Use of a lactobacillus plantarum zx028 in combination with horseradish powder for increasing conversion of glucoraphanin to sulforaphane

Abstract

The application discloses a kind of Lactobacillus plantarum ZX028 and horseradish powder composition in the application and method for improving radish glucosinolate conversion rate, and the present application is combined with Lactobacillus plantarum ZX028 and horseradish powder, endogenous enzyme system in horseradish powder is fully combined with the metabolic activity of Lactobacillus plantarum ZX028, and the hydrolysis and conversion of radish glucosinolate are promoted together, so that radish glucosinolate can be efficiently catalyzed under mild conditions to be converted into more active sulforaphane.In the composition, after further adding curcumin, the conversion efficiency is significantly enhanced, the reaction process is green and environmentally friendly, easy to operate, suitable for industrial production and practical application in the field of food and health products, and has good industrial application value and market promotion potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a composition containing *Lactobacillus plantarum* ZX028 and horseradish powder in improving the conversion rate of glucoraphane, and also to a method for promoting the conversion of glucoraphane into sulforaphane. Background Technology

[0002] Sulforaphane (SFN) is one of the most potent natural anticancer products found in vegetables to date, possessing various physiological activities including antioxidant, anti-inflammatory, cancer-preventive, and neuroprotective effects. However, sulforaphane is chemically unstable and present in extremely low amounts in the natural environment, primarily existing as its stable precursor, glucoraphane (GRA), in cruciferous plants such as broccoli. After ingestion, human beings rely on gut microbiota or exogenous enzymes (such as myrosinase) to convert GRA into bioactive sulforaphane. However, the conversion efficiency of the human gut microbiota varies greatly due to individual differences, which significantly impacts the health benefits of GRA.

[0003] To overcome this deficiency, existing technologies have developed two main approaches: 1) Using exogenous myrosinase: By adding raw materials rich in myrosinase (such as horseradish powder or mustard powder), sulforaphane can be efficiently converted in vitro or in vivo. 2) Utilizing specific microbial strains: By screening probiotics capable of metabolizing sulforaphane, probiotic preparations are made for in vivo conversion. Each approach has its advantages, but the conversion rate still has room for improvement when using horseradish powder alone, and the conversion efficiency of a single strain usually requires a long time (e.g., more than 24 hours). How to rapidly and efficiently convert sulforaphane into sulforaphane remains a continuously pursued goal in this field. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an application of a composition containing *Lactobacillus plantarum* ZX028 and horseradish powder in promoting the conversion of glucoraphane to sulforaphane; a second objective of the present invention is to provide a composition for promoting the conversion of glucoraphane to sulforaphane; a third objective of the present invention is to provide a formulation containing the aforementioned composition; and a fourth objective of the present invention is to provide a method for promoting the conversion of glucoraphane to sulforaphane.

[0005] To achieve the above objectives, the present invention provides the following technical solution: 1. The application of a composition containing Lactobacillus plantarum ZX028 and horseradish powder in promoting the conversion of glucoraphane to sulforaphane.

[0006] Preferably, the mass ratio of *Lactobacillus plantarum* ZX028 to horseradish powder is 1:10 to 10:1. More preferably, the mass ratio is 1:10 to 1:1, even more preferably, 1:10; 1:5; 1:2; 1:1. Most preferably, the mass ratio is 1:5.

[0007] Preferably, the composition of the present invention further contains curcumin.

[0008] Preferably, the curcumin is present in a mass ratio of 0.25 to 1.25:1 with horseradish powder. More preferably, the curcumin is present in a mass ratio of 0.25:1 with horseradish powder. 2. A composition for promoting the conversion of glucosinolates into sulforaphane, comprising Lactobacillus plantarum ZX028 and horseradish powder.

[0009] Preferably, the present invention also includes curcumin.

[0010] Preferably, the present invention also includes sulforaphane or sulforaphane-containing raw materials.

[0011] 3. A formulation containing the composition, wherein the formulation is a solid beverage, a probiotic preparation, a capsule, or granules.

[0012] 4. A method for promoting the conversion of sulforaphane to sulforaphane, comprising mixing an extract containing sulforaphane with a composition containing Lactobacillus plantarum ZX028 and horseradish powder and reacting the mixture under suitable conditions.

[0013] Preferably, the suitable conditions for this invention are a shaking reaction at 35-37°C under artificial simulated intestinal fluid conditions for 2-3 hours.

[0014] The beneficial effects of this invention are as follows: This invention provides a method that utilizes *Lactobacillus plantarum* ZX028 in combination with horseradish powder, fully integrating the endogenous enzyme system in the horseradish powder with the metabolic activity of *Lactobacillus plantarum* ZX028 to jointly promote the hydrolytic conversion of sulforaphane. Therefore, it can efficiently catalyze the conversion of sulforaphane into more active sulforaphane under mild conditions. In the composition... The addition of curcumin significantly enhanced the conversion efficiency, indicating that curcumin can effectively activate or assist the enzymatic reaction system, greatly improving the yield of the target product. The reaction process is mild, requiring no harsh conditions such as high temperatures, strong acids or alkalis, or organic solvents. The process is environmentally friendly, simple to operate, and suitable for industrial production and practical applications in the food and health product industries. It can also be formulated into various dosage forms such as solid beverages, probiotic preparations, capsules, or granules, making it convenient to carry and consume. Furthermore, it can be directly combined with raw materials containing sulforaphane (such as broccoli extract), expanding its application prospects in functional foods and dietary supplements. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0016] Horseradish powder was a commercially available freeze-dried product, passed through an 80-mesh sieve. Broccoli seed extract (sulforaphane ≥13%) was commercially available. Curcumin (purity ≥95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and MRS culture medium was purchased from Beijing Luqiao Technology Co., Ltd.

[0017] Artificial gastric fluid (pH 3.5): Take an appropriate amount of dilute hydrochloric acid, add about 800ml of water and 10g of pepsin, adjust the pH to 3.5, shake well, and then dilute with water to 1000ml. Artificial intestinal fluid (pH 6.8): Take 6.8g of potassium dihydrogen phosphate and 10g of trypsin, and add water to a final volume of 1000mL.

[0018] In this invention, *Lactiplantibacillus plantarum* ATCC 14917 was commercially available. *Lactiplantibacillus plantarum* ZX028 was obtained by isolating from the intestines of healthy humans; it was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20232705, located at Wuhan University, Wuhan, Hubei Province, China, on December 28, 2023, and classified as *Lactiplantibacillus plantarum*, as detailed in Chinese Patent Publication No. CN117987319B.

[0019] Example 1: Activation and freeze-dried powder preparation of Lactobacillus plantarum ZX028 (1) Activation of strains: Inoculate Lactobacillus plantarum ZX028 and Lactobacillus plantarum ATCC 14917 glycerol tubes into MRS liquid medium and incubate at 37℃ for 24 h to activate two generations.

[0020] (2) Preparation of bacterial suspension: After activation and culture, the bacterial suspension was centrifuged at 4℃ and 6000 rpm for 10 min to collect the bacterial cells, and then resuspended and washed twice with sterile physiological saline.

[0021] (3) Preparation of freeze-dried powder: The bacterial suspension was centrifuged to collect bacterial sludge, which was then mixed with a freeze-drying protectant (e.g., containing one or more of skim milk powder, trehalose, and monosodium glutamate) (freeze-drying protectant: 10% monosodium glutamate, 16% skim milk powder, 10% trehalose, with the remainder being water. Bacterial sludge: protectant = 1:4 (m / v)). The mixture was then freeze-dried using a conventional vacuum freeze-drying process. The viable count of the obtained freeze-dried powder was determined by plate counting to be no less than 6 × 10⁻⁶. 11 CFU / g (i.e., 600 billion / g).

[0022] Example 2: Verification of the conversion effect of the combination of Lactobacillus plantarum ZX028 and horseradish powder Experimental Groups: Group A (composition group): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 + 0.4g horseradish powder, dissolved in 20ml artificial intestinal fluid.

[0023] Group B (horseradish powder group): 1g broccoli seed extract + 0.4g horseradish powder, dissolved in 20ml artificial intestinal fluid.

[0024] Group C (Lactobacillus plantarum ZX028 group): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028, dissolved in 20ml artificial intestinal fluid.

[0025] Group D (Lactobacillus plantarum ATCC 14917 group): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ATCC 14917, dissolved in 20ml artificial intestinal fluid.

[0026] Group E (ATCC 14917 + Horseradish Powder Group): 1g broccoli seed extract + 0.08g Bacillus plantarum ATCC14917 lyophilized powder + 0.4g horseradish powder, dissolved in 20ml artificial intestinal fluid.

[0027] Group F (blank group): 1g of broccoli seed extract dissolved in 20ml of artificial intestinal fluid.

[0028] Reaction and Detection: Each group was reacted at 37℃ under simulated intestinal fluid conditions with shaking for 2 hours. Immediately after the reaction, the samples were heated in a boiling water bath for 5 min to terminate the reaction. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane. The concentration of glucosinolates was determined by HPLC: the chromatographic column was Amethyst C18-H (4.6 × 250 mm, 5 μm), the mobile phase was acetonitrile-water (gradient elution: 0-10 min 5%-15% acetonitrile, 10-20 min 15%-30% acetonitrile, 20-25 min 30%-15% acetonitrile, 25-35 min 15%-5% acetonitrile), the detection wavelength was 230 nm, the flow rate was 0.6 mL / min, and the injection volume was 10 μL. The degradation rate of glucosinolates was calculated based on the standard curve. The results are shown in Table 1.

[0029] Table 1. Degradation rate of glucosinolates in different treatment groups after 2 hours

[0030] The results showed that the degradation rate of the combination of Lactobacillus plantarum ZX028 and horseradish powder (83.98%) was significantly higher than that of horseradish powder alone (48.47%) and ZX028 alone (31.60%), indicating that the combination of the two can make full use of the conversion capacity of the two components and achieve efficient conversion in a short time.

[0031] Example 3: Effects of Compositions with Different Weight Ratios and Discovery of Critical Ratios Following the method of Example 2, the amount of glucoraphane (1g of broccoli seed extract) and reaction conditions were fixed, while the weight ratio of Lactobacillus plantarum ZX028 freeze-dried powder to horseradish powder was changed. The degradation rate of glucoraphane was measured after 2 hours, and the results are shown in Table 2.

[0032] Table 2. Degradation rate of compositions with different ratios

[0033] The results showed that the degradation rate of the composition reached its highest value of 83.18% when the weight ratio of *Lactobacillus plantarum* ZX028 to horseradish powder was 1:5. When the ratio deviated from 1:5, the degradation rate showed a significant decreasing trend: 72.03% at 1:10, 68.86% at 2:1, 59.38% at 5:1, and 58.44% at 10:1. It is particularly noteworthy that the 1:5 ratio is not the midpoint of a broad effective range, but rather an optimal weight ratio—the adjacent ratios on either side (74.90% at 1:2 and 72.03% at 1:10) could not reach the 83.18% level.

[0034] Example 4: Verification of the synergistic effect of curcumin Experimental Groups: Group A (curcumin alone): 1g broccoli seed extract + 0.1g curcumin, dissolved in 20ml artificial intestinal fluid.

[0035] Group B (ZX028 + horseradish powder group): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 + 0.4g horseradish powder, dissolved in 20ml artificial intestinal fluid.

[0036] Group C (ZX028 + horseradish powder + curcumin group): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 + 0.4g horseradish powder + 0.1g curcumin, dissolved in 20ml artificial intestinal fluid.

[0037] Group D (blank group): 1g of broccoli seed extract dissolved in 20ml of artificial intestinal fluid.

[0038] Reaction and detection: The reaction conditions and detection methods were the same as in Example 2, and the results are shown in Table 3.

[0039] Table 3. Effect of curcumin on the conversion efficiency of the composition

[0040] The results showed that curcumin alone had almost no conversion effect on glucosinolates (degradation rate <5%). When curcumin was added to the ZX028+ horseradish powder composition, the degradation rate significantly increased from 79.21% to 94.12%, a relative increase of 14.91%. Since the effect of curcumin alone was similar to that of the control group, this synergistic effect cannot be explained by simple additive methods and is an unexpected technical effect.

[0041] While this invention is not limited to any specific mechanism of action, it is speculated that the unexpected synergistic effect of curcumin may stem from one or more of the following mechanisms: curcumin transiently captures newly generated sulforaphane through non-covalent interactions, weakening the product inhibition effect and thus continuously shifting the reaction equilibrium towards hydrolysis; or it acts as a strong antioxidant, protecting the active conformation of myrosinase in horseradish powder and preventing its oxidative inactivation during the reaction; or it promotes the release of bacterial metabolites by regulating the cell membrane permeability of *Lactobacillus plantarum* ZX028. When used alone, curcumin lacks the core enzyme system for substrate conversion and therefore exhibits no activity. However, when it coexists with the complete enzyme-bacterial system composed of ZX028 and horseradish powder, the aforementioned auxiliary functions become apparent, constituting a significant technological advancement of this invention.

[0042] Example 5: Effects of different curcumin concentrations Following the method in Example 4, other dosages were kept constant (1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 + 0.4g horseradish powder), while the amount of curcumin added was varied (0.1g-0.5g). The degradation rate of glucosinolates after 2 hours was measured, and the results are shown in Table 4.

[0043] Table 4. Effect of different curcumin concentrations on conversion rate

[0044] Note: The baseline data differs slightly from that in Example 4, which is a normal fluctuation between different batches of experiments. The results showed that curcumin had a significant synergistic effect at a concentration of 0.1g, with the optimal concentration being 0.1g, at which the degradation rate reached 91.5%. The synergistic effect decreased slightly at higher concentrations, but still remained around 80%.

[0045] Example 6: Degradation rate of glucosinolates and formation rate of sulforaphane at the optimal ratio. Following the method in Example 5, the optimal amounts of ZX028, horseradish powder, and curcumin were prepared, and the degradation rate of glucosinolates and the formation rate of glucosinolates were measured after 2 hours. The results are shown in Table 5.

[0046] Group A (artificial intestinal fluid): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 + 0.4g horseradish powder + 0.1g curcumin, dissolved in 20ml artificial intestinal fluid.

[0047] Group B (artificial gastric juice): 1g broccoli seed extract + 0.08g Lactobacillus plantarum ZX028 lyophilized powder + 0.4g horseradish powder + 0.1g curcumin, dissolved in 20ml artificial gastric juice (pH 3.5).

[0048] Table 5. Degradation rate of glucosinolates and formation rate of sulforaphane

[0049] Note: The baseline data differs slightly from that of Example 5, which is a normal fluctuation between different batches of experiments. The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of a composition of Lactobacillus plantarum ZX028 and horseradish powder in improving the conversion rate of glucosinolates.

2. The application according to claim 1, characterized in that: The mass ratio of the composition of *Lactobacillus plantarum* ZX028 and horseradish powder is 1:10 to 10:

1.

3. The application according to claim 1, characterized in that: The composition also contains curcumin.

4. The application according to claim 3, characterized in that: The curcumin is prepared at a mass ratio of 0.25 to 1.25:1 with horseradish powder.

5. A composition for promoting the conversion of sulforaphane to glucoraphane, characterized in that: Including Lactobacillus plantarum ZX028 and horseradish powder.

6. The composition for promoting the conversion of sulforaphane to glucoraphane according to claim 5, characterized in that: It also includes curcumin.

7. The composition for promoting the conversion of sulforaphane to glucoraphane according to claim 5, characterized in that: It also includes sulforaphane or raw materials containing sulforaphane.

8. A formulation comprising the composition of claim 5 or 6, characterized in that: The preparation is a solid beverage, a probiotic preparation, a capsule, or granules.

9. A method for promoting the conversion of glucosinolates into sulforaphane, characterized in that: An extract containing glucosinolates was mixed with a composition containing Lactobacillus plantarum ZX028 and horseradish powder and reacted under suitable conditions.

10. The method according to claim 8, characterized in that: The suitable conditions are a shaking reaction at 35-37°C under artificial simulated intestinal fluid conditions for 2-3 hours.

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