A method for improving the content of active ingredients in taizheng fermentation, fermentation liquid and application

Abstract

This invention discloses a method for fermenting *Tai Sui* (a type of fungus) to increase the content of active ingredients, as well as the fermentation broth and its applications. The method involves immersing fresh *Tai Sui* in an aqueous solution of fructooligosaccharides, or by adding an appropriate amount of compound enzyme to the immersion solution for fermentation. The resulting fermentation broth is then filtered and sterilized. This invention features a short preparation cycle, is environmentally friendly, and yields a high content of active ingredients in the fermented *Tai Sui* broth.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation, specifically relating to a fermentation method for increasing the content of active ingredients in *Smilax china*, as well as the fermentation broth and its applications. Background Technology

[0002] Tai Sui ( Ganoderma lucidum It is a very rare "extra-large rare slime mold complex" in nature, rich in polysaccharides, amino acids, nucleic acids, minerals, pyrroloquinoline quinone (PQQ) and other active ingredients.

[0003] Currently, one common way to utilize *Tai Sui* (a type of fungus) is to make it into *Tai Sui water* for drinking. However, traditional methods of making *Tai Sui water* mostly involve simple soaking, which has the following problems: First, the soaking process is simple and inefficient, not only because the soaking time is too long, but also because the content of active ingredients in the *Tai Sui water* is low, affecting its efficacy; second, the soaking process is prone to microbial contamination, affecting the safety and shelf life of the *Tai Sui water*. Therefore, there is an urgent need to develop a scientific, efficient, and safe method for making *Tai Sui water* by soaking it to fully realize its value. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, this invention provides a fermentation method, fermentation broth, and application for increasing the content of active ingredients in *Smilax china* (a type of fungus). This method utilizes a specific complex enzyme to significantly increase the content of active ingredients after fermentation at low temperatures. Specifically, this invention includes the following:

[0005] A first aspect of the present invention provides a method for fermenting *Tai Sui* (a type of fungus) to increase the content of active ingredients, comprising the following steps:

[0006] Step 1: Prepare a 0.5wt%-1.5wt% oligofructose aqueous solution in a sterile container, and immerse the fresh *Pterocarya stenoptera* in the oligofructose aqueous solution at a weight ratio of 1:5-1:10, and soak at room temperature for 12-48 hours; then raise the temperature to 30℃-35℃, stir, and maintain for 12-72 hours.

[0007] Step 2: Filter to obtain the original fermented liquid of *Smilax china*.

[0008] Step 3: The Tai Sui fermentation liquid stock solution is pasteurized at 60℃-65℃ for at least 5 minutes.

[0009] In some embodiments, after soaking at room temperature for 12-48 hours as described in step 1, the complex enzyme is added, and the temperature is raised to 30℃-35℃, stirred, and maintained for 12-72 hours.

[0010] In some implementations, the stirring described in step 1 is performed every 6-8 hours, with the stirring speed controlled at 50-100 rpm and the stirring time at 5-10 minutes.

[0011] In some implementations, the filtration described in step 2 involves first performing coarse filtration using a plate and frame filter to remove larger particulate impurities, and then performing fine filtration using a membrane filtration device with a pore size of 0.2 μm to 0.5 μm.

[0012] In some embodiments, the water used to prepare the fructooligosaccharides in step 1 is purified water that has undergone reverse osmosis treatment, has a conductivity of no more than 5 μS / cm, and a pH value between 6.5 and 7.5.

[0013] In some embodiments, the amount of the compound enzyme is 0.02-0.5% of the weight of the fresh *Smilax china*.

[0014] In some embodiments, the complex enzyme consists of six enzymes: carboxypeptidase A, carboxypeptidase G, neutral protease, β-1,3-glucanase, β-1,6-glucanase, and β-glucosidase.

[0015] In some embodiments, the complex enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 200 IU neutral protease, 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase and 200 IU β-glucosidase.

[0016] A second aspect of the present invention is to provide a Tai Sui fermentation liquid, which is prepared by any of the above-described embodiments.

[0017] A third aspect of the present invention provides the application of the above-mentioned Tai Sui fermentation liquid in the pharmaceutical field.

[0018] The beneficial effects of this invention include:

[0019] 1. Highly efficient dissolution: PQQ is a novel type of water-soluble B vitamin, hailed as the "biological treasure"; it can promote human growth, enhance liver function, improve memory, and bidirectionally regulate free radicals. This invention significantly increases the content of PQQ and other active ingredients in the fermentation liquid of *Taisui* by adding fructooligosaccharides, or simultaneously adding compound enzymes, to the soaking solution.

[0020] 2. Reduced time: Compared with traditional preparation methods that require several weeks or even months, the soaking cycle of this invention is shortened to 1-5 days, which greatly improves production efficiency and is conducive to large-scale industrial production.

[0021] 3. Superior Quality: Precise temperature control and rigorous sterilization processes effectively inhibit the growth and reproduction of microorganisms, ensuring the safety and stability of the Tai Sui fermentation liquid and extending its shelf life. Simultaneously, the filtration and sterilization processes maximize the retention of active ingredients, allowing the Tai Sui fermentation liquid to fully exert its efficacy.

[0022] 4. Green and environmentally friendly: The raw materials and processes used in this method meet the requirements of green and environmental protection, do not add harmful chemicals, are environmentally friendly, and conform to modern health and environmental protection concepts. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Example

[0027] In the experimental examples of this invention, *Tai Sui* (a type of fungus) was used. High-quality *Tai Sui* from Alxa League, Inner Mongolia, were selected that were firm, smooth, uniform in color, and free from mold. Their polysaccharide content needed to be ≥15%, protein content ≥8%, and they had been tested by a professional institution to ensure they had no pathogenic bacteria or excessive heavy metals. The *Tai Sui* were rinsed with sterile water to remove attached impurities and microorganisms, and then dried for later use.

[0028] In the embodiments of this invention, the containers used for soaking *Tai Sui* are all food-grade glass or ceramic containers to ensure that the containers are clean and sterile.

[0029] The fructooligosaccharides, protein hydrolysates, and polysaccharide hydrolysates used in the embodiments of this invention are all food-grade.

[0030] In this embodiment of the invention, the water used to dissolve the fructooligosaccharides is purified water that has undergone reverse osmosis treatment, with a conductivity not exceeding 5 μS / cm and a pH value between 6.5 and 7.5.

[0031] In this embodiment of the invention, the NBT-Gly method was used to determine the content of pyrroloquinoline quinone (PQQ) in the fermentation broth of *Smilax china*. The phenol-sulfuric acid method was used to determine the polysaccharide content. A colorimetric method was used to determine the free amino acid content. High-performance liquid chromatography (HPLC) was used to determine the glucose content.

[0032] In this invention, the concentration of the fructooligosaccharide aqueous solution can be selected from 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, or 1.5wt%, preferably 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, or 1.2wt%, and most preferably 1.0wt%.

[0033] In this invention, the weight ratio of fresh *Tai Sui* to the above-mentioned fructooligosaccharide aqueous solution can be selected as 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, preferably 1:6, 1:7, 1:8 or 1:9, and most preferably 1:8.

[0034] In this invention, fresh *Pterocarya stenoptera* is immersed in an oligofructose aqueous solution for an unlimited time at room temperature. The main purpose is to allow it to gradually adapt to the immersion environment. Preferably, the immersion time is 12-48 hours, for example, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48 hours.

[0035] In this invention, after the fresh *Tai Sui* (a type of fungus) has adapted to the soaking environment, the temperature is raised to 30℃-35℃ and maintained for an unlimited time. The main purpose is to allow the effective components in the *Tai Sui* to gradually dissolve, and for the dissolved polysaccharides and proteins to further degrade and biosynthesize into more effective components under the soaking environment. Preferably, the soaking time is 12-72 hours, for example, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, or 72 hours.

[0036] The composite enzyme of this invention is a composite enzyme developed for the specific composition of *Saccharomyces cerevisiae*, and includes a variety of specific enzymes. Exemplarily, the composite enzyme of this invention includes proteolytic enzymes and polysaccharide hydrolytic enzymes. Exemplarily, the proteolytic enzymes can produce a large number of amino acids, particularly the combination of carboxypeptidase A and G and neutral protease, which can produce beneficial amino acids, providing the necessary nitrogen source for *Saccharomyces cerevisiae*. In this invention, β-1,3-glucanase and β-1,6-glucanase are mainly used to produce small-molecule glucans, which are further converted into free glucose by β-glucosidase, providing the necessary carbon source for *Saccharomyces cerevisiae*. In summary, the composite enzyme of this invention, through synergistic action, achieves the hydrolysis of proteins and polysaccharides, yielding short peptides, small-molecule free amino acids, and small-molecule polysaccharides. Simultaneously, the small-molecule substances obtained from hydrolysis can also serve as nutrients for *Saccharomyces cerevisiae*, enhancing the biosynthesis of the small-molecule active ingredient pyrroloquinoline quinone.

[0037] In this invention, the amount of compound enzyme used is not limited, but is generally less than 1% of the weight of fresh *Ulva lactucis*, preferably less than 0.5%. If the amount is too high, it may adversely affect the activity of the *Ulva lactucis* itself. On the other hand, if the amount is too low, the efficacy of the resulting *Ulva lactucis* fermentation broth tends to decrease. Exemplary amounts include, for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10%.

[0038] In this invention, the pore size of the filter membrane in step 2 can be selected from 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm or 0.5μm, preferably 0.3μm, 0.35μm or 0.4μm, and most preferably 0.35μm.

[0039] Examples 1-3

[0040] Prepare an oligofructose aqueous solution of a specified concentration in a sterile container; immerse the fresh *Tai Sui* (a type of fungus) in the above oligofructose aqueous solution at a weight ratio of 1:5, and soak at room temperature for 24 hours to allow the *Tai Sui* to gradually adapt to the soaking environment; then raise the temperature to 35°C, stir every 6 hours at a stirring speed of 100 rpm for 10 minutes, and maintain for 12 hours.

[0041] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a 0.2μm pore size membrane filter is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 5 minutes.

[0042] Comparative Example 1

[0043] Fresh Tai Sui was immersed in a sterile container at a weight ratio of 1:5 to water and soaked at room temperature for 24 hours to allow the Tai Sui to gradually adapt to the soaking environment. Then the temperature was raised to 35°C and stirred every 6 hours at a speed of 100 rpm for 10 minutes, and kept for 12 hours.

[0044] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.2μm is used for fine filtration to obtain the Tai Sui water stock solution. The above Tai Sui water stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 5 minutes.

[0045] Table 1. Effect of adding fructooligosaccharides on PQQ content

[0046]

[0047] As shown in Table 1, compared with the comparative example, adding an appropriate amount of fructooligosaccharides to the soaking water can increase the PQQ content, and the peak value is reached when the concentration is 1.0 wt%. Further increasing the concentration has no substantial effect on the increase of PQQ.

[0048] The following experiment used 1.0 wt% fructooligosaccharide.

[0049] Example 4

[0050] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container; immerse the fresh *Tai Sui* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8 and soak at room temperature for 24 hours to allow the *Tai Sui* to gradually adapt to the soaking environment; then raise the temperature to 35°C and stir every 7 hours at a stirring speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours.

[0051] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0052] Example 5

[0053] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8 at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.05% of the weight of the *Pterocarya stenoptera* compound enzyme, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The compound enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 200 IU neutral protease, 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase, and 200 IU β-glucosidase.

[0054] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0055] Example 6

[0056] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8 at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.1% of the weight of the *Pterocarya stenoptera* to the solution, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The complex enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 200 IU neutral protease, 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase, and 200 IU β-glucosidase.

[0057] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0058] Comparative Example 2

[0059] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8, and soak at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.05% of the weight of the *Pterocarya stenoptera* compound enzyme, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The compound enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase, and 200 IU β-glucosidase.

[0060] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0061] Comparative Example 3

[0062] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8, and soak at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.05% of the weight of the *Pterocarya stenoptera* compound enzyme, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The compound enzyme consists of 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase, and 200 IU β-glucosidase.

[0063] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0064] Comparative Example 4

[0065] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8, and soak at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.05% of the weight of the *Pterocarya stenoptera* compound enzyme, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The compound enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 200 IU neutral protease, and 200 IU β-glucosidase.

[0066] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0067] Comparative Example 5

[0068] Prepare a 1.0 wt% fructooligosaccharide aqueous solution in a sterile container. Immerse the fresh *Pterocarya stenoptera* (a type of fungus) in the above fructooligosaccharide aqueous solution at a weight ratio of 1:8, and soak at room temperature for 24 hours to allow the *Pterocarya stenoptera* to gradually adapt to the soaking environment. Then, add 0.05% of the weight of the *Pterocarya stenoptera* compound enzyme, and raise the temperature to 35°C. Stir every 7 hours at a speed of 50 rpm for 5 minutes, maintaining this temperature for 24 hours. The compound enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, and 200 IU neutral protease.

[0069] First, a plate and frame filter press is used for coarse filtration to remove larger particulate impurities. Then, a membrane filtration device with a pore size of 0.5μm is used for fine filtration to obtain the Tai Sui fermentation broth stock solution. The above Tai Sui fermentation broth stock solution is sterilized by low-temperature pasteurization at 60℃-65℃ for 10 minutes.

[0070] Table 2. Results of substance content in Tai Sui fermentation broth

[0071]

[0072] Note: - indicates that no detection was detected.

[0073] As can be seen from Table 2, the composite enzyme of the present invention can not only produce a large amount of beneficial components such as free amino acids and polysaccharides, but more importantly, the composite enzyme of the present invention can greatly increase the content of PQQ.

[0074] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for increasing the content of active ingredients in a Gynostemma pentaphyllum fermentation, characterized in that, The active ingredient includes pyrroloquinoline quinone, and the method includes the following steps: Step 1: Prepare a 0.5wt%-1.5wt% fructooligosaccharide aqueous solution in a sterile container. Soak the fresh *Pterocarya stenoptera* (a type of fungus) in the fructooligosaccharide aqueous solution at a weight ratio of 1:5 to 1:10, and soak at room temperature for 12-48 hours. Add a compound enzyme, then raise the temperature to 30℃-35℃, stir, and maintain for 12-72 hours. The amount of the compound enzyme is 0.02-0.5% of the weight of the fresh *Pterocarya stenoptera*, and the compound enzyme consists of 100 IU carboxypeptidase A, 100 IU carboxypeptidase G, 200 IU neutral protease, 100 IU β-1,3-glucanase, 100 IU β-1,6-glucanase, and 200 IU β-glucosidase. Step 2: Filter to obtain the original Tai Sui fermentation liquid; Step 3: The Tai Sui fermentation liquid stock solution is pasteurized at 60℃-65℃ for at least 5 minutes.

2. The method of claim 1, wherein, The stirring described in step 1 is to stir once every 6-8 hours, with the stirring speed controlled at 50-100 rpm, and each stirring time lasting 5-10 minutes.

3. The method according to claim 1, characterized in that, The filtration described in step 2 involves first using a plate and frame filter press for coarse filtration to remove larger particulate impurities, and then using a membrane filtration device with a pore size of 0.2μm to 0.5μm for fine filtration.

4. The method according to claim 1, characterized in that, The water used to prepare the fructooligosaccharides in step 1 is purified water that has undergone reverse osmosis treatment, with a conductivity of no more than 5 μS / cm and a pH value between 6.5 and 7.5.

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