Trulopsis torulosa ZZ4 and application thereof

Abstract

The invention discloses torulopsis sp. ZZ4 and application thereof, and relates to the technical field of microbial fermentation. The torulopsis torulopsis ZZ4 is preserved in the China General Microbiological Culture Collection Center on May 19, 2022, the preservation name is ZZ4, and the preservation number of the torulopsis torulopsis ZZ4 is CGMCC NO.24921. The torulopsis torulopsis ZZ4 is applied to seasoning fermentation, including fermentation of dark soy sauce, based on this, the torulopsis torulopsis ZZ4 is inoculated in the soy sauce mash fermentation process, squeezing is performed after fermentation, and semi-finished soy sauce is obtained. Adding cane sugar into the semi-finished soy sauce, performing temperature-controlled boiling, and performing cooling so as to obtain a dark soy sauce finished product; according to the method, the torulopsis toruloides ZZ4 is adopted for fermentation, and the sucrose is adopted for temperature-controlled boiling, so that the color plumpness and flavor level of the dark soy sauce are improved under the condition that no chemical caramel color is added, and the quality of the dark soy sauce is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to Torulopsis yeast ZZ4 and applications thereof. Background Art

[0002] Traditional dark soy sauce, an important condiment in Chinese cooking, is characterized by its deep brown appearance and rich color, imparted by the addition of caramel color. Currently, commercially available dark soy sauce generally relies on the addition of exogenous caramel color, such as those produced using conventional, ammonia, and sulfite methods. While these processes are highly effective in coloring, chemically synthesized caramel color carries potential health risks and runs counter to consumer demand for natural, healthy foods.

[0003] In recent years, with the upgrading of consumption and the increasing awareness of health, the market demand for "clean label" products has increased significantly. Consumers prefer condiments with simple ingredients and no chemical additives, while also placing higher demands on the naturalness and flavor complexity of traditional fermentation processes.

[0004] However, existing dark soy sauce production processes, while removing the chemical caramel color, struggle to reproduce its stable color and flavor through purely physical methods (such as high-temperature boiling). For example, while high-temperature boiling of simple sugars can produce natural coloring substances such as melanoidins, their color, antioxidant properties, and compatibility with the soy sauce base do not meet dark soy sauce quality standards, resulting in a dull product color or unbalanced flavor. Furthermore, traditional yeast used in soy sauce production suffers from low salt tolerance and ester production capacity, further limiting the quality improvement of soy sauce without the added chemical caramel color. Summary of the Invention

[0005] The present invention aims to solve the technical problem that the color and flavor of existing soy sauce without adding chemical caramel color still need to be improved. The purpose is to provide a Torulopsis yeast ZZ4 and its application. By using Torulopsis yeast ZZ4 for fermentation and coordinating with sucrose to control the temperature and cook, the color fullness and flavor level of dark soy sauce are improved without adding chemical caramel color, thereby improving the quality of dark soy sauce.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a Torulopsis sp. ZZ4, which was deposited in the General Microbiology Center of the China Microorganism Culture Management Committee on May 19, 2022, with the deposit name ZZ4 and the deposit number: CGMCC NO.24921.

[0008] The Torulopsis ZZ4 of the present invention has a strong total ester production capacity, which can reach 2-3 g / L, and has good salt tolerance.

[0009] Furthermore, the Torulopsis yeast ZZ4 is obtained by mutagenesis of CICC 1708.

[0010] The second object of the present invention is to provide an application of Torulopsis ZZ4, including application in condiment fermentation; specifically, using Torulopsis ZZ4 as a fermentation bacterium in the preparation process of condiments.

[0011] Furthermore, the condiment includes dark soy sauce, and specifically, in the preparation of the dark soy sauce, Torulopsis ZZ4 is used as a fermentation bacterium.

[0012] A third object of the present invention is to provide a method for preparing dark soy sauce, comprising the steps of inoculating Torulopsis ZZ4 during fermentation of a soy mash, squeezing the soy sauce after fermentation to obtain a semi-finished product, adding sucrose to the semi-finished product, controlling the temperature and boiling the soy sauce, and cooling the soy sauce to obtain a finished product.

[0013] This invention proposes a method for preparing dark soy sauce based on fermentation with Torulopsis ZZ4 and sucrose cooking. By inoculating the salt-tolerant Torulopsis ZZ4 yeast with high ester production, the raw materials undergo multi-stage deep fermentation, enhancing the biological activity of sucrase and promoting the enzymatic conversion of proteins and sugars, thereby generating natural pigment precursors (such as red yeast pigment and melanin) and non-volatile ester flavor substances. Simultaneously, combined with a temperature-controlled cooking process for sucrose, the Maillard reaction and caramelization reaction synergize to produce high-chroma, highly stable natural pigment substances. This method not only completely eliminates the addition of chemical caramel color, but also enhances the color richness, flavor depth, and nutritional benefits of the dark soy sauce through the combined effects of microbial metabolites and cooking products, thus meeting the consumer trend towards healthier and more natural products.

[0014] Furthermore, the method comprises the following preparation steps:

[0015] S1. Mixing the steamed soybeans and the fried wheat to obtain soy sauce koji;

[0016] S2, mixing the soy sauce koji and the brine in a mass volume ratio of 1g: (1.8-2.0)mL to obtain a soy sauce mash;

[0017] S3, fermenting the mash, and inoculating Torulopsis ZZ4 on the 20th to 35th day of fermentation;

[0018] S4, squeezing and extracting oil to obtain semi-finished soy sauce;

[0019] S5. Add sucrose with a mass concentration of 50-60% to the semi-finished soy sauce, control the temperature and boil it. After boiling, cool it to ≤15° C., let it settle for 3-7 days, and collect the supernatant to obtain the finished soy sauce.

[0020] The steamed soybeans and the fried wheat are mixed in a mass ratio of (5-9):1 to make koji, the temperature of the whole fermentation process is controlled at 18-22°C, and the fermentation time is 5-7 months, preferably 6 months.

[0021] The preparation method of the present invention provides a semi-finished soy sauce with amino acid nitrogen of 0.8-1.35 g / 100 mL and sucrase activity of ≥50 U / mL. The finished soy sauce has amino acid nitrogen of 0.6-0.9 g / 100 mL, a total ester content of 1.5-1.8 g / L, a red index of >6.0, a yellow index of >10, and a color rate controlled at 3500-6000 EBC. Compared with traditional dark soy sauce, the product quality is better.

[0022] Furthermore, the amino acid nitrogen content of the brine is 0.2-0.6 g / 100 mL, and the NaCl concentration is 21-22%.

[0023] Furthermore, the inoculation concentration of the Torulopsis yeast ZZ4 was 1×10 5 ~5×10 5 Pieces / g of soy sauce.

[0024] Furthermore, the boiling process is carried out in a gradient temperature control manner: the temperature is raised to 50-60°C for reaction, and when the sucrose concentration reaches 10-30g / L, the temperature is again raised to 120-130°C for boiling for 3-4h, and then the temperature is lowered for cooling.

[0025] The fourth object of the present invention is to provide a dark soy sauce prepared by the above method.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. The Torulopsis ZZ4 of the present invention has a strong total ester production capacity, which can reach 2-3 g / L, and has good salt tolerance.

[0028] 2. The present invention proposes a method for preparing dark soy sauce based on fermentation with Torulopsis ZZ4 and synergistic sucrose boiling. By inoculating the salt-tolerant Torulopsis ZZ4 yeast with high ester production, the raw materials undergo multi-stage deep fermentation, enhancing the biological activity of sucrase, promoting the enzymatic hydrolysis and conversion of proteins and sugars, and generating natural pigment precursors (such as red yeast pigment and melanin) and non-volatile ester flavor substances. Simultaneously, combined with the temperature-controlled boiling process of sucrose, the Maillard reaction and caramelization reaction synergistically generate high-chroma, highly stable natural pigment substances. This method not only completely eliminates the addition of chemical caramel color, but also enhances the color richness, flavor layering, and nutritional function of the dark soy sauce through the combined action of microbial metabolites and boiling products, thus meeting the consumer trend towards healthier and more natural products.

[0029] 3. The present invention adopts Torulopsis yeast ZZ4 and cooperates with sucrose to control the temperature and cook the soy sauce. The amount of sucrose added is controlled at 50-60%, and the cooking time is 3-4 hours. Under the condition of not adding chemical caramel color, the amino acid nitrogen of the finally obtained soy sauce product is 0.6-0.9 g / 100 mL, the total ester content is 1.5-1.8 g / L, the red index is greater than 6.0, the yellow index is greater than 10, and the color rate can reach 3500-6000 EBC. Compared with traditional dark soy sauce, the product quality is better. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments. Obviously, the schematic implementation modes of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0031] The following describes in detail embodiments of the Torulopsis yeast ZZ4 and its applications according to the present invention. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions may be omitted. This is done to avoid unnecessary redundancy and to facilitate understanding by those skilled in the art.

[0032] The "ranges" disclosed herein are defined in terms of lower and upper limits. A given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner may be inclusive or exclusive and may be arbitrarily combined, i.e., any lower limit may be combined with any upper limit to form a range.

[0033] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0034] Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0035] Unless otherwise specified, the terms "include" and "comprising" used in the present invention may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other substances not listed may also be included or that only the listed substances are included.

[0036] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0037] The technical solution of the present invention is further described in detail below with reference to the embodiments.

[0038] It should be noted that the experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art, unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0039] The detection method used in the embodiment is as follows:

[0040] Ammonia nitrogen detection refers to GB / T 18186.

[0041] The sucrose detection method refers to GB 5009.8.

[0042] The method for determining the activity of sucrase was based on the method established by Liu Zhongying et al. (Liu Zhongying, Wang Xiaoping, Lu Yang, He Yexin, Wang Xin, & Huang Taorui, 2022. Establishment of a method for simultaneous determination of sucrase and fructanase activity by ion chromatography-pulsed amperometry. Food Industry Science and Technology, 43(24), 8.).

[0043] The method for determining the total ester content of soy sauce is as follows:

[0044] Accurately draw 10.00mL of soy sauce sample after the above-mentioned dilution, in a 250mL conical flask, add 60mL freshly made distilled water, add 2~3 drops of phenolphthalein indicator, titrate to solution with 0.1mol / LNaOH standard solution and show light red, record the volume consumed, calculate total acid content in the soy sauce; Then accurately add 25.00mL of 0.1mol / L NaOH standard solution, reflux 0.5h (start timing when the 1st liquid drips after boiling) in a boiling water bath, carry out saponification, add 0.1mol / L H after the cooling SO4 standard solution 25.00mL, titrate to solution with 0.1mol / L NaOH standard solution and show light red, calculate the content of total esters in the soy sauce, and do reagent blank test simultaneously.

[0045] The methods for determining the red index, yellow index and color rate of soy sauce are as follows:

[0046]

[0047] Example 1

[0048] Comparison of performance between Torulopsis ZZ4 and CICC 1708

[0049] Torulopsis ZZ4 and CICC 1708 were cultured and inoculated into a culture medium to compare fermentation performance. The culture medium consisted of 25% soy sauce (ammonia nitrogen content 1.0 g / 100 mL), 3% glucose, and salt (added according to the final salt concentration in the culture medium). The pH was kept at a natural pH. Total ester content was measured after 5 days of incubation. As shown in Table 1, Torulopsis ZZ4 exhibited improved salt tolerance compared to CICC 1708, with total ester content increasing by 56% to 2.5 g / L.

[0050] Table 1. Comparison of performance of CZ2 and CICC 1708 strains

[0051]

[0052]

[0053] Example 2

[0054] A preparation method of dark soy sauce:

[0055] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25g / 100mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. CICC 1708 was added on the 25th day of fermentation. Fermentation was controlled throughout, maintaining a temperature of 20°C for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 60%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 4 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0056] Example 3

[0057] A preparation method of dark soy sauce:

[0058] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25 g / 100 mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. On the 25th day of fermentation, Torulopsis ZZ4 was added. Fermentation was controlled at 20°C throughout the fermentation process, and the fermentation lasted for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 60%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 4 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0059] Example 4

[0060] A preparation method of dark soy sauce:

[0061] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25 g / 100 mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. On the 25th day of fermentation, Torulopsis ZZ4 was added. Fermentation was controlled at 20°C throughout the fermentation process, and the fermentation lasted for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 50%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 4 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0062] Example 5

[0063] A preparation method of dark soy sauce:

[0064] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25 g / 100 mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. On the 25th day of fermentation, Torulopsis ZZ4 was added. Fermentation was controlled at 20°C throughout the fermentation process, and the fermentation lasted for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 40%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 4 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0065] Example 6

[0066] A preparation method of dark soy sauce:

[0067] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25 g / 100 mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. On the 25th day of fermentation, Torulopsis ZZ4 was added. Fermentation was controlled at 20°C throughout the fermentation process, and the fermentation lasted for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 50%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 3 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0068] Example 7

[0069] A preparation method of dark soy sauce:

[0070] Steamed soybeans and roasted wheat were mixed in a 9:1 ratio and subjected to 42 hours of ventilation to create a soy sauce koji. The koji was then mixed with brine (ammonia nitrogen content 0.25 g / 100 mL, salt content 22-23%) at a mass-to-volume ratio of 1:1.9. The resulting mash was fermented at a controlled temperature. On the 25th day of fermentation, Torulopsis ZZ4 was added. Fermentation was controlled at 20°C throughout the fermentation process, and the fermentation lasted for 180 days. After fermentation, the mash was pressed. The crude soy sauce oil, after measuring sucrase ≥50 U / mL, was then added with sucrose to a sucrose concentration of 50%. The reaction was then heated to 50°C until the sucrose concentration reached 20 g / L, then heated to 125°C to terminate the reaction. The reaction was maintained at 125°C for 2 hours, then cooled to 15°C. The supernatant was allowed to settle naturally for 7 days, and parameters were measured.

[0071] Example 8

[0072] A preparation method of dark soy sauce:

[0073] The steamed soybeans and the fried wheat were mixed in a ratio of 9:1, and the soy sauce koji was obtained after 42 hours of ventilation and koji making. The koji and brine (ammonia nitrogen content 0.25g / 100mL, salt content 22-23%) were mixed in a mass volume ratio of 1:1.9. The obtained sauce mash was subjected to temperature-controlled fermentation. On the 25th day of fermentation, Torulopsis ZZ4 was added. The whole fermentation process was temperature-controlled fermentation, the temperature was controlled at 20°C, and the fermentation was completed for 180 days. After the fermentation was completed, the sauce mash was squeezed. The squeezed soy sauce crude oil was measured to have a sucrase content of <50U / mL. Sucrose was added to a sucrose concentration of 50%, and then the temperature was raised to 50°C for the same reaction time as in Example 6. After that, the temperature was raised to 125°C to terminate the reaction, and 125°C was maintained for 2h. Then the temperature was lowered to 15°C, and the supernatant was taken for parameter measurement after natural sedimentation for 7 days.

[0074] Table 2. Comparison of soy sauce indicators in various examples

[0075]

[0076] From Table 2 we can see that:

[0077] Comparison of Example 2 and Example 3 shows that the total ester content of soy sauce is significantly increased when inoculated with Torulopsis ZZ4 compared to when inoculated with CICC 1708. This shows that inoculation with Torulopsis ZZ4 can significantly increase the ester flavor of soy sauce.

[0078] Comparison of Examples 3, 4, and 5 shows that increasing the amount of sucrose added consumes amino acids in the soy sauce, thereby reducing the ammonia nitrogen content of the soy sauce. However, when the sucrose amount is reduced to 40%, although the ester content and amino acid nitrogen content are high, the red index, yellow index, and color rate are significantly reduced. Therefore, the preferred sucrose addition amount is 50-60%.

[0079] Comparison of Examples 4, 6, and 7 shows that the longer the cooking time, the more amino acids in the soy sauce are consumed and the lower the ammonia nitrogen content of the soy sauce. However, when the cooking time is reduced to 2 h, although the ester content and amino acid nitrogen content are higher, the red index, yellow index, and color rate are significantly reduced. Therefore, the preferred cooking time is 3-4 h.

[0080] Comparison of Examples 7 and 8 shows that when the sucrase content in the semi-finished soy sauce is less than 50 U / mL, the soy sauce's red index, yellow index, and color yield are low. Low sucrase activity prevents the added sucrose from breaking down into reducing sugars. The reducing sugars in the soy sauce undergo complex coloring reactions during cooking, increasing the red index, yellow index, and color yield. However, sucrose, a non-reducing sugar, is less susceptible to coloring reactions.

[0081] In summary, the present invention adopts Torulopsis yeast ZZ4 and cooperates with sucrose to control the temperature during boiling, controls the addition amount of sucrose to 50-60%, and boils for 3-4 hours. This achieves the goal of achieving an amino acid nitrogen content of 0.6-0.9 g / 100 mL, a total ester content of 1.5-1.8 g / L, a red index greater than 6.0, a yellow index greater than 10, and a color rate of 3500-6000 EBC in the final soy sauce product without adding chemical caramel color.

[0082] Finally, it should be noted that the above specific embodiments are only used to explain in detail the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific implementation method of the present invention and is not used to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above specific embodiments, ordinary technicians in this field should understand that it is still possible to modify the technical solutions described in the above embodiments, or to replace or improve some or all of the technical features therein. These modifications, equivalent replacements, and improvements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and description of the present invention.

Claims

1. A Torulopsis yeast ZZ4, characterized in that It was deposited in the General Microbiology Center of China Microorganism Culture Management Committee on May 19, 2022, with the deposit name ZZ4 and the deposit number: CGMCC NO.24921.

2. The Torulopsis yeast ZZ4 according to claim 1, characterized in that The Torulopsis yeast ZZ4 was obtained by mutagenesis of CICC1708.

3. The use of Torulopsis ZZ4 according to claim 1 or 2, characterized in that Including applications in condiment fermentation.

4. The use of Torulopsis ZZ4 according to claim 3, characterized in that The condiment includes dark soy sauce.

5. A method for preparing dark soy sauce, characterized in that: During the fermentation process of mash, Torulopsis yeast ZZ4 is inoculated, and after fermentation, semi-finished soy sauce is obtained by squeezing. Sucrose is added to the semi-finished soy sauce, the temperature is controlled and boiled, and the finished soy sauce is obtained after cooling.

6. The method for preparing dark soy sauce according to claim 5, wherein: The following steps are involved: S1. Mixing the steamed soybeans and the fried wheat to obtain soy sauce koji; S2, mixing the soy sauce koji and the brine in a mass volume ratio of 1g: (1.8-2.0)mL to obtain a soy sauce mash; S3, fermenting the mash, and inoculating Torulopsis ZZ4 on the 20th to 35th day of fermentation; S4, squeezing and extracting oil to obtain semi-finished soy sauce; S5. Adding sucrose with a mass concentration of 50-60% to the semi-finished soy sauce, controlling the temperature and boiling, and cooling to obtain a finished soy sauce product.

7. The method for preparing dark soy sauce according to claim 6, wherein: The amino acid nitrogen content of the brine is 0.2-0.6 g / 100 mL, and the NaCl concentration is 21-22%.

8. The method for preparing dark soy sauce according to claim 6, wherein: The inoculation concentration of Torulopsis ZZ4 was 1×10 5 ~5×10 5 Pieces / g of soy sauce.

9. The method for preparing dark soy sauce according to claim 6, wherein: The boiling process is carried out in a gradient temperature control manner: the temperature is raised to 50-60° C. for reaction, and when the sucrose concentration reaches 10-30 g / L, the temperature is again raised to 120-130° C. for boiling for 3-4 hours, and then the temperature is lowered for cooling.

10. Dark soy sauce prepared by the method according to any one of claims 5 to 9.

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