Method for preparing ethyl hexanoate through yellow water closed loop fermentation and esterification

Abstract

The present application relates to the technical field of resource utilization of by-products of liquor brewing, and discloses a method for preparing ethyl hexanoate through closed-loop fermentation and esterification of yellow water, which comprises the following steps: preparing an acidic concentrated solution by pre-concentrating yellow water; adjusting the pH of the acidic yellow water, and then introducing the yellow water into an anaerobic fermentation of a domesticated composite microbial population for producing hexanoic acid from lactic acid as a substrate to obtain yellow water fermentation liquor containing hexanoic acid; performing acidic esterification on the yellow water concentrated solution as an acidifying solution after adjusting the pH of the solution to obtain a component containing ethyl hexanoate through physical separation; and recycling the remaining liquid after distillation to the hexanoic acid fermentation end. Since the lactic acid type route for producing hexanoic acid is adopted, the pH is increased by fermentation itself, and the amount of alkaline adjusting agent is less than that in the ethanol type route; the reflux liquid is still rich in fermentable substrates such as lactic acid and sugar as well as buffer components, and recycling can further reduce the alkali consumption and support fermentation, so as to form a closed-loop process of "concentration-acidification-esterification-separation-reflux-fermentation", which is stable after continuous operation for multiple rounds and has high material utilization rate.

Description

Technical Field

[0001] This invention relates to the field of resource utilization technology of by-products in liquor brewing, specifically to a method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water. Background Technology

[0002] Yellow water is an acidic liquid that precipitates from the bottom of the fermentation pits during the solid-state fermentation of baijiu. It typically contains lactic acid, acetic acid, ethanol, residual sugars, organic nitrogen, inorganic salts, and various flavor precursors. Yellow water is characterized by high acidity, high COD, and complex composition. Direct discharge of yellow water would cause environmental pollution; however, simply returning it to the fermentation pits for maintenance or treating it as a low-value product would fail to fully utilize the resource value of its lactic acid, ethanol, and hexanoic acid precursors.

[0003] Ethyl hexanoate is one of the key aroma components of strong-aroma baijiu. Fermentation with hexanoic acid bacteria using yellow water as a substrate can convert the high-content lactic acid, ethanol, sugar, and other components in the yellow water into hexanoic acid, thereby obtaining hexanoic acid bacteria for cultivation in cellar mud. Furthermore, esterifying hexanoic acid with ethanol to produce ethyl hexanoate can combine the resource utilization pathway of yellow water with the flavor requirements of the main baijiu business.

[0004] However, in existing technologies, hexanoic acid fermentation and ethyl hexanoate preparation are often separated into different process units: one type of technology focuses on the production of medium-chain fatty acids such as hexanoic acid from yellow water or organic waste through lactic acid fermentation and carbon chain elongation; the other type of technology uses resin adsorption to enrich the yellow water hexanoic acid fermentation broth, elutes it with ethanol, and then uses lipase or other catalytic methods to carry out esterification.

[0005] While the aforementioned technologies can achieve resource recovery of some components in yellow water, they still have the following shortcomings: First, before hexanoic acid fermentation, an alkaline regulator is usually added to increase the pH of the acidic yellow water. Based on sodium bicarbonate, in the first round or without residual liquid reflux, the amount added is generally 3%-6% of the mass or volume of the acidic yellow water, resulting in high alkali consumption. Furthermore, the specific dosage varies depending on the initial acidity of different batches of yellow water, and there is no uniform optimal addition amount. Second, acidification before esterification often relies on additional exogenous acidity regulators such as citric acid, lactic acid reagents, or inorganic acids. While organic acids with good food applicability, such as citric acid and lactic acid, can be used for acidification, they introduce additional organic carbon. When the acidified liquid or separation residue enters the anaerobic treatment unit, it increases COD and organic load, making subsequent treatment more difficult. Using inorganic acids such as hydrochloric acid and sulfuric acid has lower applicability and acceptance in food production scenarios, and its procurement, storage, transportation, and usage management requirements are high. Even with acidification using concentrated yellow water, existing technologies mostly involve one-time, unidirectional utilization. The organic matter contained in the concentrated yellow water may still end up in the end-of-pipe treatment system, failing to adequately reduce the organic pollution load. Third, the residual liquid after esterification and physical separation still contains reusable fermentable substrates such as lactic acid and sugars, as well as organic acid salts, buffer substances, and nutrients. If not recycled, it not only results in the loss of usable materials but also increases the burden on end-of-pipe treatment. This invention recycles the separated residual liquid to the hexanoic acid fermentation stage, allowing the lactic acid, sugars, and other organic matter in it to continue fermentation and be converted into hexanoic acid. At the same time, the liquid diverted to control salt and COD accumulation is a fermentation broth rich in hexanoic acid, which can be further used for pit mud cultivation, recycled to the pit, or used for downstream hexanoic acid extraction, thereby reducing the total amount of organic matter that needs to enter the end-of-pipe treatment system, reducing the organic pollution load, and improving material utilization efficiency. Fourth, routes such as resin adsorption and ethanol elution increase the cost of separation materials and eluents and do not fully utilize the acid-base buffering properties of yellow water itself. Fifth, the above routes are mostly unidirectional utilization, without recycling the distillation residue or fermentation broth. This is because it is generally believed in the field that refluxing the distillation residue or fermentation broth will cause inhibitory substances such as sodium ions, salts and COD to accumulate in the system, reaching the inhibition threshold and harming the caproic acid-producing bacteria. Therefore, existing technologies generally avoid reflux operations, and long-term closed-loop operation is considered difficult to achieve.

[0006] Therefore, it is necessary to provide a closed-loop process for producing yellow water: acidic yellow water is fermented with hexanoic acid using lactic acid as a substrate, and then acidified before esterification is completed using the concentrated yellow water. After acidic esterification and physical separation, a component containing ethyl hexanoate is obtained, and the remaining liquid is recycled back to the hexanoic acid fermentation system. Since the amount of alkaline regulator required for the lactic acid-based hexanoic acid production route is lower than that for the ethanol-based route, and with the recycling of fermentable substrates such as lactic acid and sugars and buffer components in the reflux liquid, it is possible to achieve stable operation for multiple rounds and efficient closed-loop resource utilization of yellow water while reducing the consumption of alkaline regulators and exogenous acidity regulators. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing ethyl hexanoate through closed-loop fermentation and esterification of yellow water. Using yellow water as raw material, the method involves hexanoic acid fermentation, yellow water source lactic acid acidification, acidic esterification, physical separation, and residual liquid recirculation to achieve the preparation of components containing ethyl hexanoate. This method utilizes the components of yellow water itself to achieve synergistic effects across multiple processes: First, lactic acid and other substances in the yellow water are used as substrates, which are converted into hexanoic acid by a domesticated complex of bacteria that produce hexanoic acid from lactic acid substrates. This lactic acid-based route reduces the consumption of alkaline regulators compared to the ethanol-based route. Second, concentrated yellow water (or concentrated yellow water nanofiltration lactic acid) is used as an acidifying liquid to adjust the hexanoic acid fermentation broth to an acidic range suitable for esterification, reducing the amount of exogenous acidity regulators required. Third, the residual liquid after physical separation is recirculated back to the hexanoic acid fermentation system, recycling the residual lactic acid, sugars, and other fermentable substrates and buffer components, further reducing subsequent alkali consumption and maintaining fermentation stability, forming a long-term operable closed-loop yellow water process.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention discloses a method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water, comprising the following steps:

[0010] (1) After adjusting the pH of the acidic yellow water to 4-7, a domesticated complex of bacteria that produce hexanoic acid using lactic acid as a substrate is introduced and hexanoic acid fermentation is carried out under anaerobic conditions to obtain yellow water fermentation liquid containing hexanoic acid.

[0011] (2) Add yellow water source acidification liquid to the yellow water fermentation broth containing hexanoic acid, adjust the pH value to 3.0-4.5 to obtain acidified fermentation broth, and then directly carry out acid esterification, and extract the hexanoic acid ethyl ester component from the acid esterification system by physical separation method; wherein, the yellow water source acidification liquid is yellow water concentrate and / or yellow water membrane filtration lactic acid concentrate;

[0012] (3) The remaining liquid after physical separation in step (2) is returned to the hexanoic acid fermentation step in step (1) to continue fermentation, and the cycle of steps (1)-(3) is repeated to achieve continuous fermentation-acidification-esterification-physical separation.

[0013] Preferably, in step (1), the initial pH of the acidic yellow water is 3.0-4.5, and the pH value of the acidic yellow water is adjusted by an alkaline regulator, which is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium carbonate, calcium bicarbonate, and calcium hydroxide.

[0014] Preferably, in step (1), the alkaline regulator adjusts the pH of the acidic yellow water to 5.5.

[0015] Preferably, in step (1), the domesticated complex microbial community is a complex microbial community obtained by enriching and domesticating one or more of yellow water, pit mud, pit fermentation products, and yellow water fermentation liquid containing hexanoic acid; or it is isolated from the pit, or it is a commercial hexanoic acid fermentation complex microbial community, or a natural hexanoic acid synthesis functional mixed / pure microbial community, or a genetically modified microbial community.

[0016] Preferably, in step (2), the yellow water concentrate is an acidic concentrate obtained by concentrating yellow water 5-10 times; the lactic acid concentrate after yellow water membrane filtration is an acidic concentrate rich in lactic acid obtained by separating yellow water by nanofiltration.

[0017] Preferably, in step (2), the acid esterification temperature is 20-60℃ and the esterification time is 5-72h; the physical separation method includes one or more of distillation, rectification, steam distillation, extraction, membrane separation, and phase separation.

[0018] Preferably, in step (2), during the acid esterification process, one or more of the following are added: edible alcohol, base wine, and tail wine; the amount of edible alcohol added is 5%-30% of the volume of the acidified fermentation liquid, the alcohol content of the base wine is not less than 50°, and the alcohol content of the tail wine is 10°-50°.

[0019] Preferably, in step (2), during the acid esterification process, a liquid / immobilized lipase or an enzyme / microorganism capable of esterification is added.

[0020] Preferably, in step (3), when the remaining liquid after physical separation is returned to the hexanoic acid fermentation system in step (1), for the route of directly producing hexanoic acid with lactic acid as substrate, the amount of alkaline regulator used in step (1) is 0.3%-1.0% of the mass or volume of the liquid entering the fermentation system, and for most cycles it is 0.5%-1.0%.

[0021] Preferably, in step (3), while recirculating the remaining liquid after physical separation, 10%-30% of the volume of the yellow fermentation liquid containing hexanoic acid is discharged to maintain the sodium ion concentration in the hexanoic acid fermentation system below 10 g / L and the total COD below 200 g / L; the discharged yellow fermentation liquid containing hexanoic acid is used for pit mud cultivation, returned to the pit, or used for downstream hexanoic acid extraction.

[0022] The present invention has the following beneficial effects:

[0023] (1) The present invention constructs a closed-loop process for yellow water by “fermentation-acidification of concentrated liquid-acid esterification-physical separation-recirculation of separation residue”, which is different from the one-way utilization mode of the existing yellow water esterification technology for making flavored wine.

[0024] (2) The present invention adopts the route of producing hexanoic acid with lactic acid as substrate, which can reduce the amount of alkaline regulator compared with the route with ethanol as substrate.

[0025] (3) In this invention, the remaining liquid after physical separation is returned to the hexanoic acid fermentation system, and the residual lactic acid, sugar and other fermentable substrates, organic acid salts and buffer components are recycled to further reduce the amount of alkaline regulator used in subsequent cycles and continue to support fermentation. The amount of sodium bicarbonate added under non-circulation conditions is generally 3%-6% of the mass or volume of acidic yellow water. The specific amount is determined according to the initial acidity of different batches of yellow water. No other preferred amount is set in this range. The amount of sodium bicarbonate added in the return circulation round is 0.3%-1.0%, of which the lowest round is 0.3% and most circulation rounds are 0.5%-1.0%, thereby forming a clear alkali consumption reduction effect.

[0026] (4) This invention addresses the industry's technical bias that "reflux can easily lead to the accumulation of inhibitory substances and harm bacteria." During reflux, 10-30% of the fermentation liquid containing hexanoic acid is discharged according to the volume of the fermentation liquid. The sodium ion concentration in the hexanoic acid fermentation system is controlled below 10 g / L and the total COD is controlled below 200 g / L, so that the inhibitory substances are always kept below the inhibition threshold. After at least 22 rounds of continuous fermentation-acidification-esterification-separation cycle verification using distillation as the physical separation method, the hexanoic acid generation in the system is stable, the alkali consumption is significantly reduced, and there is no obvious inhibition instability. This breaks through the technical bottleneck that traditional processes cannot operate in a closed loop for a long time. The discharged fermentation liquid containing hexanoic acid can also be used for pit mud cultivation, refluxed to the pit, or used for downstream hexanoic acid extraction.

[0027] (5) The present invention uses concentrated yellow water as acidification liquid, which can reduce the use of exogenous acidity regulators; the obtained product can be used as raw material for the preparation of flavoring components containing ethyl hexanoate, strong aroma baijiu flavoring components or flavoring wine, realizing the synergistic utilization of yellow water resources and the preparation of baijiu flavor substances.

[0028] (6) After the self-circulation begins, no fresh (dilute) yellow water is added to the system. The fermentation substrate is usually provided by the refluxed separation residue. The concentrated yellow water is mainly used for acid adjustment before esterification and is generally not added directly at the fermentation end. However, under certain operating conditions, a portion of the concentrated yellow water can be added according to the substrate and acidity requirements. Through the dynamic balance between substrate supply, salt discharge and product concentration, the hexanoic acid concentration is maintained above 12 g / L during the steady-state phase of the cycle, meeting the company's demand for hexanoic acid. Attached Figure Description

[0029] Figure 1 A schematic diagram of the process for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water. Detailed Implementation

[0030] 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.

[0031] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0032] This invention addresses common challenges in existing one-way resource utilization processes for yellow water, such as the easy accumulation of inhibitory substances, mismatched conditions among multiple process units, and easy instability during cyclic operation. By combining hexanoic acid fermentation, yellow water source acidification, acid esterification, physical separation, and partial reflux of the separation residue, a multi-unit synergistic dynamic equilibrium system is constructed. This system achieves control over the accumulation of inhibitory substances and has been verified to be stable through multiple rounds of continuous cycling, thus breaking through the technical bottleneck of traditional processes that cannot operate in a closed loop for a long time.

[0033] Unlike existing technologies that only utilize the fermented yellow liquid as a raw material for flavoring wine, the core breakthrough of this invention lies in:

[0034] To overcome the industry's common concern that direct reflux of separation residue can easily lead to the accumulation of inhibitory substances, this method replaces exogenous acidifiers with acidified yellow water source, diverts and utilizes hexanoic acid bacteria solution and partially refluxes separation residue, and discharges 10-30% (preferably 20%) of hexanoic acid-containing fermentation broth per round based on the volume of fermentation broth (the discharged fermentation broth can be used for pit mud cultivation, returned to the pit, or used for downstream hexanoic acid extraction). This controls the sodium ion concentration in the system below 10 g / L, the total COD below 200 g / L, and the conductivity below 40 ms / cm, maintaining the acidity, salinity, organic acids, and bacterial activity in the system within suitable ranges, thus achieving inhibitory substance accumulation control from the process pathway.

[0035] The synergistic matching of different conditions required for hexanoic acid fermentation, acid esterification, and pit mud cultivation establishes a dynamic balance between lactic acid conversion, hexanoic acid synthesis, ethanol esterification, acid-base buffering, and microbial metabolism. This is not a simple material reflux, but a steady-state regulation with multiple parameters and objectives.

[0036] Through multiple rounds of continuous fermentation-acidification-esterification-separation cycle verification using distillation as the physical separation method, the system showed stable hexanoic acid production, controllable esterification efficiency, significantly reduced alkaline regulator dosage, and no obvious inhibition of instability, forming a new closed-loop resource utilization process for yellow water that can operate stably for a long time.

[0037] This invention upgrades yellow water from unidirectional discharge and low-value utilization to closed-loop circulation, steady-state transformation, and high-value utilization. It truly solves the fermentation instability problem caused by the accumulation of salt, organic acids, and inhibitory substances in multiple cycles, and has significant technical originality and industrial application value.

[0038] refer to Figure 1 As shown, this invention provides a method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water, comprising the following steps:

[0039] (1) Mix the acidic yellow water with the alkaline regulator, adjust the pH, and then put it into the hexanoic acid fermentation tank;

[0040] In a preferred embodiment, the initial pH of the acidic yellow water is 3.0-4.5. An alkaline regulator is added based on the initial acidity of the yellow water to adjust the pH to 4-7, preferably 5.5. Based on sodium bicarbonate, the amount of alkaline regulator added in the first round or under non-circulating conditions is generally 3%-6% of the mass or volume of the acidic yellow water. Since the initial acidity varies between different batches of yellow water, no preferred addition amount is specified within this range; the actual amount used is based on adjusting the yellow water to the target pH. When using other alkaline regulators, the equivalent alkalinity is used for conversion. After pH adjustment, the solution enters the hexanoic acid fermenter. In the circulation operation after the residual liquid is refluxed, the amount of sodium bicarbonate added is 0.3%-1.0% of the mass or volume of the liquid entering the fermentation system, with the lowest round being 0.3% and most circulation rounds being 0.5%-1.0%.

[0041] The alkalinity regulator is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium carbonate, calcium bicarbonate, and calcium hydroxide, preferably sodium bicarbonate.

[0042] (2) Introduce a domesticated complex of bacteria that produce hexanoic acid using lactic acid as a substrate into the hexanoic acid fermentation tank, and carry out hexanoic acid fermentation under anaerobic conditions to obtain a yellow fermentation liquid containing hexanoic acid.

[0043] In a preferred embodiment, substrates such as lactic acid in yellow water are converted into hexanoic acid and its salts through carbon chain elongation.

[0044] The domesticated complex microbial community is a complex microbial community obtained by enriching and domesticating one or more of the following: yellow water, pit mud, pit fermentation products, and yellow water fermentation liquid containing hexanoic acid; or it can be isolated from pits, or it can be commercially available hexanoic acid fermentation complex microbial community, or natural hexanoic acid synthesis functional mixed / pure microbial community, or genetically modified microbial community.

[0045] This experiment used a highly efficient mixed microbial community of caproic acid produced from pit mud through adaptive evolution, in which lactic acid-producing caproic acid bacteria had an average relative abundance of about 65%. The remaining bacteria were common in yellow water, such as lactic acid bacteria, acetic acid bacteria, and fungi such as yeast and mold, and sometimes methanogens were also included. The abundance of these bacteria varied depending on the type of yellow water.

[0046] (3) Add yellow water source acidification solution to the yellow water fermentation broth containing hexanoic acid and adjust its pH to 3.0-4.5, preferably 3.0-4.0; the amount of yellow water source acidification solution added is based on adjusting the pH of the yellow water fermentation broth containing hexanoic acid to 3.0-4.5.

[0047] The acidified yellow water source solution is a concentrated yellow water solution and / or a concentrated lactic acid solution obtained by membrane filtration of yellow water. The concentrated yellow water solution is an acidic concentrate obtained by concentrating yellow water 5-10 times, i.e., an acidic concentrate obtained by concentrating yellow water 5-10 times through evaporation, membrane concentration, or other food-processing acceptable methods. The concentrated lactic acid solution obtained by membrane filtration of yellow water is an acidic concentrate rich in lactic acid obtained by nanofiltration separation of yellow water. Both can be used as acidified solutions before esterification, replacing exogenous acidifiers such as citric acid, lactic acid, hydrochloric acid, and sulfuric acid.

[0048] (4) Without adding exogenous acidifiers, the acidified fermentation broth obtained in step (3) is subjected to acid esterification, and the ethyl hexanoate-containing component is extracted from the acid esterification system using physical separation methods; the acid esterification temperature is 20-60℃, and the esterification time is 5-72h. The physical separation methods include, but are not limited to, distillation, rectification, steam distillation, extraction, membrane separation, phase separation, or a combination of the above methods, preferably distillation or rectification. Distillation is atmospheric distillation or vacuum distillation. The atmospheric distillation can be carried out using direct or indirect steam heating under atmospheric or slightly positive pressure conditions to collect the condensed component containing ethyl hexanoate. The vacuum distillation is carried out under conditions below ambient pressure, with a preferred temperature of 50-70°C. The operating pressure can be determined based on the boiling point, composition, and equipment capacity of the acidic esterification system. When expressed in absolute pressure, it is preferably 10-30 kPa. The steam distillation can be carried out by direct contact between steam and the acidic esterification system, allowing the ethyl hexanoate-containing component to be distilled out along with the steam. The rectification can be carried out using a plate column, packed column, or other rectification device, operating in an intermittent or continuous manner. Other physical separation methods such as extraction, membrane separation, and phase separation can be performed based on the distribution, permeation, or phase separation characteristics of ethyl hexanoate and other components in the system. The specific operation methods are existing technologies and will not be described in detail here.

[0049] During acidic esterification, liquid or immobilized lipase, or related esterifying bacteria / enzymes, need to be added to carry out the esterification step. It should be noted that in this invention, the ethanol required for esterification comes from endogenous ethanol in yellow water, yellow water fermentation broth containing hexanoic acid, or acidified yellow water source broth. When it is necessary to improve esterification efficiency, one or more of edible alcohol, base wine, and tails can be added. The amount of edible alcohol added is 5%-30% of the volume of the acidified fermentation broth, the alcohol content of the base wine is not less than 50°, and the alcohol content of the tails is 10°-50°. This added ethanol is not an exogenous acidifier.

[0050] The exogenous acidifier mentioned here is one or more of citric acid, lactic acid, acetic acid, hydrochloric acid, sulfuric acid, and phosphoric acid. Not adding an exogenous acidifier means not adding the above-mentioned exogenous acidifiers, but using lactic acid and other acidic components in the yellow water source acidification solution for acidification.

[0051] (5) The remaining liquid after physical separation in step (4) is returned to the hexanoic acid fermentation system in step (2) for continued fermentation; the above steps (1)-(5) are repeated. While returning the remaining liquid after physical separation, 10%-30% (preferably 20%) of the volume of the yellow water fermentation liquid containing hexanoic acid in step (2) is discharged to keep the sodium ion concentration in the hexanoic acid fermentation system below 10 g / L, the total COD below 200 g / L, and the conductivity below 40 ms / cm; the discharged yellow water fermentation liquid containing hexanoic acid is used for pit mud cultivation, returned to the pit, or used for downstream hexanoic acid extraction.

[0052] The remaining liquid is the liquid remaining after the extraction of the ethyl hexanoate component by physical separation method in step (4). The remaining liquid contains unseparated organic acid salts, buffer components, micronutrient components and water-soluble organic matter.

[0053] In the recirculated hexanoic acid fermentation after the residual liquid is refluxed, for routes that directly produce hexanoic acid using lactic acid as a substrate, the amount of alkaline regulator added is 0.3%-1.0% of the mass or volume of the liquid entering the fermentation system, and 0.5%-1.0% in most recirculation cycles. The fermentation system liquid here mainly refers to the refluxed separation residue; after the start of the recirculation, no fresh (diluted) yellow water is added to the system. The fermentation substrate is usually provided by the refluxed separation residue, and approximately 10% water can be added to maintain volume balance. Under certain operating conditions, a portion of concentrated yellow water can be added to the fermentation system according to substrate and acidity requirements.

[0054] The method disclosed in this invention can perform at least two fermentation-acidification-esterification-physical separation cycles, either continuously or semi-continuously, and the amount of alkaline regulator used in the second and subsequent cycles is lower than that in the first cycle.

[0055] In a preferred embodiment, after the cycle begins, no fresh (diluted) yellow water is added to the system. The fermentation substrate is typically provided by the refluxed separation residue, with approximately 10% water added to maintain volume balance. The concentrated yellow water is mainly used as the yellow water source acidification solution before esterification and is generally not added directly at the fermentation end. Under certain operating conditions, a portion of the concentrated yellow water can be added to the fermentation system according to substrate and acidity requirements. By refluxing the separation residue, partially discharging the fermentation broth, and adding concentrated yellow water when necessary, a dynamic balance is established between substrate supply, hexanoic acid-producing bacterial growth, and product concentration. On the one hand, the substrate supply sufficiently supports microbial growth; on the other hand, the hexanoic acid concentration is maintained above 12 g / L during the steady-state cycle, meeting the enterprise's hexanoic acid requirements. Since fresh dilute yellow water is typically not added during the cycle and the system is not diluted by low-concentration yellow water, it is beneficial to maintain a high product concentration. This closed-loop operation places high demands on the balance between substrate supply, salt discharge, and product concentration, which is key to achieving long-term stable closed-loop operation in this invention.

[0056] The method disclosed in this invention does not include the steps of enriching hexanoic acid with macroporous adsorption resin and eluting hexanoic acid with ethanol.

[0057] The method provided by this invention can be used in the resource recovery of yellow water. It can also be used in the preparation of ethyl hexanoate from yellow water. Furthermore, it can be used in the preparation of flavoring fractions or flavoring wines of strong-aroma baijiu.

[0058] The present invention will be further described below with reference to specific embodiments.

[0059] Example 1: First-round fermentation of yellow water with hexanoic acid and acidification and esterification of concentrated yellow water.

[0060] The steps are as follows:

[0061] (1) Acidic yellow water is used as raw material, and the initial pH of the acidic yellow water is 3.0-4.5. Sodium bicarbonate is added according to the initial acidity of the batch of acidic yellow water and mixed evenly to adjust the pH of the acidic yellow water to 4-7, preferably 5.5. The amount of sodium bicarbonate added in the first round is generally 3%-6% of the mass or volume of the acidic yellow water; since the initial acidity of different batches of yellow water is different, no additional preferred amount is set in this range, and the actual amount used is based on adjusting the batch of yellow water to the target pH.

[0062] (2) The adjusted yellow water was transferred to a hexanoic acid fermentation tank, and a domesticated complex of bacteria that produce hexanoic acid using lactic acid as a substrate was introduced. Hexanoic acid fermentation was carried out under anaerobic conditions. After fermentation, a yellow water fermentation broth containing hexanoic acid was obtained.

[0063] (3) Drain the fermentation broth and add concentrated yellow water solution obtained by concentrating yellow water 5-10 times to adjust the pH of the fermentation broth to 3.0-4.0. Do not add exogenous acidifiers such as citric acid, lactic acid, hydrochloric acid, or sulfuric acid during the acidification process.

[0064] (4) Add liquid lipase to the acidified fermentation broth at a ratio of 1.5‰ to carry out acid esterification at a temperature of 30-40°C for 24 hours. Collect the fraction containing ethyl hexanoate by atmospheric or vacuum distillation. Collect the remaining liquid after distillation for use as reflux in the next round of hexanoic acid fermentation.

[0065] In this embodiment, the original yellow water contained 60 g / L lactic acid, 30 g / L ethanol, 0.2 g / L hexanoic acid, and 205 g / L total COD. After hexanoic acid fermentation, the hexanoic acid concentration in the fermentation broth was 14.5 g / L. When adding concentrated yellow water for acidification, the proportion of concentrated yellow water added was 10% of the fermentation broth volume. After acid esterification and distillation, the concentration of ethyl hexanoate in the distillate fraction was 38 g / L. The above concentration of ethyl hexanoate can be obtained from the gas chromatographic peak table by adjusting the sample dilution factor.

[0066] Example 2: Recirculating the distillate to reduce sodium bicarbonate usage

[0067] The steps are as follows:

[0068] (1) Take the remaining liquid after distillation in Example 1 and reflux it into the hexanoic acid fermentation tank; from this round onwards, no fresh (dilute) yellow water will be added. The fermentation substrate is usually supplied by the refluxed distillation residue, and about 10% water is added to maintain volume balance; under individual operating conditions, some concentrated yellow water can be added according to the substrate and acidity requirements. According to the acidity of the reflux liquid, add 1.0% sodium bicarbonate according to the mass or volume of the reflux liquid.

[0069] (2) Introduce or retain a domesticated complex microbial community that produces hexanoic acid using lactic acid as a substrate, and continue hexanoic acid fermentation under anaerobic conditions to obtain yellow fermentation broth containing hexanoic acid.

[0070] (3) After fermentation, the fermentation broth is discharged, and concentrated yellow water is added to adjust the pH to 3.0-4.0. Then, acid esterification and distillation are carried out without the addition of exogenous acidifiers, and the fraction containing ethyl hexanoate is collected. The remaining distillate is refluxed to the next round of fermentation.

[0071] (4) During the above-mentioned reflux cycle, approximately 20% of the fermentation broth containing hexanoic acid is discharged each round, controlling the sodium ion concentration in the hexanoic acid fermentation system to below 10 g / L and the total COD to below 200 g / L, thereby maintaining the inhibitory substances below the inhibition threshold. The discharged hexanoic acid-containing fermentation broth is used for pit mud cultivation, refluxed back to the pit, or used for downstream hexanoic acid extraction. It is precisely because this discharge operation avoids the accumulation of salt and inhibitory substances to the inhibition threshold from the fermentation end that the reflux cycle described in this embodiment can run continuously and stably for more than 22 rounds without significant inhibition instability.

[0072] Representative cyclic fermentation data show that in 1-22 rounds of reflux fermentation, the lactic acid concentration in the reflux liquid was 47.3-64.8 g / L; the sodium bicarbonate addition was 0.3%-1.0%, with the lowest round at 0.3% and most rounds at 0.5%-1.0%. The hexanoic acid concentration in the later stages of each fermentation round was approximately 12.4-15.8 g / L. This result indicates that under the condition of distillation residue reflux, the cyclic fermentation stage can maintain hexanoic acid production with a relatively low sodium bicarbonate addition. After acidic esterification and distillation, the ethyl hexanoate fraction concentration under atmospheric distillation typically reaches above 10 g / L, while under vacuum distillation, the average ethyl hexanoate fraction concentration typically reaches 30-40 g / L, with the head fraction reaching 100 g / L.

[0073] Example 3: Acidification and esterification of concentrated lactic acid solution after nanofiltration of yellow water

[0074] The steps are as follows:

[0075] (1) Take acidic yellow water with an initial pH of 3.0-4.5, add sodium bicarbonate according to its initial acidity, and adjust the pH of the acidic yellow water to 5-6, preferably 5.5; the amount of sodium bicarbonate added in the first round is generally 3%-6% of the mass or volume of the acidic yellow water, and the specific amount is determined according to the initial acidity of the batch of yellow water. Then, introduce a domesticated complex bacterial group that produces hexanoic acid with lactic acid as a substrate, and carry out hexanoic acid fermentation under anaerobic conditions to obtain yellow water fermentation broth containing hexanoic acid. Take another batch of yellow water for nanofiltration treatment, collect the nanofiltration concentrate rich in lactic acid, and use it as the yellow water source acidification liquid.

[0076] (2) Add the concentrated lactic acid solution from the nanofiltration of yellow water to the fermentation broth containing hexanoic acid, and adjust the pH to 3.0-4.0. Do not add exogenous acidifiers such as citric acid, lactic acid, hydrochloric acid, or sulfuric acid during the acidification process. After acidification, perform acid esterification and distillation, collect the fraction containing ethyl hexanoate, and reflux the remaining distillate to the hexanoic acid fermentation system.

[0077] This embodiment illustrates that nanofiltration lactic acid concentrate from yellow water can replace yellow water concentrate as an acidifying solution and can be used to construct a closed-loop process of yellow water hexanoic acid fermentation-esterification distillation-residue reflux. Representative data show that the lactic acid concentration in yellow water source lactic acid concentrate or distillation residue can reach approximately 80-400 g / L, which can be used as an acid-adjusting solution before esterification. After adjusting the pH using this type of acidifying solution, acidic esterification and distillation are carried out. Under normal pressure distillation, the concentration of ethyl hexanoate fraction can usually reach above 10 g / L, and under reduced pressure distillation, the average concentration of ethyl hexanoate fraction can usually reach 30-40 g / L, with the concentration of ethyl hexanoate in the head fraction reaching 100 g / L.

[0078] Example 4: Preparation of hexanoic acid by combined fermentation of lactic butyric acid bacteria and alcohol hexanoic acid bacteria (ethanol-type hexanoic acid production route)

[0079] This embodiment is used to illustrate that the hexanoic acid-producing domestication complex microbial community of the present invention is not limited to microbial communities that directly produce hexanoic acid using lactic acid as a substrate (i.e., lactic acid hexanoic acid bacteria). A complex microbial community combination that undergoes carbon chain elongation via butyric acid intermediate can also be used. Similarly, the stable preparation of hexanoic acid and ethyl hexanoate can be achieved in the closed-loop process of "fermentation-acidification-esterification-physical separation-reflux" described in the present invention.

[0080] The steps are as follows:

[0081] (1) Acidic yellow water is used as raw material, and the initial pH of the acidic yellow water is 3.0-4.5. Sodium bicarbonate is added according to the initial acidity of the acidic yellow water and mixed evenly to adjust the pH of the acidic yellow water to 4-7, preferably 5.5; the amount of sodium bicarbonate added in the first round is generally 3%-6% of the mass or volume of the acidic yellow water, and the specific amount is determined according to the initial acidity of the batch of yellow water, and then it is put into the hexanoic acid fermentation tank.

[0082] (2) Introduce a domesticated complex of bacteria, mainly lactic acid butyric acid bacteria (which produce butyric acid using lactic acid as a substrate) and alcohol hexanoic acid bacteria (which produce hexanoic acid using ethanol, acetic acid, and butyric acid as substrates through carbon chain elongation), and carry out hexanoic acid fermentation under anaerobic conditions: lactic acid in yellow water is first converted into butyric acid by lactic acid butyric acid bacteria, and butyric acid is then converted into hexanoic acid by carbon chain elongation of alcohol hexanoic acid bacteria with ethanol in yellow water, thus obtaining yellow water fermentation broth containing hexanoic acid.

[0083] (3) After fermentation, the fermentation liquid is discharged, and the pH is adjusted to 3.0-4.0 by adding concentrated yellow water (or concentrated lactic acid after nanofiltration of yellow water). Acid esterification and distillation are carried out without adding exogenous acidifiers, and the fraction containing ethyl hexanoate is collected. The remaining distillate is refluxed to the next round of hexanoic acid fermentation system.

[0084] (4) In the cycle of distillation residue reflux, fresh (dilute) yellow water will no longer be added from this round onwards. The fermentation substrate is supplied by the refluxed distillation residue (with yellow water concentrate if necessary), and about 10% water is added to maintain volume balance. About 20% of the fermentation liquid containing hexanoic acid is discharged in each round according to the fermentation liquid volume, and the sodium ion of the system is controlled below 10 g / L and the total COD is controlled below 200 g / L.

[0085] In this embodiment, the concentration of hexanoic acid obtained is similar to that in Examples 1-2. The concentration of hexanoic acid in the fermentation broth during the later stage of fermentation is approximately 12-16 g / L. After acid esterification and distillation, the concentration of ethyl hexanoate fraction is similar to that in Examples 1-2. It should be noted that since the hexanoic acid production step in this combination uses ethanol as the main substrate, the required amount of alkaline regulator is relatively high: the actual amount of sodium bicarbonate used in the first round of this embodiment is 4.0%-5.0%, which is the actual data of this embodiment and does not constitute a fixed percentage limit; the actual amount used in the circulating wheel after the distillation residue is 3.0%-4.0%.

[0086] Example 5: Preparation of hexanoic acid by combined fermentation of ethanol butyric acid bacteria and lactic acid hexanoic acid bacteria (lactate-type hexanoic acid production route)

[0087] This embodiment illustrates that a domesticated composite microbial community, mainly composed of ethanol butyric acid bacteria (producing butyric acid from ethanol) and lactic acid hexanoic acid bacteria (producing hexanoic acid from lactic acid), can also stably prepare hexanoic acid and ethyl hexanoate in the closed-loop process described in this invention.

[0088] The steps are as follows:

[0089] (1) Acidic yellow water is used as raw material, and the initial pH of the acidic yellow water is 3.0-4.5. Sodium bicarbonate is added according to the initial acidity of the acidic yellow water and mixed evenly to adjust the pH of the acidic yellow water to 4-7, preferably 6.5; the amount of sodium bicarbonate added in the first round is generally 3%-6% of the mass or volume of the acidic yellow water, and the specific amount is determined according to the initial acidity of the batch of yellow water, and then it is put into the hexanoic acid fermentation tank.

[0090] (2) Introduce a domesticated complex of bacteria, mainly composed of ethanol butyric acid bacteria and lactic acid hexanoic acid bacteria, and carry out hexanoic acid fermentation under anaerobic conditions: the ethanol in the yellow water is first converted into butyric acid by ethanol butyric acid bacteria, and the lactic acid is then converted into hexanoic acid by lactic acid hexanoic acid bacteria (with butyric acid as needed) through carbon chain elongation, so as to obtain yellow water fermentation broth containing hexanoic acid.

[0091] (3) The acidification, esterification, distillation and reflux of the distillation residue after fermentation are the same as in Example 4. In the reflux cycle, about 20% of the fermentation liquid containing hexanoic acid is discharged each round according to the volume of the fermentation liquid to maintain the sodium ion and total COD of the system below the inhibition threshold.

[0092] In this embodiment, the concentration of hexanoic acid obtained is similar to that in Examples 1-2. Since the hexanoic acid production step of this combination uses lactic acid as the main substrate, the amount of alkaline regulator used is lower than that of the ethanol-type hexanoic acid production combination in Example 4: the actual amount of sodium bicarbonate used in the first round of this embodiment is 3.0%-4.0%, which is the actual data of this embodiment and does not constitute a fixed percentage limit; the actual amount used in the circulating wheel after the distillation residue is 2.5%-3.0%.

[0093] Example 6: Adding ethanol to improve the matching degree of esterification substrate

[0094] An acidified fermentation broth containing hexanoic acid was obtained according to Example 1 or Example 2. When the endogenous ethanol in the acidified fermentation broth was insufficient to meet the requirements for preparing the target ethyl hexanoate fraction, one or more of edible alcohol, base liquor, or tails were added to the acidified fermentation broth, followed by acid esterification and distillation. The amount of edible alcohol added could be 5%-30% of the volume of the acidified fermentation broth; the base liquor could be a low-quality base liquor with an alcohol content of 50° or higher; and the tails could be tails with an alcohol content of 10°-50°.

[0095] In this embodiment, the added ethanol is used to improve the matching degree of the esterification substrate, not to adjust the pH, and is not an exogenous acidifier. When using the above-described ethanol addition process, the esterification rate can reach over 60%; for example, without external ethanol addition, relying on the ethanol esterification of the yellow liquor itself, the esterification rate is between 50-68%; when 70° base liquor is added at a volume ratio of 5-20% to the fermentation liquid, the esterification rate can be increased to 60-85%; when tails are added at a volume ratio of 20-30% to the fermentation liquid, the esterification rate is 65-70%.

[0096] Under reduced pressure distillation conditions, the tails and hexanoic acid fermentation liquid are esterified, and the resulting ethyl hexanoate fraction typically reaches a concentration of around 20 g / L. Adding 70° base spirits increases the ethyl hexanoate concentration to between 30-50 g / L, with the head fraction reaching 110 g / L.

[0097] Esterification is performed using ethanol from the yellow water. After esterification, base wine / tails are added, and distillation is carried out using a bottom-pot steam distillation method. The ethyl hexanoate concentration is typically 9.8-15 g / L; if a tails extractor is used, the ethyl hexanoate concentration is typically 15-20 g / L; if specialized distillation equipment such as a distillation column is used, the ethyl hexanoate concentration is typically 30-40 g / L. This example is suitable for production scenarios where the endogenous ethanol content of the yellow water fluctuates greatly or where a higher esterification rate is required.

[0098] Comparative Example 1: Exogenous Citric Acid Esterification

[0099] Take the same hexanoic acid-containing yellow water fermentation broth as in Example 1, but without adding yellow water concentrate or yellow water nanofiltration lactic acid concentrate. Instead, use flake citric acid to adjust the pH of the fermentation broth to 3.0-4.0. The amount of flake citric acid added is 4%-5% of the fermentation broth volume. Subsequently, esterification distillation is carried out under the same temperature and distillation conditions, and the fraction is collected.

[0100] This comparative example is used to compare the effect of the acidified yellow water source solution of the present invention as a substitute for exogenous citric acid for acidification. Under the same distillation conditions, the ethyl hexanoate concentration obtained in this comparative example can be maintained at a similar level as in Example 1, but an additional 4%-5% of flake citric acid needs to be added, which cannot reflect the utilization value of the lactic acid component of the yellow water itself as an acidification resource.

[0101] Comparative Example 2: Reflux without distillation residue

[0102] Take acidic yellow water with an initial pH of 3.0-4.5, and add sodium bicarbonate according to the initial acidity of the yellow water, adjusting the pH of the acidic yellow water to 4-7, preferably 5.5, as described in Example 1. The amount of sodium bicarbonate added is generally 3%-6% of the mass or volume of the acidic yellow water, and the specific amount is determined according to the initial acidity of this batch of yellow water. Subsequently, hexanoic acid fermentation, acidification esterification, and distillation are carried out. The remaining liquid after distillation is not refluxed. The next round of fermentation still uses fresh acidic yellow water with an initial pH of 3.0-4.5, and sodium bicarbonate is added again, generally 3%-6%, to adjust the pH of the acidic yellow water to 4-7, preferably 5.5.

[0103] This comparative example illustrates that when no residual liquid is refluxed, each round using fresh acidic yellow water requires the addition of sodium bicarbonate, typically 3%-6%, depending on its initial acidity. In contrast, the sodium bicarbonate addition in the reflux circulation round of this invention is 0.3%-1.0%, with most circulation rounds using 0.5%-1.0%, which significantly reduces alkali consumption.

[0104] Comparative Example 3: Fixed high dose of sodium bicarbonate per round

[0105] In the unidirectional fermentation of Example 1, the amount of alkali used in each round is 3-6%. However, in the case of residual liquid reflux as in Example 2, the addition of 0.5%-1.0% sodium bicarbonate in each round is sufficient to maintain hexanoic acid fermentation, which further illustrates that the present invention can significantly reduce the amount of alkali regulator used by utilizing the reflux liquid.

[0106] This invention uses yellow water, a byproduct of baijiu (Chinese liquor) brewing, as the main raw material. It utilizes the yellow water's own lactic acid, ethanol, hexanoic acid precursors, and buffer components. Through hexanoic acid fermentation, yellow water source lactic acid acidification, acid esterification, physical separation, and recirculation of the separation residue, it achieves the preparation of hexanoic acid bacteria, the preparation of hexanoic acid-containing components, and the closed-loop resource utilization of yellow water. This method does not rely on macroporous resin adsorption and enrichment or ethanol elution processes; the pathway is simple and suitable for the resource utilization of yellow water and the preparation of flavoring fractions in baijiu enterprises.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water, characterized in that: Includes the following steps: (1) After adjusting the pH of the acidic yellow water to 4-7, a domesticated complex of bacteria that produce hexanoic acid is introduced and hexanoic acid fermentation is carried out under anaerobic conditions to obtain yellow water fermentation liquid containing hexanoic acid. (2) Add yellow water source acidification liquid to the yellow water fermentation broth containing hexanoic acid, adjust the pH value to 3.0-4.5 to obtain acidified fermentation broth, and then directly carry out acid esterification, and extract the hexanoic acid ethyl ester component from the acid esterification system by physical separation method; wherein, the yellow water source acidification liquid is yellow water concentrate and / or yellow water membrane filtration lactic acid concentrate; (3) The remaining liquid after physical separation in step (2) is returned to the hexanoic acid fermentation step in step (1) to continue fermentation, and the cycle of steps (1)-(3) is repeated to achieve continuous fermentation-acidification-esterification-physical separation.

2. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (1), the initial pH of the acidic yellow water is 3.0-4.5, and the pH value of the acidic yellow water is adjusted by an alkaline regulator, which is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium carbonate, calcium bicarbonate, and calcium hydroxide.

3. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 2, characterized in that: In step (1), the alkaline regulator adjusts the pH of the acidic yellow water to 5.

5.

4. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (1), the domesticated complex microbial community is a complex microbial community obtained by enriching and domesticating one or more of the following: yellow water, pit mud, pit fermentation products, and yellow water fermentation liquid containing hexanoic acid. Alternatively, it can be separated from fermentation pits, commercially available hexanoic acid fermentation compound bacteria, natural hexanoic acid synthesis functional mixed / pure bacteria, or genetically modified bacteria.

5. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 2, characterized in that: In step (2), the yellow water concentrate is an acidic concentrate obtained by concentrating yellow water 5-10 times; the lactic acid concentrate after yellow water membrane filtration is an acidic concentrate rich in lactic acid obtained by separating yellow water by nanofiltration.

6. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (2), the acid esterification temperature is 20-60℃ and the esterification time is 5-72h; the physical separation method includes one or more of distillation, rectification, steam distillation, extraction, membrane separation, and phase separation.

7. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (2), during the acid esterification process, one or more of the following are added: edible alcohol, base wine, and tail wine; the amount of edible alcohol added is 5%-30% of the volume of the acidified fermentation liquid, the alcohol content of the base wine is not less than 50°, and the alcohol content of the tail wine is 10°-50°.

8. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (2), during the acid esterification process, liquid / immobilized lipase, or enzyme / microorganism capable of esterification, is added.

9. A method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 2 or 3, characterized in that: In step (3), when the remaining liquid after physical separation is returned to the hexanoic acid fermentation system in step (1), for the route that directly produces hexanoic acid using lactic acid as a substrate, the amount of alkaline regulator used in step (1) is 0.3%-1.0% of the mass or volume of the liquid entering the fermentation system, and for most cycles it is 0.5%-1.0%.

10. The method for preparing ethyl hexanoate by closed-loop fermentation and esterification of yellow water according to claim 1, characterized in that: In step (3), while recirculating the remaining liquid after physical separation, 10%-30% of the volume of the yellow fermentation liquid containing hexanoic acid is discharged to maintain the sodium ion concentration in the hexanoic acid fermentation system below 10 g / L and the total COD below 200 g / L. The discharged yellow fermentation liquid containing hexanoic acid is used for pit mud cultivation, returned to the pit, or used for downstream hexanoic acid extraction.

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