Acetobacter cocoa and application thereof, fermentation method of vinegar and flavor modifier

Abstract

The invention discloses Acetobacter cacteus and application thereof, a fermentation method of vinegar and a flavor modifier, and relates to the technical field of microorganisms, the Acetobacter cacteus is named as Acetobacter cacteus JJ-1, the preservation number of the Acetobacter cacteus JJ-1 is CCTCCNO: M 20242128, the preservation time is September 30, 2024, and the preservation number of the Acetobacter cacteus JJ-1 is CCTCCNO: M 20242128. The preservation address is China Center for Type Culture Collection. The acetobacter cocoa JJ-1 has higher acid production capacity in the presence of exogenous selenium, and compared with vinegar without selenium, the fermented vinegar has better flavor.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to cocoa bean acetobacter and application thereof, a vinegar fermentation method and a flavor improver. Background Art

[0002] In nature, selenium mainly exists in an inorganic form, which is highly toxic, has low biological activity, and is not easily absorbed and utilized by the human body. Physical and chemical means are used to convert inorganic selenium into an organic form to solve this problem. However, most physical and chemical production methods are complex and costly, which is not conducive to large-scale use.

[0003] Microbial synthesis of selenium is a method with good biocompatibility and low cost. At the same time, microbial enrichment of selenium is one of the most promising technologies in the emerging selenium industry. This bio-enrichment method uses inorganic selenium as raw material to produce highly nutritious organic selenium and edible products containing organic selenium to meet the nutritional needs of the public, especially those in selenium-deficient areas. Among them, edible products containing organic selenium mainly include selenium-enriched vinegar and other selenium-enriched beverages fermented by microorganisms.

[0004] At present, selenium-enriched vinegar is mainly produced by adding selenium from outside and fermenting selenium-enriched raw materials. However, the addition of selenium from outside will affect the activity of microorganisms during the fermentation process, limiting their ability to produce acid and affecting the quality of the final product. Summary of the invention

[0005] The main purpose of the invention is to provide a cocoa bean acetobacter and application thereof, a vinegar fermentation method and a flavor improver, aiming to solve the problem in the prior art that exogenous selenium limits the acid production capacity of fermentation microorganisms.

[0006] To achieve the above object, the present invention provides a cocoa bean acetobacter, the cocoa bean acetobacter is named as cocoa bean acetobacter (Acetobacter fabarum) JJ-1, the preservation number of the cocoa bean acetobacter JJ-1 is CCTCC NO: M 20242128, the preservation time is September 30, 2024, and the preservation address is China Center for Type Culture Collection.

[0007] In one embodiment, the 16S rDNA gene sequence of Acetobacter cocoa is shown as SEQ ID No.1.

[0008] The present invention also provides an application of cocoa bean acetobacter in selenium-enriched fermentation, wherein the cocoa bean acetobacter includes the aforementioned cocoa bean acetobacter.

[0009] In one embodiment, the cocoa bean Acetobacter is mixed with an acid promoter for fermentation, and the acid promoter includes 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 cocoa JJ-1 is 10%, and the acid production of Acetobacter cocoa JJ-1 is 16-41%.

[0011] The present invention also provides a vinegar fermentation method, comprising the following steps:

[0012] Mixing cocoa bean acetobacter with an acid promoter for fermentation;

[0013] Wherein, the cocoa bean acetobacter includes the aforementioned cocoa bean acetobacter, and the acid promoter includes sodium selenite and / or sodium selenate.

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

[0015] The fermentation time is 9 to 12 days.

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

[0017] In one embodiment, the flavor improver further comprises an acid promoter.

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

[0019] In the technical solution of the present invention, the cocoa bean acetobacter is named as cocoa bean acetobacter (Acetobacterfabarum) JJ-1, the deposit number of the cocoa bean acetobacter JJ-1 is CCTCCNO: M 20242128, the deposit time is September 30, 2024, and the deposit address is China Center for Type Culture Collection. The cocoa bean acetobacter JJ-1 has a higher acid production capacity in the presence of exogenous selenium, and the fermented vinegar has a better flavor than the vinegar without selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0021] Figure 1 This is a graph showing the acid production capacity of the CL12 strain in Example 1 provided by the present invention when exogenous selenium is added;

[0022] Figure 2This is a graph showing the acid production capacity of the PT6 strain in Example 1 provided by the present invention when exogenous selenium is added;

[0023] Figure 3 This is a graph showing the acid production capacity of the JJ-1 strain in Example 1 provided by the present invention when an external strain is added;

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

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

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

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

[0028] Figure 8 This is a graph showing the acid production results of the JJ-1 strain in Example 3 provided by the present invention when exogenous selenium is added.

[0029] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0030] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0031] In nature, selenium mainly exists in an inorganic form, which is highly toxic, has low biological activity, and is not easily absorbed and utilized by the human body. Physical and chemical means are used to convert inorganic selenium into an organic form to solve this problem. However, most physical and chemical production methods are complex and costly, which is not conducive to large-scale use.

[0032] Microbial synthesis of selenium is a method with good biocompatibility and low cost. At the same time, microbial enrichment of selenium is one of the most promising technologies in the emerging selenium industry. This bio-enrichment method uses inorganic selenium as raw material to produce highly nutritious organic selenium and edible products containing organic selenium to meet the nutritional needs of the public, especially those in selenium-deficient areas. Among them, edible products containing organic selenium mainly include selenium-enriched vinegar and other selenium-enriched beverages fermented by microorganisms.

[0033] At present, selenium-enriched vinegar is mainly produced by adding selenium from outside and fermenting selenium-enriched raw materials. However, the addition of selenium from outside will affect the activity of microorganisms during the fermentation process, limiting their ability to produce acid and affecting the quality of the final product.

[0034] In view of this, the present invention provides a cocoa bean acetobacter, the cocoa bean acetobacter is named as cocoa bean acetobacter (Acetobacter fabarum) JJ-1, the preservation number of the cocoa bean acetobacter JJ-1 is CCTCC NO: M20242128, the preservation time is September 30, 2024, and the preservation address is China Center for Type Culture Collection.

[0035] In the technical solution of the present invention, the cocoa bean acetobacter is named as cocoa bean acetobacter (Acetobacterfabarum) JJ-1, the deposit number of the cocoa bean acetobacter JJ-1 is CCTCCNO: M 20242128, the deposit time is September 30, 2024, and the deposit address is China Center for Type Culture Collection. The cocoa bean acetobacter JJ-1 has a higher acid production capacity in the presence of exogenous selenium, and the fermented vinegar has a better flavor than the vinegar without selenium.

[0036] In some embodiments of the present invention, the 16S rDNA gene sequence of Acetobacter cocoa is shown as SEQ ID No.1.

[0037] SEQ ID NO.1:

[0038]

[0039] The present invention also provides an application of cocoa bean acetobacter in selenium-enriched fermentation, wherein the cocoa bean acetobacter includes the aforementioned cocoa bean acetobacter. The cocoa bean acetobacter can produce acid under the condition of exogenous selenium, and the acid production is relatively high, and is suitable for selenium-enriched fermentation to produce fermented products.

[0040] In some embodiments of the present invention, the cocoa bean acetobacter is used for mixed fermentation with an acid promoter, and the acid promoter includes sodium selenite and / or sodium selenate. The cocoa bean acetobacter can produce acid under the condition of exogenous selenium such as sodium selenite and 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 the acid production of cocoa bean acetobacter.

[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 cocoa bean acetobacter JJ-1 is 10%, and the acid production of the cocoa bean acetobacter JJ-1 is 16-41%. This shows that under the above conditions, the cocoa bean acetobacter JJ-1 of the present invention has a strong acid production capacity. The acid is the total acid, that is, the sum of all acid substances in the sample, and the determination method is based on the "GB 12456-2021 National Food Safety Standard Determination of Total Acid in Food".

[0042] The present invention also provides a vinegar fermentation method, comprising the following steps: mixing and culturing cocoa bean acetobacter and an acid promoter for fermentation; wherein the cocoa bean acetobacter includes the aforementioned cocoa bean acetobacter, and the acid promoter includes sodium selenite and / or sodium selenate. The vinegar obtained by 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 promoter is 20-80 mg / L; and / or the fermentation time is 9-12 days. The content of the acid promoter 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 its content within the above range can ensure a high acid production; the fermentation time is 9-12 days, and the fermentation time can be 9 days, 10 days, 11 days or 12 days, and the fermentation time within the above range can ensure a high acid production. At the same time, the acid promoter content and fermentation time are within the above range, and compared with not adding the acid promoter, the acid production is higher and the flavor is better.

[0044] The present invention also provides a flavor improver, comprising the aforementioned Acetobacter cocoa, and thus having all the beneficial effects of the aforementioned Acetobacter cocoa, which will not be described in detail here.

[0045] In some embodiments of the present invention, the flavor improver further comprises an acid promoter, which can increase the acid production of Acetobacter cocoa.

[0046] In some embodiments of the present invention, the acid promoter includes sodium selenite and / or sodium selenate, preferably sodium selenite, which can effectively increase the acid production of cocoa bean Acetobacter and increase the types and contents of beneficial flavor substances.

[0047] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0048] Experimental Materials

[0049] Kumquats were purchased from Taobao and were produced in Rong'an, Guangxi.

[0050] Calcium carbonate separation medium: 1% yeast powder, 1% glucose, 2% CaCO3, 2% agar, sterilize at 121°C for 20 min, wait until the temperature cools to 70°C, add 4% (v / v) anhydrous ethanol, mix well and pour into a culture dish to prepare a calcium carbonate separation medium.

[0051] Basic fermentation medium: 1% yeast powder, 1% glucose, sterilize at 121°C for 20 min, cool to 70°C, and add 4% (v / v) anhydrous ethanol.

[0052] Fermentation medium: 1% yeast powder, 1% glucose, sterilize at 121°C for 20 min, cool to 70°C, and add 6% (v / v) anhydrous ethanol.

[0053] Example 1 Isolation and preliminary identification of cocoa bean acetobacter JJ-1

[0054] 1. Strain enrichment

[0055] Wash the purchased kumquats with clean water and dry them in the air, chop them into pieces and put them into a 250 mL conical flask, add sterile water to cover the raw materials, seal the flask with a sealing film, and put it into a 28°C incubator for fermentation for 30 days to obtain kumquat fermentation liquid.

[0056] 2. Strain Isolation

[0057] The kumquat fermentation liquid was diluted and applied to the calcium carbonate separation solid medium, and inverted at 30°C to form colonies. Single colonies with typical acetic acid bacteria characteristics, which were smooth, moist, beige in color, and had a calcium-dissolving ring, were selected for streaking separation and purification. The purified strains were numbered, and 20% (volume fraction) glycerol was added and stored in a -80°C refrigerator for later use. A total of 3 strains were isolated from the kumquat fermentation liquid.

[0058] 3. Strain Screening

[0059] Use an inoculation loop to pick a single colony from the strain isolated in step 2, and inoculate it in 100mL of basic fermentation medium. Perform three parallel experiments for each strain, and shake and culture at 30°C and 180r / min for 5d. Take 25mL of fermentation broth, centrifuge at 8000r / min for 10min to remove the bacteria, take 5mL of supernatant for acetic acid qualitative test, that is, adjust the pH of the supernatant to 7.0 with saturated sodium hydroxide solution, heat and boil, add 6 drops of 5% ferric chloride solution, if a reddish-brown precipitate appears, acetic acid is produced during the culture of the strain, and it is preliminarily identified as acetic acid bacteria. After preliminary identification, the three strains screened by the present invention are all acetic acid bacteria, and they are named CL12, PT6 and JJ-1 respectively.

[0060] 4. Effect of exogenous selenium on the acid production capacity of three bacterial strains

[0061] The bacterial liquid (i.e., seed liquid) of strains CL12, PT6, and JJ-1 screened in step 3 was cultured for 36 h at 30°C, 180 rpm, and basic fermentation medium. 10% of the seed liquid was added to the fermentation medium containing 0, 10, and 20 mg / L Na2SeO3, respectively. The culture was then carried out in a constant temperature shaker at 30°C and 180 r / min for 7 days. The total acid content was determined by sampling and titration. The results were as follows: Figure 1 , Figure 2 and Figure 3 shown.

[0062] Depend on Figure 1 , Figure 2 and Figure 3 It can be seen that when the sodium selenite concentration increases, the total acid content produced by the CL12 and PT6 strains is low, that is, the acid production ability of the CL12 and PT6 strains is inhibited by sodium selenite, while when the sodium selenite concentration increases to 20 mg / L, the total acid content produced by the JJ-1 strain increases by about 3 times compared with the group without sodium selenite, and its acid production ability is stronger under high concentration sodium selenite conditions. Example 2 Morphological and molecular biological identification of cocoa bean acetic acid bacteria JJ-1

[0063] 1. Morphological identification

[0064] The strain JJ-1 obtained in Example 1 was inoculated on a calcium carbonate separation plate and cultured at 30°C for 48 hours. The cultured colonies were subjected to morphological observation, Gram staining and spore staining. The results were as follows: Figure 4 and Figure 5 shown.

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

[0066] By Gram staining ( Figure 5 ) and spore staining showed that the bacteria were Gram-negative and spore-free. Individual bacteria were short rods or elliptical in shape, most of them were arranged singly or in pairs, and some appeared in chains.

[0067] 2. Molecular Biological Identification

[0068] The single colony PCR method was used: 20 μL of sterile water was added to a 1.5 mL centrifuge tube, and a single JJ-1 colony 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 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 as follows: 94°C for 3 min; 94°C for 15 s, 55°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min; and storage at 4°C.

[0073] After PCR sequencing, first, a homologous sequence search was performed in the online database of the National Center for Biotechnology Information (NCBI). Second, the target strain and other acetic acid bacteria model strains of the same genus were compared for maximum homology. Finally, a phylogenetic tree was constructed using the Neighbor-Joining method using MEGA 7.01 software, and a Bootstrap test was performed with 1000 repetitions. The results are shown in Figure 2. Figure 6 The results showed that the highest homology between the 16S rDNA of the JJ-1 strain and the Acetobacter fabarum strain LMG 24244 was 100%, confirming that the strain was Acetobacter fabarum.

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

[0075] Example 3 Effect of exogenous selenium on the acid production capacity of Acetobacter cocoa JJ-1

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

[0077] Take a single colony of JJ-1 and inoculate it into the basic fermentation medium. Take samples every 4 hours to measure the OD 600 The monitoring time is 48 hours. The test results are as follows: Figure 7 shown.

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

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

[0080] Take 10 mL of the bacterial solution (i.e., seed solution) of JJ-1 strain cultured at 30°C, 180 rpm, and basic fermentation medium for 36 h, and add it to the fermentation medium containing 0, 10, 20, 30, 40, 50, 60, and 80 mg / L Na2SeO3, and then culture it in a constant temperature shaker at 30°C and 180 r / min. Take samples and titrate them every 24 h to determine the total acid content. The results are as follows: Figure 8 shown.

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

[0082] 3. Response Surface Optimization

[0083] Taking acid production (Y) as an indicator, four factors, namely, acetic acid bacteria fermentation temperature (A), inoculation amount (B), initial sodium selenite concentration (C), and initial ethanol concentration (D), were selected as independent variables, as shown in Table 1. Using Design Expert 13.0 software, a response surface optimization experiment with 4 factors and 3 levels and a total of 29 test points was designed to optimize the fermentation process conditions of the JJ-1 strain. The results are shown in Table 2.

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

[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 variance analysis of 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 test results

[0092] Model Item sum of squares Degrees of Freedom Mean Square F-number 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-Inoculum size 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 Lack of Fit 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, and the optimal process conditions were obtained as follows: acetic acid bacteria inoculation amount 10.483 mL, fermentation temperature 27.968 ° C, initial alcohol content 6.029%, initial sodium selenite concentration 22.783 mg / L. Under these conditions, the fermentation broth acid production of the predicted model is 39.463 g / L.

[0094] According to the actual situation, the process conditions were adjusted as follows: inoculation volume 10 mL, fermentation temperature 28 °C, initial alcohol content 6%, initial sodium selenite concentration 23 mg / L, and the final acid production was 40.22 g / L, which was basically consistent with the theoretical prediction value, indicating that the prediction model fits the actual fermentation conditions well.

[0095] Example 4 Effect of Acetobacter cocoa JJ-1 on the aroma of fermented products under the condition of exogenous selenium addition

[0096] 1. Solid phase microextraction conditions

[0097] Using the headspace solid phase microextraction (HS-SPME) method, 4 mL of the fermentation broth sample was taken into a 20 mL headspace bottle, 1 g of sodium chloride and 30 μL of the internal standard octanol (dilution ratio 1:1000) were added, the bottle cap was pressed tightly, and it was placed on the MPS injection pretreatment station for 20 minutes of equilibrium. The extraction head was inserted into the sample headspace bottle and placed on a magnetic stirrer for adsorption at 45°C for 30 minutes. After adsorption, the extraction head was taken out and inserted into the gas chromatography injection port, and desorbed at 250°C for 5 minutes. Three parallel experiments were performed for each sample.

[0098] There are two groups of fermentation broth samples, one of which is a selenium-added group, and the specific fermentation conditions are: seed liquid inoculation volume 10mL, fermentation temperature 28℃, initial alcohol content 6%, initial sodium selenite concentration 23mg / L, and fermentation for 10 days. The other group is a fermentation broth without exogenous selenium, except that the selenium content is 0mg / L, and other fermentation conditions are the same as those of the selenium-added group. The seed liquid refers to the bacterial liquid of the JJ-1 strain cultured at 30℃, 180rpm, and basic fermentation medium for 36h.

[0099] 2. Gas chromatography (GC) conditions

[0100] A DB-5MS elastic quartz capillary column (30m×0.25mm×0.25μm) was selected; the temperature program was as follows: the initial temperature was 40°C, maintained for 4 min, then increased to 90°C at a rate of 5°C / min, maintained for 3 min, then increased to 230°C at a rate of 5°C / min, and maintained for 5 min; the injection port temperature was 250°C; 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 capacity 70 eV, ion source temperature 230 ° C, quadrupole temperature 150 ° C, full scan mode, full scan, scan range 40 ~ 500 m / z.

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

[0104] The collected mass spectra were searched and analyzed by computer spectral library NIST (national institute of standards and technology) mass spectral library and Wiley Library, and the volatile substances with a matching degree greater than or equal to 80% were searched and determined for verification and confirmation. According to 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, and the test results are shown in Table 4.

[0105]

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

[0107] Table 4 Main volatile compounds in JJ-1 fermentation broth of the group without selenium addition and the group with selenium addition

[0108]

[0109]

[0110] As shown in Table 1, through HS-SPME-GC-MS detection technology, it was determined that the JJ-1 strain of the present invention detected a total of 32 volatile compounds after fermentation with the addition of exogenous selenium, and its main aroma substances were acetic acid, nonanoic acid, 2,4-di-tert-butylphenol, diisobutyl terephthalate, N-hydroxyphenylcarboximidic acid methyl ester and benzaldehyde, etc., and the types of volatile compounds of myristic acid and 2-methylpropionic acid were increased compared with the group without selenium addition, and 2-phenyl-2-butenal was reduced compared with the group without selenium addition. The total content of volatile compounds in the group with selenium addition (2937.28μg / L) was increased by 4.7% compared with the group without selenium addition (2803.37μg / L).

[0111] 3-Methylbutyric acid, 2-methylbutyric acid, 2-methylpropionic acid and octanoic acid have a pleasant fruity aroma at low concentrations. Acetic acid has a pungent taste. Nonanoic acid has a light fat and coconut aroma. Acetic acid and nonanoic acid accounted for 93.17% in the non-selenium group and 88.33% in the selenium group, and are the main flavor and aroma substances of vinegar. The content of acid substances in the non-selenium group was 1084.77μg / L, which was less than 1312.09μg / L in the selenium group. The acid production of the selenium group was 20.95% higher than that of the non-selenium group. Specifically, the addition of selenium increased the content and types of acid substances.

[0112] Esters are the main components of vinegar with aromatic characteristics, and most esters have aromatic smells. Diisobutyl terephthalate and methyl N-hydroxyphenylcarboximidate accounted for 71.59% in the non-selenium-added group and 75.59% in the selenium-added group. They are characteristic esters of vinegar and give vinegar a fruity aroma.

[0113] The unique aroma characteristics of aldehydes and ketones in the fermentation broth are an indispensable part of the vinegar aroma system. Benzaldehyde has a special cherry smell and has antibacterial effects. Benzaldehyde is a key component of natural fruit flavor. The selenium-added group increased by 44.65% compared with the non-selenium-added group. Nonanal has a strong grease smell and sweet orange flavor. 2-Phenyl-2-butenal has a sweet water and honey taste. 2,6-Di-tert-butyl-4-hydroxy-4-methylcyclohexa-2,5-diene-1-one is an important antioxidant, widely used in a variety of industrial and food fields, and has the function of extending the shelf life.

[0114] There was no significant difference in the significance difference test (p>0.05) between the group without selenium addition and the group with selenium addition for 2,4-di-tert-butylphenol. It has a strong aromatic smell and is a natural product with strong antibacterial activity and certain anti-cancer and anti-inflammatory effects. Selenium addition did not affect its synthesis. It is one of the main volatile compound components of the vinegar, accounting for 25.40% in the group without selenium addition and 23.56% in the group with selenium addition.

[0115] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A cocoa bean acetobacter, characterized in that The cocoa bean acetobacter is named as cocoa bean acetobacter (Acetobacter fabarum) JJ-1, the preservation number of the cocoa bean acetobacter JJ-1 is CCTCC NO: M20242128, the preservation time is September 30, 2024, and the preservation address is China Center for Type Culture Collection.

2. The cocoa bean Acetobacter according to claim 1, characterized in that The 16S rDNA gene sequence of the Acetobacter cocoa is shown as SEQ ID No.

1.

3. An application of cocoa bean acetobacter in selenium-enriched fermentation, characterized in that: The cocoa bean Acetobacter includes the cocoa bean Acetobacter according to claim 1.

4. The use according to claim 3, characterized in that The cocoa bean acetobacter is used for mixed fermentation with an acid promoter, and the acid promoter includes sodium selenite and / or sodium selenate.

5. The use according to claim 4, characterized in that 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 the cocoa bean acetobacter JJ-1 is 10%, and the acid production of the cocoa bean acetobacter JJ-1 is 16-41%.

6. A vinegar fermentation method, characterized in that: The following steps are involved: Mixing cocoa bean acetobacter with an acid promoter for fermentation; Wherein, the cocoa bean Acetobacter includes the cocoa bean Acetobacter as claimed in claim 1, and the acid promoter includes sodium selenite and / or sodium selenate.

7. The vinegar fermentation method according to claim 6, characterized in that: The content of the acid promoter is 20-80 mg / L; and / or, The fermentation time is 9 to 12 days.

8. A flavor improver, characterized in that The method comprises the cocoa bean Acetobacter as claimed in claim 1.

9. The flavor improving agent according to claim 8, characterized in that The flavor improver also includes an acid promoter.

10. The flavor improving agent according to claim 9, characterized in that The acid promoter includes sodium selenite and / or sodium selenate.

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