A method for constructing a complex microbial community suitable for fruit vinegar fermentation

Abstract

This invention discloses a method for constructing a complex microbial community suitable for fruit vinegar fermentation, belonging to the field of fruit vinegar fermentation. The method includes pre-treating fruit juice or pulp raw materials and then introducing them into a fermentation container to construct an initial fermentation system comprising a first functional microbial community, a second functional microbial community, and a third functional microbial community. The first functional microbial community is used for alcohol production, the second functional microbial community is used for acetic acid production, and the third functional microbial community is used for flavor modification. By detecting the fermentation state at different stages during fruit vinegar fermentation and adjusting the fermentation conditions when preset switching conditions are reached, the first, second, and third functional microbial communities sequentially acquire metabolic dominance. This solves the problem in existing technologies where different functional microbial communities mainly rely on natural succession, and the timing and conditions for switching dominance relationships are difficult to actively control.

Description

Technical Field

[0001] This invention relates to the field of fruit vinegar fermentation technology, and more specifically, to a method for constructing a complex microbial community suitable for fruit vinegar fermentation. Background Technology

[0002] Fruit vinegar fermentation typically uses fruit juice or pulp as raw material, undergoing alcohol production and acetic acid production stages to obtain the target fermentation product. In some processes, subsequent flavor modification processes are introduced to improve the final flavor. Most existing fruit vinegar fermentation methods employ single-strain fermentation, sequential inoculation of two types of bacteria, or co-cultivation of multiple strains in the same system. In practical applications, these methods often rely on the natural succession of the microbial community during fermentation to establish stage dominance. When the functional microbial community weakens in the previous stage and when it becomes dominant in the next stage is largely determined by the fermentation system itself, lacking active control over the timing and conditions of the shift in the dominant microbial community relationship.

[0003] Because parameters such as sugar, ethanol, acetic acid, dissolved oxygen, and acidity continuously change during fruit vinegar fermentation, the growth and metabolic advantages of different functional microbial communities in the same fermentation system will constantly change. If the fermentation conditions are not switched and controlled at appropriate times, it is easy for the microbial community to remain dominant in the previous stage and for the microbial community to fail to establish a dominant position in the later stage. This leads to an unstable transition between the alcohol production stage and the acetic acid production stage, large fluctuations in the acetic acid accumulation rate, insufficient subsequent flavor modification, and ultimately affects the stability of fruit vinegar fermentation and the consistency of product flavor. Summary of the Invention

[0004] To address the problems mentioned in the background section, the present invention provides the following technical solution: A method for constructing a complex microbial community suitable for fruit vinegar fermentation includes the following steps: S1, after pre-treating the fruit juice or pulp raw material, it is introduced into a fermentation container to construct an initial fermentation system containing a first functional microbial community, a second functional microbial community and a third functional microbial community, wherein the first functional microbial community is used to produce alcohol, the second functional microbial community is used to produce acetic acid, and the third functional microbial community is used for flavor modification. S2, the initial fermentation system is cultured under the first fermentation conditions, so that the first functional microbial community is in a state dominated by alcohol production metabolism in the fermentation system; S3, detect at least two of the following in the fermentation system: ethanol concentration, residual sugar concentration, and dissolved oxygen level; when the detection results reach the first switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the first functional bacterial community and to make the second functional bacterial community in the fermentation system in a state dominated by acetic acid production metabolism. S4, the fermentation system was further cultivated under the second fermentation conditions; S5, detect at least two of the following in the fermentation system: acetic acid concentration, ethanol concentration, total acid change rate, and dissolved oxygen level; when the detection results reach the second switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the second functional microbial community and to make the third functional microbial community dominate the flavor modification metabolism in the fermentation system. S6, under the third fermentation condition, continue to cultivate the fermentation system to complete the construction of a complex microbial community suitable for fruit vinegar fermentation; Furthermore, both the first switching condition and the second switching condition are combined state conditions; The combined state conditions include at least the following two types of conditions simultaneously: In the previous stage, the production state of the target metabolites corresponding to the functional microbial community enters a plateau or decline stage, and in the next stage, the substrates utilized by the functional microbial community reach a usable state in the fermentation system.

[0005] Furthermore, the fermentation condition adjustments in steps S3 and S5 do not include the removal of metabolites formed in the previous stage. The fermentation conditions are adjusted so that the metabolites formed in the previous stage are retained as substrates or precursors for the next stage, while the continuous expansion capacity of the functional microbial community in the previous stage is weakened and the expansion capacity of the functional microbial community in the next stage is enhanced.

[0006] Furthermore, after steps S3 and S5 are completed, the switched fermentation conditions continue to dominate the stable culture time. The next stage of the decision to switch to the next stage is only made when the target metabolites of the functional microbiota in the next stage continue to increase for two to four consecutive detection cycles, and the target metabolites of the functional microbiota in the previous stage no longer continue to increase.

[0007] Furthermore, the second switching condition includes at least: The second functional microbial community corresponds to the target metabolite entering the plateau growth stage, and ethanol and acetic acid that can be utilized by the third functional microbial community are also present in the fermentation system.

[0008] Furthermore, the cultivation of the third functional microbial community in step S6 is carried out without the addition of any exogenous flavor precursor substances. The third functional microbial community utilizes the ethanol left over from the first stage and the acetic acid generated in the second stage for flavor modification metabolism.

[0009] Furthermore, steps S2 to S6 are completed continuously in the same fermentation broth without changing the main body of the fermentation broth in between; The second and third functional bacterial groups are sequentially enriched in the fermentation broth by adjusting the fermentation conditions in steps S3 and S5, respectively.

[0010] Furthermore, after step S6 is completed, a portion of the fermentation liquid from the previous batch is taken as the inoculum source for the next batch, and fermentation is repeated without fully replenishing the first functional bacterial group, the second functional bacterial group, and the third functional bacterial group. When the first functional microbial community, the second functional microbial community, and the third functional microbial community are successively formed in multiple consecutive batches, the construction of the complex microbial community is considered complete.

[0011] In summary, the present invention has the following beneficial effects: By detecting the fermentation status at different stages of fruit vinegar fermentation and adjusting the fermentation conditions when preset switching conditions are met, the first, second, and third functional bacterial groups are made to successively gain metabolic dominance. This solves the problem in the prior art that different functional bacterial groups mainly rely on natural succession and that the timing and conditions for switching dominance are difficult to actively control. Attached Figure Description

[0012] 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 these drawings without creative effort.

[0013] Figure 1 This is a flowchart illustrating the overall process of the composite microbial community construction method of the present invention. Figure 2 This is a schematic diagram of the three-stage fermentation conditions and switching logic of the present invention; Figure 3 This is a diagram showing the dynamic changes in the metabolic dominance of functional microbiota during the three stages of this invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0015] Example 1 The following is in conjunction with the appendix Figure 1-3 The present invention will be described in further detail below.

[0016] This invention provides a technical solution: a method for constructing a complex microbial community suitable for fruit vinegar fermentation, comprising the following steps: S1, after pre-treating the fruit juice or pulp raw material, it is introduced into a fermentation container to construct an initial fermentation system containing a first functional microbial community, a second functional microbial community and a third functional microbial community, wherein the first functional microbial community is used to produce alcohol, the second functional microbial community is used to produce acetic acid, and the third functional microbial community is used for flavor modification. S2, the initial fermentation system is cultured under the first fermentation conditions, so that the first functional microbial community is in a state dominated by alcohol production metabolism in the fermentation system; S3, detect at least two of the following in the fermentation system: ethanol concentration, residual sugar concentration, and dissolved oxygen level; when the detection results reach the first switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the first functional bacterial community and to make the second functional bacterial community in the fermentation system in a state dominated by acetic acid production metabolism. S4, the fermentation system was further cultivated under the second fermentation conditions; S5, detect at least two of the following in the fermentation system: acetic acid concentration, ethanol concentration, total acid change rate, and dissolved oxygen level; when the detection results reach the second switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the second functional microbial community and to make the third functional microbial community dominate the flavor modification metabolism in the fermentation system. S6, under the third fermentation condition, continue to cultivate the fermentation system to complete the construction of a complex microbial community suitable for fruit vinegar fermentation; Both the first switching condition and the second switching condition are combined state conditions; The combined state conditions include at least the following two types of conditions simultaneously: In the previous stage, the production state of the target metabolites corresponding to the functional microbial community enters a plateau or decline stage, and in the next stage, the substrates utilized by the functional microbial community reach a usable state in the fermentation system. The fermentation condition adjustments in steps S3 and S5 do not include the removal of metabolites formed in the previous stage. The fermentation conditions are adjusted so that the metabolites formed in the previous stage are retained as substrates or precursors for the next stage, while the continuous expansion capacity of the functional microbial community in the previous stage is weakened and the expansion capacity of the functional microbial community in the next stage is enhanced. After steps S3 and S5 are completed, the switched fermentation conditions continue to dominate the stable culture time. The next stage of switching judgment will only be entered when the target metabolites corresponding to the functional flora in the next stage continue to increase for 2 to 4 consecutive detection cycles, and the target metabolites corresponding to the functional flora in the previous stage no longer continue to increase. In this embodiment: Clarified apple juice is used as the fruit base raw material. It is first filtered through a 120-mesh filter to remove coarse particulate matter, and then heat-treated at 85°C for 15 minutes. After cooling to room temperature, it is ready for use. The sugar content of the fruit base raw material is adjusted to 16°Bx and the pH is 4.0. It is then placed into a 5L fermentation tank, with each tank containing 3.5L of liquid. The fermentation tank is equipped with a temperature probe, a dissolved oxygen probe, and a sampling port. The main body of the fermentation liquid is not replaced during the fermentation process. When constructing the initial fermentation system, three types of functional microbial communities were simultaneously introduced into the fruit-based raw material; the first functional microbial community was commercially available brewer's yeast, and the initial viable cell concentration after inoculation was controlled at 1.0 × 10⁻⁶. 7 CFU / mL; the secondary functional bacterial group used was commercially available Acetobacter pasteurellium inoculum, with the initial viable bacterial concentration controlled at 1.0 × 10⁻⁶ CFU / mL after inoculation. 5 CFU / mL; the third functional bacterial group consisted of a mixed bacterial group of commercially available Pichia pastoris and Lactobacillus plantarum inoculum, with the viable cell concentrations of Pichia pastoris and Lactobacillus plantarum controlled at 5.0 × 10⁻⁶ CFU / mL. 4 CFU / mL and 5.0×10 4 CFU / mL; In the third functional bacterial group, Pichia pastoris is used to generate ester flavor substances such as ethyl acetate, and Lactobacillus plantarum is used to generate lactic acid. Both participate in the flavor modification metabolism in the later stage of fruit vinegar fermentation; The first fermentation stage begins immediately after inoculation. The culture conditions for the first fermentation stage were set as follows: temperature 25℃, stirring speed 100 r / min, and aeration rate 0.05 vvm. Culture under these conditions was intended to promote the dominance of the first functional bacterial group in alcohol production metabolism within the fermentation system. During the culture process, ethanol concentration, residual sugar concentration, and dissolved oxygen level were measured every 30 min. Ethanol concentration was determined by gas chromatography, residual sugar concentration by high-performance liquid chromatography, and dissolved oxygen level by an online dissolved oxygen probe. Simultaneously, samples were taken every 2 h, and the culturable bacterial count of the three functional bacterial groups was determined using selective plate counting. The first switching condition was deemed met when the ethanol concentration reached 8.2% vol, the residual sugar concentration decreased to 4.1 g / L, the dissolved oxygen level decreased to 1.8 mg / L, and the ethanol production rate no longer increased in three consecutive measurements. After the first switching condition is met, without removing the ethanol and other metabolites already formed in the fermentation system, the fermentation conditions are directly adjusted to allow the fermentation system to enter the second fermentation stage. The adjustment method is as follows: increase the aeration rate from 0.05 vvm to 0.80 vvm, increase the stirring speed from 100 r / min to 250 r / min, adjust the fermentation temperature from 25℃ to 30℃, and stop the addition of exogenous sugar. After the switch, continue to maintain the above conditions for dominant stable culture for 3 hours. During the dominant stable culture, the acetic acid concentration, ethanol concentration, and dissolved oxygen level are detected every 30 minutes, and the culturable number of the three functional bacterial groups is detected every 2 hours. When the acetic acid concentration continues to increase and the ethanol concentration continues to decrease in three consecutive tests, and the culturable number of the second functional bacterial group accounts for more than 60% of the total culturable number, it is determined that the second functional bacterial group has formed a dominant state of acetic acid production metabolism in the fermentation system. The high aeration and higher culture temperature in the second fermentation stage are more conducive to the proliferation and metabolism of the second functional bacterial group, while the third functional bacterial group is in a relatively inhibited state in this stage. The second fermentation stage continued under the conditions of 30℃, 250 r / min, and 0.80 vvm. During the cultivation process, the acetic acid concentration, ethanol concentration, total acid change rate, and dissolved oxygen level were measured every 30 min. The acetic acid concentration was verified by acid-base titration combined with high-performance liquid chromatography, and the total acid change rate was calculated based on the difference in total acid between two consecutive measurements and the detection time interval. When the acetic acid concentration reached 4.8 g / 100 mL, the ethanol concentration dropped to 1.6% vol, and the total acid change rate decreased by more than 40% from the maximum total acid change rate reached in the second fermentation stage in three consecutive measurements, the second switching condition was determined to be met. At this time, ethanol and acetic acid were still retained in the fermentation system, and no distillation, filtration, liquid replacement, or sterilization was performed. After the second switching condition is met, the fermentation conditions are directly adjusted to allow the fermentation system to enter the third fermentation stage. The adjustment method is as follows: reduce the aeration rate from 0.80 vvm to 0.20 vvm, reduce the stirring speed from 250 r / min to 120 r / min, adjust the fermentation temperature from 30℃ to 24℃, and control the pH of the fermentation broth between 3.2 and 3.4. In the third fermentation stage, no additional exogenous flavor precursors, ethanol, acetic acid, or amino acid precursors are added. Flavor modification and metabolism are carried out solely using the ethanol remaining from the first fermentation stage and the acetic acid generated in the second fermentation stage. The third fermentation stage is incubated for 8 hours, with the contents of ethyl acetate, lactic acid, ethanol, and acetic acid measured every hour. Ethyl acetate is determined by gas chromatography, and lactic acid is determined by high-performance liquid chromatography. Simultaneously, the culturable number of the third functional microbial community is measured every 2 hours. When ethyl acetate and lactic acid both increase continuously in two consecutive tests, and the culturable number of the third functional microbial community accounts for more than 50% of the total culturable number of microorganisms, it is determined that the third functional microbial community has formed a flavor-modifying metabolic dominant state in the fermentation system; after the aeration rate decreases and the culture temperature drops in the third fermentation stage, the competitive advantage of the third functional microbial community is enhanced; thus, the construction of a compound microbial community suitable for fruit vinegar fermentation is completed. The conditions for the above three-stage training and switching are shown in Table 1; Table 1: Three-stage fermentation conditions and switching parameters

[0017] Two control groups were set up; Control group 1 adopted the traditional two-stage sequential inoculation process, that is, only the first functional group was initially inoculated, and the second functional group was added after the ethanol concentration reached 8.0% vol, without setting a third fermentation stage; Control group 2 adopted a process in which three functional bacterial groups were simultaneously inoculated but constant fermentation conditions were maintained throughout the process, namely, the temperature was kept constant at 27℃, the stirring speed was kept constant at 180r / min, and the aeration rate was kept constant at 0.30vvm, without setting stage detection triggers or condition switching throughout the process; The control group 2 was used to simulate a fermentation method in which three types of functional microbial communities were continuously co-cultured under the same compromise conditions without switching between stages; The experimental group adopted the method described in the above embodiments; the three groups fermented under the same fruit substrate conditions until the end of the third stage, and the test results are shown in Table 2; Table 2: Comparison of fermentation results between the experimental group and the control group

[0018] After the third fermentation stage of the experimental group was completed, the fermentation broth sample was taken for sensory stability testing. During the test, a distinct fruit vinegar aroma and a mild ester aroma could be smelled, without any obvious pungent acetic acid odor. No obvious stratification occurred after the fermentation broth was left to stand for 12 hours. The fermentation broth of the experimental group was further used for subsequent batch inoculation verification, as detailed in Example 3.

[0019] Example 2 like Figure 1-3 As shown, the second switching condition includes at least: The second functional microbial community corresponds to the target metabolite entering the plateau growth stage, and ethanol and acetic acid that can be utilized by the third functional microbial community are also present in the fermentation system. In step S6, the third functional microbial community is cultured without the addition of any exogenous flavor precursors. The third functional microbial community utilizes the ethanol left over from the first stage and the acetic acid generated in the second stage for flavor modification metabolism. Steps S2 to S6 are completed continuously in the same fermentation broth without changing the main body of the fermentation broth in between; The second and third functional bacterial groups are sequentially enriched in the fermentation broth by adjusting the fermentation conditions in steps S3 and S5, respectively.

[0020] In this embodiment, clarified apple juice is used as the fruit base raw material. It is first filtered through a 120-mesh filter to remove coarse particulate matter, and then heat-treated at 85°C for 15 minutes. After cooling to room temperature, it is used for later use. The sugar content of the fruit base raw material is adjusted to 16°Bx and the pH is 4.0. It is then loaded into a 5L fermentation tank, with each tank containing 3.5L of liquid. The fermentation tank is equipped with a temperature probe, dissolved oxygen probe, pH probe, and sampling port. During the fermentation process, the main body of the fermentation liquid is not replaced, and no intermediate liquid replacement, centrifugation sterilization, filtration sterilization, or distillation to remove alcohol is performed. When constructing the initial fermentation system, three types of functional microbial communities were simultaneously introduced; the first functional microbial community was commercially available brewer's yeast inoculum, and the initial viable cell concentration after inoculation was controlled at 1.0 × 10⁻⁶. 7 CFU / mL; the secondary functional bacterial group used was commercially available Acetobacter pasteurellium inoculum, with the initial viable bacterial concentration controlled at 1.0 × 10⁻⁶ CFU / mL after inoculation. 5 CFU / mL; the third functional bacterial group consisted of a mixed bacterial group of commercially available Pichia pastoris and Lactobacillus plantarum inoculum, with the viable cell concentrations of Pichia pastoris and Lactobacillus plantarum controlled at 5.0 × 10⁻⁶ CFU / mL. 4 CFU / mL and 5.0×10 4 CFU / mL; In the third functional bacterial group, Pichia pastoris is used to generate ester flavor substances such as ethyl acetate, and Lactobacillus plantarum is used to generate lactic acid. Both participate in the flavor modification metabolism in the later stage of fruit vinegar fermentation. The culture conditions for the first fermentation stage were set as follows: temperature 25℃, stirring speed 100 r / min, and aeration rate 0.05 vvm. Under these conditions, the first functional bacterial group was allowed to establish a dominant state of alcohol production metabolism in the fermentation system. During the culture process, the ethanol concentration, residual sugar concentration, and dissolved oxygen level were measured every 30 min. The ethanol concentration was determined by gas chromatography, the residual sugar concentration by high performance liquid chromatography, and the dissolved oxygen level by an online dissolved oxygen probe. Simultaneously, samples were taken every 2 h, and the culturable bacterial count of the three functional bacterial groups was determined by selective plate counting. When the ethanol concentration reached 8.0% vol to 8.5% vol, the residual sugar concentration dropped to 3.5 g / L to 5.0 g / L, and the dissolved oxygen level dropped to below 2.0 mg / L, and the ethanol production rate no longer increased in three consecutive measurements, the first switching condition was deemed to have been met. After the first switching condition is met, without removing the ethanol and other metabolites already formed in the fermentation system, the fermentation conditions are directly adjusted to allow the fermentation system to enter the second fermentation stage. The adjustment method is as follows: increase the aeration rate from 0.05 vvm to 0.80 vvm, increase the stirring speed from 100 r / min to 250 r / min, adjust the fermentation temperature from 25℃ to 30℃, and stop the addition of exogenous sugar. After the switch, continue to maintain the above conditions for dominant stable culture for 3 hours. During the dominant stable culture, the acetic acid concentration, ethanol concentration, and dissolved oxygen level are detected every 30 minutes, and the number of culturable bacteria of the three functional groups is detected every 2 hours. When the acetic acid concentration continues to increase and the ethanol concentration continues to decrease in three consecutive tests, and the number of culturable bacteria of the second functional group accounts for more than 60% of the total number of culturable bacteria, it is determined that the second functional group has formed a dominant state of acetic acid production metabolism in the fermentation system. The second fermentation stage continued under the conditions of 30℃, 250 r / min, and 0.80 vvm. During the cultivation process, the concentrations of acetic acid, ethanol, total acid change rate, and dissolved oxygen level were measured every 30 min. The acetic acid concentration was verified by acid-base titration combined with high-performance liquid chromatography. The total acid change rate was calculated based on the difference in total acid concentration between two consecutive measurements and the time interval between measurements. When the acetic acid concentration reached 4.5 g / 100 mL to 5.2 g / 100 mL, the ethanol concentration decreased to 0.8% vol to 1.8% vol, and the total acid change rate decreased by 35% to 50% compared to the maximum total acid change rate reached in the second fermentation stage in three consecutive measurements, the second switching condition was deemed to be met. When the second switching condition was met, ethanol and acetic acid that could be utilized by the third functional microbial community were simultaneously present in the fermentation system, with the ethanol concentration not lower than 0.8% vol and the acetic acid concentration not lower than 4.5 g / 100 mL. Once the second switching condition is met, the fermentation conditions are directly adjusted to initiate the third fermentation stage. The adjustment method is as follows: the aeration rate is reduced from 0.80 vvm to 0.20 vvm, the stirring speed is reduced from 250 r / min to 120 r / min, the fermentation temperature is adjusted from 30℃ to 24℃, and the pH of the fermentation broth is controlled between 3.2 and 3.4. During the third fermentation stage, no additional exogenous flavor precursors, ethanol, acetic acid, esterification precursors, or amino acid precursors are added; only the residues from the first fermentation stage are utilized. Ethanol and acetic acid generated in the second fermentation stage are used for flavor modification metabolism. In the third fermentation stage, the culture is carried out for 8 hours, and the contents of ethyl acetate, lactic acid, ethanol and acetic acid are detected every 1 hour. Ethyl acetate is determined by gas chromatography and lactic acid is determined by high performance liquid chromatography. At the same time, the number of culturable bacteria of the third functional group is detected every 2 hours. When ethyl acetate and lactic acid continue to increase in two consecutive tests, and the number of culturable bacteria of the third functional group accounts for more than 50% of the total number of culturable bacteria, it is determined that the third functional group has formed a dominant state of flavor modification metabolism in the fermentation system. To verify the necessity of the simultaneous presence of ethanol and acetic acid in the defined second switching condition, experimental group E1 and control group E2 were set up. Experimental group E1 was switched between the second and third stages as described above. After the second switching condition was met, control group E2 used vacuum evaporation to reduce the ethanol concentration in the fermentation broth to below 0.2% vol, while the other conditions remained the same as experimental group E1. No exogenous flavor precursors were added to either group. The test results are shown in Table 3. Table 3: Effect of coexistence of ethanol and acetic acid during the second switching on the flavor modification results in the third stage.

[0021] To verify the conditions under which no additional exogenous flavor precursors were added, experimental group F1 and control group F2 were set up. Experimental group F1 was not supplemented with any exogenous flavor precursors during the third fermentation stage as described above. Control group F2 was supplemented with 0.5% vol of edible ethanol and 0.3 g / 100 mL of acetic acid at the beginning of the third fermentation stage. All other conditions were the same as those of experimental group F1. The test results are shown in Table 4. Table 4: The effect of adding exogenous flavor precursors in the third stage on the flavor modification results

[0022] In Table 4, experimental group F1, without the addition of exogenous flavor precursors, could still obtain 182 mg / L of ethyl acetate and 1.26 g / L of lactic acid in the third stage after 8 hours, indicating that the third functional microbial community can utilize the ethanol and acetic acid remaining from the first two stages to complete flavor modification metabolism. To verify the defined technical characteristics of "three stages being completed continuously in the same fermentation broth without changing the main body of the fermentation broth, and sequential enrichment achieved through condition switching", experimental group G1 and control group G2 were set up. Experimental group G1 completed the three-stage culture continuously in the same fermentation broth according to the above method. After the first stage, control group G2 centrifuged to remove the original fermentation broth, resuspended the bacterial body in freshly prepared culture medium for the second stage culture, and changed the fermentation broth again after the second stage to enter the third stage. The remaining culture temperature, stirring and aeration conditions were kept the same as those of experimental group G1. The test results are shown in Table 5. Table 5. The impact of changing the main fermentation broth on the results of the three-stage dominant phase switching.

[0023] After the third fermentation stage of experimental group G1, samples were taken to detect changes in the number of culturable bacteria of the three functional bacterial groups. The results showed that at the end of the first fermentation stage, the number of culturable bacteria of the first functional bacterial group accounted for 68% of the total number of culturable bacteria; at the end of the stable culture in the second fermentation stage, the number of culturable bacteria of the second functional bacterial group accounted for 63% of the total number of culturable bacteria; and at the end of the third fermentation stage, the number of culturable bacteria of the third functional bacterial group accounted for 52% of the total number of culturable bacteria. This indicates that the three functional bacterial groups can be enriched sequentially and form a metabolically dominant state after the switching of conditions in the same fermentation broth. The sample of the fermentation liquid at the end of the third fermentation stage of experimental group G1 was subjected to sensory testing after standing at 4℃ for 12 hours. It had a distinct fruit vinegar aroma and a mild ester aroma, with no obvious pungent or sharp sour smell. The overall color of the fermentation liquid was uniform, with no obvious layering or turbidity or flocculation. The fermentation liquid was then used for cross-batch inoculation verification, as detailed in Example 3.

[0024] Example 3 like Figure 1-3 As shown, after step S6 is completed, a portion of the fermentation liquid from the previous batch is taken as the inoculum source for the next batch, and fermentation is repeated without fully replenishing the first functional bacterial group, the second functional bacterial group, and the third functional bacterial group. When the first functional microbial community, the second functional microbial community, and the third functional microbial community are sequentially formed in multiple consecutive batches, the construction of the complex microbial community is determined to be complete. In this embodiment, the fermentation broth from the experimental group G1 after the third fermentation stage in Example 2 was used as the previous batch of fermentation end broth. After the fermentation end broth was stirred evenly, coarse suspended particles were removed through an 80-mesh sieve, and 10% of its total volume was used as the inoculum source for the next batch. The next batch still used the same clarified apple juice as in Example 1 as the fruit base material. After the fruit base material was filtered through a 120-mesh filter, it was heat-treated at 85°C for 15 minutes, cooled to room temperature, and the sugar content was adjusted to 16°Bx and the pH to 4.0. Then it was put into a 5L fermentation tank, with each tank containing 3.5L of liquid. During inoculation, the first functional bacterial group, the second functional bacterial group, and the third functional bacterial group were not replenished in full. Only the previous batch of fermentation end broth was added as the inoculum source. Subsequently, fermentation was carried out in the same three-stage culture method as in Examples 1 and 2. The first fermentation stage was conducted at 25°C, with a stirring speed of 100 r / min and an aeration rate of 0.05 vvm. Ethanol concentration, residual sugar concentration, and dissolved oxygen level were measured every 30 min during the fermentation process. Ethanol concentration was determined by gas chromatography, residual sugar concentration by high-performance liquid chromatography, and dissolved oxygen level by an online dissolved oxygen probe. Simultaneously, samples were taken every 2 h, and the culturable bacterial counts of the three functional bacterial groups were determined using selective plate counting. The first functional bacterial group was counted using YPD selective medium, the second functional bacterial group using acetic acid bacteria selective medium containing calcium carbonate, and the third functional bacterial group (Pichia pastoris) using WL nutrient medium and Lactobacillus plantarum using MRS medium. The results of both counts were combined to determine the culturable bacterial count of the third functional bacterial group. When the ethanol concentration, residual sugar concentration, and dissolved oxygen level reached the first switching condition of Example 1, and the ethanol production rate no longer increased in three consecutive measurements, the first functional bacterial group was determined to have formed a dominant alcohol-producing metabolic state and entered the first switching phase. After the first switch, the aeration rate was increased to 0.80 vvm, the stirring speed was increased to 250 r / min, the fermentation temperature was adjusted to 30℃, and the addition of exogenous sugar was stopped, so that the fermentation system entered the second fermentation stage. During the second fermentation stage, the acetic acid concentration, ethanol concentration, total acid change rate, and dissolved oxygen level were measured every 30 minutes. At the same time, the number of culturable bacteria of the three functional groups was measured every 2 hours. When the acetic acid concentration, ethanol concentration, and total acid change rate reached the second switch conditions in Example 1, and the acetic acid concentration continued to increase and the ethanol concentration continued to decrease in three consecutive measurements, and the number of culturable bacteria of the second functional group accounted for more than 60% of the total number of culturable bacteria, it was determined that the second functional group had formed a state dominated by acetic acid production metabolism, and the second switch was initiated. After the second switch, the aeration rate was reduced to 0.20 vvm, the stirring speed was reduced to 120 r / min, the fermentation temperature was adjusted to 24℃, and the pH of the fermentation broth was controlled between 3.2 and 3.4, so that the fermentation system entered the third fermentation stage. No additional exogenous flavor precursors were added during the third fermentation stage. During the cultivation process, the contents of ethyl acetate, lactic acid, ethanol, and acetic acid were measured every 1 hour, and the number of culturable bacteria of the third functional group was measured every 2 hours. When ethyl acetate and lactic acid continued to increase in two consecutive measurements, and the number of culturable bacteria of the third functional group accounted for more than 50% of the total number of culturable bacteria, it was determined that the third functional group had formed a flavor-modifying metabolic dominant state. Following the above method, three batches of repeated fermentation were carried out using the fermentation end broth as the inoculum source. The first batch of inoculum source was taken from the fermentation end broth of experimental group G1 in Example 2, the second batch of inoculum source was taken from the fermentation end broth of the first batch, and the third batch of inoculum source was taken from the fermentation end broth of the second batch. The three types of functional microbial groups were not fully replenished in each of the three batches. The results of the formation of the three dominant states in each batch are shown in Table 6. Table 6 shows the formation results of the dominant state of each batch when the previous batch of fermentation end broth was used as the inoculum source.

[0025] During three consecutive batches of repeated fermentation, the peak ethanol content, acetic acid endpoint, ethyl acetate content, lactic acid content, and total fermentation time of each batch were measured simultaneously. The results are shown in Table 7. Table 7 shows the fermentation results of each batch when the previous batch of fermentation broth was used as the inoculum.

[0026] To compare the effects of re-supplementing the three functional bacterial groups on the fermentation results, a control group was set up. Before each batch of fermentation, the first, second, and third functional bacterial groups were re-supplemented in full according to the inoculation method in Example 1, and the culture conditions were kept consistent with those of the experimental group. The main results of the experimental group and the control group for three consecutive batches were compared, and the results are shown in Table 8. Table 8. The impact of whether or not the three types of functional microbial communities were fully replenished on the results of continuous batch fermentation.

[0027] As shown in Table 8, without replenishing the first, second, and third functional bacterial groups, three batches of fermentation were continuously repeated using the previous batch of fermentation broth as the inoculum. Each batch successively formed the metabolically dominant state of the first, second, and third functional bacterial groups. The peak ethanol content, endpoint acetic acid content, ethyl acetate content, and lactic acid content of the experimental group in the three consecutive batches were within the same range as those of the control group, with small fluctuations between batches. Specifically, the peak ethanol content of the experimental group fluctuated from 8.0%vol to 8.3%vol, the endpoint acetic acid content fluctuated from 5.1g / 100mL to 5.3g / 100mL, the ethyl acetate content fluctuated from 176mg / L to 183mg / L, and the lactic acid content fluctuated from 1.19g / L to 1.24g / L.

[0028] The fermentation broth from the third batch was used as an inoculum for the fourth batch of pre-validation fermentation in the same manner. The test results showed that the dominant formation time of the first functional microbial community was 13.6 h, the dominant formation time of the second functional microbial community was 28.1 h, and the dominant formation time of the third functional microbial community was 38.7 h. The peak ethanol concentration was 8.1% vol, the endpoint acetic acid concentration was 5.2 g / 100 mL, the ethyl acetate concentration was 179 mg / L, and the lactic acid concentration was 1.22 g / L. The results were consistent with those of the first three batches.

[0029] After completing continuous batch fermentation under the above conditions, the construction of the complex microbial community is deemed complete.

[0030] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0031] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for constructing a complex microbial community suitable for fruit vinegar fermentation, characterized in that, Includes the following steps: S1, after pre-treating the fruit juice or pulp raw material, it is introduced into a fermentation container to construct an initial fermentation system containing a first functional microbial community, a second functional microbial community and a third functional microbial community, wherein the first functional microbial community is used to produce alcohol, the second functional microbial community is used to produce acetic acid, and the third functional microbial community is used for flavor modification. S2, the initial fermentation system is cultured under the first fermentation conditions, so that the first functional microbial community is in a state dominated by alcohol production metabolism in the fermentation system; S3, detect at least two of the following in the fermentation system: ethanol concentration, residual sugar concentration, and dissolved oxygen level; when the detection results reach the first switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the first functional bacterial community and to make the second functional bacterial community in the fermentation system in a state dominated by acetic acid production metabolism. S4, the fermentation system was further cultivated under the second fermentation conditions; S5, detect at least two of the following in the fermentation system: acetic acid concentration, ethanol concentration, total acid change rate, and dissolved oxygen level; when the detection results reach the second switching condition, adjust the fermentation conditions to inhibit the continuous expansion of the second functional microbial community and to make the third functional microbial community dominate the flavor modification metabolism in the fermentation system. S6, under the third fermentation condition, continues to cultivate the fermentation system to complete the construction of a complex microbial community suitable for fruit vinegar fermentation.

2. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 1, characterized in that, Both the first switching condition and the second switching condition are combined state conditions; The combined state conditions include at least the following two types of conditions simultaneously: In the previous stage, the production state of the target metabolites corresponding to the functional microbial community enters a plateau or decline stage, and in the next stage, the substrates utilized by the functional microbial community reach a usable state in the fermentation system.

3. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 2, characterized in that, The fermentation condition adjustments in steps S3 and S5 do not include the removal of metabolites formed in the previous stage. The fermentation conditions are adjusted so that the metabolites formed in the previous stage are retained as substrates or precursors for the next stage, while the continuous expansion capacity of the functional microbial community in the previous stage is weakened and the expansion capacity of the functional microbial community in the next stage is enhanced.

4. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 3, characterized in that, After steps S3 and S5 are completed, the switched fermentation conditions continue to dominate the stable culture time. The next stage of the decision to switch to the next stage is only made when the target metabolites of the functional microbiota in the next stage continue to increase for two to four consecutive detection cycles, and the target metabolites of the functional microbiota in the previous stage no longer continue to increase.

5. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 4, characterized in that, The second switching condition includes at least: The second functional microbial community corresponds to the target metabolite entering the plateau growth stage, and ethanol and acetic acid that can be utilized by the third functional microbial community are also present in the fermentation system.

6. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 5, characterized in that, In step S6, the third functional microbial community is cultured without the addition of any exogenous flavor precursors. The third functional microbial community utilizes the ethanol left over from the first stage and the acetic acid generated in the second stage for flavor modification metabolism.

7. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 6, characterized in that, Steps S2 to S6 are completed continuously in the same fermentation broth without changing the main body of the fermentation broth in between; The second and third functional bacterial groups are sequentially enriched in the fermentation broth by adjusting the fermentation conditions in steps S3 and S5, respectively.

8. The method for constructing a complex microbial community suitable for fruit vinegar fermentation according to claim 7, characterized in that, After step S6 is completed, take a portion of the fermentation liquid from the previous batch as the inoculum source for the next batch, and repeat the fermentation without fully replenishing the first functional bacterial group, the second functional bacterial group, and the third functional bacterial group. When the first functional microbial community, the second functional microbial community, and the third functional microbial community are successively formed in multiple consecutive batches, the construction of the complex microbial community is considered complete.

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