 and carotenoids can be used for flavor preparation. Therefore, Aeromonas intermedius HHR-1-2 has application value in flavor preparation.

[0025] In some embodiments of the present invention, the 16S rDNA gene sequence of Aeromonas intermedius HHR-1-2 is shown in SEQ ID NO. 1.

[0026] The present invention also provides an application of the aforementioned Aeromonas intermedius HHR-1-2 in the degradation of carotenoids.

[0027] In the technical solution of this invention, Aeromonas intermedius HHR-1-2, which can efficiently degrade carotenoids, was screened from the soil environment, effectively solving the problem of homogeneity of strain resources for biodegradation of carotenoids.

[0028] In some embodiments of the present invention, the carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, the degradation rate of carotenoids by Aeromonas intermedius HHR-1-2 is 85% to 95%.

[0029] In the technical solution of this invention, the *Aeromonas intermedius* HHR-1-2 can grow on a culture medium with β-carotene as the sole carbon source, degrading β-carotene in the medium and forming a clear zone. When *Aeromonas intermedius* HHR-1-2 is inoculated into a β-carotene fermentation medium and fermented at 30°C for 24 hours, the degradation rate of carotenoids by *Aeromonas intermedius* HHR-1-2 can reach 85%~95%. Therefore, using *Aeromonas intermedius* HHR-1-2 for carotenoid degradation has advantages such as short production cycle, low cost, and simple cultivation method. This technical solution is not limited to the fermentation time and temperature; that is, by optimizing fermentation time, temperature, and other conditions, the degradation rate of carotenoids by *Aeromonas intermedius* HHR-1-2 can be further improved.

[0030] In some embodiments of the present invention, the products of *Aeromonas intermedius* HHR-1-2 that degrade carotenoids include aldehydes, ketones, alcohols, and esters; the aldehydes include at least one of hexanal, heptanal, trans-2-hexenal, octanal, trans-2-heptanal, nonanal, trans-2-octenal, trans-2-nonenal, ethyl-cyclocitral, trans-2-decenal, and trans-2-undecenal; the ketones include at least one of 2,2,6-trimethylcyclohexanone, isophorone, ethyl-ionone, and 5,6-epoxy-β-ionone; the alcohols include at least one of hexanol, heptanol, octanol, and nonanol; and the esters include dihydroactinol.

[0031] In the technical solution of this invention, the products of *Aeromonas intermedius* HHR-1-2 that degrade carotenoids can simultaneously contain the above-mentioned products, wherein the aldehyde compounds include saturated aldehydes, such as hexanal, heptanal, octanal, nonanal, and ethyl citral; and also include unsaturated aldehydes, such as trans-2-hexenal, trans-2-heptanal, trans-2-octenal, trans-2-nonenal, trans-2-decenal, and trans-2-undecenal; and also include alcohols, ketones, and lipids. Therefore, the products of *Aeromonas intermedius* HHR-1-2 that degrade carotenoids together constitute a complex aroma spectrum with rich layers and combining green, fruity, fatty, and woody aromas, which has unique value in the development of high-end natural fragrances.

[0032] In some embodiments of the present invention, in the products of the degradation of carotenoids by Aeromonas intermedius HHR-1-2: the peak area of ​​the aldehyde compound accounts for 65% to 75% of the peak area of ​​the product; the total peak area of ​​β-ionone, 5,6-epoxy-β-ionone and dihydroactinolone accounts for less than 2% of the peak area of ​​the product.

[0033] In the technical solution of this invention, the peak area of ​​the aldehyde compound accounts for 65% to 75% of the peak area of ​​the product, and the product is mainly C6-C9 aldehyde; the total peak area of ​​the products β-ionone, 5,6-epoxy-β-ionone and dihydroactinolone accounts for less than 2% of the peak area of ​​the product, and its degradation products of carotenoids are different from those of the traditional CCD degradation pathway. This indicates that Aeromonas intermedius HHR-1-2 has a specific enzymatic reaction pathway for the degradation of β-carotene, and Aeromonas intermedius HHR-1-2 adopts a degradation pathway different from the traditional CCD pathway.

[0034] The present invention also provides the application of the aforementioned Aeromonas intermedius HHR-1-2 in the preparation of fragrances.

[0035] In the technical solution of this invention, Aeromonas intermedius HHR-1-2 is applied in the preparation of fragrances, which can simultaneously improve the aroma quantity, freshness, naturalness and harmony of the fragrances.

[0036] In some embodiments of the present invention, the application includes the following steps: providing a culture medium in which the carbon source includes carotenoids; culturing the Aeromonas intermedius HHR-1-2 in the culture medium to obtain a fermentation broth; and preparing the fermentation broth into a flavoring.

[0037] In the technical solution of this invention, Aeromonas intermedius HHR-1-2 is cultured in a carotenoid-containing medium to obtain a fermentation broth. The fermentation broth contains products from the degradation of carotenoids by Aeromonas intermedius HHR-1-2, which exhibit a rich and complex aroma profile, combining green, fruity, fatty, and woody notes. The fermentation broth can significantly enhance the overall aroma of fragrances, possessing significant application value in the fields of daily chemicals and food flavorings, and providing a new technical pathway for developing high-quality fragrance products. This application process is simple to operate, operates under mild conditions, and can effectively enrich aroma active ingredients.

[0038] In some embodiments of the present invention, the step of preparing the fermentation broth into a flavoring includes: preparing a fermentation concentrate from the fermentation broth; and mixing the fermentation concentrate with mango ester, pineapple ester, ethyl butyrate, vanillin, and a solvent to obtain the flavoring.

[0039] By preparing a fermentation concentrate, impurities in the fermentation broth can be removed, resulting in a significant increase in the concentrations of aldehydes, ketones, alcohols, and esters. The fermentation concentrate is then mixed with mango ester, pineapple ester, ethyl butyrate, vanillin, and a solvent to obtain a flavoring. The fermentation concentrate synergistically enhances the aroma of mango ester, pineapple ester, ethyl butyrate, and vanillin in the flavoring.

[0040] In some embodiments of the present invention, the flavoring contains: the fermentation concentrate accounts for 0.90-1.10% by mass, the pineapple ester accounts for 6-10% by mass, the ethyl butyrate accounts for 8-12% by mass, and the vanillin accounts for 1-3% by mass; and / or, the solvent includes propylene glycol, glycerol, and ethanol.

[0041] In the technical solution of this invention, the fermentation concentrate, pineapple ester, ethyl butyrate, and vanillin in the fragrance are simultaneously controlled within the specified range. This not only fully utilizes the aroma spectrum advantages of the fermentation broth, laying a rich aroma foundation for the fragrance, but also avoids the overall aroma being ruined by an overly prominent aroma due to an excessively high mass proportion of a certain component. Solvents such as propylene glycol, glycerin, and ethanol have good dissolving properties and can fully dissolve the fermentation concentrate, pineapple ester, ethyl butyrate, vanillin, and other components in the fragrance to form a homogeneous and stable fragrance system.

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example 1: Initial screening of β-carotene-degrading strains 1. Preparation of β-carotene stock solution: Weigh 20 mg of β-carotene under light-protected conditions, dissolve it in 10 mL of analytical grade dichloromethane, mix slowly and thoroughly, add 1 g of Tween 80 and sonicate in an ice-water bath to dissolve. Concentrate under reduced pressure at 30 °C to remove dichloromethane, then add 100 mL of sterile water to dissolve. Filter under sterile conditions through a 0.22 μm bacterial filter membrane to obtain a 0.2 mg / mL β-carotene stock solution. Store at 4 °C in the dark and it is recommended to use within 24 hours.

[0044] 2. Preparation of culture medium with β-carotene as the sole carbon source: Add 2.0 g / L dipotassium hydrogen phosphate, 4.0 g / L ammonium sulfate, 0.4 g / L magnesium sulfate heptahydrate, 0.2 g / L sodium chloride, 0.02 g / L calcium chloride, and 15 g / L agar powder to 200 mL of deionized water, and sterilize at 121 °C for 15 min. After cooling to 30–35 °C, add 100 mL of β-carotene stock solution, gently shake to mix, pour into plates, and cool for later use.

[0045] 3. The specific steps for the initial screening of β-carotene-degrading strains are as follows: Topsoil from the Yellow River basin in Lanzhou (36.068191°N 103.854621°E) was serially diluted and spread onto a medium with β-carotene as the sole carbon source. The culture was then incubated at 30°C for 24 hours. After single colonies emerged, colonies with a clear zone were picked and inoculated onto a medium with β-carotene as the sole carbon source. These colonies were then incubated at 30°C for another 24 hours. Two strains, exhibiting significant β-carotene degradation (producing a large clear zone), were preliminarily screened as β-carotene-degrading strains and named HHR-1-2 and HHR-1-3, respectively. Figure 1 As shown. Figure 1 The two negative control bacteria on the right side of the middle section did not have a clear zone, indicating that they are essentially unable to degrade β-carotene.

[0046] Example 2: Secondary screening of β-carotene-degrading strains 1. Preparation of β-carotene fermentation medium: Add 4 g / L glucose, 0.5 g / L dipotassium hydrogen phosphate, 2.0 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 0.04 g / L ferrous sulfate heptahydrate, and 0.02 g / L calcium chloride to 200 mL of deionized water, and sterilize at 121 °C for 15 min. After cooling to 30–35 °C, add 30 mL of β-carotene stock solution and set aside for later use.

[0047] 2. The specific steps for the secondary screening of β-carotene-degrading strains are as follows: 1) Preparation of HHR-1-2 and HHR-1-3 fermentation broth: HHR-1-2 and HHR-1-3 were activated twice with liquid culture medium and then centrifuged (8000 rpm, 15 min). The cell pellet was taken and diluted with sterile physiological saline to an OD600 value of 1.5, with a viable cell count of 1.0 × 10⁻⁶. 8 CFU / mL was inoculated into β-carotene fermentation medium at a 4% inoculum rate. The control group was the uninoculated group. The cultures were incubated at 30℃ for 24 hours to obtain the control group fermentation broth, HHR-1-2 fermentation broth, and HHR-1-3 fermentation broth. Figure 4 As shown.

[0048] 2) Plot the β-carotene standard curve as follows: Measure 0, 0.125, 0.25, 0.5, 1, 1.5, 2, and 2.5 mL of β-carotene stock solution into eight 10 mL volumetric flasks, respectively. Add distilled water to the final volume. Pipette 200 μL of each concentration of β-carotene solution into a 96-well cell culture plate. Measure the absorbance at OD450 nm using a microplate reader. Use distilled water as a blank control. Based on the obtained data, construct the standard curve. Figure 2 As shown, the linear equation obtained is Y = 5.405X - 0.0015 (R²). 2 =0.99627), where X is the concentration of β-carotene aqueous solution, mg / mL; Y is the absorbance of β-carotene aqueous solution.

[0049] 3) Determine the degradation rate of β-carotene. Measure the absorbance at OD450 of the HHR-1-2 fermentation broth from step 1), then calculate the β-carotene content in the sample based on the standard curve from step 2), and finally obtain the degradation rate using the following formula. Results are as follows: Figure 3 As shown.

[0050]

[0051] Experimental results showed that HHR-1-2 had a significantly higher ability to degrade β-carotene than HHR-1-3 (e.g., Figure 3 As shown). After 24 hours of fermentation, the fermentation broth clearly changed from orange-red to light yellow (as shown). Figure 4(As shown). After fermentation, the absorbance (OD450) of the control group fermentation broth, HHR-1-2 fermentation broth, and HHR-1-3 fermentation broth at 450 nm was measured to be 0.172, 0.004, and 0.0200, respectively. According to the β-carotene standard curve, the degradation amount of HHR-1-2 was 0.02812 mg / mL, with a degradation rate of 89.68%. Simultaneously, the fermentation broth exhibited a distinct sweet, green fruit aroma, ultimately confirming HHR-1-2 as the target strain.

[0052] Example 3 Identification of β-carotene-degrading strains HHR-1-2 colonies, after being streaked on LB medium and incubated for 24 hours, showed colony diameters between 1.5 and 2.8 mm, a creamy-yellowish-white color, neat edges, and a smooth, moist surface. Figure 5 As shown in the figure. DNA was extracted from single colonies of HHR-1-2 and amplified using the universal 16S primers 27F and 1492R. The amplified DNA was sent to the testing center of Universal Biotechnology (Anhui) Co., Ltd. for sequencing. The 16S rDNA nucleotide sequence of HHR-1-2 is shown in SEQ ID NO. 1. After sequencing, the 16S rDNA sequencing results of HHR-1-2 were compared with the NCBI database using Standard Nucleotide Blast. The results showed that HHR-1-2 had 99.79% homology with Aeromonas media RM. Therefore, strain HHR-1-2 was identified as Aeromonas media.

[0053] The 16S rDNA nucleotide sequence of Aeromonas intermedius HHR-1-2 is shown in SEQ ID NO. 1.

[0054] Example 4: Analysis of volatile components in the fermentation broth of Aeromonas intermedius HHR-1-2 The fermentation broth of Aeromonas intermedius HHR-1-2 was prepared according to the steps in Example 2. After centrifugation, 1 mL of the supernatant was collected and placed in a 20 mL headspace vial, which was immediately sealed for headspace-gas chromatography-mass spectrometry (HS-GC-MS) analysis. The mass spectra of each detected component were compared with the standard mass spectral library, and the relative percentage content of each volatile component was calculated using the peak area normalization method.

[0055] 1. Headspace injection conditions: An automated headspace sampler was used. Equilibrium temperature 80℃; cycle time 200 min; needle temperature 105℃; injection volume 1.0 mL.

[0056] 2. Gas chromatography-mass spectrometry conditions: Agilent 122-7157 column; high-purity helium carrier gas (purity >99.999%), constant flow mode, flow rate 1.0 mL / min; temperature program: initial column temperature 50℃ held for 0 min, increased to 100℃ at 5℃ / min, then increased to 260℃ at 15℃ / min, held for 10 min; MS ion source temperature 230℃, quadrupole temperature 150℃, acquisition mode scan.

[0057] 3. Experimental Results: HS-GC-MS analysis of the fermentation broth of Aeromonas intermedius HHR-1-2 revealed 20 volatile components. The GC-MS chromatogram is shown below. Figure 6 As shown in the table. The components are listed in order of retention time, along with their names, retention times, peak areas, and relative percentage contents. Taking hexanal as an example, the percentage content of hexanal is the percentage of its peak area relative to the total peak area of ​​all compounds.

[0058] Table 1. Analysis results of volatile components in the fermentation broth of Aeromonas intermedius HHR-1-2

[0059] Table 1 shows that the volatile components produced by Aeromonas intermedius HHR-1-2 after degrading β-carotene have the following significant characteristics: (1) Aldehyde compounds dominate: There are a total of 11 aldehyde compounds (11 / 20), with a total relative content of up to 70.81%. The products are mainly C6-C9 aldehydes, accompanied by corresponding alcohols and trans-2-enal. The products show a coordinated and regular metabolic pattern, indicating that Aeromonas intermedius HHR-1-2 has a specific enzymatic reaction pathway for the degradation of β-carotene; (2) In stark contrast to the products of the traditional CCD pathway: β-ionone, 5,6-epoxy-β-ionone and dihydroactinolone are the hallmark products of the traditional CCD pathway, but their total content in this example is only 1.80%, indicating that Aeromonas intermedius HHR-1-2 may have adopted a degradation pathway different from the traditional CCD pathway. (3) Application prospects: The product is mainly saturated aldehydes, while also containing abundant unsaturated enaldehydes (such as trans-2-hexenal and trans-2-nonenal), a small amount of corresponding alcohols and ketones, which together constitute a complex aroma spectrum with rich layers and a combination of green, fruity, fatty and woody aromas, which has unique value in the development of high-end natural fragrances.

[0060] Example 5: Application of Aeromonas intermedius HHR-1-2 fermentation broth in flavorings 1. Preparation of fermentation concentrate: The fermentation broth of Aeromonas intermedius HHR-1-2 was obtained according to the steps in Example 2. After centrifugation, 500g of supernatant was taken and extracted three times with dichloromethane (150mL of dichloromethane was added each time). The organic phases were combined, and 10% propylene glycol was added. The mixture was concentrated by vacuum distillation at 30°C until the dichloromethane was completely evaporated to obtain a fermentation concentrate with a sweet green aroma and an amber color, which was then set aside for use.

[0061] 2. Fragrance preparation: (1) Control group flavoring: 8% mango ester, 10% pineapple ester, 3% ethyl butyrate, 1% vanillin, propylene glycol to make up to 100%, mixed and prepared.

[0062] (2) Flavoring for the experimental group: 8% mango ester, 10% pineapple ester, 3% ethyl butyrate, 1% vanillin, 1% fermentation concentrate, and propylene glycol to make up to 100%, mixed and prepared.

[0063] 3. Sensory Evaluation: A double-blind sensory evaluation was conducted by an evaluation panel of 5 professional fragrance tasters. A 5-point scoring system was used, with the specific scoring criteria as follows: 1 point represents very poor, 2 points represent poor, 3 points represent average, 4 points represent good, and 5 points represent excellent. The following indicators were evaluated for the prepared fragrance: Fragrance quality: The type of fragrance; Aroma quantity: The intensity and power of the aroma; Freshness: The prominence of fresh aromas such as green and fruity notes; Naturalness: The degree to which the aroma closely resembles the actual aroma of the fruit; Harmony: The degree of integration of the overall aroma.

[0064] 4. Sensory evaluation results: The sensory evaluation results are the average of the scores given by 5 perfume tasters. See Table 2 for specific data.

[0065] Table 2. Sensory Evaluation Results of Flavors (Points)

[0066] Table 2 shows that the experimental group of flavorings exhibited improved aroma quality, aroma quantity, freshness, naturalness, and harmony compared to the control group, demonstrating a significant improvement in flavoring quality. This indicates that the fermentation broth of Aeromonas intermedius HHR-1-2 can significantly enhance the overall aroma of flavorings, possessing important application value in the fields of daily chemicals and food flavorings, and providing a new pathway for developing high-quality flavoring products.

[0067] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. An intermediate Aeromonas hydrophila HHR-1-2, characterized in that, The intermediate Aeromonas (Aeromonas intermedia) Aeromonas The deposit number of the intermediate Aeromonas (Aeromonas intermedia) HHR-1-2 is CCTCC NO: M 20252744, which was deposited at the China Center for Type Culture Collection on December 01, 2025.

2. The Aeromonas intermedius HHR-1-2 as described in claim 1, characterized in that, The 16S rDNA gene sequence of Aeromonas intermedius HHR-1-2 is shown in SEQ ID NO.

1.

3. The use of Aeromonas intermedius HHR-1-2 as described in any one of claims 1 to 2 in the degradation of carotenoids.

4. The application as described in claim 3, characterized in that, The carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, The degradation rate of carotenoids by Aeromonas intermediate HHR-1-2 is 85%~95%.

5. The application as described in claim 4, characterized in that, The products of *Aeromonas intermedius* HHR-1-2 that degrade carotenoids include aldehydes, ketones, alcohols, and esters. The aldehyde compounds include at least one of hexanal, heptanal, trans-2-hexenal, octanal, trans-2-heptenal, nonanal, trans-2-octenal, trans-2-nonenal, ethyl citral, trans-2-decenal, and trans-2-undecenal. The ketone compounds include at least one of 2,2,6-trimethylcyclohexanone, isophorone, ethyl ionone, and 5,6-epoxy-β-ionone; The alcohols include at least one of hexanol, heptanol, octanol, and nonanol; The ester compounds include dihydroactinolone.

6. The application as described in claim 5, characterized in that, The products of carotenoid degradation by Aeromonas intermediate HHR-1-2 include: The peak area of ​​the aldehyde compound accounts for 65% to 75% of the peak area of ​​the product; The total peak area of ​​β-ionone, 5,6-epoxy-β-ionone and dihydroactinolone accounts for less than 2% of the peak area of ​​the product.

7. The use of Aeromonas intermedius HHR-1-2 as described in any one of claims 1 to 2 in the preparation of flavorings.

8. The application as described in claim 7, characterized in that, The application includes the following steps: A culture medium is provided, wherein the carbon source in the culture medium includes carotenoids; The Aeromonas intermedia HHR-1-2 was cultured in the culture medium to obtain a fermentation broth; The fermentation broth is used to prepare a flavoring.

9. The application as described in claim 8, characterized in that, The step of preparing flavoring from fermentation broth includes: The fermentation broth was used to prepare a concentrated fermentation broth. The fermentation concentrate is mixed with mango ester, pineapple ester, ethyl butyrate, vanillin, and solvent to obtain the flavoring.

10. The application as described in claim 9, characterized in that, In the flavoring: the fermentation concentrate accounts for 0.90~1.10% by mass, the pineapple ester accounts for 6~10% by mass, the ethyl butyrate accounts for 8~12% by mass, and the vanillin accounts for 1~3% by mass; and / or, The solvents include propylene glycol, glycerol, and ethanol.

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