Preparation method of catalpol-rich rehmannia root fermented food

Abstract

The invention discloses a preparation method of catalpol-rich rehmannia root fermented food. The catalpol-rich rehmannia root fermented food is prepared from the following raw materials: fresh rehmannia root, lactobacillus reuteri, aspergillus oryzae and bacillus subtilis. The fresh rehmannia is subjected to microbial fermentation treatment, the catalpol content in a fermentation product is detected, and the result shows that the catalpol content of the rehmannia fermented food prepared by the method is obviously higher than that of a traditional process product, and the rehmannia fermented food has a good market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a preparation method of a rehmannia glutinosa fermented food rich in catalpol. BACKGROUND

[0002] As a traditional Chinese medicinal material, rehmannia glutinosa was included in the national food and medicine homologous material directory in August 2024, and its rich active ingredients endow it with high nutritional and health care value, and it has broad development prospects in the field of functional food. However, the current research on fermented food of fresh rehmannia is still in its infancy, and the existing processing methods are mainly concentrated on traditional processing or single form product development, which fails to fully exert the advantages of food and medicine homology.

[0003] As a typical iridoid active ingredient in rehmannia glutinosa, catalpol has multiple biological activities such as antioxidant, anti-inflammatory and immune regulation, and the content of catalpol in rehmannia glutinosa is one of the core indicators for measuring the quality of rehmannia glutinosa and its processed products according to the Chinese Pharmacopoeia. However, in the existing food fermentation technology, when rehmannia glutinosa is treated by traditional fermentation process, catalpol is easily degraded or transformed due to the action of microbial metabolic enzymes and the influence of fermentation environment, resulting in a significant decrease in its content and affecting the efficacy and quality of the product. Therefore, it is of application value to develop a fermentation food preparation method that can efficiently maintain or improve the content of catalpol. SUMMARY

[0004] In view of this, the present application provides a high catalpol rehmannia glutinosa fermented food preparation method.

[0005] The technical solution of the present application is as follows:

[0006] The present application provides a high catalpol rehmannia glutinosa fermented food, which is prepared by fermenting fresh rehmannia glutinosa, lactobacillus reuteri, aspergillus oryzae and bacillus subtilis. Preferably, the content of catalpol in the food is ≥0.35% or ≥0.38%. Preferably, the content of catalpol is 0.35-0.42%.

[0007] On the other hand, the present application provides a method for extracting catalpol from rehmannia glutinosa, which is obtained by jointly fermenting fresh rehmannia glutinosa with lactobacillus reuteri, aspergillus oryzae and bacillus subtilis. Preferably, the method further comprises the step of pretreating fresh rehmannia glutinosa by pulse electric field-ultrasound combination before fermentation.

[0008] Preferably, the fermentation strains include lactobacillus reuteri 10 9 CFU / mL, aspergillus oryzae 10 7 CFU / mL and bacillus subtilis 1.0x10 8 - 1.0x10 10 CFU / mL.

[0009] On the other hand, the present invention provides a method for preparing fermented Rehmannia glutinosa food with high-purity argentine, comprising the following raw materials: fresh Rehmannia glutinosa, Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis.

[0010] Furthermore, the strains mentioned above were Lactobacillus reuteri LR08, Aspergillus oryzae purchased from Hezhong Kangyuan Company, and Bacillus subtilis QK02.

[0011] Furthermore, the viable count of the *Lactobacillus reuteri* is 10. 8 -10 10 CFU / mL, preferably 10. 9 CFU / mL; Aspergillus oryzae was 10. 6 -10 8 CFU / mL, preferably 10. 7 CFU / mL; Bacillus subtilis was 10 6 -10 8 CFU / mL, preferably 10. 7 CFU / mL.

[0012] A method for preparing a fermented Rehmannia glutinosa food with high-calcium alcohol content, wherein the preparation method of the fresh Rehmannia glutinosa extract includes the following steps:

[0013] S1. Select fresh Rehmannia glutinosa, clean it, and air-dry it;

[0014] S2. Cut the fresh Rehmannia glutinosa into small sections;

[0015] S3. Add deionized water;

[0016] S4. The mixture is placed in a pulsed electric field-ultrasound combined processing device for processing;

[0017] S5, activated strain;

[0018] S6. Place the pretreated fresh Rehmannia glutinosa slurry into a fermentation tank and inoculate it with Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis strains for fermentation.

[0019] S7. Take fresh Rehmannia glutinosa fermentation liquid and test the catalpol content;

[0020] S8. Dry and store in an oven.

[0021] Another aspect of the present invention provides a method for preparing a fermented Rehmannia glutinosa food with high catalpol content, comprising the following steps:

[0022] S1. Select fresh Rehmannia glutinosa, and clean and air-dry it;

[0023] S2. Cut the dried fresh Rehmannia root into small pieces;

[0024] S3. Add deionized water to the fresh Rehmannia glutinosa segments;

[0025] S4. The mixture of fresh Rehmannia glutinosa and deionized water is placed in a pulsed electric field-ultrasound combined treatment device for treatment;

[0026] S5. Activate Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis;

[0027] S6. Transfer the pretreated fresh Rehmannia glutinosa slurry into a fermentation tank, and inoculate it with activated Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis for fermentation;

[0028] S7. Sample and test the catalpol content in the fermentation broth of fresh Rehmannia glutinosa;

[0029] S8. Dry the fermentation products in an oven and then store them.

[0030] Further, in step S2, the thickness of the fresh Rehmannia glutinosa ranges from 0.5 to 1.8 cm.

[0031] Further, in step S3, the amount of deionized water added is 1:3-1:6 g / mL (material-to-liquid ratio).

[0032] Further, in step S4, the pulse electric field processing parameters are: electric field strength 25-30kV / cm, pulse width 20-30μs, pulse frequency 50-60Hz, and processing time 3-5min.

[0033] Further, in step S4, the ultrasonic processing parameters are power 300-400W and frequency 40-50kHz.

[0034] Further, in step S5, the *Lactobacillus reuteri* is inoculated into MRS medium and anaerobically cultured at 37°C for 24 hours, activating it for two generations to achieve a viable count of 102. 9 CFU / mL.

[0035] Further, in step S5, the *Aspergillus oryzae* is inoculated into PDA medium and cultured aerobically at 30°C for 48 hours to prepare a spore suspension with a concentration of 10. 7 CFU / mL.

[0036] Further, in step S6, the inoculation ratio of Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis is 6:2:1, and the inoculation amount is 5-8% (v / v).

[0037] Further, in step S6, the initial fermentation parameters are: pH 5.5-6.0; temperature controlled at 30-32℃ and oxygen partial pressure at 30-35% for the first 12 hours; temperature increased to 35-37℃ and oxygen partial pressure decreased to 15-20% for 12-24 hours; and temperature maintained at 32-34℃ and oxygen partial pressure maintained at 10-15% for 24-48 hours. The stirring rate during fermentation is 60-80 r / min, and the pH value is measured every 8 hours. If the pH value is below 5.0, 0.1 mol / L Na₂CO₃ solution is added to adjust it to 5.0-5.5.

[0038] Furthermore, in step S7, the catalpol content is detected every 24 hours starting from the second day of fermentation. When the catalpol content shows no significant increase (increase <1%) in two consecutive tests and there is no downward trend, fermentation can be terminated.

[0039] Further, in step S8, the drying temperature of the oven is 60°C.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. This invention innovatively uses pulsed electric field-ultrasound combined treatment to pretreat fresh Rehmannia glutinosa, which effectively destroys the cell wall structure of fresh Rehmannia glutinosa, improves the dissolution efficiency of active ingredients such as catalpol, and lays the foundation for the retention of catalpol in the subsequent fermentation process.

[0042] 2. Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis are selected and fermented in a specific ratio. By optimizing the strain combination and fermentation parameters, the degradation and transformation of catalpol during the fermentation process are reduced, and the catalpol content of the final product is significantly increased.

[0043] 3. The entire preparation process is clear, the parameters are well-defined, and it is highly operable. The prepared Rehmannia glutinosa fermented food has a high content of catalpol, which meets the quality requirements of functional foods and has good market application prospects. Attached Figure Description

[0044] Figure 1 The Rehmannia glutinosa products obtained by this invention. The top row 1-5 are the products obtained by the preparation methods of Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively; the bottom row 1-5 are the products obtained by the preparation methods of Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8, respectively. Detailed Implementation

[0045] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0046] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0047] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods, and the reagents used in the embodiments are all commercially available.

[0048] The Lactobacillus reuteri is manufactured by Zhuochen Biotechnology Co., Ltd., and is food grade with a live count of 1.0 × 10⁻⁶. 10 -2.0×10 11 CFU / g indicates *Lactobacillus reuteri* LR08; *Aspergillus oryzae* is manufactured by Zhongkangyuan Biotechnology Co., Ltd., food grade, with a spore content of approximately 5.0 × 10⁻⁶ per gram. 9 Bacillus subtilis QK02 is manufactured by SuperSmart, is food grade, and has a viable count of 1.0 × 10⁻⁶. 8 CFU / g.

[0049] Example 1: Preparation of Fermented Food from Fresh Rehmannia glutinosa

[0050] The steps include:

[0051] Step (1): Select fresh Rehmannia glutinosa with a diameter of 3.5cm, remove the roots and rotten parts, wash with clean water, and air dry naturally;

[0052] Step (2): Cut the fresh Rehmannia glutinosa into small pieces 0.5 cm thick;

[0053] Step (3): Weigh the cut Rehmannia glutinosa and add deionized water at a material-to-liquid ratio of 1:3 g / mL;

[0054] Step (4): Set the pulse electric field processing parameters to electric field strength of 30kV / cm, pulse width of 20μs, pulse frequency of 60Hz, and processing time of 5min; set the ultrasonic processing parameters to power of 400W and frequency of 50kHz; put the mixture into the pulse electric field-ultrasonic combined processing device for processing.

[0055] Step (5): Inoculate Lactobacillus reuteri LR08 into MRS medium, anaerobic culture at 37℃ for 24 h, activate for 2 generations, and bring the viable count to 10. 9CFU / mL; Aspergillus oryzae purchased from Hezhong Kangyuan Company was inoculated into PDA medium and cultured aerobically at 30℃ for 48 h to prepare a spore suspension with a concentration of 10 CFU / mL; 7 CFU / mL;

[0056] Step (6): Place the pretreated fresh Rehmannia glutinosa slurry into a fermenter; mix Lactobacillus reuteri LR08, Aspergillus oryzae, and Bacillus subtilis QK02 in a 6:2:1 ratio and inoculate the mixed strain at 8% (v / v); the initial fermentation parameters are: pH 5.5, temperature controlled at around 30℃ and oxygen partial pressure at 30% for the first 12 hours; temperature increased to 35℃ and oxygen partial pressure decreased to 20% for 12-24 hours; temperature maintained at 34℃ and oxygen partial pressure maintained at 10% for 24-48 hours. The stirring rate during fermentation is 60 r / min, and the pH value is measured every 8 hours. If the pH value is lower than 5.0, add 0.1 mol / L Na2CO3 solution to adjust it to about 5.5.

[0057] Step (7): Starting from the second day of fermentation, take 1 mL of fresh Rehmannia glutinosa fermentation broth every 24 hours, add 2.5 mL of methanol, sonicate for 20 min, cool, add weight, and filter. Take the filtrate as the test solution and use high performance liquid chromatography to detect the catalpol content. When the catalpol content does not increase significantly in two consecutive tests (increase <1%), terminate the fermentation.

[0058] Step (8): Dry the fresh Rehmannia glutinosa and its fermentation liquid at 60℃.

[0059] Example 2: Preparation of Fermented Food from Fresh Rehmannia glutinosa

[0060] The steps include:

[0061] Step (1): Select fresh Rehmannia glutinosa with a diameter of 3.5cm, remove the roots and rotten parts, wash with clean water, and air dry naturally;

[0062] Step (2): Cut the fresh Rehmannia glutinosa into small pieces 0.8 cm thick;

[0063] Step (3): Weigh the cut Rehmannia glutinosa and add deionized water at a material-to-liquid ratio of 1:4 g / mL;

[0064] Step (4): Set the pulse electric field processing parameters to electric field strength 25kV / cm, pulse width 25μs, pulse frequency 60Hz, and processing time 5min; set the ultrasonic processing parameters to power 350W and frequency 45kHz; put the mixture into the pulse electric field-ultrasonic combined processing device for processing.

[0065] Step (5): Inoculate Lactobacillus reuteri into MRS medium, anaerobic culture at 37°C for 24 h, activate for 2 generations, and bring the viable count to 10^6.9 CFU / mL; Aspergillus oryzae was inoculated onto PDA medium and cultured aerobically at 30°C for 48 h to prepare a spore suspension with a concentration of 10. 7 CFU / mL;

[0066] Step (6): Place the pretreated fresh Rehmannia glutinosa slurry into a fermenter; mix Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis QK02 in a 6:2:1 ratio and inoculate the mixed strain at 8% (v / v); the initial fermentation parameters are: pH 5.5, temperature controlled at 32℃ and oxygen partial pressure at 35% for the first 12 hours; temperature increased to 37℃ and oxygen partial pressure decreased to 15% for 12-24 hours; temperature maintained at 32℃ and oxygen partial pressure maintained at 15% for 24-48 hours. The stirring rate during fermentation is 80 r / min, and the pH value is measured every 8 hours. If the pH value is lower than 5.0, add 0.1 mol / L Na2CO3 solution to adjust it to about 5.0.

[0067] Step (7): Starting from the second day of fermentation, take 1 mL of fresh Rehmannia glutinosa fermentation broth every 24 hours, add 2.5 mL of methanol, sonicate for 20 min, cool, add weight, and filter. Take the filtrate as the test solution and use high performance liquid chromatography to detect the catalpol content. When the catalpol content does not increase significantly in two consecutive tests (increase <1%), terminate the fermentation.

[0068] Step (8): Dry the fresh Rehmannia glutinosa and its fermentation liquid at 60℃.

[0069] Comparative Example 1

[0070] The difference from Example 1 is that the raw materials are different; fresh Rehmannia glutinosa is replaced with raw Rehmannia glutinosa.

[0071] Comparative Example 2

[0072] The difference from Example 1 is the raw material; fresh Rehmannia glutinosa is replaced with mature Rehmannia glutinosa.

[0073] Comparative Example 3

[0074] The difference from Example 1 is that the raw material processing method is different. The pulse electric field-ultrasound combined treatment in step (4) of Example 1 is removed and replaced with high temperature sterilization treatment.

[0075] Comparative Example 4

[0076] The difference from Example 1 is that it does not ferment.

[0077] Comparative Example 5

[0078] The difference from Example 1 is that the fermentation strain is different; the mixed strain is replaced with a single strain of Lactobacillus reuteri for fermentation.

[0079] Comparative Example 6

[0080] The difference from Example 1 is that the fermentation strain is different; the mixed strain is replaced with Aspergillus oryzae single-strain fermentation.

[0081] Comparative Example 7

[0082] The difference from Example 1 is that the fermentation strain is different; the mixed strain is replaced with Bacillus subtilis single-strain fermentation.

[0083] Comparative Example 8

[0084] The difference from Example 1 is that the fermentation strain is different; Lactobacillus reuteri is replaced with Lactobacillus plantarum, while the rest is the same as Example 1.

[0085] Test Example: Test for Detection of Catalpol Content

[0086] The content of catalpol in the fermentation broth of Rehmannia glutinosa fermented food prepared in Examples 1-2 and Comparative Examples 1-8 was tested. The fermentation broth of Rehmannia glutinosa was taken and detected by high performance liquid chromatography. The test results are shown in Table 1.

[0087] Table 1. Catasolic alcohol content (%)

[0088]

[0089] The results in Table 1 show that the Rehmannia glutinosa fermentation liquid prepared in Examples 1-2 of the present invention has a high catalpol content. Compared with Comparative Examples 1-8, the present invention has a better effect on increasing the catalpol content in Rehmannia glutinosa fermented food.

[0090] As can be seen from the above, the main raw materials of the Rehmannia glutinosa fermented food with high catalpol content prepared by the present invention are fresh Rehmannia glutinosa, Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis. By optimizing the preparation process, the prepared Rehmannia glutinosa fermented food has a high catalpol content. Comparative Example 1 replaced fresh Rehmannia glutinosa with raw Rehmannia glutinosa; Comparative Example 2 replaced fresh Rehmannia glutinosa with processed Rehmannia glutinosa; Comparative Example 3 changed the pulsed electric field-ultrasound combined treatment to high temperature sterilization treatment; Comparative Example 4 removed the fermentation process; Comparative Examples 5-7 changed the mixed bacteria to Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis single-strain fermentation respectively, while keeping the total bacterial count unchanged; Comparative Example 8 replaced Lactobacillus reuteri with Lactobacillus plantarum. The catalpol content in the fermentation broth of the above comparative examples all decreased significantly, indicating that the Rehmannia glutinosa fermented food prepared by the raw materials, raw material pretreatment method, and preparation method of the present invention has a high catalpol content.

[0091] Further analysis and speculation: Compared with Comparative Examples 1 and 2, it shows that using fresh Rehmannia glutinosa as raw material is the basis for ensuring catalpol content; compared with Comparative Example 3, it shows that "pulsed electric field-ultrasound combined treatment" can effectively improve catalpol dissolution and retention, which is superior to traditional high-temperature sterilization treatment; compared with Comparative Example 4, it shows that the fermentation process of this invention can further improve catalpol content, while the catalpol content is lower without fermentation; compared with Comparative Examples 5-7, it shows that the strain combination of "Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis mixed fermentation in a 6:2:1 ratio" is superior to single-strain fermentation in terms of catalpol retention; compared with Comparative Example 8, it shows that "Lactobacillus reuteri LR08" is the key strain in the mixed strain, and the catalpol content decreases significantly after being replaced with other lactobacilli.

[0092] In summary, this invention, through the selection of specific raw materials, combined pulsed electric field-ultrasound pretreatment, mixed strain fermentation, and precise parameter control, can effectively increase the catalpol content in Rehmannia glutinosa fermented foods, demonstrating significant technical advantages.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0094] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A fermented food product containing high-purine Rehmannia glutinosa, characterized in that, The food is prepared by fermentation of fresh Rehmannia glutinosa, Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis.

2. The fermented Rehmannia glutinosa food as described in claim 1, characterized in that, The food is prepared by fermentation of fresh Rehmannia glutinosa, Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis, and the content of catalpol in the food is ≥0.35%.

3. A method for preparing the high-calcium ol Rehmannia glutinosa fermented food as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Select fresh Rehmannia glutinosa, and clean and air-dry it; S2. Cut the dried fresh Rehmannia glutinosa into small pieces and add deionized water to them; S3. The mixture of fresh Rehmannia glutinosa and deionized water is placed in a pulsed electric field-ultrasound combined treatment device for treatment; S4. Activate Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis; S5. Transfer the pretreated fresh Rehmannia glutinosa slurry into a fermenter and inoculate it with the activated strain for fermentation; S6. Detect the catalpol content in the fermentation broth, and stop fermentation after the termination condition is met; S7. Dry the fermentation product in an oven to obtain the fermented Rehmannia glutinosa product.

4. The preparation method according to claim 3, characterized in that, In step S2, the thickness of the fresh Rehmannia glutinosa segments is 0.5-1.8 cm, and the amount of deionized water added meets the material-to-liquid ratio (mass of fresh Rehmannia glutinosa: volume of deionized water) of 1:3-1:6 g / mL.

5. The preparation method according to claim 3, characterized in that, In step S3, the pulse electric field processing parameters are: electric field strength 25-30kV / cm, pulse width 20-30μs, pulse frequency 50-60Hz, and processing time 3-5min; the ultrasonic processing parameters are: power 300-400W and frequency 40-50kHz.

6. The preparation method according to claim 3, characterized in that, In step S4, the activation conditions for Lactobacillus reuteri are: inoculated into MRS medium, anaerobic culture at 37°C for 24 h, and continuously activated for 2 generations; the activation conditions for Aspergillus oryzae are: inoculated into PDA medium, aerobic culture at 30°C for 48 h, and spore suspension is prepared.

7. The preparation method according to claim 3, characterized in that, In step S5, the inoculation ratio of Lactobacillus reuteri, Aspergillus oryzae, and Bacillus subtilis is 6:2:1, and the total inoculation amount is 5-8% (v / v).

8. The preparation method according to claim 3, characterized in that, In step S5, the fermentation parameters are controlled as follows: initial pH 5.5-6.0; temperature 30-32℃ and oxygen partial pressure 30-35% for the first 12 hours; temperature 35-37℃ and oxygen partial pressure 15-20% for 12-24 hours; temperature 32-34℃ and oxygen partial pressure 10-15% for 24-48 hours; stirring rate 60-80 r / min; pH is checked every 8 hours; if pH < 5.0, it is adjusted to 5.0-5.5 with 0.1 mol / L Na2CO3.

9. The preparation method according to claim 3, characterized in that, In step S6, the catalpol content is detected every 24 hours starting from the second day of fermentation. Fermentation is terminated when the catalpol content increases by less than 1% twice consecutively and shows no downward trend. In step S7, the oven drying temperature is 60℃.

10. The use of a fermented Rehmannia glutinosa food product as described in claim 1 or 2, or a product prepared by any of the preparation methods described in claims 3-9, in the preparation of foods with antioxidant, anti-inflammatory, or immunomodulatory functions.

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