Mulberry leaf and kudzu vine fermented powder, and preparation method and application thereof

Abstract

This invention discloses a mulberry leaf and kudzu root fermented powder, its preparation method, and its application. The preparation method includes: extracting a mixture of mulberry leaf powder and kudzu root powder using hot water; adding a complex enzyme composed of cellulase, pectinase, and protease in a 5:3:2 ratio to the aqueous extract for enzymatic hydrolysis; sterilizing and cooling to obtain the enzymatic hydrolysate; then inoculating the hydrolysate with a complex bacterial agent composed of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 in a 3-6:2-4:1 ratio, and fermenting at 35-39℃ for 18-36 hours; finally, after solid-liquid separation, sterilization, and drying, the mulberry leaf and kudzu root fermented powder is obtained. This invention achieves efficient release and conversion of active ingredients through the organic combination of extraction, complex enzymatic hydrolysis, and synergistic fermentation with three specific lactic acid bacteria strains. The resulting fermented powder exhibits synergistic and enhanced effects in lowering blood sugar, lowering blood lipids, anti-oxidation, anti-inflammation, and improving sleep. Furthermore, the process is stable and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of mulberry leaf and kudzu root fermentation product processing technology, and in particular to a mulberry leaf and kudzu root fermented powder with anti-inflammatory, hypoglycemic, and sleep-improving effects, and its preparation method. Background Technology

[0002] Mulberry leaves are rich in active ingredients such as deoxynojirimycin, flavonoids, and polysaccharides, which have effects such as regulating glucose and lipid metabolism, anti-oxidation, and anti-inflammation. Kudzu root is rich in puerarin, isoflavones, and polysaccharides, which have effects such as improving microcirculation, anti-oxidation, and soothing and anti-inflammatory properties. Theoretically, combining the two can achieve complementary active ingredients, showing development potential in the field of functional foods for lowering blood sugar and lipids, anti-oxidation, and improving sleep. However, the deep processing of the mulberry leaf and kudzu root composite system faces unique technical obstacles that cannot be solved by simply adding up the processes for a single raw material. First, mulberry leaf cell walls are rich in cellulose and pectin, while kudzu root cell walls are mainly composed of cellulose and starch-based polysaccharides, with flavonoids and isoflavones existing mostly in bound form. Conventional pulverization, hot water extraction, or single enzymatic hydrolysis often only partially release the effective components of one of the raw materials, making it difficult to achieve simultaneous and efficient dissolution of mulberry leaf flavonoids and kudzu root isoflavones. This results in an imbalance in the proportion of active ingredients after compounding, and the synergistic effect cannot be exerted.

[0003] Secondly, the flavonoids and volatile components in mulberry leaves produce a distinct grassy and bitter taste, while kudzu root has a thick powdery texture and a bland taste. When the two are combined, their unpleasant flavors overlap, and it is difficult to effectively improve them by simply relying on physical mixing or conventional fermentation. The product has poor palatability, which limits its application as a food ingredient.

[0004] Third, current fermentation methods for mulberry leaves or kudzu root mostly follow single-raw-material or general lactic acid bacteria protocols, without considering the unique characteristics of the carbon and nitrogen source composition, phenolic content, and probiotic / antibiotic-promoting factors in the enzymatic hydrolysate of mulberry leaves and kudzu root. Single strains or conventional bacterial groups have low metabolic efficiency in this system, making it difficult to effectively convert bound flavonoids, rutin, and other functional components, and also failing to simultaneously achieve multiple functional goals such as lowering blood sugar, lowering blood lipids, anti-oxidation, and GABA accumulation. Furthermore, it is difficult to degrade bitter substances and improve flavor. Summary of the Invention

[0005] The purpose of this invention is to provide a fermented mulberry leaf and kudzu root powder and its preparation method, so as to overcome the shortcomings of the prior art mentioned in the background art above.

[0006] To achieve the above objectives, the solution of the present invention is: a method for preparing mulberry leaf and kudzu root fermented powder, comprising the following steps: Raw material pretreatment: Mulberry leaves are crushed and passed through a 55-65 mesh sieve, and kudzu root is crushed and passed through a 5-10 mesh sieve. The sieve-underfill material is removed to obtain mulberry leaf powder and kudzu root powder. Then, the mulberry leaf powder and kudzu root powder are mixed at a mass ratio of 8:2 to 9:1. The mulberry leaf and kudzu root mixture is then mixed with water at a mass ratio of 1:20 to 30 to obtain a mulberry leaf and kudzu root mixture. The mixture is then extracted at 90-100℃ for 150-180 minutes to obtain a mulberry leaf and kudzu root aqueous extract.

[0007] Enzymatic hydrolysis: Add 0.3% to 1% of the aqueous extract of mulberry leaf and kudzu root to the aqueous extract of mulberry leaf and kudzu root, and hydrolyze at 50 to 60°C for 2 to 3 hours to obtain mulberry leaf and kudzu root enzymatic hydrolysate; the composite enzyme is a mixture of cellulase, pectinase and protease in a mass ratio of 5:3:2, and the enzyme activity of the three enzymes is 20,000 to 30,000 U / g. Next, the mulberry leaf and kudzu root enzymatic hydrolysate was heated to boiling (100-110℃) and kept at that temperature for 20-30 minutes for sterilization. After sterilization, the mulberry leaf and kudzu root enzymatic hydrolysate was cooled to 37±2℃.

[0008] Fermentation: A compound microbial agent is added to the cooled mulberry leaf and kudzu root enzymatic hydrolysate at 0.01%–0.03% of the liquid system for fermentation. The fermentation temperature is 35–39℃, and the fermentation time is 18–36 h, yielding a mulberry leaf and kudzu root fermentation broth. The compound microbial agent is composed of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentatus* B153 in a mass ratio of 3–6:2–4:1. A preferred mass ratio is 4.5:3:1.

[0009] The *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 are all provided in the form of bacterial powder; the viable counts of these three bacteria in the bacterial powder are 500 billion CFU / g, 400 billion CFU / g, and 400 billion CFU / g, respectively.

[0010] Solid-liquid separation: The fermentation broth of mulberry leaves and kudzu root was subjected to solid-liquid separation. First, the fermentation broth of mulberry leaves and kudzu root was filtered through a 300-mesh sieve. Then, it was centrifuged at 5000 r / min for 5 to 10 min. Finally, it was microfiltered through a membrane with a pore size of 0.22 to 0.45 μm to obtain the fermentation supernatant of mulberry leaves and kudzu root.

[0011] Sterilization after fermentation: The supernatant of mulberry leaf and kudzu root fermentation is sterilized to obtain sterilized mulberry leaf and kudzu root fermentation liquid.

[0012] Spray drying: The mulberry leaf and kudzu root fermented powder is obtained by drying using a spray dryer.

[0013] This invention also provides a mulberry leaf and kudzu root fermented powder, prepared using the method described above. The mulberry leaf and kudzu root fermented powder provided by this invention possesses at least two of the following functional characteristics: Flavonoid content ≥2.8g / 100g and rutin content ≥1.8g / 100g; The α-glucosidase inhibitory activity is ≥94%, and the α-amylase inhibitory activity is ≥16%. Pancreatic lipase inhibitory activity ≥23%; DPPH free radical scavenging rate ≥90%, ABTS scavenging rate ≥74%, and superoxide anion scavenging rate ≥68%; The content of γ-aminobutyric acid is ≥50 mg / 100g.

[0014] The mulberry leaf and kudzu root fermented powder provided by this invention can be used to prepare products with anti-inflammatory, hypoglycemic, and sleep-improving effects.

[0015] After adopting the above solution, the beneficial effects of the present invention are as follows: This invention uses mulberry leaf powder and kudzu root powder in a mass ratio of 8:2 to 9:1 as raw materials. Through hot water extraction and synergistic enzymatic hydrolysis by a complex enzyme composed of cellulase, pectinase, and protease in a mass ratio of 5:3:2, the cellulose, pectin, and protein macromolecules in the cell walls of mulberry leaves and kudzu roots are degraded stepwise. This effectively overcomes the barrier to simultaneous cell wall disruption caused by the differences in cell wall structure between the two raw materials, and promotes the synergistic release of active ingredients such as mulberry leaf flavonoids and kudzu root isoflavones.

[0016] Based on this, the present invention uses a compound microbial agent of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 in a mass ratio of 3~6:2~4:1 for fermentation. This specific tri-microbial combination can adapt to the matrix characteristics of mulberry leaf and kudzu root enzymatic hydrolysate, and simultaneously achieves the conversion and release of bound flavonoids and rutin, and the degradation of bitter substances under mild fermentation conditions. The resulting mulberry leaf and kudzu root fermented powder has a distinct flavor, harmonious taste, and no bitter or astringent aftertaste. In addition, this mulberry leaf and kudzu root fermented powder has a high content of γ-aminobutyric acid (GABA), which can be used to develop products that improve sleep function.

[0017] The mulberry leaf and kudzu root fermented powder prepared by this invention has excellent α-glucosidase inhibitory activity and α-amylase inhibitory activity, which can effectively delay the decomposition of carbohydrates and the absorption of glucose in the intestine, and help stabilize postprandial blood sugar. At the same time, its pancreatic lipase inhibitory activity is also significantly improved, which can reduce the decomposition and absorption of dietary fat and assist in weight management.

[0018] The mulberry leaf and kudzu root fermented powder prepared by this invention also has a high free radical scavenging rate, albumin denaturation inhibition activity and significant promotion of macrophage proliferation. It is evident that the product also has antioxidant, anti-inflammatory and immune-enhancing activities, and has unique application value in the fields of anti-aging, skin care and functional foods.

[0019] The method of this invention uses a combination of unit operations such as hot water extraction, enzymatic hydrolysis, microbial fermentation, solid-liquid separation and spray drying. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0020] In summary, this invention, through a synergistic technical solution of extraction, enzymatic hydrolysis, and fermentation with a specific compound microbial agent, produces a high-quality mulberry leaf and kudzu root fermented powder that integrates multiple effects such as lowering blood sugar, lowering blood lipids, anti-oxidation, anti-inflammation, immune enhancement, and improving sleep. At the same time, it solves key technical problems such as low content of active ingredients, poor flavor, insufficient functional activity, and difficulty in mass production of traditional products, and has significant economic and social benefits.

[0021] Information on the preservation of biological materials: All bacterial strains used in this invention are publicly available strains, preserved and viable in biological collection centers. Technicians can obtain them from the collection center or from the applicant based on the accession number. Therefore, this application does not involve the preservation of bacterial strains and does not require proof of their viability.

[0022] The plant lactobacillus BXM2, Latin scientific name: Lactiplantibacillus plantarum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO.16436. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: September 6, 2018. The Lactobacillus paracasei YYS-K1, Latin scientific name: Lactobacillus paracasei The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 26405. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: January 6, 2023. The fermenting *Lactobacillus mucilaginosus* B153, Latin scientific name: Lactobacillus fermentum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 16454. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: September 10, 2018. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. 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.

[0024] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0025] Example 1 (1) Raw material pretreatment: Mulberry leaves are crushed and passed through a 60-mesh sieve, and kudzu root is crushed and passed through a 5-mesh sieve. The sieve-less material is removed to obtain mulberry leaf powder and kudzu root powder. Mix 80g of mulberry leaf powder and 20g of kudzu root powder evenly, add to 2500mL of pure water, and extract at 95±2℃ for 160min to obtain an aqueous extract; (2) Enzymatic hydrolysis: The aqueous extract was cooled to 50℃, and 0.6% of the aqueous extract mass of the compound enzyme was added. The mixture was enzymatically hydrolyzed at 50℃±2℃ for 150 min. The mass ratio of cellulase, pectinase and protease in the compound enzyme was 5:3:2, that is, 0.3% of the aqueous extract mass of cellulase, 0.18% of the pectinase and 0.12% of the protease were added respectively. Next, the enzymatic hydrolysate was sterilized at 100°C for 20 minutes and then cooled to 37°C. (3) Fermentation: Add compound microbial agent to the cooled mulberry leaf and kudzu root enzymatic hydrolysate at 0.02% of the liquid system and ferment at 37℃ for 20h to obtain fermentation liquid; the mass ratio of Lactobacillus plantarum BXM2, Lactobacillus paracasei YYS-K1 and Lactobacillus fermentum B153 in the compound microbial agent is 4.5:3:1.

[0026] (4) Solid-liquid separation: The fermentation broth is first passed through a 300-mesh sieve and then centrifuged to separate the solid and liquid. The supernatant is then microfiltered through a filter membrane with a pore size of 0.22-0.45 μm to obtain the fermentation supernatant.

[0027] (5) Sterilization after fermentation: Sterilize the fermentation supernatant to obtain sterilized fermentation liquid; (6) Spray drying: Spray drying the fermentation supernatant to obtain the yeast powder.

[0028] Example 2 The difference between this embodiment and Embodiment 1 is that the raw material ratio is different, the amount of compound enzyme added is different, the amount of compound bacteria added is different, and the strain ratio is different.

[0029] (1) Raw material pretreatment: Mulberry leaves are crushed and passed through a 60-mesh sieve, and kudzu root is crushed and passed through a 5-mesh sieve. The sieve-less material is removed to obtain mulberry leaf powder and kudzu root powder. Mix 90g of mulberry leaf powder and 10g of kudzu root powder evenly, add to 2500mL of pure water, and extract at 95±2℃ for 160min to obtain an aqueous extract; (2) Enzymatic hydrolysis: Cool the aqueous extract to 50℃, add 1% of the complex enzyme by mass of the aqueous extract, and enzymatically hydrolyze for 150 min at 50℃±2℃; the mass ratio of cellulase, pectinase and protease in the complex enzyme is 5:3:2. Next, the enzymatic hydrolysate was sterilized at 100°C for 20 minutes and then cooled to 37°C. (3) Fermentation: Add compound microbial agent to the cooled mulberry leaf and kudzu root enzymatic hydrolysate at 0.03% of the liquid system, and ferment at 37℃ for 20h to obtain fermentation liquid; the mass ratio of Lactobacillus plantarum BXM2, Lactobacillus paracasei YYS-K1 and Lactobacillus fermentum B153 in the compound microbial agent is 6:4:1.

[0030] Steps (4) and (5) are the same as in Example 1.

[0031] Example 3 The difference between this embodiment and Embodiment 1 is that the amount of compound enzyme added, the amount of compound bacteria added, and the ratio of strains are different.

[0032] (1) Raw material pretreatment: Mulberry leaves are crushed and passed through a 60-mesh sieve, and kudzu root is crushed and passed through a 5-mesh sieve. The sieve-less material is removed to obtain mulberry leaf powder and kudzu root powder. Mix 80g of mulberry leaf powder and 20g of kudzu root powder evenly, add to 2500mL of pure water, and extract at 95±2℃ for 160min to obtain an aqueous extract; (2) Enzymatic hydrolysis: Cool the aqueous extract to 50℃, add 0.3% of the complex enzyme by mass of the aqueous extract, and enzymatically hydrolyze for 150 min at 50℃±2℃; the mass ratio of cellulase, pectinase and protease in the complex enzyme is 5:3:2. Next, the enzymatic hydrolysate was sterilized at 100°C for 20 minutes and then cooled to 37°C. (3) Fermentation: Add compound microbial agent to the cooled mulberry leaf and kudzu root enzymatic hydrolysate at 0.01% of the liquid system and ferment at 37℃ for 20h to obtain fermentation liquid; the mass ratio of Lactobacillus plantarum BXM2, Lactobacillus paracasei YYS-K1 and Lactobacillus fermentum B153 in the compound microbial agent is 3:2:1.

[0033] Steps (4) and (5) are the same as in Example 1.

[0034] Comparative Example 1 The difference between this comparative example and Example 1 is that the weight of the mulberry leaf powder was too low, while the weight of the kudzu root powder was too high, at 40g and 60g respectively. All other operations and steps are the same as in Example 1.

[0035] Comparative Example 2 The difference between this comparative example and Example 1 is that the raw materials contain only mulberry leaf powder and not kudzu root powder; that is, 100g of mulberry leaf powder is weighed and mixed with 2500mL of pure water. All other operations and steps are the same as in Example 1.

[0036] Comparative Example 3 The difference between this comparative example and Example 1 is that only the mixed powder of mulberry leaves and kudzu root is extracted and enzymatically hydrolyzed, without microbial fermentation. All other operations and steps are the same as in Example 1.

[0037] Comparative Example 4 The difference between this comparative example and Example 1 is that in the fermentation step, 0.02% of a single Lactobacillus paracasei YYS-K1 was added to the liquid system to ferment the enzymatic hydrolysate. All other operations and steps were the same as in Example 1.

[0038] Comparative Example 5 The difference between this comparative example and Example 1 is that in the fermentation step, 0.02% of a single fermenting Lactobacillus mucilaginosus B153 was added to the liquid system as a fermentation agent to ferment the enzymatic hydrolysate. All other operations and steps are the same as in Example 1.

[0039] Comparative Example 6 The difference between this comparative example and Example 1 is that in the fermentation step, an equal amount of compound microbial agent is added. The compound microbial agent is a mixture of Lactobacillus plantarum BXM2 and Lactobacillus fermentum B153 in a mass ratio of 4.5:1 to ferment the enzymatic hydrolysate. All other operations and steps are the same as in Example 1.

[0040] Comparative Example 7 The difference between this comparative example and Example 1 is that in the fermentation step, an equal amount of compound microbial agent is added. The compound microbial agent is a mixture of Lactobacillus paracasei YYS-K1 and Lactobacillus fermentatus B153 in a mass ratio of 3:1 to ferment the enzymatic hydrolysate. All other operations and steps are the same as in Example 1.

[0041] Comparative Example 8 The difference between this comparative example and Example 1 is that in the fermentation step, an equal amount of compound microbial agent is added. The compound microbial agent is composed of Lactobacillus plantarum BXM2, Lactobacillus paracasei YYS-K1 and Lactobacillus fermentum B153 mixed in a mass ratio of 1:1:3. The enzymatic hydrolysate is fermented. Other operations and steps are the same as in Example 1.

[0042] Comparative Example 9 The difference between this comparative example and Example 1 is that in the fermentation step, an equal amount of compound microbial agent is added. The compound microbial agent is composed of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus paracasei* B20 mixed in a mass ratio of 4.5:3:1. That is, *Lactobacillus paracasei* YYS-K1 is replaced with *Lactobacillus paracasei* B20 to ferment the enzymatic hydrolysate. All other operations and steps are the same as in Example 1.

[0043] Performance testing The performance of the mulberry leaf and kudzu root powder products prepared in the above embodiments and comparative examples was tested to verify the synergistic contribution of each key feature in the technical solution of the present invention to the functional activity of the products.

[0044] 1. Rutin and flavonoid content The amounts of rutin and flavonoids extractable from 100 grams of mulberry leaf and kudzu root powder under different process conditions in each example and comparative example were determined. The rutin content was determined by high performance liquid chromatography (General Rule 0512); the flavonoid content was determined by method two of the "Technical Guidelines for the Detection and Evaluation of Physicochemical and Hygienic Indicators of Health Foods" (2020 Edition). Specific results are shown in Table 1.

[0045] Table 1. Flavonoid and Rutin Content in Different Mulberry Leaf and Kudzu Root Powder Products

[0046] As shown in Table 1, the total flavonoid and rutin contents in the mulberry leaf and kudzu root fermented powders prepared in each example were higher than those in all comparative examples. Among them, the total flavonoid content of the sample in Example 1 was 3.19±0.24g / 100g, and the rutin content was 2.35±0.35g / 100g, which was the highest among all groups; the active ingredients in Examples 2 and 3 were the next highest.

[0047] Comparing Comparative Examples 1 and 2, it can be seen that the ratio of mulberry leaf to kudzu root has a significant impact on the yield of active substances. If the proportion of kudzu root is too high (Comparative Example 1) or only mulberry leaf is used as the single raw material (Comparative Example 2), the total flavonoid and rutin content will decrease significantly. This indicates that the mulberry leaf and kudzu root compound ratio specified in this invention is conducive to the synergistic dissolution of flavonoids in the two raw materials.

[0048] Comparative Example 3, which did not undergo microbial fermentation but only water extraction and enzymatic hydrolysis, had the lowest flavonoid and rutin content among all groups. This indicates that the fermentation process can promote the release and transformation of flavonoid active substances in the raw materials.

[0049] As can be seen from Comparative Examples 4 to 9, the flavonoid and rutin contents in the samples were lower than those in Examples 1 to 3 when using single strains, combinations of two strains, strain ratio imbalances, and replacement of functional strains. This indicates that the specific three-strain lactic acid bacteria compound system of the present invention is more conducive to cell wall degradation and the retention and transformation of flavonoids.

[0050] The above structure indicates that appropriate raw material compounding ratios, compound enzyme addition amounts, and co-fermentation with a compound microbial agent of a specific ratio of Lactobacillus plantarum BXM2, Lactobacillus paracasei YYS-K1, and Lactobacillus fermentum B153 can significantly increase the content of total flavonoids and rutin in the product, and these three specific strains have a synergistic effect.

[0051] 2. Inhibitory activities of α-glucosidase and α-amylase The inhibition rates of mulberry leaf and kudzu root powder products on α-glucosidase and α-amylase in each example and comparative example were detected, and the specific results are shown in Table 2.

[0052] Table 2. Inhibitory activities of α-glucosidase and α-amylase in different mulberry leaf and kudzu root powder products.

[0053] As shown in Table 2, the samples from Examples 1 to 3 all exhibited excellent inhibitory effects on α-glucosidase and α-amylase. Among them, the α-glucosidase inhibitory activity of Example 1 reached 98.28±2.36%, and the α-amylase inhibitory activity was 19.83±2.45%, which were the best among the groups.

[0054] After changing the raw material ratios in Comparative Examples 1 and 2, the inhibitory activities of both enzymes decreased significantly, indicating that the proper combination of mulberry leaves and kudzu root is the basis for obtaining better inhibitory activity of carbohydrate digestive enzymes. In Comparative Example 3, which did not undergo a fermentation process, the inhibitory activities of both enzymes were the lowest, indicating that the fermentation process can enhance the inhibitory ability of the product on carbohydrate digestive enzymes.

[0055] Comparative Examples 4 to 9 show that the inhibitory activities of α-glucosidase and α-amylase are all lower than those in Examples 1 to 3 of this invention, regardless of whether the strain is a single strain, a combination of two strains, an imbalanced strain ratio, or a replacement strain. Even with a similar total bacterial count, an unreasonable strain combination cannot achieve the same inhibitory effect. The product fermented with the specific complex enzyme of this invention not only increases the level of flavonoids but also significantly enhances the inhibitory ability against α-glucosidase and α-amylase, thereby achieving a stronger inhibitory effect on sugar-digesting enzymes and possessing the potential to regulate carbohydrate absorption.

[0056] 3. Pancreatic lipase inhibitory activity Pancreatic lipase plays a crucial role in digestion, breaking down dietary fats into glycerol and fatty acids for absorption in the small intestine. Substances that inhibit pancreatic lipase activity can reduce fat absorption by suppressing lipase activity; this inhibitory activity is an important indicator for evaluating anti-obesity effects. The inhibitory activity of mulberry leaf and kudzu root powder products on pancreatic lipase in each example and comparative example was tested, and the specific results are shown in Table 3.

[0057] Table 3. Pancreatic lipase inhibitory activity of different mulberry leaf and kudzu root powder products

[0058] As shown in Table 3, all samples from Examples 1 to 3 had higher pancreatic lipase inhibitory activity, with Example 1 reaching 27.09±3.36%, which was superior to all other comparative groups.

[0059] Comparative Example 1, with its excessive kudzu root content, and Comparative Example 2, which used only mulberry leaves, showed lower pancreatic lipase inhibitory activities than Examples 1 to 3. Comparative Example 3, lacking a fermentation step, had a pancreatic lipase inhibitory activity of only 14.21 ± 1.28%, indicating that fermentation treatment can effectively enhance the product's ability to inhibit fat decomposition.

[0060] Comparative Examples 4 to 9, using single-strain, dual-strain, imbalanced, or replaced strains, showed lower pancreatic lipase inhibitory activity than Examples 1 to 3. Combined with the detection results of flavonoid and rutin content, it can be inferred that the higher flavonoid activity in the process of this invention, along with the small-molecule metabolites produced by the synergistic metabolism of the three compound lactic acid bacteria, synergistically enhanced the inhibitory effect on pancreatic lipase.

[0061] 4. Antioxidant properties The superoxide anion scavenging rate, DPPH free radical scavenging rate, and ABTS free radical scavenging rate of the mulberry leaf and kudzu root powder products of each example and comparative example were tested.

[0062] Table 4 Antioxidant capacity of different mulberry leaf fermentation powders

[0063] As shown in Table 4, the superoxide anion scavenging rate, DPPH free radical scavenging rate, and ABTS free radical scavenging rate of Examples 1 to 3 were significantly higher than those of the comparative examples. Example 1 had the best antioxidant capacity, with a superoxide anion scavenging rate of 74.25±3.45%, a DPPH scavenging rate of 99.81±0.19%, and an ABTS scavenging rate of 80.91±3.54%.

[0064] In Comparative Examples 1 and 2, the free radical scavenging abilities significantly decreased due to changes in the raw material ratios. Comparative Example 3, which had no fermentation process, had the lowest antioxidant index among all groups, indicating that microbial fermentation plays a crucial role in enhancing the antioxidant properties of the product. In Comparative Examples 4 to 9, after altering the strain composition or ratios, the free radical scavenging abilities were all lower than in the examples.

[0065] Analysis suggests that flavonoids and rutin in mulberry leaves and kudzu root are the main antioxidants. This invention promotes the release of antioxidant active substances by enzymatically breaking down plant cell walls; the synergistic fermentation of specific three bacteria further transforms large-molecule polyphenols into smaller-molecule derivatives with stronger antioxidant activity, resulting in a multi-faceted effect that enhances the product's ability to scavenge various free radicals.

[0066] 5. Albumin denaturation inhibitory activity Albumin, as an important endogenous antioxidant, can effectively scavenge free radicals in the body, thereby reducing oxidative stress, which is crucial for alleviating the inflammatory process. Abnormal changes in endogenous proteins have been proven to be a significant factor in inducing chronic inflammatory diseases. In particular, the inhibitory activity of albumin is closely related to the body's anti-inflammatory capacity and can maintain healthy skin.

[0067] Take 2g of each powder from each example and comparative example, and test its albumin inhibitory activity. See Table 5 for detailed data.

[0068] Table 5. Albumin denaturation inhibitory activity of mulberry leaf and kudzu root powder products under different processing conditions

[0069] As shown in Table 5, Examples 1 to 3 all exhibited strong inhibitory activity against albumin denaturation, with Example 1 reaching 84.25 ± 3.39%, significantly superior to the other examples. Albumin denaturation is associated with the occurrence of inflammation in the body, and the inhibitory activity against albumin denaturation can reflect the potential anti-inflammatory activity level of the sample.

[0070] With changes in the raw material ratios of Comparative Examples 1 and 2, the albumin denaturation inhibitory activity was significantly reduced; Comparative Example 3, which did not undergo fermentation, had an inhibitory activity of only 40.25±3.65%, the lowest among all groups. Comparative Examples 4 to 9, after using single-strain, dual-strain, or replacing the strain, all showed significantly lower albumin denaturation inhibitory activities than the Example groups.

[0071] The above results indicate that the appropriate combination of mulberry leaf and kudzu root with the synergistic fermentation of specific compound bacteria can significantly enhance the product's ability to inhibit protein denaturation, providing a material basis for its anti-inflammatory effects.

[0072] 6. Influence on the proliferation and phagocytic capacity of RAW264.7 macrophages Phagocytosis is an important defense mechanism for vertebrates against pathogen invasion, and enhanced phagocytosis is a significant feature of macrophage activation.

[0073] (1) The effect of mulberry leaf fermentation powder on the proliferation of RAW264.7 cells was determined by the CCK-8 assay. RAW264.7 cells were activated and passaged in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% penicillin and streptomycin. After reaching the logarithmic growth phase, cell counts were performed, and the cells were resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% penicillin and streptomycin to a cell concentration of 1×10⁻⁶ cells / mL. 5 Cells / mL. 100 μL of RAW264.7 cells were seeded into 96-well cell culture plates. After the cells adhered, fresh DMEM medium was added to replace the original culture medium.

[0074] The experiment included a blank control group (DMEM culture medium) and an experimental group (with added mulberry leaf fermentation powder), with three replicates in each group. Cells were incubated at 37℃ in a 5% CO2 incubator for 24 h. After incubation, the old culture medium was removed, and 100 μL of DMEM medium containing 10% CCK-8 was added to each well. The cells were then protected from light and incubated in a CO2 incubator for another 30 min. The OD value of each well was measured at 450 nm using a microplate reader, and the relative cell proliferation rate was calculated.

[0075] Relative cell proliferation rate % = OD value of experimental group / OD value of blank group.

[0076] (2) The effect of different mulberry leaf fermentation powders on the phagocytic ability of RAW264.7 cells was determined by the neutral red method.

[0077] RAW264.7 cells were activated and passaged in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% penicillin and streptomycin. After reaching the logarithmic growth phase, cell counts were performed, and the cells were resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% penicillin and streptomycin to a cell concentration of 1×10⁻⁶ cells / mL. 5Cells / mL. 100 μL of RAW264.7 cells were seeded into 96-well cell culture plates. After the cells adhered, fresh DMEM medium was added to replace the original culture medium. The experimental groups were the same as in (1), with 3 replicates per group. The cells were cultured at 37℃ and 5% CO2 for 24 h. The supernatant in the wells was discarded, and then 0.1% neutral red-PBS solution was added under light-protected conditions. The cells were incubated at 37℃ and 5% CO2 for 1 h. After incubation, the supernatant was discarded, and the cells were washed three times with PBS at room temperature. Then, lysis buffer (acetic acid: anhydrous ethanol = 1:1) was added to each well and the cells were allowed to stand in the dark. After the cells were completely lysed, the absorbance of each well was measured at 540 nm using a microplate reader.

[0078] Cell phagocytosis index = OD value of experimental group / OD value of blank group.

[0079] Table 6. Effects of different mulberry leaf fermentation powders on the proliferation and phagocytic capacity of RAW264.7 macrophage cells.

[0080] As shown in Table 6, the samples from Examples 1 to 3 all significantly promoted the proliferation of RAW264.7 macrophages and increased the macrophage phagocytic index; among them, Example 1 showed the best effect with a cell proliferation rate of 158% and a phagocytic index of 1.37.

[0081] After the raw material ratios were changed in Comparative Examples 1 and 2, the macrophage proliferation rate and phagocytic index decreased significantly. In Comparative Example 3, which only underwent water extraction and enzymatic hydrolysis without fermentation, the cell proliferation rate was 106% and the phagocytic index was 1.12, showing the weakest enhancement effect. Comparative Examples 4 to 9, due to the use of a single strain, two strains, or an unbalanced strain ratio, showed weaker macrophage proliferation and phagocytic enhancement effects compared to Examples 1 to 3.

[0082] The proliferative and phagocytic abilities of macrophages are important manifestations of the body's non-specific immunity. The above results indicate that the mulberry leaf and kudzu root compound fermented powder of the present invention can activate macrophages and enhance their phagocytic function, thereby achieving an immunomodulatory effect. This effect depends on the compounding of mulberry leaf and kudzu root, enzymatic hydrolysis to break up cell walls, and the synergistic fermentation of three specific strains of lactic acid bacteria. If only raw material extraction and enzymatic hydrolysis or simple fermentation by a single strain are used, it is difficult to achieve the same immune activation effect.

[0083] 7. Production of γ-aminobutyric acid (GABA) γ-Aminobutyric acid (GABA) has a certain regulatory effect on sleep. It can help the body enter a sleep state and improve sleep quality by inhibiting excessive excitation of the central nervous system. The yield of GABA in various mulberry leaf fermentation powders was determined by high performance liquid chromatography (HPLC), and the results are shown in Table 7.

[0084] Table 7. γ-aminobutyric acid yield of different mulberry leaf fermentation powders

[0085] As shown in Table 7, the γ-aminobutyric acid (GABA) yield in the products of Examples 1 to 3 was significantly higher than that in all comparative examples. Specifically, Example 1 had the highest GABA yield at 56.93 ± 3.36 mg / 100g. In Comparative Examples 1 and 2, the GABA production decreased significantly after changing the raw material ratios. Comparative Example 3, which did not undergo microbial fermentation, had the lowest GABA content at only 34.59 ± 2.02 mg / 100g, indicating that GABA mainly originates from the biotransformation during lactic acid bacteria fermentation. In Comparative Examples 4 to 9, the GABA yield was lower than that of the examples of this invention after changing the strain combination and ratios.

[0086] The above results indicate that the three strains of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 selected in this invention, under specific ratios, can efficiently convert substrates into γ-aminobutyric acid (GABA) under the given process conditions, thus providing the product with a potential basis for calming and sleep-aiding functions.

[0087] Based on the above performance test results, it is clear that: This invention achieves efficient release and transformation of active ingredients through a synergistic process involving a specific ratio of mulberry leaf and kudzu root, hot water extraction, synergistic enzymatic hydrolysis with compound enzymes, and fermentation with a specific combination of three lactic acid bacteria. The resulting product is significantly superior to the original formulations in terms of flavonoid and rutin content, α-glucosidase and α-amylase inhibitory activity, pancreatic lipase inhibitory activity, antioxidant capacity, albumin denaturation inhibitory activity, macrophage proliferation and phagocytic capacity, and GABA production.

[0088] The comparative analysis further demonstrates that the ratio of mulberry leaves to kudzu root, the presence or absence of fermentation steps, the combination of strains, and their ratios all significantly affect the functional activity of the product. The absence or deviation of any key feature leads to a significant decrease in one or more functional indicators, proving that the technical solution of this invention is not a simple superposition of existing technologies, but rather that the various technical features have a significant synergistic relationship, collectively constituting a complete inventive concept.

Claims

1. A method for preparing mulberry leaf and kudzu root fermented powder, characterized in that, Includes the following steps: Raw material pretreatment: Mulberry leaves and kudzu root are crushed and sieved separately to obtain mulberry leaf powder and kudzu root powder; then the mulberry leaf powder and kudzu root powder are mixed at a mass ratio of 8:2 to 9:1; then the mixed powder is mixed with water at a mass ratio of 1:20 to 30 to obtain a mulberry leaf and kudzu root mixture, which is then extracted with hot water to obtain a mulberry leaf and kudzu root aqueous extract. Enzymatic hydrolysis: 0.3% to 1% of a compound enzyme by weight of the water extract of mulberry leaf and kudzu root is added to the water extract of mulberry leaf and kudzu root for enzymatic hydrolysis. After enzymatic hydrolysis, the mulberry leaf and kudzu root enzymatic hydrolysate is obtained, which is then sterilized and cooled before fermentation. The compound enzyme is composed of cellulase, pectinase and protease mixed in a mass ratio of 5:3:

2. Fermentation: A compound microbial agent was added to the cooled mulberry leaf and kudzu root enzymatic hydrolysate at 0.01%–0.03% of the liquid system for fermentation. The fermentation temperature was 35–39℃, and the fermentation time was 18–36 h, yielding a mulberry leaf and kudzu root fermentation broth. The compound microbial agent was composed of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum BXM2, Lactobacillus paracasei ( Lactobacillus paracasei YYS-K1 and Lactobacillus fermentum ( Lactobacillus fermentum B153 is a mixture in a mass ratio of 3-6:2-4:1; The preservation number of the *Lactobacillus plantarum* BXM2 is: CGMCC NO.16436; The preservation number of Lactobacillus paracasei YYS-K1 is: CGMCC No. 26405; The preservation number of the fermenting Lactobacillus mucinus B153 is: CGMCC No. 16454; Finally, the mulberry leaf and kudzu root fermentation liquid was subjected to solid-liquid separation, sterilization, and drying to obtain mulberry leaf and kudzu root fermentation powder.

2. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: In the raw material pretreatment step, the hot water extraction conditions are: extraction at 90-100℃ for 150-180 min.

3. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: In the raw material pretreatment step, the mass ratio of mulberry leaf powder to kudzu root powder is 8:

2.

4. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: The compound microbial agent is composed of *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 in a mass ratio of 4.5:3:

1.

5. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis at 50-60℃ for 2-3 hours to obtain mulberry leaf and kudzu root enzymatic hydrolysate; The cellulase, pectinase, and protease in the complex enzyme all have enzyme activities of 20,000–30,000 U / g.

6. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: The *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-K1, and *Lactobacillus fermentum* B153 are all provided in the form of bacterial powder; the viable counts of these three bacteria in the bacterial powder are 500 billion CFU / g, 400 billion CFU / g, and 400 billion CFU / g, respectively.

7. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: The sterilization conditions before fermentation are as follows: heat the mulberry leaf and kudzu root enzymatic hydrolysate to 100-110℃ and maintain it for 20-30 minutes for sterilization. After sterilization, cool the mulberry leaf and kudzu root enzymatic hydrolysate to 37±2℃.

8. The method for preparing mulberry leaf and kudzu root fermented powder as described in claim 1, characterized in that: The solid-liquid separation and sterilization steps are as follows: First, the fermentation liquid of mulberry leaves and kudzu root is initially filtered using a 300-mesh sieve, then centrifuged at 5000 r / min for 5-10 min, and then microfiltered through a membrane with a pore size of 0.22-0.45 μm to obtain the fermentation supernatant of mulberry leaves and kudzu root; then the fermentation supernatant of mulberry leaves and kudzu root is sterilized to obtain the sterilized fermentation liquid of mulberry leaves and kudzu root.

9. A mulberry leaf and kudzu root fermented powder, characterized in that: The mulberry leaf and kudzu root fermented powder is prepared by the method according to any one of claims 1 to 8; the fermented powder possesses at least two of the following functional characteristics: Flavonoid content ≥2.8g / 100g and rutin content ≥1.8g / 100g; The α-glucosidase inhibitory activity is ≥94%, and the α-amylase inhibitory activity is ≥16%. Pancreatic lipase inhibitory activity ≥23%; DPPH free radical scavenging rate ≥90%, ABTS scavenging rate ≥74%, and superoxide anion scavenging rate ≥68%; The content of γ-aminobutyric acid is ≥50 mg / 100g.

10. The use of the mulberry leaf and kudzu root fermented powder as described in claim 9 in the preparation of products with anti-inflammatory, hypoglycemic, and sleep-improving effects.

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