Soluble leucine-containing mixture with reduced bitterness and application of leucine-containing mixture

Abstract

The invention discloses a leucine-containing dipeptide mixture easy to dissolve and reduced in bitterness as well as a preparation method and application of the leucine-containing dipeptide mixture. The mixture comprises the following components in percentage by mass: not less than 90% of alanyl-leucine, 0-5% of free leucine, less than 1% of water and not more than 0.10% of other impurities. The solubility of the mixture at room temperature reaches up to 500 g / L, the mixture is kept transparent and free of precipitates, the bitterness is reduced by 65% or above compared with leucine, the taste can be improved without a taste masking agent, and the mixture is suitable for sports nourishment and functional food. According to the preparation method, an enzyme catalysis reaction is adopted, and the steps of solid-liquid separation, pH adjustment, crystallization, nanofiltration concentration, drying and the like are performed, so that the total yield is not lower than 90%, the purity is not lower than 99.5%, and high solubility, low bitter taste, instant solubility and environmental friendliness are achieved; and the method has a wide industrial application scene.

Description

Technical Field

[0001] This invention relates to the field of amino acid product preparation technology, specifically to a leucine-containing mixture that is easily soluble and has reduced bitterness, and its application. Background Technology

[0002] Leucine belongs to the branched-chain amino acids (BCAAs) and has multiple functions in the body, including promoting muscle recovery, controlling blood sugar, providing energy, increasing growth hormone production, and helping to burn visceral fat. Leucine also helps heal bones, skin, and damaged muscles in surgical patients. Furthermore, leucine can be used to treat some diseases, such as hepatic encephalopathy, chronic hepatitis, chronic persistent hepatitis, and cirrhosis, because it promotes synthesis, inhibits catabolism, maintains a positive nitrogen balance, and reduces ammonia production. For the elderly, especially those prone to sarcopenia, leucine supplementation has a beneficial effect on weight, body mass index, and muscle mass. Simultaneously, leucine is more likely than other amino acids to form acetyl-CoA, directly providing energy to the body. Acetyl-CoA can also be used to produce ketone bodies, which helps with fat consumption. In addition, leucine is significantly effective in muscle recovery, improving training endurance, and stabilizing blood sugar. Recent studies have shown that most of the end products of protein digestion in the digestive tract are small peptides, while oligopeptides can pass intact through intestinal mucosal cells into the human circulation. Small molecule peptides are absorbed quickly, consume less energy, and are less prone to carrier saturation. Oligopeptides are less prone to further hydrolysis in the intestines and can be absorbed more completely into the bloodstream. Small peptides in the bloodstream can directly participate in the synthesis of tissue proteins. Furthermore, the liver, kidneys, skin, and other tissues can also fully utilize small peptides. Compared to free amino acids, oligopeptides have a lower osmotic pressure. Some dipeptides and tripeptides have also been shown to be directly absorbed and exert more bioregulatory effects than free amino acids. Because they avoid competition with free amino acids during absorption, dipeptides can make the intake of amino acids more balanced, improving the efficiency of protein synthesis in the body. For infants with immature digestive systems, elderly people whose digestive systems are beginning to degenerate, athletes who urgently need nitrogen supplementation but cannot increase the burden on their gastrointestinal function, and those with poor digestion, nutritional deficiencies, weakness, or multiple illnesses, supplementing amino acids in the form of dipeptides can improve amino acid absorption and meet the body's needs for amino acids and nitrogen. Although WO2019204633 reported that leucine dipeptides had a plasma concentration 2.2 times higher than free leucine within 30 minutes, this patent did not address the following issues: The existing technology (WO2019204633 patent) only proposes the theoretical advantage that dipeptides may be well absorbed, but it completely avoids the solubility and flavor issues that must be faced as an actual product. In fact, it is these two defects that hinder the application of dipeptides in the field of oral supplements.

[0003] On the one hand, the aforementioned patent only mentions a mixture of leucine and leucine, without providing other dipeptides, so it cannot be explained whether the dipeptides are better absorbed or whether it is simply an effect of supplementing leucine. Secondly, the WO2019204633 patent avoids mentioning the bitterness of the product, which may indicate a lack of compliance (the prior art did not determine the bitterness threshold). In fact, leucine is one of the most bitter amino acids among branched-chain amino acids (BCAAs), with a low bitterness threshold (approximately 0.1% w / v), meaning even low concentrations can significantly affect taste. In oral nutritional supplements, large amounts of masking agents (such as sucralose and vanillin) are needed to mask bitterness, increasing formulation complexity and cost. Masking agents may introduce additional chemicals, affecting the product's naturalness and consumer acceptability.

[0004] Furthermore, the aforementioned patent WO2019204633 did not consider the solubility of dipeptides containing leucine. Leucine has low solubility in water (approximately 24 g / L at 25°C), and is prone to precipitation in high-concentration formulations, affecting product stability and user experience. Microencapsulation can improve the solubility and bitterness of leucine, but may reduce its bioavailability (due to the influence of the encapsulation material on the release rate). Moreover, the microencapsulation process is complex, requiring the use of encapsulation materials (such as gelatin or gum arabic), increasing production costs. Microencapsulated products may experience reduced solubility due to the presence of encapsulation materials, affecting ease of use.

[0005] In summary, a dipeptide product containing leucine that is orally compliant, easily soluble, and readily absorbed by the human body is urgently needed for development. Summary of the Invention

[0006] The product of this invention truly enables the commercialization of high-concentration, palatable dipeptide oral supplements, solving a long-standing pain point in the industry.

[0007] This invention discloses a soluble dipeptide mixture containing leucine with reduced bitterness, which is composed of the following components by mass percentage: alanyl-leucine (Ala-Leu, alanyl-leucine dipeptide) of not less than 90%; more preferably not less than 95%; most preferably not less than 97%; free leucine of 0-5%; more preferably 0-2%; moisture content of less than 1%; and total other impurities of not more than 0.10%. The mixture has good solubility and remains transparent without precipitation or floating particles at room temperature and at a concentration not exceeding 500 g / L.

[0008] For the technical solution described above, a further preferred embodiment is that the mixture contains 97-99% alanine-leucine.

[0009] With regard to the technical solution described above, a further preferred embodiment is that the bitterness of the mixture is significantly reduced relative to leucine, specifically, by more than 50%, and more preferably, by more than 65%.

[0010] For the technical solution described above, a further preferred embodiment is that the solubility of the mixture is such that it remains transparent, without precipitation or floating particles, at room temperature and a concentration not exceeding 200 g / L. A more preferred concentration is 500 g / L. Example data demonstrates that the product of this invention exhibits "transparent solution, no precipitation, and no floating particles" at concentrations of 20, 50, 100, 200, and 500 g / L.

[0011] For the technical solution described above, it is further preferred that the dissolution time of the mixture at a concentration of 20 g / L is 180-250 seconds; more preferably 192 seconds.

[0012] Another aspect of the present invention discloses a method for preparing the above mixture, comprising the following steps: The mixture after the enzyme-catalyzed reaction is subjected to solid-liquid separation to obtain the filtrate; The pH of the filtrate was adjusted to 5.5–7.0, and after concentration, crystallization and solid-liquid separation were performed to obtain the primary crystallized product and the first mother liquor. The primary crystallization product is recrystallized in aqueous phase to obtain a recrystallized product and a second mother liquor. The first mother liquor and / or the second mother liquor are concentrated by permeation through a nanofiltration membrane with a molecular weight cutoff of less than 500 Daltons to remove inorganic salts and small molecule impurities with a molecular weight smaller than the target dipeptide, thereby obtaining a concentrated solution. The recrystallization product was combined with the concentrate and dried to obtain a dipeptide product containing leucine. The total yield of the product is not less than 90%, and the purity of the leucine-containing peptide is not less than 99.5%.

[0013] For the technical solution described above, a further preferred embodiment of the method includes the following steps: the molecular weight cutoff of the nanofiltration membrane is 100–300 Daltons; The percolation concentration is a multi-stage operation, which includes adding water to the nanofiltration system during the concentration process and continuing concentration at least once more. The product has an alanyl-leucine content of 90%–99%, a free leucine content of no more than 5%, a total of other impurities of no more than 0.10%, and a moisture content of less than 1%.

[0014] In a further preferred embodiment of the above-described technical solution, the mixture in the method is prepared by an enzyme-catalyzed condensation reaction. The enzyme-catalyzed reaction is carried out in an aqueous system, using free amino acids or their activated derivatives as substrates. Under suitable pH and temperature, a biocatalyst selected from transesterases or peptide ligases catalyzes the formation of peptide bonds to generate a dipeptide mixture with alanyl-leucine as the main product. For the technical solution described above, a further preferred embodiment of the method is that the method includes: (a) Using the engineered bacterium BL21-pET29(a)-Ssaet-G226S disclosed in CN116970587A as a catalyst, with L-alanine C1–C4 alkyl ester hydrochloride and L-leucine as substrates, wherein the concentration of L-alanine C1–C4 alkyl ester hydrochloride is 200–300 mM and the concentration of L-leucine is 150–250 mM, the pH of the reaction system is 8.8–9.2, the temperature is 20–30℃, and the reaction time is 0.5–2 hours, to generate a mixture containing alanyl-leucine; or (b) Using Cg-pXMJ-iapV69H-YWFE-SS prepared in ZL202211481398.6 and / or Cg-pXMJ-iapV69H-ppk prepared in ZL202211368802.9 as immobilized enzymes, with L-alanine and L-leucine as substrates, wherein the concentration of L-alanine is 150–250 mM and the concentration of L-leucine is 200–300 mM, the total amount of immobilized enzyme is 0.5–2.0 g / L, the pH of the reaction system is 9.8–10.2, the temperature is 20–30℃, and the reaction time is 3–8 hours, to generate a mixture containing alanyl-leucine.

[0015] The present invention also protects the use of the mixture in the preparation of sports nutrition products and functional foods.

[0016] To address the shortcomings of existing technologies, this invention provides a dipeptide mixture containing leucine, which has the following beneficial effects: 1. High solubility: High-concentration solutions can be prepared without complex processes.

[0017] 2. Low bitterness: No additional masking agents are needed, improving taste compliance.

[0018] 3. Instant solubility: No additional processing is required; it can directly form an instant product.

[0019] 4. Environmental friendliness: It is prepared by bio-enzyme catalysis, and no solvent is introduced during the separation process, so there is no chemical pollution. Attached Figure Description

[0020] Figure 1Chromatogram of a transesterase-catalyzed reaction solution for the production of leucine-containing dipeptides; Figure 2 Chromatogram of the reaction solution for the synthesis of leucine-containing dipeptides catalyzed by ligase; Figure 3 Evaluation of the bitterness intensity of the mixture; the results shown in the figure are that leucine, leucine dipeptide, leucine + alanine, the product of the present invention, and the raw materials formulated with reference to the product of the present invention were scored for bitterness intensity, which proved the advantages of the product of the present invention in reducing bitterness and improving user compliance. Detailed Implementation

[0021] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0022] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the materials and reagents used can be purchased commercially.

[0023] Unless otherwise specified, all raw materials and reagents used in the specific embodiments of this invention are commercially available products. Their nominal purity is typically greater than 98%. Those skilled in the art should understand that, as long as the purity meets experimental requirements, similar products from different brands or manufacturers can be used equivalently to implement this invention, and the choice of specific brands should not be construed as a limitation of this invention. The raw materials and reagents are typically not further purified before the experiment, which sufficiently demonstrates the good applicability and industrialization potential of the method of this invention.

[0024] Example 1: Preparation of a mixture containing leucine dipeptides – transesterase 230 mM alanine methyl ester hydrochloride and 200 mM leucine were dissolved in an aqueous solution at 25°C and pH 9. Then, 2 g / L of wet cells of the engineered bacterium BL21-pET29(a)-Ssaet-G226S (CN116970587A, i.e., a mutant of the enzyme) was added to initiate the catalytic process. After 0.5 h, HPLC analysis showed that the yield of alanine-leucine dipeptide reached 190 mM, with 25 mM of alanine remaining, 15 mM of leucine remaining, and 5 mM of alanine-leucine dipeptide. HPLC analysis showed... Figure 1 No other substance peaks were detected.

[0025] Example 2 Preparation of a mixture containing leucine dipeptides – ligase 200 mM alanine and 250 mM leucine were dissolved in an aqueous solution at 25°C and pH 10. Immobilized enzyme Cg-pXMJ-iapV69H-YWFE-SS prepared in ZL202211481398.6 (1.2 g / L) and immobilized enzyme Cg prepared in ZL202211368802.9 (0.25 g / L) were then added. pXMJ iapV69H The catalytic process was initiated by PPK. After 3-6 hours, HPLC analysis revealed that the yields of propionyl leucine dipeptide reached 190 mM, propionyl dipeptide 2 mM, leucine dipeptide 10 mM, alanine remaining 5 mM, and leucine remaining 35 mM. HPLC analysis showed... Figure 2 No other substance peaks were detected.

[0026] Example 3 Crystallization process of the final product The mixture product from Example 1 was subjected to the following crystallization operation: Each reaction solution was filtered through a ceramic membrane with a pore size of 0.22 μm to remove the cell catalyst. The pH of the enzyme-removed reaction solution was adjusted to approximately 6, and then concentrated by rotary evaporation to remove approximately 90% of the original volume, followed by filtration to obtain crystal cake I and filtrate I. Crystal cake I was reconstituted with water and then rotary evaporated to approximately 1.2 times its original mass, followed by filtration to obtain a recrystallized filter cake. The primary recrystallization filtrate and filtrate I were concentrated through a 150 Dalton nanofiltration membrane to remove salts and small molecule amino acids at an operating pressure of 2.7 MPa, yielding a primary supernatant and a primary concentrate. Water was added to the nanofiltration unit for further nanofiltration, yielding a secondary supernatant and a secondary concentrate. The recrystallized cake and the two concentrates were combined and evaporated to dryness to obtain the product, with a total yield of over 90% of the dipeptide containing leucine. Its composition was analyzed by HPLC. The peptides containing leucine account for 99.9% of the total content, the content of proleucide dipeptide is between 97% and 99%, the leucine content is no higher than 2%, the content of other impurities is no higher than 0.10%, and the water content is <1%.

[0027] The mixture product from Example 2 was subjected to the following crystallization operation: Each reaction solution was filtered through a ceramic membrane with a pore size of 0.22 μm to remove the cell catalyst. The pH of the enzyme-removed reaction solution was adjusted to approximately 6, and then concentrated by rotary evaporation to remove approximately 90% of the original volume, followed by filtration to obtain crystal cake I and filtrate I. Crystal cake I was reconstituted with water and then rotary evaporated to approximately 1.2 times its original mass, followed by filtration to obtain a recrystallized filter cake. The primary recrystallization filtrate and filtrate I were concentrated through a 150 Dalton nanofiltration membrane to remove salts and small molecule amino acids at an operating pressure of 2.7 MPa, yielding a primary supernatant and a primary concentrate. Water was added to the nanofiltration device for further nanofiltration, yielding a secondary supernatant and a secondary concentrate. The recrystallized cake and the two concentrates were combined and evaporated to dryness to obtain the product, with a total yield of over 90% of the dipeptide containing leucine. Its composition was analyzed by HPLC. The peptides containing leucine account for 99.9% of the total content, the content of proleucide dipeptide is between 97% and 99%, the leucine content is no higher than 1%, the leucide dipeptide content is no higher than 0.5%, the content of other impurities is no higher than 0.10%, and the water content is <1%.

[0028] The final product is a dipeptide containing leucine, with a propionyl leucine dipeptide content between 97% and 99%, other amino acids ≤2%, other impurities not exceeding 0.10%, and a water content <1%. No organic reagents are introduced.

[0029] Example 4: Final Product Solubility Solubility refers to the feasibility of dissolving in water to a degree invisible to the naked eye. Easily soluble supplements can increase users' willingness to drink them. Test method: Weigh 2.0g of each sample from test groups 1-5 and add them to 150mL beakers containing 100mL of water at 25℃. Stir at a constant speed of 140-160rpm for 10 minutes and observe whether the solution is completely soluble. Complete dissolution is characterized by a clear, transparent solution without sediment or floating particles.

[0030] The raw material content of the proleucide purchased from multiple sources was only 96%-98%, and there was obvious solvent residue, which affected various properties and made it unsuitable as an evaluation object, especially for sensory evaluation.

[0031] Test group 1: Leucine-containing dipeptide of the present invention The crystalline powder product prepared according to the method described in Example 3 of this invention was directly weighed and tested.

[0032] Test group 2: Raw materials formulated according to the composition of this invention The raw materials were physically mixed according to the same composition and proportion as in Example 3, but without undergoing the process described in this invention. The physical mixture was then weighed directly for testing.

[0033] Test group 3: 1g alanine raw material + 1g leucine raw material Weigh out 1g of alanine raw material and 1g of leucine raw material directly, mix them physically at a mass ratio of 1:1 and use the mixture as the test sample.

[0034] Test group 4: Leucine raw materials Commercially available leucine raw materials without any processing were weighed directly for testing.

[0035] Test Group 5: Bright Dipeptide Raw Material Weigh out commercially available Bright Bright Dipeptide raw material directly without any processing for testing.

[0036] Table 1 Comparison of dissolving abilities

[0037] Table 1 shows that the crystalline powders of Examples 1 and 2 of the present invention can be completely dissolved at all tested concentrations (20 g / L to 500 g / L), and the solutions are always transparent, without precipitation or floating. In contrast, the control group (including physical mixtures prepared according to the ingredients of the present invention, physical mixtures of alanine and leucine, and single leucine or leucine dipeptide raw materials) showed floating particles at low concentrations (e.g., 50 g / L), and the solubility deteriorated sharply with increasing concentration. This highlights the unexpected and significant improvement in solubility achieved by the crystallization process of Examples 1 and 2.

[0038] Example 5: Evaluation of the instant solubility of the final product "Dispersibility in water" refers to the rate at which a mixture disperses into water, which is also the rate at which particles become wetted and "sink." Experimental method: Take a 150 mL beaker and add 100 mL of 25°C water. Let it stand for 15-45 seconds until no bubbles appear and the water surface is calm. Take 2.0 g of each sample from test groups 1-5 described in Example 4, and quickly pour it into the water from 50 mm above the center of the beaker. Time the time it takes for the sample to be completely wetted and "sink" to the bottom of the beaker, recording the required time T1, s, which is the dispersion time in water.

[0039] "Water dissolution clarification time" refers to the time it takes for a mixture of amino acid particles to dissolve in water until it becomes clear and transparent to the naked eye under stirring or shaking conditions.

[0040] Table 2 Comparison of instant solubility

[0041] The rapid solubility data in Table 2 further confirms the significant advantages of the crystalline powder of the present invention. Its dispersion time in water is only 6.7 seconds, and it can be completely clarified within 3 minutes and 12 seconds. In contrast, the dispersion and clarification times of the physical mixture raw materials prepared according to the ingredients of the present invention are extended to 30.2 seconds and 9 minutes, respectively, and other control raw materials cannot even be effectively tested due to severe floating. This clearly shows that the processes of Examples 1 and 2 not only endow the final product with excellent instant dispersibility, but also greatly shorten its complete dissolution time. This rapid solubility performance represents a qualitative leap.

[0042] Example 6: Sensory Evaluation of the Final Product Twenty members were selected for the sensory evaluation panel. Evaluation experiments were conducted in a standard sensory evaluation laboratory, and the results were statistically analyzed. The highest and lowest scores were removed, and the average was taken. The sensory evaluation experiments were conducted in a sensory evaluation room at 25°C ± 2°C. For each substance to be evaluated, 0.5g of powder was weighed and dissolved in 100mL of drinking water. The sensory evaluators tasted each substance and scored it. The scoring range was 0-10 points, with higher scores indicating stronger intensity.

[0043] Evaluation results as follows Figure 1 As shown, the average bitterness score of leucine is 4.85±0.81; the bitterness score of leucine dipeptide is 8.1±1.1; and the bitterness score of the leucine + alanine combination is 3.10±1.2. The average bitterness score of the product is 1.6±0.99, which is 67% lower than that of leucine. The average bitterness score of the raw materials formulated according to the ingredients of this invention is 3.25±1.99, indicating a large number of outliers and unstable product mixing uniformity. The low bitterness of the leucine + alanine combination is due to the low leucine content.

[0044] Taking all factors into consideration, this product is a mixture containing leucine dipeptides, rich in leucine required for sports nutrition, easy to dissolve and with significantly reduced bitterness, has an easily acceptable miso flavor, and is well-tolerated orally.

[0045] Example 7: Evaluation of bitterness using an electronic tongue The test group samples prepared in Example 4 were set aside for later use; 0.5g of powder was weighed from each group sample, dissolved in 100mL of drinking water (concentration 5mg / mL), and filtered for later use.

[0046] Bitterness was evaluated using the InsentTS-5000Z electronic tongue, equipped with a BT04 (bitterness-sensitive sensor) and a CT03 (cation sensor). Calibration: The sensor was calibrated using a 0.1 mM quinine hydrochloride solution to ensure linear response (R² > 0.95). Detection conditions: Solution temperature 25 ± 1℃; the sensor was cleaned three times before detection; each detection was repeated three times and the average value was taken.

[0047] Bitterness Index (BI) = 0.6 × BT04 response value + 0.4 × CT03 response value (weighting coefficients are based on the sensor's preset sensitivity to bitter substances) The test results are shown in Table 3 below: Table 3. Test Results

[0048] The test results show that the electronic tongue's objective evaluation is consistent with the results of human sensory evaluation. Leucine has the highest BI value (0.68), corresponding to a strong bitter taste; leucine dipeptide has the highest bitterness BI value, and the bitterness index of the mixture of equal masses of leucine and alanine is 0.3, lower than leucine but higher than the product's bitterness index. The target product has a BI value of 0.21, with significantly reduced bitterness, 69% lower than leucine. However, the poor uniformity of the raw materials in the formulation resulted in a slightly higher overall score than the product.

[0049] This uniformity difference stems precisely from the core process of this invention. Physically mixed raw materials (even with correct composition) cannot achieve a uniform distribution at the molecular level, resulting in excessively high concentrations of bitter substances (leucine) in certain areas. This leads to a stronger bitterness response measured by the electronic tongue and greater data fluctuations (see the standard deviation of the response value for "Raw Materials Prepared According to the Ingredients of this Invention" ±0.08, far higher than the ±0.01 for crystalline powders). In contrast, the process of this invention fundamentally achieves uniformity and effective masking of bitter components, thus obtaining a lower and more stable bitterness index.

[0050] In summary, the data in Table 3 not only confirms that the crystalline powder of the present invention has the lowest bitterness, but more importantly, it reveals that there is an essential difference in key technologies between simple physical mixing (comparative example) and the crystallization process of the present invention (example), which leads to a significant difference in the bitterness masking effect of the final product.

[0051] Comparative Example 1: Preparation of a mixture containing leucine dipeptides – Transesterase 200 mM alanine methyl ester hydrochloride and 250 mM leucine were dissolved in an aqueous solution at 25 °C and pH 9. The resulting solution was then added to the BL21-pET29(a)-Ssaet-G226A engineered bacteria (i.e., the enzyme mutant) from OD2 CN116970587A to initiate the catalytic process. After 0.5 h, HPLC analysis revealed that the yield of alanine-leucine dipeptide reached over 150 mM, with alanine remaining at 35–45 mM, leucine remaining at ~100 mM, and alanine-alanine dipeptide at ~3 mM. No other peaks were detected.

[0052] The reaction solution was treated according to Example 3, and the resulting product composition was the same. However, a large amount of alanine and leucine entered the waste liquid during the process, resulting in significant waste.

[0053] Comparative Example 2: Preparation of a mixture of leucine-containing dipeptides – ligase 250 mM alanine and 200 mM leucine were dissolved in an aqueous solution at 25 °C and pH 10. Immobilized enzyme Cg-pXMJ-iapV69H-YWFE-SS prepared in ZL202211481398.6 (1.2 g / L) and immobilized enzyme Cg prepared in ZL202211368802.9 (0.25 g / L) were then added. pXMJ iapV69H The catalytic process was initiated by PPK. After 3-6 hours, HPLC analysis showed that the yield of propionyl leucine dipeptide reached over 160 mM, propionyl dipeptide ~10 mM, leucine dipeptide ~1 mM, alanine remaining 50-60 mM, leucine remaining 30-35 mM, and no other peaks were observed.

[0054] The reaction solution was treated according to Example 3, and the resulting product composition was the same. However, a large amount of alanine and leucine entered the waste liquid during the process, resulting in significant waste.

[0055] This invention recognizes a long-standing, unresolved common problem in the enzymatic preparation of leucine-containing dipeptides: regardless of whether efficient synthetic tools such as transesterases (as shown in Comparative Example 1) or ligases (as shown in Comparative Example 2) are used, the reaction system inevitably retains a large amount of unreacted, expensive raw materials, resulting in significant waste in subsequent conventional processing. This makes this technical route uncompetitive in terms of economic cost and environmental protection. The contribution of this invention lies not in the selection of a specific enzyme, but in the unexpected development of a universal integrated crystallization and purification process (Example 3). This process can serve as a 'universal solution' to effectively process crude products generated from different synthetic pathways (such as the transesterase in Example 1 and the ligase in Example 2), and overcomes the aforementioned common challenges: it not only achieves efficient recovery of raw materials (total yield >90%), but also yields a final product with high purity, high solubility, and low bitterness.

[0056] The stark contrast between Comparative Examples 1 and 2 and Examples 1 and 2 demonstrates that even if those skilled in the art possess various enzyme catalytic tools for synthesizing dipeptides, they cannot readily resolve their inherent economic and product performance deficiencies without the core process inspiration of this invention.

[0057] Only by combining the front-end synthesis with the back-end refining process specific to this invention can a holistic technical solution produce significant improvements in recovery rate and product performance, albeit non-obviously.

[0058] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A soluble dipeptide mixture containing leucine with reduced bitterness, characterized in that, The mixture is composed of the following components by mass percentage: alanine-leucine at least 90%; free leucine at 0-5%; moisture content less than 1%; and total other impurities not exceeding 0.10%. The mixture has good solubility and remains transparent without precipitation or floating particles at room temperature and a concentration not exceeding 500 g / L.

2. The mixture according to claim 1, characterized in that, Alanyl-leucine 90-99%.

3. The mixture according to claim 1, characterized in that, The bitterness of the mixture is significantly reduced by more than 65% compared to the leucine raw material.

4. The mixture according to claim 1, characterized in that, The solubility refers to the ability to maintain transparent, non-precipitating floating particles at room temperature and a concentration not exceeding 200 g / L. A more preferred concentration is 500 g / L.

5. The mixture according to claim 1, characterized in that, The mixture dissolves in 180-250 seconds at a concentration of 20 g / L.

6. A method for preparing the mixture according to claims 1-5, characterized in that, Includes the following steps: The mixture after the enzyme-catalyzed reaction is subjected to solid-liquid separation to obtain the filtrate; The pH of the filtrate was adjusted to 5.5–7.0, and after concentration, crystallization and solid-liquid separation were performed to obtain the primary crystallized product and the first mother liquor. The primary crystallization product is recrystallized in aqueous phase to obtain a recrystallized product and a second mother liquor. The first mother liquor and / or the second mother liquor are concentrated by permeation through a nanofiltration membrane with a molecular weight cutoff of less than 500 Daltons to remove inorganic salts and small molecule impurities with a molecular weight smaller than the target dipeptide, thereby obtaining a concentrated solution. The recrystallization product was combined with the concentrate and dried to obtain the final product.

7. The method according to claim 6, characterized in that, Includes the following steps: The nanofiltration membrane has a molecular weight cutoff of 100–300 Daltons; The percolation concentration is a multi-stage operation, which includes adding water to the nanofiltration system during the concentration process and continuing concentration at least once more. The product has an alanyl-leucine content of 90%–99%, a free leucine content of no more than 5%, a total of other impurities of no more than 0.10%, and a moisture content of less than 1%.

8. The method according to claim 6, characterized in that, The mixture is prepared by an enzyme-catalyzed condensation reaction in an aqueous system, using free amino acids or their activated derivatives as substrates. Under suitable pH and temperature, a biocatalyst selected from transesterases or peptide ligases catalyzes the formation of peptide bonds, generating a dipeptide mixture with alanyl-leucine as the main product.

9. The method according to claim 8, characterized in that, The method includes: (a) Using the engineered bacterium BL21-pET29(a)-Ssaet-G226S disclosed in CN116970587A as a catalyst, with L-alanine C1–C4 alkyl ester hydrochloride and L-leucine as substrates, wherein the concentration of L-alanine C1–C4 alkyl ester hydrochloride is 200–300 mM and the concentration of L-leucine is 150–250 mM, the pH of the reaction system is 8.8–9.2, the temperature is 20–30℃, and the reaction time is 0.5–2 hours, to generate a mixture containing alanyl-leucine; or (b) Using Cg-pXMJ-iapV69H-YWFE-SS prepared in ZL202211481398.6 and / or Cg-pXMJ-iapV69H-ppk prepared in ZL202211368802.9 as immobilized enzymes, with L-alanine and L-leucine as substrates, wherein the concentration of L-alanine is 150–250 mM and the concentration of L-leucine is 200–300 mM, the total amount of immobilized enzyme is 0.5–2.0 g / L, the pH of the reaction system is 9.8–10.2, the temperature is 20–30℃, and the reaction time is 3–8 hours, to generate a mixture containing alanyl-leucine.

10. The use of the mixtures as described in claims 1-5 in the preparation of sports nutrition products or functional foods.

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