Gastrodin fermentation product based on lactobacillus reuteri and preparation method thereof

Abstract

The application discloses a gastrodia elata fermentation product based on lactobacillus reuteri and a preparation method thereof, and relates to the technical field of gastrodia elata fermentation product preparation.The gastrodia elata fermentation product is prepared by fermenting gastrodia elata by lactobacillus reuteri with a preservation number of CICC6119, and the lactobacillus reuteri is preserved in the China Industrial Microbial Culture Collection Center.The lactobacillus reuteri (CICC6119) is used to ferment gastrodia elata for the first time, the "horses urine smell" of the gastrodia elata after the fermentation treatment is obviously weakened, special aroma is generated, and the sensory quality is obviously improved.The fermentation process can improve the content of effective components in the gastrodia elata, enhance the biological activity, and thus a health-care product with better functionality is obtained.

Description

Technical Field

[0001] This invention relates to the field of Gastrodia elata fermentation product preparation technology, and in particular to a Gastrodia elata fermentation product based on Lactobacillus reuteri and its preparation method. Background Technology

[0002] Gastrodia elata, also known as Chijian, Shencao, Helicao, and Limu, is the dried tuber of the orchid Gastrodia elata Bl. It is rich in active ingredients such as gastrodin, p-hydroxybenzyl alcohol, and barisonin. It has the effects of calming wind and stopping spasms, suppressing liver yang, and dispelling wind and unblocking meridians. It is mainly used to treat internal liver wind, epilepsy, convulsions, dizziness, headache, numbness of limbs, hemiplegia, and rheumatic pain. Gastrodia elata was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and was considered a rare traditional Chinese medicine. With the increasing demand for Gastrodia elata in the food and health product industries, existing products can no longer meet market needs, and more new Gastrodia elata products urgently need to be developed.

[0003] Microbial fermentation technology offers advantages in the food and health product industries, including energy conservation, environmental protection, and preservation of active ingredients. *Lactobacillus reuteri* is a beneficial bacterium commonly found in the intestines of humans and animals, harmless to the host, and possessing excellent biocompatibility. Therefore, using *Lactobacillus reuteri* as the fermentation starter for *Gastrodia elata* can improve its sensory quality and increase its biological activity, resulting in health products with superior functionality. This invention provides a new solution for the deep processing of *Gastrodia elata*; it also establishes process parameters and flavor quality standards for *Lactobacillus reuteri* fermentation of *Gastrodia elata*, providing guidance for the industrial production of fermented *Gastrodia elata* products and laying a solid foundation for the emergence of functional products related to *Gastrodia elata*.

[0004] Currently, there are no publicly available technical solutions for using Lactobacillus reuteri to ferment Gastrodia elata to improve its sensory quality and bioactivity. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a fermented Gastrodia elata product based on Lactobacillus reuteri and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A fermented product of Gastrodia elata based on Lactobacillus reuteri, wherein the fermented product is obtained by fermenting Gastrodia elata with Lactobacillus reuteri with accession number CICC6119, which is deposited at the China Industrial Microbial Culture Collection Center.

[0007] A method for preparing the *Lactobacillus reuteri* fermented *Gastrodia elata* product includes the following steps: (1) Sterilize the gastrodia elata by standing it at 121℃ for 15-20 minutes. (2) Inoculate the Lactobacillus reuteri culture with preservation number CICC6119 into sterilized Gastrodia elata; (3) Ferment at 31-43℃ in the dark for 6-14 days; (4) The fermentation product is dried by freeze drying for 2-3 days to obtain the fermented Gastrodia elata product of Lactobacillus reuteri.

[0008] Preferably, the concentration of the *Lactobacillus reuteri* bacterial culture is not less than 3.0 × 10⁻⁶. 8 CFU / mL.

[0009] Preferably, the inoculation amount of the bacterial solution is 1%-5% of the dry weight of the Gastrodia elata.

[0010] A fermented product prepared according to the above preparation method, which improves the content of effective components in Gastrodia elata by fermentation with Lactobacillus reuteri.

[0011] The beneficial effects of this invention are as follows:

[0012] 1. This invention is the first to use Lactobacillus reuteri (CICC 6119) to ferment Gastrodia elata. After fermentation, the "horse urine smell" of Gastrodia elata is significantly reduced and a special aroma is produced, and the sensory quality is significantly improved.

[0013] 2. The fermentation process described in this invention can increase the content of effective components in Gastrodia elata and enhance its biological activity, thereby obtaining a health product with better functionality.

[0014] 3. This invention establishes the optimal process parameters and flavor quality standards for fermentation of Gastrodia elata by Lactobacillus reuteri through single-factor experiments and response surface methodology, providing guidance for the industrial production of fermented Gastrodia elata products.

[0015] 4. This invention provides a new technical approach for the deep processing of Gastrodia elata and lays the foundation for the development of Gastrodia elata-related functional products. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the effect of the inoculum amount on the overall score of fermented Gastrodia elata. Figure 2 This is a schematic diagram illustrating the effect of fermentation temperature on fermented Gastrodia elata according to the present invention; Figure 3 This is a schematic diagram illustrating the effect of fermentation time on the overall score of fermented Gastrodia elata. Figure 4 This is a schematic diagram illustrating the influence of the interaction of various factors in this invention on the overall score of fermented Gastrodia elata. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0018] Example 1: A fermented product made from Lactobacillus reuteri to increase the content of active ingredients in Gastrodia elata.

[0019] The fermented product described above, which uses Lactobacillus reuteri fermentation to increase the content of effective components in Gastrodia elata, is made from the following raw materials: Gastrodia elata and Lactobacillus reuteri (CICC 6119, China Industrial Microbial Culture Collection Center).

[0020] Preparation of Lactobacillus reuteri culture: Lactobacillus reuteri from previously stored MRS plates was inoculated onto MRS plates and cultured at 37°C for 2-3 days. Then, Lactobacillus reuteri was inoculated into LB liquid medium and cultured at 37°C with a shaker at 180 r / min for 2-3 days. After that, it was taken out and counted using a hemocytometer.

[0021] Example 2: A method for preparing a fermented product that increases the content of effective components in Gastrodia elata through Lactobacillus reuteri fermentation, comprising the following steps: S1: First, a single-factor experiment was conducted on the fermentation process of Gastrodia elata: using the comprehensive score as the corresponding index, fermentation temperature, fermentation time, and inoculum volume as single-factor variables, with the inoculum concentration not less than 3.0 × 10⁻⁶. 8 CFU / mL, with other factors kept constant during the experiment.

[0022] S2: The method for adjusting the bacterial concentration is as follows: Mix the cultured bacterial solution thoroughly and take 10 μL for counting. Count the bacteria in the counting chamber of the hemocytometer. The counting rule is: select 5 cells from the middle grid of each counting chamber for counting, and average the counts from the upper and lower counting chambers to obtain the final count. For cells located on the grid lines, generally only the upper and right grid lines are counted. Count a total of 3 times, take the average value, and calculate using the following formula: .

[0023] S3: Under the conditions of 8 days of fermentation time and 37℃ fermentation temperature, the inoculum volume is set to 1%, 2%, 3%, 4%, and 5%; under the conditions of 2% inoculum volume and 37℃ fermentation temperature, the fermentation time is set to 6 days, 8 days, 10 days, 12 days, and 14 days; under the conditions of 2% inoculum volume and 8 days of fermentation time, the fermentation temperature is set to 31℃, 34℃, 37℃, 40℃, and 43℃.

[0024] S4: Select the optimal conditions from the single-factor experiments and use DesignExpert13 software to conduct a Box-Behnken experimental design. A 3-factor, 3-level experimental design was used, with fermentation temperature, fermentation time, and inoculum volume as the three factors, and the overall score of *Gastrodia elata* as the corresponding value.

[0025] S5: Comprehensive scoring method for Gastrodia elata: The content of effective components in fermented Gastrodia elata is detected by HPLC, electronic nose detection, and electronic tongue detection. The results of these three tests are then analyzed by multiple indicators and weighted by CRITIC scoring.

[0026] S6: First, pulverize each fermentation sample into powder, accurately weigh 1.0g, add 25mL of acetonitrile-water solution (3:97), weigh, soak for 20min, ultrasonically treat at 30℃ (power 200W, frequency 40kHz) for 1h, cool, weigh again, make up the weight loss with acetonitrile-water solution (3:97), filter through a 0.22μm microporous membrane to obtain the sample solution. HPLC chromatographic conditions: Hypersil C18 (Gemini 5μC18 110A, 4.6mm × 250mm; Phernomex C18 guard column); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution was used (0–10 min, A:B = 2:98; 10–20 min, A:B = 8:92; 20–30 min, A:B = 12:88; 30–47 min, A:B = 25:75; 47–53 min, A:B = 90:10; 53–60 min, A:B = 2:98); flow rate 1.0 mL / min; column temperature 35℃; detection wavelength 220 nm; injection volume 10 μL. Preparation of standard solutions: Accurately weigh appropriate amounts of gastrodin, p-hydroxybenzyl alcohol, balithinoside A, balithinoside B, balithinoside C, and balithinoside E standards, dissolve them in acetonitrile-water (3:97) mixed solution to prepare mixed standard solutions with concentration gradients of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL. Filter the solutions using a 0.22 μm organic microporous membrane and store them in a refrigerator at 4℃ for later use.

[0027] S7: Detection was performed using a Shimadzu LC-20 high-performance liquid chromatograph. A standard curve was plotted with the standard concentration (X, mg / mL) on the x-axis and the peak area (Y) on the y-axis. The equation of the standard curve is shown below:

[0028] S8: The samples were analyzed using a Shimadzu LC-20 high-performance liquid chromatograph, and the content of active ingredients in each sample was obtained according to the standard curve.

[0029] S9: Crush each fermentation sample into powder, accurately weigh 1.0g, add it to a 20mL headspace vial, use an ultra-fast gas chromatography electronic nose to detect the fermented Gastrodia elata, perform component analysis on the results, and quantify the content of the components with good sensory evaluation.

[0030] S10: Crush each fermentation sample into powder, accurately weigh 1.0g, add 80mL of pure water, sonicate at 30℃ (power 200W, frequency 40kHz) for 1h, cool, and filter. Use an electronic tongue to test the filtrate and score the results by taste.

[0031] S11: Summarize the results from S8, S9, and S10, normalize each indicator using the multi-indicator experimental formula method to obtain a comprehensive score, and select the optimal value among the measured values ​​of each indicator. The score is set as the maximum score (100 points), and then the remaining measured values ​​are converted into scores. The formula is:

[0032] In the formula: For the first Group 1 trial The score for each indicator shall not exceed 100. For the first Group 1 trial The measured values ​​of each indicator.

[0033] S12: Objectively assign weights to each indicator using the coefficient of variation method, as shown in the formula:

[0034] In the formula: Let be the weight coefficient of the i-th indicator; Let be the coefficient of variation of the j-th index.

[0035] S13: Calculate the overall score Pi for each group of trials, as shown in the formula:

[0036] Example 3: The effect of the single-factor experiment of fermented Gastrodia elata in Example 1 on the overall score

[0037] Under the conditions of 8 days of fermentation time and 37℃, the inoculum volume was set to 1%, 2%, 3%, 4%, and 5%; under the conditions of 2% inoculum volume and 37℃ fermentation temperature, the fermentation time was set to 6 days, 8 days, 10 days, 12 days, and 14 days; under the conditions of 2% inoculum volume and 8 days of fermentation time, the fermentation temperature was set to 31℃, 34℃, 37℃, 40℃, and 43℃.

[0038] 1. For example Figure 1 As shown, the inoculum size affects the overall score of fermented Gastrodia elata. With increasing inoculum size, the overall score initially rises and then declines. At an inoculum size of 5%, it rises again, but does not exceed the overall score of 3%, which is 100. Therefore, a 3% inoculum size is determined to be the appropriate inoculum size.

[0039] 2. For example Figure 2 As shown, the effect of fermentation temperature on fermented Gastrodia elata shows that as the fermentation temperature gradually increases, the overall score of fermented Gastrodia elata first increases and then decreases, reaching its highest value of 100 at a fermentation temperature of 37℃. Therefore, a fermentation temperature of 37℃ is determined to be the suitable fermentation temperature.

[0040] 3. For example Figure 3 As shown, the fermentation time affects the overall score of fermented Gastrodia elata. With increasing fermentation time, the overall score reaches its highest point on day 12. After day 12, the overall score decreases, reaching 99 on day 12. Therefore, a fermentation time of 12 days can be determined as the most suitable fermentation time.

[0041] Example 4: Response surface methodology optimization experiment of fermented Gastrodia elata in Example 1 Based on the results of the single-factor experiments, response surfaces were used to optimize the design for three factors: inoculum size, fermentation temperature, and fermentation time. The levels and codes of the experimental design factors are as follows:

[0042] Based on the above data, a three-factor, three-level Box-Behnken experimental design was performed in Design Expert 13 software, resulting in 15 experimental schemes. Fermentation experiments were designed for each scheme, with three replicates per scheme. HPLC, electronic nose, and electronic tongue measurements were performed, and the overall score was calculated. The 15 experimental conditions and corresponding overall scores are as follows:

[0043] The data above was analyzed and fitted using Design Expert 13 software to test the effectiveness of the regression model. The analysis of variance is as follows:

[0044] Note: * indicates a significant difference (P<0.05); ** indicates an extremely significant difference (P<0.01).

[0045] The quadratic regression equation of the comprehensive score of fermented Gastrodia elata on A, B, and C is: Y = 77.81 - 0.6721A + 6.11B - 2.09C - 1.46D + 0.3349AB - 0.4065AC - 6.42BC - 26.85A 2 -21.16B 2 -27.91C 2 Quadratic term A 2 B 2 C 2 The effect was extremely significant (P < 0.01). This indicates that the inoculum size, fermentation temperature, and fermentation time have a significant impact on the overall score of fermented Gastrodia elata.

[0046] As shown above, the model is highly significant (P < 0.01), while the lack-of-fit term (P = 0.9387) is insignificant (P > 0.05), which is favorable for the model. The R-squared value of the model is shown below. 2 =0.9317, indicating that the model fits well and the experiment is relatively stable, and can be used to predict and analyze the fermentation conditions of Gastrodia elata.

[0047]

[0048] The impact of the interaction of various factors on the overall score of fermented Gastrodia elata, such as Figure 4 As shown, the optimal fermentation conditions obtained through Design Expert 13 software analysis are: inoculum size 2.989%, fermentation temperature 37.457℃, fermentation time 11.889d, and the overall predicted response score is 78.342.

[0049] Based on the fermentation conditions obtained from response surface methodology and taking into account actual conditions, the final optimal fermentation conditions were: 3% inoculum, 37℃ fermentation temperature, and 11 days fermentation time, with a final comprehensive score of 78.677.

[0050] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermented Gastrodia elata product based on Lactobacillus reuteri, characterized in that, The fermented Gastrodia elata product is obtained by fermenting Gastrodia elata with Lactobacillus reuteri.

2. A method for preparing a fermented product of Gastrodia elata based on Lactobacillus reuteri, characterized in that, Includes the following steps: Inoculate Gastrodia elata with Lactobacillus reuteri bacterial solution; Ferment at a certain temperature in the dark for a certain period of time; The fermented Gastrodia elata by Lactobacillus reuteri was obtained.

3. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 2, characterized in that, The gastrodia elata was sterilized by static treatment before inoculation.

4. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 3, characterized in that, The sterilization conditions for the static sterilization process are sterilization at 121°C for 15-20 minutes.

5. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 2, characterized in that, The concentration of the Lactobacillus reuteri culture is not less than 3.0 × 10⁻⁶. 8 CFU / mL.

6. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 2, characterized in that, The inoculation amount of the bacterial solution is 1%-5% of the dry weight of the Gastrodia elata.

7. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 2, characterized in that, The fermentation temperature is 31-43℃.

8. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 7, characterized in that, The fermentation time is 6-14 days.

9. The method for preparing a fermented Gastrodia elata product based on Lactobacillus reuteri according to claim 8, characterized in that, Before obtaining the fermented Gastrodia elata by Lactobacillus reuteri, the fermented Gastrodia elata is further dried by freeze-drying for 2-3 days.

10. A fermented product prepared by the preparation method according to any one of claims 2-9, which improves the content of effective components of Gastrodia elata by fermentation with Lactobacillus reuteri.

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