Common fenugreek seed extract and preparation method thereof

Abstract

The invention discloses a fenugreek seed extract preparation method, which comprises: taking fenugreek seeds, crushing to obtain a fenugreek seed crushing material, adding ethanol to the fenugreek seed crushing material, and pre-soaking to obtain a first mixture; performing pulsed electric field treatment on the first mixture to obtain a second mixture; the second mixture is put into an extraction tank for extraction, and an extracting solution is obtained; the extracting solution is firstly subjected to microfiltration through a ceramic membrane and then subjected to ultrafiltration through an organic membrane, and dialysate is obtained; concentrating the dialysate through an organic nanofiltration membrane to obtain a concentrated solution; adding gamma-cyclodextrin into the concentrated solution to carry out inclusion treatment, so as to obtain an inclusion solution; and carrying out spray drying treatment on the inclusion liquid to obtain the fenugreek seed extract. According to the method, the production cycle is shortened, the production cost is reduced, the content of 4-hydroxyisoleucine is increased, the stability of 4-hydroxyisoleucine is enhanced, and the redissolved clarity and bitter taste of the extract are improved.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a fenugreek seed extract and a preparation method thereof. Background Art

[0002] Fenugreek (Trigonella foenum-graecum L.) is a plant of the genus Fenugreek in the Fabaceae family. It is an annual herb, 30 to 80 cm tall, with light yellow triangular flowers and triangular leaves. The seeds are slightly rhombic, 3 to 5 mm long, 2 to 3 mm wide, and about 2 mm thick. The surface is yellow-brown or reddish-brown, with a little gray short hair, and there is a deep oblique groove on each side. The junction of the two grooves is the seed hilum, and the texture is hard. It has a fragrant smell and a slightly bitter taste. It is commonly known as bitter beans, fragrant beans, and fenugreek. When the fruit is ripe, the whole plant is cut, the seeds are beaten, dried, and used raw or slightly fried. The medicinal history of fenugreek is about 4,000 years. At present, the main producing country is India. In my country, it is mainly distributed in Henan, Qinghai, Sichuan and other places. Fenugreek can warm the kidney and assist yang, dispel cold and relieve pain. It is mainly used to treat kidney yang deficiency, lower deficiency cold, lower abdominal cold pain, cold hernia abdominal pain, and cold and damp athlete's foot. At present, many chemical components have been extracted from fenugreek stems, leaves, young shoots, seeds and other parts. Most of them are extracted and separated from seeds. They can be roughly divided into the following categories: steroidal saponins, flavonoids, triterpenes, alkaloids, organic acids and oils, coumarins and lignins, etc.

[0003] Fenugreek has multiple effects such as lowering blood sugar, lowering blood lipids, lowering cholesterol, anti-oxidation and anti-inflammatory. Modern studies have found that 4-hydroxyisoleucine, the active ingredient of fenugreek, can significantly improve insulin resistance in fat cells and stimulate insulin secretion. Its specific mechanism of action may be related to the inhibition of TNF-α expression. 4-hydroxyisoleucine is a hydroxylated product of L-isoleucine and a non-protein amino acid. Studies have shown that 4-hydroxyisoleucine has the activity of promoting insulin secretion in a blood sugar concentration-dependent manner and can enhance the sensitivity of receptors to insulin; at the same time, 4-hydroxyisoleucine also has the effects of promoting blood sugar absorption by muscle cells, accelerating fat metabolism, lowering blood lipids and protecting liver function. As an ideal potential drug for the effective prevention and treatment of type 2 diabetes and obesity, 4-hydroxyisoleucine has broad application prospects and market demand.

[0004] The main production methods of 4-hydroxyisoleucine currently include seed extraction, chemical synthesis, chemical-enzyme, enzyme, microbial transformation and fermentation. The extraction and separation method is used for the industrial production of 4-hydroxyisoleucine, but it has the disadvantages of large demand for raw materials, difficulty in separation and purification, low extraction rate and high cost. The chemical method uses glucose, menthone or α-methylacetoacetic acid ethyl ester as raw materials to produce 4-hydroxyisoleucine through multi-step chemical reactions. The chemical method is difficult to separate and has a low yield, so it still remains in the laboratory stage. The chemical-enzyme method first uses a chemical method to synthesize a 4-hydroxyisoleucine precursor, and then synthesizes 4-hydroxyisoleucine by an enzyme method. The enzyme method can specifically generate (2S, 3R, 4S)-4-hydroxyisoleucine, which solves the problem that the chemical method and the chemical-enzyme method produce more 4-hydroxyisoleucine configurations. However, the substrate (2S, 3R)-2-amino-3-methyl-4-ketoglutaric acid used in this method is not easy to obtain, resulting in high production costs. The biological synthesis of 4-hydroxyisoleucine was developed based on the discovery of L-isoleucine dioxygenase (IDO). It can be divided into microbial conversion and fermentation methods according to whether the substrate L-isoleucine and (or) α-ketoglutarate is added. The above methods have shortcomings such as long cycle and the need to add 1 to 2 precursors, which limits the large-scale production of 4-hydroxyisoleucine. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of fenugreek seed extract, which shortens the production cycle and reduces the production cost, increases the content of 4-hydroxyisoleucine, enhances the stability of 4-hydroxyisoleucine, and improves the clarity and bitter taste of the re-dissolved extract.

[0006] Another technical problem to be solved by the present invention is to provide a fenugreek seed extract.

[0007] In order to solve the above technical problems, the present invention provides a method for preparing a fenugreek seed extract, comprising the following steps:

[0008] Taking fenugreek seeds and crushing them to obtain crushed fenugreek seeds, adding ethanol to the crushed fenugreek seeds for pre-soaking to obtain a first mixture;

[0009] The first mixture is subjected to a pulse electric field treatment to obtain a second mixture;

[0010] Putting the second mixture into an extraction tank for extraction to obtain an extract;

[0011] The extract is first microfiltered through a ceramic membrane and then ultrafiltered through an organic membrane to obtain a dialysate;

[0012] The dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0013] adding γ-cyclodextrin to the concentrated solution for inclusion treatment to obtain an inclusion solution;

[0014] The inclusion liquid is spray-dried to obtain the fenugreek seed extract.

[0015] In some embodiments, fenugreek seeds are taken and crushed to obtain fenugreek seed crushed material, and the fenugreek seed crushed material is sieved through a 30-80 mesh sieve and then pre-soaked in 20 wt % to 50 wt % ethanol for 30 min to 60 min to obtain a first mixture.

[0016] In some embodiments, the parameters of the pulsed electric field treatment include: an electric field strength of 0.1 kV / cm to 10 kV / cm;

[0017] The number of pulses is 125 to 2500;

[0018] Pulse width is 0.5μs~10μs;

[0019] The liquid-to-solid ratio is 1:(8-15);

[0020] The single electric field treatment time is 3min to 6min;

[0021] The pulse frequency is fixed at 10Hz.

[0022] In some embodiments, the step of putting the second mixture into an extraction tank for extraction is achieved by the following method:

[0023] The second mixture is put into an extraction tank, heated to 30°C to 50°C, stirred, and the first extraction and the second extraction are performed in sequence;

[0024] The first extraction uses 8-10 times ethanol solution to extract for 1h-2h;

[0025] The second extraction uses 6-8 times ethanol solution for 1h-2h.

[0026] In some embodiments, the microfiltration through a ceramic membrane is achieved by the following method:

[0027] Microfiltration is performed using food safety grade ceramic membranes. During the microfiltration process, the membrane pressure is lower than 0.3 Bar, the cooling water is circulated, and the liquid circulation temperature is lower than 30°C.

[0028] The ceramic film is one or more of a zirconium oxide film, an aluminum oxide film and a silicon nitride film;

[0029] The pore size of the ceramic membrane is 500nm-800nm.

[0030] In some embodiments, the ultrafiltration through an organic membrane is achieved by the following method:

[0031] Use food safety grade organic membrane for ultrafiltration. During the ultrafiltration process, the membrane pressure is 0.1MPa~2MPa, cooling water is circulated, and the liquid circulation temperature is lower than 30℃;

[0032] The organic membrane is one or more of a polyvinylidene fluoride membrane, a polysulfone membrane, and a polyamide membrane;

[0033] The pore size of the organic membrane is 3KDa to 300KDa.

[0034] In some embodiments, the concentration by organic nanofiltration membrane is achieved by the following method:

[0035] A food safety grade polyamide nanofiltration membrane is used for concentration. During the concentration process, the membrane pressure is 0.1MPa to 2MPa, and the cooling water is circulated for cooling. The circulating temperature of the liquid medicine is lower than 30°C.

[0036] The pore size of the organic nanofiltration membrane is 200Da to 300Da.

[0037] In some embodiments, the inclusion treatment of adding γ-cyclodextrin to the concentrated solution is achieved by the following method:

[0038] Firstly, γ-cyclodextrin is dissolved in hot water to obtain a cyclodextrin mixture, and then the cyclodextrin mixture is added to the concentrated solution, stirred evenly, and then inclusion treatment is performed using a high-speed shearing machine or an inclusion machine.

[0039] In some embodiments, γ-cyclodextrin is added at 20% to 40% of the dry cream amount;

[0040] The shearing speed of the high-speed shearing machine is not less than 8000rpm / min, and the shearing time is 10min to 20min;

[0041] The cycle grinding time of the encapsulating machine is not less than 5 minutes.

[0042] In order to solve the above problems, the present invention also provides a fenugreek seed extract, which is prepared by the preparation method of the fenugreek seed extract described above.

[0043] The implementation of the present invention has the following beneficial effects:

[0044] The present invention uses a high-voltage pulse electric field to pretreat the fenugreek seed medicinal material, destroys the plant seed cell wall under low temperature, increases cell permeability, promotes the dissolution of 4-hydroxyisoleucine, and induces molecules to accelerate movement and produce collisions, thereby improving the yield. At the same time, the membrane integration technology is used to replace the traditional alcohol precipitation technology and cyclodextrin inclusion technology to enhance the thermal stability of 4-hydroxyisoleucine, shorten the production cycle and reduce the production cost, increase the content of 4-hydroxyisoleucine, enhance the stability of 4-hydroxyisoleucine, and improve the clarity and bitter taste of the re-dissolved extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The present invention provides a process flow chart of a method for preparing a fenugreek seed extract. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] In the description of the present invention, it is necessary to understand that the terms "length", "width", "up", "down", "left", "right", "horizontal", "top", "bottom", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0048] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0049] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0051] The disclosure below provides many different embodiments or examples to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the examples of various specific processes and materials provided by the present invention, but those of ordinary skill in the art can be aware of the application of other processes and / or the use of other materials.

[0052] In the present invention, "preferred" and "better" are only used to describe implementation methods or embodiments with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features. In the present invention, when involving numerical ranges, unless otherwise specified, the two endpoints of the numerical range are included.

[0053] In order to solve the above technical problems, Figure 1 As shown, the present invention provides a method for preparing a fenugreek seed extract, comprising the following steps:

[0054] S1, taking fenugreek seeds and crushing them to obtain a crushed fenugreek seed, and adding ethanol to the crushed fenugreek seeds for pre-soaking to obtain a first mixture;

[0055] In some embodiments, fenugreek seeds are taken and crushed to obtain fenugreek seed crushed material, and the fenugreek seed crushed material is sieved through a 30-80 mesh sieve and then pre-soaked in 20 wt % to 50 wt % ethanol for 30 min to 60 min to obtain a first mixture.

[0056] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0057] In some embodiments, the parameters of the pulsed electric field treatment include: an electric field strength of 0.1 kV / cm to 10 kV / cm;

[0058] The number of pulses is 125 to 2500;

[0059] Pulse width is 0.5μs~10μs;

[0060] The liquid-to-solid ratio is 1:(8-15);

[0061] The single electric field treatment time is 3min to 6min;

[0062] The pulse frequency is fixed at 10Hz.

[0063] High-voltage pulse electric field extraction technology is a non-thermal processing technology. The material to be processed is placed in a processing chamber with two electrodes, and a high-voltage pulse is applied to the electrodes. The "electric field compression" and "electroporation effect" generated by the high-voltage pulse are used to form "aperture channels" in molecules, cells, and tissues, changing the osmotic dissolution system of intracellular molecules, forming "channel bridges" inside and outside cells, breaking the inefficient mechanism of the traditional osmotic pressure extraction mode, reducing the energy consumption of mass transfer inside and outside cells, and achieving efficient dissolution of target active molecules and functional molecules inside cells and tissues. It has technical advantages such as "non-thermal processing, precise wall breaking, and directional extraction". 4-Hydroxyisoleucine is a heat-sensitive substance. The technology of the present invention uses a high-voltage pulse electric field to pre-treat fenugreek seed medicinal materials, destroy the plant seed cell wall under low temperature conditions, increase cell permeability, promote the dissolution of 4-hydroxyisoleucine, and induce molecules to accelerate motion and produce collisions, thereby increasing the yield.

[0064] S3, putting the second mixture into an extraction tank for extraction to obtain an extract;

[0065] In some embodiments, the step of putting the second mixture into an extraction tank for extraction is achieved by the following method:

[0066] The second mixture is put into an extraction tank, heated to 30°C to 50°C, stirred, and the first extraction and the second extraction are performed in sequence;

[0067] The first extraction uses 8-10 times ethanol solution to extract for 1h-2h;

[0068] The second extraction uses 6-8 times ethanol solution for 1h-2h.

[0069] S4, the extract is first microfiltered through a ceramic membrane, and then ultrafiltered through an organic membrane to obtain a dialysate;

[0070] In some embodiments, the microfiltration through the ceramic membrane is achieved by the following method:

[0071] Microfiltration is performed using food safety grade ceramic membranes. During the microfiltration process, the membrane pressure is lower than 0.3 Bar, the cooling water is circulated, and the liquid circulation temperature is lower than 30°C.

[0072] The ceramic film is one or more of a zirconium oxide film, an aluminum oxide film and a silicon nitride film;

[0073] The pore size of the ceramic membrane is 500nm-800nm.

[0074] In some embodiments, when the circulating volume of the drug solution is close to the dead volume of the equipment, purified water with a volume 2 to 5 times the dead volume of the equipment is added to the circulating liquid to dialyze the active ingredients in the circulating liquid, and the operation is repeated once.

[0075] In some embodiments, the ultrafiltration through an organic membrane is achieved by the following method:

[0076] Use food safety grade organic membrane for ultrafiltration. During the ultrafiltration process, the membrane pressure is 0.1MPa~2MPa, cooling water is circulated, and the liquid circulation temperature is lower than 30℃;

[0077] The organic membrane is one or more of a polyvinylidene fluoride membrane, a polysulfone membrane, and a polyamide membrane;

[0078] The pore size of the organic membrane is 3KDa to 300KDa.

[0079] In some embodiments, when the circulating volume of the drug solution is close to the dead volume of the equipment, purified water with a volume 2 to 4 times the dead volume of the equipment is added to the circulating liquid to dialyze the active ingredients in the circulating liquid.

[0080] The membrane integration technology integrates coarse filtration, microfiltration, ultrafiltration and concentration into one technical integration line. According to the molecular weight of 4-hydroxyisoleucine, low-pore size microfiltration membrane and low-molecular-weight ultrafiltration membrane can be selected to remove large molecular impurities. The extract has good solubility and high clarity.

[0081] S5, the dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0082] In some embodiments, the concentration by organic nanofiltration membrane is achieved by the following method:

[0083] A food safety grade polyamide nanofiltration membrane is used for concentration. During the concentration process, the membrane pressure is 0.1MPa to 2MPa, and the cooling water is circulated for cooling. The circulating temperature of the liquid medicine is lower than 30°C.

[0084] The pore size of the organic nanofiltration membrane is 200Da to 300Da.

[0085] In some embodiments, the organic nanofiltration membrane is used to concentrate the solid content to 12% to 20%.

[0086] Organic nanofiltration membrane low-temperature concentration enriches the effective ingredients, effectively avoiding thermal oxidation of 4-hydroxyisoleucine and retaining the flavor substances of fenugreek seeds to a large extent.

[0087] The membrane integration technology process uses microfiltration through a ceramic membrane, ultrafiltration through an organic membrane, and finally concentration through an organic nanofiltration membrane. It can significantly improve production efficiency, shorten production cycle, reduce production costs, and significantly increase the content of target ingredients and product stability.

[0088] S6, adding γ-cyclodextrin to the concentrated solution for inclusion treatment to obtain an inclusion solution;

[0089] In some embodiments, the following method is used to implement the inclusion treatment by adding γ-cyclodextrin to the concentrated solution:

[0090] Firstly, γ-cyclodextrin is dissolved in hot water to obtain a cyclodextrin mixture, and then the cyclodextrin mixture is added to the concentrated solution, stirred evenly, and then inclusion treatment is performed using a high-speed shearing machine or an inclusion machine.

[0091] In some embodiments, γ-cyclodextrin is added at 20% to 40% of the dry cream amount;

[0092] The shearing speed of the high-speed shearing machine is not less than 8000rpm / min, and the shearing time is 10min to 20min;

[0093] The cycle grinding time of the encapsulating machine is not less than 5 minutes.

[0094] 4-Hydroxyisoleucine is a heat-sensitive substance. Cyclodextrin molecules have a cavity structure that is hydrophilic on the outside and hydrophobic on the inside. Cyclodextrin inclusion compounds have more stable chemical and physical properties, which can delay the degradation and oxidation of effective substances and improve stability. In addition, fenugreek seed extract contains flavonoid bitter substances. Cyclodextrin inclusion compounds can significantly mask the bitterness and improve the taste. They can effectively increase the solubility and dissolution rate of this product in water, greatly improving the compliance of downstream preparations.

[0095] S7. The inclusion solution is spray-dried to obtain a fenugreek seed extract.

[0096] Correspondingly, the present invention also provides a fenugreek seed extract, which is prepared by the preparation method of the fenugreek seed extract described above.

[0097] In summary, the present invention utilizes high-voltage pulse electric field assisted dissolution, integrated membrane separation technology and cyclodextrin inclusion combined technology, ceramic membrane microfiltration and organic membrane ultrafiltration to enrich the effective components of the fenugreek seed extract, and organic nanofiltration membrane concentration, which effectively prevents the thermal decomposition of the heat-sensitive substance 4-hydroxyisoleucine due to excessive heating. Compared with traditional alcohol precipitation, the yield and content of 4-hydroxyisoleucine are increased, the clarity of the extract is improved, the production cycle is significantly shortened, and the production cost is reduced; cyclodextrin is used to include the effective components, the stability of 4-hydroxyisoleucine is improved, and the bitter taste of the extract is reduced.

[0098] The following is a further description in conjunction with embodiments.

[0099] Example 1

[0100] This embodiment is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0101] S1, screening 1 kg of mildew-free and insect-free fenugreek seeds and crushing them through a 30-mesh sieve to obtain a crushed fenugreek seed, adding 20 wt % ethanol to the crushed fenugreek seed for pre-soaking for 30 min to obtain a first mixture;

[0102] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0103] The pulse electric field treatment parameters are electric field intensity (1 kV / cm), number of pulses (200), pulse width (10 μs), liquid-to-solid ratio (1:8) and each electric field treatment time of 3 min.

[0104] S3, putting the second mixture into an extraction tank for extraction, the extraction process is to heat and stir and extract twice at 50°C, the first decoction is extracted with 8 times 20% ethanol solution for 1 hour, the second decoction is extracted with 6 times 20% ethanol solution for 1 hour, the extract is filtered through a 350 mesh sieve, and the supernatant is centrifuged at 3000 rpm to quickly remove coarse particles and visible flocs to obtain an extract;

[0105] S4, the extract is first microfiltered through a ceramic membrane, and then ultrafiltered through an organic membrane to obtain a dialysate;

[0106] The pore size of the ceramic membrane is 800nm, the membrane pressure is lower than 0.3Bar, and the temperature is lower than 30℃. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add deionized water with the same volume of the remaining liquid to replace the dry paste.

[0107] Organic membrane ultrafiltration uses polyvinylidene fluoride membrane elements made of food safety grade materials, with a pore size of 300KDa, a membrane pressure of 0.1-1MPa, and a temperature below 30°C. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add 2 deionized water with the same volume as the remaining liquid to replace the effective ingredients.

[0108] S5, the dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0109] The organic nanofiltration membrane uses a polyamide membrane element made of food safety grade material, with a membrane pressure of 0.1 to 2 MPa and a temperature below 30°C.

[0110] S6, adding γ-cyclodextrin to the concentrated solution for inclusion treatment, adding γ-cyclodextrin for inclusion treatment according to a dry cream rate of 30%, first dissolving γ-cyclodextrin in hot water, then adding it to the concentrated solution, and inclusion treatment with a high-speed shearing machine to obtain an inclusion solution;

[0111] S7. The inclusion solution is spray-dried to obtain a fenugreek seed extract.

[0112] Example 2

[0113] This embodiment is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0114] S1, screening 10 kg of mildew-free and insect-free fenugreek seeds and crushing them through a 30-mesh sieve to obtain a crushed fenugreek seed, adding 20 wt % ethanol to the crushed fenugreek seed for pre-soaking for 60 min to obtain a first mixture;

[0115] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0116] The pulse electric field treatment parameters are electric field intensity (3 kV / cm), number of pulses (200), pulse width (10 μs), liquid-to-solid ratio (1:8) and electric field treatment time of 3 min each time (two treatments).

[0117] S3, putting the second mixture into an extraction tank for extraction, the extraction process is to heat and stir and extract twice at 50°C, the first decoction is extracted with 8 times 20% ethanol solution for 1 hour, the second decoction is extracted with 6 times 20% ethanol solution for 1 hour, the extract is filtered through a 350 mesh sieve, and the supernatant is centrifuged at 3000 rpm to quickly remove coarse particles and visible flocs to obtain an extract;

[0118] S4, the extract is first microfiltered through a ceramic membrane, and then ultrafiltered through an organic membrane to obtain a dialysate;

[0119] The pore size of the ceramic membrane is 500nm, the membrane pressure is lower than 0.3Bar, and the temperature is lower than 30℃. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add deionized water with the same volume of the remaining liquid to replace the dry paste.

[0120] The organic membrane ultrafiltration step uses a food safety grade polyvinylidene fluoride membrane element with a pore size of 50KDa, a membrane pressure of 0.1-1MPa, and a temperature below 30°C. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add deionized water with the same volume of the remaining liquid to replace the effective ingredients.

[0121] S5, the dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0122] The organic nanofiltration membrane uses a food safety grade polyamide membrane element with a pore size of 200Da, a membrane pressure of 0.1 to 2MPa, and a temperature below 30°C.

[0123] S6, adding γ-cyclodextrin to the concentrated solution for inclusion treatment, adding γ-cyclodextrin for inclusion treatment according to a dry cream rate of 30%, first dissolving γ-cyclodextrin in hot water, then adding it to the concentrated solution, and inclusion treatment with a high-speed shearing machine to obtain an inclusion solution;

[0124] S7. The inclusion solution is spray-dried to obtain a fenugreek seed extract.

[0125] Example 3

[0126] This embodiment is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0127] S1, screening 100 kg of mildew-free and insect-free fenugreek seeds and crushing them through a 30-mesh sieve to obtain a crushed fenugreek seed, adding 30 wt % ethanol to the crushed fenugreek seed for pre-soaking for 60 min to obtain a first mixture;

[0128] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0129] The pulse electric field treatment parameters are electric field intensity (3 kV / cm), number of pulses (200), pulse width (10 μs), liquid-to-solid ratio (1:9) and electric field treatment time of 3 min each time (two treatments).

[0130] S3, putting the second mixture into an extraction tank for extraction, the extraction process is to heat and stir and extract twice at 50°C, the first decoction is extracted with 10 times 30% ethanol solution for 1 hour, the second decoction is extracted with 8 times 30% ethanol solution for 1 hour, the extract is filtered through a 350 mesh sieve, and the supernatant is centrifuged at 4000rpm to quickly remove coarse particles and visible flocs to obtain an extract;

[0131] S4, the extract is first microfiltered through a ceramic membrane, and then ultrafiltered through an organic membrane to obtain a dialysate;

[0132] The pore size of the ceramic membrane is 500nm, the membrane pressure is lower than 0.3Bar, and the temperature is lower than 30℃. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add deionized water with the same volume of the remaining liquid to replace the dry paste.

[0133] The organic membrane ultrafiltration step uses polyvinylidene fluoride membrane elements made of food safety grade materials, with a pore size of 3KDa, a membrane pressure of 0.1-1MPa, and a temperature below 30°C. When the circulating liquid is microfiltrated to only about 10% of the original liquid volume, deionized water with the same volume as the remaining liquid is added to replace the effective ingredients.

[0134] S5, the dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0135] The organic nanofiltration membrane uses a polyamide membrane element made of food safety grade material, with a membrane pressure of 0.1 to 2 MPa and a temperature below 30°C.

[0136] S6, adding γ-cyclodextrin to the concentrated solution for inclusion treatment, adding γ-cyclodextrin for inclusion treatment according to a dry cream rate of 40%, first dissolving γ-cyclodextrin in hot water, then adding it to the concentrated solution, and inclusion treatment with a high-speed shearing machine to obtain an inclusion solution;

[0137] S7. The inclusion solution is spray-dried to obtain a fenugreek seed extract.

[0138] Comparative Example 1

[0139] This comparative example is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0140] 1 kg of non-mildew and insect-free fenugreek is selected, crushed, and put into an extraction tank. It is heated and stirred at 50°C for extraction twice. The first decoction is extracted with 10 times 30% ethanol solution for 1 hour, and the second decoction is extracted with 8 times 30% ethanol solution for 1 hour. The extract is filtered through a 350-mesh sieve, concentrated under reduced pressure at low temperature, and anhydrous ethanol is added to 5 times the volume of the concentrate for alcohol precipitation for 12 hours. The clear liquid is centrifuged at 3000 rpm to quickly remove coarse particles and visible floccules, filtered, and concentrated under reduced pressure until there is no alcohol taste. γ-cyclodextrin is added for inclusion according to a dry paste rate of 30%. γ-cyclodextrin is first dissolved in hot water and then added to the concentrate. High-speed shearing machine is used for inclusion to obtain inclusion liquid, and spray drying is performed.

[0141] Comparative Example 2

[0142] This comparative example is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0143] S1, screening 1 kg of mildew-free and insect-free fenugreek seeds and crushing them through a 30-mesh sieve to obtain a crushed fenugreek seed, adding 20 wt % ethanol to the crushed fenugreek seed for pre-soaking for 30 min to obtain a first mixture;

[0144] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0145] The pulse electric field treatment parameters are electric field intensity (1 kV / cm), number of pulses (200), pulse width (10 μs), liquid-to-solid ratio (1:8) and each electric field treatment time of 3 min.

[0146] S3, putting the second mixture into an extraction tank for extraction, the extraction process is to heat and stir and extract twice at 50°C, the first decoction is extracted with 8 times 20% ethanol solution for 1 hour, the second decoction is extracted with 6 times 20% ethanol solution for 1 hour, the extract is filtered through a 350 mesh sieve, and the supernatant is centrifuged at 3000 rpm to quickly remove coarse particles and visible flocs to obtain an extract;

[0147] S4, the extract is first microfiltered through a ceramic membrane to obtain a dialysate;

[0148] The pore size of the ceramic membrane is 800nm, the membrane pressure is lower than 0.3Bar, and the temperature is lower than 30℃. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, add deionized water with the same volume of the remaining liquid to replace the dry paste to obtain the dialysate.

[0149] S5, the dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution;

[0150] The organic nanofiltration membrane uses a polyamide membrane element made of food safety grade material, with a membrane pressure of 0.1 to 2 MPa and a temperature below 30°C.

[0151] S6. The concentrated solution is spray-dried to obtain a fenugreek seed extract.

[0152] Comparative Example 3

[0153] This embodiment is a method for preparing a fenugreek seed extract, which comprises the following steps:

[0154] S1, screening 1 kg of mildew-free and insect-free fenugreek seeds and crushing them through a 30-mesh sieve to obtain a crushed fenugreek seed, adding 20 wt % ethanol to the crushed fenugreek seed for pre-soaking for 30 min to obtain a first mixture;

[0155] S2, subjecting the first mixture to pulse electric field treatment to obtain a second mixture;

[0156] The pulse electric field treatment parameters are electric field intensity (1 kV / cm), number of pulses (200), pulse width (10 μs), liquid-to-solid ratio (1:8) and each electric field treatment time of 3 min.

[0157] S3, putting the second mixture into an extraction tank for extraction, the extraction process is to heat and stir and extract twice at 50°C, the first decoction is extracted with 8 times 20% ethanol solution for 1 hour, the second decoction is extracted with 6 times 20% ethanol solution for 1 hour, the extract is filtered through a 350 mesh sieve, and the supernatant is centrifuged at 3000 rpm to quickly remove coarse particles and visible flocs to obtain an extract;

[0158] S4, the extract is microfiltered through a ceramic membrane to obtain a dialysate;

[0159] The pore size of the ceramic membrane is 100 nm, the membrane pressure is lower than 0.3 Bar, and the temperature is lower than 30°C. When the circulating liquid is microfiltered to only about 10% of the original liquid volume, the dialysate is collected.

[0160] S5, after the dialysate is concentrated under reduced pressure, γ-cyclodextrin is added to the concentrated solution for inclusion treatment, and γ-cyclodextrin is added for inclusion treatment according to a dry cream rate of 30%, γ-cyclodextrin is first dissolved in hot water, and then added to the concentrated solution, and inclusion treatment is performed by a high-speed shearing machine to obtain an inclusion solution;

[0161] S6. The inclusion solution is spray-dried to obtain a fenugreek seed extract.

[0162] The 4-hydroxyisoleucine content, transfer rate, stability and properties of the fenugreek seed extracts obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were tested respectively, and the specific results are as follows:

[0163] 1. Trait comparison

[0164] Take about 10g of the sample and spread it flat on a clean white porcelain plate. Under natural light, observe its color with the naked eye, smell its fragrance, put a small amount on the tip of the tongue, spread it all over your mouth, and taste it carefully. Take 1.5g of the sample and put it in 100ml of hot water to observe its solubility.

[0165]

[0166] From the results, it can be seen that the fenugreek seed extract prepared by the methods of Examples 1 to 3 has a fenugreek aroma, a slightly bitter taste, good fluidity, good solubility, and a bright color, which is better than Comparative Examples 1 to 3. Examples 1 to 3 use a cyclodextrin inclusion process to reduce bitterness, which has obvious effects and is easy to operate.

[0167] 2. 4-Hydroxyisoleucine content

[0168] 2.1 Sample 4-hydroxyisoleucine determination

[0169] 2.1.1 Chromatographic conditions

[0170] Use Kromasil 100-5C18 (250mm×4.6μm, 5μm) as the chromatographic column, acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, and perform gradient elution according to the table below; the flow rate is 1.5ml per minute, the column temperature is 30°C, the detection wavelength is 254nm, and the injection volume is 20μl.

[0171] Table 1 Gradient elution table

[0172]

[0173] 2.1.2 Preparation of reference solution

[0174] Weigh 3.88 mg of 4-hydroxyisoleucine reference substance, accurately weigh, place in a 5 ml volumetric flask, add 50% methanol to dissolve and dilute to scale. Accurately measure 4 ml of the above reference substance to a 50 ml volumetric flask, add 5 ml of acetonitrile: water: triethylamine (40:12:8), add 250 μl of phenyl isothiocyanate (PITC), shake for 5 minutes, add 30 ml of methanol, add water to scale, shake well, filter, and obtain the reference substance solution.

[0175] 2.1.3 Sample determination

[0176] Weigh about 0.1g of fenugreek powder sample, accurately weigh it, place it in a 25ml volumetric flask, add 20ml of 50% methanol solution, ultrasonicate for 25 minutes, let it cool to room temperature, add 50% methanol to the scale, shake well, filter, take 4ml of the filtrate, place it in a 50ml volumetric flask, add 5ml of acetonitrile: water: triethylamine (40:12:8), add 250μl of phenyl isothiocyanate (PITC), shake for 5 minutes, add 30ml of methanol, add water to the scale, shake well, filter, and obtain.

[0177] Weigh about 0.1 g of fenugreek liquid sample, accurately weigh it, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile: water: triethylamine (40:12:8), add 250 μl of phenyl isothiocyanate (PITC), shake for 5 minutes, add 30 ml of methanol, and add water to the scale, shake well, filter, and obtain.

[0178] 2.2 Measurement results

[0179] 2.2.1 The 4-hydroxyisoleucine content of the fenugreek seed raw material selected in this embodiment is 1.1%.

[0180] 2.2.2 Results of content determination in examples and comparative examples

[0181]

[0182]

[0183] From the results, it can be seen that the content of 4-hydroxyisoleucine in the fenugreek seed extract prepared by the methods of Examples 1 to 3 can reach up to 13.56%, and the transfer rate can reach up to 93.10%;

[0184] The content of 4-hydroxyisoleucine in the fenugreek seed extract prepared by the method of Comparative Example 1 was 4.27%, the transfer rate was 21.35%, and the average finished product yield was 5.5%, which were much lower than those of the method of this embodiment; the content of 4-hydroxyisoleucine in the fenugreek seed extract prepared by the method of Comparative Example 2 was 8.92%, the transfer rate was 63.25%, and the average finished product yield was 7.8%, indicating that the pulsed electric field treatment can significantly increase the content of 4-hydroxyisoleucine; the content of 4-hydroxyisoleucine in the fenugreek seed extract prepared by the method of Comparative Example 3 was 7.75%, and the transfer rate was 47.91%, indicating that the membrane integration process can increase the content of 4-hydroxyisoleucine, a heat-sensitive substance.

[0185] In summary, pulsed electric field treatment PEF assisted low-temperature extraction increased the permeability of fenugreek seed cell walls and accelerated the dissolution of target components. At the same time, the combination of ceramic membrane filtration and organic membrane ultrafiltration can effectively improve the transfer rate and finished product yield of 4-hydroxyisoleucine. Therefore, the fenugreek seed extract prepared by the method of this embodiment has a high 4-hydroxyisoleucine content, a high transfer rate, a high finished product yield, and is more environmentally friendly.

[0186] 3. Stability inspection

[0187] The samples were placed in a constant temperature and humidity chamber at 40°C ± 2°C for a 3-month accelerated stability test. The 4-hydroxyisoleucine content was used as the evaluation index. The results of the accelerated stability test are shown in the table below.

[0188] project 0 months 1 month 2 months 3 months Example 1 11.93％ 11.65％ 11.38％ 10.89％ Example 2 13.56％ 13.29％ 13.01％ 12.87％ Example 3 13.30％ 13.03％ 12.73％ 12.66％ Comparative Example 1 4.27％ 4.02％ 3.85％ 3.75％ Comparative Example 2 8.92％ 8.33％ 7.81％ 7.05％ Comparative Example 3 7.75％ 7.51％ 7.35％ 7.24％

[0189] From the results of the accelerated stability test, it can be seen that after the fenugreek seed extract in Example 2 was included in the cyclodextrin, the 4-hydroxyisoleucine content decreased by only 4.8% after 3 months of acceleration, and the 4-hydroxyisoleucine content in Comparative Example 2 decreased by 21.0% after 3 months of acceleration. The extract included in the cyclodextrin had good stability and was not easily degraded.

[0190] In summary, the present invention utilizes pulsed electric field treatment to assist low-temperature extraction, which can significantly improve the dissolution of the target component, prevent the thermal decomposition of the heat-sensitive substance 4-hydroxyisoleucine due to excessive heating, and further purify and separate the target component by combining integrated membrane technology. At the same time, cyclodextrin, which is hydrophilic on the outside and hydrophobic on the inside, is used to encapsulate 4-hydroxyisoleucine, thereby improving the stability and flavor of the extract.

[0191] In the description of this specification, the description with reference to the terms "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0192] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for preparing a fenugreek seed extract, characterized in that: The following steps are involved: Taking fenugreek seeds and crushing them to obtain crushed fenugreek seeds, adding ethanol to the crushed fenugreek seeds for pre-soaking to obtain a first mixture; The first mixture is subjected to a pulse electric field treatment to obtain a second mixture; Putting the second mixture into an extraction tank for extraction to obtain an extract; The extract is first microfiltered through a ceramic membrane and then ultrafiltered through an organic membrane to obtain a dialysate; The dialysate is concentrated by an organic nanofiltration membrane to obtain a concentrated solution; adding γ-cyclodextrin to the concentrated solution for inclusion treatment to obtain an inclusion solution; The inclusion liquid is spray-dried to obtain the fenugreek seed extract.

2. The method for preparing the fenugreek seed extract according to claim 1, wherein Fenugreek seeds are taken and crushed to obtain fenugreek seed crushed material, and the fenugreek seed crushed material is sieved through a 30-80 mesh sieve and then pre-soaked in 20 wt % to 50 wt % ethanol for 30 min to 60 min to obtain a first mixture.

3. The method for preparing the fenugreek seed extract according to claim 1, wherein The parameters of the pulse electric field treatment include: the electric field strength is 0.1 kV / cm to 10 kV / cm; The number of pulses is 125 to 2500; Pulse width is 0.5μs~10μs; The liquid-to-solid ratio is 1:(8-15); The single electric field treatment time is 3min to 6min; The pulse frequency is fixed at 10Hz.

4. The method for preparing the fenugreek seed extract according to claim 1, wherein The step of putting the second mixture into an extraction tank for extraction is achieved by using the following method: The second mixture is put into an extraction tank, heated to 30°C to 50°C, stirred, and the first extraction and the second extraction are performed in sequence; The first extraction uses 8-10 times ethanol solution to extract for 1h-2h; The second extraction uses 6-8 times ethanol solution for 1h-2h.

5. The method for preparing the fenugreek seed extract according to claim 1, wherein The microfiltration through the ceramic membrane is achieved by the following method: Microfiltration is performed using food safety grade ceramic membranes. During the microfiltration process, the membrane pressure is lower than 0.3 Bar, the cooling water is circulated, and the liquid circulation temperature is lower than 30°C. The ceramic film is one or more of a zirconium oxide film, an aluminum oxide film and a silicon nitride film; The pore size of the ceramic membrane is 500nm-800nm.

6. The method for preparing the fenugreek seed extract according to claim 1, wherein The ultrafiltration through the organic membrane is achieved by the following method: Use food safety grade organic membrane for ultrafiltration. During the ultrafiltration process, the membrane pressure is 0.1MPa~2MPa, cooling water is circulated, and the liquid circulation temperature is lower than 30℃; The organic membrane is one or more of a polyvinylidene fluoride membrane, a polysulfone membrane, and a polyamide membrane; The pore size of the organic membrane is 3KDa to 300KDa.

7. The method for preparing the fenugreek seed extract according to claim 1, wherein The concentration by organic nanofiltration membrane is achieved by the following method: A food safety grade polyamide nanofiltration membrane is used for concentration. During the concentration process, the membrane pressure is 0.1MPa to 2MPa, and the cooling water is circulated for cooling. The circulating temperature of the liquid medicine is lower than 30°C. The pore size of the organic nanofiltration membrane is 200Da to 300Da.

8. The method for preparing the fenugreek seed extract according to claim 1, wherein The following method is used to implement the inclusion treatment by adding γ-cyclodextrin to the concentrated solution: Firstly, γ-cyclodextrin is dissolved in hot water to obtain a cyclodextrin mixture, and then the cyclodextrin mixture is added to the concentrated solution, stirred evenly, and then inclusion treatment is performed using a high-speed shearing machine or an inclusion machine.

9. The method for preparing the fenugreek seed extract according to claim 8, wherein: Add γ-cyclodextrin according to 20% to 40% of the dry paste; The shearing speed of the high-speed shearing machine is not less than 8000rpm / min, and the shearing time is 10min to 20min; The cycle grinding time of the encapsulating machine is not less than 5 minutes.

10. A fenugreek seed extract, characterized in that The fenugreek seed extract is prepared by the preparation method of the fenugreek seed extract according to any one of claims 1 to 9.

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