Extracts enriched in garlic characteristic sulfur amino acids and methods of preparation

Abstract

The present application provides an extract rich in multiple garlic characteristic sulfur-containing amino acids and a preparation method thereof, the method separates and concentrates polysaccharides, characteristic sulfur-containing amino acids and oligosaccharides in garlic through multi-stage membrane filtration, obtains the extract rich in multiple garlic characteristic sulfur-containing amino acids, and is simple in operation, friendly to the environment, rich in product, and high in yield.

Description

Technical Field

[0001] This invention belongs to the field of natural substance separation and extraction, and more specifically, relates to an extract rich in sulfur-containing amino acids characteristic of garlic and its preparation method. Background Technology

[0002] In recent years, many scholars at home and abroad have devoted themselves to the study of garlic, confirming that garlic has the effects of preventing and treating cardiovascular diseases, anti-tumor, antibacterial and anti-inflammatory, and long-term consumption can prevent and treat diseases.

[0003] Garlic is rich in beneficial substances, including various amino acids and oligosaccharides. The main sulfur-containing amino acids in garlic include S-allyl-L-cysteine ​​sulfoxide (alliin), which contains a sulfoxide group, as well as a variety of other sulfur-containing amino acids. Different characteristic sulfur-containing amino acids in garlic have their own unique functions, such as antioxidant activity.

[0004] The main oligosaccharide component in garlic is fructo-oligosaccharide (FOS), also known as oligofructose or sucrose trisaccharide oligosaccharide. It is a fructose polymer composed of fructose monomers linked by β-glycosidic bonds that is not hydrolyzed by endogenous enzymes. FOS has been recognized as a safe substance by the US FDA and has become a widely commercialized prebiotic. Studies have shown that FOS can promote the growth of beneficial bacteria (such as Bifidobacterium and Lactobacillus), regulate the immune system and thus improve immunity, improve lipid metabolism, lower cholesterol and blood sugar levels, promote the absorption of minerals, and regulate the gut microbiota.

[0005] In my country, garlic sales are mainly based on fresh garlic, with a lack of deep-processed garlic products. This is likely due to the low content of active ingredients in garlic and the difficulty in extraction. Currently, there are three main methods for obtaining the unique active ingredients of garlic: chemical synthesis, cell culture, and natural extraction and purification. The first two methods can only yield single sulfur-containing amino acids (such as allicin).

[0006] In addition, natural extraction and purification methods mainly involve water extraction or water-organic solvent mixture extraction followed by purification using alcohol precipitation, ultrafiltration, or ion exchange. These methods yield relatively simple amino acid products; and many amino acids are lost during the extraction process, resulting in low extraction content. Furthermore, the use of organic solvents can easily cause environmental pollution.

[0007] Therefore, there is an urgent need in this field to develop a preparation process for an extract rich in various sulfur-containing amino acids characteristic of garlic, so as to efficiently extract active substances from garlic. Summary of the Invention

[0008] The purpose of this invention is to provide an extract rich in various sulfur-containing amino acids characteristic of garlic and its preparation method.

[0009] In a first aspect, the present invention provides a method for preparing an extract rich in various sulfur-containing amino acids characteristic of garlic, comprising the steps of:

[0010] (a) Garlic is subjected to enzyme inactivation treatment to obtain enzyme-inactivated garlic cloves;

[0011] (b) After grinding the enzyme-inactivated garlic cloves into a homogenate, centrifuge to remove the residue and obtain the centrifuged liquid;

[0012] (c) The centrifuged liquid is filtered through a 40-60nm ceramic membrane to obtain the first filtrate;

[0013] (d) The first filtrate is ultrafiltered through a 900-1100 Da molecular weight membrane to obtain the second filtrate and the first chopped liquid;

[0014] (e) The second filtrate is ultrafiltered through a 550-650 Da molecular weight membrane to obtain the third filtrate and the second chopped liquid;

[0015] (f) The third filtrate is concentrated by an 80-150 Da molecular weight filter membrane to obtain the third retentate, which is an extract rich in various sulfur-containing amino acids characteristic of garlic.

[0016] In another preferred embodiment, the extract comprises:

[0017] Allicin, methyl allicin, isoalliin, γ-glutamyl-S-allyl-L-cysteine, γ-glutamyl-S-propenyl-L-cysteine, γ-glutamyl-S-methyl-L-cysteine, S-allyl-L-cysteine, and S-methyl-L-cysteine, aspartic acid, arginine, threonine, alanine, proline, phenylalanine, tryptophan, lysine, and other amino acids and oligosaccharides.

[0018] In another preferred embodiment, the first and second throttling liquids are freeze-dried to obtain garlic polysaccharide freeze-dried powder.

[0019] In another preferred embodiment, the third throttling liquid is freeze-dried to obtain a freeze-dried powder of an extract rich in a variety of sulfur-containing amino acids characteristic of garlic.

[0020] In another preferred embodiment, the total content of garlic polysaccharides in the freeze-dried garlic polysaccharide powder is greater than 92 wt% (on dry weight).

[0021] In another preferred embodiment, the alliin content of the method is accurately determined using immunochromatography.

[0022] In another preferred embodiment, the final yield of allicin by the method is greater than or equal to 75%, preferably 75%-90%.

[0023] In another preferred embodiment, the method concentrates alliin by a factor of 5 or greater, preferably 5-15, and more preferably 5-10.

[0024] In another preferred embodiment, the method concentrates methylalliin to a factor of 6 or greater, preferably 6-15, and more preferably 6-10.

[0025] In another preferred embodiment, the method concentrates S-allyl-L-cysteine ​​by a factor of 10 or greater, preferably 10-20, and more preferably 10-15.

[0026] In another preferred embodiment, the method concentrates tryptophan by a factor of 6 or greater, preferably 6-15, and more preferably 6-10.

[0027] In another preferred embodiment, the enzyme inactivation method is selected from the group consisting of: microwave inactivation, boiling inactivation, or a combination thereof.

[0028] In another preferred embodiment, the method results in a water loss rate of 20%-26% for garlic cloves after enzyme inactivation treatment.

[0029] In another preferred embodiment, the method retains at least 97% alliin after enzyme inactivation treatment.

[0030] In another preferred embodiment, the microwave enzyme inactivation time of the method is 5-15 minutes.

[0031] In another preferred embodiment, during the homogenization process of garlic cloves, the mass ratio of garlic cloves to water is 1:5-1:7, preferably 1:6.

[0032] In another preferred embodiment, freeze drying includes a freezing process and a drying process, wherein the temperature of the drying process is less than or equal to 30°C.

[0033] In another preferred embodiment, the method can be used for large-scale production of garlic active substances.

[0034] In another preferred embodiment, garlic polysaccharides and extracts rich in various sulfur-containing amino acids characteristic of garlic can be used in the preparation of garlic preparations and health products.

[0035] In another preferred embodiment, the garlic preparation and health product include garlic polysaccharide compound prebiotic solid beverage, Shuangjin tablets, and Shuangshen tablets.

[0036] A second aspect of the present invention provides an extract rich in a variety of sulfur-containing amino acids characteristic of garlic, the extract comprising:

[0037] Allicin, methyl allicin, isoalliin, γ-glutamyl-S-allyl-L-cysteine, γ-glutamyl-S-propenyl-L-cysteine, γ-glutamyl-S-methyl-L-cysteine, S-allyl-L-cysteine, and S-methyl-L-cysteine.

[0038] In another preferred embodiment, the extract further comprises amino acids such as aspartic acid, arginine, threonine, alanine, proline, phenylalanine, tryptophan, and lysine, as well as oligosaccharides.

[0039] In another preferred embodiment, the oligosaccharide comprises:

[0040] Fructosaccharides, fructosaccharides, fructosaccharides.

[0041] In another preferred embodiment, the extract rich in various sulfur-containing amino acids characteristic of garlic has a total sulfur-containing amino acid content greater than 30 wt% (on dry weight), preferably 30-50 wt%, more preferably 30-40 wt%.

[0042] In another preferred embodiment, the extract rich in various sulfur-containing amino acids characteristic of garlic has a total essential amino acid content of more than 30 wt% (on dry weight), preferably 30-50 wt%, more preferably 30-40 wt%.

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0044] Figure 1 The diagram shows a process flow chart in one embodiment of the present invention: the pore size of membrane A is 40-60 nm, and the molecular weights of membranes B, C and D are 900-1100 Da, 550-650 Da and 80-150 Da, respectively.

[0045] Figure 2 The detection principle of background fluorescence quenching-competitive chromatography in one embodiment of the present invention is shown.

[0046] Figure 3 A schematic diagram of an immunochromatographic plate according to one embodiment of the present invention is shown.

[0047] Figure 4 A series of alliin concentration standard curves are shown in one embodiment of the present invention. Detailed Implementation

[0048] Through extensive and in-depth research and screening, the inventors have developed for the first time a process for preparing garlic extracts rich in characteristic sulfur-containing amino acids of garlic. This method efficiently extracts garlic extracts rich in numerous different characteristic sulfur-containing amino acids of garlic (especially rare functional ones), achieving a total content of up to approximately 32% characteristic sulfur-containing amino acids. Furthermore, this method is energy-efficient, yield-efficient, and environmentally friendly, requiring no addition of organic solvents (such as ethanol or methanol). This invention is based on these findings.

[0049] Specifically, the method of this invention safely and environmentally separates components of different molecular weights (including polysaccharides, amino acids, and oligosaccharides) in garlic through multi-stage membrane filtration, and selectively and efficiently concentrates and enriches the characteristic sulfur-containing amino acids of garlic, thereby obtaining an extract rich in the characteristic sulfur-containing amino acids of garlic. The method of this invention exhibits high yields or enrichment efficiencies for the extraction of various characteristic sulfur-containing amino acids of garlic, further facilitating the utilization of beneficial substances in garlic. Furthermore, this invention also provides for the first time a precise method for determining the alliin content in the extract of this invention, namely, quantitative determination of alliin using immunochromatography with specific antibodies.

[0050] the term

[0051] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0052] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0053] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0054] sulfur-containing amino acids

[0055] The characteristic sulfur-containing amino acids in garlic involved in this invention mainly include S-allyl-L-cysteine ​​sulfoxide (alliin), S-methyl-L-cysteine ​​sulfoxide (methylalliin), and S-propenyl-L-cysteine ​​sulfoxide (isoalliin), which contain sulfoxide groups. These substances can react with alliinase to produce thiosulfinates with anticancer, antifungal, and antitumor biological activities. In addition, it also contains characteristic sulfides without sulfoxide groups, such as γ-glutamyl-S-allyl-L-cysteine ​​(GSAC), γ-glutamyl-S-propenyl-L-cysteine ​​(GSPC), γ-glutamyl-S-methyl-L-cysteine ​​(GSMC), S-allyl-L-cysteine ​​(SAC), S-propenyl-L-cysteine ​​(SPC), and S-methyl-L-cysteine ​​(SMC), all of which have certain antioxidant activities.

[0056] Fructooligosaccharides

[0057] Functional oligosaccharides with α-D-glucose groups linked by β-glycosidic bonds and a degree of polymerization of 3-9, or without α-D-glucose groups and a degree of polymerization of 2-9.

[0058] Degree of aggregation

[0059] The sum of the number of fructose groups and glucose groups in the fructooligosaccharide molecule chain.

[0060] Background fluorescence quenching - competitive immunochromatography

[0061] Background fluorescence quenching-immunochromatography (bFQICA) is an analytical technique built upon colloidal gold immunochromatography (GICA). The detection method depends on the structure of the target analyte, typically employing either a sandwich method or a competitive method. The competitive method is suitable for detecting small molecules. Allicin is a small molecule; due to its small molecular weight and single antigenic determinant, it can only be detected using the competitive method. This method allows for rapid interpretation of positive or negative results by observing the color intensity of the T-line with the naked eye, and also enables quantitative detection by measuring the relative intensity of the background fluorescence and the fluorescence of the detection line (T-line). Figure 2 As shown in the figure. Furthermore, since it detects relative fluorescence intensity, interference from factors such as protein conjugates in the sample is significantly suppressed, resulting in a marked improvement in the sensitivity of quantitative detection. It also avoids interference from impurities in the sample, offering advantages such as strong anti-interference capability, accurate measurement results, high sensitivity, wide linear range, short detection time, simple operation, and good precision.

[0062] Complete antigen

[0063] Typically, haptens need to be covalently coupled with macromolecules such as KLH (hemocyanin) or BSA (bovine serum albumin) to become complete antigens that are both immunoreactive and immunogenic.

[0064] As used herein, the term "complete antigen" refers to the product of the hapten of the present invention bound to a suitable protein carrier.

[0065] As used herein, "protein carrier" in this invention refers to any immunologically acceptable protein for forming a complete antigen, such as hemocyanin, bovine serum albumin, etc.

[0066] The complete antigen (such as allicin-KLH) prepared by this invention has excellent immunogenicity. In one embodiment, it can stimulate a strong immune response in New Zealand white rabbits. After one basic immunization and three booster immunizations, the antiserum titer can reach 1:32K. The complete antigen allicin-KLH retains the immunoreactivity of allicin very well.

[0067] In a preferred embodiment of the present invention, allicin-BSA complete antigen is used on the T line of the immunochromatographic slide, and a specific antibody against allicin prepared based on allicin-KLH complete antigen is used, thereby significantly improving the specificity of antigen-antibody binding.

[0068] Allicin antibody

[0069] New Zealand white rabbits were immunized with the alliin-KLH complete antigen, resulting in the production of specific monoclonal antibodies against alliin.

[0070] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0071] The amino acid sequence of the allicin antibody described in this invention is as follows:

[0072] Antibody 1A10 sequence

[0073]

[0074]

[0075]

[0076] Immune probes

[0077] Immunological probes can bind to antigens or antibodies and achieve qualitative or quantitative analysis of target substances through different signal detection methods.

[0078] The immunoprobe described in this invention is a colloidal gold enhancer-allicin antibody, which binds to the allicin complete antigen on an immunochromatographic slide. The allicin sample is then qualitatively or quantitatively analyzed by detecting the fluorescence signal.

[0079] Colloidal gold enhancers are core-shell structured composite nanomaterials prepared by attaching a silver shell to colloidal gold as the base material and then mixing it with small molecule compounds. They have a strong molar absorption coefficient and can be used as tracers in immunochromatography.

[0080] The immune probe described in this invention is prepared from allicin antibody and colloidal gold enhancer in a certain ratio.

[0081] Colloidal gold

[0082] Colloidal gold is produced by polymerizing chloroauric acid into gold particles of a specific size under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, and tannic acid. Due to electrostatic interaction, it becomes a stable colloidal state. Colloidal gold carries a negative charge in a weakly alkaline environment and can form a strong bond with the positively charged groups of protein molecules. Since this bond is an electrostatic bond, it does not affect the biological characteristics of the protein.

[0083] Immunochromatographic slides

[0084] The immunochromatographic slide of the present invention includes a substrate plate and a sample pad, a fluorescent membrane, and absorbent paper located on the substrate plate. The fluorescent membrane is provided with a detection line (T line) and a control line (C line), wherein the T line is 0.5 mg / mL allicin-BSA protein, and the C line is 0.5 mg / mL goat anti-rabbit IgG antibody.

[0085] Compared with the prior art, the advantages of the present invention are as follows:

[0086] 1. It consumes little energy and is environmentally friendly, as no organic solvents or other polluting reagents are added during the extraction process;

[0087] 2. The garlic solution was subjected to multi-stage filtration and concentration. The final extract was rich in various sulfur-containing amino acids, with a total content of up to about 32%, of which allicin yield was up to about 76%.

[0088] 3. The final extract is freeze-dried using a low-temperature freeze-drying method, which greatly preserves the characteristic sulfur-containing amino acids and oligosaccharides and other active substances in the extract;

[0089] 4. This invention is the first to apply background fluorescence quenching-immunochromatography to the detection of allicin. This method has high sensitivity and fast detection speed.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0091] Example 1. Investigation of various parameters during the extraction of active substances from garlic

[0092] 1. Transmission speed of enzyme inactivation unit

[0093] The main process parameters for microwave enzyme inactivation are power and the conveyor belt speed. To improve efficiency and save costs, the microwave power was set to the maximum power of the unit. The conveyor belt speed was then examined. Since the machine's conveyor speed range is 0-900 r / min, the microwave enzyme inactivation performance at speeds of 0-900 r / min was investigated. Initially, a speed of 550 r / min was tested. The results showed that the conveyor speed was too fast, the contact time was short, the generated temperature was insufficient, and the garlic cloves were too hard, failing to achieve the desired enzyme inactivation effect. Therefore, the conveyor speed was later reduced to the range of 200-400 r / min.

[0094] The conveyor belt has a size of 11.0m * 0.6m. For each test rotation speed, the amount of garlic used is 20kg, and the thickness of the garlic layer is 1.5cm to 2.5cm.

[0095] 350r / min

[0096] Turn on the tunnel microwave sterilizer and set the conveyor belt speed to 350 rpm. Weigh 20.0 kg of fresh garlic, spread it evenly on a towel, and then put the garlic cloves into the sterilization chamber. Turn on all microwave transformers and magnetrons. After discharge, the garlic cloves feel firm to the touch and have a spicy taste. Weigh and record the weight of the garlic cloves after enzyme inactivation, take samples, and test the alliin content.

[0097] 300r / min

[0098] Turn on the tunnel microwave sterilizer and set the conveyor belt speed to 300 rpm. Weigh 20.0 kg of fresh garlic, spread it evenly on a towel, and then put the garlic cloves into the sterilization chamber. Turn on all microwave transformers and magnetrons. Observe the state of the material after enzyme inactivation, weigh and record the weight of the garlic cloves after enzyme inactivation, take samples, and test the alliin content.

[0099] 250r / min

[0100] Turn on the tunnel microwave sterilizer and set the conveyor belt speed to 250 r / min. Weigh 20.0 kg of fresh garlic, spread it evenly on a towel, and then put the garlic cloves into the sterilization chamber. Turn on all microwave transformers and magnetrons. Observe the state of the material after enzyme inactivation, weigh and record the weight of the garlic cloves after enzyme inactivation, take samples, and test the alliin content.

[0101] 200r / min

[0102] Turn on the tunnel microwave sterilizer and set the conveyor belt speed to 200 rpm. Weigh 20.0 kg of fresh garlic, spread it evenly on a towel, and then put the garlic cloves into the sterilization chamber. Turn on all microwave transformers and magnetrons. Observe the state of the material after enzyme inactivation, weigh and record the weight of the garlic cloves after enzyme inactivation, take samples, and test the alliin content.

[0103] Table 1. Microwave enzyme inactivation effect of fresh garlic at different conveying speeds

[0104]

[0105]

[0106] Experimental results show that when the conveying speed is set at 250 r / min, the garlic cloves are very soft, have no spiciness, and the allicin content is 0.00%, resulting in the best enzyme inactivation effect and the lowest alliin loss rate. Therefore, the microwave enzyme inactivation process parameters can be set as follows: power 39KW, conveying speed 250 r / min.

[0107] 2. Slurry liquid-to-material ratio

[0108] A colloid mill homogenizer was used in the production department. Garlic cloves were added in water at mass ratios of 1:3, 1:4, and 1:5, with a homogenization time of 2 minutes. After homogenization, the homogenate was pumped out, and the mill was rinsed with twice the volume of water (determined based on previous experiments). The rinsing liquid was then combined with the homogenate. The homogenate was centrifuged at 16,000 rpm using a tubular centrifuge. The supernatant and garlic residue were collected to determine the alliin content.

[0109] Table 2 Results of the study on the ratio of homogenized slurry to liquid content

[0110]

[0111] Experimental results showed that the yield of the homogenization step was greater than 98% for different ratios of garlic cloves to water, but it affected the efficiency of centrifugation. When the ratio of garlic cloves to water was 1:6, the residual amount of alliin in the garlic residue was small, which was consistent with the previous experimental results. Considering that there was residue in the pipe after homogenization, it was necessary to rinse with water. The subsequent homogenization process was set at a mass ratio of fresh garlic to water of 1:4. After homogenization, water with a mass of 2 times that of fresh garlic was added to backflush the pipe. The backflushing liquid and the homogenizing liquid were combined, mixed, and then centrifuged.

[0112] 3. Centrifuge feed pump frequency

[0113] When the tubular centrifuge speed is 16000 rpm, the feed pump frequency is adjusted to 15Hz, 20Hz and 25Hz respectively. When the feed pump frequency is 15Hz, the supernatant can pass through the 120-mesh sieve. When the feed pump frequency is 20Hz and 25Hz, the supernatant cannot pass through the 120-mesh sieve. 15Hz is selected as the feed frequency in the future.

[0114] Example 2. Extraction of active substances from garlic

[0115] 1.42 kg of garlic bulbs were separated into cloves, peeled, washed, and dried to obtain 37.8 kg of peeled garlic cloves. The alliin content was determined to be 1.37 wt% and the moisture content to be 62.8% by background fluorescence quenching-competitive immunochromatography.

[0116] 2. Microwave enzyme inactivation: The conveyor belt rotates at 250 r / min, yielding 29.11 kg of enzyme-inactivated garlic cloves.

[0117] 3. Homogenization with colloid mill: Add 116.44 kg of water to the garlic after enzyme inactivation, homogenize, and then add 58.22 kg of water to rinse the pipe. Combine the rinsing liquid with the homogenate (the mass ratio of garlic cloves to water is 1:6) to obtain a total of 202.77 kg of homogenate.

[0118] 4. Centrifugation for residue removal: The tubular centrifuge operates at 16,000 rpm, and the feed pump frequency is 15 Hz. 202.77 kg of homogenate is pumped into the tubular centrifuge, and the centrifugation time is 40 minutes. The entire homogenate is centrifuged to obtain 192 kg of supernatant and 11.65 kg of garlic residue. Both the centrifuged supernatant and the garlic residue can pass through a 120-mesh sieve, which meets the requirements of ceramic membrane filtration.

[0119] 5. Ceramic membrane (50nm) filtration: The supernatant from centrifugation was continuously added to the ceramic membrane material tank for filtration. The working pressure was 2.4 MPa, the temperature was 15-25℃, and the filtration time was 2 hours. The final weight of the retentate was 12.8 kg, and the weight of the permeate was 178.6 kg.

[0120] 6. Ultrafiltration (1000 Da): 178.6 kg of permeate was continuously ultrafiltered through a ceramic membrane at a working pressure of 0.4 MPa, a temperature of 15-25℃, and an ultrafiltration time of 2 hours and 10 minutes. The final ultrafiltration yielded 10.90 kg of retentate and 167.2 kg of permeate. The permeate and retentate were collected separately. The retentate was freeze-dried to obtain garlic polysaccharide extract, and the permeate was used for further extraction of sulfur-containing amino acids.

[0121] 7. Nanofiltration membrane (600Da): Add 162.7kg of ultrafiltration permeate to the nanofiltration membrane material tank for nanofiltration. The working pressure is 1.6Mpa, the temperature is 15-25℃, and the nanofiltration time is 1 hour. The resulting retentate is 13.8kg and the permeate is 148.2kg.

[0122] 8. Concentrating membrane (100Da): Add 148.2kg of permeate from a 600Da nanofiltration membrane to the concentrating membrane material tank. The working pressure is 1.6Mpa, the temperature is 15-25℃, and the concentration time is 1 hour and 50 minutes to obtain 27.65kg of retentate.

[0123] 9. Freeze-drying

[0124] Table 3 Freeze-drying operation and process parameters

[0125]

[0126] (1) 24.7 kg of ultrafiltration and nanofiltration retentate was freeze-dried using a freeze dryer according to the freeze-drying operation and process parameters in Table 1 to obtain 2.72 kg of garlic polysaccharide freeze-dried powder.

[0127] (2) 27.65 kg of concentrated membrane retentate was freeze-dried using a freeze dryer according to the freeze-drying operation and process parameters in Table 1 to obtain 2.00 kg of freeze-dried powder of extract rich in sulfur-containing amino acids characteristic of garlic.

[0128] Example 3. Determination of the yield and content of active substances in garlic

[0129] 1. The yield of alliin in each extraction step was determined by background fluorescence quenching-competitive immunochromatography.

[0130] Table 4 Reagents

[0131]

[0132] Preparation of BSA-Allicin

[0133] (1) Dialyze / dissolve 1 mg of BSA protein into 1 ml of 25 mM 2-(N-morpholine) (MES), and add 1 mg of allicin small molecules, mix well and dissolve;

[0134] (2) Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 1 ml of the mixture from the previous step;

[0135] (3) Add 0.16 ml of 25 mM, pH 5 MES to the mixture from the previous step;

[0136] (4) Mix and incubate at 4℃ for 2 hours;

[0137] (5) Dialyze at 4℃ three times, 2 hours each time, to remove unbound small molecules in a free state.

[0138] Preparation of antibody 1A10

[0139] (1) Preparation of alliin-KLH complete antigen

[0140] Dialyze / dissolve 1 mg of KLH protein in 1 ml of 25 mM 2-(N-morpholino)(MES) ethanesulfonic acid, and add 1 mg of allicin small molecules, mix well and dissolve.

[0141] Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 1 ml of the mixture from the previous step;

[0142] Add 0.16 ml of 25 mM MES (pH = 5) to the mixture from the previous step;

[0143] Mix and incubate at 4℃ for 2 hours;

[0144] Dialysis was performed three times at 4℃ for 2 hours each time to remove unbound alliin molecules in a free state.

[0145] (2) Animal Immunization

[0146] Allicin-KLH complete antigen was injected into rabbits for immunization. After immunization, peripheral blood B cells were collected, and positive B cells were screened by flow cytometry and antigen specificity.

[0147] (3) Cloning

[0148] The selected positive B cells were cultured for 15 days, and the supernatant was used for indirect ELISA. Clones with high positive values ​​(OD values) were selected, and RNA was extracted from their B cells. The RNA was then reverse transcribed and amplified to obtain the heavy and light chain sequences, which were then sequenced to obtain the monoclonal antibody sequence (see Table 2, antibody 1A10). Subsequently, the target genes of the monoclonal antibody (coding sequences of VH and VL) were transfected (transiently transfected) into HEK293 cells (human embryonic kidney 239 cells) for protein expression. The antibody was then purified and collected.

[0149] Preparation of immunochromatographic slides

[0150] like Figure 3 As shown, a YNHS-120B fluorescent membrane was attached to a PVC substrate. The C line was scratched with 0.5 mg / mL goat anti-rabbit IgG, and the T line was scratched with 0.5 mg / mL BSA-alliin. A 21 mm long T1 sample pad was placed at one end of the membrane strip, and an 18 mm long absorbent paper was placed at the other end.

[0151] Detection method:

[0152] The sample to be tested was combined with an immunoprobe solution containing allicin antibody to form a mixture. A certain amount of the mixture was spotted onto an immunochromatographic membrane strip containing BSA-allicin complete antigen. After chromatographic analysis at room temperature for 15 minutes, the F1 / F2 ratio (F1 is the fluorescence intensity between the C line and the T line, and F2 is the fluorescence intensity of the T line) was measured using a background fluorescence-immunoassay analyzer.

[0153] (1) Preparation of standard curves

[0154] Take 70 μl of a series of alliin reference solutions (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μg / ml) at 1 μg / ml, add 2 μl of immunoprobe solution, mix well, and spot 50 μl onto the sample pad. Chromatography at room temperature for 15 min. Measure the F1 / F2 ratio using a background fluorescence-immunoassay analyzer (Table 5) (F1 is the fluorescence intensity between the C and T lines, and F2 is the fluorescence intensity of the T line). Plot alliin concentration on the x-axis and F1 / F2 on the y-axis, and fit using a Logistic model to obtain the standard curve as shown below. Figure 4 As shown, r = 0.9994, indicating that F1 / F2 has a good correlation with allicin concentration in the range of 0.1 to 1.0 μg / ml.

[0155] Table 5. F1 / F2 values ​​of alliin reference standards at various concentrations (n=3)

[0156]

[0157]

[0158] (2) Sample determination

[0159] Take 70 μl of the test solution, add 2 μl of the immunoprobe solution, mix well, and spot 50 μl onto the sample pad. Incubate at room temperature for 15 min, and then determine the F1 / F2 ratio using a background fluorescence-immunoassay analyzer. Figure 4 The amount of allicin in the sample was calculated using the standard curve, and the results are shown in Table 6.

[0160] Table 6 Summary of alliin yield at each step

[0161]

[0162] Note: Yield (%) is the ratio of alliin content in the material of this step to the alliin content in the material of the previous step. Overall yield (%) is the ratio of alliin content in the material of this step to the alliin content in the peeled garlic cloves.

[0163] After peeling garlic cloves are filtered through multiple filter membranes, a freeze-dried powder containing sulfur-containing amino acids and oligosaccharides characteristic of garlic is obtained. The final yield of alliin is as high as 75.80%, indicating that the method of the present invention results in very little loss of alliin.

[0164] 2. Determination of the content of various active substances in garlic

[0165] Based on the above process, this invention successfully extracts, separates, concentrates, and enriches active substances (such as sulfur-containing amino acids, other amino acids, and oligosaccharides) from garlic. The extraction process is free from organic reagent contamination, maximizing the preservation of the active ingredients in garlic. Specific components are shown in Tables 7 and 8.

[0166] (1) The total sugar content of the freeze-dried garlic polysaccharide powder was as high as 92.57% (calculated as glucose) as determined by UV-vis; the water content was 3.2% and the particle size was 120 mesh, and it was light yellow.

[0167] (2) Garlic characteristics: The freeze-dried powder of sulfur-containing amino acids and oligosaccharides was determined by HPLC-DAD to contain 32.06% sulfur-containing amino acids and 30.03% other amino acids; the content of oligosaccharides was determined by UV-vis to be 26.64% (calculated as glucose); the water content was 2.8% and the particle size was 120 mesh, and it was milky white.

[0168] As can be seen from the table:

[0169] The content of allicin in peeled garlic cloves is about 3.68%. After concentration by the method described above, the content in the freeze-dried powder of characteristic sulfur-containing amino acids and oligosaccharides in garlic is about 19.68%, achieving a concentration of >5 times.

[0170] The content of methyl allicin in peeled garlic cloves is about 0.73%. After concentration by the method described above, the content in the freeze-dried powder of characteristic sulfur-containing amino acids and oligosaccharides in garlic is about 4.67%, achieving a concentration of >6 times.

[0171] The content of S-allyl-L-cysteine ​​in peeled garlic cloves is about 0.07%. After concentration by the method described above, the content in the freeze-dried powder of characteristic sulfur-containing amino acids and oligosaccharides in garlic is about 0.74%, achieving a concentration of >10 times.

[0172] The content of tryptophan in peeled garlic cloves is about 0.24%. After concentration by the method described above, the content in the freeze-dried powder of characteristic sulfur-containing amino acids and oligosaccharides in garlic is about 1.63%, achieving a concentration of >6 times.

[0173] Sulfur-containing amino acids can serve as precursor amino acids in the antioxidant process within organisms and play a vital role in metabolism. The intake of essential amino acids is also crucial for maintaining health. The method described in this invention achieves highly efficient concentration of active substances in garlic, significantly improving the utilization rate of garlic.

[0174] Table 7. Content of sulfur-containing amino acids before and after extraction.

[0175]

[0176] Table 8. Content of other amino acids before and after extraction

[0177]

[0178] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for detecting an extract rich in various characteristic sulfur-containing amino acids of garlic, characterized in that, The method described above uses immunochromatography with specific antibodies to achieve quantitative detection of allicin. The VH sequence of the antibody is shown in SEQ ID NO. 1, and the VL sequence is shown in SEQ ID NO.

8. The extract was obtained through the following steps: (a) Garlic is subjected to enzyme inactivation treatment to obtain enzyme-inactivated garlic cloves; (b) After grinding the enzyme-inactivated garlic cloves into a homogenate, centrifuge to remove the residue and obtain the centrifuged liquid; (c) The centrifuged liquid is filtered through a 40-60 nm ceramic membrane to obtain the first filtrate; (d) The first filtrate is ultrafiltered through a 900-1100 Da molecular weight membrane to obtain the second filtrate and the first chopped liquid; (e) The second filtrate is ultrafiltered through a 550-650 Da molecular weight membrane to obtain the third filtrate and the second chopped liquid; (f) The third filtrate is concentrated through an 80-150 Da molecular weight filter membrane to obtain the third retentate, which is an extract rich in various sulfur-containing amino acids characteristic of garlic. In step (a), the enzyme inactivation process is microwave inactivation, the microwave inactivation time is 5-15 minutes, and the microwave inactivation process parameters are: power 39KW, conveying speed 250r / min. The extract described in step (f) comprises: Allicin, methyl allicin, isoalliin, γ-glutamyl-S-allyl-L-cysteine, γ-glutamyl-S-propenyl-L-cysteine, S-allyl-L-cysteine, and S-methyl-L-cysteine; Furthermore, the concentration factor of S-allyl-L-cysteine ​​in the extract is greater than or equal to 10. The concentration factor of methylalliin is greater than or equal to 6. Allicin has a concentration factor of 5 or greater.

2. The method as described in claim 1, characterized in that, The extract contains: Aspartic acid, arginine, threonine, alanine, proline, phenylalanine, tryptophan, lysine, and oligosaccharides.

3. The method as described in claim 1, characterized in that, The final yield of allicin in the extract is greater than or equal to 75%.

4. The method as described in claim 1, characterized in that, The concentration factor of S-allyl-L-cysteine ​​in the extract is 10-20.

5. The method as described in claim 1, characterized in that, The concentration factor of methylalliin in the extract is 6-15.

6. The method as described in claim 1, characterized in that, The concentration factor of allicin in the extract is 5-15.

7. The method as described in claim 1, characterized in that, The extract contains tryptophan at a concentration factor of 6 or greater.

8. The method as described in claim 1, characterized in that, The extract, rich in various sulfur-containing amino acids characteristic of garlic, has a total sulfur-containing amino acid content of ≥30 wt% on a dry weight basis.

9. The method as described in claim 1, characterized in that, The extract, rich in various sulfur-containing amino acids characteristic of garlic, contains a total sulfur-containing amino acid content of 30-50 wt% on a dry weight basis.

10. The method as described in claim 1, characterized in that, The extract, rich in various sulfur-containing amino acids characteristic of garlic, contains other amino acids with a total content of 30 wt% or more, based on dry weight.

11. The method as described in claim 1, characterized in that, The extract, rich in various sulfur-containing amino acids characteristic of garlic, contains 30-50 wt% of other amino acids on a dry weight basis.

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