Method for preparing fermentation broth with high yield of gamma-aminobutyric acid and monascus pigment

Abstract

The invention discloses a method for preparing a fermentation liquid of high-yield γ-aminobutyric acid and monascus pigment, wherein the monascus slant strains stored in a refrigerator are accessed into a monascus slant seed culture medium and statically cultured for 7 days, the newly cultured monascus slant strains are cut into small pieces and accessed into a monascus seed liquid culture medium for activation, and then the activated seed liquid is inoculated into a liquid culture medium with mulberry leaves as a substrate, and fermented at 26°C to 36°C and 180r / min speed for 192h to obtain a fermentation liquid. The fermentation liquid obtained by this method is rich in GABA and monascus pigment. Compared with traditional monascus fermentation, the content of GABA and monascus pigment is increased by more than 100%, and it has good application prospects in the fields of biology and food.

Description

Technical Field

[0001] The invention relates to the field of biology and food technology, and in particular to a method for preparing a fermentation broth with high yield of gamma-aminobutyric acid and monascus pigment. Background Art

[0002] Monascus is a fungus used in fermented foods in China for thousands of years. It produces many nutritionally valuable secondary metabolites, including monascus pigments and gamma-aminobutyric acid (GABA). Its fermentation products can effectively lower blood pressure and plasma cholesterol levels, and exhibit antibacterial activity. Monascus pigments are traditional Chinese food colorants, composed of a mixture of yellow, orange, and red compounds. Studies have shown that monascus pigments can lower total cholesterol and low-density lipoprotein cholesterol levels, while also exhibiting anti-inflammatory and immune-boosting properties. They have become a hot topic for research and development in the fields of medicine, health preservation, and food coloring. GABA is a functional non-protein amino acid produced by glutamate decarboxylase from glutamate. It is the primary inhibitory neurotransmitter in the central nervous system, exhibiting physiological functions such as blood pressure reduction, sedation, and diuresis. It can also slow brain degeneration, alleviate anxiety, and treat neurological diseases. In 1979, Endo Japan first discovered that GABA, a specific substance synthesized by Monascus, exhibits antihypertensive activity. Monascus has thus become another microorganism, following lactic acid bacteria and yeast, that has been widely studied and applied in GABA production.

[0003] Existing red yeast rice pigment production processes include liquid fermentation and liquid-solid fermentation. The liquid fermentation process follows the following process: plate and slant culture (7-8 days) → seed liquid culture (24-26 hours) → primary seed tank culture (7-8 hours) → secondary seed tank culture (7-8 hours) → fermentation tank culture (70-75 hours) → plate and frame filter press → soaking the filter residue (soaking and stirring in 70%-80% alcohol for approximately 26-38 hours) → re-filter press → vacuum concentration (to recover the alcohol) → spray drying (to produce powdered red yeast rice) → finished product packaging. Liquid-solid fermentation involves first using liquid seed tanks to culture the seeds, replacing solid seed inoculation, followed by solid culture and fermentation. This process, referred to as the liquid-solid method, is suitable for high-volume red yeast rice plants. It differs from traditional production processes in that it produces pure seed; increases fermentation thickness; utilizes ventilation measures and automatic control equipment to control the culture temperature; and utilizes drying equipment to dry the finished product. However, low yields of monascus metabolites are a key challenge in monascus fermentation. At present, the main way to increase the production of metabolites is to improve the strain and optimize the fermentation parameters. For example, Dong Yongsheng in "Study on the process conditions for the production of Monacolin K by solid-state fermentation of Monascus purpureus" ("Food Research and Development" 2007.Vol28.No.06) Abstract: Through the study of the process conditions for the production of Monacolin K by solid-state fermentation of Monascus purpureus TQ-57, the process conditions for solid-state fermentation were determined to be: culture medium moisture 50%, relative humidity 75%, initial pH value 6.0, fermentation temperature 25°C, fermentation time 16 days, peptone dosage 3.0%, glycerol dosage 0.2%, Monacolin K content can reach 2.243 mg / g; the Monacolin K content is 2.563 mg / g dry substrate during ventilation culture. In his article "Optimization of Conditions for Liquid-Phase Fermentation of Monascus Pigment by Monascus," Wang Yuanzhuo determined the optimal fermentation conditions to be 30 g / L glucose, 15 g / L sodium nitrate, and 5.5 pH by varying the carbon source (glucose, sucrose, soluble starch), nitrogen source (sodium nitrate, ammonium chloride, urea), pH (3.5, 4.5, 5.5, 6.5, 7.5, and 8.0), and liquid volume (75, 100, 125, 150, 175, and 200 mL). In addition, a growing number of substrates have been explored, such as sugarcane bagasse, potato residue, corn cobs, and rice water. Most researchers tend to optimize existing fermentation methods or modify specific strains to increase nutrient content, but these efforts have had limited success. Therefore, there is an urgent need to develop methods to increase the nutrient content of Monascus fermentation products.

[0004] Mulberry leaves are the leaves of the Moraceae family, rich in nutritional value and possessing a variety of medicinal properties. Mulberry leaves contain a variety of active ingredients, including flavonoids, amino acids, alkaloids, and steroids, resulting in numerous therapeutic benefits, including lowering blood pressure, lowering blood lipids, and providing anti-inflammatory and anti-aging benefits. Studies have shown that the average GABA content in mulberry leaves is approximately 2260 mg / kg (Gong Shuo et al., "Effects of Anaerobic Treatment on GABA Content in Mulberry Leaves," Anhui Agricultural Sciences, 2016, 44(15): P71-72, 89), significantly higher than that of other natural plants. Therefore, developing GABA-rich products using mulberry leaves as raw materials holds great promise.

[0005] Due to the excellent biological activity and high nutritional value of mulberry leaves, this patent is the first to use them as a substrate for Monascus fermentation. A method for preparing a Monascus fermentation broth with high yields of GABA and Monascus pigment is proposed, providing a new approach for the efficient production of GABA and Monascus pigment by Monascus. The method described in this patent has not been reported before. Summary of the Invention

[0006] Purpose of the invention: In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a fermentation liquid with high yield of γ-aminobutyric acid and monascus pigment, which uses mulberry leaves as the substrate for Monascus fermentation, and comprehensively improves the GABA and monascus pigment production of Monascus through liquid fermentation.

[0007] Technical solution: In order to achieve the above-mentioned invention objectives, the inventors adopted the following technical solutions:

[0008] A method for preparing a fermentation liquid with high yield of gamma-aminobutyric acid and monascus pigment comprises the following steps: preparing monascus into seed liquid, inoculating the liquid into a liquid culture medium with mulberry leaves as a substrate, and fermenting the liquid under certain conditions to obtain the fermentation liquid.

[0009] The seed liquid preparation method is as follows: (Refer to the method reported by Gao Jiajia to prepare red yeast rice seed liquid (Study on the process of mixed fermentation of black tea with Ganoderma lucidum, Monascus and Kombucha [D]. Zhejiang University of Technology, 2013))

[0010] 1. Prepare culture medium

[0011] (1) Monascus slant seed culture medium:

[0012] 12brix malt agar medium: Take malt and water (m:V) = 1:4, stir well, and saccharify in a 65℃ water bath for 4 hours. The degree of saccharification is titrated with iodine. After saccharification is complete, filter the saccharified liquid through 4-6 layers of gauze. If the filtrate is turbid, add egg white to the saccharified liquid, boil it, and then filter it. Test the sugar content of the clear liquid. If it is high, dilute it; if it is low, add glucose. After reaching 12brix, add 2% agar, heat and stir to dissolve, and divide it into test tubes. Sterilize at 121℃ for 20 minutes. Place it on a slope (the slope height is about one-third of the test tube height) and cool until solidified.

[0013] (2) Monascus seed liquid culture medium:

[0014] Glucose 30g / L, peptone 20g / L, sodium nitrate 2g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 1g / L, initial pH natural, add appropriate amount of distilled water, heat and stir to dissolve, divide into 500mL Erlenmeyer flasks, 100mL of liquid in each bottle, sterilize at 121℃ for 20min, cool and set aside.

[0015] 2. Activation of Monascus strains: After the Monascus slant strain (from the Institute of Microbiology, Chinese Academy of Sciences) stored in the refrigerator was placed at room temperature for 5 h, it was inoculated into a freshly prepared Monascus slant seed culture medium and cultured in a constant temperature incubator at 30°C for 7 days; the cultured Monascus slant strain was cut into 5 mm × 5 mm blocks, 5 blocks of strains were taken and inoculated into 100 mL of freshly prepared Monascus seed liquid culture medium (packed in a 500 mL Erlenmeyer flask), and cultured at 30°C, 150 rpm for 2 days before use.

[0016] 3. Preparation of liquid culture medium and fermentation conditions are as follows: the liquid fermentation medium is 20-60 g / L mulberry leaf powder, 15-75 g / L glucose, 5-25 g / L peptone, 1 g / L magnesium sulfate heptahydrate and 2 g / L potassium dihydrogen phosphate. The seed liquid is inoculated into the liquid fermentation medium at an inoculum amount of 8% (v / v), and fermented at 26°C to 36°C and 180 r / min for 192 h to obtain the fermentation liquid.

[0017] The indicator active substances of red yeast liquid fermented mulberry leaves are GABA and red yeast pigment. The determination methods of GABA and red yeast pigment are as follows:

[0018] 1) Determination of GABA content (Comparison of colorimetry and HPLC for determination of γ-aminobutyric acid in mulberry leaf tea [J]. Food Science, 2018, 39(24): 256-260.)

[0019] Take 10mL of fermentation broth, ultrasonically extract for 10min, centrifuge at 10000rpm for 10min, and pass the supernatant through a 0.45μm millipore filter. Finally, dilute 10 times in 60% ethanol solution. Take 1.0mL of sample extract, add 1mL 0.1mol / L sodium tetraborate buffer, 1.2mL 6% redistilled phenol solution, 0.6mL 7% sodium hypochlorite solution, mix well, boil in water bath for 10min, then immediately ice bath for 5min, leave at room temperature until it turns blue-green, and add 2mL 60% ethanol solution. Use no sample added as blank control, measure absorbance at 640nm, and use standard curve method to calculate the concentration of GABA in the extract. Draw a standard curve with absorbance value as vertical coordinate and GABA concentration (g / L) as horizontal coordinate. The standard curve equation is: y=0.7332x+0.3933, R 2 =0.9909.

[0020] 2) Determination of color scale of Monascus pigments (Analysis of secondary metabolites produced by fermentation of four Monascus strains [J]. Journal of Food Safety and Quality, 2014, 5(1): 148-153; Production of citrinin-free Monascus pigments by submerged culture at low pH [J]. Enzyme and Microbial Technology, 2014, 55)

[0021] 5 mL of fermentation broth was mixed with 45 mL of 70% ethanol and sonicated for 2 hours at 60°C. The mixture was then centrifuged at 4000 rpm for 15 minutes. After dilution 20-fold with 70% ethanol, the yellow, orange, and red color scales were analyzed at 410 nm, 470 nm, and 510 nm, respectively. Finally, the three color scales were superimposed to form the red yeast rice pigment color scale.

[0022] Beneficial effects:

[0023] 1) The present invention uses mulberry leaves as the main substrate for Monascus liquid fermentation for the first time, significantly increasing the yields of GABA and Monascus pigment.

[0024] 2) The present invention uses mulberry leaves as a liquid fermentation substrate for Monascus, providing a reference for the development of mulberry leaf medicinal and food products and the production of GABA and Monascus pigments by Monascus fermentation.

[0025] 3) The GABA content in the fermentation broth of the present invention is 11.239±0.103 g / L, and the color level of the monascus pigment is 311.083±0.231 U / mL. Compared with traditional Monascus fermentation, the contents of GABA and monascus pigment are increased by more than 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison chart of red yeast rice mulberry leaf fermentation liquid and red yeast rice fermentation liquid (control).

[0027] Figure 2 Comparison of GABA content in red yeast rice fermented with mulberry leaves, red yeast rice fermented with mulberry leaves, and GABA content in red yeast rice fermented with mulberry leaves

[0028] Figure 3 This is a comparison chart of the red yeast rice pigment color levels of red yeast rice fermented with mulberry leaves and red yeast rice fermented with red yeast rice. DETAILED DESCRIPTION

[0029] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0030] Example 1

[0031] This example illustrates the effect of the amount of mulberry leaf powder added on the content of GABA and monascus pigment in the fermentation broth.

[0032] Mulberry leaf liquid culture media containing 20, 30, 40, 50, and 60 g / L of mulberry leaf powder were prepared, maintaining the constant levels of glucose (40 g / L), peptone (15 g / L), magnesium sulfate heptahydrate (1 g / L), and potassium dihydrogen phosphate (2 g / L). Activated Monascus seed liquid was inoculated into the mulberry leaf liquid culture media at an 8% (v / v) inoculum. The culture was then fermented at 30°C and 180 rpm for 192 hours to obtain fermentation broth. The GABA and monascus pigment contents in the fermentation broths with varying amounts of mulberry leaf powder added are shown in Table 1. As can be seen from Table 1, the addition of mulberry leaf powder increased the GABA and monascus pigment contents in the fermentation broth. When 40 g / L of mulberry leaf powder was added, the GABA and monascus pigment contents in the fermentation broth were the highest, significantly higher than those in Control 1 and Control 2. Control 1 consisted of a 40 g / L mulberry leaf powder solution; Control 2 consisted of a fermentation broth without mulberry leaf powder.

[0033] Table 1 Effect of mulberry leaf powder addition on GABA and monascus pigment content in fermentation broth

[0034]

[0035]

[0036] Example 2

[0037] This example illustrates the effect of glucose addition on the GABA and monascus pigment contents in the fermentation broth.

[0038] The fixed medium contained mulberry leaf powder (40 g / L), peptone (15 g / L), magnesium sulfate heptahydrate (1 g / L), and potassium dihydrogen phosphate (2 g / L), and glucose contents of 15, 30, 45, 60, and 75 g / L were prepared. Activated Monascus seed liquid was inoculated into the mulberry leaf liquid medium at an 8% (v / v) inoculum, and fermented at 30°C and 180 rpm for 192 hours to obtain a fermentation broth. The GABA and monascus pigment contents in the fermentation broths with varying glucose additions are shown in Table 2. As can be seen from Table 2, changes in glucose content have an impact on the GABA and monascus pigment contents in the fermentation broth. At a glucose content of 45 g / L, the GABA and monascus pigment contents in the fermentation broth were highest and significantly higher than those in the control group.

[0039] Table 2 Effect of glucose addition on GABA and monascus pigment content in fermentation broth

[0040] Different fermentation broths GABA (g / L) Monascus pigment (U / mL) Containing 0g / L glucose (control) 4.319±0.171 144.567±0.611 Contains 15g / L glucose 9.207±0.159 157.567±0.202 Contains 30g / L glucose 10.343±0.102 149.167±0.454 Contains 45g / L glucose 10.707±0.055 255.110±0.328 Contains 60g / L glucose 8.288±0.144 250.050±0.614 Contains 75g / L glucose 3.001±0.450 230.500±0.100

[0041] Example 3

[0042] This example illustrates the effect of the amount of peptone added on the content of GABA and monascus pigment in the fermentation broth.

[0043] The fixed medium contained mulberry leaf powder (40 g / L), glucose (45 g / L), magnesium sulfate heptahydrate (1 g / L), and potassium dihydrogen phosphate (2 g / L) at constant levels, and mulberry leaf liquid culture media with peptone contents of 5, 10, 15, 20, and 25 g / L were prepared. Activated Monascus seed liquid was inoculated into the mulberry leaf liquid culture media at an 8% (v / v) inoculum, and fermented at 30°C and 180 rpm for 192 hours to obtain a fermentation broth. The GABA and monascus pigment contents in the fermentation broths with different peptone additions are shown in Table 3. As can be seen from Table 3, changes in peptone content have an impact on the GABA and monascus pigment contents in the fermentation broth. At a peptone content of 15 g / L, the GABA and monascus pigment contents in the fermentation broth were highest and significantly higher than those in the control group.

[0044] Table 3 Effect of peptone addition on GABA and monascus pigment content in fermentation broth

[0045] Different fermentation broths GABA (g / L) Monascus pigment (U / mL) Contains 0g / L peptone 4.701±0.250 141.733±0.247 Contains 5g / L peptone 11.334±0.089 234.100±0.150 Contains 10g / L peptone 11.112±0.197 270.750±0.050 Contains 15g / L peptone 11.643±0.089 297.550±0.180 Contains 20g / L peptone 10.339±0.127 292.433±0.355 Contains 25g / L peptone 7.484±0.085 248.233±0.462

[0046] Example 4

[0047] This example illustrates the effect of fermentation temperature on the content of GABA and monascus pigment in the fermentation broth.

[0048] A mulberry leaf liquid culture medium was prepared containing 40 g / L mulberry leaf powder, 45 g / L glucose, 15 g / L peptone, 1 g / L magnesium sulfate heptahydrate, and 2 g / L potassium dihydrogen phosphate. Activated Monascus purpureus seed solution was inoculated into the mulberry leaf liquid culture medium at an inoculum rate of 8% (v / v). The culture was then fermented at 26°C, 28°C, 30°C, 32°C, 34°C, and 36°C for 192 hours at 180 rpm to obtain a fermented liquid. Table 4 shows the GABA and monascus pigment contents in the fermented liquid at different fermentation temperatures. As can be seen from Table 4, changes in fermentation temperature have an impact on the GABA and monascus pigment contents in the fermented liquid. At a fermentation temperature of 32°C, the GABA and monascus pigment contents in the fermented liquid were highest.

[0049] In summary, the optimal composition and fermentation temperature of mulberry leaf liquid culture medium were determined to be: 40 g / L mulberry leaf powder, 45 g / L glucose, 15 g / L peptone, 1 g / L magnesium sulfate heptahydrate, 2 g / L potassium dihydrogen phosphate, and fermentation temperature of 32°C; under these conditions, the GABA content in the mulberry leaf fermentation liquid was 11.239 ± 0.103 g / L, and the red yeast rice pigment color level was 311.083 ± 0.231 U / mL; the performance indicators are shown in Figure 2. Figure 1 、 Figure 2 and Figure 3 .

[0050] Table 4 Effect of fermentation temperature on GABA and monascus pigment content in fermentation broth

[0051] Fermentation temperature GABA (g / L) Monascus pigment (U / mL) 26℃ 6.674±0.512 239.317±0.225 28℃ 9.557±0.170 251.001±1.276 30℃ 10.371±0.088 291.083±0.104 32℃ 11.239±0.103 311.083±0.231 34℃ 10.516±0.232 309.333±0.491 36℃ 8.925±0.199 298.533±0.247 .

Claims

1. A method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment, characterized by: (1) Preparing Monascus into seed liquid; (2) The obtained Monascus seed liquid was inoculated into a liquid culture medium with mulberry leaves as a substrate at an inoculum amount of 8% (v / v), and fermented to obtain a fermentation liquid.

2. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 1, characterized in that: The liquid culture medium using mulberry leaves as substrate contains 20-60 g / L mulberry leaf powder, 15-75 g / L glucose, 5-25 g / L peptone, 1 g / L magnesium sulfate heptahydrate and 2 g / L potassium dihydrogen phosphate.

3. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 1, characterized in that: The fermentation conditions in step (2) are 26°C~36°C and 180 r / min for 192 hours.

4. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 1, characterized in that: The liquid culture medium with mulberry leaves as substrate contains 40 g / L mulberry leaf powder, 45 g / L glucose, 15 g / L peptone, 1 g / L magnesium sulfate heptahydrate and 2 g / L potassium dihydrogen phosphate.

5. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 1, characterized in that: The fermentation conditions were 32°C and 180 r / min for 192 h.

6. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 1, characterized in that: The specific steps of step (1) preparing Monascus into seed liquid are as follows: (1.1) Activation of Monascus strains: After the preserved Monascus strains were placed at room temperature for 5 hours, they were inoculated into a freshly prepared Monascus slant seed culture medium and left to activate in a constant temperature incubator for 7 days before use; (1.2) Monascus seed culture: Cut the activated Monascus into 5 mm × 5 mm pieces. Take 5 pieces of the culture medium and inoculate them into 100 mL of freshly prepared Monascus seed culture medium. Incubate at 30°C and 150 rpm for 2 days.

7. The method for preparing a fermentation broth with high yield of γ-aminobutyric acid and monascus pigment according to claim 6, characterized in that: The composition and preparation process of the Monascus slant seed culture medium are as follows: 12brix malt agar medium: Mix malt and water (m:v) in a ratio of 1:4, stir well, and saccharify in a 65°C water bath for 4 hours. Determine the degree of saccharification with iodine titration. After saccharification is complete, filter the saccharified liquid through 4-6 layers of gauze. If the filtrate is turbid, add egg white, boil, and filter again. Test the clear liquid for sugar content. Dilute if high; add glucose if low. Once the sugar content reaches 12brix, add 2% agar, heat, and stir to dissolve. Aliquot into test tubes, sterilize at 121°C for 20 minutes, and cool on a slant until solidified. The preparation process of the Monascus seed liquid culture medium is as follows: Glucose 30g / L, peptone 20g / L, sodium nitrate 2g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 1g / L, initial pH natural. Add appropriate amount of distilled water, heat and stir to dissolve, divide into 500mL Erlenmeyer flasks, 100mL of liquid in each bottle, sterilize at 121℃ for 20min, and cool for use.

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