Pseudomonas sp. producing fruity flavor and use thereof

Abstract

This invention belongs to the field of food flavor engineering technology, specifically relating to a *Pseudomonas* bacterium that produces fruit flavor compounds and its applications. The *Pseudomonas* FM1106, which produces fruit flavor compounds, is classified and named *Pseudomonas* (…). Pseudomonas The *Pseudomonas* FM1106 strain of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34667, deposited on May 23, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Fermentation of this *Pseudomonas* FM1106 in a specific vitamin-amino acid-mineral complex culture medium produces fruit flavor compounds such as methyl palmitate and methyl glyoxylate through microbial metabolic transformation of the culture medium components. These compounds can be added to beverages as natural flavor enhancers to improve their flavor.

Description

Technical Field

[0001] This invention belongs to the field of food flavor engineering technology, specifically relating to a Pseudomonas aeruginosa that produces fruit flavor compounds and its applications. Background Technology

[0002] As consumers become less resistant to chemical products in beverages and prefer natural flavorings, traditional synthetic flavorings are struggling to meet the future needs of the beverage market. Existing flavoring technology has become a technological bottleneck restricting the development of my country's beverage industry.

[0003] Compared to natural products, chemically synthesized flavoring substances have significant disadvantages. First, chemically synthesized flavorings cannot obtain "clean label" certification. For example, flavoring substances using DBP / DIBP as a carrier do not comply with Chinese GB9685-2016 and EU No. 10 / 2011 regulations for beverage contact materials. Second, traditional flavoring substances can only achieve simple aroma simulation, failing to meet consumers' demands for natural flavors and multi-layered tastes. For example, traditional linear aroma structures rely on single compounds (such as ethyl hexanoate) to simulate fruit aromas, resulting in a simple aroma structure lacking complexity. Finally, synthetic flavorings cannot form molecular interactions with beverage matrices (acids / sugars / colloids), exhibiting poor compatibility. This leads to easily volatile top notes, rapid flavor decay after opening, a superficial aroma, and short-lasting aftertaste, creating a disconnect between taste and aroma.

[0004] Currently, my country's beverage industry is undergoing a critical transformation in flavor compound production, shifting from traditional chemically synthesized products to natural, personalized, and long-lasting flavor compounds. Adding natural products to enhance the taste and aroma persistence of beverages is a hot topic in the beverage additive industry. Therefore, developing novel flavor compounds, especially natural products obtained through microbial cultivation that can add unique flavors to low-alcohol drinks and dairy-based fermented beverages, has significant market value and application prospects. Summary of the Invention

[0005] The purpose of this application is to provide a novel method for producing natural flavor compounds for the beverage industry, specifically involving a Pseudomonas FM1106 strain that produces fruity flavor compounds and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A pseudomonospora that produces natural fruity flavor compounds, wherein the pseudomonospora is FM1106 and is classified as Pseudomonas ( Pseudomonas The specimen (sp.) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34667, deposited on May 23, 2025, in Beijing, China.

[0008] An application of the aforementioned fruit flavoring Pseudomonas FM1106 in the production of beverage flavoring additives.

[0009] A beverage flavoring additive containing the aforementioned Pseudomonas FM1106.

[0010] A beverage flavoring additive containing natural metabolites of the aforementioned Pseudomonas FM1106.

[0011] The beverage flavoring additive is the fermentation broth, fermentation culture, fermentation concentrate or its isolated and purified product of the Pseudomonas FM1106.

[0012] The fermentation broth, fermentation culture, and fermentation concentrate of Pseudomonas FM1106 contain methyl palmitate and methyl glyoxylate.

[0013] Pseudomonas FM1106 was fermented for 48-72 hours at a temperature of 25-30℃. After centrifugation to remove the bacterial cells, the supernatant was filtered and concentrated to 1 / 5 of its original volume to obtain the concentrate. This product can be used as a beverage additive with special fruit flavorings.

[0014] The centrifugation and filtration steps specifically include: centrifuging the fermentation broth (8000 rpm, 10 min) to remove the bacterial cells, filtering the supernatant through a 0.22 μm membrane, and concentrating it under reduced pressure at 60℃ to 1 / 5 of the original volume to obtain the concentrate.

[0015] An application of a beverage flavoring additive, wherein the beverage flavoring additive is used to impart a special fruity flavor to a beverage.

[0016] The beverages include, but are not limited to, mead, fruit juice, kombucha, low-alcohol drinks, or dairy-based fermented beverages.

[0017] Compared with the prior art, the advantages of the present invention are as follows:

[0018] 1. The Pseudomonas FM1106 of the present invention ferments in a specific vitamin-amino acid-mineral complex culture medium. Through microbial metabolic transformation of the culture medium components, fruit flavor compounds such as methyl palmitate and methyl glyoxylate are generated, which can be added to beverages as pure natural flavoring agents to enhance the flavor of beverages.

[0019] 2. This invention provides a new pathway for flavor compound generation by optimizing the synergistic system of microbial strains and culture medium, breaking through the bottleneck of low efficiency in traditional flavor generation.

[0020] 3. The fermentation product of Pseudomonas FM1106 of this invention conforms to the ISO / TS19657:2017 definition of natural, avoiding the regulatory risks of synthetic flavorings. The flavor compounds generated during fermentation synergistically construct a three-dimensional aroma framework of "creamy aroma - ripe fruit honey aroma - green fruit aldehyde aroma", which is far more complex than single extracts. It breaks through the limitations of traditional flavoring and is suitable for the development of innovative categories such as high-end fruit juice beverages, kombucha, low-alcohol beverages, and dairy-based fermented beverages. Attached Figure Description

[0021] Figure 1 The colony morphology of Pseudomonas FM1106 on Luria-Bertani liquid medium;

[0022] Figure 2 Phylogenetic tree of 16S rRNA of Pseudomonas FM1106;

[0023] Figure 3 This is a graph showing the GC-MS detection results of the flavor concentrate in Example 2 of the present invention;

[0024] Figure 4 This is a graph showing the GC-MS detection results of the fermentation broth products in Example 3 of the present invention;

[0025] Figure 5 This is a graph showing the GC-MS detection results of the fermentation broth products in Example 4 of the present invention;

[0026] Figure 6 This is a graph showing the GC-MS detection results of the fermentation broth product in Example 5 of the present invention. Detailed Implementation

[0027] Example 1: Isolation and identification of Pseudomonas FM1106

[0028] Pseudomonas FM1106 was isolated from capped honeycomb of *Apis chinensis* from the Shuipingxi Chinese honeybee farm in Huitong County, Huaihua City, Hunan Province. The specific steps are as follows:

[0029] Capped honeycomb from Chinese honeybees was collected, and beeswax was removed in a sterile environment, leaving only the honey. 5g of the honey was added to 400ml of Luria-Bertani liquid culture medium and incubated at 30°C with shaking for 24 hours until OD (Organic Depth) was reached. 600 =1.2-1.5, and then purified to obtain purified Pseudomonas FM1106.

[0030] After cultivation, samples were taken for microscopic examination to observe the morphology of the bacteria, such as... Figure 1 As shown, Pseudomonas FM1106 colonies in Luria-Bertani liquid medium are round or oval, raised, smooth, moist, pale orange, and have spores.

[0031] Sequencing analysis was performed on Pseudomonas FM1106, and the 16S rRNA gene sequence was determined as shown in SEQ ID No. 1. A phylogenetic tree of FM1106 was constructed, and sequence similarity analysis was performed with other Pseudomonas strains. The results are as follows: Figure 2 As shown. Based on the obtained 16S rDNA sequence sequencing results and colony morphology observation, strain FM1106 was identified to the genus. After comparison with NCBI, the selected strain FM1106 belongs to the genus *Pseudomonas*. Pseudomonas sp.).

[0032] The *Pseudomonas* species mentioned is FM1106, which was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34667, and classified as *Pseudomonas* (…). Pseudomonas sp.)FM1106, located in Beijing, China.

[0033] SEQ ID No.1

[0034]

[0035] Example 2 Preparation of Beverage Flavor Additives

[0036] S1: Prepare the raw materials for the culture medium: Weigh out 18.43 μg of vitamin B12, 6.18 mg of vitamin B1, 0.61 mg of vitamin B6, 35 mg of L-lysine, 18.56 mg of nicotinamide, 702.12 mg of dicalcium phosphate, 871 mg of monosodium glutamate, 185.87 mg of magnesium sulfate heptahydrate, 49.5 g of sucrose, and 1000 mL of distilled water;

[0037] S2: Preparation of culture medium: Add the above raw materials into a glass container and heat to 90°C, stirring until completely dissolved;

[0038] S3: Adjust pH: Adjust the pH of the culture medium obtained in S2 to 7.0;

[0039] S4: High-pressure sterilization: The culture medium obtained in S3 is sterilized by high-pressure steam sterilization (sterilization at 121℃ for 20 minutes).

[0040] S5: Inoculate the *Pseudomonas* FM1106 at a 3% inoculum into the culture medium and ferment at 28°C and 150-200 rpm for 48 hours to obtain the fermentation broth. Centrifuge the fermentation broth (8000 rpm, 10 min) to remove the bacterial cells. Filter the supernatant through a 0.22 μm membrane and concentrate it under reduced pressure at 60°C to 1 / 5 of the original volume to obtain the flavor concentrate.

[0041] The GC-MS results of the flavor concentrate are shown in the figure. Figure 3 ,Depend on Figure 3 It is evident that the Pseudomonas FM1106 concentrate contains methyl glyoxylate and methyl palmitate. Methyl glyoxylate has a green apple and fresh aldehyde aroma, used to impart a fresh fruity top note. FEMA 2966 lists it as a "Generally Recognized As Safe" (GRAS) food flavoring, which also complies with FEMA 4003 (FDA approval). Methyl palmitate (methyl palmitate) has a sweet fruity and creamy aroma, as well as a waxy base note, used to enhance the persistence of the beverage finish. FEMA 2718 lists it as a food flavoring approved.

[0042] Example 3 Preparation of Beverage Flavor Additives

[0043] S1: Prepare raw materials: Weigh 18.43μg vitamin B12, 6.18mg vitamin B1, 0.61mg vitamin B6, 35mg L-lysine, 18.56mg nicotinamide, 702.12mg dicalcium phosphate, 1025mg monosodium glutamate, 185.87mg magnesium sulfate heptahydrate, 49.5g sucrose, and 1L distilled water;

[0044] S2: Preparation of culture medium: Add the above raw materials into a glass container and heat to 90°C, stirring until completely dissolved;

[0045] S3: Adjust pH: Adjust the pH of the culture medium obtained in S2 to 7.0;

[0046] S4: High-pressure sterilization: The culture medium obtained in S3 is sterilized by high-pressure steam sterilization (sterilization at 121℃ for 20 minutes).

[0047] S5: Inoculate the Pseudomonas FM1106 into the culture medium at an inoculation rate of 3%, and ferment at 28°C and 150-200 rpm for 48 hours.

[0048] The GC-MS results of the fermentation broth products are shown in the figure. Figure 4 ,Depend on Figure 4 It is evident that the fermentation broth of Pseudomonas FM1106 contains methyl glyoxylate and methyl palmitate, and the fermentation broth has an aromatic odor.

[0049] Example 4: Preparation of Beverage Flavor Additives

[0050] S1: Prepare raw materials: Weigh 18.43μg vitamin B12, 6.18mg vitamin B1, 0.61mg vitamin B6, 35mg L-lysine, 18.56mg nicotinamide, 702.12mg dicalcium phosphate, 185.87mg magnesium sulfate heptahydrate, 49.5g sucrose, and 1L distilled water;

[0051] S2: Preparation of culture medium: Add the above raw materials into a glass container and heat to 90°C, stirring until completely dissolved;

[0052] S3: Adjust pH: Adjust the pH of the culture medium obtained in S2 to 7.0;

[0053] S4: High-pressure sterilization: The culture medium obtained in S3 is sterilized by high-pressure steam sterilization (sterilization at 121℃ for 20 minutes).

[0054] S5: Inoculate the Pseudomonas FM1106 into the culture medium at an inoculation rate of 3%, and ferment at 28°C and 150-200 rpm for 48 hours.

[0055] The GC-MS results of the fermentation broth products are shown in the figure. Figure 5 ,Depend on Figure 5 It is evident that the fermentation broth of Pseudomonas FM1106 contains methyl glyoxylate and methyl palmitate, and the fermentation broth has an aromatic odor.

[0056] Example 5: Using a traditional bacterial culture medium formula

[0057] S1: Prepare raw materials: Weigh 3g of beef extract, 10g of peptone, and 5g of NaCl. Add the above substances to 1L of distilled water in sequence and stir until completely dissolved.

[0058] S2: Preparation of culture medium: Add the above raw materials into a glass container and heat to 90°C, stirring until completely dissolved;

[0059] S3: Adjust the pH to 7.4 to obtain the culture medium.

[0060] S4: High-pressure sterilization: The culture medium obtained in S3 is sterilized by high-pressure steam sterilization (sterilization at 121℃ for 20 minutes).

[0061] S5: Inoculate the Pseudomonas FM1106 at a 3% inoculation rate into the culture medium and ferment at 28°C and 150-200 rpm for 48 hours.

[0062] The GC-MS results of the fermentation broth products are shown in the figure. Figure 6 .

[0063] Depend on Figure 6 GC-MS analysis showed that the fermentation culture of Pseudomonas FM1106 contained methyl glyoxylate and methyl palmitate, and the fermentation broth had an aromatic odor.

[0064] Application example: blending of fresh fruit cream, honey, and mead.

[0065] The fermentation broth from Example 2 was centrifuged at 8000 rpm for 15 min to remove bacteria. The supernatant was collected, and the pH was adjusted to 2.0 with HCl to precipitate proteins. The mixture was allowed to stand at 4°C for 2 h, and then centrifuged again to collect the supernatant. After decolorization with activated carbon (1% w / v 40°C × 30 min), impurities were removed using a 0.22 μm filter membrane. The extracts were then extracted with dichloromethane (pH = 7.0, 4°C) and diethyl ether (pH = 2.0, 0°C), respectively, to obtain the extracts. The extracts were subjected to silica gel column chromatography with gradient elution to obtain fractions of methyl hexadecanoate and methyl glyoxylate (purified by reversed-phase HPLC-ELSD on a C18 column).

[0066] Base spirit processing: Select citrus honey (bright fruit acid) base spirit with low tannin content. Spirit parameters: alcohol content 12-14% vol, residual sugar 3-5 g / L, total acid 4-6 g / L (calculated as lactic acid). The base spirit is clarified with bentonite, filtered with diatomaceous earth, and then filtered with a 0.45 μm membrane to remove colloids and impurities.

[0067] Mother liquor preparation: Methyl glyoxylate: Dissolved in 10% ethanol aqueous solution (avoiding excessive evaporation due to pure alcohol), concentration 1.0 g / L, stored at 4℃ protected from light, and used within 7 days. Methyl palmitate: Dissolved in food-grade glycerol (to improve solubility and prevent precipitation), concentration 5.0 g / L, stored stably at room temperature (≤25℃).

[0068] Blending Process: During the mid-aging stage of the base spirit (3 months after fermentation), 50 μg / L of methyl glyoxylate and 200 μg / L of methyl palmitate are added, stirred and blended, then transferred to oak barrels and aged at 15°C in the dark to promote the integration of esters with the body of the spirit. The basic flavor integration is completed in approximately 30 days. 15 days before bottling, 30 μg / L of methyl glyoxylate is added to enhance freshness, and 100 μg / L of methyl palmitate is added to enhance the finish. If the methyl palmitate results in an overly waxy texture, ethyl lactate (100-150 μg / L) is added to increase creamy sweetness. Finally, the pH is adjusted to 3.8-4.0 with a citric acid-malic acid buffer to enhance the brightness of the fruit aromas. The optimized mead exhibits a sensory profile where the initial aroma transforms from a single honey note to a green apple and honey note; the mid-palate transitions from a flat to a slightly creamy note with a lingering fruity finish; and the finish changes from a short, astringent note to a waxy honey aroma that is persistent and rounded. Overall, it smells like fresh apples and leaves a warm, honey-like aftertaste without the harshness of artificial flavorings.

[0069] This application example demonstrates an industrial production process for fruit-flavored mead using a pre-fermentation method followed by post-fermentation for aroma development. During the aroma development stage, a mother liquor containing target flavor compounds is added, resulting in a final product with a three-dimensional flavor profile of "fresh fruit aromas and creamy honey aromas," free of artificial flavorings. This satisfies consumers' dual demands for "naturalness" and "complexity." Through a combination of targeted microbial metabolism and meticulous blending, this application precisely enhances the fruit aroma dimension while preserving the natural properties of mead, making it particularly suitable for upgrading the flavor of high-end, elegant mead.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of pseudomonospora that produces fruity flavor compounds ( Pseudomonas sp.)FM1106, characterized in that: The aforementioned Pseudomonas FM1106 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34667, deposited on May 23, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the *Pseudomonas FM1106*, which produces fruity aroma compounds as described in claim 1, characterized in that: The Pseudomonas FM1106 is used to produce beverage flavoring additives.

3. A beverage flavoring additive, characterized in that: The beverage flavoring additive is the fermentation broth, fermentation culture, or fermentation concentrate of Pseudomonas FM1106 as described in claim 1.

4. The beverage flavoring additive according to claim 3, characterized in that: The Pseudomonas FM1106 of claim 1 is fermented and cultured for 48-72 hours at a fermentation temperature of 25-30°C. The fermentation broth is then filtered and concentrated to 1 / 5 of its original volume to obtain a fruity flavoring, which is a beverage flavoring additive.

5. The application of the beverage flavoring additive according to claim 3, characterized in that: The beverage flavoring additives are used to impart fruity flavors to the beverage.

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