Application of paecilomyces variotii in lactic acid degradation
By fermenting and culturing the *Paecilomyces wanensis* strain CGMCC NO.7903 under aerobic conditions, the gap in mold degradation of lactic acid was filled, achieving efficient lactic acid degradation and enriching the role of molds in the solid-state fermentation process of baijiu.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KWEICHOW MOUTAI COMPANY
- Filing Date
- 2022-08-18
- Publication Date
- 2026-05-22
AI Technical Summary
Research on the degradation of lactic acid by molds is still in its infancy. Traditional physical methods are not very effective, and microbial screening of biological pathways has not been effectively applied to lactic acid degradation.
The strain of *Paecilomyces wani* CGMCC NO.7903 was used for fermentation under aerobic conditions to degrade lactic acid in the fermentation substrate. The fermentation conditions were 28℃-45℃, the addition amount was 1%-5%, and the initial concentration was 104-105 cfu/ml. It is suitable for fermentation media or mash.
The *Penicillium wanyi* can achieve a 100% degradation rate when the lactic acid concentration is ≤1.2%, with the highest efficiency at 40℃. The lactic acid degradation rate in the mash reaches 53%, providing rich information on the role of molds in the solid-state fermentation of baijiu and offering a reference for the brewing process.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of brewing technology and relates to the application of *Paecilomyces wanensis* in the degradation of lactic acid. Background Technology
[0002] Currently, there are two main approaches to reducing lactic acid in fermented mash: one is the physical approach, which mainly involves extending the distillation time and increasing the steam pressure to remove acid. However, lactic acid is a non-volatile acid, and this approach is not very effective in degrading it. The other approach is the biological approach, which involves screening for microorganisms that can utilize lactic acid for acid reduction. This method is energy-efficient, pollution-free, and highly efficient, and is currently the mainstream research direction.
[0003] For example, domestic patent CN105420168A, "A Rumen Clostridium that Produces Hexanoic Acid Using Lactic Acid and Its Uses," discloses a strain of rumen Clostridium that can produce hexanoic acid using lactic acid; domestic patent CN105586293A, "A New Clostridium Utilizing Lactic Acid and Its Uses," discloses a new type of Clostridium utilizing lactic acid; domestic patent CN111254085A, "A Microbial Compound Agent for Degrading Lactic Acid," discloses a strain of Pichia kudrica that degrades lactic acid; and domestic patent CN104059854A, "A Strain of Paecilomyces wanensis and Its Applications," discloses a strain of Paecilomyces wanensis that has the function of degrading cellulose. However, research on the degradation of lactic acid by molds is currently in its infancy. Summary of the Invention
[0004] To address the above problems, this invention provides an application of *Paecilomyces wanensis* in the degradation of lactic acid and a method for degrading lactic acid.
[0005] On the one hand, the present invention provides an application of *Paecilomyces wanensis* in the degradation of lactic acid.
[0006] In some implementations, the degradation of lactic acid in the fermentation substrate, which includes a fermentation medium or mash, is included.
[0007] On the other hand, the present invention also provides a method for degrading lactic acid, the method comprising the step of adding *Paecilomyces wanensis* to a fermentation substrate.
[0008] In some implementations, the method includes the following steps:
[0009] S1 Preparation of Penicillium wani seed culture;
[0010] S2. Add the *Penicillium wani* seed liquid to the fermentation substrate for fermentation culture;
[0011] The fermentation substrate includes fermentation culture medium or fermented mash.
[0012] In some embodiments, the amount of *Paecilomyces wanensis* seed culture added relative to the fermentation substrate is 1%-5%; the initial concentration of the *Paecilomyces wanensis* seed culture is 10. 4 -10 5 cfu / ml; the fermentation culture conditions are: under aerobic conditions, 28℃-45℃, culture for 2-12 days; preferably, the fermentation culture conditions are: under aerobic conditions, 30℃-40℃, culture for 3-10 days.
[0013] In some embodiments, when the fermentation substrate is a fermentation medium, the amount of *Paecilomyces wani* seed solution added relative to the fermentation substrate is 1%-3%; the initial concentration of the *Paecilomyces wani* seed solution is 10... 4 -10 5 cfu / ml; the fermentation culture conditions are: under aerobic conditions, 38℃-41℃, culture for 2-5 days; preferably, the fermentation culture conditions are: under aerobic conditions, 40℃, culture for 3 days.
[0014] In some embodiments, when the fermentation substrate is brewing mash, the amount of *Paecilomyces wanensis* seed liquid added relative to the fermentation substrate is 3%-5%; the initial concentration of the *Paecilomyces wanensis* seed liquid is 10... 4 -10 5 cfu / ml; the fermentation culture conditions are: under aerobic conditions, 28℃-35℃, culture for 8-12 days; preferably, the fermentation culture conditions are: under aerobic conditions, 30℃, culture for 10 days.
[0015] In some embodiments, the *Penicillium wanensis* is selected from *Penicillium wanensis* MM2 with accession number CGMCC NO.7903.
[0016] In some embodiments, the lactic acid content in the fermentation medium is 0.1-4%; preferably, the lactic acid content in the fermentation medium is 0.4-3%.
[0017] In some embodiments, the fermentation medium further comprises the following components: peptone and yeast extract; the weight ratio of lactic acid to peptone and yeast extract is 4-40:20:10; preferably, the weight ratio of lactic acid to peptone and yeast extract is 10-20:20:10; preferably, the weight ratio of lactic acid to peptone and yeast extract is 20:20:10.
[0018] In some implementations, the fermented mash is either from the first round of fermentation in the brewing process or from the third round of fermentation in the brewing process; the lactic acid content in the fermented mash is 0.5%-3%.
[0019] In some implementations, the fermented mash is obtained by sterilizing fermented mash from the first or third round of fermentation in the brewing process at high temperature; the lactic acid content in the fermented mash is 0.5%-3%; the high temperature sterilization conditions are: 121℃, sterilization for 15-20 minutes.
[0020] In some embodiments, the fermentation medium is prepared by: using distilled water according to the formula of the fermentation medium, and then sterilizing it at high temperature; the high temperature sterilization conditions are: 121℃, sterilization for 15-20 minutes.
[0021] In some embodiments, the preparation method of the *Paecilomyces wani* seed solution includes the following steps: streaking *Paecilomyces wani* strain on YPD medium, culture conditions: 30℃, 3d; after culture, picking single colonies of *Paecilomyces wani* from the plate, uniformly mixing them into sterile water, preparing a bacterial suspension, and obtaining the *Paecilomyces wani* bacterial solution.
[0022] In summary, this application has at least one of the following beneficial effects:
[0023] (1) Molds are indispensable microorganisms in the solid-state fermentation process of baijiu. Currently, existing research on molds mainly focuses on enzyme provision and flavor substance metabolism. Research on the degradation of lactic acid by molds during fermentation is still in its infancy. *Paecilomyces wanyi* is a dominant mold in the solid-state fermentation stage of baijiu. This invention utilizes *Paecilomyces wanyi* to degrade lactic acid in the fermentation substrate and achieves good lactic acid degradation, further enriching the role of molds in the stacking stage of baijiu solid-state fermentation. This provides a reference for the study of the mechanism of microbial action in baijiu brewing. Moreover, the strains used are screened from the fermentation environment of the mash and will not affect the brewing process.
[0024] (2) The present invention utilizes *Paecilomyces wanensis* to degrade lactic acid in a fermentation substrate. When the lactic acid content in the fermentation substrate, with lactic acid as the sole carbon source, is less than or equal to 1.2%, the lactic acid degradation rate of *Paecilomyces wanensis* can reach 100%. *Paecilomyces wanensis* grows well at lactic acid concentrations of 1.2% and below, and its growth is gradually inhibited as the lactic acid concentration increases. When the culture temperature is 40℃, the lactic acid degradation efficiency of *Paecilomyces wanensis* in the fermentation substrate can reach 100%. When the lactic acid concentration in the fermentation substrate is less than or equal to 2%, the lactic acid degradation efficiency of *Paecilomyces wanensis* reaches 100%, and when the lactic acid concentration in the fermentation substrate is 3%, the lactic acid degradation efficiency of *Paecilomyces wanensis* reaches 53%. Attached Figure Description
[0025] Figure 1 This is an example of how *Penicillium wani* degrades lactic acid under aerobic culture conditions in this invention.
[0026] Figure 2 This is a colony morphology diagram of *Penicillium wani* in an embodiment of the present invention.
[0027] Figure 3 The lactic acid degradation rate of *Paecilomyces wani* under different lactic acid concentrations is shown in the embodiments of the present invention.
[0028] Figure 4 The growth of *Paecilomyces wani* under different lactic acid concentrations is shown in the embodiments of the present invention.
[0029] Figure 5 This describes the lactic acid degradation capacity of *Paecilomyces wani* at different temperatures in the embodiments of the present invention.
[0030] Figure 6 This illustrates the lactic acid degradation of *Paecilomyces wani* at different lactic acid concentrations under 40°C culture conditions in this embodiment of the invention.
[0031] Figure 7 This invention illustrates the lactic acid degradation effect of *Paecilomyces wanensis* on solid-state fermented mash in an embodiment of the invention.
[0032] Figure 8 This invention illustrates the effect of *Paecilomyces wani* on lactic acid degradation in simulated solid-state fermentation in an embodiment of the present invention. Detailed Implementation
[0033] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.
[0034] The Paecilomyces varioti strain used in this embodiment of the invention can be obtained from the authorized patent document CN104059854B. The Paecilomyces varioti strain used in this embodiment of the invention has the accession number CGMCC NO.7903, which is strain MM2 and is classified as Paecilomyces varioti. It has been deposited at the China General Microbiological Culture Collection Center, located at the Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing, on July 8, 2013. The Paecilomyces varioti strain used in this embodiment of the invention is a strain that is directly available to the public and does not require preservation.
[0035] Preparation of fermentation medium: The fermentation medium was prepared according to the following formula: lactic acid 4-40 g / L, peptone 20 g / L, yeast extract 10 g / L, with distilled water added to a final volume of 1 L. After sterilization at 121°C for 15 min, the fermentation medium was obtained. In the embodiments of this invention, the fermentation mediums used differed only in lactic acid content; all other components and amounts were the same. For example: the lactic acid content in the fermentation medium used in Example 3 was 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L; the lactic acid content in the fermentation mediums used in Examples 2 and 5 was 20 g / L; the lactic acid content in the fermentation medium used in Example 4 was 0 g / L, 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L; and the lactic acid content in the fermentation medium used in Example 6 was 12 g / L, 20 g / L, 30 g / L, and 40 g / L.
[0036] The specific method for determining lactic acid in fermentation broth using HPLC is as follows:
[0037] The fermentation broth was centrifuged at 4℃ and 12000 rpm for 10 min. After centrifugation, 0.1 mL of the supernatant was added to 2.9 mL of ultrapure water and analyzed by chromatography under the following conditions: A 4.6 × 50 mm, 1.8 μm Agilent ZORBX RRHT SB-C18 column was used; 0.12% phosphoric acid solution and 2% methanol were used as mobile phase A, and 100% methanol was used as mobile phase B, with gradient elution (mobile phase A for 8 min, mobile phase B for 5 min, mobile phase A for 1 min); the column temperature was 30℃, the detection wavelength was 214 nm, the flow rate was 0.8 mL / min, and the injection volume was 10 μL.
[0038] Example 1: Preparation method of Penicillium wanyi seed liquid
[0039] Activation culture of *Paecilomyces wanensis* strain: *Paecilomyces wanensis* strain was streaked onto YPD medium and cultured at 30℃ for 3 days. After culture, a single colony of *Paecilomyces wanensis* was picked from the plate and evenly mixed into 1 mL of sterile water. 100 μL of this mixture was then added to 10 mL of sterile water to prepare a spore concentration of 10... 5 A bacterial suspension of cfu / ml was used to obtain the seed culture of *Penicillium wanyi*.
[0040] Example 2: Detection of lactic acid degradation by *Penicillium wanensis*
[0041] The *Paecilomyces wani* seed culture obtained above was inoculated into sterilized fermentation medium at an inoculation rate of 1%, and cultured at 30°C for 2 days under aerobic / anaerobic conditions. After the culture, the lactic acid content of the fermentation broth was determined by HPLC. Specific results are shown below. Figure 1 As shown. According to Figure 1The results showed that the above-mentioned *Penicillium wanensis* can degrade lactic acid under aerobic conditions. The above-mentioned fermentation medium formula was prepared by adding 20 g / L lactic acid, 20 g / L peptone, 10 g / L yeast extract, and distilled water to a final volume of 1 L.
[0042] Example 3: Determination of the lactic acid degradation ability of *Penicillium wanensis* at different lactic acid concentrations.
[0043] The seed culture of *Paecilomyces wani* strain prepared in Example 1 was inoculated at a rate of 1% into fermentation media with lactic acid concentrations of 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L (lactic acid content of 0.4%, 0.8%, 1.2%, 1.6%, and 2%, respectively). The pH values of the fermentation media with lactic acid concentrations of 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L were 4.14, 3.67, 3.45, 3.28, and 3.15, respectively. The cultures were incubated at 30°C for 3 days under aerobic conditions. The lactic acid degradation rate was then measured, and the lactic acid degradation rate was the rate of change in lactic acid concentration in the fermentation media before and after inoculation with *Paecilomyces wani*. The results are shown in [Figure 1]. Figure 3 .
[0044] according to Figure 3 The results showed that when the lactic acid content in the fermentation medium with lactic acid as the sole carbon source was less than or equal to 1.2%, the lactic acid degradation rate of *Paecilomyces wani* could reach 100%.
[0045] Example 4: Growth of *Penicillium wanyi* under different lactic acid concentrations
[0046] The seed culture of *Paecilomyces wani* strain prepared in Example 1 was inoculated at a rate of 1% into fermentation media with lactic acid contents of 0 g / L, 4 g / L, 8 g / L, 12 g / L, 16 g / L, and 20 g / L, respectively. The cultures were incubated at 30°C for 3 days under aerobic conditions. After incubation, the cultures were filtered through filter paper, dried, and weighed. Specific results are shown below. Figure 4 As shown.
[0047] according to Figure 4 The results showed that *Paecilomyces wani* grew well at lactic acid concentrations of 1.2% and below, but its growth was gradually inhibited as the lactic acid concentration increased.
[0048] Example 5: The ability of *Penicillium wani* to degrade lactic acid at different temperatures.
[0049] The seed culture of *Paecilomyces wani* strain prepared in Example 1 was inoculated into a fermentation medium with a lactic acid concentration of 20 g / L at an inoculation rate of 1%. The culture was then incubated under aerobic conditions at 30℃, 37℃, 40℃, 42℃, and 45℃ for 3 days. After the inoculation, the lactic acid degradation rate was measured. Figure 5 As shown.
[0050] according to Figure 5 The results showed that as the culture temperature increased, the lactic acid degradation efficiency of *Paecilomyces wanensis* first increased and then decreased. When the culture temperature was 40℃, the lactic acid degradation efficiency of *Paecilomyces wanensis* reached 100%.
[0051] Example 6: Effect of *Paecilomyces wanensis* on lactic acid degradation at different lactic acid concentrations under 40℃ culture conditions.
[0052] The seed culture of *Paecilomyces wani* strain prepared in Example 1 was inoculated at a rate of 1% into fermentation media with lactic acid contents of 12 g / L, 20 g / L, 30 g / L, and 40 g / L (pH 3.45, 3.15, 2.95, and 2.86, respectively). The cultures were incubated at 40°C for 3 days under aerobic conditions. The lactic acid degradation rate was then measured, and the results are shown below. Figure 6 .
[0053] according to Figure 6 The results showed that Paecilomyces wani exhibited good lactic acid degradation at a culture temperature of 40℃. When the lactic acid concentration in the culture medium was less than or equal to 2%, the lactic acid degradation efficiency of Paecilomyces wani reached 100%, and when the lactic acid concentration in the culture medium was 3%, the lactic acid degradation efficiency of Paecilomyces wani reached 53%.
[0054] Example 7: Detection of the lactic acid degradation ability of *Penicillium wanensis* in solid-state fermented mash.
[0055] The seed culture of *Paecilomyces wanensis* strain was inoculated at a rate of 5% into fermented mash from the first and third rounds of brewing (lactic acid content 0.93 g / 100 g mash) and the third round of brewing (lactic acid content 2.50 g / 100 g mash). Fermentation was carried out at 30℃, 37℃, 40℃, 42℃, and 45℃ for 10 days, respectively. The changes in lactic acid content in the mash before and after fermentation were measured. The experimental steps are as follows:
[0056] (1) Weigh 20g of fermented mash from the first and third rounds of fermentation for 2 days into 250mL Erlenmeyer flasks, sterilize at 121℃ for 15min, and set up three parallel samples for each type of mash for later use.
[0057] (2) Take *Paecilomyces wani* colonies that have been cultured at 30°C for 3 days on YPD agar plates, scrape off colonies from the plates with an inoculation loop, and mix them evenly into 1 mL of sterile water. Take 100 μL of this mixture and add it to 10 mL of sterile water to prepare a spore content of 10... 5A CFU / ml bacterial suspension was added to sterilized fermented mash at a 5% inoculum concentration, stirred thoroughly, and then statically cultured at 30℃, 37℃, 40℃, 42℃, and 45℃ for 10 days, with stirring once daily. The changes in lactic acid content in the fermented mash under different temperature gradient fermentation conditions were measured. Based on the results of lactic acid content changes under different temperature gradient fermentation conditions, it was found that *Penicillium wani* exhibited the best lactic acid degradation effect when fermented in fermented mash at 30℃.
[0058] Under 30℃ incubation conditions, the effect of *Penicillium wanyi* on lactic acid degradation in solid-state fermented mash is as follows: Figure 7 As shown, according to Figure 7 The results show that under 30℃ culture conditions, *Paecilomyces wani* can completely degrade lactic acid in the mash, indicating that *Paecilomyces wani* has a good degradation effect on lactic acid in the mash.
[0059] Example 8: Detection of the lactic acid degradation ability of *Penicillium wanensis* in simulated solid-state fermented mash.
[0060] The main difference between this embodiment and Embodiment 7 is that the mash is fermented directly without sterilization, simulating the real fermentation environment of solid-state mash, as detailed below:
[0061] (1) Weigh 20g of fermented mash from the first and third rounds of fermentation for 2 days into 250mL Erlenmeyer flasks, and divide them into a simulated experimental group and a blank control group for later use.
[0062] (2) The simulated experimental group was treated as follows: *Paecilomyces wani* colonies were cultured on YPD agar plates at 30°C for 3 days. Colonies were scraped from the plates using an inoculation loop and mixed evenly into 1 mL of sterile water. 100 μL of this mixture was then added to 10 mL of sterile water to prepare a spore content of 10... 5 A suspension of *Paecilomyces wani* at a concentration of 5 cfu / ml was added to the Erlenmeyer flasks containing fermented mash prepared in step (1). The mixture was stirred thoroughly and incubated statically at 30°C for 10 days, with stirring once daily. Three replicates were prepared for each group, and the lactic acid content in the fermented mash was determined.
[0063] The blank control group was treated as follows: *Penicillium wannii* was not added to the fermented mash. The prepared triangular flask containing the fermented mash (from step (1)) was directly incubated at 30°C for 10 days with stirring once daily. Three replicates were performed for each group, and the lactic acid content in the fermented mash was determined.
[0064] The above-mentioned effect of *Penicillium wanensis* on lactic acid degradation in simulated solid-state fermented mash is as follows: Figure 8 As shown, according to Figure 8 The results show that *Penicillium wani* also has a good degradation effect on lactic acid in mash during the actual stacking process.
[0065] It should be noted that the above embodiments and test examples are only for further elaboration and explanation of the technical solutions of the present invention, so that those skilled in the art can more accurately understand the inventive concept and operation scheme of the present invention, and are not further limitations on the present invention. Any non-prominent substantive features and non-significant improvements made by those skilled in the art on this basis shall fall within the protection scope of the present invention.
Claims
1. The application of *Penicillium wani* in degrading lactic acid in fermentation culture medium or mash, wherein the lactic acid content in the fermentation culture medium is 0.4-3 (w / v)%; the mash is mash from a single-stage or triple-stage fermentation process in winemaking, wherein the lactic acid content in the mash is 0.5-3 (w / w)%; or, the mash is obtained by high-temperature sterilization of mash from a single-stage or triple-stage fermentation process in winemaking, wherein the lactic acid content in the mash is 0.5-3 (w / w)%; the *Penicillium wani* is selected from *Penicillium wani* MM2 with accession number CGMCCNO.7903; the fermentation culture medium further comprises the following components: peptone and yeast extract; the weight ratio of lactic acid to peptone and yeast extract is 4-40:20:
10.
2. A method for degrading lactic acid, characterized in that, The method includes the following steps: S1 Preparation of Penicillium wani seed culture; S2. Add the seed liquid of *Penicillium wani* to the fermentation medium or mash for fermentation culture; The lactic acid content in the fermentation medium is 0.4-3 (w / v)%. The fermented mash is either from the first or third round of fermentation in the brewing process, and the lactic acid content in the mash is 0.5-3 (w / w)%. Alternatively, the fermented mash is obtained by sterilizing the fermented mash from the first or third round of fermentation in the brewing process at high temperature, and the lactic acid content in the mash is 0.5-3 (w / w)%. The fermentation medium also contains the following components: peptone and yeast extract; the weight ratio of lactic acid to peptone and yeast extract is 4-40:20:10; the *Penicillium wanyi* is selected from *Penicillium wanyi* MM2 with accession number CGMCC NO.7903.
3. The method as described in claim 2, characterized in that, The amount of *Paecilomyces wanensis* seed solution added relative to the fermentation medium is 1%-3%; the initial concentration of the *Paecilomyces wanensis* seed solution is 10... 4 -10 5 cfu / ml; the fermentation conditions are: under aerobic conditions, 38℃-41℃, culture for 2-5 days; or, The amount of *Paecilomyces wanensis* seed solution added relative to the fermented mash is 3%-5%; the initial concentration of the *Paecilomyces wanensis* seed solution is 10. 4 -10 5 cfu / ml; the fermentation conditions are: under aerobic conditions, 28℃-35℃, for 8-12 days.
4. The application as described in claim 1 or the method as described in claim 2, characterized in that, The high-temperature sterilization conditions are: 121℃, sterilization for 15-20 minutes.