Cistanche composite enzyme drink and preparation method thereof

Abstract

The present application relates to the technical field of biological fermentation, and particularly relates to a Cistanche deserticola composite enzyme drink and a preparation method thereof, and specifically comprises the following steps: after extracting and concentrating traditional Chinese medicine raw materials such as Cistanche deserticola, double-wheel short-time fermentation is carried out: in the first round, a composite inoculant containing yeast and lactobacillus is inoculated and fermented for 2-3 hours, and in the second round, a composite inoculant containing bifidobacterium is inoculated and fermented for 2-3 hours. After the fermentation liquid is subjected to enzyme inactivation at 75 DEG C to terminate fermentation, ultrafiltration treatment is carried out, and finally, the product is filled and subjected to short-time sterilization at 100 DEG C. The present application initiates double-wheel short-time relay fermentation, efficiently enriches postbiotics and avoids excessive acid production; in combination with a gradient precision filtration and sterilization process, the problems of easy precipitation of traditional enzyme and easy damage of heat-sensitive components are solved. The obtained drink is clear and bright, the echinacoside retention rate reaches more than 91%, and has significant anti-fatigue and intestinal regulation effects.

Description

Technical Field

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

[0002] Cistanche deserticola, known as "desert ginseng," is rich in phenylethanoid glycosides such as echinacoside and verbascoside, as well as polysaccharides and trace elements. It is believed to have effects such as tonifying kidney yang, nourishing essence and blood, moistening the intestines and promoting bowel movements, and combating fatigue. Currently, most Cistanche deserticola products on the market are produced by directly soaking in alcohol, decocting, or simply grinding and pressing into tablets. However, traditional water extraction methods have low dissolution rates for large molecular active ingredients such as polysaccharides and proteins, and the bioavailability of phenylethanoid glycosides is limited for direct oral consumption. Alcohol-soaked products have limited appeal and are unsuitable for people with alcohol allergies or those who need to drink alcohol frequently. Direct grinding and tableting result in a rough texture and a strong bitter taste, seriously affecting consumer compliance.

[0003] In recent years, enzymes (plant fermentation broths) have attracted much attention due to their ability to degrade macromolecules, improve flavor, and be rich in beneficial metabolites. In particular, the rise of the "post-biotic" concept has made small-molecule active substances such as cell lysates, extracellular polysaccharides, and short-chain fatty acids in fermentation products a research hotspot. However, existing traditional Chinese medicine enzyme preparation processes have the following drawbacks: First, the fermentation methods are outdated, resulting in low post-biotic yields and flavor degradation. Most existing traditional Chinese medicine enzymes use natural inoculation or single-strain fermentation, with fermentation cycles lasting several weeks or even months. Prolonged fermentation not only makes them susceptible to contamination by other microorganisms and poor batch stability, but also causes a sharp drop in pH as fermentation time increases, leading to excessive acid production and a sour, astringent taste that is difficult to swallow. Simultaneously, as the microorganisms enter their decline phase, they consume the already produced metabolites, resulting in a decrease in the accumulation of "post-biotics" such as extracellular polysaccharides. Second, the post-processing and sterilization processes are crude, making it difficult to simultaneously ensure product quality and activity. The fermentation broth contains a large number of dead bacteria, large molecular proteins, and crude plant fibers, which cannot be completely removed by traditional plate and frame filtration or centrifugal coarse filtration. This causes the product to easily develop sediment and turbidity during its shelf life. In order to achieve commercial sterility, traditional processes often use high-temperature sterilization at 121°C or long-term sterilization at 100°C. This will seriously damage the heat-sensitive core components (such as echinacoside) in Cistanche deserticola and the heat-sensitive post-biotics produced by fermentation, resulting in a significant reduction in the product's efficacy.

[0004] Therefore, how to develop a Cistanche deserticola compound enzyme beverage with a short fermentation cycle, high enrichment of post-genetic agents, excellent taste and flavor, and maximum retention of heat-sensitive active ingredients is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a Cistanche deserticola compound enzyme beverage and its preparation method, optimize its preparation and processing technology, improve the quality of the Cistanche deserticola compound enzyme beverage, and solve the problems mentioned in the background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a Cistanche deserticola compound enzyme beverage and its preparation method, comprising the following steps: S1. Extraction and Concentration: The herbal raw material composition is soaked in water twice for extraction. The soaking liquids obtained from the two extractions are combined and filtered to obtain a mixed extract. The mixed extract is then concentrated under vacuum to obtain a concentrated extract.

[0007] S2. Preparation of compound microbial agents: Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. The viable cell count of each bacterial solution was measured. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the first compound microbial agent. Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. The viable cell count of each bacterial solution was measured. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the second compound microbial agent.

[0008] S3. Stepwise fermentation: Add functional excipients to the concentrated extract and inoculate with the first compound microbial agent to make the initial viable cell concentration of the first compound microbial agent in the fermentation system 10. 7 -10 9 The culture was incubated at CFU / mL at 35-38℃ for 2-3 hours to obtain the first fermentation broth. A second compound microbial agent was then inoculated into the first fermentation broth to achieve an initial viable cell concentration of 10 CFU / mL. 7 -10 9 The fermentation solution was cultured at CFU / mL for a second round at 35-38℃ for 2-3 hours to obtain the fermentation stock solution. The pH of the fermentation stock solution was 3.8-4.5 and the total acidity (calculated as lactic acid) was 0.6-1.2%.

[0009] S4. Post-treatment and sterilization: The fermentation broth is coarsely filtered through a high-level filter to obtain the initial filtrate; the initial filtrate is heated to 75°C and kept at that temperature for 10-15 minutes to inactivate the enzyme to obtain the enzyme-inactivated solution; the enzyme-inactivated solution is then subjected to ultrafiltration through an ultrafiltration machine to obtain the ultrafiltrate.

[0010] S5. Filling and secondary sterilization: The ultrafiltrate is filled into bottles and then sterilized at 100-105℃ for 15-20 minutes to obtain the Cistanche deserticola compound enzyme beverage.

[0011] As a preferred technical solution of the present invention, the traditional Chinese medicine raw material composition in step S1 includes the following components: 10-30 parts by weight of Cistanche deserticola, 1-5 parts by weight of ginseng, 5-15 parts by weight of Polygonatum sibiricum, 5-15 parts by weight of Lycium barbarum, 5-10 parts by weight of Rubus idaeus, 5-10 parts by weight of Cornus officinalis, 5-15 parts by weight of Rehmannia glutinosa, 1-5 parts by weight of cinnamon, 5-15 parts by weight of Codonopsis pilosula, 5-15 parts by weight of Astragalus membranaceus, 3-8 parts by weight of peach kernel, 5-15 parts by weight of mulberry, 5-10 parts by weight of lotus seed, 1-3 parts by weight of clove, 3-8 parts by weight of pumpkin seed, and 2-6 parts by weight of licorice; the Cistanche deserticola, ginseng, Polygonatum sibiricum, Cornus officinalis, Rehmannia glutinosa, cinnamon, Codonopsis pilosula, Astragalus membranaceus, peach kernel, lotus seed, clove, pumpkin seed, and licorice are all pulverized to a particle size of 1.0-8.0 mm.

[0012] As a preferred embodiment of the present invention, the method of two water-soaking extractions in step S1 is as follows: the amount of water added for the first time is 8-10 times the mass of the herbal raw material composition, and the extraction is carried out at 60-80℃ for 1-2 hours; the amount of water added for the second time is 5-8 times the mass of the herbal raw material composition, and the extraction is carried out at 60-80℃ for 0.5-1 hours; the conditions for vacuum concentration are: temperature 50-60℃, vacuum degree 0.08-0.09MPa, and concentration until the soluble solids content is 15-20%.

[0013] As a preferred technical solution of the present invention, the preparation method of the first compound microbial agent in step S2 is as follows: Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulans are independently cultured according to the following culture media and conditions: Lactobacillus johnsonii, Lactobacillus plantarum, and Lactobacillus casei are cultured on MRS medium (the preparation method of MRS medium is as follows: weigh 10.0g of casein peptone, 10.0g of beef extract, 5.0g of yeast extract, 5.0g of glucose, 5.0g of sodium acetate, 2.0g of diammonium citrate, 1.0g of Tween 80, and dihydrogen phosphate). Potassium 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate monohydrate 0.05g, and agar 15.0g were mixed, and then distilled water was added to bring the volume to 1000mL. The pH was adjusted to 6.5-6.8. The resulting culture medium was dispensed into Erlenmeyer flasks, sealed with cotton plugs, and autoclaved at 115-125℃ for 20-30 minutes. After sterilization, the culture medium was allowed to cool to room temperature to obtain MRS culture medium. The medium was then anaerobically cultured at 35-38℃ for 18-24 hours. The Kluyveromyces martensii was cultured on YPD medium (the YPD medium was prepared by weighing 10.0g glucose, 10.0g peptone, 5.0g yeast extract, and 20g agar). 0g, mix, then add distilled water to a final volume of 1000mL, adjust the pH to 6.5-7.0, dispense the resulting culture medium into Erlenmeyer flasks, plug with cotton, and autoclave at 115-125℃ for 20-30 minutes. After sterilization, allow the culture medium to cool to room temperature to obtain YPD culture medium. Culture aerobacterium animalis subspecies aerobically at 28-30℃ and 150-200r / min for 24-36h. The animal subspecies of Bifidobacterium animalis is cultured on modified MRS medium supplemented with 0.05-0.1% L-cysteine ​​hydrochloride at 36-38℃ under strictly anaerobic conditions for 24-48h. The *Weizmannii coagulans* is cultured on LB medium (the L... The preparation method of B medium is as follows: Weigh 10.0g of tryptone, 5.0g of yeast extract, and 10.0g of sodium chloride, mix them, and then add distilled water to a final volume of 1000mL. Adjust the pH to 6.8-7.2. Dispense the resulting medium into Erlenmeyer flasks, seal them with cotton plugs, and autoclave at 115-125℃ for 20-30 minutes. After sterilization, allow the medium to cool to room temperature to obtain LB medium. Incubate aerobically with shaking at 35-38℃ and 150-200r / min for 18-24h. After incubation, determine the viable cell count of each culture. The viable cell count of each culture after expansion should reach the following range: Lactobacillus johnsonii 1.0×10⁻⁶. 9 -5.0×10 9 CFU / mL, Kluyveromyces martensii is 5.0 × 10⁻⁶. 8 -3.0×10 9CFU / mL, Lactobacillus plantarum was 1.0 × 10⁻⁶. 9 -6.0×10 9 CFU / mL, Lactobacillus casei 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / mL, Bifidobacterium animalis subspecies: 5.0 × 10⁻⁶ CFU / mL. 8 -3.0×10 9 CFU / mL, with 1.0 × 10⁻⁶ Weizmannia coagulans. 9 -5.0×10 9 CFU / mL; the first compound bacterial agent is obtained by mixing Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans in a volume ratio of 1-2:2-3:1-2:1-2:2-3:1-2.

[0014] As a preferred embodiment of the present invention, the preparation method of the second compound bacterial agent in step S2 is as follows: *Lactobacillus helveticus*, *Bifidobacterium breve*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus casei*, *Bifidobacterium animalis* subsp. *animal*, and *Weizmannii coagulans* are independently cultured according to the following culture media and conditions: The culture conditions for each bacterial strain are as follows: *Lactobacillus helveticus* is cultured anaerobically at 37-42℃ for 18-24 hours using MRS medium; *Bifidobacterium breve* is cultured strictly anaerobicly at 36-38℃ for 24-48 hours using a modified MRS medium supplemented with 0.05-0.1% L-cysteine ​​hydrochloride; *Lactococcus lactis* subsp. *lactococcus* is cultured anaerobically at 36-38℃ using MRS medium. The cultures were anaerobic at 28-32℃ for 16-20 hours; *Lactobacillus casei* was cultured on MRS medium at 35-38℃ for 18-24 hours; *Bifidobacterium animalis* subspecies was cultured on modified MRS medium supplemented with 0.05-0.1% L-cysteine ​​hydrochloride at 36-38℃ under strictly anaerobic conditions for 24-48 hours; *Weizmannii coagulans* was cultured on LB medium at 35-38℃ with aerobic shaking at 150-200 r / min for 18-24 hours. After culture, the viable cell counts of each culture were measured, and the viable cell counts of each culture after expansion reached the following ranges: *Lactobacillus helveticus* 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, Bifidobacterium breve was 5.0 × 10⁻⁶. 8 -3.0×10 9 CFU / mL, Lactococcus lactis subsp. lactis was 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, Lactobacillus casei 1.0 × 10⁻⁶ 9 -5.0×10 9CFU / mL, Bifidobacterium animalis subspecies: 5.0 × 10⁻⁶ CFU / mL. 8 -3.0×10 9 CFU / mL, with 1.0 × 10⁻⁶ Weizmannia coagulans. 9 -5.0×10 9 CFU / mL; the second compound bacterial agent is obtained by mixing Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans in a volume ratio of 1-2:2-3:1-2:2-3:1-2:1-2.

[0015] As a preferred embodiment of the present invention, the functional excipients in step S3 include the following raw materials: 1-5 parts by weight of oyster peptide, 5-15 parts by weight of concentrated apple juice, 1-5 parts by weight of arabinose, 1-5 parts by weight of xylitol, 1-5 parts by weight of trehalose, 0.5-2 parts by weight of L-theanine, and 2-10 parts by weight of honey.

[0016] As a preferred embodiment of the present invention, the high-level filter in step S3 is a device that relies on the gravity of the fermentation broth to perform coarse filtration through a filter screen with a mesh size of 50-200 mesh; the microporous membrane inside the ultrafiltration machine has a pore size of 0.1-0.22 μm, the ultrafiltration operating pressure is 0.2-0.4 MPa, and the temperature is 25-35℃; the surface of the microporous membrane inside the ultrafiltration machine is pre-coated with a dynamic filter layer formed by a diatomaceous earth suspension with a mass concentration of 1-3%.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention breaks away from the traditional long-cycle fermentation model, employing a dual-stage, short-duration (2-3 hours each) logarithmic-phase directional fermentation with a high inoculum size. The first stage utilizes yeast and specific lactobacilli for rapid cell wall disruption and acid production, while the second stage introduces bifidobacteria for further deep metabolism. Fermentation is terminated at the end of the logarithmic growth phase, when the microbial community is most metabolically active, enriching exopolysaccharides and short-chain fatty acids, as well as other metabiotics. This perfectly avoids excessive acid production and flavor degradation caused by prolonged fermentation, maintaining the product's pH within the optimal sweet and sour range, without any bitter or astringent medicinal taste.

[0018] 2. This invention employs a tiered process of "75℃ mild inactivation + diatomaceous earth and microporous membrane ultrafiltration + 100-105℃ short-time sterilization". The 75℃ inactivation aims to deactivate proteolytic enzymes produced during fermentation, preventing them from further degrading peptides during subsequent ultrafiltration, rather than for sterilization. Diatomaceous earth combined with a 0.1-0.22μm microporous membrane thoroughly traps dead bacteria and large molecular impurities, ensuring the product remains clear and free of sediment during long-term storage. Finally, short-time sterilization at 100℃ ensures commercial sterility. This process achieves a retention rate of over 91% for echinacoside, a core indicator component of Cistanche deserticola, representing a dual breakthrough in product clarity and active ingredient retention.

[0019] 3. This invention uses Cistanche deserticola as the principal ingredient, scientifically combined with ginseng, Polygonatum sibiricum, and other kidney-tonifying and essence-boosting traditional Chinese medicines, and introduces oyster peptides and L-theanine. The synergistic effect of food-medicine homology and functional excipients results in significant health benefits. The fermentation process degrades the macromolecules of the traditional Chinese medicine into easily absorbed small-molecule aglycones, which, in conjunction with excipients and post-biotics, produce significant synergistic effects in anti-fatigue, sleep improvement, and regulation of intestinal microecology. Attached Figure Description

[0020] Figure 1 Flowchart of the preparation process for Cistanche deserticola compound enzyme beverage. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] like Figure 1 As shown, a method for preparing a Cistanche deserticola compound enzyme beverage specifically includes the following steps: S1. Extraction and Concentration: The herbal raw material composition is soaked and extracted twice, the soaking liquids obtained from the two extractions are combined and filtered to obtain a mixed extract; then the mixed extract is concentrated under vacuum to obtain a concentrated extract.

[0023] S2. Preparation of compound microbial agents: Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. The viable cell count of each bacterial solution was measured. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the first compound microbial agent. Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. The viable cell count of each bacterial solution was measured. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the second compound microbial agent.

[0024] S3. Stepwise fermentation: Add functional excipients to the concentrated extract and inoculate with the first compound microbial agent to make the initial viable cell concentration of the first compound microbial agent in the fermentation system 10. 7 -10 9 The culture was incubated at CFU / mL at 35-38℃ for 2-3 hours to obtain the first fermentation broth. A second compound microbial agent was then inoculated into the first fermentation broth to achieve an initial viable cell concentration of 10 CFU / mL. 7-10 9 The fermentation solution was cultured at CFU / mL for a second round at 35-38℃ for 2-3 hours to obtain the fermentation stock solution. The pH of the fermentation stock solution was 3.8-4.5 and the total acidity (calculated as lactic acid) was 0.6-1.2%.

[0025] S4. Post-treatment and sterilization: The fermentation broth is coarsely filtered through a high-level filter to obtain the initial filtrate; the initial filtrate is heated to 75°C and kept at that temperature for 10-15 minutes to inactivate the enzyme to obtain the enzyme-inactivated solution; the enzyme-inactivated solution is then subjected to ultrafiltration through an ultrafiltration machine to obtain the ultrafiltrate.

[0026] S5. Filling and secondary sterilization: The ultrafiltrate is filled into bottles and then sterilized at 100-105℃ for 15-20 minutes to obtain the Cistanche deserticola compound enzyme beverage.

[0027] Lactobacillus johnsonii, Kluyveromyces martensii, Lactobacillus plantarum, and Lactobacillus casei were purchased from Shandong Pingju Biotechnology Co., Ltd.; Bifidobacterium animalis subsp. animalis was purchased from Guangdong Mingtong Biotechnology Co., Ltd.; Weizmann's coagulans was purchased from Hebei Hongtao Bioengineering Co., Ltd.; Lactobacillus helveticus, Bifidobacterium breve, and Lactococcus lactis subsp. lactis were purchased from Shandong Pingju Biotechnology Co., Ltd.

[0028] All other raw materials used in this invention are commercially available.

[0029] Preparation of the first compound microbial agent Lactobacillus johnsonii, Kluyveromyces martensii, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans were independently expanded using the following culture media and conditions: (1) Lactobacillus johnsonii: cultured anaerobically at 37°C for 20 h using MRS liquid medium; (2) Kluyveromyces martensii: YPD liquid medium was used for aerobic shaking culture at 29℃ and 180r / min for 30h; (3) Lactobacillus plantarum: MRS liquid medium was used for anaerobic culture at 37℃ for 20h; (4) Lactobacillus casei: cultured anaerobically at 37°C for 20 h using MRS liquid medium; (5) Animal subspecies of Bifidobacterium animalis: The modified MRS liquid medium supplemented with 0.08% L-cysteine ​​hydrochloride was cultured at 37°C under strict anaerobic conditions for 36 h. (6) Coagulating Weizmannii: LB liquid medium was used for aerobic shaking culture at 37℃ and 180r / min for 20h.

[0030] After incubation, the viable cell count of each culture was measured. The viable cell count of each culture reached the following range: Lactobacillus johnsonii 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, Kluyveromyces martensii is 5.0 × 10⁻⁶. 8 -3.0×10 9 CFU / mL, Lactobacillus plantarum was 1.0 × 10⁻⁶. 9 -6.0×10 9 CFU / mL, Lactobacillus casei 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / mL, Bifidobacterium animalis subspecies: 5.0 × 10⁻⁶ CFU / mL. 8 -3.0×10 9 CFU / mL, with 1.0 × 10⁻⁶ Weizmannia coagulans. 9 -5.0×10 9 CFU / mL; the first compound bacterial agent is obtained by mixing Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans in a volume ratio of 1-2:2-3:1-2:1-2:2-3:1-2.

[0031] Preparation of the second compound microbial agent Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulans were independently cultured according to the following culture media and conditions: (1) Lactobacillus helveticus: cultured anaerobically at 40°C for 20 h using MRS liquid medium; (2) Bifidobacterium breve: It was cultured in modified MRS liquid medium supplemented with 0.08% L-cysteine ​​hydrochloride for 36 h under strict anaerobic conditions at 37 °C; (3) Lactococcus lactis subsp. lactis: M17 liquid medium was used for static anaerobic culture at 30℃ for 18h; (4) Lactobacillus casei: cultured anaerobically at 37°C for 20 h using MRS liquid medium; (5) Animal subspecies of Bifidobacterium animalis: The modified MRS liquid medium supplemented with 0.08% L-cysteine ​​hydrochloride was cultured at 37°C under strict anaerobic conditions for 36 h. (6) Coagulating Weizmannii: LB liquid medium was used for aerobic shaking culture at 37℃ and 180r / min for 20h.

[0032] After incubation, the viable cell count of each culture was measured. The viable cell count of each culture reached the following range: Lactobacillus helveticus 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, Bifidobacterium breve was 5.0 × 10⁻⁶. 8 -3.0×10 9 CFU / mL, Lactococcus lactis subsp. lactis was 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, Lactobacillus casei 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / mL, Bifidobacterium animalis subspecies: 5.0 × 10⁻⁶ CFU / mL. 8 -3.0×10 9 CFU / mL, with 1.0 × 10⁻⁶ Weizmannia coagulans. 9 -5.0×10 9 CFU / mL; the second compound bacterial agent is obtained by mixing Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans in a volume ratio of 1-2:2-3:1-2:2-3:1-2:1-2.

[0033] Example 1 A method for preparing a Cistanche deserticola compound enzyme beverage includes the following steps: S1. Extraction and Concentration: Weigh out 20 parts by weight of Cistanche deserticola, 3 parts by weight of ginseng, 10 parts by weight of Polygonatum sibiricum, 10 parts by weight of Lycium barbarum, 8 parts by weight of Rubus idaeus, 8 parts by weight of Cornus officinalis, 10 parts by weight of Rehmannia glutinosa, 3 parts by weight of cinnamon, 10 parts by weight of Codonopsis pilosula, 10 parts by weight of Astragalus membranaceus, 5 parts by weight of peach kernel, 10 parts by weight of mulberry, 8 parts by weight of lotus seed, 2 parts by weight of clove, 5 parts by weight of pumpkin seed, and 4 parts by weight of licorice. After pulverizing, extract for the first time with 8 times the amount of water at 70℃ for 1.5 hours, and for the second time with 6 times the amount of water at 70℃ for 0.5 hours. Combine the filtrates and concentrate under vacuum at 55℃ and -0.085 MPa until the soluble solids content is 18%, to obtain a concentrated solution.

[0034] S2. Preparation of compound microbial agents: A first compound microbial agent was obtained by mixing *Lactobacillus johnsonii*, *Kluyveromyces martensii*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Bifidobacterium animalis* subsp. *animal*, and *Weizmann's coagulans* at a volume ratio of 1:2:1:2:2:1. A second compound microbial agent was obtained by mixing *Lactobacillus helveticus*, *Bifidobacterium breve*, *Lactococcus lactis* subsp. *animal*, *Lactobacillus casei*, *Bifidobacterium animalis* subsp. *animal*, and *Weizmann's coagulans* at a volume ratio of 1:2:1:2:1:1.

[0035] S3. Stepwise Fermentation: Add 3 parts by weight of oyster peptides, 10 parts by weight of concentrated apple juice, 3 parts by weight of arabinose, 3 parts by weight of xylitol, 3 parts by weight of trehalose, 1 part by weight of L-theanine, and 5 parts by weight of honey to the concentrated liquid. First Round of Cultivation: Inoculate with *Lactobacillus johnsonii*, *Kluyveromyces martensii*, *Lactobacillus plantarum*, *Lactobacillus casei*, *Bifidobacterium animalis* subsp. *animal*, and *Weizmannii coagulans* at a bacterial volume ratio of 1:2:1:2:2:1 (total inoculum 10...). 8 The culture was first fermented at 37°C for 2.5 hours (CFU / mL). Second round of culture: *Lactobacillus helveticus*, *Bifidobacterium breve*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus casei*, *Bifidobacterium animalis* subsp. *animal*, and *Weizmannii coagulans* were inoculated at a bacterial volume ratio of 1:2:1:2:1:1, and fermentation continued at 37°C for 2.5 hours. The pH was measured to be 4.2, and the total acidity was 0.8%.

[0036] S4. Post-treatment: The fermentation broth was filtered through a high-level filter to remove coarse impurities, and then treated with enzyme inactivation at 75℃ for 15 minutes to terminate fermentation. Subsequently, it was pumped into an ultrafiltration machine (pre-coated with diatomaceous earth, microporous membrane with a pore size of 0.22μm, pressure of 0.3MPa, and temperature of 30℃) for ultrafiltration.

[0037] S5. Filling and Sterilization: After the ultrafiltrate is filled, it is sterilized at 100℃ for 15 minutes to obtain the finished product.

[0038] Example 2 The difference from Example 1 is that in this example, the first fermentation time in step S3 is 2 hours and the second fermentation time is 3 hours; in step S4, the pore size of the ultrafiltration microfiltration membrane is 0.1 μm. Other steps and parameters are the same as in Example 1.

[0039] Example 3 The difference from Example 1 is that in this example, the first fermentation time in step S3 is 3 hours and the second fermentation time is 2 hours; in step S4, the pore size of the ultrafiltration microfiltration membrane is 0.15 μm. Other steps and parameters are the same as in Example 1.

[0040] Comparative Example 1 The difference from Example 1 is that the second round of cultivation was omitted in this comparative example, and only the first compound bacterial agent was used for continuous fermentation at 37°C for 5 hours. The other steps are the same as in Example 1.

[0041] Comparative Example 2 The difference from Example 1 is that the enzyme inactivation at 75°C and ultrafiltration in S4 are omitted in this comparative example. Instead, the fermentation broth is directly filtered through a plate and frame filter press and then sterilized at 100°C for 20 minutes. The other steps are the same as in Example 1.

[0042] Comparative Example 3 The difference from Example 1 is that in this comparative example, the fermentation time in step S3 is significantly extended, with the first round of culture fermentation lasting 12 hours and the second round lasting 12 hours (total fermentation time 24 hours). Other steps and parameters are the same as in Example 1.

[0043] The beverages prepared in each embodiment and comparative example were subjected to the following tests: Determination of the content of post-biotic active ingredients: The content of extracellular polysaccharides was determined by the anthrone-sulfuric acid colorimetric method, and the total amount of short-chain fatty acids (calculated as acetic acid and lactic acid) was determined by HPLC.

[0044] Sensory evaluation: 10 professional tasters will score the color, clarity, and taste (balance of sweet and sour, presence of bitterness) (out of 100).

[0045] Retention rate of active ingredients: The retention rate of echinacoside (the core indicator component of Cistanche deserticola) in the sterilized beverage was determined by HPLC.

[0046] Stability test: Let the product stand at room temperature for 6 months and observe whether sediment or turbidity occurs.

[0047] Table 1: Results of various performance tests Determination of soluble protein content in bacterial cell lysates: The Bradford method was used. Beverage samples were filtered through a 0.22 μm filter to remove bacterial cell debris. The supernatant was then mixed with Coomassie Brilliant Blue G-250 staining solution and allowed to stand at room temperature for 5 min. The absorbance was measured at 595 nm. A standard curve was plotted using bovine serum albumin (BSA) as a standard, and the soluble protein content (mg / mL) in the sample was calculated. Soluble proteins mainly originate from intracellular proteins and lysate fragments released during bacterial autolysis or enzymatic lysis during fermentation. Higher soluble protein content indicates more complete bacterial lysis and better metagenic enrichment.

[0048] Determination of small molecule active peptide content: The trichloroacetic acid (TCA)-soluble nitrogen method (OPA method) was used. An equal volume of 10% TCA solution was added to the beverage sample, mixed well, and allowed to stand for 15 min. The mixture was then centrifuged at 4℃ and 12000 r / min for 15 min. The supernatant was collected, and the absorbance was measured at 340 nm using the o-phthalaldehyde (OPA) derivatization method. A standard curve was plotted using glutathione as a standard, and the TCA-soluble peptide content (expressed in μmol / mL) was calculated. This indicator reflects the accumulation level of small molecular weight oligopeptides and free amino acids in the fermentation system. A higher content indicates that the large molecule protein is more fully degraded, and the product is more easily absorbed by the human body.

[0049] Table 2: Detection results of bacterial cell lysates and small molecule bioactive peptides Results analysis: As shown in Table 1, the extracellular polysaccharide content of Examples 1-3 (4.72-4.85 mg / mL) was significantly higher than that of Comparative Example 1 (2.15 mg / mL) and Comparative Example 3 (2.50 mg / mL). In terms of short-chain fatty acids, Examples 1-3 (12.2-12.6 mg / mL) was significantly higher than that of Comparative Example 1 (6.8 mg / mL). However, although Comparative Example 3 (18.5 mg / mL) had a higher value, its pH value dropped sharply, its taste was severely sour and astringent (sensory score of only 45 points), and its extracellular polysaccharide content dropped to 2.50 mg / mL due to excessive acid hydrolysis.

[0050] In Comparative Example 1, the total fermentation time was 5 hours, completely consistent with the total fermentation time of Examples 1-3. Under the same total fermentation time, the metagenic enrichment efficiency of the double-round relay fermentation was significantly better than that of the single-round continuous fermentation, indicating that the technical effect of this invention stems from the "relay" stepwise fermentation mode itself, rather than the extension of the total fermentation time. Comparative Example 3 further shows that while simply extending the fermentation time can further increase the absolute value of short-chain fatty acids, the pH value of the fermentation system drops excessively, and extracellular polysaccharides are largely lost due to acid hydrolysis, resulting in a sensory score of 45 points and rendering the product commercially unviable. In contrast, this invention, with a total fermentation time of only 5 hours, achieved a short-chain fatty acid concentration of 12.6 mg / mL, an extracellular polysaccharide enrichment to 4.85 mg / mL, and a sensory score of 95 points, finding the optimal balance between fermentation efficiency and overall product quality.

[0051] This invention controls the pH value at the fermentation endpoint to 3.8-4.5 and the total acidity to 0.6-1.2% (calculated as lactic acid), which has the following technical significance: On the one hand, this pH range can effectively inhibit the growth of miscellaneous bacteria while avoiding the problem of excessive acid production leading to a sharp sour taste, resulting in a balanced sweet and sour taste and a sensory score of 93-95 points. On the other hand, this acidity condition is conducive to the stable survival of exopolysaccharides and other metabiotics, avoiding the degradation and loss of polysaccharides due to acid hydrolysis when pH < 3.8 (e.g., in Comparative Example 3, the pH value was too low, causing the exopolysaccharide to drop to 2.50 mg / mL). When the total acidity is > 1.2%, the product is too sour, and the sensory score drops significantly; when the total acidity is < 0.6%, the fermentation degree is insufficient, the accumulation of short-chain fatty acids and other metabiotics is low, and the intestinal regulatory effect of the product cannot be fully exerted. Therefore, this invention limits the pH value and total acidity of the fermentation broth to the above range, which is the optimal balance point that takes into account the sensory quality of the product, the efficiency of metabiotic enrichment, and the shelf-life stability.

[0052] As shown in Table 1, the echinacoside retention rates (91.8-92.4%) and 6-month room temperature stability (clear and transparent, no precipitation) of Examples 1-3 were significantly better than those of Comparative Example 2 (85.2% and a large amount of precipitation and turbidity, respectively). Comparative Example 2 used conventional plate and frame filter press instead of the diatomaceous earth pre-coated microporous membrane ultrafiltration of the present invention, and sterilized at 100°C for 20 min instead of sterilizing at 100°C for 15 min of the present invention. Since plate and frame filter press cannot effectively remove the residual dead bacteria (approximately 0.5-10 μm in size) and large molecular protein aggregates in the fermentation broth, the product produced a large amount of precipitation due to particulate matter aggregation during the 6-month shelf life; at the same time, the residual bacteria significantly increased the initial microbial load of the system, and the sterilization time needed to be extended to 20 min to achieve commercial sterility, which is a 33.3% increase in heat exposure time compared to Example 1 (15 min), resulting in accelerated thermal degradation of echinacoside and a decrease in retention rate to 85.2%.

[0053] As shown in Table 2, the soluble protein content (3.31-3.42 mg / mL) and small molecule bioactive peptide content (8.52-8.76 μmol / mL) of Examples 1-3 were significantly higher than those of Comparative Example 1 (single-round fermentation, soluble protein 1.85 mg / mL, small molecule bioactive peptide 4.63 μmol / mL) and Comparative Example 3 (long-term fermentation, soluble protein 2.10 mg / mL, small molecule bioactive peptide 6.85 μmol / mL). This indicates that fermentation was terminated at the end of the logarithmic growth phase (2-3 h), at which point the cells began partial autolysis, releasing abundant intracellular proteins and enzyme systems. This avoided the problems of excessive autolysis in long-term fermentation, which led to inhibition of proteolytic enzymes by the acidic environment and degradation and consumption of soluble proteins. This fully demonstrates the unique advantages of the "short-time relay" fermentation mode in releasing cell lysates and enriching small molecule bioactive peptides.

[0054] Although the soluble protein content (2.96 mg / mL) and small molecule active peptide content (7.2 μmol / mL) of Comparative Example 2 were higher than those of Comparative Examples 1 and 3, they were still lower than those of the other examples. Combined with the significant precipitation observed in Comparative Example 2 after 6 months of stability in Table 1, it can be seen that traditional coarse filtration cannot effectively remove dead bacteria and large molecule proteins, causing some soluble proteins to precipitate due to aggregation during storage, thus reducing the actual effective content of soluble postbiotic active ingredients during the shelf life. In contrast, this invention uses a diatomaceous earth pre-coated 0.1-0.22 μm microporous membrane for ultrafiltration, which effectively retains large molecule impurities while allowing small molecule active peptides and soluble proteins released from bacterial lysis to pass through smoothly, thereby ensuring a high degree of consistency between clarity and retention rate of active ingredients during long-term storage.

[0055] In summary, this invention solves the industry pain points of traditional Chinese medicine enzymes, such as long fermentation cycles, poor taste, easy sedimentation, and easy destruction of active ingredients, through the synergistic innovation of dual-wheel short-time relay fermentation and tiered post-processing technology.

[0056] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A method for preparing a Cistanche deserticola compound enzyme beverage, characterized in that, Includes the following steps: S1. Extraction and Concentration: The herbal raw material composition is soaked in water twice for extraction. The soaking liquids obtained from the two extractions are combined and filtered to obtain a mixed extract. The mixed extract is then concentrated under vacuum to obtain a concentrated extract. S2. Preparation of compound microbial agents: Lactobacillus johnsonii, Kluyveromyces marsupialis, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the first compound microbial agent. Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmann's coagulant were independently cultured to the logarithmic growth phase in their respective culture media. Then, the bacterial solutions were mixed evenly according to the volume ratio of the bacterial solutions to obtain the second compound microbial agent. S3. Stepwise fermentation: Add functional excipients to the concentrated extract and inoculate with the first compound microbial agent to make the initial viable cell concentration of the first compound microbial agent in the fermentation system 10. 7 -10 9 The culture was incubated at CFU / mL at 35-38℃ for 2-3 hours to obtain the first fermentation broth. A second compound microbial agent was then inoculated into the first fermentation broth to achieve an initial viable cell concentration of 10 CFU / mL. 7 -10 9 The concentration of CFU / mL was increased, and a second round of fermentation was carried out at 35-38℃ for 2-3 hours to obtain the fermentation stock solution. The pH of the fermentation stock solution was 3.8-4.5, and the total acidity (calculated as lactic acid) was 0.6-1.2%. S4. Post-treatment and sterilization: The fermentation broth is coarsely filtered through a high-level filter to obtain the initial filtrate; the initial filtrate is heated to 75°C and kept at that temperature for 10-15 minutes to inactivate the enzymes, thereby obtaining the enzyme-inactivated solution. The enzyme-inactivating solution was subjected to ultrafiltration using an ultrafiltration machine to obtain ultrafiltrate; S5. Filling and secondary sterilization: The ultrafiltrate is filled and then sterilized at 100-105℃ for 15-20 minutes to obtain Cistanche deserticola compound enzyme beverage.

2. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 1, characterized in that, In step S1, the traditional Chinese medicine raw material composition includes the following components: 10-30 parts by weight of Cistanche deserticola, 1-5 parts by weight of ginseng, 5-15 parts by weight of Polygonatum sibiricum, 5-15 parts by weight of Lycium barbarum, 5-10 parts by weight of Rubus idaeus, 5-10 parts by weight of Cornus officinalis, 5-15 parts by weight of Rehmannia glutinosa, 1-5 parts by weight of cinnamon, 5-15 parts by weight of Codonopsis pilosula, 5-15 parts by weight of Astragalus membranaceus, 3-8 parts by weight of peach kernel, 5-15 parts by weight of mulberry, 5-10 parts by weight of lotus seed, 1-3 parts by weight of clove, 3-8 parts by weight of pumpkin seed, and 2-6 parts by weight of licorice.

3. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 1, characterized in that, In step S1: The method of two-stage water soaking and extraction is as follows: the amount of water added for the first time is 8-10 times the mass of the Chinese herbal raw material composition, and the soaking and extraction is carried out at 60-80℃ for 1-2 hours; the amount of water added for the second time is 5-8 times the mass of the Chinese herbal raw material composition, and the soaking and extraction is carried out at 60-80℃ for 0.5-1 hours. The conditions for vacuum concentration are: temperature 50-60℃, vacuum degree 0.08-0.09MPa, and concentration to a soluble solids content of 15-20%.

4. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 1, characterized in that, In step S2, the preparation method of the first compound microbial agent is as follows: Lactobacillus johnsonii, Kluyveromyces marxi, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans are mixed in a volume ratio of 1-2:2-3:1-2:1-2:2-3:1-2 to obtain the first compound microbial agent. The preparation method of the second compound microbial agent is as follows: Lactobacillus helveticus, Bifidobacterium breve, Lactococcus lactis subsp. lactis, Lactobacillus casei, Bifidobacterium animalis subsp. animalis, and Weizmannii coagulans are mixed in a volume ratio of 1-2:2-3:1-2:2-3:1-2:1-2 to obtain the second compound microbial agent.

5. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 1, characterized in that, In step S3, the functional excipients include the following raw materials: 1-5 parts by weight of oyster peptide, 5-15 parts by weight of concentrated apple juice, 1-5 parts by weight of arabinose, 1-5 parts by weight of xylitol, 1-5 parts by weight of trehalose, 0.5-2 parts by weight of L-theanine, and 2-10 parts by weight of honey.

6. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 1, characterized in that, In step S4, the high-level filter is a device that relies on the gravity of the fermentation broth to perform coarse filtration through a filter screen with a mesh size of 50-200 mesh; the microporous membrane inside the ultrafiltration machine has a pore size of 0.1-0.22 μm, an ultrafiltration operating pressure of 0.2-0.4 MPa, and a temperature of 25-35℃; the surface of the microporous membrane inside the ultrafiltration machine is pre-coated with a dynamic filter layer formed by a diatomaceous earth suspension with a mass concentration of 1-3%.

7. The method for preparing a Cistanche deserticola compound enzyme beverage according to claim 2, characterized in that, The ingredients, including Cistanche deserticola, ginseng, Polygonatum sibiricum, Cornus officinalis, Rehmannia glutinosa, cinnamon, Codonopsis pilosula, Astragalus membranaceus, peach kernel, lotus seed, clove, pumpkin seed, and licorice, were all pulverized to a particle size of 1.0-8.0 mm.

8. A Cistanche deserticola compound enzyme beverage, characterized in that, It is prepared by the preparation method of any one of claims 1-7.

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