Method for increasing content of sulforaphane in broccoli through magnetic field assisted blanching

Abstract

The invention discloses a method for increasing the content of sulforaphane in broccoli through magnetic field assisted blanching, and belongs to the technical field of food processing. The method for improving the content of sulforaphane in broccoli through magnetic field assisted blanching comprises the following steps: (1) preparing raw materials; (2) magnetic field assisted blanching pretreatment; (3) cooling and structure keeping treatment; (4) carrying out enzymatic incubation based on a magnetic field-assisted blanching regulation state; and (5) obtaining the broccoli product with high sulforaphane content. Exogenous enzymes, chemical reagents or additives are not introduced in the process, and the activity of endogenous myrosinase and the catalytic hydrolysis path of the endogenous myrosinase are regulated by synchronously applying a physical regulation means in the blanching pretreatment process, so that the generation level of sulforaphane in a treated sample is improved. The method is suitable for a food processing system, has the advantages of mild process conditions, strong controllability, high safety and easiness in industrial amplification, and has a wide application prospect in the field of food processing.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching. Background Technology

[0002] Sulforaphane is an important isothiocyanate bioactive substance derived from cruciferous vegetables. It possesses significant antioxidant, anti-inflammatory, anti-tumor, and detoxification enzyme regulation functions, and has broad application prospects in functional foods, nutritional interventions, and the health industry. Broccoli, rich in glucoraphane, a direct precursor to sulforaphane, is considered one of the main dietary sources of sulforaphane.

[0003] In broccoli tissue, sulforaphane does not exist in a free form, but is generated by the hydrolysis of sulforaphane glycosides catalyzed by endogenous myrosinase. This enzymatic reaction first forms an unstable thiohydroxyoxime intermediate, which then rearranges under physiological pH conditions to form sulforaphane. However, in actual processing and consumption, this hydrolysis system is highly complex and easily affected by various factors such as temperature, pH, metal ions, and epithiospecifier protein (ESP). The presence of ESP significantly alters the hydrolysis pathway, preferentially generating nitrile byproducts from the intermediate, thereby reducing the efficiency of sulforaphane formation.

[0004] To enhance sulforaphane formation in broccoli, heat treatment (such as blanching and steaming) is widely used to regulate enzymatic reaction systems. Moderate blanching can promote sulforaphane production. However, traditional blanching processes are highly sensitive to temperature and time windows; even slight deviations can lead to myrosinase inactivation or significant loss of glucosinolates, resulting in insufficient process stability and controllability. Furthermore, existing research has attempted to increase sulforaphane yield by exogenously adding myrosinase, adjusting pH conditions, or supplementing with metal ions. For example, Chinese invention patent CN110066776A discloses a method for extracting sulforaphane and myrosinase magnetic microspheres. This invention uses exogenously added, solidified myrosinase magnetic microspheres as a catalyst to hydrolyze glucosinolates into sulforaphane, thereby increasing the sulforaphane content. Chinese invention patent CN117137098A provides a method for preparing broccoli sprout powder rich in sulforaphane and glucoraphane. The process involves a combination of steaming and zinc sulfate soaking, as well as enzymatic hydrolysis of the sprouts, which increases the glucoraphane content in the sprouts.

[0005] However, the aforementioned methods suffer from high costs, complex processes, and insufficient safety and industrialization feasibility in food applications. Specifically, for exogenous black mustard enzymes, high-quality, food-grade black mustard enzymes are expensive, and the equipment and conditions required for further basic magnetic nanomaterials, functionalization reagents, and immobilization reactions will significantly increase costs. Furthermore, the process chain for introducing magnetic separation and recovery is long, with increased control points, and the microspheres are easily contaminated and deactivated in the food matrix due to the adsorption of impurities such as oils and proteins. In actual production, exogenously added enzymes and their immobilization carriers require rigorous safety assessments, which also restricts the widespread application of the process. For adjusting process parameters or supplementing metal ions, the addition of metal ions such as zinc must be strictly controlled within the safety limits of food additive standards, increasing the burden of raw material testing and process control. Moreover, metal ions can easily degrade the flavor of broccoli or react with other components in food to produce precipitation and discoloration, severely affecting the sensory quality of the product. Existing methods for increasing sulforaphane content involve additional costs and process complexity that far exceed their market value, making them difficult to promote and apply in actual food processing.

[0006] In conclusion, there is an urgent need to develop a simple, controllable technology suitable for food processing conditions to significantly improve the formation efficiency of sulforaphane in broccoli while ensuring product safety, so as to meet the needs of the functional food and related industries. Summary of the Invention

[0007] Given the shortcomings of existing technologies in broccoli, such as low sulforaphane formation efficiency, poor process stability, and poor controllability, especially the high sensitivity of traditional blanching to temperature and time, which easily leads to inactivation of myrosinase or an increase in the proportion of nitrile byproducts, thus limiting the efficiency of sulforaphane formation and its industrial application in the food industry, the purpose of this invention is to provide a green processing method based on endogenous enzymes to promote the efficient formation of sulforaphane in broccoli and achieve the technical effect of increasing sulforaphane content, without introducing exogenous enzymes or chemical reagents, while meeting the requirements of food processing safety and industrialization. This method utilizes reasonable physical means to synergistically regulate myrosinase activity and hydrolysis reaction pathways.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows: In a first aspect of the present invention, a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching is provided, comprising the following steps: (1) Raw material preparation: Select fresh plants as samples to be treated; (2) Magnetic field-assisted blanching pretreatment: The sample to be treated is placed in a liquid blanching medium and blanched at 50~70 ℃ for 5~20 min. During the heat treatment process, a magnetic field with an intensity of 0.1~10 mT is applied to the entire blanching system simultaneously. (3) Cooling and structure preservation treatment: Collect broccoli samples that have undergone magnetic field-assisted blanching pretreatment and cool them to terminate further heat treatment; (4) Enzymatic incubation based on magnetic field-assisted blanching: Cooled and crushed broccoli samples were placed under certain conditions for enzymatic incubation to promote the hydrolysis of glucosinolates. (5) Obtain broccoli products with high sulforaphane content: After incubation, broccoli products with significantly increased sulforaphane content are obtained.

[0009] This invention provides a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching. This method is proposed based on an in-depth analysis of the reaction characteristics of the endogenous myrosinase-sulforaphane hydrolysis system in broccoli and the limitations of existing processing techniques. The core of this method lies in the synergistic regulatory effect of the magnetic field and blanching. Blanching utilizes the significant difference in thermal stability between myrosinase and epidermal-specific sulfur proteins, achieving preferential inactivation of epidermal-specific sulfur proteins through controlled temperature and time. Based on this, a static magnetic field of specific intensity is introduced to improve the conversion efficiency of sulforaphane to sulforaphane by influencing myrosinase activity and the microenvironment of the enzymatic hydrolysis system. By simultaneously applying a static magnetic field of specific intensity during the blanching pretreatment of broccoli, the magnetic field treatment and heat treatment synergistically act on the endogenous enzymatic hydrolysis system of broccoli, effectively inhibiting the activity of epidermal-specific proteins while increasing the activity of myrosinase, thereby promoting the conversion of sulforaphane to sulforaphane and improving the formation efficiency. The corresponding process flow is described below: In the raw material preparation stage of step (1), those skilled in the art can select fresh broccoli plants, remove inedible parts, and cut the florets with stems into uniformly sized clusters. If necessary, they can be washed and drained. The broccoli mentioned above is a plant of the Brassica genus in the Brassicaceae family. Without prior high-temperature treatment or enzyme inactivation treatment, it can be ensured that its endogenous myrosinase and glucosinolates are in a reactive state.

[0010] In the magnetic field-assisted blanching pretreatment in step (2), the blanching heat treatment is first used to disrupt the broccoli cell structure, which is conducive to the release and contact of sulforaphane and myrosinase from different cell compartments. At the same time, the blanching temperature and time are controlled within a range where endogenous myrosinase is stable but epidermal-specific sulfur proteins are significantly inactivated, so as to ensure the conversion of sulforaphane to sulforaphane and inhibit the formation of nitrile byproducts. The simultaneous application of a magnetic field on the basis of the above blanching treatment can act on the endogenous enzymatic reaction microenvironment in plant tissues, and have a regulatory effect on enzyme protein molecular conformation, enzyme-substrate interaction and reaction kinetics. This magnetic field action is carried out in synergy with the heat treatment, and is designed to regulate the catalytic activity of myrosinase to a higher level during the blanching stage; inhibit the competitive role of epidermal-specific sulfur proteins in the hydrolysis and rearrangement of sulforaphane; and guide the hydrolysis reaction of sulforaphane to develop in a direction that is conducive to the formation of isothiocyanate products in the early stage. Through the above-mentioned synergistic regulation process of magnetic field and blanching, the endogenous sulforaphane hydrolysis system of broccoli is pre-adjusted to a reaction state more conducive to the subsequent generation of sulforaphane while blanching pretreatment is completed, providing a stable and repeatable reaction basis for the subsequent enzymatic incubation steps.

[0011] In the cooling and structure preservation process of step (3), the broccoli sample pretreated by magnetic field-assisted blanching is quickly removed from the heating system and cooled to room temperature or lower by cold water to terminate further thermal action. This cooling step is not only used to stop the blanching process, but also to fix and maintain the state of the endogenous enzymatic system formed in step (2). Since the activity of myrosinase and the glucosinolate hydrolysis system have been pre-regulated under magnetic field-assisted blanching conditions, rapid cooling can effectively avoid the decrease in myrosinase activity or the deviation of the reaction path caused by residual heat, thereby maintaining its high catalytic potential in subsequent reaction stages. After cooling, those skilled in the art can crush, chop or homogenize the sample as needed to further improve the contact efficiency between the enzyme and the substrate without introducing exogenous substances; in this context, the crushing operation mainly plays the role of amplifying the enzyme activity advantage established in step (2) in this step, rather than a simple physical homogenization process.

[0012] Step (4), based on the magnetic field-assisted blanching regulation state, relies solely on the endogenous myrosinase system activated by the magnetic field-assisted blanching in the broccoli sample, without adding any additional myrosinase or other catalysts. Unlike the enzymatic hydrolysis process in the prior art that relies solely on the regulation of incubation conditions, this step is not an independent conventional enzymatic hydrolysis step, but rather a continuation and amplification of the magnetic field-assisted blanching regulation effect in step (2). The two have a clear causal relationship in terms of time sequence and technical effect, that is, the enzymatic incubation is carried out on the basis that the myrosinase activity and reaction pathway have been pre-regulated in step (2), and the activity of epidermal-specific sulfur proteins has been effectively inhibited. Therefore, under the same or similar incubation temperature and time conditions, this method can exhibit higher sulforaphane conversion efficiency and make the reaction more inclined to rearrange to generate sulforaphane rather than generate nitrile byproducts.

[0013] After incubation, step (5) can directly yield broccoli products with significantly increased sulforaphane content, or be further used for subsequent processing steps such as drying, extraction, and pulverization.

[0014] Preferably, in step (2), the liquid blanching medium includes a buffer solution or liquid medium that meets the food processing safety requirements; no additional metal salts, organic solvents or enzyme preparations are added to the liquid medium.

[0015] More preferably, the buffer solution includes at least one of phosphate buffer solution, citrate-sodium citrate buffer solution, and acetate-sodium acetate buffer solution, and the pH value of the buffer solution is 5.5 to 7.5; the liquid medium includes at least one of water, deionized water, softened water, and low ionic strength aqueous solution.

[0016] Preferably, in step (2), the magnetic field includes one of a static magnetic field, a low-frequency alternating magnetic field, and a pulsed magnetic field.

[0017] More preferably, the frequency of the low-frequency alternating magnetic field is 0.1~50 Hz; the pulse width of the pulsed magnetic field is 0.1~5 s, the pulse interval is 0.1~10 s, and the pulse duty cycle is preferably 10%~90%.

[0018] Preferably, in step (2), the change in magnetic field strength includes one of continuous adjustment within a set range, phased adjustment, or gradient adjustment.

[0019] More preferably, the number of stages of the phased adjustment is 2 to 4, the change range of magnetic field strength between adjacent stages is 0.1 to 1.5 mT, and the change rate of the gradient adjustment is 0.05 to 1.0 mT / min.

[0020] Preferably, in step (2), the magnetic field is applied either continuously or intermittently.

[0021] More preferably, the intermittent application method includes: a single magnetic field application time of 0.5 to 10 minutes, a blank time between two adjacent magnetic field applications of 0.5 to 10 minutes, and the magnetic field application and blank time cycled once or more, with the total magnetic field action time controlled within the bleaching treatment time range.

[0022] Those skilled in the art can apply a magnetic field using appropriate devices according to actual conditions or needs. For example, the magnetic field can be generated by a Helmholtz coil, an electromagnetic coil, or a functionally equivalent magnetic field generator. The magnetic field can be unidirectional or multidirectional, covering the entire blanching system, so that the broccoli sample is continuously in a uniform magnetic field environment during the blanching process.

[0023] The setting of low-frequency alternating magnetic fields or pulsed magnetic fields is based on the premise of not damaging the structural stability and catalytic activity of black mustard enzyme. Its technical effect is equivalent to the regulatory effect of static magnetic fields on the enzymatic hydrolysis system during blanching. Under the optimal conditions of low-frequency alternating magnetic fields, the magnetic field action mode can be ensured to be close to that of quasi-static magnetic fields on a time scale, without introducing significant induced currents or thermal effects.

[0024] The magnetic field strength can be continuously adjusted within the range described in this invention, or it can be adjusted in stages or gradients. All of the above-mentioned staged or gradient application methods are based on the premise of not inactivating black myrosinase and not weakening the inhibitory effect on epidermal specific sulfur proteins during blanching. As long as stable control of the enzymatic hydrolysis reaction system can be achieved, they are all alternative solutions of this invention. Specifically, staged application refers to dividing the magnetic field strength into two or more stages during the blanching process, with the magnetic field strength remaining relatively stable in each stage; gradient application refers to the magnetic field strength increasing or decreasing in a stepwise or linear manner over time during the blanching process.

[0025] The magnetic field can be applied continuously or intermittently. Intermittent application is used to create a periodic magnetic field environment during the blanching process. Its technical effect is to effectively regulate the behavior of endogenous black mustard enzymes without increasing the magnetic field strength. This effect is functionally equivalent to that of continuous application.

[0026] The above-mentioned preferred magnetic field parameter forms are all engineering modifications or equivalent substitutions of the magnetic field application method within the parameter range of magnetic field strength, action time and application method described in this invention. Those skilled in the art can select within the preferred range, and all of them can effectively regulate the endogenous black mustard enzyme activity of broccoli during magnetic field-assisted blanching.

[0027] Preferably, in step (4), the incubation temperature is 30~45 ℃ and the incubation time is 30~120 min.

[0028] In a second aspect of the invention, an application is provided to the method of increasing sulforaphane content in broccoli by magnetic field-assisted blanching according to the first aspect of the invention, comprising: treating broccoli or other cruciferous vegetables containing glucosinolates and having endogenous myrosinase to increase sulforaphane content.

[0029] The method of this invention has excellent applicability and is suitable for cruciferous vegetables, such as broccoli, which are rich in glucosinolates and possess endogenous myrosinase. In practical applications, this method can be implemented in laboratory-scale, semi-industrial, or industrial food processing processes, and can be used as a pretreatment step for subsequent nutritional fortification, functional food processing, or pre-processed vegetable processing.

[0030] Based on the above technical solutions, the design concept and principle of this invention are as follows: Under existing technological approaches, improving the efficiency of sulforaphane production in broccoli through enzymatic hydrolysis has long faced the following technical challenges that are difficult to address through conventional methods: 1) The activity of myrosinase in plant tissues is limited, resulting in low hydrolysis efficiency; 2) The hydrolysis and rearrangement pathways of sulforaphane are complex, and the presence of epidermal-specific sulfur proteins significantly alters the hydrolysis pathways, leading to a high proportion of nitrile byproducts; 3) Myrosinase, epidermal-specific sulfur proteins, and substrates are highly sensitive to process parameters such as temperature and time, resulting in an extremely narrow process window; 4) Existing solutions often rely on exogenous enzymes, chemical reagents, or complex condition control, making it difficult to meet the safety and industrialization requirements of food processing.

[0031] In recent years, magnetic fields, as a green and low-energy physical regulation method, have been reported to potentially affect enzyme activity or reaction efficiency in a few free enzyme systems. Magnetic field technology, as a green, non-thermal, and low-energy physical regulation method, has gradually gained attention in the fields of enzyme activity regulation, bioreaction system enhancement, and food processing. Magnetic fields of appropriate strength may alter enzyme-catalyzed reaction efficiency by affecting enzyme molecular conformation, reaction system microenvironment, or substrate diffusion behavior. However, current magnetic field technology mainly focuses on a few free enzyme systems (such as catalase, pectinase, and laccase), and is mostly conducted in idealized solution systems. There is a lack of applied research in glucosinolate hydrolysis systems in plant tissues. Similarly, the effects of magnetic fields on the activity, conformation, and kinetics of black mustard enzymes, or their catalytic effect on the hydrolysis of glucosinolates in plant tissues, lack practical guidance. More importantly, existing magnetic field-enzyme research has not addressed the highly complex multi-enzyme synergistic systems in cruciferous plant tissues, nor has it considered the strong interference of epidermal-specific sulfur proteins on reaction pathways. Because the effects of magnetic fields on the synergistic multi-enzyme system within plant tissues are highly uncertain—potentially acting on both myrosinase and epidermal-specific sulfur proteins simultaneously—a lack of proper process design could exacerbate byproduct formation. Therefore, designing a process to positively regulate the myrosinase-sulforaphane system in broccoli is the core challenge of this invention. Against this background, the main technical obstacles encountered when using magnetic fields to increase sulforaphane content are: (i) the effectiveness of magnetic fields on myrosinase; (ii) whether magnetic fields simultaneously enhance epidermal-specific sulfur protein activity, thus producing a negative effect; (iii) the type, intensity, and application method of the magnetic field applied to plant tissues; and (iv) how the magnetic field can be used synergistically with feasible heat treatment methods in food processing.

[0032] The inventors discovered that in the process of preparing high sulforaphane products under food processing conditions, the key factor affecting the efficiency of sulforaphane formation is not a single factor, but rather the simultaneous existence of multiple mutually restrictive technical contradictions.

[0033] Specifically, on the one hand, the formation of sulforaphane depends on the catalytic hydrolysis of glucoraphane by endogenous myrosinase. However, the activity of myrosinase itself in plant tissues is limited and highly sensitive to processing conditions (such as temperature, time, pH, and metal ions), easily leading to a decrease in activity during processing. This results in low hydrolysis efficiency of glucoraphane and limited sulforaphane yield. On the other hand, epidermal-specific proteins, which are ubiquitous in plant tissues, competitively interfere with the reaction pathway after hydrolysis, causing intermediates to recombine and generate nitrile byproducts, thus significantly reducing the proportion of sulforaphane formed. Although existing blanching processes can inactivate epidermal-specific sulfur proteins through heat treatment, their control window for temperature and time is relatively narrow. Slight deviations can simultaneously damage myrosinase activity, making it difficult to maintain a high level of myrosinase catalytic efficiency while effectively inhibiting epidermal-specific sulfur proteins. This limits further improvements in sulforaphane formation efficiency and process stability.

[0034] Due to the aforementioned technical obstacles, simply relying on temperature or time regulation cannot simultaneously resolve the contradiction between the inactivation of epidermal-specific sulfur proteins and the efficient catalysis of black myrosinase. Therefore, a non-chemical, non-thermal physical regulation method must be introduced to finely control the enzymatic reaction system. Based on this, this invention proposes introducing a static magnetic field into the blanching pretreatment stage, allowing the magnetic field effect to be applied simultaneously with heat treatment to the enzymatic hydrolysis system within the plant tissue.

[0035] The inventive concept of this invention lies in first inactivating the epidermal specific sulfur protein through heat under blanching conditions, thereby eliminating the main influencing factor for the formation of nitrile byproducts in the reaction pathway; then, under this premise, by applying a static magnetic field of a specific intensity range, the hydrolysis process dominated by black myrosinase is physically regulated, so that it exhibits higher enzyme activity, reaction efficiency and stability without further increasing the blanching intensity and without adding other chemical components, thereby promoting the selective conversion of glucoraphane to sulforaphane.

[0036] Guided by the aforementioned inventive concept, the magnetic field strength, blanching temperature, and time parameters of this invention are based on objective laws such as the intensity-dependent effect of the magnetic field and the dual impact of heat treatment on the enzyme system. A parameter range suitable for food processing conditions has been pre-set, and the feasibility and technical effects of this technical solution have been verified through examples and comparative examples. As presented by the test results of one or more embodiments of this invention, only under the process combination conditions established by this invention can the comprehensive technical effects of simultaneously increasing black mustard enzyme activity, significantly increasing sulforaphane production, and maintaining nitrile byproducts at extremely low levels for a long period be achieved.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching. The process does not introduce exogenous enzymes, chemical reagents, or additives. By applying physical control measures to the blanching pretreatment process, the endogenous enzymatic reaction pathway is regulated, thereby increasing the sulforaphane content of the treated samples. This method is suitable for food processing systems and has the advantages of mild process conditions, strong controllability, high safety, and easy industrial scale-up.

[0038] This invention provides an application of a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching, which has broad application prospects in the food processing field. Attached Figure Description

[0039] Figure 1 Schematic diagram of a magnetic field-assisted bleaching and hot water treatment device; Figure 2 The curves showing the changes in black myrosinase activity in broccoli under different magnetic field intensities; Figure 3 The curves showing the changes in sulforaphane content under different magnetic field intensities; Figure 4 A graph showing the change in residual glucosinolate content after magnetic field-assisted blanching treatment; Figure 5 Images of a method for increasing sulforaphane content in broccoli through magnetic field-assisted blanching; (a) corresponds to a broccoli sample during the raw material preparation stage, (b) corresponds to magnetic field-assisted blanching pretreatment, (c) corresponds to a broccoli sample obtained after cooling and structure preservation treatment, (d) corresponds to an enzymatic incubation process based on magnetic field-assisted blanching regulation, and (e) corresponds to sampling and testing of broccoli products with high sulforaphane content. Detailed Implementation

[0040] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0041] In the following embodiments: The method of increasing the sulforaphane content in broccoli through magnetic field-assisted blanching uses a magnetic field-assisted blanching device such as... Figure 1As shown, the device includes a heating container, a magnetic field generating device, and a temperature control system. The magnetic field can be generated by a Helmholtz coil, but is not limited to this. Alternatively, the magnetic field can be obtained by: using an electromagnetic coil, solenoid, or other electromagnetic device capable of generating a stable magnetic field; using permanent magnets or arrays of permanent magnets to form a stable or quasi-stable magnetic field around the blanching container; or using a combined magnetic field device that combines electromagnetic and permanent magnetic fields. The blanching device can be a conventional water bath device, a continuous blanching device, or other devices suitable for heat treatment of vegetables. The magnetic field device can be integrated into the exterior, bottom, or sidewall of the blanching container, or it can be integrated into the blanching equipment as a single unit.

[0042] The method for increasing the sulforaphane content in broccoli using magnetic field-assisted blanching in this embodiment includes the following steps: (1) Raw material preparation: Select fresh broccoli, remove the inedible parts, cut the florets into evenly sized clusters, and wash and drain them if necessary; (2) Magnetic field-assisted blanching pretreatment: The broccoli sample obtained in step (1) is placed in a container filled with water and blanched at 50~70 ℃ for 5~20 min under heating conditions. During the blanching process, a static magnetic field with an intensity of 0.1~10 mT is applied to the entire blanching system simultaneously. The magnetic field effect and the heat treatment are consistent in time and space, so that the broccoli sample is continuously in a uniform magnetic field environment during the blanching process. (3) Cooling and structure preservation treatment: After blanching, the broccoli sample treated by magnetic field assisted blanching in step (2) is quickly removed from the heating system and cooled with cold water to reduce the sample temperature to room temperature or lower temperature to terminate further heat treatment. (4) Enzymatic incubation based on magnetic field-assisted blanching control: Broccoli samples treated in steps (2) and (3) were crushed and placed under suitable conditions for enzymatic incubation. The incubation temperature was 30-45 °C and the incubation time was 30-120 min to promote further hydrolysis of glucosinolates. The incubation system relied only on the endogenous myrosinase system activated by magnetic field-assisted blanching in the broccoli samples, without adding myrosinase or other catalysts. (5) Obtain broccoli products with high sulforaphane content: After incubation, broccoli products with significantly increased sulforaphane content are obtained.

[0043] The basic objective of this invention can also be achieved under other alternative options or preferred parameter ranges provided by this invention.

[0044] The test methods involved in the embodiments are as follows: 1. Determination of black mustard enzyme activity Broccoli samples that had undergone different treatments were pulverized under low temperature conditions, and the samples were extracted with a buffer solution to obtain crude black mustard enzyme solution.

[0045] A certain volume of crude enzyme solution was mixed with a substrate solution of known concentration, and the reaction was terminated after reacting for a certain time under constant temperature conditions.

[0046] The catalytic activity of black mustard enzyme is characterized by measuring the amount of glucose generated in the reaction system, with the amount of glucose generated per unit time serving as an evaluation index of enzyme activity.

[0047] Black mustard enzyme activity is expressed as enzyme activity per unit mass of sample.

[0048] This method can reflect the changes in black mustard enzyme activity under different magnetic field-assisted blanching conditions.

[0049] 2. Determination of sulforaphane and sulforaphane content Broccoli samples that had been blanched and incubated with magnetic field assistance were pulverized, and the target compound was extracted using an organic solvent. The extract was then purified, filtered, and analyzed.

[0050] Sulforaphane and thiophanate-methyl were qualitatively and quantitatively analyzed using liquid chromatography-mass spectrometry, and the content of the target substances in the samples was calculated by external standard method or equivalent standard curve method.

[0051] This method can simultaneously distinguish and quantify sulforaphane and its byproduct sulforaphane, thereby evaluating the effect of magnetic field-assisted blanching on product distribution.

[0052] 3. Determination of residual glucosinolate content The treated broccoli samples were extracted using a polar organic solvent to inhibit further reactions by endogenous enzymes. The extract was then concentrated, reconstituted, and filtered before analysis.

[0053] Residual glucosinolates in the samples were detected using liquid chromatography-mass spectrometry (LC-MS), and the content of each glucosinolate component was calculated by comparing it with standard substances or standard curves.

[0054] The effects of magnetic field-assisted blanching and subsequent incubation treatment on the hydrolysis efficiency of glucosinolates were evaluated by measuring the residual glucosinolate content.

[0055] Example 1 This embodiment provides a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching, the steps of which are as follows: (1) Raw material preparation: Select fresh broccoli, remove old leaves and inedible parts, take the floret part, wash and cut into floret segments of uniform size, 5 cm long and 0.7~0.9 cm wide; (2) Magnetic field-assisted blanching pretreatment: The treated broccoli was placed in a sample container, and an appropriate amount of water was added to the container to completely submerge the broccoli. Under heating conditions, blanching was performed at 60 °C for 12 min. During the blanching process, a static magnetic field with an intensity of 1.0 mT was applied to the entire blanching system simultaneously. The magnetic field effect and the heat treatment were consistent in time and space, so that the broccoli sample was continuously in a uniform magnetic field environment during the blanching process. (3) Cooling and structure preservation treatment: After blanching, the sample is quickly removed from the heating system and cooled with cold water to reduce the temperature of the sample to room temperature or lower and drain the water to terminate further heat treatment; after cooling, drain the surface water and perform a light crushing treatment on the sample to cause partial structural rupture of the flower head tissue without forming a paste, so as to increase the contact area between the intracellular substrate and black myrosinase, while maintaining the integrity of the overall tissue structure of the sample. (4) Enzymatic incubation based on magnetic field-assisted blanching control: Incubation treatment was carried out under suitable conditions, with the incubation temperature controlled at 38 °C and the incubation time being 1 h, in order to promote the enzymatic hydrolysis of glucosinolates by black myrosinase. (5) Obtain broccoli products with high sulforaphane content: After incubation, broccoli products with significantly increased sulforaphane content are obtained.

[0056] Example 2 The method steps in this embodiment are basically the same as those in Embodiment 1, the only difference being the intensity of the magnetic field applied during the blanching process. Specifically, in this embodiment, the static magnetic field intensities applied during the blanching process are set to 0.5 mT, 3.0 mT, 5 mT, and 10 mT, respectively.

[0057] Example 3 This embodiment is basically the same as the method and steps in Embodiment 1. The difference is that in this embodiment, broccoli is subjected to magnetic field-assisted blanching under a magnetic field strength of 1.0 mT, and the blanching temperature is adjusted to examine its effect on the treatment effect. Specifically, the blanching temperature is set to 50 ℃, 65 ℃, and 70 ℃, and the blanching time is kept at 12 min, while the other process conditions remain the same.

[0058] Example 4 This embodiment is basically the same as the method and steps in Embodiment 1. The difference is that in this embodiment, broccoli is subjected to magnetic field-assisted blanching under the conditions of a magnetic field strength of 1.0 mT and a constant blanching temperature, and the blanching time is adjusted to examine its effect on the treatment effect. Specifically, the blanching time is set to 10 min, 15 min, and 20 min, respectively, and the blanching temperature is maintained at 60 ℃, while the other process conditions remain the same.

[0059] Comparative Example 1 This comparative example is a conventional bleaching method without the application of a magnetic field. The process is carried out according to the steps of Example 1, but without the application of a magnetic field during the bleaching process. The steps are as follows: (1) Raw material preparation: Select fresh broccoli, remove old leaves and inedible parts, take the floret part, wash and cut into floret segments of uniform size, 5 cm long and 0.7~0.9 cm wide; (2) Blanching pretreatment: Place the treated broccoli in a sample container, add an appropriate amount of water to the container to completely submerge the broccoli, and blanch it at 60 ℃ for 12 min under heating conditions. (3) Cooling and structure preservation treatment: After blanching, the sample is quickly removed from the heating system and cooled with cold water to reduce the temperature of the sample to room temperature or lower and drain the water to terminate further heat treatment. (4) Enzymatic incubation: Broccoli samples were incubated under suitable conditions. The incubation temperature was controlled at 38 °C and the incubation time was 1 h to promote the enzymatic hydrolysis of glucosinolates by myrosinase. (5) Obtain broccoli products: After incubation, broccoli products are obtained.

[0060] Comparative Example 2 This comparative example illustrates the method without blanching, i.e., directly incubating the broccoli without blanching. The steps are as follows: (1) Raw material preparation: Select fresh broccoli, remove old leaves and inedible parts, take the floret part, wash and cut into floret segments of uniform size, 5 cm long and 0.7~0.9 cm wide; (2) Enzymatic incubation: Incubation treatment was carried out under suitable conditions, with the incubation temperature controlled at 38 °C and the incubation time being 1 h, in order to promote the enzymatic hydrolysis of glucosinolates by black myrosinase. (3) Obtain broccoli products: After incubation, broccoli products are obtained.

[0061] The contents of myrosinase, sulforaphane, and thiouracil in broccoli samples treated in Examples 1-4 and Comparative Examples 1 and 2 were determined using the aforementioned test methods. The results are shown in Table 1. Correspondingly, the curves showing the changes in myrosinase activity in broccoli under different magnetic field intensities are shown in Table 1. Figure 2 As shown; the curves of sulforaphane content variation under different magnetic field intensities are as follows. Figure 3 As shown in the figure; the change in residual glucosinolate content after magnetic field-assisted blanching treatment is shown in the figure. Figure 4 As shown.

[0062] Table 1: Effects of different treatments on the activity of myrosinase, sulforaphane, and thiophanate-methyl in broccoli

[0063] Based on the test results in Table 1, it can be seen that the activity of myrosinase in broccoli treated in Example 1 was significantly increased, the content of sulforaphane was significantly higher than that of the control sample without a magnetic field, and the content of nitrile byproducts was at a low level, indicating that this method can effectively improve the efficiency of sulforaphane production.

[0064] The experimental results of Example 2 show that, within the above-mentioned magnetic field strength range, magnetic field-assisted blanching can promote the increase of black myrosinase activity and significantly reduce the content of residual glucosinolates, with the effect being particularly obvious under low to medium magnetic field strength.

[0065] The results of Example 3 show that, within the above temperature range, magnetic field-assisted blanching treatment can improve the production level of sulforaphane while ensuring the activity of myrosinase, indicating that the method of the present invention has good applicability and stability within a certain blanching temperature range.

[0066] The results of Example 4 show that, within the above time range, magnetic field-assisted blanching treatment can effectively maintain the activity of myrosinase and promote the generation of sulforaphane, indicating that the method of the present invention also has good applicability within a certain blanching time range.

[0067] The results of Comparative Example 1 showed that the myrosinase activity and sulforaphane content in the Comparative Example 1 sample were significantly lower than those in Example 1, while the residual glucosinolate content was higher, indicating that magnetic field assistance is a key technical feature for improving sulforaphane production.

[0068] Figures 2-4 The results of magnetic field-assisted blanching treatment of broccoli samples under different magnetic field strengths (including samples without magnetic field (Comparative Example 1) and sample samples of the example with static magnetic field applied at 0.5~10 mT) under the same blanching temperature, blanching time and subsequent incubation conditions are used to illustrate the effect of magnetic field strength changes on the endogenous enzymatic reaction system and the distribution of its hydrolysis products.

[0069] like Figure 2 As shown, within the magnetic field strength range of 0–10 mT, the activity of myrosinase in broccoli samples exhibited a trend of first increasing and then decreasing with the change in magnetic field strength. Among them, the myrosinase activity of the samples reached the highest level (86.32 ± 3.09 U / g fresh weight, Table 1) under a magnetic field strength of about 1 mT. Throughout the entire test range, the blanched samples subjected to the magnetic field showed significantly higher myrosinase activity than the samples that underwent only conventional blanching treatment, indicating that introducing a low-intensity static magnetic field of appropriate strength during the blanching process is beneficial to maintaining or enhancing the catalytic activity of endogenous myrosinase.

[0070] like Figure 3As shown, the distribution of hydrolysis products in broccoli samples changed accordingly with varying magnetic field strengths under different magnetic field intensities. The trend of sulforaphane production with magnetic field strength was basically consistent with the trend of myrosinase activity, reaching its highest level (61.14 ± 1.21 μmol / 100 g fresh weight) at approximately 1 mT. Meanwhile, the byproduct sulforaphane remained at extremely low levels throughout the entire magnetic field strength range, indicating that under the magnetic field-assisted blanching conditions described in this invention, magnetic field regulation did not induce any adverse shifts in the hydrolysis rearrangement pathway.

[0071] like Figure 4 As shown, after blanching with magnetic fields of different intensities, the residual content of glucosinolates in broccoli samples generally decreased with the increase of magnetic field strength, and decreased to an extremely low level within the range of magnetic field strength. This indicates that magnetic field-assisted blanching can promote the enzymatic hydrolysis of glucosinolates and increase the degree of conversion of precursor substances into target products.

[0072] In conclusion, Figures 2-4 The results show that introducing a low-intensity static magnetic field of appropriate strength during the blanching pretreatment process can effectively regulate the endogenous enzymatic hydrolysis system of broccoli without changing the subsequent incubation conditions, thereby increasing the activity of myrosinase, promoting the generation of sulforaphane, and reducing the amount of precursor substances remaining. This provides a direct illustration of the effectiveness of the magnetic field-assisted blanching technology of the present invention.

[0073] In addition, the implementation and sampling process of the method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching are as follows: Figure 5 As shown. Figure 5 In the image, (a) is a photo of a broccoli sample during the raw material preparation stage, (b) corresponds to the magnetic field-assisted blanching pretreatment, (c) corresponds to the broccoli sample obtained after cooling and structure preservation treatment, (d) is the enzymatic incubation process based on the magnetic field-assisted blanching state regulation, and (e) corresponds to the sampling and testing of broccoli products with high sulforaphane content after obtaining them.

[0074] Based on the above process and test results, the method of magnetic field-assisted blanching to increase the sulforaphane content in broccoli has the following advantages: (1) Significantly improves the efficiency of sulforaphane production This invention introduces a low-intensity static magnetic field during the blanching pretreatment process, allowing the magnetic field treatment and heat treatment to work synergistically on the endogenous enzymatic hydrolysis system of broccoli, thereby significantly improving the generation efficiency of sulforaphane without adding exogenous enzymes or chemical reagents.

[0075] Experimental results show that under magnetic field-assisted blanching conditions, the sulforaphane content in broccoli is significantly higher than that in the control group treated only by conventional blanching, reaching a maximum of 61.14±1.21 μmol / 100 g fresh weight, achieving an increase of 1.89 times. This proves that the method can effectively solve the problem of limited sulforaphane generation efficiency in existing technologies.

[0076] (2) Enhance black mustard enzyme activity and promote full substrate conversion. One of the core technical features of this invention lies in the regulatory effect of magnetic field on the activity of myrosinase. Experimental results show that, within a suitable range of magnetic field strength, magnetic field-assisted blanching can significantly increase the activity of myrosinase, up to 3.11 times, thereby significantly enhancing its catalytic efficiency and promoting the conversion of glucosinolates to sulforaphane.

[0077] Compared with the prior art methods that rely solely on heat treatment or the addition of exogenous enzymes, the present invention can improve the utilization efficiency of endogenous enzymes and reduce production costs while maintaining process simplification.

[0078] (3) Effectively reduces the content of residual glucosinolates and improves the adequacy of hydrolysis. After treatment with the method of this invention, the residual glucoraphane content in broccoli was reduced to a low level. The glucoraphane content in the 0.5–3.0 mT treated broccoli was reduced by approximately 97.5–98.3% compared to the 0 mT control, verifying the positive effect of this method in improving the completeness of enzymatic hydrolysis.

[0079] (4) Byproduct generation is controlled, resulting in high product safety. Although the content of nitrile byproducts (such as thiocyanate) remained at a low level (0.21-3.01 μmol / 100 g fresh weight) under magnetic field-assisted blanching conditions, far lower than the corresponding sulforaphane content, and the overall hydrolysis pathway was still dominated by isothiocyanate formation, these results indicate that the method of this invention can increase the content of the target functional components without significantly increasing the formation of undesirable byproducts, demonstrating good food safety and application feasibility.

[0080] (5) The process is simple and safe, making it suitable for industrial application. This invention uses a magnetic field as an auxiliary physical means, without introducing any chemical additives or genetic modification, thus meeting the safety requirements of food processing. The magnetic field strength is low, energy consumption is low, and it is easily integrated with existing blanching equipment, exhibiting good process compatibility and scale-up potential.

[0081] Therefore, the method of the present invention is not only applicable to laboratory conditions, but also suitable for widespread application in the fields of food processing and functional food preparation.

[0082] In summary, this invention does not introduce exogenous enzymes, chemical reagents, or additives. Through the synergistic design of a magnetic field and a blanching process, and by fully utilizing the difference in thermal stability between myrosinase and epidermal-specific sulfur proteins, the invention introduces a magnetic field to physically regulate the enzymatic hydrolysis system. This effectively guides the hydrolysis of sulforaphane in broccoli, effectively inhibiting the activity of epidermal-specific proteins while increasing the activity of myrosinase. Under the premise of ensuring food safety and processing feasibility, it promotes the conversion of sulforaphane to sulforaphane, significantly improving the efficiency of sulforaphane formation. This invention has the advantages of mild process conditions, strong controllability, high safety, and ease of industrial scale-up. This invention provides a simple, highly controllable, and industrially viable new technological path for the development of functional foods and related health products, with broad application prospects.

[0083] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching, characterized in that, Includes the following steps: (1) Raw material preparation: Select fresh plants as samples to be treated; (2) Magnetic field-assisted blanching pretreatment: The sample to be treated is placed in a liquid blanching medium and blanched at 50~70 ℃ for 5~20 min. During the heat treatment process, a magnetic field with an intensity of 0.1~10 mT is applied to the entire blanching system simultaneously. (3) Cooling and structure preservation treatment: Collect broccoli samples that have undergone magnetic field-assisted blanching pretreatment and cool them to terminate further heat treatment; (4) Enzymatic incubation based on magnetic field-assisted blanching: Cooled and crushed broccoli samples were placed under certain conditions for enzymatic incubation to promote the hydrolysis of glucosinolates. (5) Obtain broccoli products with high sulforaphane content: After incubation, broccoli products with significantly increased sulforaphane content are obtained.

2. The method for increasing the sulforaphane content in broccoli by magnetic field-assisted blanching according to claim 1, characterized in that: In step (2), the liquid blanching medium includes a buffer solution or liquid medium that meets food processing safety requirements; no additional metal salts, organic solvents or enzyme preparations are added to the liquid medium; the buffer solution includes at least one of phosphate buffer solution, citric acid-sodium citrate buffer solution, and acetic acid-sodium acetate buffer solution, and the pH value of the buffer solution is 5.5~7.5; the liquid medium includes at least one of water, deionized water, softened water, and low ionic strength aqueous solution.

3. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 1, characterized in that: In step (2), the magnetic field includes one of the following: static magnetic field, low-frequency alternating magnetic field, and pulsed magnetic field.

4. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 3, characterized in that: The frequency of the low-frequency alternating magnetic field is 0.1~50 Hz; the pulse width of the pulsed magnetic field is 0.1~5 s, the pulse interval is 0.1~10 s, and the pulse duty cycle is preferably 10%~90%.

5. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 1, characterized in that: In step (2), the magnetic field strength can be changed in one of the following ways: continuous adjustment within a set range, phased adjustment, or gradient adjustment.

6. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 5, characterized in that: The number of stages in the phased adjustment is 2 to 4, and the change range of magnetic field strength between adjacent stages is 0.1 to 1.5 mT; the change rate of the gradient adjustment is 0.05 to 1.0 mT / min.

7. The method for increasing the sulforaphane content in broccoli by magnetic field-assisted blanching according to claim 1, characterized in that: In step (2), the magnetic field is applied either continuously or intermittently.

8. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 7, characterized in that, The intermittent application method includes: a single magnetic field application time of 0.5 to 10 minutes, a blank time between two adjacent magnetic field applications of 0.5 to 10 minutes, and the magnetic field application and blank time are repeated once or more, with the total magnetic field action time controlled within the bleaching treatment time range.

9. The method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching according to claim 1, characterized in that: In step (4), the incubation temperature is 30~45 ℃ and the incubation time is 30~120 min.

10. The application of a method for increasing the sulforaphane content in broccoli through magnetic field-assisted blanching as described in any one of claims 1 to 9, characterized in that, include: This treatment is used on broccoli or other cruciferous vegetables containing glucosinolates and endogenous myrosinase to increase sulforaphane content.

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