Pseudomonas alcaligenes HHR-1-1 strain and application thereof in degrading carotenoids and preparing essence

Abstract

This invention discloses a strain of *Alcaligenes* HHR-1-1 and its application in the degradation of carotenoids and the preparation of flavorings, relating to the field of microbial fermentation technology. The *Alcaligenes* strain of this invention (… Pseudomonas alcaligenes The HHR-1-1 strain has the accession number CCTCC NO: M 20252745, and the *Alcaligenes* HHR-1-1 strain was deposited at the China Center for Type Culture Collection on December 1, 2025. The *Alcaligenes* HHR-1-1 strain can degrade carotenoids with a degradation rate of 80%–97%, and its main degradation products are β-ionone and its oxides, and dihydroactinolone. Furthermore, the fermentation broth of *Alcaligenes* HHR-1-1 strain and carotenoids can be used in flavor preparation, effectively improving the quality of the flavors.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to an Alcaligenes Pseudomonas strain HHR-1-1 and its application in the degradation of carotenoids and the preparation of flavorings. Background Technology

[0002] Ionone, dihydroactinolone, and other substances are key aroma components that make up many natural floral and fruity aromas, and are in huge demand in the high-end food, cosmetics, and fragrance industries.

[0003] Currently, the main methods for obtaining such aromatic compounds include plant extraction and chemical synthesis. Plant extraction suffers from problems such as strong dependence on raw materials, low extraction rate, and high cost, making it difficult to meet the needs of industrial production; while chemical synthesis often uses harsh reaction conditions and may produce undesirable byproducts, which does not meet the modern consumer demand for "clean labels" and natural products.

[0004] While microbial transformation has the advantages of mild reaction conditions and good specificity, it still has the disadvantages of complex product profiles and insufficient aroma purity. Summary of the Invention

[0005] The main objective of this invention is to propose a strain of Alcaligenes Pseudomonas HHR-1-1 and its application in the degradation of carotenoids and the preparation of flavorings, aiming to solve the problem of low purity of product spectrum in the production of aroma components such as dihydroactinolone by microbial transformation in the prior art.

[0006] To achieve the above objectives, this invention proposes a strain of *Alcaligenes* HHR-1-1, wherein *Alcaligenes* (… Pseudomonas alcaligenes The accession number of HHR-1-1 is CCTCC NO: M 20252745. The alkali-producing Pseudomonas HHR-1-1 was deposited at the China Center for Type Culture Collection on December 1, 2025.

[0007] In one embodiment, the 16S rDNA gene sequence of the *Alcaligenes* HHR-1-1 strain is shown in SEQ ID NO. 1.

[0008] The present invention also provides the application of Alcaligenes Pseudomonas alkaloidus strain HHR-1-1 in the degradation of carotenoids.

[0009] In one embodiment, the application includes the following steps: A culture medium is provided, the culture medium comprising carotenoids; The alkaloid Pseudomonas albogene HHR-1-1 strain was inoculated onto the culture medium and cultured to obtain degradation products.

[0010] In one embodiment, the culture medium further includes a regulator: The regulator includes at least one of soybean phospholipids, lecithin, oleic acid, linoleic acid, β-sitosterol, and cholesterol; and / or, The regulator is present in the culture medium at a mass ratio of 0.01% to 0.2%.

[0011] In one embodiment, the regulator includes soybean lecithin, oleic acid, and β-sitosterol, wherein the soybean lecithin has a mass percentage of 0.01% to 0.1% in the culture medium, the oleic acid has a mass percentage of 0.01% to 0.1% in the culture medium, and the β-sitosterol has a mass percentage of 0.01% to 0.1% in the culture medium.

[0012] In one embodiment, the carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, The inoculation amount of the *Alcaligenes* HHR-1-1 strain is 0.5% to 1.5%; and / or, The culture temperature is 28~32℃, and the culture time is 20~30h; and / or, The culture was conducted at a pH of 6.5–7.5; and / or, The *Alcaligenes* strain HHR-1-1 exhibits a carotenoid degradation rate of 80%–97%; and / or, The degradation products include at least one of β-ionone, 5,6-epoxy-β-ionone, 3-oxo-β-ionone, and dihydroactinolone.

[0013] The present invention also provides the application of Alcaligenes Pseudomonas alkaliformis strain HHR-1-1 in the preparation of fragrances.

[0014] In one embodiment, the application includes the following steps: A culture medium is provided, wherein the carbon source in the culture medium includes carotenoids; The *Alcaligenes* strain HHR-1-1 was cultured in the culture medium to obtain a fermentation broth. The fermentation broth was mixed with triacetin, ethyl acetate, linalool, dihydrocoumarin, and a solvent to obtain the flavoring.

[0015] In one embodiment, the fragrance comprises: 65-75% by mass of the solvent, 4-6% by mass of the triacetyl ester, 2-4% by mass of the ethyl acetate, 0.04-0.06% by mass of the linalool, and 1.9-2.0% by mass of the dihydrocoumarin; and / or, The solvent includes at least one of propylene glycol and ethanol.

[0016] In the technical solution of this invention, the alkaloid-producing Pseudomonas aeruginosa ( Pseudomonas alcaligenes The HHR-1-1 strain has the accession number CCTCC NO: M 20252745, and the *Alcaligenes* HHR-1-1 strain was deposited at the China Center for Type Culture Collection on December 1, 2025. The *Alcaligenes* HHR-1-1 strain can degrade carotenoids with a degradation rate of 80%–97%, and its main degradation products are β-ionone and its oxides, and dihydroactinolone. Furthermore, the fermentation broth of *Alcaligenes* HHR-1-1 strain and carotenoids can be used in flavor preparation, effectively improving the quality of the flavors. 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 diagram showing the growth of strain HHR-1-1 in Example 1 of the present invention on a culture medium with β-carotene as the sole carbon source. Figure 2 This is a diagram showing the degradation of β-carotene by strain HHR-1-1 in Example 2 of the present invention; Figure 3 The graph shows the degradation rate of β-carotene by strain HHR-1-1 in Example 2 of this invention. Figure 4 This is a GC-MS analysis result of the products of β-carotene degradation by strain HHR-1-1 in Example 3 of the present 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] Compared to plant extraction techniques, biotransformation using microorganisms or enzymes is considered a highly promising alternative due to its advantages such as mild reaction conditions, high specificity, and the ability to produce "natural" products. Currently, certain fungi (such as Penicillium and Aspergillus) and Escherichia coli can degrade β-carotene. However, these strains generally suffer from low degradation efficiency, complex product profiles (leading to impure aromas), or food safety risks inherent in the strains themselves (such as the potential production of mycotoxins by some fungi).

[0022] More importantly, the types of aroma-producing strains that have been developed so far are relatively limited, mostly concentrated in a few common genera. Their degradation pathways and aroma-producing characteristics have been thoroughly studied, making it difficult to achieve breakthroughs in aroma quality or conversion efficiency.

[0023] Therefore, there is an urgent need in this field to screen and discover new, safe and efficient microbial resources from nature, especially those uncommon strains with unique degradation capabilities and excellent aroma-producing characteristics, in order to solve the problems of low conversion efficiency, poor aroma quality and doubts about the safety of strains in existing technologies, and to provide new solutions for the production of natural fragrances.

[0024] In view of this, the present invention proposes a strain of Alcaligenes HHR-1-1, wherein Alcaligenes ( Pseudomonas alcaligenes The accession number of HHR-1-1 is CCTCC NO: M 20252745. The alkali-producing Pseudomonas HHR-1-1 was deposited at the China Center for Type Culture Collection on December 1, 2025.

[0025] In the technical solution of this invention, the alkaloid-producing Pseudomonas aeruginosa ( Pseudomonas alcaligenesThe HHR-1-1 strain has the accession number CCTCC NO: M 20252745. The *Alcaligenes* HHR-1-1 strain was deposited on December 1, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province. The *Alcaligenes* HHR-1-1 strain can degrade carotenoids with a degradation rate of 80%–97%. Its main carotenoid degradation products are β-ionone and its oxides, and dihydroactinolone. Furthermore, the fermentation broth of *Alcaligenes* HHR-1-1 strain and carotenoids can be used in flavor preparation, effectively improving the quality of the flavors.

[0026] In some embodiments, the 16S rDNA gene sequence of the *Alcaligenes* HHR-1-1 strain is shown in SEQ ID NO. 1.

[0027] This invention also provides the application of *Alcaligenes f. hHR-1-1* strain in the degradation of carotenoids. The *Alcaligenes f. hHR-1-1* strain provided by this invention exhibits good degradation activity against carotenoids and can grow on culture media where carotenoids are the sole carbon source.

[0028] In some embodiments, the application includes the following steps: providing a culture medium comprising carotenoids; inoculating *Alcaligenes f. alkalitomyces* strain HHR-1-1 onto the culture medium and culturing it to obtain degradation products. This method can obtain degradation products relatively quickly.

[0029] In some embodiments, the culture medium further includes a regulator, which includes at least one selected from soybean lecithin, lecithin, oleic acid, linoleic acid, β-sitosterol, and cholesterol. It is understood that the culture medium may contain regulators in addition to carotenoids. The regulator may be any one of soybean lecithin, lecithin, oleic acid, linoleic acid, β-sitosterol, and cholesterol, or may contain two or more of these substances, all within the scope of this application. Under specific conditions, the above-mentioned regulators can effectively improve the degradation efficiency of carotenoids by the *Alcaligenes f. sp.* HHR-1-1 strain.

[0030] In some embodiments, the regulator is present in the culture medium at a mass ratio of 0.01% to 0.2%. The mass ratio of the regulator can be any two values ​​within a range of 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, or higher. This can effectively improve the degradation efficiency of carotenoids by the *Alcaligenes* HHR-1-1 strain.

[0031] In some embodiments, the regulator is preferably any one of soybean lecithin, oleic acid, and β-sitosterol. When soybean lecithin is used alone, its mass percentage in the culture medium is preferably 0.05%; when oleic acid is used alone, its mass percentage in the culture medium is preferably 0.05%; and when β-sitosterol is used alone, its mass percentage in the culture medium is preferably 0.01%, resulting in a higher degradation rate of carotenoids.

[0032] In some embodiments, the culture medium further includes soybean lecithin, oleic acid, and β-sitosterol. The soybean lecithin, oleic acid, and β-sitosterol are present in the culture medium at a mass ratio of 0.01% to 0.1% by mass. It is understood that the culture medium may contain soybean lecithin, oleic acid, and β-sitosterol. The mass ratio of soybean lecithin in the culture medium can be any two values ​​within a range of 0.01%, 0.05%, 0.1%, or higher. The mass ratio of oleic acid in the culture medium can also be any two values ​​within a range of 0.01%, 0.05%, 0.1%, or higher. The mass ratio of β-sitosterol in the culture medium can also be any two values ​​within a range of 0.01%, 0.05%, 0.1%, or higher. Simultaneously controlling the mass ratios of soybean lecithin, oleic acid, and β-sitosterol within the above ranges ensures a high efficiency in the degradation of carotenoids by the *Pseudomonas alkaloidosa* strain HHR-1-1.

[0033] Preferably, when the mass percentages of soybean phospholipids, oleic acid, and β-sitosterol in the culture medium are simultaneously controlled at 0.05%, the degradation rate of carotenoids by *Pseudomonas alkaloidosa* strain HHR-1-1 can reach over 95%.

[0034] In some embodiments, the carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, the inoculum size of the *Alcaligenes f. f. HHR-1-1* strain is 0.5% to 1.5%; and / or, the culture temperature is 28 to 32°C, and the culture time is 20 to 30 hours; and / or, the culture pH is 6.5 to 7.5; and / or, the degradation rate of carotenoids by the *Alcaligenes f. f. HHR-1-1* strain is 80% to 97%; and / or, the degradation products include at least one of β-ionone, 5,6-epoxy-β-ionone, 3-oxo-β-ionone, and dihydroactinolone. Simultaneously controlling the types of carotenoids, the inoculum size of the strains, and the culture conditions within the above ranges can ensure the rapid degradation of carotenoids to obtain products such as β-ionone, 5,6-epoxy-β-ionone, 3-oxo-β-ionone, and dihydroactinolone, and the products are relatively pure in composition and have a purer aroma.

[0035] This invention also provides the application of *Alcaligenes f. hHR-1-1* strain in the preparation of flavorings. In some embodiments, the application includes the following steps: providing a culture medium in which the carbon source includes carotenoids; culturing the *Alcaligenes f. hHR-1-1* strain in the culture medium to obtain a fermentation broth; and mixing the fermentation broth with triacetin, ethyl acetate, linalool, dihydrocoumarin, and a solvent to obtain the flavoring. Because *Alcaligenes f. hHR-1-1* strain can rapidly degrade carotenoids on a culture medium with carotenoids as the sole carbon source to obtain a fermentation broth containing β-ionone and its oxides and dihydroactinol, this fermentation broth has a simple composition and can directly yield flavorings after mixing with other aroma components and solvents, without the need for purification of the fermentation broth.

[0036] In some embodiments, the fragrance comprises: 65-75% by mass of the solvent, 4-6% by mass of the triacetyl ester, 2-4% by mass of the ethyl acetate, 0.04-0.06% by mass of the linalool, and 1.9-2.0% by mass of the dihydrocoumarin; and / or, the solvent includes at least one of propylene glycol and ethanol. The mass percentages of the solvent can be 65%, 70%, or 75%, the triacetyl ester can be 4%, 5%, or 6%, the ethyl acetate can be 2%, 3%, or 4%, the linalool can be 0.04%, 0.05%, or 0.06%, and the dihydrocoumarin can be 1.9%, 1.95%, or 2.0%. Controlling the mass percentages of the above components within these ranges ensures a high richness, good layering, sweetness, and good taste in the fragrance, with minimal off-flavors.

[0037] 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.

[0038] Experimental materials: 1. Solid selective culture medium with β-carotene as the sole carbon source (1) Preparation of β-carotene stock solution: Under light-protected conditions, weigh 0.1g of β-carotene (purchased from Hubei Weideli Chemical Reagent Co., Ltd., purity 98%), add 2g of Tween 80 and 40g of dichloromethane for ultrasonic dissolution, and then remove dichloromethane by vacuum distillation at 30℃ to obtain concentrated solution. Add 100mL of sterile water and mix well. Filter with a sterile 0.22μm filter membrane to obtain 1mg / mL of β-carotene stock solution.

[0039] (2) Preparation of solid selective culture medium with β-carotene as the sole carbon source: 0.8g dipotassium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 0.1g magnesium sulfate heptahydrate, 1g ammonium sulfate, 0.01g calcium chloride dihydrate, 0.02g ferric sulfate heptahydrate and 15g bacterial agar powder were added to 1L deionized water and sterilized at 121℃ for 15min. After cooling to 30℃, 10% of β-carotene stock solution was added (that is, the volume ratio of β-carotene stock solution in the final system is 10%). After slowly shaking and mixing, the medium was poured into plates, cooled and set aside for use to obtain solid plates.

[0040] 2. Liquid culture medium with β-carotene as the sole carbon source: Add 0.8g dipotassium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 0.1g magnesium sulfate heptahydrate, 1g ammonium sulfate, 0.01g calcium chloride dihydrate, and 0.02g ferric sulfate heptahydrate to 1L of deionized water. Sterilize at 121℃ for 15min, then cool to 30℃. Add 10% of the β-carotene stock solution (i.e., the volume percentage of the β-carotene stock solution in the final system is 10%) and slowly shake to mix.

[0041] 3. Preparation of LB liquid culture medium (1L): 10g sodium chloride, 5g yeast powder, 10g peptone, add 1L distilled water, autoclave at 115℃ for 15min, and cool.

[0042] 4. Preparation of LB solid culture medium (1L): 10g sodium chloride, 5g yeast powder, 10g peptone and 15g bacterial agar powder, add 1L distilled water, autoclave at 115℃ for 15min, pour into plates, cool and set aside to use, to obtain solid plates.

[0043] 5. Liquid fermentation medium containing β-carotene: Add 10g glucose, 0.8g dipotassium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 0.1g magnesium sulfate heptahydrate, 1g ammonium sulfate, 0.01g calcium chloride dihydrate, and 0.02g ferric sulfate heptahydrate to 1L deionized water. Sterilize at 115℃ for 15min, then cool to 30℃. Add 10% of β-carotene stock solution (i.e., the volume percentage of β-carotene stock solution in the final system is 10%) and slowly shake to mix.

[0044] Example 1: Isolation, primary screening, secondary screening and molecular identification of Alcaligenes HHR-1-1 1. Separation and primary screening The samples used for isolation and screening of strains were soil samples collected from Huanghe Lijingyuan, Chengguan District, Lanzhou City, Gansu Province.

[0045] Soil samples were weighed and added to 200 mL of sterile water. The mixture was incubated at 37°C with shaking at 200 rpm for 2 hours to obtain a soaking solution. Then, 1 mL of the soaking solution was added to a glass tube containing 9 mL of sterile water for serial dilution, resulting in three concentration gradients: 10-fold, 100-fold, and 1000-fold. 100 μL of each dilution was plated onto solid selective medium plates using β-carotene as the sole carbon source and incubated at 30°C for 24 hours. Each concentration gradient was performed in triplicate. Single colonies with distinct morphological characteristics and a clear degradation zone were then selected and enriched using LB solid medium.

[0046] like Figure 1 As shown in the left figure, after 24 hours of enrichment culture, a strain (marked by a red box) was found to have a large and obvious degradation zone on a solid selective medium with β-carotene as the sole carbon source, which contrasts sharply with the undegraded area. This indicates that the strain can effectively degrade β-carotene and can grow on a solid selective medium with β-carotene as the sole carbon source. This strain was named HHR-1-1.

[0047] 2. Rescreening and verification HHR-1-1 strain colonies enriched on LB solid medium plates were purified by streak plating three times to obtain pure cultures. Single colonies of the pure cultures were then streaked onto solid selective medium plates with β-carotene as the sole carbon source and incubated at 30°C for 24 hours. Figure 1 The right figure shows that there are still obvious degradation zones.

[0048] 3. Molecular identification The 16S rDNA sequence of this strain was obtained by amplification using universal bacterial sequencing primers 27F (AGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) (as shown in SEQ ID No. 1). The 16S rDNA sequence was then aligned with the gene sequence on the NCBI website. The results showed that strain HHR-1-1 is similar to... Pseudomonas alcaligenes The strain HHR-1-1 was identified as *Alcaligenes faecalis* because it was most closely related to the bacteria, with a homology of 99.79%. Pseudomonas alcaligenes ).

[0049] SEQ ID No. 1:

[0050] Example 2: Test of the ability of HHR-1-1 strain to degrade β-carotene The HHR-1-1 single colony verified in Example 1 was activated twice with 100 mL LB liquid medium and centrifuged (9000 rpm for 15 min). The bacterial pellet was collected and diluted with sterile physiological saline to OD. 600 The value is 1, and the viable bacterial count using a cell counting chamber is 1.0 × 10⁻⁶. 8 CFU / mL was used to obtain HHR-1-1 bacterial suspension. The HHR-1-1 bacterial suspension was inoculated into 5 mL of liquid culture medium with β-carotene as the sole carbon source at a 4% inoculation rate. For the control group, the same volume of sterile water as the bacterial suspension was added to 5 mL of liquid culture medium with β-carotene as the sole carbon source. The mixture was incubated at 30℃, in the dark, and at a shaker speed of 200 rpm for 24 h before observation. Results are as follows: Figure 2 As shown.

[0051] like Figure 2 As shown in the left image, the control group remains yellow. Figure 2 As shown in the right figure, the β-carotene color in the test tubes containing the HHR-1-1 strain has largely faded.

[0052] Subsequently, 1 mL of each of the two culture media groups was centrifuged, and the supernatant was retained. 200 μL of the supernatant was then pipetted into each well of a 96-well plate, and the OD was measured using a microplate reader. 450 The absorbance values ​​are as follows: Figure 3 As shown.

[0053] Depend on Figure 3 It can be seen that the OD of the control group 450 The measured value was 0.675, and the OD of the group with added HHR-1-1 strain was... 450 The measured value was 0.117, and its degradation rate reached 81% after 24 hours. The results indicate that this strain has a strong ability to degrade β-carotene.

[0054] Example 3 Gas chromatography-mass spectrometry (GC-MS) analysis of the products of β-carotene degradation by strain HHR-1-1 1. Preparation of fermentation broth The HHR-1-1 bacterial suspension obtained in Example 2 was inoculated into 100 mL of liquid fermentation medium containing β-carotene at an inoculation rate of 4%, and fermented at 30 °C for 48 h to obtain the fermentation broth.

[0055] 2. Extraction of fermentation products The above fermentation broth was centrifuged at 5000 rpm for 10 min to remove the bacterial cells and obtain the supernatant. The supernatant was extracted with ethyl acetate at a volume ratio of 1:1 for 1 h. Then, the ethyl acetate was removed by vacuum evaporation, and 10 g of propylene glycol was added as a solvent to prepare the concentrated solution.

[0056] 3. GC-MS analysis of the components of the fermentation concentrate 1 μL of the above concentrated solution was manually injected and analyzed by GC-MS. GC-MS 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. The detected degradation products are shown in Table 1, and the relevant GC-MS spectra are shown below. Figure 4 As shown. The mass spectra of each detected component were compared with a standard mass spectral library, and the relative percentage content of each volatile component was calculated using the peak area normalization method.

[0057] Table 1. Products of β-carotene degradation by strain HHR-1-1

[0058] Table 1 shows that strain HHR-1-1 produced six degradation products from β-carotene: isophorone, cyclocitral, 5,6-epoxy-β-ionone, β-ionone, dihydroactinolone, and 3-oxo-β-ionone. Among these, β-ionone and its oxides, and dihydroactinolone, were the main degradation products, both possessing distinct and natural aroma characteristics. Dihydroactinolone exhibits a warm, sweet fruity and tea-like aroma with strong persistence, making it widely applicable in food, perfumes, skincare products, and tobacco flavorings. β-ionone exhibits a sophisticated woody, violet, and berry aroma with a rich and mellow fragrance and an extremely low aroma threshold, making it suitable for use in high-end perfumes and skincare products. Overall, dihydroactinolone and β-ionone, with their unique aroma characteristics, play an irreplaceable role in the flavoring fields of food, daily chemicals, and tobacco.

[0059] Example 5: Screening of regulators to improve the efficiency of β-carotene degradation in strain HHR-1-1 1. Screening of single regulators Soybean phospholipids, lecithin, oleic acid, linoleic acid, β-sitosterol, and cholesterol were added to a liquid fermentation medium with β-carotene as the sole carbon source, at three concentration gradients: low (0.01%), medium (0.05%), and high (0.1%). The inoculum size of strain HHR-1-1 was 1%, and the medium was cultured at 30°C and 200 rpm for 24 hours. The OD values ​​of the different culture media were determined using the enzyme-linked immunosorbent assay (ELISA) method described in Example 2. 450 The absorbance values ​​were measured, and qualitative and quantitative analyses were performed using GC-MS with dihydroactinidone as an internal standard. The results are shown in Table 2. The control group was the group without any additives.

[0060] Table 2. Effects of different concentrations of regulators on the degradation efficiency of β-carotene by strain HHR-1-1

[0061] Table 2 shows that soybean lecithin, oleic acid, and β-sitosterol alone, at specific concentrations, can significantly increase the degradation rate of β-carotene and the yield of the target aroma product, dihydroactinol. Their optimal concentrations are: soybean lecithin 0.05%, oleic acid 0.05%, and β-sitosterol 0.01%, respectively.

[0062] 2. Screening of compound regulators Orthogonal experiments were used to determine the optimal formulation: Based on the single-factor experimental results in Table 2, the degradation rate of β-carotene was used as the indicator, and the optimal formulation was determined according to L(9)3 in Table 3. 3 The optimal regulator ratio was screened using an orthogonal experiment with three factors and three levels. The experimental results are shown in Table 4.

[0063] Table 3 L(9)3 3 Orthogonal array

[0064] Table 4 Results of the orthogonal experiment

[0065] Table 4 shows that range analysis (calculation of K, k, and R values) revealed that the order of influence of each factor on the degradation rate was: β-sitosterol concentration > soybean phospholipid concentration > oleic acid concentration, indicating that β-sitosterol is the key regulatory factor. Furthermore, by comparing the mean k values ​​at each factor level, the optimal level was determined, ultimately yielding the theoretically optimal compound regulator ratio as A2B3C2, i.e., 0.05% soybean phospholipid, 0.10% oleic acid, and 0.05% β-sitosterol. This combination significantly enhances the degradation and aroma production performance of β-carotene by strain HHR-1-1. Verification showed that the actual degradation rate of the optimal combination A2B3C2 reached 95.3%.

[0066] The composite regulator of this invention mainly enhances the dispersibility of carotene in the culture medium through soybean phospholipids, optimizes the permeability of the cell membrane of HHR-1-1 strain through oleic acid, and induces the expression of enzymes that degrade carotenoids in HHR-1-1 strain through β-sitosterol, thereby achieving efficient conversion of β-carotene.

[0067] Under the technical solution of this invention, the compound regulator (0.05% soybean phospholipids, 0.10% oleic acid, and 0.05% β-sitosterol) is added to the HHR-1-1 fermentation system (1% inoculum, 30℃, pH 7.0). After 24 hours, the degradation rate of β-carotene can be increased to 95.3%, and the yield of dihydroactinolone can be increased from 5 mg / L to 7.0-8.0 mg / L.

[0068] Example 6: Application of the product of β-carotene degradation by strain HHR-1-1 in flavorings The fermentation broth obtained in Example 3 was used to prepare flavorings, and the specific formula is shown in Table 5.

[0069] Table 5 Flavor Formulas

[0070] In Table 5, all addition amounts are mass fractions, and the total system formulation mass is 100g.

[0071] Following the formulas in Table 5, the flavorings for the basic and experimental groups were prepared. 1g of each flavoring was then added to blank tobacco to prepare cigarettes for evaluation. The sensory evaluation method was as follows: a evaluation committee composed of seven experienced professional smokers used a double-blind triangular testing method. In a standard evaluation room, the smokers evaluated the coded cigarette samples. Referring to the industry standard (YC / T 145-1998), the key indicators in Table 6 were scored on a 9-point scale (1 being the worst, 9 the best), and descriptive comments were recorded. The evaluation results are shown in Table 6.

[0072] Table 6 Sensory Evaluation Results

[0073] As shown in Table 6, in the basic flavoring formula, replacing 5% of the tobacco extract with 5% of the natural flavoring concentrate prepared in this invention resulted in a significant improvement in the sensory quality of the experimental group of flavorings. Professional evaluation results showed significant improvements in key indicators such as aroma richness and layering, natural aroma harmony, sweetness in the taste, and masking effect on off-flavors (P<0.01). This improvement effect stems from natural flavor substances derived from microbial fermentation in the concentrate (such as β-ionone and dihydroactinolone), which not only endow the flavorings with complex and elegant fruity and woody aromas but also play a crucial role in taste improvement and smoke purification. This fully demonstrates that the natural flavoring concentrate provided by this invention is a highly efficient and high-quality functional flavoring module that can significantly improve the overall quality of traditional flavorings.

[0074] 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. A strain of Alcaligenes HHR-1-1, characterized in that, The Alcaligenes (Pseudomonas alcaligenes) Pseudomonas alcaligenes The accession number of HHR-1-1 is CCTCC NO: M 20252745. The alkali-producing Pseudomonas HHR-1-1 was deposited at the China Center for Type Culture Collection on December 1, 2025.

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

1.

3. The use of the *Alcaligenes* strain HHR-1-1 as described in any one of claims 1 to 2 in the degradation of carotenoids.

4. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 3 in the degradation of carotenoids, characterized in that, The application includes the following steps: A culture medium is provided, the culture medium comprising carotenoids; The alkaloid Pseudomonas albogene HHR-1-1 strain was inoculated onto the culture medium and cultured to obtain degradation products.

5. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 4 in the degradation of carotenoids, characterized in that, The culture medium also includes a regulator: The regulator includes at least one of soybean phospholipids, lecithin, oleic acid, linoleic acid, β-sitosterol, and cholesterol; and / or, The regulator is present in the culture medium at a mass ratio of 0.01% to 0.2%.

6. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 5 in the degradation of carotenoids, characterized in that, The regulators include soybean lecithin, oleic acid and β-sitosterol, wherein the soybean lecithin accounts for 0.01% to 0.1% of the mass of the culture medium, the oleic acid accounts for 0.01% to 0.1% of the mass of the culture medium, and the β-sitosterol accounts for 0.01% to 0.1% of the mass of the culture medium.

7. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 4 in the degradation of carotenoids, characterized in that, The carotenoids include at least one of β-carotene, lutein, and lycopene; and / or, The inoculation amount of the *Alcaligenes* HHR-1-1 strain is 0.5% to 1.5%; and / or, The culture temperature is 28~32℃, and the culture time is 20~30h; and / or, The culture was conducted at a pH of 6.5–7.5; and / or, The *Alcaligenes* strain HHR-1-1 exhibits a carotenoid degradation rate of 80%–97%; and / or, The degradation products include at least one of β-ionone, 5,6-epoxy-β-ionone, 3-oxo-β-ionone, and dihydroactinolone.

8. The use of the *Alcaligenes* strain HHR-1-1 as described in any one of claims 1 to 2 in the preparation of flavorings.

9. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 8 in the preparation of flavorings, 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 *Alcaligenes* strain HHR-1-1 was cultured in the culture medium to obtain a fermentation broth. The fermentation broth was mixed with triacetin, ethyl acetate, linalool, dihydrocoumarin, and a solvent to obtain the flavoring.

10. The application of the *Alcaligenes* HHR-1-1 strain as described in claim 9 in the preparation of flavorings, characterized in that, In the fragrance: the solvent accounts for 65-75% by mass, the triacetin accounts for 4-6% by mass, the ethyl acetate accounts for 2-4% by mass, the linalool accounts for 0.04-0.06% by mass, and the dihydrocoumarin accounts for 1.9-2.0% by mass; and / or, The solvent includes at least one of propylene glycol and ethanol.

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