Preparation method of oral liquid of fermented and processed caulis ephedrae

Abstract

The present application belongs to the field of traditional Chinese medicine processing and microbial fermentation technology, and particularly relates to a preparation method of a fermented oral liquid of leaf of caesia. The present application uses caesia dry leaves and wild chrysanthemum as a basic culture medium, and obtains special fermentation strains through an induction training program. The metabolic activity of the strains is more conducive to the full use and release of effective components in the medicinal material tissue and cells during the fermentation process, and a series of bioactive components can be produced. The prepared caesia oral liquid adds bioactive products and flavor substances after lactic acid bacteria fermentation, the taste is improved, and the caesia oral liquid has multiple health care functions and natural flavor.

Description

Technical Field

[0001] This invention relates to the fields of processing and preparation of traditional Chinese medicinal materials and microbial fermentation technology, specifically to a method for preparing an oral liquid made from fermented and processed leaves of *Gynostemma pentaphyllum*. Background Technology

[0002] Oral liquid preparations are sterile or semi-sterile oral liquid formulations made from traditional Chinese medicine decoctions. These preparations are based on decoctions, extracting the active ingredients, adding flavoring agents, antibacterial agents, and other additives, and then filling them using the same ampoule filling process as injections. Therefore, they are also called oral ampoules. Their production generally employs mature and traditional preparation processes. In particular, the health-promoting oral liquid based on water-soluble traditional Chinese medicine decoctions, as described in this invention, involves washing and soaking two kinds of Chinese medicinal herbs, decocting them three times, combining the decoctions, concentrating them by heating, and then further processing, filtering, and filling. It is evident that this production process, involving three decoctions with added water, only yields some water-soluble components, resulting in insufficient utilization. Furthermore, the combined decoctions require further heating and vacuum distillation to concentrate them to approximately one-sixth of their original volume. Therefore, this process consumes a significant amount of energy. In order to overcome the shortcomings of the above-mentioned oral liquid production methods, the invention of "a method for preparing oral liquid by fermentation of wild golden firewood leaves" was developed. This invention is based on the following theories and discoveries: 1. The number of lactic acid bacteria that are safe and beneficial to the human body is huge, and their heredity and variation are constantly occurring, making them highly adaptable to the environment. Therefore, by homogenizing and crushing the leaves of *Gynostemma pentaphyllum* and wild chrysanthemum to make a medicinal culture solution, and by gradually increasing the proportion of this medicinal culture solution in the MRS culture solution, a lactic acid bacteria strain that is tolerant to *Gynostemma pentaphyllum* and wild chrysanthemum and can use these two medicinal materials as its own growth culture medium can be induced and trained. 2. During the production fermentation process, the process of water soaking, homogenizing and crushing, sterilizing at 121℃ for 20 minutes and fermenting at 37℃ for 30-36 hours is also a process of accelerated decoction, extraction, bacterial metabolism degradation and release of medicinal components of Chinese medicinal materials under high temperature and high pressure, without changing the efficacy and safety. 3. During the fermentation process, 0.02% magnesium sulfate, 0.5% sucrose and 0.2% corn steep liquor powder (all food-grade materials) are added to compensate for the lack of nutrients in the medicinal materials as a fermentation culture medium and to protect the polysaccharides and total flavonoids of the medicinal materials. Through a series of life activities such as growth, metabolism, synthesis, emulsification, rearrangement, transformation, and accumulation of lactic acid bacteria, the tissues and cellular components of medicinal materials are released and utilized more fully and comprehensively than those extracted and decocted with water; 4. Lactic acid and unique flavor substances are synthesized; the flavonoids contained in the medicinal materials are converted into flavonoid aglycones that are more easily absorbed and utilized by the human body; lactic acid bacteria synthesize and secrete extracellular polysaccharides (whose degradation temperature Td is at least 290.44℃, higher than the conventional processing and sterilization temperature of food). Therefore, while maintaining the functions of the two medicinal materials, this oral liquid also adds bioactive products and flavor substances after lactic acid bacteria fermentation, improving the taste and providing multiple health benefits and natural flavor. Therefore, it can improve product production efficiency and active ingredients; 5. Combining the above four points, the method of lactic acid bacteria fermentation of these two traditional Chinese medicines is superior to their traditional oral liquid production methods. Summary of the Invention

[0003] This invention provides a lactic acid bacteria strain. The strain is produced by gradually increasing the proportion of the medicinal herb culture solutions of dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum in the MRS culture medium from 0:1 to 1:3, then to 1:1, 2:1, 3:1, 4:1 to 1:0, until they completely replace each other. This induces and trains a lactic acid bacteria strain that is tolerant to *Gynostemma pentaphyllum* and wild chrysanthemum and can use these two herbs as its own growth medium.

[0004] Preferably, the strain is used in the preparation of fermented oral liquid made from wild goldenrod leaves.

[0005] Preferably, the preparation method of the application includes the following steps:

[0006] (1) After removing impurities, washing and soaking the dried leaves of wild golden firewood and wild chrysanthemum in hot water, add MgSO4·7H2O, sucrose and corn syrup powder, mix them together and pour them into a 100L fermentation tank and add water to 52L. Stir evenly, heat to 121℃ for 20 minutes for sterilization, cool to 37℃, add the condensate produced by steam sterilization, and control the culture medium at 60L.

[0007] (2) Inoculate the culture medium of step (1) with activated lactic acid bacteria culture medium, stir at 120 rpm for 1 to 5 minutes, ferment, sample and examine the fermentation broth under a microscope, and stop fermentation when the pH value reaches 4.0 to 4.5;

[0008] (3) The fermentation broth from step (2) is filtered through 80-120 mesh to obtain 56L of filtrate. The phlorizin content of the filtrate is then tested.

[0009] (4) The filtrate from step (3) is prepared and further filtered to obtain 52L of fine filtrate. It is then filled into ampoules at 10ml / bottle, sterilized at 115℃ for 20 minutes, packaged, and inspected to obtain the finished oral liquid product.

[0010] Preferably, in step (1), there are 4200g of dried leaves of wild golden firewood, 2400g of wild chrysanthemum, 10-14g of MgSO4·7H2O, 280-320g of sucrose, and 100-140g of corn syrup powder.

[0011] Preferably, in step (2), the culture medium is inoculated with lactic acid bacteria activated inoculum. This is because the dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum are fermented and processed through the growth, metabolism, synthesis, emulsification, rearrangement, transformation and accumulation of lactic acid bacteria, which allows the dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum to be released and utilized more fully and comprehensively than by water extraction and decoction.

[0012] Preferably, in step (2), the amount of activated lactic acid bacteria culture solution added is 3.3–5 L, and the viable bacteria count is 1 × 10⁻⁶. 8 ~3×108 CFU / mL.

[0013] Preferably, the fermentation in step (2) is carried out at a constant temperature of 30-37°C for 40-32 hours, with intermittent stirring 3-8 times during the period.

[0014] Preferably, the oral liquid, while ensuring that the prescription and efficacy remain basically unchanged, adds bioactive products and flavor substances produced by lactic acid bacteria fermentation, thus possessing multiple health benefits and a natural flavor.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1) The raw materials used in this process remain basically unchanged. 93.86% of the fermentation culture medium is composed of the Chinese medicinal herbs themselves, and 6.14% is composed of nutrients and growth factors to supplement the fermentation bacteria. Therefore, the formula and efficacy of the oral liquid can be kept basically unchanged.

[0017] 2) During the fermentation and processing of wild chrysanthemum leaves and wild chrysanthemum, the two Chinese medicinal herbs undergo homogenization, sterilization at 121℃ for 20 minutes, and constant-temperature fermentation at 37℃ for 30-36 hours, replacing the traditional decoction process. In particular, during the fermentation and processing of Chinese medicinal herbs, the tissues and cellular components of the herbs are affected by a series of life activities such as growth, metabolism, synthesis, emulsification, rearrangement, transformation, and accumulation of lactic acid bacteria, resulting in a more complete and comprehensive release and utilization than water extraction and decoction. This includes an increase in phlorizin content, the conversion of flavonoids contained in the herbs into flavonoid aglycones that are more easily absorbed and utilized by the human body, the synthesis and secretion of extracellular polysaccharides (with good thermal stability), the synthesis of lactic acid, and unique flavor substances. Therefore, the oral liquid produced by this invention has added bioactive products and flavor substances after lactic acid bacteria fermentation, improved taste, and multiple health care functions and natural flavor.

[0018] 3) The strains used in fermentation are lactic acid bacteria that have undergone special training and induction. They are edible strains and there are no biosafety issues.

[0019] 4) Based on the content of the active ingredient in the product—phlorizin, the fermentation broth can be used as an oral liquid after filtration, without the need for concentration. Detailed Implementation

[0020] The invention will be further illustrated below with reference to examples.

[0021] Example 1

[0022] Induction and training steps of production strains

[0023] 1) Preparation of culture medium for two Chinese medicinal materials: Weigh 42g of dried leaves of wild golden firewood and 24g of wild chrysanthemum, wash them with water, soak them in hot water until they expand, then homogenize them together and add water to make up to 600ml.

[0024] 2) Preparation of medicinal herb culture medium and MRS mixed culture medium in different proportions; ① 0:1 culture medium: take only 100ml of MRS culture medium (a special culture medium for lactic acid bacteria), ② 1:3 culture medium: mix 25ml of medicinal herb culture medium and 75ml of MRS culture medium, ③ 1:1 culture medium: mix 50ml of medicinal herb culture medium and 50ml of MRS culture medium, ④ 2:1 culture medium: mix 60ml of medicinal herb culture medium and 30ml of MRS culture medium, ⑤ 3:1 culture medium: mix 75ml of medicinal herb culture medium and 25ml of MRS culture medium, ⑥ 4:1 culture medium: mix 80ml of medicinal herb culture medium and 20ml of MRS culture medium, ⑦ 1:0 culture medium: take only 100ml of medicinal herb culture medium;

[0025] 3) Stepwise induction and acclimatization: Inoculate the lactic acid bacteria into culture medium ① from step 2), and incubate at 37°C for 30–36 hours until the pH value is less than 4.5. Then, transfer the bacteria to culture medium ② at a 3% inoculation rate, and incubate at 37°C until the pH value is less than 4.5. Then, transfer the bacteria to culture medium ③ at a 3% inoculation rate, and so on, stepwise transferring the bacteria to culture media ④, ⑤, and ⑥ until the strain has adapted to growth in culture medium ⑦, and the pH value can be less than 4.5 after incubation at 37°C for 30–36 hours. This process induces and acclimates a lactic acid bacteria strain that is tolerant to *Gynostemma pentaphyllum* (phlorizin) and *Chrysanthemum indicum* (chlorogenic acid and volatile oils) and can utilize these two medicinal materials as components of its own growth medium. Then, streak the culture medium ⑦ to isolate single colonies of the lactic acid bacteria and prepare fermentation and preservation strains.

[0026] Example 2

[0027] 1) After removing impurities, washing, and soaking 4200g of dried leaves of *Gynostemma pentaphyllum* and 2400g of wild chrysanthemum flowers in hot water, 12g of MgSO4·7H2O, 300g of sucrose, and 120g of corn steep liquor powder (all food-grade ingredients) are added. The mixture is homogenized and poured into a 100L fermentation tank, with water added to the 52L mark. The mixture is stirred thoroughly, heated to 121℃ for sterilization for 20 minutes, cooled to 37℃, and the condensate from the steam sterilization is added, maintaining the culture medium at approximately 60L. This process also accelerates the decoction of the medicinal herbs.

[0028] 2) Inoculate the culture medium from step 1) with 3.3 liters of fermentation inoculum, with a viable count of 1–3 × 10⁻⁶ cells / day. 8 CFU / mL, stir at 120 rpm for 1 minute, and ferment at a constant temperature of 37℃ for 36 hours, with intermittent stirring 3 times during the period. Sample and examine the fermentation broth under a microscope. Fermentation can be stopped when the pH value reaches 4.5.

[0029] 3) Filter the fermentation broth from step 2) above through 80-120 mesh to obtain about 56 liters of filtrate. The phlorizin content of the filtrate is 5600 mg / L. As a control, the phlorizin content of the culture medium that has not undergone fermentation is 5040 mg / L.

[0030] 4) The filtrate from step 3) above is prepared and further filtered to obtain approximately 52 liters of fine filtrate. It is then filled into ampoules at a rate of 10 ml / bottle, sterilized at 115°C for 20 minutes, packaged, and inspected to obtain the finished oral liquid product.

[0031] Based on the phlorizin content detection results of Specific Example 1, the phlorizin content before and after fermentation differed by approximately 11.11%. This demonstrates that the fermentation processing technology of this invention has a significant effect on enhancing the effective components of the health oral liquid.

[0032] Example 3

[0033] 1) After removing impurities, washing, and soaking 4200g of dried leaves of *Gynostemma pentaphyllum* and 2400g of wild chrysanthemum flowers in hot water, 12g of MgSO4·7H2O, 300g of sucrose, and 120g of corn steep liquor powder (all food-grade ingredients) are added. The mixture is homogenized and poured into a 100L fermentation tank, with water added to the 52L mark. The mixture is stirred thoroughly, heated to 121℃ for sterilization for 20 minutes, cooled to 37℃, and the condensate from the steam sterilization is added, maintaining the culture medium at approximately 60L. This process also accelerates the decoction of the medicinal herbs.

[0034] 2) Inoculate the culture medium from step 1) with 3.3 liters of fermentation inoculum, with a viable count of 1–3 × 10⁻⁶ cells / day. 8 CFU / mL, stir at 120 rpm for 1 minute, and ferment at a constant temperature of 37℃ for 30 hours, with intermittent stirring 3 times during the period. Sampling and microscopic examination of the fermentation broth until the pH value reaches 4.5 can end the fermentation.

[0035] 3) Filter the fermentation broth from step 2) above through 80-120 mesh to obtain about 56 liters of filtrate. The phlorizin content of the filtrate was found to be 4200 mg / L. As a control, the phlorizin content of the culture medium that has not undergone fermentation was 3790 mg / L.

[0036] 4) The filtrate from step 3) is prepared and further filtered to obtain approximately 52 liters of refined filtrate. This refined filtrate is then bottled into ampoules at 10 ml / bottle, sterilized at 115°C for 20 minutes, packaged, and inspected to obtain the finished oral liquid product. From the phlorizin content test results of specific Example 3, the phlorizin content before and after fermentation differs by approximately 10.82%. This demonstrates that the fermentation processing technology of this invention significantly enhances the effective components of this health-promoting oral liquid.

Claims

1. A method for preparing a fermented oral liquid from *Gynostemma pentaphyllum* leaves, characterized in that: Includes the following steps: (1) After removing impurities, washing and soaking the dried leaves of wild golden firewood and wild chrysanthemum in hot water, add MgSO4·7H2O, sucrose and corn syrup powder, mix them together and pour them into a 100L fermentation tank and add water to 52L. Stir evenly, heat to 121℃ for 20 minutes for sterilization, cool to 37℃, add the condensate produced by steam sterilization, and control the culture medium at 60L. (2) Inoculate the culture medium of step (1) with activated lactic acid bacteria culture medium, stir at 120 rpm for 1 to 5 minutes, ferment, sample and examine the fermentation broth under a microscope, and stop fermentation when the pH value reaches 4.0 to 4.5; (3) The fermentation broth from step (2) is filtered through 80-120 mesh to obtain 56L of filtrate. The phlorizin content of the filtrate is then tested. (4) The filtrate from step (3) is prepared and then finely filtered to obtain 52L of fine filtrate. It is then filled into ampoules at 10ml / bottle, sterilized at 115℃ for 20 minutes, packaged, and inspected to obtain the finished oral liquid product. The strain in the activated lactic acid bacteria inoculum was obtained by gradually increasing the proportion of the medicinal culture of dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum in the MRS culture medium until it completely replaced the original culture medium by mixing the medicinal culture medium of dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum with the MRS culture medium from 0:1 to 1:3, then to 1:1, 2:1, 3:1, 4:1 to 1:0, thereby inducing and cultivating a lactic acid bacteria strain that is tolerant to dried leaves of *Gynostemma pentaphyllum* and wild chrysanthemum and can use these two medicinal materials as its own growth medium components. In step (1), 4200g of dried leaves of wild golden firewood, 2400g of wild chrysanthemum, 10-14g of MgSO4·7H2O, 280-320g of sucrose, and 100-140g of corn syrup powder are used.

2. The preparation method according to claim 1, characterized in that: In step (2), the amount of activated lactic acid bacteria culture solution added is 3.3–5 L, and the viable bacteria count is 1 × 10⁻⁶. 8 ~3×10 8 CFU / mL.

3. The preparation method according to claim 1, characterized in that: The fermentation in step (2) is carried out at a constant temperature of 30-37°C for 40-32 hours, with intermittent stirring 3-8 times during the period.