A method for preparing a rum with a high total ester content

Abstract

This invention discloses a method for preparing rum with high total ester content. The method includes the following steps: pretreating molasses; preparing a first fermentation medium using the pretreated molasses; expanding the culture of fissile yeast seed liquid in the first fermentation medium to obtain fissile yeast fermentation broth; continuously adding the pretreated molasses to the fissile yeast fermentation broth, culturing to obtain a fermentation product, wherein the residual sugar content of the fermentation product is controlled at 0.5-2%; preparing a second fermentation medium using the pretreated molasses; inoculating the fermentation bacteria into the second fermentation medium, culturing to obtain a fermentation broth; the fermentation bacteria include at least one of Clostridium acetic acid and Acetobacter pasteurella; adding the fermentation broth to the fermentation product, culturing to obtain a mash; continuously distilling the mash, using a kettle heating method for three-part distillation, mixing the second fraction with oak shavings, and aging; obtaining the base spirit, which is then blended to obtain rum with high total ester content. This method can produce mash with high alcohol content and high ester content in a short time.

Description

Technical Field

[0001] This invention relates to the field of brewing, and in particular to a method for preparing rum with a high total ester content. Background Technology

[0002] Rum is the specific name for a distilled spirit made from sugarcane juice or molasses. Like whiskey made from grain and brandy made from grapes, sugarcane juice or molasses, after being treated with lime and heated, undergoes oxidation to produce a series of mixed aromas of sugarcane and sugar—the characteristic aroma of rum. Because esters are key components in the formation of rum's aroma, adding ester-rich microorganisms during fermentation will enhance the flavor of the spirit. Generally, the longer the ester carbon chain produced during fermentation or aging, the richer and more persistent the aroma. Internationally, there is a tradition of using fermentation processes different from conventional methods to produce extra-rich rum, known as flavored rum. Jamaica, for example, has a process for producing extra-rich ester rum through natural yeast fermentation (i.e., without added yeast, fermentation relies entirely on microorganisms in the air), resulting in a final product with an alcohol content of up to 86% and an ester content exceeding 1000 grams per hectoliter of ethanol. There are already a few patents in China related to rum production processes. For example, CN2012105308626 describes a method for producing blended rum from molasses. This method uses molasses as a carbon source, brewer's yeast as the fermentation strain, and adds tandoori and schine, prepared from by-products through anaerobic fermentation, as the main fermentation process. The preparation of the key raw materials, tandoori and schine, is relatively complex, with unclear composition and processes. It requires external addition at the initial stage of production and demands a high alcohol content, but does not have requirements for acid and ester content. Generally speaking, in the production of distilled spirits, aromatic substances in the fermented mash are more important than alcohol. CN2019101384105 relates to a method for improving the flavor compounds in rum. The key to the process is to treat molasses with a compound enzyme and microwave process, use multi-strain fermentation and double distillation to remove most of the undesirable aldehydes and higher alcohols. In the example, after aging for 2 years, the total acidity is 0.28-0.29 g / L and the total esters are 0.42-0.44 g / L. In this process, the molasses pretreatment conditions are not conducive to fully removing impurities and improving sugar utilization. At the same time, the distillation stage may lose a lot of aroma compounds. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing rum with a high total ester content.

[0004] The objective of this invention is achieved through the following technical solution: A method for preparing a rum with high total ester content includes the following steps: (1) Molasses is treated by hot acid method, then enzyme is added, stirred, centrifuged, and the supernatant is taken to obtain pretreated molasses; the enzyme includes at least one of amylase, pullulanase, glucanase and sucrose hydrolase. (2) Take the pretreated molasses obtained in step (1), dilute it to 20-40°Bx, add nutrients, and prepare the first fermentation medium; use the first fermentation medium to expand the culture of the fission yeast seed liquid to obtain the fission yeast fermentation broth. (3) Continuously add the pretreated molasses obtained in step (1) to the fission yeast fermentation broth obtained in step (2), culture, and continue culturing at 10-16℃ to obtain the fermentation product. The residual sugar content of the fermentation product is controlled at 0.5-2%. (4) Take the pretreated molasses obtained in step (1), add bleaching powder, let stand, take the upper sugar solution and dilute to 5-10°Bx, adjust the pH to 6-6.5 to obtain the second fermentation medium; inoculate the fermentation bacteria into the second fermentation medium, culture, and obtain the fermentation broth; the fermentation bacteria include at least one of Clostridium acetic acid and Acetobacter pasteurellium. (5) Add the fermentation liquid of the fermentation bacteria obtained in step (4) to the fermentation product obtained in step (3), cultivate it to obtain mash, and continuously distill the mash to obtain the first distillate. Dilute the first distillate to 40° and add it to the distillation kettle. Heat the kettle in a pot and reflux 1-1.5% for esterification. Distill in three parts. Take the cooled second fraction and mix it with oak shavings. Aged in a bourbon barrel. (6) After aging, the original wine is obtained, and after blending, a rum with a high total ester content is obtained.

[0005] Furthermore, the hot acid treatment of molasses described in step (1) is as follows: Add glutinous rice flour to molasses, then add concentrated sulfuric acid, heat and stir, adjust the pH to 4.5-5.5, and then add magnesium sulfate; Furthermore, the ratio of glutinous rice flour to molasses is 1–5 kg: 1000 L; Furthermore, the volume ratio of the concentrated sulfuric acid to molasses is 1-5:10000; Furthermore, the heating and stirring is performed by heating to 80-85°C and stirring for 4-8 hours; Furthermore, the pH adjustment is achieved by adding a sodium hydroxide solution; Furthermore, the final concentration of the magnesium sulfate is 0.05–0.2 g / L.

[0006] Furthermore, the concentration of molasses in step (1) is 45–60°Bx.

[0007] Further, the amount of enzyme added in step (1) is calculated as 0.2 to 40 U / mL of enzyme per 1 mL of molasses; even further, it is calculated as 0.2 to 10 U / mL of each enzyme per 1 mL of molasses.

[0008] Further, the enzymes mentioned in step (1) are amylase, pullulanase, glucanase and sucrose hydrolase; even further, the amount of enzymes added in step (1) is calculated as 0.2-10 U / mL amylase, 0.2-10 U / mL pullulanase, 0.2-10 U / mL glucanase and 0.2-10 U / mL sucrose hydrolase per 1 mL of molasses.

[0009] Furthermore, the stirring described in step (1) is for 2 to 4 hours.

[0010] Furthermore, the centrifugation described in step (1) is centrifugation at 5000-15000 rpm for 10-20 min.

[0011] Further, the dilution described in step (2) is a dilution to 30°Bx.

[0012] Furthermore, the nutrients in step (2) include at least one of (NH4)2SO4 and KH2PO4; Furthermore, the ratio of (NH4)2SO4 to diluted pretreated molasses is 1-10 kg: 1000 L; Furthermore, the ratio of KH2PO4 to diluted pretreated molasses is 2-20 kg: 1000 L.

[0013] Furthermore, the concentration of the fission yeast seed solution is 0.7 × 10⁻⁶. 7 ~0.9×10 9 Cells / mL; even further, 0.8 × 10⁻⁶. 8 per mL.

[0014] Furthermore, in the expanded culture described in step (2), the inoculation amount of each fission yeast culture is 20% to 30% of the fermentation medium volume, and the number of fermentation stages is two to five; even further, the inoculation amount of each fission yeast culture is 25% of the fermentation medium volume.

[0015] Furthermore, in the expanded culture described in step (2), after inoculation, aeration is stopped after 1 h of culture.

[0016] Further, in step (3), the total feed volume in the continuous feed is 0-20% of the volume of the fission yeast fermentation broth, excluding 0; even further, the total feed volume in the continuous feed is 20% of the volume of the fission yeast fermentation broth.

[0017] Furthermore, the cultivation described in step (3) is carried out for 36 to 60 hours.

[0018] Furthermore, the 10-16℃ incubation step (3) is continued for 12-24 h.

[0019] Furthermore, in step (4), the ratio of bleaching powder to pretreated molasses is 1-5 kg: 1000 L.

[0020] Furthermore, the settling time mentioned in step (4) is 6 to 18 hours.

[0021] Further, in step (4), the upper sugar solution is diluted to 7-8°Bx.

[0022] Furthermore, the pH adjustment mentioned in step (4) is to adjust the pH to 6.5.

[0023] Further, in step (4), the fermentation bacteria are inoculated into the second fermentation medium, and the concentration of the inoculated fermentation bacteria solution is 5 × 10⁻⁶. 5 ~5×10 7 The concentration of the inoculated fermentation bacteria is 5 × 10⁶ / mL, and the volume of the inoculated fermentation bacteria solution is 5–15% of the volume of the second fermentation medium; furthermore, the concentration of the inoculated fermentation bacteria solution is 5 × 10⁶ / mL. 6 The volume of the inoculated fermentation culture was 10% of the volume of the second fermentation medium.

[0024] Furthermore, the culture described in step (4) is carried out at 20-40℃ for 24-72 h; even further, it is carried out at 37℃ for 48 h.

[0025] Furthermore, the volume of the fermentation broth of the fermenting bacteria in step (5) is 3% to 10% of the volume of the fermentation product.

[0026] Furthermore, the culture described in step (5) is for 12 to 36 hours.

[0027] Furthermore, in the three-part distillation described in step (5), the first fraction accounts for 3% to 4% of the total fraction volume, and the second fraction accounts for 1% to 2% of the total fraction volume.

[0028] Compared with the prior art, the present invention has the following advantages and effects: the fermented mash can maintain a high alcohol yield, and under the condition of short fermentation time, the alcohol yield reaches 9-11°. The fermented mash has a high ester content, with total acid at 0.58-1.41 g / L and total ester at 0.25-0.42 g / L. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0030] Example 1 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 2 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 1.0 U of amylase, 1.0 U of pullulanase, 1.0 U of dextranase and 1.0 U of sucrase per 1 mL of molasses A, stir for 3 h, centrifuge at 5000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0031] 2. Fermentation using fission yeast: (1) Preparation of *Schizophyllum commune* seed culture: *Schizophyllum commune* (CCIC1056) lyophilized powder was dissolved in approximately 0.5 mL of YPD medium using a sterile pipette and transferred to a test tube containing 5 mL of medium. The mixture was then stirred, and 1-2 drops of the bacterial suspension were transferred to solid YPD medium (yeast extract peptone glucose medium, including 10 g / L yeast extract (purchased from Sangon Biotech (Shanghai) Co., Ltd.), 20 g / L peptone (purchased from Sangon Biotech (Shanghai) Co., Ltd.), 20 g / L glucose (purchased from Guangzhou Chemical Reagent Factory), and 15 g / L agar (Shanghai Aladdin Biochemical Technology Co., Ltd.). After incubation at 28℃ for 24 h, a single colony was picked and inoculated into liquid YPD medium (including 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose). The culture was incubated at 28℃ for 24 h to obtain the *Schizophyllum commune* seed culture, with a concentration of 0.8 × 10⁻⁶. 8 per mL.

[0032] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0033] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 25% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation liquid was obtained through four stages of fermentation.

[0034] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar of fermentation product A was controlled at 1.5%.

[0035] 4. Fermentation using *Acetobacter pasteurization*: (1) Preparation of fermentation medium for Acetobacter pasteurellii: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 7°Bx, adjust pH to 6.5, and obtain fermentation medium for Acetobacter pasteurellii.

[0036] (2) Preparation of Acetobacter pasteurellium fermentation broth: Acetobacter pasteurellium (CICC22762) bacterial culture (concentration of 5×10⁻⁶) 6 The inoculum was 10% of the volume of the *Acetobacter pasteurellium* fermentation medium (10 cells / mL) and incubated at 30°C for 48 hours to obtain the *Acetobacter pasteurellium* fermentation broth.

[0037] 5. Add 5% (by volume) of *Acetobacter pasteurization* fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 9%, total acidity of 0.58 g / L, and total ester content of 0.25 g / L. Perform stepwise distillation, first distilling the initial distillate (approximately 4-98%) in a continuous column, diluting to 40°C, and then refluxing at 1-1.5% in a kettle. Distill in three fractions: the first fraction (heads, approximately 4%), the third fraction (tails, approximately 2%), and the second fraction (main product). After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0038] 6. After aging for half a year, a mellow and fragrant base wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 1.21 g / L, a total acid content of 0.22 g / L, and a higher alcohol content of 0.08 g / L.

[0039] Example 2 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 5 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 10.0 U of amylase, 10.0 U of pullulanase, 10.0 U of dextranase and 10.0 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0040] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0041] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0042] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0043] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 1.0%.

[0044] 4. Fermentation using Clostridium acetic acid: (1) Preparation of Clostridium acetic acid fermentation medium: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 8°Bx, adjust pH to 6.5, and obtain Clostridium acetic acid fermentation medium.

[0045] (2) Preparation of Clostridium acetic acid fermentation broth: Clostridium acetic acid (DSM1946) bacterial culture (concentration of 5×10⁻⁶)6 The seed culture was inoculated into Clostridium acetate fermentation medium at a volume of 10% of the fermentation medium volume, and cultured at 37°C for 48 h to obtain Clostridium acetate fermentation broth.

[0046] 5. Add 5% (by volume) of Clostridium acetic acid fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 11%, total acidity of 1.41 g / L, and total ester content of 0.42 g / L. Perform stepwise distillation. In a continuous column, first distill off the initial distillate (approximately 4-98%), dilute to 40°, and then perform esterification reflux in a kettle at 1-1.5% before three-part distillation. The first fraction is the heads (approximately 3.5%), the third fraction is the tails (approximately 1.5%), and the second fraction is the main product. After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0047] 6. After aging for half a year, a mellow and fragrant base wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 1.88 g / L, a total acid content of 0.18 g / L, and a higher alcohol content of 0.02 g / L.

[0048] Example 3 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 1 kg:1000 L; then add 0.05% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 85°C and stir for 6 h; adjust pH to 4.2 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 0.2 U of amylase, 0.2 U of pullulanase, 0.2 U of dextranase and 0.2 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0049] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0050] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0051] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 37℃ and the culture was carried out for 72 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation liquid was obtained through four stages of fermentation.

[0052] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar of fermentation product A was controlled at 1.5%.

[0053] 4. Fermentation using *Acetobacter pasteurization*: (1) Preparation of fermentation medium for Acetobacter pasteurellii: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 8°Bx, adjust pH to 6.5 to obtain fermentation medium for Acetobacter pasteurellii.

[0054] (2) Preparation of Acetobacter pasteurellium fermentation broth: Acetobacter pasteurellium (CICC22762) bacterial culture (concentration of 5×10⁻⁶) 6 Inoculate the culture medium with 10% of the volume of the fermentation medium (10 cells / mL) of *Acetobacter pasteurellium* and incubate at 30°C for 48 hours to obtain the *Acetobacter pasteurellium* fermentation broth.

[0055] 5. Add 5% (by volume) of *Acetobacter pasteurization* fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 9%, total acidity of 0.88 g / L, and total ester content of 0.16 g / L. Perform stepwise distillation. In a continuous column, first distill the initial distillate (approximately 4-98%), dilute to 40°, and then perform esterification reflux in a kettle bath at 1-1.5%. Distill in three fractions: the first fraction (heads, approximately 3.5%), the third fraction (tails, approximately 1.5%), and the second fraction (main product). After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0056] 6. After aging for half a year, a mellow and fragrant base wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.82 g / L, a total acid content of 0.35 g / L, and a higher alcohol content of 0.08 g / L.

[0057] Comparative Example 1: Fermentation using only fission yeast 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 5 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 10.0 U of amylase, 10.0 U of pullulanase, 10.0 U of dextranase and 10.0 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0058] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0059] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0060] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0061] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 1.0%.

[0062] 4. Fermentation product A has a purity of 12%, total acidity of 0.4 g / L, and total esters of 0.1 g / L. It undergoes fractional distillation. In a continuous column, the initial distillate (approximately 4-98%) is distilled off, diluted to 40°C, and then refluxed in a kettle at 1-1.5% esterification before being distilled in three fractions. The first fraction is the heads (approximately 4%), the third fraction is the tails (approximately 1.5%), and the second fraction is the main product. The second fraction is cooled, mixed with oak shavings, and then aged in bourbon barrels.

[0063] 5. After aging for half a year, a mellow and fragrant base wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.86 g / L, a total acid content of 0 g / L, and a higher alcohol content of 0.76 g / L.

[0064] Comparative Example 2: Pretreatment without enzyme treatment 1. Molasses pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour, the ratio of glutinous rice flour to 50°Bx molasses is 5 kg: 1000 L; then add 0.01% concentrated sulfuric acid by volume of 50°Bx molasses, heat to 80℃ and stir for 4 h; add sodium hydroxide solution to adjust pH to 4.5, then add magnesium sulfate to make the final concentration of magnesium sulfate 0.1 g / L; molasses A is obtained.

[0065] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0066] (2) Preparation of fission yeast fermentation medium: Take molasses A, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses A is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses A is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0067] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0068] 3. Add concentrated molasses: Molasses A obtained in step 1 was continuously fed into the four-stage fermentation broth of fissile yeast. The total volume of molasses A fed into the broth was 20% of the volume of the fissile yeast fermentation broth. The broth was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 1.0%.

[0069] 4. Fermentation using Clostridium acetic acid: (1) Preparation of Clostridium acetic acid fermentation medium: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 8°Bx, adjust pH to 6.5, and obtain Clostridium acetic acid fermentation medium.

[0070] (2) Preparation of Clostridium acetic acid fermentation broth: Clostridium acetic acid (DSM1946) bacterial culture (concentration of 5×10⁻⁶) 6 (10% of the volume of the fermentation medium) was inoculated into the Clostridium acetate fermentation medium and cultured at 37°C for 48 h to obtain the Clostridium acetate fermentation broth.

[0071] 5. Add 5% (by volume) of Clostridium acetic acid fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 6.5%, total acidity of 0.18 g / L, and total ester content of 0.24 g / L. Perform stepwise distillation, first distilling the initial distillate (approximately 4-98%) in a continuous column, diluting to 40°C, and then refluxing at 1-1.5% in a kettle. Distill in three fractions: the first fraction (heads, approximately 4%), the third fraction (tails, approximately 1.5%), and the second fraction (main product). After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0072] 6. After aging for half a year, a mellow and fragrant base wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.58 g / L, a total acid content of 0 g / L, and a higher alcohol content of 0.25 g / L.

[0073] Comparative Example 3: Fermentation using brewer's yeast 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 5 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 10.0 U of amylase, 10.0 U of pullulanase, 10.0 U of dextranase and 10.0 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0074] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0075] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0076] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0077] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 1.0%.

[0078] 4. Fermentation using brewer's yeast: (1) Preparation of fermentation medium for brewing yeast: Take molasses B, add 0.03% of bleaching powder by volume of molasses B, let stand for 10 h, take the upper sugar solution and dilute to 20°Bx, adjust pH to 5.5 to obtain fermentation medium for brewing yeast.

[0079] (2) Preparation of brewing yeast fermentation broth: Brewing yeast (CICC 31154) bacterial culture (concentration of 5×10⁻⁶) 6 Inoculate the yeast fermentation medium with yeast cells / mL at a rate of 10% of the fermentation medium volume, and incubate at 30℃ for 48 h to obtain the yeast fermentation broth.

[0080] 5. Add 50% of the volume of brewer's yeast fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 4.8%, total acidity of 0.3 g / L, and total ester content of 0.1 g / L. Perform stepwise distillation, first distilling the initial distillate (approximately 4-98%) in a continuous column, diluting to 40°C, and then refluxing in a kettle at 1-1.5% for esterification before three-part distillation. The first fraction is the heads (approximately 4%), the third fraction is the tails (approximately 2%), and the second fraction is the main product. After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0081] 6. After aging for half a year, a light-smelling but lacking-character original wine is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.28 g / L, a total acid content of 0.31 g / L, and a higher alcohol content of 0.08 g / L.

[0082] Comparative Example 4: The final residual sugar content of fermentation product A was controlled at 4.0%. 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 5 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 10.0 U of amylase, 10.0 U of pullulanase, 10.0 U of dextranase and 10.0 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0083] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0084] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0085] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0086] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 20% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 4.0%.

[0087] 4. Fermentation using Clostridium acetic acid: (1) Preparation of Clostridium acetic acid fermentation medium: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 8°Bx, adjust pH to 6.5, and obtain Clostridium acetic acid fermentation medium.

[0088] (2) Preparation of Clostridium acetic acid fermentation broth: Clostridium acetic acid (DSM1946) bacterial culture (concentration of 5×10⁻⁶) 6 (10% of the volume of the fermentation medium) was inoculated into the Clostridium acetate fermentation medium and cultured at 37°C for 48 h to obtain the Clostridium acetate fermentation broth.

[0089] 5. Add 5% (by volume) of Clostridium acetic acid fermentation broth to fermentation product A obtained in step 3, and continue culturing for 12 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 6.2%, total acidity of 0.45 g / L, and total ester content of 0.25 g / L. Perform stepwise distillation, first distilling the initial distillate (approximately 4-98%) in a continuous column, diluting to 40°, and then refluxing in a kettle at 1-1.5% for esterification before three-part distillation. The first fraction is the heads (approximately 4%), the third fraction is the tails (approximately 2%), and the second fraction is the main product. After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0090] 6. After aging for half a year, a base wine with a balanced aroma and fruity fragrance is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.62 g / L, a total acid content of 1.17 g / L, and a higher alcohol content of 0.04 g / L.

[0091] Comparative Example 5: The feed volume was controlled at 40%. 1. Molasses Pretreatment: Dilute molasses to 50°Bx, add glutinous rice flour at a ratio of 5 kg:1000 L; then add 0.01% concentrated sulfuric acid (by volume of 50°Bx molasses), heat to 80°C and stir for 4 h; adjust pH to 4.5 with sodium hydroxide solution, then add magnesium sulfate to a final concentration of 0.1 g / L; molasses A is obtained. Take molasses A, add 10.0 U of amylase, 10.0 U of pullulanase, 10.0 U of dextranase and 10.0 U of sucrose hydrolase to each 1 mL of molasses A, stir for 6 h, centrifuge at 10000 rpm for 10 min, and collect the supernatant; molasses B is obtained.

[0092] 2. Fermentation using fission yeast: (1) Preparation of fission yeast seed culture: The concentration of 0.8 × 10⁻⁶ was prepared according to the method in Example 1. 8 Schizomyces yeast seed culture (number of cells / mL).

[0093] (2) Preparation of fission yeast fermentation medium: Take molasses B, dilute it to 30°Bx, add nutrients (in this example, add (NH4)2SO4 and KH2PO4, the ratio of (NH4)2SO4 to 30°Bx molasses B is 5 kg:1000 L, and the ratio of KH2PO4 to 30°Bx molasses B is 1 kg:100 L) to obtain fission yeast fermentation medium.

[0094] (3) Expanded culture: The fissilla seed liquid was inoculated into the fissilla fermentation medium. The inoculation amount of each fissilla seed liquid was 20% of the fermentation medium volume. The culture temperature was 28℃ and the culture was carried out for 24 h (aeration was stopped 1 h after inoculation during the culture process). In this example, the fissilla fermentation broth was obtained after four stages of fermentation.

[0095] 3. Add concentrated molasses: Molasses B obtained in step 1 was continuously added to the four-stage fermentation broth of fissile yeast. The total added volume of molasses B was 40% of the volume of the fermentation broth of fissile yeast. The mixture was then cultured for another 48 h. The temperature was then lowered to 16℃ and cultured for another 12 h to obtain fermentation product A. The final residual sugar content of fermentation product A was controlled at 1.0%.

[0096] 4. Fermentation using Clostridium acetic acid: (1) Preparation of Clostridium acetic acid fermentation medium: Take molasses B, add 0.03% of bleaching powder (by volume of molasses B), let stand for 10 h, take the upper sugar solution and dilute to 8°Bx, adjust pH to 6.5, and obtain Clostridium acetic acid fermentation medium.

[0097] (2) Preparation of Clostridium acetic acid fermentation broth: Clostridium acetic acid (DSM1946) bacterial culture (concentration of 5×10⁻⁶) 6 (Inoculate with 10% of the Clostridium acetate fermentation medium at a concentration of 1 / mL) and incubate at 37°C for 48 h to obtain the Clostridium acetate fermentation broth.

[0098] 5. Add 5% (by volume) of *Acetobacter pasteurization* fermentation broth to fermentation product A obtained in step 3, and continue culturing for 24 hours before transferring to a fermentation tank. The fermented mash has an alcohol content of 5.4%, total acidity of 0.29 g / L, and total ester content of 0.05 g / L. Perform stepwise distillation, first distilling the initial distillate (approximately 4-98%) in a continuous column, diluting to 40°C, and then refluxing at 1-1.5% in a kettle. Distill in three fractions: the first fraction (heads, approximately 4%), the third fraction (tails, approximately 2%), and the second fraction (main product). After cooling, the second fraction is mixed with oak shavings and then aged in bourbon barrels.

[0099] 6. After aging for half a year, a light-bodied base wine with floral and fruity aromas is obtained. It is then blended with distilled water to an alcohol content of 40% and bottled. The finished wine has a total ester content of 0.44 g / L, a total acid content of 0.88 g / L, and a higher alcohol content of 0.02 g / L.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a rum with high total ester content, characterized in that, Includes the following steps: (1) Molasses is treated by hot acid method, then enzyme is added, stirred, centrifuged, and the supernatant is taken to obtain pretreated molasses; the enzyme includes at least one of amylase, pullulanase, glucanase and sucrose hydrolase. (2) Take the pretreated molasses obtained in step (1), dilute it to 20-40°Bx, add nutrients, and prepare the first fermentation medium; use the first fermentation medium to expand the culture of the fission yeast seed liquid to obtain the fission yeast fermentation broth. (3) Continuously add the pretreated molasses obtained in step (1) to the fission yeast fermentation broth obtained in step (2), culture, and continue culturing at 10-16℃ to obtain the fermentation product. The residual sugar content of the fermentation product is controlled at 0.5-2%. (4) Take the pretreated molasses obtained in step (1), add bleaching powder, let stand, take the upper sugar solution and dilute to 5-10°Bx, adjust the pH to 6-6.5 to obtain the second fermentation medium; inoculate the fermentation bacteria into the second fermentation medium, culture, and obtain the fermentation broth; the fermentation bacteria include at least one of Clostridium acetic acid and Acetobacter pasteurellium. (5) Add the fermentation liquid of the fermentation bacteria obtained in step (4) to the fermentation product obtained in step (3), cultivate it to obtain mash, and continuously distill the mash to obtain the first distillate. Dilute the first distillate to 40° and add it to the distillation kettle. Heat the kettle in a pot and reflux 1-1.5% for esterification. Distill in three parts. Take the cooled second fraction and mix it with oak shavings. Aged in a bourbon barrel. (6) After aging, the original wine is obtained, and after blending, a rum with a high total ester content is obtained.

2. The method according to claim 1, characterized in that, The steps for treating molasses using the hot acid method described in step (1) are as follows: Add glutinous rice flour to molasses, then add concentrated sulfuric acid, heat and stir, adjust the pH to 4.5-5.5, and then add magnesium sulfate; The concentration of molasses mentioned in step (1) is 45–60°Bx; The amount of enzyme added in step (1) is calculated as 0.2 to 40 U / mL of enzyme per 1 mL of molasses.

3. The method according to claim 2, characterized in that, The ratio of glutinous rice flour to molasses is 1-5 kg: 1000 L; The volume ratio of concentrated sulfuric acid to molasses is 1-5:10000; The heating and stirring process involves heating to 80–85°C and stirring for 4–8 hours. The pH is adjusted by adding sodium hydroxide solution; The final concentration of the magnesium sulfate is 0.05–0.2 g / L.

4. The method according to claim 1, characterized in that, The enzymes mentioned in step (1) are amylase, pullulanase, glucanase and sucrose hydrolase; The stirring described in step (1) is for 2 to 4 hours; The centrifugation in step (1) is 5000-15000 rpm for 10-20 min.

5. The method according to claim 1, characterized in that, The dilution mentioned in step (2) is a dilution to 30°Bx; The nutrients in step (2) include at least one of (NH4)2SO4 and KH2PO4; The concentration of the fission yeast seed culture is 0.7 × 10⁻⁶. 7 ~0.9×10 9 cells / mL; In the expanded culture described in step (2), the inoculation amount of each stage of fission yeast liquid is 20% to 30% of the fermentation medium volume, and the number of fermentation stages is two to five. In the expanded culture described in step (2), aeration was stopped after 1 h of inoculation and culture was continued.

6. The method according to claim 5, characterized in that, The concentration of the fission yeast seed culture is 0.8 × 10⁻⁶. 8 cells / mL; In the expanded culture described in step (2), the inoculation amount of each fission yeast culture is 25% of the fermentation medium volume.

7. The method according to claim 1, characterized in that, In step (3), the total feed volume in the continuous feed is 0-20% of the volume of the fission yeast fermentation broth, excluding 0. The culture described in step (3) is for 36–60 h; The incubation at 10-16℃ in step (3) is to continue incubation for 12-24 h.

8. The method according to claim 1, characterized in that, In step (4), the ratio of bleaching powder to pretreated molasses is 1-5 kg: 1000 L; The settling time mentioned in step (4) is 6 to 18 hours; The step (4) involves diluting the upper sugar solution to a concentration of 7-8°Bx. The pH adjustment mentioned in step (4) is to adjust the pH to 6.5; In step (4), the fermentation bacteria are inoculated into the second fermentation medium, and the concentration of the inoculated fermentation bacteria solution is 5 × 10⁻⁶. 5 ~5×10 7 The volume of the inoculated fermentation culture was 5-15% of the volume of the second fermentation medium; The culture described in step (4) is carried out at 20-40℃ for 24-72 h.

9. The method according to claim 8, characterized in that, In step (4), the fermentation bacteria are inoculated into the second fermentation medium, and the concentration of the inoculated fermentation bacteria solution is 5 × 10⁻⁶. 6 The volume of the inoculated fermentation culture was 10% of the volume of the second fermentation medium.

10. The method according to claim 1, characterized in that, The volume of the fermentation broth of the fermenting bacteria mentioned in step (5) is 3% to 10% of the volume of the fermentation product; The culture described in step (5) is for 12 to 36 hours; In the three-part distillation described in step (5), the first fraction accounts for 3% to 4% of the total fraction volume, and the second fraction accounts for 1% to 2% of the total fraction volume.