Gluconacetobacter cocoa and use thereof, fermentation method of vinegar, and flavor modifier

Abstract

This invention discloses a cacao-bean acetic acid bacterium and its application, a vinegar fermentation method, and a flavor improver, relating to the field of microbial technology. The cacao-bean acetic acid bacterium is named *Acetobacter fabarum* JJ-1, with the accession number CCTCCNO: M 20242128, a deposit date of September 30, 2024, and a deposit address at the China Center for Type Culture Collection. The cacao-bean acetic acid bacterium JJ-1 exhibits high acid-producing capacity in the presence of exogenous selenium, and the fermented vinegar produced has a better flavor compared to vinegar without added selenium.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a cacao acetic acid bacillus and its application, a method for vinegar fermentation, and a flavor modifier. Background Technology

[0002] In nature, selenium exists primarily in an inorganic form, which is highly toxic, has low biological activity, and is not easily absorbed and utilized by the human body. This problem can be addressed by converting inorganic selenium into its organic form through physical and chemical methods. However, most physical and chemical production methods are complex and costly, hindering large-scale application.

[0003] Microbial synthesis of selenium is a biocompatible and low-cost method, and microbial enrichment of selenium is one of the most promising technologies in the emerging selenium industry. This bioenrichment method uses inorganic selenium as a raw material to produce highly nutritious organic selenium and edible products containing organic selenium to meet the nutritional needs of the public, especially people in selenium-deficient areas. Among these, edible products containing organic selenium mainly include selenium-enriched beverages such as selenium-enriched vinegar, which are produced through microbial fermentation.

[0004] Currently, selenium-enriched vinegar is mainly produced through the addition of exogenous selenium and fermentation of selenium-enriched raw materials. However, the addition of exogenous selenium can affect the activity of microorganisms during fermentation, limiting their ability to produce acid and other substances, thus affecting the quality of the final product. Summary of the Invention

[0005] The main objective of this invention is to propose a cocoa bean acetic acid bacterium and its application, a method for vinegar fermentation, and a flavor modifier, aiming to solve the problem of exogenous selenium limiting the acid-producing capacity of fermenting microorganisms in the prior art.

[0006] To achieve the above objectives, this invention proposes a cacobacter fabarum, named cacobacter fabarum JJ-1, with accession number CCTCC NO: M 20242128, accession date September 30, 2024, and accession address China Center for Type Culture Collection.

[0007] In one embodiment, the 16S rDNA gene sequence of the cocoa bean acetic acid bacillus is shown in SEQ ID No. 1.

[0008] The present invention also provides the application of Acetobacter acnes in selenium-enriched fermentation, wherein the Acetobacter acnes includes the aforementioned Acetobacter acnes.

[0009] In one embodiment, the cocoa bean acetic acid bacteria is used for fermentation in combination with an acid promoter, the acid promoter including sodium selenite and / or sodium selenate.

[0010] In one embodiment, the concentration of the acid promoter is 20-80 mg / L, the fermentation temperature is 28°C, the fermentation time is 11 days, the initial alcohol content is 6%, the concentration of Acetobacter JJ-1 in cocoa beans is 10%, and the acid production of Acetobacter JJ-1 in cocoa beans is 16-41%.

[0011] This invention also provides a method for fermenting vinegar, comprising the following steps:

[0012] Acetobacterium acnes in cocoa beans is mixed with an acid-promoting agent and cultured for fermentation.

[0013] The cacao acetic acid bacteria mentioned above include the cacao acetic acid bacteria mentioned above, and the acid promoter includes sodium selenite and / or sodium selenate.

[0014] In one embodiment, the content of the acid-promoting agent is 20–80 mg / L; and / or,

[0015] Fermentation time is 9–12 days.

[0016] The present invention also provides a flavor improver, comprising the aforementioned cocoa bean acetic acid bacteria.

[0017] In one embodiment, the flavor improver further includes an acidifier.

[0018] In one embodiment, the acid promoter includes sodium selenite and / or sodium selenate.

[0019] In the technical solution of this invention, the *Acetobacter faberi* of cocoa beans is named *Acetobacter faberi* JJ-1, and its accession number is CCTCCNO: M 20242128, the accession date is September 30, 2024, and the accession address is the China Center for Type Culture Collection. *Acetobacter faberi* JJ-1 exhibits high acid-producing capacity in the presence of exogenous selenium, and the fermented vinegar produced has a better flavor compared to vinegar without added selenium. Attached Figure Description

[0020] 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.

[0021] Figure 1 This is a graph showing the acid production capacity of strain CL12 in Example 1 of the present invention under the condition of exogenous selenium addition;

[0022] Figure 2The diagram shows the acid production capacity of strain PT6 in Example 1 of this invention under the condition of exogenous selenium addition;

[0023] Figure 3 This is a graph showing the acid production capacity of strain JJ-1 in Example 1 of the present invention under the condition of addition to other departments;

[0024] Figure 4 This is a colony diagram of strain JJ-1 in Example 2 provided by the present invention;

[0025] Figure 5 Gram staining image of strain JJ-1 in Example 2 provided by the present invention;

[0026] Figure 6 This is a phylogenetic tree diagram of strain JJ-1 in Example 2 provided by the present invention;

[0027] Figure 7 The growth curve of strain JJ-1 in Example 3 provided by the present invention;

[0028] Figure 8 The figure shows the acid production results of strain JJ-1 in Example 3 of this invention under the condition of exogenous selenium addition.

[0029] 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

[0030] 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.

[0031] In nature, selenium exists primarily in an inorganic form, which is highly toxic, has low biological activity, and is not easily absorbed and utilized by the human body. This problem can be addressed by converting inorganic selenium into its organic form through physical and chemical methods. However, most physical and chemical production methods are complex and costly, hindering large-scale application.

[0032] Microbial synthesis of selenium is a biocompatible and low-cost method, and microbial enrichment of selenium is one of the most promising technologies in the emerging selenium industry. This bioenrichment method uses inorganic selenium as a raw material to produce highly nutritious organic selenium and edible products containing organic selenium to meet the nutritional needs of the public, especially people in selenium-deficient areas. Among these, edible products containing organic selenium mainly include selenium-enriched beverages such as selenium-enriched vinegar, which are produced through microbial fermentation.

[0033] Currently, selenium-enriched vinegar is mainly produced through the addition of exogenous selenium and fermentation of selenium-enriched raw materials. However, the addition of exogenous selenium can affect the activity of microorganisms during fermentation, limiting their ability to produce acid and other substances, thus affecting the quality of the final product.

[0034] In view of this, the present invention provides a cacao-bean acetic acid bacterium, named cacao-bean acetic acid bacterium (Acetobacter fabarum) JJ-1, the accession number of cacao-bean acetic acid bacterium JJ-1 is CCTCC NO: M20242128, the accession date is September 30, 2024, and the accession address is China Center for Type Culture Collection.

[0035] In the technical solution of this invention, the *Acetobacter faberi* of cocoa beans is named *Acetobacter faberi* JJ-1, and its accession number is CCTCCNO: M 20242128, the accession date is September 30, 2024, and the accession address is the China Center for Type Culture Collection. *Acetobacter faberi* JJ-1 exhibits high acid-producing capacity in the presence of exogenous selenium, and the fermented vinegar produced has a better flavor compared to vinegar without added selenium.

[0036] In some embodiments of the present invention, the 16S rDNA gene sequence of the cocoa bean acetic acid bacillus is shown in SEQ ID No. 1.

[0037] SEQ ID NO.1:

[0038]

[0039] This invention also provides the application of *Acetobacterium cocoa* in selenium-enriched fermentation, wherein the *Acetobacterium cocoa* includes the aforementioned *Acetobacterium cocoa*. The *Acetobacterium cocoa* can produce acid under exogenous selenium conditions, and the acid production is relatively high, making it suitable for selenium-enriched fermentation to produce fermented products.

[0040] In some embodiments of the present invention, the *Acetobacterium cocoa* is used for fermentation in combination with an acid-promoting agent, which includes sodium selenite and / or sodium selenate. The *Acetobacterium cocoa* can produce acid under conditions of exogenous selenium such as sodium selenite or sodium selenate, and the acid production is relatively high. That is, compared with not adding exogenous selenium, adding a certain concentration of exogenous selenium can promote acid production by *Acetobacter cocoa*.

[0041] In some embodiments of the present invention, the concentration of the acid promoter is 20–80 mg / L, the fermentation temperature is 28°C, the fermentation time is 11 days, the initial alcohol content is 6%, the concentration of *Acetobacterium acnes* JJ-1 in cocoa beans is 10%, and the acid production of *Acetobacterium acnes* JJ-1 in cocoa beans is 16–41%. This indicates that under the above conditions, the *Acetobacterium acnes* JJ-1 in cocoa beans of the present invention has a strong acid-producing capacity. The acid is total acid, that is, the sum of all acidic substances in the sample, and the determination method is in accordance with the "GB 12456-2021 National Food Safety Standard - Determination of Total Acid in Food".

[0042] This invention also provides a method for fermenting vinegar, comprising the following steps: culturing and fermenting a mixture of *Acetobacterium cocoa* and an acid-promoting agent; wherein the *Acetobacterium cocoa* includes the aforementioned *Acetobacterium cocoa*, and the acid-promoting agent includes sodium selenite and / or sodium selenate. The vinegar obtained through the above steps has a high total acid content and a good flavor.

[0043] In some embodiments of the present invention, the content of the acid-promoting agent is 20–80 mg / L; and / or, the fermentation time is 9–12 days. The content of the acid-promoting agent can be 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, or 80 mg / L, and a content within the above range ensures a high acid production. The fermentation time is 9–12 days, which can be 9 days, 10 days, 11 days, or 12 days, and a fermentation time within the above range ensures a high acid production. Simultaneously, by ensuring that the acid-promoting agent content and fermentation time are within the above ranges, compared to not adding an acid-promoting agent, the acid production is higher and the flavor is better.

[0044] The present invention also provides a flavor improver comprising the aforementioned *Acetobacterium cocoa*. Therefore, it possesses all the beneficial effects of the aforementioned *Acetobacterium cocoa*, which will not be elaborated upon here.

[0045] In some embodiments of the present invention, the flavor improver further includes an acid promoter. The acid promoter can increase the acid production of *Acetobacter acetobacter* from cocoa beans.

[0046] In some embodiments of the present invention, the acid-promoting agent includes sodium selenite and / or sodium selenate. Sodium selenite is preferred, as it can effectively increase the acid production of *Acetobacter acetobacter* from cocoa beans and increase the variety and content of beneficial flavor compounds.

[0047] 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.

[0048] Experimental materials

[0049] The kumquats were purchased from Taobao and originated from Rong'an, Guangxi.

[0050] Calcium carbonate separation medium: 1% yeast powder, 1% glucose, 2% CaCO3, 2% agar, sterilized at 121℃ for 20 min, cooled to 70℃, 4% (v / v) anhydrous ethanol added, mixed well and poured into petri dishes to prepare calcium carbonate separation medium.

[0051] Basic fermentation medium: 1% yeast powder, 1% glucose, sterilized at 121℃ for 20 min, and after cooling to 70℃, add 4% (v / v) anhydrous ethanol.

[0052] Fermentation medium: 1% yeast powder, 1% glucose, sterilized at 121℃ for 20 min, and after cooling to 70℃, 6% (v / v) anhydrous ethanol was added.

[0053] Example 1: Isolation and preliminary identification of Acetobacter JJ-1 from cocoa beans

[0054] 1. Strain enrichment

[0055] Wash the purchased kumquats with clean water and let them dry. Chop them and put them into a 250mL Erlenmeyer flask. Add sterile water to cover the raw materials, seal the flask with sealing film, and put it into a 28℃ incubator for fermentation for 30 days to obtain kumquat fermentation liquid.

[0056] 2. Strains Isolation

[0057] The kumquat fermentation broth was serially diluted and spread onto calcium carbonate separation solid medium. Colonies were formed by incubation at 30°C under inverted conditions. Single colonies exhibiting typical acetic acid bacteria characteristics—smooth, moist, pale yellow in color, and displaying a calcium-dissolving zone—were selected for streak plating for purification. The purified strains were numbered and stored at -80°C with 20% (v / v) glycerol for later use. A total of three strains were isolated from the kumquat fermentation broth.

[0058] 3. Strains screening

[0059] Single colonies were picked from the strains isolated in step 2 using an inoculation loop and inoculated into 100 mL of basal fermentation medium. Three parallel experiments were performed for each strain, and the cultures were incubated at 30°C and 180 rpm for 5 days with shaking. 25 mL of the fermentation broth was centrifuged at 8000 rpm for 10 min to remove the bacterial cells. 5 mL of the supernatant was used for acetic acid qualitative testing. This involved adjusting the pH of the supernatant to 7.0 with saturated sodium hydroxide solution, heating to boiling, and adding 6 drops of 5% ferric chloride solution. If a reddish-brown precipitate appeared, acetic acid was produced during the bacterial culture, and the strain was preliminarily identified as acetic acid bacteria. After preliminary identification, the three strains screened in this invention were all acetic acid bacteria, and were named CL12, PT6, and JJ-1, respectively.

[0060] 4. Effects of exogenous selenium on the acid-producing capacity of three bacterial strains

[0061] The bacterial cultures (i.e., seed cultures) of strains CL12, PT6, and JJ-1 selected in step 3 were cultured for 36 hours at 30℃, 180 rpm, and on basal fermentation medium. 10% of each seed culture was added to fermentation medium containing 0, 10, and 20 mg / L Na2SeO3, respectively. The cultures were then incubated for 7 days in a constant temperature shaker at 30℃ and 180 rpm. Samples were then taken for titration to determine the total acid content. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown.

[0062] Depend on Figure 1 , Figure 2 and Figure 3 It can be seen that the total acid content produced by strains CL12 and PT6 was lower when the sodium selenite concentration increased, meaning that the acid-producing ability of strains CL12 and PT6 was inhibited by sodium selenite. However, when the sodium selenite concentration was increased to 20 mg / L, the total acid content produced by strain JJ-1 increased by about three times compared to the group without sodium selenite, indicating a stronger acid-producing ability under high concentrations of sodium selenite. Example 2: Morphological and molecular biological identification of *Acetobacter acetic acid bacillus* JJ-1 from cocoa beans.

[0063] 1. Morphological identification

[0064] The strain JJ-1 screened in Example 1 was inoculated onto a calcium carbonate isolation plate and cultured at 30°C for 48 h. Morphological observation, Gram staining, and spore staining of the cultured colonies were performed. The results are as follows: Figure 4 and Figure 5 As shown.

[0065] Depend on Figure 4It can be seen that the colonies are round, with a smooth and flat surface, neat edges, and are opaque or translucent light yellow in color, with a transparent calcium soluble ring, which is a typical colony morphology of acetic acid bacteria.

[0066] Gram staining ( Figure 5 Staining with spores indicates that the bacteria are Gram-negative and do not form spores. Individual bacteria are short rod-shaped or oval, mostly arranged singly or in pairs, but some appear in chains.

[0067] 2. Molecular biological identification

[0068] Single colony PCR was used: 20 μL of sterile water was added to a 1.5 mL centrifuge tube, and a single colony of JJ-1 was picked from the activated culture dish and placed in sterile water to prepare a DNA template.

[0069] Upstream primer: 27F (5'-AGAGTTTGATCCTGGCTCAG-3');

[0070] Downstream primer: 1492R (5'-TACGACTTAACCCCAATCGC-3').

[0071] 16S rDNA amplification system: 1 μL each of upstream and downstream primers, 2 μL of DNA template, 12.5 μL of Taq PCR Mix premix (2×), and 8.5 μL of sterile water.

[0072] The PCR reaction conditions were: 94℃ for 3 min; 94℃ for 15 s, 55℃ for 30 s, 72℃ for 2 min, for 30 cycles; 72℃ for 5 min; and stored at 4℃.

[0073] After PCR sequencing, firstly, homology sequences were searched in the National Center for Biotechnology Information (NCBI) online database. Secondly, the target strain and other acetic acid bacteria type strains of the same genus were compared for maximum homology. Finally, a phylogenetic tree was constructed using MEGA 7.01 software with Neighbor-Joining, and a Bootstrap test with 1000 replicates was performed. The results are as follows: Figure 6 As shown in the figure. The results showed that the 16S rDNA of strain JJ-1 had the highest homology (100%) with Acetobacter fabarumstrain LMG 24244, confirming that this strain is Acetobacter fabarum.

[0074] It should be noted that after PCR sequencing, the 16S rDNA of strains CL12 and PT6 showed the highest homology with Acetobacter fabarumstrain LMG 24244, confirming that strains CL12 and PT6 are also Acetobacter fabarum.

[0075] Example 3: Effect of exogenous selenium on the acid-producing ability of Acetobacter JJ-1 from cocoa beans

[0076] 1. Growth curve of strain JJ-1

[0077] A single colony of JJ-1 was inoculated into the basal fermentation medium, and samples were taken every 4 hours to measure the OD. 600 The monitoring time was 48 hours, and the test results were as follows: Figure 7 As shown.

[0078] Depend on Figure 7 It can be seen that the logarithmic growth phase of strain JJ-1 is between 8 and 36 hours. Since the strain has the strongest reproductive growth capacity and is most sensitive to external environmental stimuli during the logarithmic growth phase, the fermentation broth of strain JJ-1 cultured for 36 hours was selected as the seed culture.

[0079] 2. Effects of exogenous selenium on the acid production capacity of strain JJ-1

[0080] The seed culture of strain JJ-1, cultured at 30℃, 180 rpm on basal fermentation medium for 36 h, was used as a basis for fermentation. 10 mL of each seed culture was added to fermentation media containing 0, 10, 20, 30, 40, 50, 60, and 80 mg / L Na₂SeO₃, respectively. The cultures were then incubated in a constant temperature shaker at 30℃ and 180 rpm. Every 24 h, samples were taken and titrated to determine the total acid content. The results are as follows: Figure 8 As shown.

[0081] Depend on Figure 8 It can be seen that strain JJ-1 produced a relatively large amount of acid, 35.12 g / L, after 12 days of culture under the condition of sodium selenite concentration of 20 mg / L.

[0082] 3. Response surface optimization

[0083] Using acid production (Y) as the indicator, four factors were selected as independent variables: acetic acid bacteria fermentation temperature (A), inoculum size (B), initial sodium selenite concentration (C), and initial ethanol concentration (D), as shown in Table 1. A response surface methodology (RSM) experiment with 29 experimental points across 3 levels and 4 factors was designed using Design Expert 13.0 software to optimize the fermentation conditions of strain JJ-1. The results are shown in Table 2.

[0084] After performing least squares regression fitting on the response surface experimental data using Design Expert 13 software, the regression equation was obtained as follows:

[0085] Y=37.3388+1.02138A+0.921067B+10.6789C+1.78742D-1.1217AB+1.36792AC+0.0820753AD+0.793394BC+0.0820753BD+0.984903CD-4.31534A 2 -2.24977B 2 -13.3026C-3.08421D 2 R 2 =0.9886, R Adj 2 =0.9771. The results of the analysis of variance for the regression equation are shown in Table 3.

[0086] Table 1 Factor Level Table

[0087]

[0088] Table 2 Response Surface Optimization Experiment

[0089]

[0090]

[0091] Table 3 Analysis of Response Surface Experiment Results

[0092] Model Item sum of squares Degrees of freedom Mean Square F value p-value Significance Model 2621.09 14 187.22 86.42 <0.0001 ** A-Temperature 12.52 1 12.52 5.78 0.0306 * B-Vaccination Dose 10.18 1 10.18 4.7 0.0479 * C-sodium selenite concentration 1368.47 1 1368.47 631.69 <0.0001 ** D-ethanol concentration 38.34 1 38.34 17.7 0.0009 ** AB 5.03 1 5.03 2.32 0.1497 N AC 7.48 1 7.48 3.46 0.0842 N AD 0.0269 1 0.0269 0.0124 0.9128 N BC 2.52 1 2.52 1.16 0.2992 N BD 0.0269 1 0.0269 0.0124 0.9128 N CD 3.88 1 3.88 1.79 0.2021 N A2 120.79 1 120.79 55.76 <0.0001 ** B2 32.83 1 32.83 15.15 0.0016 ** C2 1147.84 1 1147.84 529.85 <0.0001 ** D2 61.7 1 61.7 28.48 0.0001 ** residual 30.33 14 2.17 Dissimilarity 24.62 10 2.46 1.72 0.3158 N Pure error 5.71 4 1.43 sum 2651.42 28

[0093] The vinegar fermentation process conditions were optimized using Design Expert 13 software. The optimal process conditions were: acetic acid bacteria inoculum of 10.483 mL, fermentation temperature of 27.968℃, initial alcohol content of 6.029%, and initial sodium selenite concentration of 22.783 mg / L. Under these conditions, the predicted acid production of the fermentation broth was 39.463 g / L.

[0094] The process conditions were adjusted based on the actual situation as follows: inoculum volume 10 mL, fermentation temperature 28℃, initial alcohol content 6%, initial sodium selenite concentration 23 mg / L, and the final acid production was 40.22 g / L, which basically matched the theoretical prediction value, indicating that the prediction model fits the actual fermentation conditions well.

[0095] Example 4: Effect of Acetobacter JJ-1 on the aroma of cocoa bean fermentation products under exogenous selenium addition.

[0096] 1. Solid-phase microextraction conditions

[0097] Headspace solid-phase microextraction (HS-SPME) was used. 4 mL of fermentation broth sample was placed in a 20 mL headspace vial, along with 1 g of sodium chloride and 30 μL of the internal standard 2-octanol (diluted 1:1000). The vial was capped tightly and placed on an MPS pretreatment stage for equilibration for 20 min. The extraction head was then inserted into the headspace vial, and the vial was placed on a magnetic stirrer at 45 °C for 30 min. After adsorption, the extraction head was removed and inserted into the gas chromatograph injection port for desorption at 250 °C for 5 min. Three parallel experiments were performed for each sample.

[0098] The fermentation broth samples were divided into two groups. One group consisted of selenium-added broth, with the following fermentation conditions: 10 mL seed culture inoculation, fermentation temperature 28℃, initial alcohol content 6%, initial sodium selenite concentration 23 mg / L, and fermentation for 10 days. The other group consisted of fermentation broth without exogenous selenium, with the same fermentation conditions as the selenium-added group except for a selenium content of 0 mg / L. The seed culture refers to the bacterial culture of strain JJ-1, cultured at 30℃, 180 rpm, and on a basic fermentation medium for 36 hours.

[0099] 2. Gas Chromatography (GC) Conditions

[0100] A DB-5MS flexible quartz capillary column (30m×0.25mm×0.25μm) was used; the temperature program was as follows: initial temperature 40℃, hold for 4 min, increase to 90℃ at a rate of 5℃ / min, hold for 3 min, then increase to 230℃ at a rate of 5℃ / min, hold for 5 min; the injection port temperature was 250℃; the carrier gas was high-purity helium (He) at a flow rate of 1 mL / min.

[0101] 3. Mass Spectrometry (MS) Conditions

[0102] Electron ionization source, electron capability 70eV, ion source temperature 230℃, quadrupole temperature 150℃, full scan mode, full scan, scan range 40~500m / z.

[0103] 4. Qualitative and quantitative methods for volatile substances

[0104] The acquired mass spectra were searched and analyzed using the NIST (National Institute of Standards and Technology) mass spectrometry library and the Wiley Library. Volatile substances with a matching degree greater than or equal to 80% were identified and verified. Based on the mass concentration of the 2-octanol internal standard, the content of each volatile component in the fermentation broth was calculated according to the following formula. The detection results are shown in Table 4.

[0105]

[0106] In the above formula: X, the relative mass concentration of the analyte, μg / L; Ai , analyte peak area; A0, internal standard peak area; m, internal standard mass, μg; V, sample volume before treatment, L.

[0107] Table 4. Main volatile compounds in the JJ-1 fermentation broth of the unselenium-added and selenium-added groups.

[0108]

[0109]

[0110] Table 1 shows that, using HS-SPME-GC-MS detection technology, a total of 32 volatile compounds were detected in the JJ-1 strain described in this invention after fermentation with exogenous selenium addition. The main aroma substances were acetic acid, nonanoic acid, 2,4-di-tert-butylphenol, diisobutyl terephthalate, methyl N-hydroxyphenylcarboxyimide, and benzaldehyde. Compared with the selenium-free group, the group showed an increase of two volatile compounds, myristic acid and 2-methylpropionic acid, while showing a decrease of 2-phenyl-2-butenal. The total content of volatile compounds in the selenium-added group (2937.28 μg / L) was 4.7% higher than that in the selenium-free group (2803.37 μg / L).

[0111] 3-Methylbutyric acid, 2-methylbutyric acid, 2-methylpropionic acid, and octanoic acid exhibit pleasant fruity aromas at low concentrations. Acetic acid has a pungent odor. Nonanoic acid has a faint fatty and coconut aroma. Acetic acid and nonanoic acid accounted for 93.17% of the unselenium-added group and 88.33% of the selenium-added group, making them the main flavor and aroma compounds in vinegar. The acid content in the unselenium-added group was 1084.77 μg / L, which was lower than the 1312.09 μg / L in the selenium-added group. The acid production in the selenium-added group was 20.95% higher than that in the unselenium-added group. Specifically, selenium addition increased the content and variety of acidic substances.

[0112] Esters are the main components that give vinegar its aromatic characteristics, and most esters have an aromatic odor. Diisobutyl terephthalate and methyl N-hydroxyphenylcarboxyimide accounted for 71.59% in the unselenium-added group and 75.59% in the selenium-added group. They are characteristic esters of vinegar and give it a fruity aroma.

[0113] The unique aroma characteristics of aldehydes and ketones in fermentation broth are an indispensable part of the vinegar's aroma system. Benzaldehyde has a distinctive cherry aroma and antibacterial properties; it is a key component of natural fruit flavor, with a 44.65% increase in aroma intensity compared to the selenium-added group. Nonanal has a strong oily odor and a sweet orange scent. 2-Phenylon-2-butenal has a sweet, honey-like aroma. 2,6-Di-tert-butyl-4-hydroxy-4-methylcyclohexane-2,5-dien-1-one is an important antioxidant widely used in various industrial and food sectors, and it has the function of extending shelf life.

[0114] There was no significant difference in 2,4-di-tert-butylphenol between the selenium-free and selenium-added groups (p>0.05). It has a strong aromatic odor, is a natural product, has strong antibacterial activity, and has certain anticancer and anti-inflammatory effects. Selenium addition did not affect its synthesis. It is one of the main volatile compounds in this vinegar, accounting for 25.40% in the selenium-free group and 23.56% in the selenium-added group.

[0115] 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 cacao bean acetic acid bacterium, characterized in that, The Acetobacter cocoa is named as Acetobacter cocoa (JJ-1), the preservation number of the Acetobacter cocoa (JJ-1) is CCTCC NO: M20242128, the preservation time is September 30, 2024, and the preservation address is China Center for Type Culture Collection. Acetobacter fabarum ) The 16S rDNA gene sequence of the cacao acetic acid bacillus is shown in SEQ ID No.

1.

2. The application of the cocoa bean acetic acid bacteria as described in claim 1 in selenium-enriched fermentation.

3. The application as described in claim 2, characterized in that, The cocoa bean acetic acid bacteria are used for fermentation in combination with an acid promoter, which is selected from sodium selenite and / or sodium selenate, and the concentration of the acid promoter is 20-80 mg / L.

4. The application as described in claim 3, characterized in that, The fermentation temperature was 28℃, the fermentation time was 11 days, the initial alcohol content was 6%, and the concentration of Acetobacter JJ-1 in cocoa beans was 10%.

5. A method for fermenting vinegar, characterized in that, Includes the following steps: The cocoa bean acetic acid bacteria as described in claim 1 are mixed with an acid-promoting agent and cultured for fermentation. The acid-promoting agent is selected from sodium selenite and / or sodium selenate, and the content of the acid-promoting agent is 20-80 mg / L.

6. The vinegar fermentation method according to claim 5, characterized in that, Fermentation time is 9–12 days.

7. A flavor improver, characterized in that, Including the cocoa bean acetic acid bacteria as described in claim 1.

8. The flavor improver as described in claim 7, characterized in that, The flavor improver also includes an acid promoter, which is selected from sodium selenite and / or sodium selenate, and the concentration of the acid promoter is 20-80 mg / L.

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