Pea seedling extract, preparation method thereof, fingerprint spectrum construction method and application thereof

Abstract

This invention discloses a pea sprout extract, its preparation method, fingerprint chromatogram construction method, and its applications. The preparation method includes freezing fresh pea sprouts, short-time heat treatment to inactivate enzymes, extraction with medium-temperature hot water under nitrogen protection, filtration, vacuum concentration, and drying. This reduces oxidation and heat loss of active ingredients and improves the stability of the extract composition. The obtained pea sprout extract retains polyphenolic active substances well and has a high amino acid content with a distinctive amino acid composition, including aspartic acid, glutamic acid, and various functional amino acids such as arginine, tyrosine, and methionine. A reference fingerprint chromatogram containing 13 common characteristic peaks is constructed using high-performance liquid chromatography (HPLC), with the phenylalanine peak as the reference peak. The quality evaluation and control of the pea sprout extract are achieved by comparing the relative retention time and relative peak area of ​​the test samples. The method of this invention is simple to operate, requires low equipment, and is suitable for full-process quality monitoring in industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant extract processing and analysis technology, and particularly relates to a pea sprout extract and its preparation method, fingerprint spectrum construction method and application. Background Technology

[0002] Pea sprouts ( Piam sativm L Pea sprouts, also known as dragon's beard vegetable, butterfly vegetable, and pea shoots, are a vegetable whose tender shoots and leaves are the edible part. They are low in fiber, fragrant and delicious, tender and smooth, bright green in color, and rich in nutrients. They are rich in crude protein, crude fat, and various essential amino acids, as well as enzymes that can break down nitrosamines in the body. They also contain abundant calcium, vitamin B, and vitamin C. They have high development value in the fields of functional foods and raw materials for hair care cosmetics.

[0003] Existing pea sprout-related patents can be mainly divided into three categories: The first category is pea sprout "planting and cultivation patents," which mainly optimize seed germination, light, water and fertilizer conditions to improve the yield and nutrition of fresh pea sprouts; the second category, which accounts for the largest proportion, is "composition formulation patents," which use commercially available pea sprout extract as one of the active ingredients, and combine it with ginger, various plant extracts, peptides, and vitamins to prepare anti-hair loss, nutritional food, and skin care compositions. This type of patent hardly improves the extraction, purification, and drying processes of the pea sprout extract itself, and directly uses commercially available extract raw materials for formulation; the third category is a few simple primary extraction processes of juicing-filtration-concentration, which lack in-depth process optimization schemes for controlling the intrinsic quality of the extract.

[0004] Overall, existing patents focus on compound formulations for end products, with few patents specifically addressing the complete preparation process of pea sprout extract, including enzyme inactivation, purification, desaccharification, and degradation inhibition. Furthermore, no publicly available patent literature reports on quality control methods for pea sprout extract based on its proprietary fingerprint spectrum. The industry generally purchases commercially available pea sprout extract directly for formulation development, and the extract's production process and quality standards are highly dependent on raw material suppliers. Downstream formulation patents cannot address the instability in efficacy caused by batch fluctuations in the extract. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is that existing pea sprout extracts suffer from large fluctuations in chemical composition, poor batch consistency, lack of dedicated HPLC fingerprint spectra, and difficulty in achieving full-process quality monitoring. This invention proposes a pea sprout extract, its preparation method, fingerprint spectra construction method, and its application.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing pea sprout extract, comprising the following steps: S1. After freezing the whole fresh pea shoots, put them into water at 75±5℃, let the water temperature drop to 65±5℃, and keep warm for 10~20 minutes. S2. Nitrogen gas is introduced into the system and the temperature is lowered to 45~55℃. Extraction is carried out for 1~4 h. After extraction, the filtrate is obtained by filtration. S3. The filtrate obtained in step S2 is concentrated under vacuum to obtain an extract. Polydextrose is then added to the extract and stirred. The extract is then dried to obtain pea sprout extract.

[0007] Preferably, in step S3, the temperature of vacuum concentration is 55°C to 65°C, and the solid content of the resulting extract is 70% to 80%.

[0008] Preferably, in step S3, the solid content of the obtained extract is 75%.

[0009] Preferably, in step S3, the mass percentage of polydextrose is 10% to 50%, based on the total mass of dry matter and polydextrose in the extract.

[0010] Preferably, in step S1, the freezing temperature is -4℃ to -18℃ and the freezing time is 2 to 8 hours.

[0011] Preferably, the freezing temperature is -15℃ to -18℃ and the freezing time is 8 hours.

[0012] Preferably, in step S1, the mass ratio of pea shoots to water is 1:6 to 1:30.

[0013] Preferably, in step S1, the mass ratio of pea shoots to water is 1:10.

[0014] Preferably, in step S1, the heat preservation time is 15 minutes.

[0015] Preferably, in step S2, after extraction, the filtrate is obtained by filtering through a 200-mesh filter cloth.

[0016] Preferably, in step S3, after adding polydextrose, the mixture is stirred at 50℃~60℃ for 30min~60min.

[0017] Preferably, in step S3, the drying method is freeze drying, vacuum drying, or spray drying.

[0018] In a second aspect, the present invention provides a pea sprout extract prepared using the above-described preparation method.

[0019] As a preferred option, pea sprout extract contains 16 amino acids, namely aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine.

[0020] In a third aspect, the present invention provides a method for constructing a fingerprint spectrum of pea sprout extract, comprising the following steps: The above pea sprout extract was prepared into a solution using purified water to obtain the sample solution to be tested. High performance liquid chromatography was used to determine the sample solution and construct a fingerprint spectrum of pea sprout extract.

[0021] As a preferred option, the chromatographic conditions are: The chromatographic column is an octadecylsilane-bonded silica gel column; Column temperature: 30~40℃; Dimensions: 250×4.6 mm, 5μm; The flow rate was 0.9–1.0 ml / min; The detection wavelength is 254~270 nm; The injection volume is 10~30 μl; Mobile phase A is methanol, and mobile phase B is an aqueous solution; The gradient elution procedure is as follows: 0~30 min, 98%B→86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

[0022] Preferably, the reference fingerprint spectrum of the pea sprout extract includes 13 common characteristic peaks, and the absolute retention time and peak area of ​​each common characteristic peak are as follows: Characteristic peak 1: Absolute retention time 4.462 min, peak area 411509; Characteristic peak 2: Absolute retention time 5.22 min, peak area 251660; Characteristic peak 3: Absolute retention time 5.738 min, peak area 1259667; Characteristic peak 4: Absolute retention time 6.557 min, peak area 1259936; Characteristic peak 5: Absolute retention time 11.584 min, peak area 1575103; Characteristic peak 6: Absolute retention time 12.41 min, peak area 672469; Characteristic peak 7: Phenylalanine reference peak, absolute retention time 16.681 min, peak area 110012; Characteristic peak 8: Absolute retention time 18.957 min, peak area 206430; Characteristic peak 9: Absolute retention time 19.927 min. Characteristic peak 10: absolute retention time 20.701 min, peak area 642599; Characteristic peak 11: absolute retention time 28.944 min, peak area 272149; Characteristic peak 12: absolute retention time 33.918 min, peak area 690775; Characteristic peak 13: absolute retention time 53.483 min, peak area 272673.

[0023] In a fourth aspect, the present invention provides an application of the above-mentioned method for constructing fingerprint patterns of pea sprout extract, the application method comprising the following steps: The pea sprout extract to be tested was detected according to the above fingerprint spectrum construction method to obtain the fingerprint spectrum of the pea sprout extract to be tested. Using the fingerprint spectrum of the pea sprout extract according to claim 9 as a reference fingerprint spectrum, and taking the phenylalanine peak No. 7 of the reference fingerprint spectrum as a reference peak S, the relative retention time and relative peak area of ​​each common characteristic peak relative to the reference peak S are calculated respectively; wherein, the relative retention time is the ratio of the retention time of each test peak to the retention time of the reference peak S, and the relative peak area is the ratio of the peak area of ​​each test peak to the peak area of ​​the reference peak S. The relative retention times and relative peak areas of each common characteristic peak in the fingerprint spectrum of the pea sprout extract to be tested are compared with the relative retention times and relative peak areas of the corresponding common characteristic peaks in the reference fingerprint spectrum. When the deviation of the relative retention time of each common characteristic peak from the relative retention time of the corresponding common characteristic peak in the reference fingerprint spectrum does not exceed ±5%, and the relative peak area of ​​each common characteristic peak is greater than 60% of the relative peak area of ​​the corresponding common characteristic peak in the reference fingerprint spectrum, the pea sprout extract to be tested is determined to meet the quality requirements.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing pea sprout extract. Addressing the problems of enzymatic degradation due to the lack of enzyme inactivation in existing pea sprout extraction processes, and the loss of heat-sensitive active components such as amino acids caused by prolonged high-temperature enzyme inactivation, leading to fluctuations in the chemical composition of the extract and poor batch-to-batch consistency, this invention involves freezing fresh, intact pea sprout plants. This causes intracellular water to form ice crystals and disrupts the plant cell walls and cell membranes. The frozen pea sprouts are then extracted directly without thawing, reducing oxidation caused by the release of cell contents and contact with oxygen during thawing. Simultaneously, short-duration, controllable heat treatment is used to inactivate enzymes, combined with hot water extraction. This effectively inactivates endogenous enzymes while minimizing heat loss of heat-sensitive components such as amino acids, thereby improving the retention of active ingredients and the stability of the extract composition across different batches. This invention also provides a pea sprout extract. Through raw material freezing, short-time enzyme inactivation, and hot water extraction processes, the obtained pea sprout extract better retains polyphenolic active substances and has a high amino acid content with a distinctive amino acid composition. The high content of aspartic acid and glutamic acid, combined with various functional amino acids (arginine, tyrosine, methionine, etc.), has a synergistic effect in anti-oxidation, nutrient supply, and improvement of hair follicle microcirculation. This distinguishes it from ordinary crude pea sprout extract, constituting the unique material basis and efficacy advantages of the extract. This invention also provides a method for constructing a fingerprint spectrum of pea sprout extract and its application. Addressing the lack of existing quality evaluation methods for pea sprout extract that can reflect its overall chemical composition and quality changes during the production process, this invention uses conventional high-performance liquid chromatography (HPLC) equipment to establish an HPLC fingerprint spectrum of pea sprout extract. By setting common characteristic peaks including endogenous characteristic components and process degradation indicator components, the chemical composition of the pea sprout extract is characterized, thereby improving the consistency of quality evaluation across different production stages and batches. Furthermore, this method can be performed using conventional HPLC equipment, reducing the requirements for testing equipment and facilitating its widespread application in actual production quality control. Attached Figure Description

[0025] Figure 1 Comparison of HPLC fingerprint chromatograms of pea sprout extracts obtained in Examples 1, 4, and 5 of this invention; Figure 2 The HPLC fingerprint of the pea sprout extract obtained in Example 2 of this invention; Figure 3 The HPLC fingerprint of the pea sprout extract obtained in Example 3 of this invention; Figure 4 This is a comparison of the HPLC fingerprints of the pea sprout extracts obtained in Example 1 of this invention and Comparative Examples 1-5; Figure 5This is a comparison of the HPLC fingerprint of pea shoots in Example 1 of the present invention and the fingerprint of pea shoots under different flow rates; Figure 6 This is a comparison of the HPLC fingerprint of pea shoots in Example 1 of the present invention and under a changed mobile phase gradient. Detailed Implementation

[0026] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0027] In a first aspect, the present invention provides a method for preparing pea sprout extract, comprising the following steps: S1. After freezing the whole fresh pea shoots, put them into water at 75±5℃, let the water temperature drop to 65±5℃, and keep warm for 10~20 minutes. S2. Nitrogen gas is introduced into the system and the temperature is lowered to 45~55℃. Extraction is carried out for 1~4 h. After extraction, the filtrate is obtained by filtration. S3. The filtrate obtained in step S2 is concentrated under vacuum to obtain an extract. Polydextrose is then added to the extract and stirred. The extract is then dried to obtain pea sprout extract.

[0028] This invention preserves the complete morphology of pea shoots, omitting pre-processing steps such as chopping and juicing that damage the intact tissue of the pea shoots. This reduces the opportunity for active ingredients to come into contact with oxygen, lowering the oxidative loss of effective components in the early stages of extraction. Simultaneously, the pea shoots, after freezing pretreatment, can be directly transported via cold chain, effectively solving the problem of fresh pea shoots being highly susceptible to spoilage, foul odor, and waterlogging during storage and transportation. This significantly extends the raw material storage and transportation cycle, reduces raw material loss, and facilitates large-scale industrial production. Fresh pea shoots are frozen at low temperatures, causing the internal moisture to form ice crystals. The expansion of these ice crystals mechanically punctures and damages the cell walls and cell membranes of the pea shoots, increasing cell permeability. The frozen material is not thawed but directly immersed in preheated hot water at 70-80℃. The significant temperature difference creates a thermal shock that further breaks down the cell structure. This dual physical action promotes the dissolution of intracellular free amino acids and other active substances, increasing the extraction rate. Simultaneously, when frozen solid materials are added to high-temperature water, they can quickly reach the enzyme-inactivating temperature, shortening the heating phase time and reducing the damage to active ingredients such as amino acids during the heating process. This treatment is a purely physical method, without the addition of chemical reagents, and does not damage the target active components, making it suitable for the preparation of food-grade pea sprout extract. Directly immersing whole fresh pea sprouts in water at 75±5℃ after freezing maintains the system temperature at 65±5℃ after addition, allowing the pea sprouts to reach the suitable enzyme-inactivating temperature in a short time. If the initial water temperature is below 70℃, the system temperature easily drops below 60℃ after the frozen material is added, making it difficult to quickly inactivate the active enzymes in the pea sprouts, leading to changes in the composition of the extract.

[0029] This invention employs a segmented process: enzyme inactivation is performed at high temperature for a short time; extraction is performed at medium temperature for a long time, and the two processes are separated. Immediately after enzyme inactivation, the temperature is lowered to 45-55℃ for prolonged extraction, avoiding prolonged high-temperature soaking of the active ingredients. Nitrogen gas is introduced to create an inert extraction environment, which is crucial for antioxidant protection. This replaces dissolved oxygen in the system, inhibiting the oxidative degradation of polyphenols and amino acids, addressing the industry pain points of easy oxidation and discoloration, and decreased activity in pea sprout water extracts. Polydextrose is used as a drying excipient. Compared to the industry-standard maltodextrin, polydextrose has almost no reducing end, thus avoiding the introduction of additional reducing sugar substrates and preventing the exacerbation of Maillard reactions; it also improves the powder's moisture resistance and powdering properties.

[0030] In this invention, in step S1, the freezing temperature is -4℃ to -18℃, and the freezing time is 2 to 8 hours. Further, the freezing temperature is -15℃ to -18℃, and the freezing time is 8 hours. In this invention, the mass ratio of pea shoots to water is 1:6 to 1:30. Further, the mass ratio of pea shoots to water is 1:10. Further, in step S1, the heat preservation time is 15 minutes.

[0031] In this invention, in step S2, after extraction, the obtained extract is filtered through a 200-mesh filter cloth to obtain filtrate.

[0032] In this invention, in step S3, the vacuum concentration temperature is 55℃~65℃, and the solid content of the resulting extract is 70%~80%. This is because when the solid content of the extract is low and the water content is high, even with the addition of polydextrose, it is difficult to effectively encapsulate and isolate the relevant components in the extract, thus failing to sufficiently inhibit the Maillard reaction. Furthermore, the resulting material is prone to hygroscopic absorption, adhesion, or clumping within 72 hours. When the solid content of the extract is too high and the water content is too low, it cannot fully integrate with the polydextrose, leading to uneven mixing and clumping of excipients. Further, the solid content of the resulting extract is 75%. Based on the total mass of dry matter and polydextrose in the extract, the mass percentage of polydextrose is 10%~50%. Excessive polydextrose addition can dilute the amino acids and active ingredients in pea sprout extract, reducing the proportion of effective components per unit mass of extract and weakening its beneficial effects. Conversely, insufficient polydextrose addition results in inadequate moisture resistance and difficulty in effectively inhibiting the Maillard reaction. Therefore, controlling the polydextrose addition ratio within the range of 10%–50% balances product physicochemical stability with retention of active ingredients, resulting in a more scientifically sound formulation. After adding polydextrose, stir at 50℃–60℃ for 30–60 minutes. Drying methods include freeze-drying, vacuum drying, or spray drying.

[0033] In a second aspect, the present invention provides a pea sprout extract prepared using the above-described preparation method.

[0034] In this invention, the pea sprout extract contains 16 amino acids, namely aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine.

[0035] The pea sprout extract of this invention contains 16 amino acids, among which glutamic acid and aspartic acid are significantly higher in content than the other monomeric amino acids, and the two dominate all amino acids; at the same time, characteristic functional amino acids such as tyrosine, phenylalanine, arginine, and histidine are also detected.

[0036] Glutamic acid and aspartic acid are the two most abundant amino acids in this extract. Both are acidic amino acids and natural flavor amino acids, contributing to the extract's fresh and crisp flavor. Physiologically, both are important neurotransmitter precursors in the body, participating in energy metabolism and antioxidant defense, and can scavenge some reactive oxygen free radicals, thus synergistically enhancing the extract's in vitro antioxidant capacity with the polyphenol components. Furthermore, the acidic amino acids also play a role in regulating the pH of the system, helping to stabilize polyphenols and other active substances and reducing the oxidative degradation rate of active ingredients during extraction, drying, and storage.

[0037] In addition to the aforementioned acidic amino acids, aromatic amino acids such as phenylalanine and tyrosine were also detected in this extract. These amino acids have a benzene ring structure, exhibit characteristic ultraviolet absorption, and are also precursors for the biosynthesis of active substances such as polyphenols and melanin in plants. Among them, tyrosine and phenylalanine have certain free radical scavenging capabilities and can synergistically enhance the DPPH antioxidant activity of the sample; at the same time, tyrosine is also an important substrate for hair melanin synthesis and an important raw material for hair physiological metabolism, playing a positive role in maintaining normal pigment homeostasis in hair.

[0038] In addition, this extract also contains arginine. Arginine is a basic functional amino acid and a precursor to nitric oxide (NO) synthesis. NO can promote microcirculation in the hair follicle, improve blood supply to the dermal papilla, and provide nutrients to the hair follicle, thereby helping to maintain the normal physiological activity of the hair follicle. Therefore, arginine is also an important active component for in vitro evaluation of hair health.

[0039] This extract also contains methionine. Methionine is an essential sulfur-containing amino acid and an important component of the body's antioxidant system. It can participate in glutathione synthesis, scavenge reactive oxygen species, and thus reduce oxidative stress damage to hair follicle cells. In addition, its sulfur-containing structure is also an important building block of hair keratin.

[0040] In addition to the aforementioned functional amino acids, this extract also contains a variety of essential amino acids, including threonine, valine, leucine, isoleucine, lysine, and histidine. The presence of these essential amino acids serves as a nutritional substrate, providing fundamental nutrition for the proliferation of hair papilla cells and synergistically contributing to improved hair health.

[0041] In summary, the pea sprout extract of this invention not only has a high total amino acid content, but also has a distinctive amino acid composition. It is based on high levels of aspartic acid and glutamic acid, and also contains a variety of functional amino acids such as arginine, tyrosine, and methionine. It can play a synergistic role in anti-oxidation, nutrient supply, and improvement of hair follicle microcirculation, which distinguishes it from ordinary crude pea sprout extracts. This constitutes the unique material basis and efficacy advantages of the extract of this invention.

[0042] In a third aspect, the present invention provides a method for constructing a fingerprint spectrum of pea sprout extract, comprising the following steps: The pea sprout extract was prepared into a solution using purified water to obtain the sample solution to be tested. High performance liquid chromatography was used to determine the sample solution and construct a reference fingerprint chromatogram for pea sprout extract.

[0043] In this invention, the chromatographic conditions are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; Column temperature: 30~40℃; Dimensions: 250×4.6 mm, 5μm The flow rate was 0.9–1.0 ml / min; The detection wavelength is 254~270 nm; The injection volume is 10~30 μl; Mobile phase A is methanol, and mobile phase B is an aqueous solution; The gradient elution procedure is as follows: 0~30 min, 98%B→86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

[0044] In this invention, the reference fingerprint spectrum of the pea sprout extract includes 13 common characteristic peaks, and the absolute retention time and peak area of ​​each common characteristic peak are as follows: Characteristic peak 1: Absolute retention time 4.462 min, peak area 411509; Characteristic peak 2: Absolute retention time 5.22 min, peak area 251660; Characteristic peak 3: Absolute retention time 5.738 min, peak area 1259667; Characteristic peak 4: Absolute retention time 6.557 min, peak area 1259936; Characteristic peak 5: Absolute retention time 11.584 min, peak area 1575103; Characteristic peak 6: Absolute retention time 12.41 min, peak area 672469; Characteristic peak 7: Phenylalanine reference peak, absolute retention time 16.681 min, peak area 110012; Characteristic peak 8: Absolute retention time 18.957 min, peak area 206430; Characteristic peak 9: Absolute retention time 19.927 min. Characteristic peak 10: absolute retention time 20.701 min, peak area 642599; Characteristic peak 11: absolute retention time 28.944 min, peak area 272149; Characteristic peak 12: absolute retention time 33.918 min, peak area 690775; Characteristic peak 13: absolute retention time 53.483 min, peak area 272673.

[0045] In a fourth aspect, the present invention provides an application of the above-mentioned method for constructing fingerprint patterns of pea sprout extract, the application method comprising the following steps: The pea sprout extract to be tested was detected according to the above fingerprint spectrum construction method to obtain the fingerprint spectrum of the pea sprout extract to be tested. The fingerprint spectrum of the above pea sprout extract was used as a reference fingerprint spectrum, and the phenylalanine peak No. 7 of the reference fingerprint spectrum was used as the reference peak S. The relative retention time and relative peak area of ​​each common characteristic peak relative to the reference peak S were calculated. The relative retention time is the ratio of the retention time of each test peak to the retention time of the reference peak S, and the relative peak area is the ratio of the peak area of ​​each test peak to the peak area of ​​the reference peak S. The relative retention times and relative peak areas of each common characteristic peak in the fingerprint spectrum of the pea sprout extract to be tested are compared with the relative retention times and relative peak areas of the corresponding common characteristic peaks in the reference fingerprint spectrum. When the deviation of the relative retention time of each common characteristic peak from the relative retention time of the corresponding common characteristic peak in the reference fingerprint spectrum does not exceed ±5%, and the relative peak area of ​​each common characteristic peak is greater than 60% of the relative peak area of ​​the corresponding common characteristic peak in the reference fingerprint spectrum, the pea sprout extract to be tested is determined to meet the quality requirements.

[0046] In this invention, the phenylalanine peak (peak 7) is used as the reference peak S. Phenylalanine, as a stable aromatic amino acid found in pea sprout extract, exhibits characteristic absorption under UV detection conditions, and its chromatographic peak has good shape, high repeatability, and high stability, making it suitable as an internal reference peak in fingerprint analysis.

[0047] To more clearly and in detail introduce the pea sprout extract, its preparation method, fingerprint spectrum construction method, and applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0048] In the following examples and comparative examples of the present invention, the polyphenol content was determined using the Folin-Ciocalteu method, in accordance with the national standard GB / T 8313—2018 "Determination Method of Tea Polyphenol and Catechin Content in Tea".

[0049] The amino acid content was determined in accordance with the national standard GB 5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food".

[0050] Example 1: Preparation method of pea sprout extract S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0051] S2. Extraction and Filtration After the enzyme inactivation treatment in step S1, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0052] S3, Concentration, Addition of Excipients and Drying The filtrate obtained in step S2 was concentrated under vacuum at 65°C to obtain 26.0 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 22.62 g of pea sprout extract.

[0053] After testing, the polyphenol yield of pea sprout extract (excluding excipients) was 1.30 g / kg, and the solids yield was 19.5 g / kg.

[0054] Example 2: Preparation method of pea sprout extract S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 4 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0055] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0056] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 65°C to obtain 25.1 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 50°C for 60 min and then dried under reduced pressure for 2 h to obtain 21.08 g of pea sprout extract.

[0057] The polyphenol yield of pea sprout extract (excluding excipients) was 1.23 g / kg, and the solids yield was 18.82 g / kg.

[0058] Example 3: Preparation method of pea sprout extract S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 20 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:20. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0059] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0060] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 65°C to obtain 27.8 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 60°C for 30 min and then dried under reduced pressure for 2 h to obtain 23.04 g of pea sprout extract.

[0061] The polyphenol yield of pea sprout extract (excluding excipients) was 1.31 g / kg, and the solids yield was 20.85 g / kg.

[0062] Example 4: Preparation method of pea sprout extract S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0063] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 1 hour under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0064] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 60°C to obtain 25.8 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 22.59 g of pea sprout extract.

[0065] The polyphenol yield of pea sprout extract (excluding excipients) was 1.28 g / kg, and the solids yield was 19.35 g / kg.

[0066] Example 5: Preparation method of pea sprout extract S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0067] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0068] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 60°C to obtain 26 g of extract with a solid content of 75%. 19.5 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 38.71 g of pea sprout extract.

[0069] After testing, the polyphenol yield of the extract (excluding excipients) was 1.30 g / kg, and the solids yield was 19.5 g / kg.

[0070] Example 6: Method for constructing fingerprint profiles of pea sprout extract The pea sprout extract was prepared into a sample test solution using purified water. Specifically, 0.1g of each of the pea sprout extracts from Examples 1-5 was accurately weighed into a 25ml volumetric flask, dissolved in a small amount of purified water, and then diluted to volume to obtain the sample test solution.

[0071] High performance liquid chromatography was used to determine the sample solution and construct a reference fingerprint chromatogram for pea sprout extract.

[0072] The chromatographic conditions for Examples 1 and 5 are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; Column temperature: 30℃; Dimensions: 250×4.6 mm, 5μm The flow rate was 1.0 ml / min; The detection wavelength is 254 nm; The injection volume was 20 μl; Mobile phase A is methanol, and mobile phase B is an aqueous solution; The gradient elution procedure is as follows: 0~30 min, 98%B→86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

[0073] The chromatographic conditions in Example 2 differed from those in Examples 1 and 5 in that the flow rate was adjusted to 0.9 mL / min, while the other chromatographic conditions remained unchanged.

[0074] The chromatographic conditions of Example 3 differ from those of Examples 1 and 5 in that the detection wavelength is 270 nm, while the other chromatographic conditions remain unchanged.

[0075] The chromatographic conditions in Example 4 differed from those in Examples 1 and 5 in that the column temperature was 40°C, while the other chromatographic conditions remained unchanged.

[0076] Comparative Example 1 The difference between this comparative example and Example 1 is that the fresh pea shoots were not frozen. The specific steps include: S1. Raw material processing and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and without freezing, directly add them to 10 L of purified water preheated to 80℃. The mass ratio of pea shoots to purified water is 1:10. After adding the pea shoots, keep them warm for 15 min to inactivate the enzymes.

[0077] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0078] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 65°C to obtain 18.6 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 17.82 g of pea sprout extract.

[0079] The polyphenol yield of pea sprout extract (excluding excipients) was 0.67 g / kg, and the solids yield was 13.95 g / kg.

[0080] Comparative Example 2 The main difference between this comparative example and Example 1 is that the shredding process is used instead of the freezing process, specifically including the following steps: S1. Raw material processing and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, then chop them. Put the chopped pea shoots into 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the pea shoots, keep them warm for 15 min to inactivate the enzymes.

[0081] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0082] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 55°C to obtain 24.8 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 21.98 g of pea sprout extract.

[0083] Tests showed that the polyphenol yield of pea sprout extract (excluding excipients) was 0.74 g / kg, and the solids yield was 18.60 g / kg.

[0084] Comparative Example 3 This comparative example differs from Example 1 in that nitrogen was not introduced during the extraction process. Specifically, it includes the following steps: S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0085] S2. Extraction and Filtration After enzyme inactivation, without introducing nitrogen, the system was cooled for extraction (temperature reduced to 50℃) for 3 hours. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0086] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 55°C to obtain 25.9 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 23.92 g of pea sprout extract.

[0087] The polyphenol yield of pea sprout extract (excluding excipients) was 0.95 g / kg, and the solids yield was 19.43 g / kg.

[0088] Comparative Example 4 This comparative example uses a traditional method to prepare pea sprout extract, specifically including the following steps: S1, Raw Material Processing Weigh 1 kg of whole fresh pea sprouts, select and wash them, add 10 kg of purified water, and heat to boil for 3 hours to extract.

[0089] S2, Filtration The filtrate was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0090] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated at 80°C to obtain 28.1 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 25.75 g of pea sprout extract.

[0091] The polyphenol yield of pea sprout extract (excluding excipients) was 0.69 g / kg, and the solids yield was 21.07 g / kg.

[0092] Comparative Example 5 The main difference between this comparative example and Example 1 is that the initial water temperature during enzyme inactivation treatment is lower, and the specific steps include: S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 60℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 50℃ and keep it at that temperature for 15 minutes.

[0093] S2. Extraction and Filtration Nitrogen gas was introduced into the system, and extraction was carried out for 3 hours. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0094] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 55°C to obtain 23.8 g of extract with a solid content of 75%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 21.06 g of pea sprout extract.

[0095] The polyphenol yield of pea sprout extract (excluding excipients) was 0.49 g / kg, and the solids yield was 17.85 g / kg.

[0096] Comparative Example 6 The main difference between this comparative example and Example 1 is that polydextrose is not added after concentration. The specific steps include: S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0097] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0098] S3, Concentration and Drying The filtrate was concentrated under vacuum at 65°C to obtain 26 g of extract with a solid content of 75%. Without the addition of polydextrose, the extract was directly dried under reduced pressure for 2 h to obtain 18.73 g of pea sprout extract.

[0099] The polyphenol yield of pea sprout extract (excluding excipients) was 1.30 g / kg, and the solids yield was 19.50 g / kg.

[0100] Comparative Example 7 This comparative example differs from Example 1 in that the concentrated extract has a different solid content, specifically including the following steps: S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0101] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0102] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 65°C to obtain 39 g of extract with a solid content of 50%. 5.00 g of polydextrose was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 23.19 g of pea sprout extract.

[0103] Comparative Example 8 This comparative example differs from Example 1 in that maltodextrin is used instead of polydextrin as an excipient, specifically including the following steps: S1, freezing treatment and enzyme inactivation Weigh 1 kg of whole fresh pea shoots, select and wash them, and freeze them at -15℃ for 8 hours. After freezing, without thawing, directly add the frozen whole pea shoots to 10 L of purified water preheated to 80℃, with a pea shoot to purified water mass ratio of 1:10. After adding the materials, lower the system temperature to approximately 70℃ and keep it at this temperature for 15 minutes to inactivate the enzymes.

[0104] S2. Extraction and Filtration After enzyme inactivation, nitrogen gas was introduced into the system, and the system was cooled for extraction (temperature dropped to 50℃) for 3 hours under nitrogen protection. After extraction, the extract was filtered through a 200-mesh filter cloth to obtain the filtrate.

[0105] S3, Concentration, Addition of Excipients and Drying The filtrate was concentrated under vacuum at 50–60°C to obtain 26.0 g of extract with a solid content of 75%. 5.00 g of maltodextrin was added to the extract, and the mixture was stirred at 55°C for 45 min and then dried under reduced pressure for 2 h to obtain 22.98 g of pea sprout extract.

[0106] Comparative Example 9 Prepare the sample test solution using purified water. Specifically, accurately weigh 0.1g of each of the pea shoot extracts from Comparative Examples 1-5 into a 25ml volumetric flask, dissolve them in a small amount of purified water, and then dilute to the final volume to obtain the sample test solution.

[0107] High performance liquid chromatography was used to determine the sample solution and construct a reference fingerprint chromatogram for pea sprout extract.

[0108] The chromatographic conditions are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; Column temperature: 30℃; Dimensions: 250×4.6 mm, 5μm The flow rate was 1.0 ml / min; The detection wavelength is 254 nm; The injection volume was 20 μl; Mobile phase A is methanol, and mobile phase B is an aqueous solution; The gradient elution procedure is as follows: 0~30 min, 98%B→86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

[0109] To investigate the effects of different treatment conditions on pea sprout extract, Example 1 was compared with the comparative example, and the results are as follows: 1. Comparison of polyphenol content, polyphenol yield, and solids yield between Example 1 and Comparative Examples 1-5: Table 1

[0110] As shown in Table 1, the polyphenol yield of the pea sprout extract obtained in Example 1 was 1.30 g / kg, which was higher than that of Comparative Examples 1-5.

[0111] The difference between Example 1 and Comparative Example 1 is that Example 1 involved freezing fresh pea shoots at -15°C for 8 hours, while Comparative Example 1 did not undergo freezing pretreatment. Compared to Example 1, the polyphenol yield of the extract obtained in Comparative Example 1 decreased from 1.30 g / kg to 0.67 g / kg, and the solids yield decreased from 19.5 g / kg to 13.95 g / kg. These results indicate that freezing pretreatment at -15°C facilitates physical disruption of plant tissue structure through ice crystal formation, increasing cell wall and cell membrane permeability, thereby promoting the release and dissolution of intracellular polyphenols and other soluble substances, and improving the extraction efficiency of active ingredients.

[0112] The difference between Example 1 and Comparative Example 2 is that Example 1 used whole fresh pea shoots that had been frozen before being directly fed into the plant, while Comparative Example 2 used chopped pea shoots. The polyphenol yield of the extract obtained in Comparative Example 2 was 0.74 g / kg, which was significantly lower than that in Example 1. Compared with chopped treatment, freezing pretreatment with whole pea shoots promotes the destruction of plant tissue structure and the release of active ingredients, while reducing oxidative losses caused by mechanical cutting, thus facilitating the retention of polyphenolic active ingredients.

[0113] The difference between Example 1 and Comparative Example 3 is that Example 1 used nitrogen gas for protection during extraction, while Comparative Example 3 did not. The polyphenol yield of the extract obtained in Comparative Example 3 was 0.95 g / kg, lower than that in Example 1; while the solids yields of Example 1 and Comparative Example 3 were 19.5 g / kg and 19.4 g / kg, respectively, which were essentially equivalent. This shows that, with a similar overall solids extraction level, the lack of nitrogen protection leads to a decrease in the retention of polyphenolic components. The results indicate that using nitrogen gas to create a low-oxygen environment during extraction can reduce the oxidative loss of polyphenolic components during extraction, thereby improving the retention rate of active polyphenolic ingredients.

[0114] Comparative Example 4 prepared pea sprout extract using a conventional process, without specific freezing pretreatment and short-term heat treatment for enzyme inactivation, and concentrated it at 80°C. The polyphenol yield of the extract obtained in Comparative Example 4 was 0.69 g / kg; its solids yield was 21.10 g / kg, which is close to and slightly higher than the 19.5 g / kg in Example 1. This result indicates that a higher solids yield does not necessarily mean that the active polyphenolic components are effectively retained. During conventional crushing and high-temperature concentration processes, factors such as oxidation caused by mechanical crushing, endogenous enzyme activity, and heat loss due to high temperatures may simultaneously exist, leading to a significant reduction in the active polyphenolic components.

[0115] The main difference in Comparative Example 5 was the lower initial water temperature during enzyme inactivation treatment; the initial feeding water temperature was 60℃, and the extraction temperature was 50℃. The polyphenol yield of the extract obtained in Comparative Example 5 was 0.68 g / kg, and the solids yield decreased from 19.5 g / kg to 17.8 g / kg. This suggests that low-temperature feeding cannot inhibit active enzymes within pea shoots, leading to the enzymatic hydrolysis of polyphenols and other active substances, resulting in a decrease in yield; furthermore, the solids yield also decreased.

[0116] In contrast, this invention employs whole pea shoots for frozen pretreatment, combined with short-time heat treatment to inactivate enzymes, nitrogen-protected extraction, and vacuum low-temperature concentration. This reduces the oxidation and heat loss of polyphenolic components during extraction and concentration, thereby improving the retention level of polyphenolic active ingredients in the pea shoot extract.

[0117] 2. Comparison of the dried product state of Example 1 and Comparative Examples 6-8 Table 2

[0118] As shown in Table 2, the pea sprout extract obtained in Example 1 is yellow after drying and has the aroma of pea sprouts. It remains yellow after being dissolved in water, and no obvious clumping phenomenon was observed after 24 h and 72 h. This indicates that the product obtained under the process conditions of the present invention has good appearance, odor and storage stability.

[0119] Comparative Example 6, without added polydextrose, was a deep yellow sample after drying with a burnt odor. After soluble in water, it turned dark brown and exhibited severe clumping after 24 hours, which persisted for 72 hours. Comparative Example 7, with a 50% solids content, also had polydextrose added, but its dried sample remained deep yellow with a burnt odor, turned dark brown after soluble in water, and showed clumping after 72 hours. These results indicate that when the solids content of the extract is low and the water content of the system is high, even with added polydextrose, effective physical inclusion is difficult to achieve, leading to the continued Maillard phenomenon and clumping within 72 hours. Comparative Example 8, using an equal amount of maltodextrin, produced a deep yellow sample with a burnt odor, which turned dark brown after soluble in water; slight clumping appeared after 24 hours, and the clumping significantly worsened after 72 hours.

[0120] Regarding moisture content, Example 1 had a moisture content of 1.50% after 2 hours of vacuum drying, and the moisture content only increased slightly to 1.57% after 24 hours, showing excellent moisture stability. Comparative Example 6 had an initial moisture content of 3.92%, and the moisture content increased to 5.63% after 24 hours, showing a large degree of moisture absorption. Comparative Examples 7 and 8 had initial moisture contents of 2.88% and 2.93%, respectively, and the moisture content continued to increase after 24 hours and 72 hours, showing a clear trend of moisture absorption.

[0121] 3. Comparison of amino acid content between Example 1 and Comparative Examples 1-5: Table 3 Comparison of amino acid content between Example 1 and Comparative Examples 1-5

[0122] As shown in Table 3, the total amino acid content of the pea sprout extract prepared in Example 1 reached 20.3 g / 100g, which was significantly higher than that of all comparative examples. The retention levels of each characteristic amino acid and easily oxidized amino acid were optimal.

[0123] Comparative Example 1 did not undergo freezing pretreatment, so the plant cell walls were not damaged by ice crystals, resulting in insufficient dissolution of intracellular amino acids. The total amino acid content was only 14.1 g / 100 g. The content of easily oxidized amino acids such as methionine, tyrosine, and lysine was significantly lower than that in Example 1. Relying solely on sufficient enzyme inactivation temperature, the amino acid retention effect was better than that of Comparative Example 5, which had insufficient enzyme inactivation.

[0124] Comparative Example 2 used a chopping method instead of cryogenic pretreatment. The chopping effect on cell walls was weaker than that of cryogenic ice crystal puncture. The total amino acid content was 15.6 g / 100g, which was higher than that of Comparative Example 1 but lower than that of Example 1. The chopping operation directly tore the cell tissue, and the intracellular oxidases came into contact with the substrate, causing oxidative loss of sensitive amino acids such as methionine and tyrosine.

[0125] Comparative Example 3 retained the freeze-crushing process, resulting in a better amino acid dissolution base and a total amino acid content of 17.4 g / 100g; however, nitrogen protection was not introduced during the extraction stage, and dissolved oxygen in the system caused oxidative degradation of methionine, tyrosine, and lysine, leading to a significantly lower content of the above functional amino acids compared to Example 1.

[0126] Comparative Example 4 used a traditional boiling high-temperature extraction process. High temperature causes thermal decomposition of amino acids and intensifies the oxidation reaction. The total amount of amino acids was the lowest, only 10.4 g / 100g. Methionine and tyrosine were almost completely lost, and the heat-sensitive amino acids were the least retained.

[0127] In Comparative Example 5, the initial water temperature for enzyme inactivation was too low, and the endogenous oxidase was not fully inactivated. During the extraction process, the enzyme continued to catalyze the oxidative degradation of amino acids, and the total amount of amino acids was 12.7 g / 100g, with methionine, tyrosine, and lysine at relatively low levels.

[0128] In summary, Example 1 of this invention utilizes ice crystal puncture to efficiently break down cell walls, promoting the full dissolution of intracellular amino acids. Furthermore, it employs nitrogen gas to isolate oxygen, inhibiting the degradation of easily oxidized amino acids such as methionine, tyrosine, and lysine. This process not only achieves the highest total amino acid content but also retains significantly higher levels of key characteristic amino acids such as aspartic acid and glutamic acid, as well as various functional amino acids, compared to the comparative examples. This maximizes the preservation of amino acid components in the pea sprout extract, ensuring its beneficial efficacy.

[0129] Experimental Example 1 Following the fingerprint chromatogram construction method described in Example 6, the pea sprout extracts prepared in Examples 1 and 2-5 were subjected to high-performance liquid chromatography (HPLC) to obtain the HPLC fingerprint chromatograms of the pea sprout extracts from each example. The phenylalanine peak (peak 7) was used as a reference peak for analysis.

[0130] Relative Retention Time (RRT) = Absolute Retention Time of the Peak ÷ Retention Time of the Reference Peak (S) Relative Peak Area (RPA) = Peak Area of ​​the Peak ÷ Peak Area of ​​the Reference Peak (S) The HPLC fingerprint of the pea sprout extract of Example 1 was used as a reference fingerprint, and the relative retention time and relative peak area of ​​the reference fingerprint were used as benchmark values.

[0131] The allowable deviation of the relative retention time of the characteristic peak is ±5% = RRT (standard) × (1 ± 5%). RPA lower limit = RPA (standard) × 60%.

[0132] Table 4. Chromatographic parameters of common characteristic peaks in the HPLC fingerprint of pea sprout extract from Example 1.

[0133] Following the fingerprint chromatogram construction method described in Example 6, the pea sprout extracts prepared in Examples 2-5 were subjected to high-performance liquid chromatography (HPLC) to obtain the HPLC fingerprint chromatograms of each group of pea sprout extracts, and then compared and analyzed.

[0134] Table 5. Relative retention time parameters of HPLC fingerprint spectra of pea sprout extracts from Examples 2-5

[0135] Table 6. Relative area parameters of HPLC fingerprint chromatograms of pea sprout extracts from Examples 2-5

[0136] The test results showed that the HPLC fingerprint of the pea sprout extract obtained in Example 1 showed 13 common characteristic peaks, of which peak 7 was the phenylalanine peak.

[0137] Depend on Figure 1 As can be seen, the overall profile of the HPLC fingerprint of the pea sprout extracts obtained in Examples 1, 4, and 5 is basically the same, and all the common characteristic peaks from 1 to 13 can be detected. The main difference is that the peak areas of some common characteristic peaks are different.

[0138] Depend on Figure 2 As can be seen, in Example 2, the HPLC fingerprint of pea shoots showed a delayed peak elution time due to changes in flow rate.

[0139] Depend on Figure 3 As can be seen, in Example 3, the HPLC fingerprint of pea shoots showed a change in peak area due to the change in detection wavelength.

[0140] As shown in Tables 4 and 5, using the chromatogram of Example 1 as the standard fingerprint chromatogram, peak 7 (phenylalanine) was selected as the reference peak S. The relative retention times of each common characteristic peak were calculated, and an allowable deviation of ±5% was set. All 13 common characteristic peaks were detected in the chromatograms of samples from Examples 2 to 5, and the peak elution order was consistent with the standard chromatogram. The relative retention times of all common peaks in Examples 2, 3, 4, and 5 all fell within the ±5% limit, indicating that this fingerprint chromatogram has strong specificity and is suitable for the qualitative quality control of pea sprout extract.

[0141] As shown in Tables 4 and 6, using the chromatogram of Example 1 as the standard fingerprint chromatogram, peak 7 (phenylalanine) was selected as the reference peak S. The relative peak areas of each common characteristic peak were calculated, and it was determined that the relative peak area of ​​the common characteristic peak should not be less than 60% of the baseline value. In Examples 2, 3, 4, and 5, the relative peak areas of all common peaks were not less than 60% of the baseline value; this indicates that the fingerprint chromatogram has strong specificity and is suitable for the qualitative quality control of pea sprout extract.

[0142] Experimental Example 2 Following the fingerprint chromatogram construction method described in Example 6, the pea sprout extracts prepared in Example 1 and Comparative Examples 1-5 were subjected to high-performance liquid chromatography (HPLC) to obtain the HPLC fingerprint chromatograms of each group of pea sprout extracts, and then compared and analyzed.

[0143] Depend on Figure 4 As can be seen, compared with Example 1, Comparative Example 1 did not undergo freeze-drying physical cell wall disruption, thus the pea sprout cell walls remained intact, resulting in greater resistance to the dissolution of intracellular polyphenolic active substances. Under the same extract, the characteristic peaks were lower, and components of peaks 9 and 12 were not dissolved, indicating a low content of active ingredients.

[0144] Depend on Figure 4 As can be seen, compared with Example 1, Comparative Example 2 uses shredding treatment instead of whole-plant freezing pretreatment in Example 1. Mechanical cutting allows the plant tissue and intracellular components to come into full contact with air before subsequent nitrogen protection, which may increase the oxidative degradation of related active ingredients. The components of peaks 6, 8 and 9 did not dissolve or oxidize and degrade, indicating that the content of effective ingredients is low.

[0145] Depend on Figure 4 As can be seen, compared with Example 1, the peak areas of some common characteristic peaks in the pea sprout extract obtained in Comparative Example 3 were reduced. The main difference between Example 1 and Comparative Example 3 is whether nitrogen protection was used during the extraction stage. Comparative Example 3 did not use nitrogen during the extraction process, and no inert environment was established. Dissolved oxygen in the system continued to oxidize, and the components of peaks 6 and 9 did not dissolve or were oxidized and degraded, indicating that the content of effective components was low.

[0146] Depend on Figure 4As can be seen, there are significant differences in the HPLC fingerprint chromatograms of the pea sprout extracts obtained in Example 1 and Comparative Example 4. Compared with Example 1, peaks 1, 2, 5, 6, 8, 9, 10, and peak 12 in Comparative Example 4 are significantly reduced or undetectable, indicating that some of their common characteristic components have been significantly lost, suggesting that the excessively high temperature in the traditional process has a very significant destructive effect on the active ingredients.

[0147] Depend on Figure 4 As can be seen, there are significant differences in the HPLC fingerprint chromatograms of the pea sprout extracts obtained in Example 1 and Comparative Example 5. Compared with Example 1, peaks 5, 6, 8, 9, 10, and 12 in Comparative Example 5 were significantly reduced or undetectable, indicating that some of their common characteristic components were significantly lost, suggesting that the low temperature did not achieve the enzyme inactivation effect and caused very significant damage to the active ingredients.

[0148] Experimental Example 3 To further investigate the effect of chromatographic conditions on the separation effect of HPLC fingerprint of pea sprout extract, the pea sprout extract prepared in Example 1 was used as the test sample. Based on the detection method described in Example 6, the flow rate and gradient elution program were adjusted. Except for the chromatographic conditions specifically described below, the preparation method of the test solution, chromatographic column, column temperature, detection wavelength, injection volume, mobile phase composition, and other detection conditions were consistent with the detection conditions of the corresponding test sample in Example 1 of Example 6.

[0149] (1) Flow velocity investigation The flow rate of the test sample corresponding to Example 1 in Example 6 was adjusted from 1.0 mL / min to 0.8 mL / min, while all other detection conditions remained the same as those for the test sample corresponding to Example 1 in Example 6. High-performance liquid chromatography (HPLC) was used for detection, and the corresponding HPLC fingerprint chromatograms were obtained. The separation of each chromatographic peak was compared and analyzed.

[0150] Depend on Figure 5 It can be seen that when the flow rate was adjusted to 0.8 mL / min, peaks 9 and 10 could not be effectively separated, indicating that the separation effect of adjacent chromatographic peaks was poor under this flow rate condition, which is not conducive to the effective separation of characteristic chromatographic peaks of pea sprout extract.

[0151] (2) Gradient elution procedure investigation Based on the detection conditions for the test sample in Example 1 of Example 6, the gradient elution program was adjusted. Methanol was still used as mobile phase A, and water as mobile phase B. The adjusted gradient elution program is as follows: 0~10 min, 98%B→98%B; 10~30 min, 98%B → 86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

[0152] Except for the gradient elution procedure mentioned above, all other detection conditions are the same as those for the test sample corresponding to Example 1 in Example 6.

[0153] The pea sprout extract from Example 1 was subjected to high-performance liquid chromatography (HPLC) under the adjusted gradient elution conditions described above to obtain the corresponding HPLC fingerprint, and the separation of each chromatographic peak was compared and analyzed.

[0154] Depend on Figure 6 It is evident that peaks 9 and 10 could not be effectively separated when the adjusted gradient elution program was used, indicating that adjacent chromatographic peaks could not be effectively separated under this gradient elution condition, resulting in poor separation performance.

Claims

1. A method for preparing pea sprout extract, characterized in that, Includes the following steps: S1. After freezing the whole fresh pea shoots, put them into water at 75±5℃, let the water temperature drop to 65±5℃, and keep warm for 10~20 minutes. S2. Nitrogen gas is introduced into the system and the temperature is lowered to 45~55℃. Extraction is carried out for 1~4 h. After extraction, the filtrate is obtained by filtration. S3. The filtrate obtained in step S2 is concentrated under vacuum to obtain an extract. Polydextrose is then added to the extract and stirred. The extract is then dried to obtain pea sprout extract.

2. The method for preparing pea sprout extract according to claim 1, characterized in that, In step S3, the vacuum concentration temperature is 55℃~65℃, and the solid content of the resulting extract is 70%~80%.

3. The method for preparing pea sprout extract according to claim 1, characterized in that, In step S3, the total mass of dry matter and polydextrose in the extract is used as the basis, and the mass percentage of polydextrose is 10% to 50%.

4. The method for preparing pea sprout extract according to claim 1, characterized in that, In step S1, the freezing temperature is -4℃ to -18℃, and the freezing time is 2 to 8 hours. In step S1, the mass ratio of pea shoots to water is 1:6 to 1:30; In step S2, after extraction, the obtained extract is filtered through a 200-mesh filter cloth to obtain the filtrate. In step S3, after adding polydextrose, stir at 50℃~60℃ for 30min~60min.

5. A pea sprout extract, characterized in that, It is prepared using the preparation method described in any one of claims 1-4.

6. The pea sprout extract according to claim 5, characterized in that, The pea sprout extract contains 16 amino acids, namely aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine.

7. A method for constructing a fingerprint spectrum of pea sprout extract, characterized in that, Includes the following steps: The pea sprout extract of claim 5 or 6 was prepared into a solution using purified water to obtain the sample test solution. High-performance liquid chromatography (HPLC) was used to determine the sample solution and construct a fingerprint spectrum for pea sprout extract.

8. The construction method according to claim 7, characterized in that, The chromatographic conditions are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; Column temperature: 30~40℃; Dimensions: 250×4.6 mm, 5 μm; The flow rate was 0.9–1.0 ml / min; The detection wavelength is 254~270 nm; The injection volume is 10~30 μl; Mobile phase A is methanol, and mobile phase B is an aqueous solution; The gradient elution procedure is as follows: 0~30 min, 98%B→86%B; 30~40 min, 86%B → 57%B; 40~60 min, 57%B → 57%B; 60~61 min, 57%B→98%B; 61~71 min, 98%B→98%B.

9. The construction method according to claim 8, characterized in that, The fingerprint spectrum of the pea sprout extract includes 13 common characteristic peaks, and the absolute retention time and peak area of ​​each common characteristic peak are as follows: Characteristic peak 1: Absolute retention time 4.462 min, peak area 411509; Characteristic peak 2: Absolute retention time 5.22 min, peak area 251660; Characteristic peak 3: Absolute retention time 5.738 min, peak area 1259667; Characteristic peak 4: Absolute retention time 6.557 min, peak area 1259936; Characteristic peak 5: Absolute retention time 11.584 min, peak area 1575103; Characteristic peak 6: Absolute retention time 12.41 min, peak area 672469; Characteristic peak 7: Phenylalanine reference peak, absolute retention time 16.681 min, peak area 110012; Characteristic peak 8: Absolute retention time 18.957 min, peak area 206430; Characteristic peak 9: Absolute retention time 19.927 min. Characteristic peak 10: absolute retention time 20.701 min, peak area 642599; Characteristic peak 11: absolute retention time 28.944 min, peak area 272149; Characteristic peak 12: absolute retention time 33.918 min, peak area 690775; Characteristic peak 13: absolute retention time 53.483 min, peak area 272673.

10. The application of the fingerprint pattern construction method for pea sprout extract according to claim 9, characterized in that, Its application method includes the following steps: The pea sprout extract to be tested was detected according to the above fingerprint spectrum construction method to obtain the fingerprint spectrum of the pea sprout extract to be tested. Using the fingerprint spectrum of the pea sprout extract according to claim 9 as a reference fingerprint spectrum, and taking the phenylalanine peak No. 7 of the reference fingerprint spectrum as a reference peak S, the relative retention time and relative peak area of ​​each common characteristic peak relative to the reference peak S are calculated respectively; wherein, the relative retention time is the ratio of the retention time of each test peak to the retention time of the reference peak S, and the relative peak area is the ratio of the peak area of ​​each test peak to the peak area of ​​the reference peak S. The relative retention times and relative peak areas of each common characteristic peak in the fingerprint spectrum of the pea sprout extract to be tested are compared with the relative retention times and relative peak areas of the corresponding common characteristic peaks in the reference fingerprint spectrum. When the deviation of the relative retention time of each common characteristic peak from the relative retention time of the corresponding common characteristic peak in the reference fingerprint spectrum does not exceed ±5%, and the relative peak area of ​​each common characteristic peak is greater than 60% of the relative peak area of ​​the corresponding common characteristic peak in the reference fingerprint spectrum, the pea sprout extract to be tested is determined to meet the quality requirements.

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