Cistanche enzyme and preparation method thereof

Abstract

The invention discloses cistanche enzyme and a preparation method thereof, and belongs to the technical field of food fermentation. The problems that in existing cistanche enzyme preparation, active ingredients are seriously lost, flora metabolism is unbalanced, the quality is unstable, and the flavor is poor are solved. A specific raw material formula and a specific preparation process are adopted; the raw materials comprise 30-50 parts of herba cistanche, 8-20 parts of an antioxidant protective agent consisting of at least two of fructus lycii and the like, and 10-25 parts of a flora adaptive slow-release carbon source consisting of at least two of red dates and the like, and are matched with a compound fermentation strain of lactobacillus plantarum, saccharomyces cerevisiae and acetic acid bacteria; the preparation process comprises the steps of segmented temperature control and dark fermentation, accurate pH adjustment, standard aging and low-temperature sterilization and sterile filling. The total content of phenylethanoid glycoside components is larger than or equal to 0.5 mg / mL, the loss rate is smaller than or equal to 10%, the activity of superoxide dismutase is larger than or equal to 100 U / mL, the viable count of lactic acid bacteria is larger than or equal to 1 * 10 CFU / mL, core functional components of cistanche deserticola and the stability of a fermentation system are reserved, the flavor and storage safety of products are improved, and high-value utilization of cistanche deserticola resources is achieved.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically to a Cistanche deserticola enzyme and its preparation method. Background Technology

[0002] Cistanche deserticola, the dried, fleshy stem with scaly leaves of a plant in the genus Cistanche of the family Orobanchaceae, is a traditional and precious tonic in Chinese medicine. It is listed in the *Pharmacopoeia of the People's Republic of China* and is known for its effects of tonifying kidney yang, nourishing essence and blood, and moistening the intestines to relieve constipation. Modern pharmacological studies have shown that phenylethyl glycosides, the core active substances in Cistanche deserticola, possess significant antioxidant, anti-aging, immunomodulatory, and neuroprotective effects, and have broad application prospects in health foods and functional beverages.

[0003] Enzymes, also known as enzymes, are products obtained through microbial fermentation that breaks down and transforms the nutrients in plant and animal raw materials. This process not only retains the active ingredients of the raw materials but also adds beneficial bacteria, organic acids, vitamins, peptides, and other bioactive substances produced during fermentation. These substances are easily absorbed and utilized by the human body and have gained widespread popularity among consumers in recent years. Combining Cistanche deserticola with enzyme fermentation technology to develop Cistanche deserticola enzyme products can achieve high-value utilization of Cistanche deserticola resources, expand its application scenarios, and meet market demand for novel tonic enzyme products.

[0004] However, existing Cistanche deserticola enzyme-related products and preparation technologies still face many problems that urgently need to be addressed: First, the phenylethanoid glycosides in Cistanche deserticola have poor stability and are easily affected by factors such as oxygen, temperature, and microbial metabolites during fermentation, leading to oxidative degradation and severe loss of active ingredients, thus failing to fully exert the efficacy of Cistanche deserticola. Existing technologies lack targeted strategies for protecting active ingredients. Second, the carbon sources used in fermentation are mostly fast-acting carbon sources such as glucose and sucrose, which can easily lead to excessive proliferation of microorganisms in the early stages of fermentation and insufficient carbon sources in the later stages, resulting in an imbalance in microbial metabolism, affecting fermentation efficiency and product quality, and making it difficult to achieve continuous and stable metabolism of the microbial community. Third, the selection of fermentation strains is often singular or the combination is unreasonable, which cannot fully degrade the macromolecules in Cistanche deserticola and can easily lead to a monotonous product flavor and low content of active ingredients. Fourth, the control of fermentation conditions in the preparation process is not precise enough, such as excessive pH fluctuations and improper control of fermentation temperature, which can easily lead to contamination by other microorganisms. At the same time, the flavor adjustment strategy is not perfect, resulting in poor product palatability and failing to meet consumers' dual demands for product flavor and quality.

[0005] Therefore, in response to the problems of severe loss of active ingredients, imbalance of microbial metabolism, unstable product quality, and poor flavor in the existing Cistanche deserticola enzyme preparation technology, it is of great practical significance and market value to develop a Cistanche deserticola enzyme and its preparation method that can efficiently retain the active ingredients of Cistanche deserticola, optimize the fermentation system, and improve product quality. Summary of the Invention

[0006] In order to solve the technical problems existing in the prior art, this application provides a Cistanche deserticola enzyme and its preparation method.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: a Cistanche deserticola enzyme, characterized in that it is prepared from raw materials comprising the following parts by weight: 30-50 parts of Cistanche deserticola, 8-20 parts of antioxidant protectant, 10-25 parts of microbial community-adapted slow-release carbon source, 20-40 parts of sugar source, 0.5-2 parts of compound fermentation strain, and 80-120 parts of sterile water; wherein, the antioxidant protectant is at least two of wolfberry, mulberry, and schisandra chinensis; the microbial community-adapted slow-release carbon source is at least two of jujube, konjac flour, and snow lotus fruit; the total content of phenylethanoid glycosides in the Cistanche deserticola enzyme is ≥0.5mg / mL, and the loss rate of phenylethanoid glycosides is ≤10%.

[0008] The aforementioned Cistanche deserticola is rich in phenylethanoid glycosides, which are key to ensuring the health benefits of enzymes. These components possess pharmacological activities such as anti-oxidation, anti-aging, immune regulation, and neuroprotection. In addition to active ingredients, Cistanche deserticola also contains nutrients such as polysaccharides, amino acids, and minerals. These components can serve as auxiliary nutrient sources for the fermentation system, providing basic support for the growth and reproduction of subsequent compound fermentation strains. Furthermore, its fleshy stem tissue can easily release nutrients after pretreatment, thereby enhancing the nutritional richness of the fermentation system.

[0009] The aforementioned antioxidants inhibit the oxidative degradation of phenylethyl glycosides through synergistic antioxidant effects, scavenging free radicals and blocking oxidation reactions. Goji berries, mulberries, and schisandra berries are all rich in natural, potent antioxidants. These substances preferentially bind to reactive oxygen species (ROS) such as superoxide anions and hydroxyl radicals generated during fermentation, undergoing redox reactions to convert ROS into harmless substances. This reduces the attack of ROS on phenylethyl glycosides, fundamentally blocking their oxidative degradation pathway. The mechanisms of action of different antioxidants are complementary. Using at least two components in combination can create a synergistic antioxidant effect. Compared to a single antioxidant, this approach can more comprehensively scavenge various ROS in the fermentation system, significantly improving the protection efficiency for phenylethyl glycosides. A ratio of 1:1-2 between goji berries and mulberries, and 2-3:1 between mulberries and schisandra berries, can further optimize the synergistic effect and precisely match the ROS generation patterns during fermentation.

[0010] Red dates are a primary source of readily available carbon. Their monosaccharides and disaccharides, such as glucose and fructose, can be rapidly absorbed and utilized by the microbial community, meeting the energy needs for rapid proliferation in the early stages of fermentation. This helps the microbial community quickly establish its dominance, effectively inhibiting the growth and reproduction of harmful bacteria in the fermentation system and ensuring stability in the early stages of fermentation. Konjac flour's main component is glucomannan, which cannot be directly and rapidly utilized by the microbial community in the fermentation system. It needs to be slowly hydrolyzed into glucose by enzymes produced by the microbial community's metabolism, thus achieving a slow release of carbon. This provides a continuous energy supply for the later stages of fermentation, preventing a decline in microbial activity due to the depletion of readily available carbon sources and ensuring the continuity and stability of the fermentation process. Yacon is rich in fructooligosaccharides, which cannot be digested and absorbed by the human body, but can act as a prebiotic for probiotics such as Lactobacillus plantarum. By selectively promoting the growth and reproduction of dominant fermenting microorganisms and inhibiting harmful bacteria, it further optimizes the microbial community structure of the fermentation system and improves the efficiency of synergistic metabolism among the microbial community. At least two components can be combined to precisely match the carbon source supply with the growth cycle of the compound microbial strain during its rapid proliferation, stable metabolism, and later maintenance phases by adjusting the ratio of fast-acting carbon source to slow-release carbon source and prebiotics. Combining red dates with konjac flour at a ratio of 3-5:1 and red dates with snow lotus fruit at a ratio of 2-3:1 can further optimize the carbon source release rate and maximize fermentation efficiency. The aforementioned supplementary carbon sources ensure the metabolism of the microbial community: the sugar source provides a large amount of readily available sugars such as sucrose, complementing the microbial-adapted slow-release carbon source, providing sufficient energy for the growth, reproduction, and metabolic activities of the compound fermentation microbial strain, ensuring that the strain can efficiently decompose and transform the nutrients in the raw materials, generating enzyme-specific components such as organic acids and esters.

[0011] Furthermore, in the antioxidant protectant, the weight ratio of wolfberry to mulberry is 1:1-2, and / or the weight ratio of mulberry to schisandra is 2-3:1; in the microbial-adapted slow-release carbon source, the weight ratio of jujube to konjac flour is 3-5:1, and / or the weight ratio of jujube to yacon is 2-3:1.

[0012] The formula uses a 1:1-2 ratio of goji berries and mulberries. Goji berries are rich in antioxidants, primarily goji polysaccharides and polyphenols, which are highly effective at scavenging hydroxyl radicals generated in the early stages of fermentation. Mulberries, on the other hand, are rich in antioxidants, primarily anthocyanins and resveratrol, which are better at scavenging superoxide anions generated in the middle stages of fermentation. In the early stages of fermentation, rapid bacterial growth is accompanied by the generation of large amounts of hydroxyl radicals. At this time, the high proportion of goji berries can preferentially and efficiently scavenge these radicals. As fermentation progresses into the middle stages, the generation of superoxide anions increases, and the higher proportion of mulberries can specifically enhance the efficiency of superoxide anion scavenging. The 1:1-2 ratio precisely matches the types and amounts of ROS generated at different stages of fermentation, avoiding antioxidant blind spots caused by insufficient targeting of a single component. This achieves continuous antioxidant protection throughout the entire fermentation cycle. Furthermore, since both components are water-soluble, this ratio ensures uniform dispersion in the fermentation system, further enhancing the synergistic effect.

[0013] A 2-3:1 ratio of mulberry to schisandra chinensis was used. Schisandra chinensis' antioxidant components are schisandrin and lignans, characterized by their long-lasting antioxidant effect and ability to inhibit the activity of oxidases in the fermentation system, thus reducing ROS formation at its source. However, their onset of action is slower than that of anthocyanins in mulberry. A 2-3 part mulberry to 1 part schisandra chinensis ratio creates a synergistic effect of rapid onset and long-term inhibition. In the early stages of fermentation, mulberry anthocyanins rapidly eliminate existing ROS, preventing the rapid oxidation of phenylethanoid glycosides. In the middle and late stages of fermentation, the antioxidant components of mulberry are gradually consumed. At this point, schisandrin and lignans begin to play a role, continuously eliminating residual ROS and reducing the formation of new ROS by inhibiting oxidase activity, thus extending the antioxidant protection period. A 2-3:1 ratio can balance the need for rapid protection and long-term maintenance. If the proportion of Schisandra chinensis is too high, it will lead to insufficient antioxidant activity in the early stage of fermentation due to slow onset of action. If the proportion is less than 1 / 3, it will not be able to achieve long-term protection in the later stage, which will eventually lead to an increased loss rate of phenylethanoid glycosides.

[0014] A 3-5:1 ratio of jujubes to konjac flour is used as a slow-release carbon source adapted to the microbial community. Jujubes contain fast-acting carbon sources such as glucose and fructose, which meet the energy requirements of the microbial community during its rapid proliferation phase. Konjac flour contains glucomannan, which needs to be slowly hydrolyzed into glucose by the microbial community's metabolic enzymes. It is a slow-release carbon source that meets the energy requirements of the microbial community during its stable metabolic phase and later maintenance phase.

[0015] Specifically, by setting a ratio of 3-5 parts red dates to 1 part konjac powder, the release rhythm of carbon source can be precisely controlled: In the early stage of fermentation, the large amount of fast-acting carbon source provided by 3-5 parts red dates can quickly meet the rapid proliferation needs of Lactobacillus plantarum and Saccharomyces cerevisiae, helping the dominant bacterial community to be established quickly and inhibiting contamination by other bacteria; as the fast-acting carbon source is gradually consumed, 1 part konjac powder begins to slowly hydrolyze and release glucose, providing continuous energy for the stable metabolism of the bacterial community and avoiding the decline in bacterial activity due to the interruption of carbon source.

[0016] If the proportion of red dates is less than 3 parts, there will be insufficient fast-acting carbon source, resulting in slow bacterial growth and an inability to quickly establish dominance. If the proportion of red dates is more than 5 parts, there will be an excess of fast-acting carbon source, which will lead to excessive bacterial growth in the early stages, rapidly consuming oxygen and nutrients, causing a sharp drop in the pH value of the fermentation system, and inhibiting metabolism in the later stages. A proportion of 1 part konjac flour can ensure a continuous supply of slow-release carbon source, while avoiding an increase in the viscosity of the fermentation system due to excessive konjac flour, which would affect the contact efficiency between the bacterial community and nutrients.

[0017] A 2-3:1 ratio of red dates to yacon is ideal. Yacon contains fructooligosaccharides, which cannot be directly utilized by the gut microbiota, but can serve as a prebiotic for *Lactobacillus plantarum*, selectively promoting its growth and optimizing the gut microbiota structure. A ratio of 2-3 parts red dates to 1 part yacon balances the need for rapid energy supply and gut microbiota optimization: the 2-3 parts red dates provide a rapid carbon source that simultaneously meets the basic energy needs of *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, and *Acetobacter*, ensuring the synergistic proliferation of these three strains; the 1 part yacon provides fructooligosaccharides that specifically promote the growth of dominant bacteria, increasing their proportion in the gut microbiota and strengthening their lactic acid production and inhibition of harmful bacteria.

[0018] If the proportion of yacon is too high, excessive fructooligosaccharides will lead to the overgrowth of *Lactobacillus plantarum*, resulting in an excessively low pH in the fermentation system. This inhibits the metabolism of *Saccharomyces cerevisiae* and *Acetic Acid Bacteria*, disrupting the synergistic balance of the microbial community. Conversely, if the proportion of yacon is too low, insufficient prebiotics will prevent effective optimization of the microbial community structure, diminishing the dominant position of *Lactobacillus plantarum* and reducing fermentation efficiency. A 2-3:1 ratio ensures precise optimization of the microbial community structure while guaranteeing basic energy supply, achieving synergistic metabolism among the three types of microorganisms and improving fermentation efficiency and product quality.

[0019] Furthermore, the compound fermentation strain consists of Lactobacillus plantarum, Saccharomyces cerevisiae, and acetic acid bacteria in a weight ratio of 5:3:2, and the viable count of the compound fermentation strain is ≥1×10⁻⁶. 9 CFU / g; sugar source is brown sugar and / or rock sugar.

[0020] The above-mentioned compound fermentation strain adopts a ratio of Lactobacillus plantarum: Saccharomyces cerevisiae: Acetic acid bacteria = 5:3:2. Based on the metabolic characteristics of the three strains, a synergistic metabolic network with anaerobic dominance, facultative synergy, and microaerobic supplementation is constructed to meet the fermentation requirements of Cistanche deserticola enzyme.

[0021] Lactobacillus plantarum accounted for the largest proportion of the bacteria. As an anaerobic fermenting bacteria, it can ferment sugar sources to produce lactic acid, rapidly reducing the pH of the fermentation system to the suitable range of 3.5-4.5. This not only inhibits contamination by other bacteria but also creates an acidic environment for the growth of other microorganisms. Saccharomyces cerevisiae is a facultative anaerobic bacterium. In the early stage of fermentation, it can rapidly proliferate using readily available carbon sources, producing ethanol and ester flavor substances. Ethanol can also serve as a substrate for subsequent acetic acid bacteria. At the same time, the vitamins and other substances produced by its metabolism can promote the growth of Lactobacillus plantarum, achieving mutual promotion among the microbial community. Acetic acid bacteria is an aerobic bacterium. In the later stage of fermentation, it can convert ethanol into acetic acid, adjusting the acidity of the product and enriching the sour taste. At the same time, the generation of acetic acid can further enhance the antibacterial effect and improve the stability of the product. The 5:3:2 ratio precisely balances the competition and synergy among the three types of bacteria: if the proportion of Lactobacillus plantarum is less than 5 parts, an acidic environment cannot be established quickly, increasing the risk of contamination by other bacteria; if the proportion of Saccharomyces cerevisiae is too high, it will consume too much carbon source and produce a large amount of ethanol, inhibiting the activity of Lactobacillus plantarum; if the proportion of Acetic Acid Bacteria is too high, more oxygen is required, which can easily lead to increased oxidation in the fermentation system and damage phenylethanol glycosides. This ratio ensures that the metabolism of the microbial community is orderly and synergistically efficient throughout the fermentation cycle.

[0022] Setting a high viable cell count is a crucial prerequisite for ensuring fermentation efficiency and stability. In the early stages of fermentation, a high concentration of inoculum can quickly dominate the fermentation system, inhibiting the growth and reproduction of other microorganisms in the environment and preventing contamination of the product by harmful substances produced by their metabolism. Simultaneously, a high viable cell count shortens the inoculum's adaptation period, rapidly initiates the fermentation process, reduces carbon source waste and excessive reactive oxygen species generation in the early stages of fermentation, and lays the foundation for protecting active ingredients and improving fermentation efficiency. If the viable cell count is below 1×10⁻⁶... 9 CFU / g indicates slow bacterial proliferation and delayed establishment of dominant bacterial groups, which can easily lead to contamination by other microorganisms, resulting in prolonged fermentation cycles and unstable product quality.

[0023] Furthermore, it also includes flavor-regulating excipients, which are at least one of dried tangerine peel, rose petals, and licorice slices, and the total amount of flavor-regulating excipients added is 3%-8% of the weight of Cistanche deserticola.

[0024] Three types of excipients are used to address the flavor defects of Cistanche deserticola enzyme, such as its fishy and gamey smell and monotonous sourness, and to play a differentiated and complementary role in flavor regulation:

[0025] The flavor components of dried tangerine peel are volatile oils, possessing a unique citrus aroma that can mask the fishy or gamey smell of Cistanche deserticola. Simultaneously, hesperidin has a certain bitterness-neutralizing ability, balancing the astringency from the organic acids produced during fermentation and resulting in a richer, more mellow flavor. Its flavor components are stable and do not easily decompose in the acidic environment and mild temperatures of fermentation, thus sustaining their flavor-regulating effect.

[0026] The flavor component of rose is rose essential oil, which has a fresh rose aroma and can give enzymes a unique floral flavor, enhancing the product's sensory appeal. At the same time, the flavonoids contained in roses can slightly neutralize the acidity, making the taste smoother. Although its aroma components are volatile, the design of the second stage of the fermentation process, which avoids light, can reduce the loss of volatilization and ensure the contribution of flavor.

[0027] The flavor component of licorice tablets is glycyrrhizin, which has a naturally sweet and mellow taste, with a high and lasting sweetness. It can gently regulate the sweetness of the product and balance the acidity of organic acids. At the same time, glycyrrhizin can suppress the sensory presentation of odor components in Cistanche deserticola, further weakening the fishy and muttony smell and making the overall taste of the product more harmonious. The flavor components of licorice have good solubility in the fermentation system and do not inhibit the activity of the complex fermentation strains, thus not interfering with the core fermentation process.

[0028] By selecting at least one auxiliary ingredient, a synergistic optimization effect can be achieved according to the product's flavor requirements: for example, combining dried tangerine peel with rose petals can achieve a complex aroma of citrus and rose fragrance, enhancing the flavor profile; combining dried tangerine peel with licorice slices can achieve the dual effect of masking aroma and neutralizing sweetness, more effectively improving fishy and astringent tastes; a combination of three auxiliary ingredients can create a richer flavor system, catering to the taste preferences of different consumer groups. This formulation design enhances the flexibility and adaptability of flavor adjustment, avoiding the problem of monotonous flavor from a single auxiliary ingredient.

[0029] Furthermore, the superoxide dismutase activity in Cistanche deserticola enzyme is ≥100 U / mL, the total organic acid content is 5-10 mg / mL, and the viable lactic acid bacteria count is ≥1×10⁻⁶. 8 CFU / mL.

[0030] The superoxide dismutase (SOD) activity in Cistanche deserticola enzyme is ≥100 U / mL. From a generation mechanism perspective, *Lactobacillus plantarum* in the compound fermentation strain can induce the synthesis of SOD during metabolism. The continuous energy supply provided by the microbial community-adapted slow-release carbon source ensures stable metabolism of *Lactobacillus plantarum*, providing a microbial community basis for the continuous generation of SOD. From a synergistic mechanism perspective, the natural antioxidant components in the antioxidant protectant can protect the spatial structure of SOD, preventing its degradation by reactive oxygen species in the fermentation system. Simultaneously, the phenylethanoid glycosides in Cistanche deserticola form a synergistic antioxidant effect with SOD. SOD can rapidly scavenge superoxide anions, while the antioxidant components scavenge hydroxyl radicals and other reactive oxygen species, jointly enhancing the product's antioxidant efficacy. Setting an activity standard of ≥100 U / mL is based on the minimum effective dose required for human antioxidant needs, ensuring that the product can exert a substantial antioxidant health-promoting effect. If the activity is lower than this value, an effective antioxidant protective effect cannot be achieved.

[0031] The total amount of organic acids is 5-10 mg / mL. From the perspective of the generation mechanism, organic acids mainly come from the synergistic metabolism of the complex strains: Lactobacillus plantarum ferments sugar sources to produce lactic acid, and Acetic Acid Bacteria convert ethanol into acetic acid in the later stage. The gradient supply of slow-release carbon sources can avoid the sudden increase or decrease of organic acids caused by the imbalance of strain metabolism, thus achieving precise control of the total amount.

[0032] Lactic acid bacteria live count ≥1×10 8 From a process protection perspective, maintaining a stable acidic environment of pH 3.5-4.5 during fermentation, low-temperature and light-protected conditions in the later stages of fermentation, and a final low-temperature pasteurization process at 60-70℃ can minimize the death of lactic acid bacteria. A concentration of ≥1×10⁻⁶ CFU / mL is recommended. 8 CFU / mL is the minimum number of live bacteria that probiotics can effectively regulate the gut microbiota. If the number is below this value, lactic acid bacteria cannot form a dominant flora in the human gut, making it difficult to achieve the effect of regulating the balance of gut microbiota.

[0033] This invention also provides a method for preparing Cistanche deserticola enzyme, comprising the following steps:

[0034] S1: After washing and slicing the Cistanche deserticola, sterilize it with steam at 105℃ for 15-20 minutes and then cool it; wash, crush, slice, pulp or sieve the antioxidant and the microbial-compatible slow-release carbon source respectively.

[0035] The Cistanche deserticola is cleaned and sliced. Cleaning removes surface mud, impurities, and some microorganisms, reducing the risk of contamination by other microorganisms. Slicing increases the contact area between the Cistanche deserticola and the fermentation system, creating conditions for the subsequent dissolution of active ingredients. Steam sterilization at 105℃ for 15-20 minutes effectively kills surface microorganisms, preventing them from competing with the complex microbial strain for nutrients and producing harmful substances. It also avoids the degradation of phenylethanoid glycosides in the Cistanche deserticola due to prolonged high-temperature sterilization. Simultaneously, the high temperature of steam softens the fleshy stem tissue of the Cistanche deserticola, further promoting the dissolution of active ingredients during subsequent fermentation. Differentiated pretreatment of antioxidants and carbon sources: Based on the characteristics of the raw materials, the appropriate pretreatment methods are selected: crushing, slicing, pulping, or sieving. For example, goji berries and mulberries are suitable for pulping to fully release antioxidants, red dates are suitable for slicing to facilitate sugar dissolution, and konjac flour is suitable for sieving to ensure uniform dispersion. This design maximizes the utilization rate of different raw materials, laying the foundation for subsequent synergistic effects. The cooling step is to prevent the inoculum from becoming inactive after the high-temperature raw materials are introduced, thus ensuring the smooth start of subsequent fermentation.

[0036] S2: Mix the pretreated Cistanche deserticola, antioxidant protectant, microbial-adapted slow-release carbon source, sugar source and sterile water in a fermenter, adjust the solid-liquid ratio of the system to 1:2-3, and then inoculate the compound fermentation strain and stir evenly.

[0037] It should be noted that mixing the pretreated raw materials with sterile water and sugar source ensures uniform dispersion of nutrients in the system, avoiding local nutrient excess or deficiency, and ensuring that the compound microbial strain can uniformly contact nutrients for simultaneous proliferation. Adjusting the solid-liquid ratio to 1:2-3 is a precisely matched parameter. An excessively high solid-liquid ratio leads to high system viscosity and low oxygen content, affecting microbial metabolism and component dissolution; an excessively low solid-liquid ratio results in low nutrient concentration, slow microbial proliferation, and decreased fermentation efficiency. A ratio of 1:2-3 balances component dissolution efficiency and the microbial growth environment, providing a suitable physical environment for fermentation. Inoculation and uniform mixing: Mixing allows the compound microbial strain to quickly and evenly disperse in the fermentation system, avoiding metabolic imbalances caused by localized microbial aggregation. Simultaneously, mixing temporarily increases dissolved oxygen in the system, helping the brewing yeast to rapidly proliferate in the early stages, quickly establishing a dominant microbial community, and laying the foundation for subsequent anaerobic fermentation.

[0038] S3: First, ferment at 25-30℃ under anaerobic conditions for 30-40 days, maintaining the pH of the fermentation system at 3.5-4.5; then add flavor-adjusting ingredients and continue fermenting at 20-25℃ under light-proof conditions for 10-20 days.

[0039] The first stage of fermentation is conducted at 25-30℃, anaerobic, for 30-40 days, with a pH of 3.5-4.5. The goal of this stage is to achieve rapid proliferation of the complex microbial strain, establish a dominant microbial community, and initially accumulate active ingredients. 25-30℃ is the optimal growth temperature for *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, ensuring efficient microbial metabolism. The anaerobic environment reduces the oxidative degradation of phenylethanol glycosides and is suitable for the anaerobic metabolic characteristics of *Lactobacillus plantarum*. The 30-40 day cycle ensures sufficient microbial proliferation and initial carbon source conversion, laying the foundation for subsequent metabolism. Maintaining a pH of 3.5-4.5 inhibits the growth of other microorganisms while meeting the metabolic needs of the complex microbial strain, avoiding contamination from excessively high pH levels and inhibition of microbial activity from excessively low pH levels.

[0040] The second stage of fermentation, conducted at 20-25℃ in the dark for 10-20 days, involves the addition of flavor enhancers. The goal of this stage is to optimize product flavor and continuously accumulate active ingredients. Lowering the temperature to 20-25℃ slows down the metabolic rate of the microorganisms, preventing over-fermentation that could lead to excessive organic acids and degradation of active ingredients. The dark environment further reduces the photo-oxidative degradation of phenylethyl glycosides and SOD, ensuring product efficacy. The addition of flavor enhancers allows for the slow dissolution of flavor components from the enhancers under mild fermentation conditions, forming a complex flavor with esters and organic acids produced during fermentation. The 10-20 day cycle ensures the flavor components are fully integrated, improving product palatability.

[0041] S4: After fermentation, the fermentation liquid is aged in a dark environment for 1-3 months, and then filtered and centrifuged to obtain a clear fermentation liquid;

[0042] Aging in the dark for 1-3 months allows for the slow degradation of macromolecules in the fermentation liquid, while simultaneously enabling the flavor compounds to fully blend and balance, reducing the product's pungent odor and enhancing its richness. The dark environment also protects the active ingredients from oxidation, ensuring the product's efficacy and stability. Filtration removes solid impurities from the fermentation liquid, and centrifugation removes fine suspended particles and some macromolecular impurities, resulting in a clarified fermentation liquid.

[0043] S5: Sterilize the clarified fermentation broth at 60-70℃ for 10-15 minutes, cool it, and then aseptically fill it to obtain the final product.

[0044] Sterilization at 60-70℃ for 10-15 minutes is designed for low-temperature pasteurization, which can effectively kill harmful bacteria in the fermentation broth and ensure the safety of the product for consumption; it can also avoid the inactivation of SOD and degradation of phenylethanol glycosides caused by high-temperature sterilization, thus maximizing the preservation of the product's active ingredients and nutritional value.

[0045] Cooling can quickly lower the temperature of the fermentation broth, preventing residual heat from causing continuous degradation of active ingredients; aseptic filling in a Class 100 cleanroom environment can prevent airborne bacteria from entering the product during the filling process, ensuring the stability of the product during storage and extending its shelf life.

[0046] Furthermore, during the first stage of fermentation in step S3, when the pH value is below 3.5, trehalose accounting for 0.5%-1% of the fermentation liquid weight is added for adjustment; when the pH value is above 4.5, sea buckthorn juice accounting for 1%-2% of the fermentation liquid volume is added for adjustment.

[0047] Trehalose's mild buffering properties are fundamental to pH regulation: Trehalose is a non-reducing disaccharide with stable chemical properties and a mild pH buffering capacity. Its aqueous solution is neutral. When added to acidic fermentation broths with a pH below 3.5, it can slowly neutralize some hydrogen ions through its buffering action, gradually raising the pH to the target range of 3.5-4.5. Compared to conventional chemical buffers, trehalose's regulatory effect is mild and slow, preventing sudden pH increases or decreases and avoiding disruption of the fermentation system's stability due to pH abrupt changes, thus ensuring the continuity of the microbial metabolic environment.

[0048] The first stage of fermentation involves *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, both of which thrive in a pH range of 3.5-4.5. When the pH falls below 3.5, high concentrations of hydrogen ions can damage the cell membranes of the microorganisms, inhibiting enzyme activity and leading to metabolic stagnation. Trehalose not only regulates pH but also acts as a microbial stress protectant. By binding to the cell membranes to form a protective film, it reduces damage to the cell membranes caused by low pH conditions, maintains intracellular osmotic pressure balance, alleviates the inhibitory effect of acid stress on microbial activity, and ensures continuous and efficient metabolism by the microorganisms.

[0049] Furthermore, the centrifugation speed in step S4 is 3000-5000 r / min, and the centrifugation time is 15-20 min; the aseptic filling in step S5 is carried out in a Class 100 cleanroom environment.

[0050] Centrifugation uses the centrifugal force generated by high-speed rotation to cause solid impurities in the fermentation broth with a density greater than that of the liquid to experience centrifugal acceleration far exceeding gravity. This causes them to quickly settle against the inner wall of the centrifuge tube / drum, achieving separation from the clarified fermentation broth. Compared to conventional filtration, centrifugation can efficiently remove fine suspended particles, improve the clarity of the fermentation broth, and prevent sensory defects such as turbidity and sedimentation in subsequent products.

[0051] The 3000-5000 r / min rotation speed range is determined based on a balance between separation efficiency and retention of active ingredients. A minimum speed of 3000 r / min generates sufficient centrifugal force to drive most solid impurities in the fermentation broth to settle rapidly. If the speed is below 3000 r / min, the centrifugal force is insufficient, and fine impurities cannot be effectively separated, resulting in poor clarity of the fermentation broth and affecting the sensory quality of the product. The maximum speed limit is 5000 r / min. Speeds exceeding 5000 r / min will lead to a surge in energy consumption of the centrifuge equipment, increasing production costs. Furthermore, excessive centrifugal force may damage the structure of some active ingredients in the fermentation broth, leading to a decrease in the number of viable lactic acid bacteria and SOD activity. It may also cause some nutrients in the fermentation broth to settle along with impurities, resulting in the loss of effective components. A speed of 3000-5000 r / min allows for efficient separation of impurities while maximizing the retention of the product's active ingredients and nutritional value.

[0052] Class 100 cleanliness refers to a clean environment where the number of dust particles with a diameter ≥0.5μm per cubic foot of air does not exceed 100, and there are no visible microorganisms. This clean environment removes airborne dust particles and microorganisms through the filtration of high-efficiency air filters, creating a clean operating space. Although most harmful bacteria are killed after the fermentation broth is sterilized at 60-70℃, airborne bacteria may still enter the product during the filling process if it comes into contact with outside air, leading to secondary contamination and spoilage. A Class 100 clean environment can block airborne bacteria and impurities at the source, providing a sterile operating atmosphere for the filling process and avoiding secondary contamination.

[0053] Furthermore, in step S3, the flavor-adjusting additives are added after the first stage of fermentation. If rose petals are used, they are added 5-7 days before the end of the second stage of fermentation and removed after fermentation is completed.

[0054] The core objective of the first stage of fermentation is to establish a dominant microbial community within the complex microbial strain, completing initial carbon source metabolism and the initial accumulation of active ingredients. If flavoring additives are added at the beginning of the first stage of fermentation, their components, such as fiber and pectin, may compete with the microbial strain for nutrients, or their complex composition may slightly inhibit microbial activity, leading to a delay in the establishment of the dominant microbial community and a decrease in fermentation efficiency. After the first stage of fermentation is completed, the fermentation system has formed a stable acidic environment and a dominant microbial community structure. Adding additives at this point can avoid interfering with the core fermentation process and ensure the stability of subsequent microbial metabolism.

[0055] The second stage of fermentation uses low-temperature, light-protected conditions of 20-25℃. Compared to the first stage's fermentation temperature of 25-30℃, this environment is milder and can reduce the excessive volatilization and decomposition of flavor components in the excipients. At the same time, the organic acids produced in the first stage of fermentation can lower the pH value of the fermentation system, promote the dissolution of flavor components such as hesperidin in dried tangerine peel and glycyrrhizin in licorice tablets, improve the dissolution efficiency of flavor substances, and ensure that the excipients can fully exert their flavor-regulating effects.

[0056] Rose essential oil has a low boiling point and is highly volatile. If added simultaneously with dried tangerine peel and licorice slices at the beginning of the second stage of fermentation, and kept in a fermentation environment of 20-25℃ for an extended period, the rose essential oil will continue to evaporate, resulting in a decrease in flavor intensity and failing to fully impart the rose fragrance to the product. Adding it 5-7 days before the end of the second stage of fermentation can minimize the high-temperature exposure time of the rose essential oil, reduce evaporation loss to the minimum, and ensure that the flavor components can fully dissolve into the fermentation liquid.

[0057] Furthermore, during the aging process described in step S4, the mixture is stirred at a speed of 50-80 r / min for 5-10 min every 15 days.

[0058] The 15-day stirring interval is determined based on the material transformation rhythm of the aging process. On the one hand, the fermentation broth enters a relatively stable transformation phase during the first 15 days of aging, with flavor substances gradually diffusing and blending without frequent disturbance. If the stirring interval is less than 10 days, it will disrupt the stable environment of aging, leading to insufficient degradation of macromolecules and disordered flavor blending rhythm. On the other hand, if the stirring interval exceeds 20 days, the slightly denser microparticles in the fermentation broth are prone to slow sedimentation, forming localized uneven concentrations. This may result in a lack of flavor substances and abnormal oxidation rates of active ingredients in the sedimentation areas, affecting the overall uniformity of product quality. The 15-day interval precisely matches the material transformation cycle of the aging process, achieving a balance between stable transformation and avoiding imbalance.

[0059] Beneficial effects:

[0060] 1. This invention specifically incorporates an antioxidant protectant composed of at least two of the following: wolfberry, mulberry, and schisandra. Its rich natural antioxidant components effectively scavenge free radicals in the fermentation system and synergistically inhibit the oxidative degradation of phenylethanoid glycosides in Cistanche deserticola. Simultaneously, the entire process of anaerobic fermentation, light-protected fermentation, and light-protected aging is designed with controlled oxygen and light protection, ensuring that the total content of phenylethanoid glycosides is stably controlled at ≥0.5mg / mL with a loss rate ≤10%. This fully preserves the core active ingredients of Cistanche deserticola, solving the critical problem of severe loss of active ingredients in existing technologies and significantly enhancing the product's nutritional and health benefits.

[0061] 2. This invention innovatively employs a microbial community-adapted slow-release carbon source composed of at least two of the following: jujube, konjac flour, and yacon. Jujube provides a fast-acting carbon source to meet the initial proliferation needs of the microbial strain, konjac flour slowly releases carbon to maintain microbial activity in the later stages of fermentation, and yacon contains fructooligosaccharides that act as prebiotics to promote probiotic growth. These three components synergistically achieve a gradient supply of carbon sources. Combined with a compound fermentation strain composed of *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, and *Acetobacter acetic acid bacteria* in a 5:3:2 ratio, this strain ratio achieves a synergistic connection between anaerobic and microaerobic metabolism, with a concentration of ≥1×10⁻⁶. 9 The high CFU / g viable cell count effectively avoids the problems of carbon source imbalance and microbial metabolic disorder in existing technologies, significantly improving fermentation efficiency and product quality stability. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0063] Figure 1 This is a process flow diagram of a method for preparing Cistanche deserticola enzyme according to the present invention. Detailed Implementation

[0064] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, the specific embodiments of the present invention are now described in detail.

[0065] Example 1

[0066] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 30 parts of Cistanche deserticola, 8 parts of antioxidant protectant, 10 parts of microbial adaptive slow-release carbon source, 20 parts of sugar source, 0.5 parts of compound fermentation strain, 80 parts of sterile water; and 3% of the weight of dried tangerine peel.

[0067] Preparation method: S1: After washing and slicing the Cistanche deserticola, sterilize it with steam at 105℃ for 15 minutes and then cool it; grind the wolfberry and mulberry into a pulp, slice the red dates, and pass the konjac powder through an 80-mesh sieve.

[0068] S2: Mix the pretreated raw materials with brown sugar and sterile water, adjust the solid-liquid ratio to 1:2, inoculate with compound fermentation bacteria and stir evenly.

[0069] S3: The first stage ferments at 25℃ under anaerobic conditions for 30 days, maintaining a pH of 3.5-4.5; after the first stage fermentation is completed, dried tangerine peel is added, and fermentation continues for 10 days at 20℃ under light-proof conditions.

[0070] S4: Aged the fermentation broth in the dark for 1 month, stirring at 50 r / min for 5 min every 15 days; after aging, filtered, and then centrifuged at 3000 r / min for 15 min to obtain a clear fermentation broth.

[0071] S5: Sterilize the clarified fermentation broth at 60℃ for 10 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0072] The total content of phenylethanoid glycosides was 0.62 mg / mL, with a loss rate of 8.5%; superoxide dismutase activity was 112 U / mL; total organic acids were 5.3 mg / mL; and the viable count of lactic acid bacteria was 1.2 × 10⁻⁶. 8 CFU / mL.

[0073] Example 2

[0074] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 35 parts of Cistanche deserticola, 12 parts of antioxidant protectant, 15 parts of microbial adaptive slow-release carbon source, 25 parts of sugar source, 1 part of compound fermentation strain, 90 parts of sterile water; rose petals, added at 5% of the weight of Cistanche deserticola.

[0075] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 17 minutes and then cool it; pulp the mulberry, crush the Schisandra chinensis, slice the red dates and pulp the snow lotus fruit.

[0076] S2: Mix the pretreated raw materials with rock sugar and sterile water, adjust the solid-liquid ratio to 1:2.2, inoculate with compound fermentation bacteria and stir evenly.

[0077] S3: The first stage fermented at 27℃ under anaerobic conditions for 33 days. On the 20th day of fermentation, the pH was measured to be 3.4. Trehalose, accounting for 0.5% of the weight of the fermentation liquid, was added to adjust it to 3.6. After the first stage of fermentation was completed, rose petals were added 5 days before the end of the second stage of fermentation. Fermentation continued for 13 days at 22℃ under dark conditions. After the fermentation was completed, the rose petals were removed.

[0078] S4: Aged the fermentation broth in the dark for 1.5 months, stirring at 60 r / min for 7 min every 15 days; filtered after aging, and then centrifuged at 3500 r / min for 17 min to obtain a clear fermentation broth.

[0079] S5: Sterilize the clarified fermentation broth at 63℃ for 12 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0080] The total content of phenylethanoid glycosides was 0.75 mg / mL, with a loss rate of 7.2%; superoxide dismutase activity was 128 U / mL; total organic acids were 6.8 mg / mL; and the viable count of lactic acid bacteria was 2.5 × 10⁻⁶. 8 CFU / mL.

[0081] Example 3

[0082] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 40 parts of Cistanche deserticola, 15 parts of antioxidant protectant, 20 parts of microbial adaptive slow-release carbon source, 30 parts of sugar source, 1.2 parts of compound fermentation strain, 100 parts of sterile water; and licorice tablets, added at 6% of the weight of Cistanche deserticola.

[0083] Preparation method: S1: After washing and slicing the Cistanche deserticola, sterilize it with steam at 105℃ for 18 minutes and cool it; grind the wolfberry and mulberry into a pulp, slice the red dates, pass the konjac powder through a 100-mesh sieve, and crush the licorice tablets.

[0084] S2: Mix the pretreated raw materials with the mixed sugar source and sterile water, adjust the solid-liquid ratio to 1:2.5, inoculate with the compound fermentation strain and stir evenly.

[0085] S3: The first stage was fermented at 28℃ under anaerobic conditions for 35 days. On the 15th day of fermentation, the pH was measured to be 4.6. Sea buckthorn juice, accounting for 1% of the fermentation liquid volume, was added to adjust it to 4.3. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued at 23℃ under light-proof conditions for another 15 days.

[0086] S4: Aged the fermentation broth in the dark for 2 months, stirring at 70 r / min for 8 min every 15 days; after aging, filtered and then centrifuged at 4000 r / min for 18 min to obtain a clear fermentation broth.

[0087] S5: Sterilize the clarified fermentation broth at 65℃ for 13 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0088] The total content of phenylethanoid glycosides was 0.83 mg / mL, with a loss rate of 6.1%; superoxide dismutase activity was 145 U / mL; total organic acids were 7.6 mg / mL; and the viable count of lactic acid bacteria was 3.8 × 10⁻⁶.8 CFU / mL.

[0089] Example 4

[0090] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 45 parts of Cistanche deserticola, 18 parts of antioxidant protectant, 22 parts of microbial adaptive slow-release carbon source, 35 parts of sugar source, 1.5 parts of compound fermentation strain, 110 parts of sterile water; 2 parts of dried tangerine peel, 1 part of licorice slices, and the total amount added is 6.7% of the weight of Cistanche deserticola.

[0091] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 19 minutes and cool it; grind wolfberry and mulberry into pulp, crush Schisandra chinensis, slice red dates, grind snow lotus fruit into pulp, and crush dried tangerine peel and licorice slices.

[0092] S2: Mix the pretreated raw materials with brown sugar and sterile water, adjust the solid-liquid ratio to 1:2.8, inoculate with compound fermentation bacteria and stir evenly.

[0093] S3: The first stage fermented at 29℃ under anaerobic conditions for 38 days. On the 25th day of fermentation, the pH was measured to be 3.3, and trehalose (0.8% of the fermentation liquid weight) was added to adjust it to 3.7. On the 30th day of fermentation, the pH was measured to be 4.7, and sea buckthorn juice (1.5% of the fermentation liquid volume) was added to adjust it to 4.2. After the first stage of fermentation was completed, dried tangerine peel and licorice slices were added, and fermentation continued for 18 days at 24℃ under light-proof conditions.

[0094] S4: Aged the fermentation broth in the dark for 2.5 months, stirring at 75 r / min for 9 min every 15 days; filtered after aging, and then centrifuged at 4500 r / min for 19 min to obtain a clear fermentation broth.

[0095] S5: Sterilize the clarified fermentation broth at 68℃ for 14 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0096] The total content of phenylethanol glycosides was 0.91 mg / mL, with a loss rate of 5.3%; superoxide dismutase activity was 162 U / mL; total organic acids were 8.9 mg / mL; and the viable count of lactic acid bacteria was 4.6 × 10⁻⁶. 8 CFU / mL.

[0097] Example 5

[0098] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 50 parts of Cistanche deserticola, 20 parts of antioxidant protectant, 25 parts of microbial adaptive slow-release carbon source, 40 parts of sugar source, 2 parts of compound fermentation strain, 120 parts of sterile water; and rose petals, added at 8% of the weight of Cistanche deserticola.

[0099] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 20 minutes and cool it; pulp mulberry, crush Schisandra chinensis, slice red dates, and pass konjac powder through a 120-mesh sieve.

[0100] S2: Mix the pretreated raw materials with rock sugar and sterile water, adjust the solid-liquid ratio to 1:3, inoculate with compound fermentation bacteria and stir evenly.

[0101] S3: The first stage fermented at 30℃ under anaerobic conditions for 40 days. On the 18th day of fermentation, the pH was measured to be 4.8. Sea buckthorn juice, accounting for 2% of the fermentation liquid volume, was added to adjust it to 4.4. After the first stage of fermentation was completed, rose petals were added 7 days before the end of the second stage of fermentation. Fermentation continued for 20 days at 25℃ under light-proof conditions. After the fermentation was completed, the rose petals were removed.

[0102] S4: Aged the fermentation broth in the dark for 3 months, stirring at 80 r / min for 10 min every 15 days; after aging, filtered and then centrifuged at 5000 r / min for 20 min to obtain a clear fermentation broth.

[0103] S5: Sterilize the clarified fermentation broth at 70℃ for 15 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0104] The total content of phenylethanoid glycosides was 1.05 mg / mL, with a loss rate of 4.8%; superoxide dismutase activity was 185 U / mL; total organic acids were 9.7 mg / mL; and the viable count of lactic acid bacteria was 5.2 × 10⁻⁶. 8 CFU / mL.

[0105] Example 6

[0106] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 32 parts of Cistanche deserticola, 10 parts of antioxidant protectant, 12 parts of microbial adaptive slow-release carbon source, 22 parts of sugar source, 0.7 parts of compound fermentation strain, 85 parts of sterile water; 1 part of dried tangerine peel, 0.8 parts of rose petals, and the total amount added is 5.6% of the weight of Cistanche deserticola.

[0107] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 16 minutes and cool it; grind wolfberry and mulberry into pulp, slice red dates and grind snow lotus fruit into pulp, and crush dried tangerine peel.

[0108] S2: Mix the pretreated raw materials with the mixed sugar source and sterile water, adjust the solid-liquid ratio to 1:2.1, inoculate with the compound fermentation bacteria and stir evenly.

[0109] S3: The first stage ferments at 26℃ under anaerobic conditions for 32 days, with the pH remaining stable at 3.8-4.2 throughout the process; after the first stage fermentation is completed, dried tangerine peel is added, and fermentation continues for 12 days at 21℃ under light-proof conditions; rose petals are added 6 days before the end of fermentation, and the rose petals are removed after fermentation is completed.

[0110] S4: Aged the fermentation broth in the dark for 1.2 months, stirring at 55 r / min for 6 min every 15 days; filtered after aging, and then centrifuged at 3200 r / min for 16 min to obtain a clear fermentation broth.

[0111] S5: Sterilize the clarified fermentation broth at 62℃ for 11 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0112] The total content of phenylethanoid glycosides was 0.68 mg / mL, with a loss rate of 7.8%; superoxide dismutase activity was 121 U / mL; total organic acids were 6.1 mg / mL; and the viable count of lactic acid bacteria was 1.9 × 10⁻⁶. 8 CFU / mL.

[0113] Example 7

[0114] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 38 parts of Cistanche deserticola, 14 parts of antioxidant protectant, 18 parts of microbial adaptive slow-release carbon source, 28 parts of sugar source, 1.1 parts of compound fermentation strain, 95 parts of sterile water; 2 parts of licorice tablets, with the amount added being 5.3% of the weight of Cistanche deserticola.

[0115] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 17 minutes and cool it; grind wolfberry and mulberry into pulp, crush Schisandra chinensis, slice red dates, pass konjac powder through a 90-mesh sieve, grind snow lotus fruit into pulp, and crush licorice tablets.

[0116] S2: Mix the pretreated raw materials with brown sugar and sterile water, adjust the solid-liquid ratio to 1:2.4, inoculate with compound fermentation bacteria and stir evenly.

[0117] S3: The first stage was fermented at 27℃ under anaerobic conditions for 34 days. On the 22nd day of fermentation, the pH was measured to be 3.4. Trehalose, accounting for 0.6% of the weight of the fermentation liquid, was added to adjust it to 3.9. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued at 22℃ under light-proof conditions for 14 days.

[0118] S4: Aged the fermentation broth in the dark for 1.8 months, stirring at 65 r / min for 7 min every 15 days; filtered after aging, and then centrifuged at 3800 r / min for 17 min to obtain a clear fermentation broth.

[0119] S5: Sterilize the clarified fermentation broth at 64℃ for 12 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0120] The total content of phenylethanoid glycosides was 0.79 mg / mL, with a loss rate of 6.5%; superoxide dismutase activity was 136 U / mL; total organic acids were 7.2 mg / mL; and the viable count of lactic acid bacteria was 3.1 × 10⁻⁶. 8 CFU / mL.

[0121] Example 8

[0122] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 42 parts of Cistanche deserticola, 16 parts of antioxidant protectant, 21 parts of microbial adaptive slow-release carbon source, 32 parts of sugar source, 1.6 parts of compound fermentation strain, 105 parts of sterile water; 1.5 parts of rose petals, 1.5 parts of licorice tablets, and the total amount added is 7.1% of the weight of Cistanche deserticola.

[0123] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 19 minutes and cool it; pulp mulberry, crush Schisandra chinensis, slice red dates, pulp snow lotus fruit, and crush licorice tablets.

[0124] S2: Mix the pretreated raw materials with rock sugar and sterile water, adjust the solid-liquid ratio to 1:2.6, inoculate with compound fermentation bacteria and stir evenly.

[0125] S3: The first stage fermented at 29℃ under anaerobic conditions for 36 days. On the 16th day of fermentation, the pH was measured to be 4.6. Sea buckthorn juice, accounting for 1.2% of the fermentation liquid volume, was added to adjust it to 4.3. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued at 24℃ under light-proof conditions for 17 days. Rose petals were added 5 days before the end of fermentation. After the fermentation was completed, the rose petals were removed.

[0126] S4: Aged the fermentation broth in the dark for 2.2 months, stirring at 72 r / min for 9 min every 15 days; filtered after aging, and then centrifuged at 4200 r / min for 18 min to obtain a clear fermentation broth.

[0127] S5: Sterilize the clarified fermentation broth at 67℃ for 14 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0128] The total content of phenylethanoid glycosides was 0.87 mg / mL, with a loss rate of 5.7%; superoxide dismutase activity was 153 U / mL; total organic acids were 8.3 mg / mL; and the viable count of lactic acid bacteria was 4.2 × 10⁻⁶. 8 CFU / mL.

[0129] Example 9

[0130] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 48 parts of Cistanche deserticola, 19 parts of antioxidant protectant, 24 parts of microbial adaptive slow-release carbon source, 38 parts of sugar source, 1.8 parts of compound fermentation strain, 115 parts of sterile water; 2 parts of dried tangerine peel, 1 part of rose petals and 1 part of licorice tablets, with a total addition amount of 8% of the weight of Cistanche deserticola.

[0131] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 20 minutes and cool it; grind wolfberry and mulberry into pulp, crush Schisandra chinensis, slice red dates, pass konjac powder through a 110-mesh sieve, and crush dried tangerine peel and licorice slices.

[0132] S2: Mix the pretreated raw materials with the mixed sugar source and sterile water, adjust the solid-liquid ratio to 1:2.9, inoculate with the compound fermentation strain and stir evenly.

[0133] S3: The first stage fermented at 30℃ under anaerobic conditions for 39 days. On the 28th day of fermentation, the pH was measured to be 3.2. Trehalose, accounting for 0.9% of the weight of the fermentation liquid, was added to adjust it to 3.8. After the first stage of fermentation was completed, dried tangerine peel and licorice slices were added, and fermentation continued at 25℃ under light-proof conditions for 19 days. Rose petals were added 7 days before the end of fermentation. After the fermentation was completed, the rose petals were removed.

[0134] S4: Aging the fermentation broth in the dark for 2.8 months, stirring at 78 r / min for 10 min every 15 days; after aging, filter and centrifuge at 4800 r / min for 19 min to obtain a clear fermentation broth.

[0135] S5: Sterilize the clarified fermentation broth at 69℃ for 15 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0136] The total content of phenylethanol glycosides was 0.98 mg / mL, with a loss rate of 5.1%; superoxide dismutase activity was 176 U / mL; total organic acids were 9.2 mg / mL; and the viable count of lactic acid bacteria was 4.9 × 10⁻⁶. 8 CFU / mL.

[0137] Example 10

[0138] Please refer to Figure 1 This embodiment provides a Cistanche deserticola enzyme, which is prepared from the following raw materials in parts by weight: 36 parts of Cistanche deserticola, 11 parts of antioxidant protectant, 16 parts of microbial adaptive slow-release carbon source, 24 parts of sugar source, 0.9 parts of compound fermentation strain, 90 parts of sterile water; 0.8 parts of dried tangerine peel, the amount added is 2.2% of the weight of Cistanche deserticola, and after supplementing to 3%, 0.2 parts of licorice tablets are added.

[0139] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 105℃ for 16 minutes and cool it; grind wolfberry and mulberry into pulp, crush Schisandra chinensis, slice red dates, pass konjac powder through an 80-mesh sieve, grind snow lotus fruit into pulp, and crush dried tangerine peel and licorice slices.

[0140] S2: Mix the pretreated raw materials with brown sugar and sterile water, adjust the solid-liquid ratio to 1:2.3, inoculate with compound fermentation bacteria and stir evenly.

[0141] S3: The first stage ferments at 26℃ under anaerobic conditions for 31 days, with the pH remaining stable at 3.9-4.4 throughout the process; after the first stage fermentation is completed, dried tangerine peel and licorice slices are added, and fermentation continues for 11 days at 21℃ under light-proof conditions.

[0142] S4: Aged the fermentation broth in the dark for 1.3 months, stirring at 58 r / min for 6 min every 15 days; filtered after aging, and then centrifuged at 3400 r / min for 16 min to obtain a clear fermentation broth.

[0143] S5: Sterilize the clarified fermentation broth at 61℃ for 11 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0144] The total content of phenylethanoid glycosides was 0.72 mg / mL, with a loss rate of 7.0%; superoxide dismutase activity was 125 U / mL; total organic acids were 6.5 mg / mL; and the viable count of lactic acid bacteria was 2.2 × 10⁻⁶. 8 CFU / mL.

[0145] Comparative Example 1

[0146] The difference from Example 1 is that only wolfberry is used as the antioxidant protectant, and the ratio of compound fermentation strains is changed to 3:3:4.

[0147] Preparation method: Same as in Example 1.

[0148] Results: The total content of phenylethanol glycosides was 0.35 mg / mL, with a loss rate of 22.3%; the viable count of lactic acid bacteria was 0.4 × 10⁻⁶. 8 CFU / mL, fermentation broth has a musty smell.

[0149] Preparation method:

[0150] S1-S2: Same as Example 1.

[0151] S3: The first stage ferments for 30 days at 25℃ under anaerobic conditions without adding any regulators. After the first stage fermentation is completed, dried tangerine peel is added, and fermentation continues for 10 days at 20℃ under light-proof conditions.

[0152] S4-S5: Same as Example 1.

[0153] The total content of phenylethanoid glycosides was 0.35 mg / mL, with a loss rate of 22.3%; superoxide dismutase activity was 68 U / mL; total organic acids were 3.1 mg / mL; and the viable count of lactic acid bacteria was 0.4 × 10⁻⁶. 8 CFU / mL (not up to standard); the fermentation broth has a slight musty smell, indicating an increased risk of contamination by other microorganisms.

[0154] Comparative Example 2

[0155] The formulation components are the same as in Example 2.

[0156] Preparation method: pH=3.4 unadjusted, rose petals added throughout the process and not removed.

[0157] Results: The total content of phenylethanol glycosides was 0.48 mg / mL, with a loss rate of 18.5%; the viable count of lactic acid bacteria was 0.9 × 10⁻⁶. 8 CFU / mL, fermentation broth was cloudy and had a bitter taste.

[0158] Preparation method:

[0159] S1-S2: Same as Example 2.

[0160] S3: The first stage was fermented at 27℃ under anaerobic conditions for 33 days. On the 20th day of fermentation, the pH was measured to be 3.4, and no regulator was added. After the first stage of fermentation was completed, rose petals were added immediately, and fermentation continued at 22℃ under light-proof conditions for 13 days. The rose petals were not removed.

[0161] S4-S5: Same as Example 2.

[0162] The total content of phenylethanoid glycosides was 0.48 mg / mL, with a loss rate of 18.5%; superoxide dismutase activity was 85 U / mL; total organic acids were 4.2 mg / mL; and the viable count of lactic acid bacteria was 0.9 × 10⁻⁶. 8 CFU / mL; the fermentation broth was cloudy with a slightly bitter taste, and the presence of rose petal residue caused uneven quality.

[0163] Comparative Example 3

[0164] Compared with Example 3, the viable count of the compound fermentation strain was only 1×10⁻⁶. 8 CFU / g.

[0165] Preparation method: pH=4.6, unadjusted.

[0166] Results: The total content of phenylethanol glycosides was 0.52 mg / mL, with a loss rate of 16.7%, and the viable count of lactic acid bacteria was 0.6 × 10⁻⁶. 8 CFU / mL, prolonged fermentation cycle and off-flavor.

[0167] Preparation method: S1-S2: Same as in Example 3.

[0168] S3: The first stage was fermented at 28℃ under anaerobic conditions for 35 days. On the 15th day of fermentation, the pH was measured to be 4.6, and no regulator was added. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued for 15 days at 23℃ under light-proof conditions.

[0169] S4-S5: Same as Example 3.

[0170] The total content of phenylethanoid glycosides was 0.52 mg / mL, with a loss rate of 16.7%; superoxide dismutase activity was 98 U / mL; total organic acids were 4.8 mg / mL; and the viable count of lactic acid bacteria was 0.6 × 10⁻⁶. 8 CFU / mL; the fermentation period was extended to 45 days, and a small amount of off-odors from bacterial metabolism appeared.

[0171] Comparative Example 4

[0172] The formulation components are the same as in Example 4; preparation method: 2% trehalose and 3% sea buckthorn juice are added, and the flavoring agents are added at the beginning of fermentation.

[0173] Results: The total content of phenylethanol glycosides was 0.61 mg / mL, with a loss rate of 14.2%. The organic acid content was 12.3 mg / mL (too high), resulting in a sour and astringent taste and disordered bacterial metabolism.

[0174] Preparation method:

[0175] S1-S2: Same as Example 4.

[0176] S3: The first stage fermented at 29℃ under anaerobic conditions for 38 days. On the 25th day of fermentation, the pH was measured to be 3.3, and trehalose accounting for 2% of the weight of the fermentation liquid was added. On the 30th day of fermentation, the pH was measured to be 4.7, and sea buckthorn juice accounting for 3% of the volume of the fermentation liquid was added. In the early stage of the first stage of fermentation, dried tangerine peel and licorice slices were added, and fermentation continued for 18 days at 24℃ under light-proof conditions.

[0177] S4-S5: Same as Example 4.

[0178] The total content of phenylethanoid glycosides was 0.61 mg / mL, with a loss rate of 14.2%; superoxide dismutase activity was 115 U / mL; total organic acids were 12.3 mg / mL; and the viable count of lactic acid bacteria was 1.8 × 10⁻⁶. 8 CFU / mL; The product has a sour and astringent taste, and the abnormal osmotic pressure of the fermentation system leads to metabolic disorders of the microorganisms.

[0179] Comparative Example 5

[0180] Compared to Example 5, the antioxidant protectant used only mulberry, and the amount of rose petals added was 10%.

[0181] Preparation method: pH=4.8, unadjusted, rose petals added throughout the process and not removed.

[0182] Results: The total content of phenylethanol glycosides was 0.72 mg / mL, the loss rate was 17.9%, the rose aroma was pungent, and the fermentation broth was turbid.

[0183] Preparation method: S1-S2: Same as in Example 5.

[0184] S3: The first stage was fermented at 30℃ under anaerobic conditions for 40 days. On the 18th day of fermentation, the pH was measured to be 4.8, and no regulator was added. After the first stage of fermentation was completed, rose petals were added at the beginning of the second stage of fermentation. Fermentation continued for 20 days at 25℃ under dark conditions. The rose petals were not removed.

[0185] S4-S5: Same as Example 5.

[0186] Product specifications: Total content of phenylethanoid glycosides: 0.72 mg / mL, loss rate: 17.9%; Superoxide dismutase activity: 132 U / mL; Total organic acids: 7.1 mg / mL; Viable lactic acid bacteria count: 2.1 × 10⁻⁶. 8 CFU / mL; the product has a pungent rose aroma that masks the natural flavor of Cistanche deserticola, and there is also a cloudiness problem caused by petal residue.

[0187] Comparative Example 6

[0188] The formulation components are consistent with those of Example 8.

[0189] Preparation method: Cistanche deserticola was sterilized at 120℃ for 25 minutes (high temperature), solid-liquid ratio was 1:1.5, rose petals were added 10 days in advance and not removed, and the aging process was not stirred.

[0190] Results: The total content of phenylethanol glycosides was 0.42 mg / mL, with a loss rate of 32.4%; the viable count of lactic acid bacteria was 0.8 × 10⁻⁶. 8 CFU / mL (not up to standard), fermentation broth has high viscosity and poor taste.

[0191] Preparation method: S1: After washing and slicing Cistanche deserticola, sterilize it with steam at 120℃ for 25 minutes and then cool it; other raw materials are pretreated in the same way as in Example 6.

[0192] S2: Mix the pretreated raw materials with the mixed sugar source and sterile water, adjust the solid-liquid ratio to 1:1.5, inoculate with the compound fermentation bacteria and stir evenly.

[0193] S3: The first stage fermented at 26℃ under anaerobic conditions for 32 days, with the pH remaining stable at 3.8-4.2 throughout the process; after the first stage fermentation was completed, dried tangerine peel was added, and fermentation continued at 21℃ under light-proof conditions for 12 days; rose petals were added 10 days before the end of fermentation, and the rose petals were not removed after the fermentation was completed.

[0194] S4: The fermentation broth was aged in the dark for 1.2 months without stirring; after aging, it was filtered and then centrifuged at 3200 r / min for 16 min to obtain a clear fermentation broth.

[0195] S5: Same as Example 6.

[0196] Product specifications: Total content of phenylethanoid glycosides 0.42 mg / mL, loss rate 32.4%; superoxide dismutase activity 76 U / mL; total organic acids 3.9 mg / mL; viable lactic acid bacteria count 0.8 × 10⁻⁶. 8 CFU / mL; the fermentation broth has high viscosity, uneven component distribution, and a disjointed taste.

[0197] Comparative Example 7

[0198] Compared with Example 7, the ratio of compound fermentation strains in the formula was changed to 5:6:2.

[0199] Preparation method: pH=3.4 unadjusted, aged with stirring interval of 10 days, speed of 100 r / min, time of 15 min.

[0200] Results: The total content of phenylethanol glycosides was 0.53 mg / mL, with a loss rate of 18.9%, and the viable count of lactic acid bacteria was 0.7 × 10⁻⁶. 8 CFU / mL, fermentation broth has an alcoholic odor.

[0201] Preparation method: S1-S2: Same as in Example 7.

[0202] S3: The first stage was fermented at 27℃ under anaerobic conditions for 34 days. On the 22nd day of fermentation, the pH was measured to be 3.4, and no regulator was added. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued for 14 days at 22℃ in the dark.

[0203] S4: Aged the fermentation broth in the dark for 1.8 months, stirring at 100 r / min for 15 min every 10 days; filtered after aging, and then centrifuged at 3800 r / min for 17 min to obtain a clear fermentation broth.

[0204] S5: Same as Example 7.

[0205] Product specifications: Total content of phenylethanoid glycosides: 0.53 mg / mL, loss rate: 18.9%; Superoxide dismutase activity: 102 U / mL; Total organic acids: 4.5 mg / mL; Viable lactic acid bacteria count: 0.7 × 10⁻⁶. 8 CFU / mL (not up to standard); excessive proliferation of Saccharomyces cerevisiae consumes a large amount of carbon source, produces excessive ethanol that inhibits the activity of Lactobacillus plantarum, and the fermentation liquid has a slight alcoholic odor.

[0206] Comparative Example 8

[0207] The raw material ratio is the same as that of Implementation 8.

[0208] Preparation method: First stage fermentation at 35℃, sea buckthorn juice added at 0.5%, second stage fermentation at 28℃ / light, aging under light, centrifugation at 2000r / min, sterilization at 80℃ / 20min.

[0209] Results: The total content of phenylethanol glycosides was 0.58 mg / mL, with a loss rate of 24.1%. The active ingredients were significantly degraded, resulting in flavor imbalance and shortened shelf life.

[0210] Preparation method: S1-S2: Same as in Example 8.

[0211] S3: The first stage was fermented at 35℃ under anaerobic conditions for 36 days. On the 16th day of fermentation, the pH was measured to be 4.6, and sea buckthorn juice accounting for 0.5% of the fermentation liquid volume was added. After the first stage of fermentation was completed, licorice tablets were added, and fermentation continued for 17 days at 28℃ under light conditions. Rose petals were added 5 days before the end of fermentation. The rose petals were not removed after the fermentation was completed.

[0212] S4: Aged the fermentation broth under light for 2.2 months, stirring at 72 r / min for 9 min every 15 days; filtered after aging, and then centrifuged at 2000 r / min for 18 min to obtain a clear fermentation broth.

[0213] S5: Sterilize the clarified fermentation broth at 80℃ for 20 minutes, cool it, and then aseptically fill it in a Class 100 cleanroom environment to obtain the final product.

[0214] Product specifications: Total content of phenylethanoid glycosides: 0.58 mg / mL, loss rate: 24.1%; Superoxide dismutase activity: 95 U / mL; Total organic acids: 5.8 mg / mL; Viable lactic acid bacteria count: 1.2 × 10⁻⁶. 8 CFU / mL; the product's active ingredients are significantly degraded, resulting in flavor imbalance and a shortened shelf life.

[0215] Comparative Example 9

[0216] Compared with Example 9, the viable count of the compound fermentation strain was only 1×10⁻⁶. 8 CFU / g, flavor additives added at 12%.

[0217] Preparation method: The flavoring agent was added at the beginning of fermentation and not removed, and then centrifuged at 6000 r / min.

[0218] Results: The total content of phenylethanol glycosides was 0.69 mg / mL, with a loss rate of 19.4%; the organic acid content was 11.5 mg / mL; the excipient flavor was too strong and the taste was bitter.

[0219] Preparation method:

[0220] S1-S2: Same as Example 9.

[0221] S3: The first stage was fermented at 30℃ under anaerobic conditions for 39 days. On the 28th day of fermentation, the pH was measured to be 3.2, and trehalose accounting for 0.9% of the weight of the fermentation liquid was added. All flavor-adjusting ingredients were added at the beginning of the first stage of fermentation, and fermentation continued for 19 days at 25℃ under light-proof conditions. The roses were not removed after the fermentation was completed.

[0222] S4: Aged the fermentation broth in the dark for 2.8 months, stirring at 78 r / min for 10 min every 15 days; filtered after aging, and then centrifuged at 6000 r / min for 19 min to obtain a clear fermentation broth.

[0223] S5: Same as Example 9.

[0224] Product specifications: Total content of phenylethanol glycosides: 0.69 mg / mL, loss rate: 19.4%; superoxide dismutase activity: 128 U / mL; total organic acids: 11.5 mg / mL; viable lactic acid bacteria count: 1.5 × 10⁻⁶. 8 CFU / mL; the strong flavor of the excipients masked the fermentation flavor, and high-speed centrifugation caused the loss of active ingredients, resulting in a bitter taste.

[0225] Comparative Example 10

[0226] The difference from the components in Example 10 is that the compound fermentation strain uses only Lactobacillus plantarum, and the amount of flavor additives is 2.2%.

[0227] Preparation method: S5 is filled in a non-sterile, ordinary environment.

[0228] Results: The total content of phenylethanol glycosides was 0.51 mg / mL, with a loss rate of 15.3%; the viable count of lactic acid bacteria was 0.9 × 10⁻⁶. 8 CFU / mL, product secondary contamination, deterioration after 5 days of storage.

[0229] Preparation method:

[0230] S1-S2: Same as Example 10.

[0231] S3: The first stage ferments at 26℃ under anaerobic conditions for 31 days, with the pH remaining stable at 3.9-4.4 throughout the process; after the first stage fermentation is completed, dried tangerine peel is added, and fermentation continues for 11 days at 21℃ under light-proof conditions.

[0232] S4: Aged the fermentation broth in the dark for 1.3 months, stirring at 58 r / min for 6 min every 15 days; filtered after aging, and then centrifuged at 3400 r / min for 16 min to obtain a clear fermentation broth.

[0233] S5: Sterilize the clarified fermentation broth at 61℃ for 11 minutes, cool it, and then fill it under normal conditions to obtain the final product.

[0234] Product specifications: Total content of phenylethanoid glycosides: 0.51 mg / mL, loss rate: 15.3%; Superoxide dismutase activity: 89 U / mL; Total organic acids: 4.2 mg / mL; Viable lactic acid bacteria count: 0.9 × 10⁻⁶. 8 CFU / mL; ordinary environmental filling leads to secondary contamination, and the product swells and deteriorates after 5 days of storage.

[0235] Experimental Example 1

[0236] Verification objective: To clarify the impact of requirements for the formulation of antioxidant protectants, the ratio and viable count of compound fermentation strains, and the requirements for the formulation of microbial community-adapted slow-release carbon sources on enzyme quality.

[0237] Experimental Groups:

[0238] Experimental group: Enzyme samples prepared in Examples 1, 3, 5 and 7 were selected.

[0239] Control group: Enzyme samples prepared by comparative examples 1, 3, 5 and 7 were selected.

[0240] Experimental methods: The total content and loss rate of phenylethanol glycosides were determined by high performance liquid chromatography, the viable number of lactic acid bacteria was determined by microbial plate counting, and the activity of superoxide dismutase was determined by spectrophotometry. Basic sensory evaluation was carried out simultaneously.

[0241] Experimental results:

[0242] Experimental group: The total content of phenylethanoid glycosides was ≥0.62mg / mL, and the loss rate was ≤8.5%; the viable count of lactic acid bacteria was ≥1.2×10⁻⁶. 8 CFU / mL; superoxide dismutase activity ≥112U / mL; sensory characteristics: brownish-red and translucent, with no abnormal odor.

[0243] Control group: Total content of phenylethanol glycosides ≤0.72mg / mL, loss rate ≥16.7%; viable lactic acid bacteria count ≤0.9×10⁻⁶. 8 CFU / mL (all below standard); superoxide dismutase activity ≤102U / mL; some samples had abnormal odors such as musty smell and alcohol smell.

[0244] Experimental conclusion: The original technical solution required "at least two antioxidants in combination" and "a 5:3:2 ratio of compound fermentation strains with a viable count ≥1×10⁻⁶". 9 The raw material formulation requirements of "CFU / g" and "at least two kinds of microbial-adapted slow-release carbon sources" are key to ensuring the retention rate of enzyme active ingredients, microbial stability and basic flavor. A single raw material or a formulation that deviates from the ratio will lead to significant deterioration in quality.

[0245] Experimental Example 2

[0246] Verification objective: To verify the effects of pH adjustment strategies, timing and dosage of flavor-enhancing additives, and fermentation temperature and light conditions on enzyme quality during fermentation.

[0247] Experimental Groups:

[0248] Experimental group: Enzyme samples prepared in Examples 2, 4, 6 and 8 were selected.

[0249] Control group: Enzyme samples prepared by comparative examples 2, 4, 6 and 8 were selected.

[0250] Experimental methods: The total amount of organic acids and the content of phenylethanoid glycosides were detected to evaluate the taste harmony and flavor layers. The turbidity and content of miscellaneous bacteria in the fermentation broth were detected, and an accelerated shelf life test at 37℃ was conducted.

[0251] Experimental results:

[0252] Experimental group: Total organic acid content was stable at 5.3-8.9 mg / mL; phenylethanol glycoside loss rate was ≤7.8%; fermentation broth was clear and transparent, and no miscellaneous bacteria were detected; the taste was mellow and harmonious, and the flavor layers were clear; the accelerated shelf life was ≥30 days.

[0253] Control group: Abnormal total organic acid content; loss rate of phenylethanol glycosides ≥18.5%; turbid fermentation broth with some detected contaminating bacteria; sour or bitter taste with unbalanced flavor; accelerated shelf life ≤20 days.

[0254] Experimental conclusion: The process requirements of "precise pH adjustment of 3.5-4.5", "addition of flavor-adjusting auxiliary materials in the later stage" and "segmented temperature-controlled and light-proof fermentation" in the original technical solution can ensure the stability of the fermentation system and avoid loss of active ingredients and deterioration of flavor. Deviation from the above parameters will lead to a comprehensive decline in enzyme quality.

[0255] Experimental Example 3

[0256] Validation objective: To verify the effects of stirring parameters, centrifugation parameters, sterilization parameters, and filling environment during the aging process on the final quality and storage stability of the enzyme.

[0257] Experimental Groups:

[0258] Experimental group: Enzyme samples prepared in Examples 1, 4, 7 (with reasonable aging and stirring parameters) and 10 were selected.

[0259] Control group: Enzyme samples prepared by comparative examples 1, 4, 7 and 10 were selected.

[0260] Experimental methods: The retention rate of phenylethanol glycosides and the number of viable lactic acid bacteria were detected, the uniformity of the fermentation broth was evaluated, and a 60-day storage stability test at room temperature was conducted to observe whether any deterioration occurred.

[0261] Experimental results:

[0262] Experimental group: phenylethanol glycoside retention rate ≥91.5%; viable lactic acid bacteria count ≥1.2×10⁻⁶. 8 CFU / mL; uniform composition of fermentation broth; no deterioration after 60 days of storage at room temperature, and stable quality.

[0263] Control group: phenylethanol glycoside retention rate ≤85%; viable lactic acid bacteria count ≤0.9×10⁻⁶. 8 CFU / mL; uneven composition of fermentation broth; Comparative Example 10 showed bloating and deterioration after 5 days of storage, while the other control groups showed flavor imbalance after 30 days of storage.

[0264] Experimental conclusion: The post-processing requirements in the original technical solution, namely "aging interval of 15 days, stirring at 50-80 r / min for 5-10 min", "centrifugation at 3000-5000 r / min", "sterilization at 60-70℃", and "aseptic filling at Class 100 cleanliness", are the key to ensuring the uniformity of enzyme components, stable activity, and safe storage. Deviation from these parameters will lead to unstable quality and storage risks.

[0265] Test Example 4

[0266] Verification objective: To verify the synergistic effect of parameters in the original technical solution, including raw material formulation, fermentation process, aging, and post-processing, on the overall quality of the enzyme.

[0267] Experimental Groups:

[0268] Experimental group: All enzyme samples prepared in Examples 1-10.

[0269] Control group: All enzyme samples prepared in comparative examples 1-10.

[0270] Test method: A comprehensive quality evaluation system was adopted, covering five dimensions: active ingredients, microbiological indicators, physicochemical indicators, sensory quality and storage stability. The system was quantitatively scored with a total score of 100 points, and ≥80 points was considered as passing.

[0271] Experimental results:

[0272] Experimental group: The overall scores were all 85-96 points, all core indicators met the requirements of the original technical solution, the sensory quality was excellent, and the storage was stable.

[0273] Control group: The overall scores were all 45-68 points, and at least two core indicators failed to meet the standards, resulting in poor sensory quality and insufficient storage stability.

[0274] Experimental conclusion: The parameters of each step in the original technical solution do not exist in isolation, but rather work synergistically to ensure the overall quality of Cistanche deserticola enzyme. Any deviation of a single step from the core parameters will disrupt the overall synergy and lead to a significant deterioration in enzyme quality, thus verifying the scientific validity and completeness of the original technical solution.

[0275] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A Cistanche deserticola enzyme, characterized in that, It is prepared from the following raw materials in parts by weight: 30-50 parts of Cistanche deserticola, 8-20 parts of antioxidant protectant, 10-25 parts of microbial adaptive slow-release carbon source, 20-40 parts of sugar source, 0.5-2 parts of compound fermentation strain, and 80-120 parts of sterile water. The antioxidant is at least two of the following: wolfberry, mulberry, and schisandra chinensis. The microbial community-adapted slow-release carbon source is at least two of the following: red dates, konjac powder, and snow lotus fruit. The total content of phenylethanoid glycosides in the Cistanche deserticola enzyme is ≥0.5mg / mL, and the loss rate of phenylethanoid glycosides is ≤10%.

2. The Cistanche deserticola enzyme according to claim 1, characterized in that, In the antioxidant protectant, the weight ratio of wolfberry to mulberry is 1:1-2, and / or the weight ratio of mulberry to schisandra is 2-3:1; in the microbial-adapted slow-release carbon source, the weight ratio of jujube to konjac powder is 3-5:1, and / or the weight ratio of jujube to snow lotus fruit is 2-3:

1.

3. The Cistanche deserticola enzyme according to claim 1, characterized in that, The compound fermentation strain is composed of Lactobacillus plantarum, Saccharomyces cerevisiae, and acetic acid bacteria in a weight ratio of 5:3:2, and the viable count of the compound fermentation strain is ≥1×10⁻⁶. 9 CFU / g; the sugar source is brown sugar and / or rock sugar.

4. The Cistanche deserticola enzyme according to claim 1, characterized in that, It also includes flavor-regulating excipients, which are at least one of dried tangerine peel, rose petals, and licorice slices, and the total amount of flavor-regulating excipients added is 3%-8% of the weight of Cistanche deserticola.

5. The Cistanche deserticola enzyme according to claim 1, characterized in that, The Cistanche deserticola enzyme contains superoxide dismutase activity ≥100 U / mL, total organic acid content of 5-10 mg / mL, and viable lactic acid bacteria count ≥1×10⁻⁶. 8 CFU / mL.

6. A method for preparing Cistanche deserticola enzyme as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: After washing and slicing the Cistanche deserticola, sterilize it with steam at 105℃ for 15-20 minutes and then cool it; wash, crush, slice, pulp or sieve the antioxidant and the microbial-compatible slow-release carbon source respectively. S2: Mix the pretreated Cistanche deserticola, antioxidant protectant, microbial-adapted slow-release carbon source, sugar source and sterile water in a fermenter, adjust the solid-liquid ratio of the system to 1:2-3, and then inoculate the compound fermentation strain and stir evenly. S3: First, ferment at 25-30℃ under anaerobic conditions for 30-40 days, maintaining the pH of the fermentation system at 3.5-4.5; then add flavor-adjusting ingredients and continue fermenting at 20-25℃ under light-proof conditions for 10-20 days. S4: After fermentation, the fermentation liquid is aged in a dark environment for 1-3 months, and then filtered and centrifuged to obtain a clear fermentation liquid; S5: Sterilize the clarified fermentation broth at 60-70℃ for 10-15 minutes, cool it, and then aseptically fill it to obtain the final product.

7. The method for preparing Cistanche deserticola enzyme according to claim 6, characterized in that, In the first stage of fermentation in step S3, when the pH value is below 3.5, trehalose of 0.5%-1% of the fermentation liquid weight is added for adjustment; when the pH value is above 4.5, sea buckthorn juice of 1%-2% of the fermentation liquid volume is added for adjustment.

8. The method for preparing Cistanche deserticola enzyme according to claim 6, characterized in that, The centrifugation speed in step S4 is 3000-5000 r / min, and the centrifugation time is 15-20 min; the aseptic filling in step S5 is carried out in a Class 100 cleanroom environment.

9. The method for preparing Cistanche deserticola enzyme according to claim 6, characterized in that, In step S3, the flavor-adjusting additives are added after the first stage of fermentation. If rose petals are used, they are added 5-7 days before the end of the second stage of fermentation and removed after fermentation is completed.

10. A method for preparing Cistanche deserticola enzyme according to claim 6, characterized in that, During the aging process described in step S4, the mixture is stirred at a speed of 50-80 r / min for 5-10 min every 15 days.

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