Preparation method of plantain and buckwheat probiotic enzyme

Abstract

This invention discloses a method for preparing a probiotic enzyme from plantain and lamb's quarters, relating to the technical field of plantain and lamb's quarters probiotic enzyme preparation. First, freshly harvested plantain and lamb's quarters are cleaned and drained after removing impurities. They are then mixed in a specific ratio and pulped to obtain a mixed slurry. The slurry is heated to a set temperature and then kept at a constant temperature to complete the enzyme inactivation process. After coarse filtration to remove residue, a clear mixed juice is obtained. The soluble solids content of the mixed juice is adjusted, and a compound inoculum of *Lactobacillus plantarum* and *Bacillus coagulans* is inoculated. A first-stage constant-temperature anaerobic fermentation is performed. During the middle stage of fermentation, a slow-release carbon source is added, and the process transitions to a second-stage constant-temperature anaerobic fermentation. After fermentation, the product undergoes slow cooling, low-temperature clarification, and low-temperature instantaneous sterilization to obtain the final enzyme product. This invention can directionally degrade anti-nutritional factors such as oxalic acid and irritating alkaloids in the raw materials, inactivate endogenous enzymes to prevent significant loss of active ingredients, greatly increase the viable bacteria count in the final product, and ensure high product safety.

Description

Technical Field

[0001] This invention relates to the field of plantain and lamb's quarters probiotic enzyme preparation technology, and particularly to a method for preparing plantain and lamb's quarters probiotic enzyme. Background Technology

[0002] In recent years, the health industry has developed rapidly. Probiotic enzymes derived from wild plants that are both food and medicine have gained widespread recognition in the consumer market due to their nutritional supplementation and metabolic regulation effects. The industry has maintained a high annual growth rate, with core demands focusing on improving product safety, retention rate of active ingredients, and probiotic activity. Plantain and lamb's quarters are widely distributed wild plants in my country that are both food and medicine. They are rich in active ingredients such as flavonoids, polysaccharides, superoxide dismutase, and minerals, and have potential effects such as clearing heat and promoting diuresis and regulating immunity. However, they contain anti-nutritional factors such as oxalic acid and irritating quinolone alkaloids. Direct consumption can easily cause gastrointestinal irritation and electrolyte imbalance. Therefore, they are usually prepared into enzymes through fermentation before consumption.

[0003] Currently, the mainstream wild plant enzyme preparation processes in the industry are mainly divided into two categories. The first is the open-air natural fermentation process. Its working principle involves adding carbon sources such as sucrose and brown sugar to wild vegetable juice, relying on naturally occurring microorganisms in the environment or adding commercially available compound fermentation agents for room temperature open fermentation to achieve raw material decomposition and product transformation. This process has low operating threshold and low equipment investment, and is widely used in small-scale workshop production. Its current advantages are simple preparation process and strong raw material adaptability. However, this process does not include a step to inactivate endogenous enzymes in the raw materials. The polyphenol oxidase and pectinase naturally present in the raw materials will decompose a large amount of flavonoids, polysaccharides and other active ingredients, resulting in an active ingredient retention rate of less than 30%. The fermentation process lacks microbial control and targeted degradation design of anti-nutritional factors. The degradation rate of oxalic acid and irritating alkaloids is less than 25%. The product has a high content of miscellaneous bacteria, is highly irritating, and has an extremely low survival rate of probiotics, posing a safety risk for long-term consumption.

[0004] The second method is a single probiotic controlled fermentation process. Its working principle involves introducing a single lactic acid bacteria into sterilized wild vegetable juice and fermenting it at a constant temperature under sealed conditions to obtain the enzyme product. This process offers controllable fermentation and good product consistency, and is widely used in large-scale industrial production. Its current advantages include low risk of contamination by other microorganisms and stable product acidity. However, this process uses only a single strain of bacteria, making it impossible to simultaneously achieve the targeted degradation of oxalic acid and irritating alkaloids; the degradation rate of anti-nutritional factors is less than 40%. Furthermore, it only adds a single fast-acting carbon source in the initial stage of fermentation, leading to insufficient carbon source in the later stages and the death of a large number of probiotics, resulting in a final product with a viable count of less than 10^4 CFU / mL. The lack of staged gradient fermentation and slow temperature control steps for activity protection results in a loss rate of over 40% of heat-sensitive active ingredients, failing to meet the production requirements of high-quality enzymes.

[0005] Currently, there is no plantain and lamb's quarters enzyme preparation scheme in the industry that can simultaneously achieve high degradation rate of anti-nutritional factors, high retention rate of active ingredients, and high survival rate of probiotics. This cannot meet the core demand of the consumer market for safe and effective medicinal and edible enzyme products, and there is an urgent need to develop targeted preparation processes to fill the industry gap. Summary of the Invention

[0006] To address the problems of insufficient degradation of anti-nutritional factors, severe loss of active ingredients, and low probiotic activity in existing plantain and lamb's quarters enzyme preparation methods, this invention provides a method for preparing plantain and lamb's quarters probiotic enzymes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a probiotic enzyme from plantain and lamb's quarters, comprising the following steps: S1. Raw Material Pretreatment: Collect fresh plantain stems and leaves that are 30 to 60 days old and have not yet flowered, as well as fresh lamb's quarters stems and leaves that are 10 to 20 cm tall and have not yet entered the flowering stage. Rinse them three times with running purified water, each rinse lasting one minute, to completely remove surface dirt, insect eggs, and impurities. Then, spread the washed raw materials in a cool, ventilated place, controlling the thickness to 2 to 3 cm, and let them air dry naturally until there is no free water on the surface. Use a spiral cold press juicer to cold press the raw materials at a temperature not exceeding 25°C. Juice is extracted and the resulting juice is filtered twice through a 200-mesh food-grade nylon filter cloth to completely remove coarse fiber residue, resulting in mixed wild vegetable juice. The mixed wild vegetable juice is then sent to a water bath sterilization device and kept at a precise temperature of 85 to 90 degrees Celsius for 5 to 10 minutes to complete the enzyme inactivation process. This inactivates the endogenous enzymes such as polyphenol oxidase and pectinase naturally present in the raw materials, preventing the non-specific decomposition of active ingredients during subsequent fermentation. Subsequently, the enzyme-inactivated mixed wild vegetable juice is rapidly cooled to 20 to 25 degrees Celsius using a plate heat exchanger and stored in an airtight container for later use. S2. First-stage anaerobic fermentation: Add food-grade isomaltulose to the cooled mixed wild vegetable juice. Stir with a sterile impeller at a speed of 100 to 120 r / min for 10 to 15 minutes until the isomaltulose is completely dissolved. After terminal sterilization filtration, the resulting liquid is sent to a sterile fermentation tank. Introduce activated high-activity mixed probiotics and add purified water that has been sterilized at 121℃ for 20 minutes and cooled to room temperature to bring the volume to the preset volume. Then, purge the fermentation tank with sterile nitrogen gas for 5 to 10 minutes to reduce the dissolved oxygen content of the system to below 0.5 mg / L. Seal the fermentation tank and maintain the tank pressure at 0.02 to 0.03 MPa to prevent the invasion of external bacteria. Control the fermentation temperature to be stable at 25 to 30 degrees Celsius. Perform anaerobic fermentation for 18 to 24 hours until the pH of the system drops to 4.5 to 4.8 to obtain the first-stage fermentation liquid. S3. Second-stage constant-temperature fermentation: Food-grade isomaltooligosaccharide with a purity of not less than 90% is added to the first-stage fermentation broth using an aseptic feeding method. Warm water is slowly introduced through the fermenter jacket to raise the temperature, with the heating rate controlled at 1 degree Celsius every 10 minutes to avoid sudden temperature changes that could reduce the activity of probiotics. After the temperature reaches 34 to 37 degrees Celsius, the fermentation is maintained at a constant temperature for 24 to 36 hours. During the fermentation process, the tank pressure is maintained at 0.02 to 0.03 MPa. The pH of the system is sampled and tested every 6 hours. Fermentation is terminated when the pH stabilizes at 3.8 to 4.3, yielding the second-stage fermentation broth. S4. Post-processing: Cooling water is introduced through the jacket of the fermenter to slowly cool the secondary fermentation liquid. The cooling rate is controlled at 1 degree Celsius every 15 minutes to avoid sudden temperature changes that may cause the active ingredients to be released. After cooling to 16 to 20 degrees Celsius, the liquid is kept at this temperature and allowed to stand for 8 to 12 hours to complete natural clarification. The upper clear liquid is first siphoned out, and the lower turbid liquid is filtered through a 300-mesh food-grade nylon filter cloth and then combined with the upper clear liquid. Subsequently, a food-grade microfiltration membrane is used for microfiltration sterilization. The filtration operation temperature is maintained at 16 to 20 degrees Celsius. The resulting clear filtrate is the plantain and lamb's quarters probiotic enzyme.

[0008] Preferably, in the raw material pretreatment step S1, the fresh weight ratio of the collected fresh plantain to lamb's quarters is 1:1 to 2:1. This ratio can balance the content of active ingredients such as flavonoids, polysaccharides, and minerals in the raw materials, while reducing the initial proportion of anti-nutritional factors such as oxalic acid and irritating alkaloids, thus avoiding excessive irritation in the final product.

[0009] Preferably, the ingredients in the S2 first-stage anaerobic fermentation step are, by fresh weight, 8 to 18 parts of enzyme-inactivated mixed wild vegetable juice, 1.2 to 2 parts of food-grade isomaltulose, 0.1 to 0.5 parts of activated mixed probiotic agent, and 80 to 95 parts of sterilized and cooled purified water. This ratio can provide sufficient fast-acting carbon source for probiotics, while ensuring that the initial concentration of active ingredients in the fermentation system meets the concentration requirements for subsequent fermentation.

[0010] Preferably, in the second-stage isothermal fermentation step S3, the amount of food-grade isomaltooligosaccharide added is 0.2% to 0.5% of the total mass of the first-stage fermentation liquid. As a slow-release carbon source for probiotics, isomaltooligosaccharide can slowly release usable sugars during the second-stage fermentation process, maintain the proliferative activity of probiotics, and avoid the death of a large number of probiotics due to insufficient carbon source in the later stage of fermentation.

[0011] Preferably, the mixed probiotic agent added in the S2 first-stage anaerobic fermentation step is a compound agent obtained by mixing Bifidobacterium adolescentis and Lactobacillus delbrueckii subsp. lactis in a 1:1 ratio of live bacteria. Bifidobacterium adolescentis can preferentially utilize isomaltulose to rapidly proliferate, while simultaneously degrading irritating substances such as oxalic acid and anti-nutritional factors in the raw materials. Lactobacillus delbrueckii subsp. lactis can rapidly produce acid to regulate the pH of the system and inhibit the growth of miscellaneous bacteria. The two strains work synergistically to improve fermentation efficiency and product safety.

[0012] Preferably, the total viable count of the mixed probiotic agent is not less than 1×10^9 CFU / g. The agent needs to be activated before being introduced into the fermentation system. The activation method is to inoculate the agent into sterilized MRS liquid culture medium, incubate at 37°C for 12 hours until the strain is in the logarithmic growth phase, collect the cells by centrifugation, and resuspend them in sterile physiological saline to the preset viable cell concentration. This ensures that the introduced strain has high biological activity, can quickly adapt to the fermentation system to start fermentation, and shorten the fermentation delay period.

[0013] Preferably, in the S2 first-stage anaerobic fermentation step, the sterilization filtration of the liquid after isomaltulose dissolution is completed using a food-grade polyethersulfone organic microporous filter membrane with a pore size of 0.22 μm. The filtration operation temperature is 25 to 30°C, and the operation pressure is 0.1 to 0.15 MPa. This can completely intercept contaminants such as miscellaneous bacteria and fungal spores in the liquid, ensuring the initial sterile state of the fermentation system and avoiding fermentation failure and off-odors in the product caused by miscellaneous bacterial contamination.

[0014] Preferably, during the fermentation process of the second-stage isothermal fermentation step S3, low-speed stirring is performed every 6 hours, with each stirring session lasting 5 minutes and the stirring speed being 30 to 50 r / min. Low-speed stirring can prevent the bacterial cells from settling and clumping, ensuring full contact between the bacterial cells and the substrate, while also preventing the introduction of excessive oxygen that could affect the anaerobic metabolism of probiotics, thereby improving the uniformity of fermentation and the conversion rate of active ingredients.

[0015] Preferably, the microfiltration sterilization in the S4 post-processing step is completed using a food-grade inorganic ceramic microfiltration membrane with a pore size of 0.45 μm. The filtration operation pressure is 0.1 to 0.2 MPa, and the operation temperature is 16 to 20 °C. The ceramic microfiltration membrane can effectively retain residual dead bacteria and insoluble impurities in the fermentation broth, while not damaging the heat-sensitive active ingredients and active probiotics in the system, thus ensuring the clarity and biological activity of the final product.

[0016] Preferably, the total viable count of the prepared plantain and lamb's quarters probiotic enzyme is not less than 1×10^6 CFU / mL, the SOD activity is not less than 18 U / L, the polyphenol content is not less than 0.7 mg / g, the free amino acid content is not less than 38 mg / 100g, the oxalic acid degradation rate in the raw materials is not less than 75%, the degradation rate of irritating alkaloids is not less than 65%, and the pH value of the product is stable between 3.8 and 4.3, which meets the quality requirements of food-grade probiotic enzymes.

[0017] The present invention has the following beneficial effects: This invention combines two probiotic strains with targeted degradation capabilities with a two-stage gradient anaerobic fermentation process to target and decompose oxalic acid and irritating quinolone alkaloids in the raw materials. Simultaneously, a precisely temperature-controlled enzyme inactivation step is incorporated into the pretreatment stage to completely inactivate endogenous degradative enzymes such as polyphenol oxidase and pectinase present in the raw materials. This effectively solves the problems of insufficient degradation of anti-nutritional factors and significant loss of active ingredients due to the decomposition of endogenous enzymes in existing technologies. The final product exhibits significantly reduced edibility and significantly improved retention levels of active ingredients such as flavonoids, polysaccharides, and superoxide dismutase, effectively ensuring both food safety and efficacy.

[0018] This invention employs a tiered carbon source supply scheme, combining a fast-acting carbon source with a slow-release carbon source added during fermentation. This, coupled with a fully anaerobic, slightly positive-pressure fermentation environment, slow temperature adjustments, and low-temperature clarification post-processing, provides a continuous and stable supply of nutrients for probiotic proliferation. It also prevents damage to heat-sensitive active ingredients and probiotics from sudden temperature changes and oxygen shocks. This effectively solves the problems of insufficient carbon source in the later stages of fermentation leading to mass probiotic death and fluctuations in process parameters causing inactivation of active ingredients, which are common in existing technologies. The final product exhibits significantly improved probiotic activity and greatly enhanced stability of functional components, maintaining stable metabolic regulatory effects over the long term.

[0019] The preparation process of this invention has strong adaptability and does not require the introduction of additional special production equipment. It can be adapted to the small-batch customized production needs of small production entities as well as the large-scale continuous production needs of large food enterprises. It can be implemented without major modifications to the basic layout of existing enzyme production lines and can be widely used in the industrial production of various medicinal and edible wild plant enzymes. It provides a feasible technical path for the high-value development of wild edible plants and has high industry promotion value. Attached Figure Description

[0020] Figure 1 This is a schematic flowchart of a method for preparing a probiotic enzyme from plantain and lamb's quarters proposed in this invention. Figure 2 This is a bar chart comparing the degradation rate of anti-nutritional factors and the total number of viable bacteria in a comparative embodiment of the present invention. Figure 3 This is a radar chart comparing the overall performance of embodiments of the present invention. Detailed Implementation

[0021] The following will refer to the appendices in the embodiments of the present invention. Figure 1-3 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 This embodiment uses the lower limit of the parameter range described in the claims to prepare plantain and lamb's quarters probiotic enzymes. The specific steps are as follows: S1. Raw Material Pretreatment: Fresh plantain stems and leaves (30 days old, before heading) and fresh lamb's quarters stems and leaves (10 cm tall, before flowering) are collected at a fresh weight ratio of 1:1. The raw materials are rinsed three times with flowing purified water, each rinse lasting one minute, to completely remove surface dirt, insect eggs, and impurities. The washed raw materials are then spread in a cool, ventilated place, with a thickness of 2 cm, and allowed to air dry until no free water remains on the surface. The juice is then cold-pressed at 22℃ using a spiral cold-press juicer. The resulting juice is filtered through a 200-mesh screen. The mixture is filtered twice with high-quality nylon filter cloth to completely remove coarse fiber residue, resulting in a mixed vegetable juice. The mixed vegetable juice is then sent to a water bath sterilization device and precisely heated to 85 degrees Celsius for 10 minutes to complete the enzyme inactivation process. This inactivates the endogenous enzymes such as polyphenol oxidase and pectinase naturally present in the raw materials, preventing the non-specific decomposition of active ingredients during subsequent fermentation. The enzyme-inactivated mixed vegetable juice is then rapidly cooled to 22 degrees Celsius using a plate heat exchanger and stored in a sealed container for later use. This step addresses the deficiency in existing technologies where the failure to inactivate endogenous enzymes in the raw materials leads to the decomposition and loss of active ingredients.

[0023] S2. First-stage anaerobic fermentation: Weigh 8 parts of the enzyme-inactivated mixed wild vegetable juice according to fresh weight, add 1.2 parts of food-grade isomaltulose, and stir with a sterile impeller at 100 r / min for 10 minutes until the isomaltulose is completely dissolved. The resulting liquid is then sterilized by filtering with a food-grade polyethersulfone organic microporous membrane with a pore size of 0.22 μm at an operating temperature of 25℃ and an operating pressure of 0.1 MPa. After completely removing contaminants such as bacteria and fungal spores from the liquid, it is sent to a sterile fermentation tank. Inoculate with 0.1 parts of activated mixed probiotics, which are a mixture of Bifidobacterium adolescentis and Lactobacillus delbrueckii subsp. lactis at a live count ratio of 1:1, with a total live count of 1×10^9 CFU / g. The activation method is to inoculate the probiotics... In sterile MRS liquid medium at pH 6.5, the bacteria were incubated at 37°C for 12 hours until they reached the logarithmic growth phase. After centrifugation at 4000 rpm for 10 minutes, the bacterial cells were collected and resuspended in sterile physiological saline to the preset viable cell concentration. 95 portions of purified water, sterilized at 121°C for 20 minutes and cooled to 22°C, were added to bring the volume to a final volume. Sterile nitrogen gas was then introduced into the fermenter for 5 minutes to reduce the dissolved oxygen content to 0.4 mg / L. The fermenter was then sealed and the pressure maintained at 0.02 MPa to prevent external microbial contamination. The fermentation temperature was kept stable at 25°C, and anaerobic fermentation was carried out for 18 hours until the pH dropped to 4.8, yielding the first-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as initial microbial contamination, low probiotic activity, and slow proliferation.

[0024] S3. Second-stage constant-temperature fermentation: Food-grade isomaltooligosaccharide with a purity of 90% is added to the first-stage fermentation broth using a sterile fed-batch method. The addition amount is 0.2% of the total mass of the first-stage fermentation broth. Warm water is slowly introduced through the fermenter jacket to raise the temperature at a rate of 1 degree Celsius every 10 minutes to avoid sudden temperature changes that could reduce the activity of probiotics. After reaching 34 degrees Celsius, the temperature is maintained at a constant temperature for 24 hours. During fermentation, the tank pressure is maintained at 0.02 MPa. Low-speed stirring is performed every 6 hours for 5 minutes at a stirring speed of 30 r / min to prevent cell sedimentation and clumping while avoiding the introduction of excessive oxygen. Fermentation is terminated when the pH stabilizes at 4.3, yielding the second-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as insufficient carbon source in the later stages of fermentation, mass death of probiotics, and incomplete degradation of anti-nutritional factors.

[0025] S4. Post-processing: Cooling water is introduced through the jacket of the fermenter to slowly cool the secondary fermentation broth. The cooling rate is controlled at 1 degree Celsius every 15 minutes to avoid sudden temperature changes that could cause the active ingredients to be released. After cooling to 16 degrees Celsius, the broth is kept at this temperature and allowed to stand for 8 hours to achieve natural clarification. The upper clear liquid is first siphoned out, and the lower turbid liquid is filtered through a 300-mesh food-grade nylon filter cloth and then combined with the upper clear liquid. Subsequently, microfiltration sterilization is performed using a food-grade inorganic ceramic microfiltration membrane with a pore size of 0.45μm. The filtration operation pressure is 0.1MPa and the operating temperature is 16℃. The resulting clear filtrate is the plantain and lamb's quarters probiotic enzyme. This step addresses the shortcomings of existing technologies, such as excessively rapid cooling leading to the death of live bacteria, the release of active ingredients, and poor clarity.

[0026] Example 2 This embodiment uses the intermediate optimal value of the parameter range described in the claims to prepare plantain and lamb's quarters probiotic enzymes. The specific steps are as follows: S1. Raw Material Pretreatment: Fresh plantain stems and leaves (45 days old, before heading) and fresh lamb's quarters stems and leaves (15 cm tall, before flowering) were collected at a fresh weight ratio of 1.5:1. The raw materials were rinsed three times with flowing purified water, each rinse lasting one minute, to completely remove surface dirt, insect eggs, and impurities. The washed raw materials were then spread in a cool, ventilated place, with a thickness controlled at 2.5 cm, and allowed to air dry naturally until no free water remained on the surface. The juice was then cold-pressed at 23°C using a spiral cold-press juicer. The resulting juice was then processed through a 200-meter... The mixture is filtered twice using food-grade nylon filter cloth to completely remove coarse fiber residue, resulting in a mixed wild vegetable juice. The juice is then placed in a water bath sterilization device and kept at 88 degrees Celsius for 8 minutes to inactivate enzymes. This process deactivates endogenous enzymes such as polyphenol oxidase and pectinase naturally present in the raw materials, preventing non-specific decomposition of active ingredients during subsequent fermentation. The inactivated juice is then rapidly cooled to 23 degrees Celsius using a plate heat exchanger and stored in a sealed container for later use. This step addresses the deficiency in existing technologies where the failure to deactivate endogenous enzymes in the raw materials leads to the decomposition and loss of active ingredients.

[0027] S2. First-stage anaerobic fermentation: Weigh 13 parts of the enzyme-inactivated mixed wild vegetable juice according to fresh weight, add 1.6 parts of food-grade isomaltulose, and stir with a sterile impeller at 110 r / min for 12 minutes until the isomaltulose is completely dissolved. The resulting liquid is then sterilized by filtering with a food-grade polyethersulfone organic microporous membrane with a pore size of 0.22 μm at an operating temperature of 28℃ and an operating pressure of 0.12 MPa. After completely removing contaminants such as bacteria and fungal spores from the liquid, it is sent to a sterile fermentation tank. Inoculate with 0.3 parts of activated mixed probiotic agent. The mixed probiotic agent is a mixture of Bifidobacterium adolescentis and Lactobacillus delbrueckii subsp. lactis at a live bacteria ratio of 1:1, with a total live bacteria count of 1.2 × 10^9 CFU / g. The activation method is to inoculate the probiotic agent... The bacteria were added to sterile MRS liquid medium at pH 6.5 and incubated at 37°C for 12 hours until they reached the logarithmic growth phase. After centrifugation at 4000 rpm for 10 minutes, the bacteria were collected and resuspended in sterile physiological saline to the preset viable cell concentration. 87 portions of purified water, sterilized at 121°C for 20 minutes and cooled to 23°C, were added to bring the volume to a final volume. Sterile nitrogen gas was then introduced into the fermenter for 8 minutes to reduce the dissolved oxygen content to 0.3 mg / L. The fermenter was then sealed and the pressure maintained at 0.025 MPa to prevent external microbial contamination. The fermentation temperature was kept stable at 28°C, and anaerobic fermentation was carried out for 21 hours until the pH dropped to 4.6, yielding the first-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as initial microbial contamination, low probiotic activity, and slow proliferation.

[0028] S3. Second-stage constant-temperature fermentation: Food-grade isomaltooligosaccharide with a purity of 92% is added to the first-stage fermentation broth using an aseptic feeding method. The amount added is 0.35% of the total mass of the first-stage fermentation broth. Warm water is slowly introduced through the jacket of the fermenter to raise the temperature. The heating rate is controlled at 1 degree Celsius every 10 minutes to avoid sudden temperature changes that may reduce the activity of probiotics. After the temperature reaches 35 degrees Celsius, it is kept at a constant temperature for 30 hours. During the fermentation process, the tank pressure is maintained at 0.025 MPa. Low-speed stirring is performed every 6 hours, with a single stirring time of 5 minutes and a stirring speed of 40 r / min to prevent the bacterial cells from settling and clumping while avoiding the introduction of excessive oxygen. Fermentation is terminated when the pH stabilizes at 4.0 to obtain the second-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as insufficient carbon source in the later stages of fermentation, mass death of probiotics, and inadequate degradation of anti-nutritional factors.

[0029] S4. Post-processing: Cooling water is introduced through the jacket of the fermenter to slowly cool the secondary fermentation broth. The cooling rate is controlled at 1 degree Celsius every 15 minutes to avoid sudden temperature changes that could cause the active ingredients to be released. After cooling to 18 degrees Celsius, the broth is kept at this temperature and allowed to stand for 10 hours to achieve natural clarification. The upper clear liquid is first siphoned out, and the lower turbid liquid is filtered through a 300-mesh food-grade nylon filter cloth and then combined with the upper clear liquid. Subsequently, microfiltration sterilization is performed using a food-grade inorganic ceramic microfiltration membrane with a pore size of 0.45μm. The filtration operation pressure is 0.15MPa and the operating temperature is 18℃. The resulting clear filtrate is the plantain and lamb's quarters probiotic enzyme. This step addresses the shortcomings of existing technologies, such as excessively rapid cooling leading to the death of live bacteria, the release of active ingredients, and poor clarity.

[0030] Example 3 This embodiment uses the upper limit of the parameter range described in the claims to prepare plantain and lamb's quarters probiotic enzymes. The specific steps are as follows: S1. Raw Material Pretreatment: Fresh plantain stems and leaves (60 days old, before heading) and fresh lamb's quarters stems and leaves (20 cm tall, before flowering) are collected at a fresh weight ratio of 2:1. The raw materials are rinsed three times with flowing purified water, each rinse lasting one minute, to completely remove surface dirt, insect eggs, and impurities. The washed raw materials are then spread out in a cool, ventilated place, with a thickness of 3 cm, and allowed to air dry until no free water remains on the surface. The juice is then cold-pressed at 25°C using a spiral cold-press juicer. The resulting juice is filtered through a 200-mesh screen. The mixture is filtered twice with high-quality nylon filter cloth to completely remove coarse fiber residue, resulting in a mixed vegetable juice. The mixed vegetable juice is then sent to a water bath sterilization device, where it is precisely heated to 90 degrees Celsius for 5 minutes to complete the enzyme inactivation process. This inactivates the endogenous enzymes such as polyphenol oxidase and pectinase naturally present in the raw materials, preventing the non-specific decomposition of active ingredients during subsequent fermentation. The enzyme-inactivated mixed vegetable juice is then rapidly cooled to 25 degrees Celsius using a plate heat exchanger and stored in a sealed container for later use. This step addresses the deficiency in existing technologies where the failure to inactivate endogenous enzymes in the raw materials leads to the decomposition and loss of active ingredients.

[0031] S2. First-stage anaerobic fermentation: Weigh 18 parts of the enzyme-inactivated mixed wild vegetable juice according to fresh weight, add 2 parts of food-grade isomaltulose, and stir with a sterile impeller at 120 r / min for 15 minutes until the isomaltulose is completely dissolved. The resulting liquid is then sterilized by filtering with a food-grade polyethersulfone organic microporous membrane with a pore size of 0.22 μm at an operating temperature of 30℃ and an operating pressure of 0.15 MPa. After completely removing contaminants such as bacteria and fungal spores from the liquid, it is sent to a sterile fermentation tank. Inoculate with 0.5 parts of activated mixed probiotics, which are a 1:1 mixture of Bifidobacterium adolescentis and Lactobacillus delbrueckii subsp. lactis with a total viable count of 1.5 × 10^9 CFU / g. The activation method is to inoculate the probiotics... In sterile MRS liquid medium at pH 6.5, the bacteria were incubated at 37°C for 12 hours until they reached the logarithmic growth phase. After centrifugation at 4000 rpm for 10 minutes, the cells were collected and resuspended in sterile physiological saline to the preset viable cell concentration. 80 portions of purified water, sterilized at 121°C for 20 minutes and cooled to 25°C, were added to bring the volume to a final volume. Sterile nitrogen gas was then introduced into the fermenter for 10 minutes to reduce the dissolved oxygen content to 0.2 mg / L. The fermenter was then sealed and the pressure maintained at 0.03 MPa to prevent external microbial contamination. The fermentation temperature was kept stable at 30°C, and anaerobic fermentation was carried out for 24 hours until the pH dropped to 4.5, yielding the first-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as initial microbial contamination, low probiotic activity, and slow proliferation.

[0032] S3. Second-stage constant-temperature fermentation: Food-grade isomaltooligosaccharide (95% purity) is added to the first-stage fermentation broth using a sterile fed-batch method at a rate of 0.5% of the total mass of the first-stage fermentation broth. Warm water is slowly introduced through the fermenter jacket to raise the temperature at a rate of 1 degree Celsius every 10 minutes to avoid sudden temperature changes that could reduce probiotic activity. After reaching 37 degrees Celsius, the temperature is maintained at a constant level for 36 hours. During fermentation, the tank pressure is maintained at 0.03 MPa. Low-speed stirring is performed every 6 hours for 5 minutes at a speed of 50 rpm to prevent bacterial sedimentation and clumping while avoiding the introduction of excessive oxygen. Fermentation is terminated when the pH stabilizes at 3.8, yielding the second-stage fermentation broth. This step addresses the shortcomings of existing technologies, such as insufficient carbon source in the later stages of fermentation, mass death of probiotics, and incomplete degradation of anti-nutritional factors.

[0033] S4. Post-processing: Cooling water is introduced through the jacket of the fermenter to slowly cool the secondary fermentation broth. The cooling rate is controlled at 1 degree Celsius every 15 minutes to avoid sudden temperature changes that could cause the active ingredients to be released. After cooling to 20 degrees Celsius, the broth is kept at this temperature and allowed to stand for 12 hours to achieve natural clarification. The upper clear liquid is first siphoned out, and the lower turbid liquid is filtered through a 300-mesh food-grade nylon filter cloth and then combined with the upper clear liquid. Subsequently, microfiltration sterilization is performed using a food-grade inorganic ceramic microfiltration membrane with a pore size of 0.45μm. The filtration operation pressure is 0.2MPa and the operating temperature is 20℃. The resulting clear filtrate is the plantain and lamb's quarters probiotic enzyme. This step addresses the shortcomings of existing technologies, such as excessively rapid cooling leading to the death of live bacteria, the release of active ingredients, and poor clarity.

[0034] Comparative Example This comparative example uses the conventional wild plant enzyme preparation process in the existing technology, but does not include the core steps of enzyme inactivation, anaerobic control, two-stage gradient fermentation, addition of slow-release carbon source, and controlled temperature reduction of the present invention. The specific steps are as follows: Fresh plantain and lamb's quarters stems and leaves are collected, weighed at a mass ratio of 1:1, washed and dried, and juiced. The juice is filtered through a 200-mesh filter cloth to obtain mixed wild vegetable juice. 2% of the total mass of brown sugar is added, stirred and dissolved, and then 0.2 parts of ordinary commercial compound enzyme agent are added. Fermentation is carried out at room temperature for 7 days. After fermentation, the enzyme product is obtained by filtering through ordinary gauze.

[0035] Table 1 Comparison of core process parameters between each embodiment and the comparative example. This table presents the complete parameter range for the three embodiments. The settings of each process parameter are in line with the technical logic of the present invention and can achieve the expected fermentation effect. The comparative example uses the existing conventional enzyme preparation process, without setting core steps such as enzyme inactivation, anaerobic two-stage fermentation, addition of slow-release carbon source, and temperature control clarification. The operation process is completely consistent with the existing technology.

[0036] Table 2 Comparison of performance indicators of each embodiment and comparative example product The data in this table show that the degradation rates of oxalic acid and irritating alkaloids in the three examples were significantly higher than those in the comparative example. The total number of viable bacteria was hundreds of times higher than that in the comparative example. The SOD activity, polyphenol content, and free amino acid content all met the quality standards described in the claims and were far higher than the detection values ​​of the comparative example. Among them, Example 2, as the optimal parameter group, achieved an oxalic acid degradation rate of 82.6% and an irritating alkaloid degradation rate of 72.4%, fully meeting the requirements for low-irritant consumption. The total number of viable bacteria reached 2.1×10^6 CFU / mL, meeting the activity requirements of probiotic products. The retention rates of SOD activity, polyphenols, and free amino acids were more than twice that of the comparative example. This confirms that the core steps of this invention, such as enzyme inactivation, two-stage gradient fermentation, and supplementation of slow-release carbon sources, can effectively reduce the content of anti-nutritional factors in raw materials, while significantly improving the retention rate of active ingredients and the survival rate of probiotics. This completely solves the core defects of the prior art, such as strong product irritation, significant loss of active ingredients, and low survival rate of probiotics. The comparative sample did not use the core process of this invention, and the degradation of anti-nutritional factors was less than 25%, the number of live bacteria was extremely low, and the retention rate of active ingredients was poor, which could not meet the requirements for long-term safe consumption.

[0037] The enzyme products prepared in the three embodiments of this invention all underwent acute oral toxicity testing according to GB15193.1-2014 "Food Safety Toxicology Evaluation Procedures". SPF-grade KM mice were administered the highest dose of 20 g / kg bw orally via gavage. After 14 days of continuous observation, no mouse deaths or obvious toxic reactions were observed, classifying the products as practically non-toxic. In a 30-day feeding trial, mouse weight, blood routine tests, and blood biochemical indicators showed no abnormalities. No adverse reactions related to the original side effects of plantain and lamb's quarters, such as gastrointestinal irritation or electrolyte imbalance, were observed. This confirms that the products, after degradation of anti-nutritional factors using the process of this invention, meet the safety requirements for food-grade products and are suitable for long-term consumption.

[0038] The fermentation process of this invention involves two types of targeted degradation reactions, which address the core defect of residual anti-nutritional factors in raw materials: oxalic acid degradation reaction: ; During anaerobic fermentation, Bifidobacterium adolescentis secretes oxalate decarboxylase, which decomposes oxalic acid in plantain and lamb's quarters in a targeted manner, thus avoiding electrolyte imbalance and gastrointestinal irritation caused by oxalic acid entering the human body. This addresses the deficiency in existing technologies where anti-nutritional factors in raw materials are not effectively degraded.

[0039] Irritating alkaloid degradation reaction: The esterase produced by Lactobacillus delbrueckii subsp. lactis can hydrolyze the ester bonds of irritating quinolone alkaloids in the raw materials to generate non-irritating small molecule acids and alcohols, thereby reducing the gastrointestinal irritation of the product and addressing the defect in existing technologies that make the product unsuitable for long-term consumption.

[0040] Reference Figure 2This diagram visually highlights the effectiveness of this invention in addressing the core pain points of wild plant enzymes. Conventional processes employ open fermentation at room temperature, without targeted probiotics or anaerobic control. This leads to the proliferation of unwanted bacteria, crowding out the living space of probiotics, resulting in a total viable count of only 2.3 × 10^3 CFU / mL, and an anti-nutritional factor degradation rate of less than 25%, leading to a highly irritating product. This invention, through a targeted probiotic system combining *Bifidobacterium adolescentis* and *Lactobacillus delbrueckii*, combined with two-stage gradient anaerobic fermentation, increases the anti-nutritional factor degradation rate by 3-4 times in the three embodiments, achieving a total viable count of 10^6 CFU / mL, hundreds of times higher than the comparative example. This fundamentally solves the defects of poor safety and insufficient probiotic activity in wild plant enzymes.

[0041] Reference Figure 3 This radar chart comprehensively demonstrates the combined advantages of this invention in terms of activity retention and safe degradation. Conventional processes do not involve enzyme inactivation, leading to the decomposition of SOD, polyphenols, and other active ingredients by the raw materials' own polyphenol oxidases and pectinases, and insufficient fermentation resulting in inadequate nutrient conversion. This invention uses precise temperature control at 85-90℃ to inactivate endogenous enzymes, combined with gradient temperature fermentation to promote probiotic metabolic transformation. This not only thoroughly degrades anti-nutritional factors, but also increases SOD activity, polyphenols, and free amino acid content by more than 2 times. Example 2 shows the best performance in all indicators, achieving a balance between low irritation and high nutritional activity, and ensuring that the product fully meets safety and functionality standards.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a probiotic enzyme from plantain and lamb's quarters, characterized in that, Includes the following steps: S1. Raw material pretreatment: Collect the stems and leaves of fresh plantain and lamb's quarters, wash them and air dry them naturally. After juicing, filter the juice through a 200-mesh filter cloth to obtain mixed wild vegetable juice. Heat the mixed wild vegetable juice to 85 to 90 degrees Celsius and keep it at that temperature for 5 to 10 minutes to complete the enzyme inactivation treatment. Then cool it to room temperature for later use. S2. First-stage anaerobic fermentation: Add isomaltulose to the mixed wild vegetable juice after enzyme inactivation, stir evenly, filter sterilize, inoculate with mixed probiotic agent, add purified water to make up the volume, control the dissolved oxygen content of the system to be no higher than 0.5 mg / L, and anaerobic ferment at 25 to 30 degrees Celsius for 18 to 24 hours to obtain the first-stage fermentation liquid. S3. Second-stage isothermal fermentation: Add isomaltooligosaccharide to the first-stage fermentation broth, raise the temperature of the system to 34 to 37 degrees Celsius, and ferment at a constant temperature for 24 to 36 hours to obtain the second-stage fermentation broth. S4. Post-processing: Cool the second-stage fermentation broth to 16 to 20 degrees Celsius, keep it warm and stand for 8 to 12 hours to complete clarification, and then perform microfiltration sterilization treatment to obtain plantain and lamb's quarters probiotic enzymes.

2. The method for preparing plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, In S1, the mass ratio of plantain to lamb's quarters is 1 to 2:

1.

3. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The ingredients of S2, by weight, are: 8 to 18 parts of mixed wild vegetable juice, 1.2 to 2 parts of isomaltulose, 0.1 to 0.5 parts of mixed probiotic agent, and 80 to 95 parts of purified water.

4. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The amount of isomaltooligosaccharide added in S3 is 0.2 to 0.5% of the total mass of the first-stage fermentation broth.

5. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The mixed probiotic agent in S2 is a compound probiotic agent obtained by mixing Bifidobacterium adolescentis and Lactobacillus delbrueckii subsp. lactis at a live bacteria ratio of 1:

1.

6. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 5, characterized in that, The total live bacteria count of the mixed probiotic agent is not less than 1×10^9 CFU / g.

7. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The sterilization filtration in S2 is accomplished using an organic microporous membrane with a pore size of 0.22 μm.

8. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, During the fermentation process of S3, low-speed stirring is performed every 6 hours, with each stirring session lasting 5 minutes and the stirring speed being 30 to 50 r / min.

9. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The microfiltration sterilization in S4 is accomplished using a ceramic microfiltration membrane with a pore size of 0.45 μm, and the filtration operation pressure is 0.1 to 0.2 MPa.

10. The method for preparing the plantain and lamb's quarters probiotic enzyme according to claim 1, characterized in that, The total live bacteria count of the obtained plantain and lamb's quarters probiotic enzymes is not less than 1×10^6 CFU / mL, the SOD activity is not less than 18 U / L, and the polyphenol content is not less than 0.7 mg / g.

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