Preparation method and application of tomato seed small molecular protein with anti-muscle attenuation activity

Abstract

The present application relates to a kind of preparation method and application of tomato seed small molecular protein with anti-muscle attenuation activity, belong to the field of active ingredient development of biotechnology.The preparation method of the present application includes: after washing and drying tomato seed, it is carried out ultrafine grinding treatment, by alkali treatment superposition biological enzyme low-temperature enzymolysis, from tomato seed, preparation small molecular protein with anti-muscle attenuation activity.The experimental results show that the tomato seed small molecular protein obtained in the present application has the effect of improving muscle attenuation, and has the advantages of safety, long-term use, no toxic side effects, etc., can be applied to the medical field for improving muscle attenuation and its related symptoms.The present application provides a theoretical basis and technical support for developing new anti-muscle attenuation products, has wide application prospect and market value.

Description

Technical Field

[0001] This invention belongs to the field of development and application of new bioactive protein resources, specifically relating to a method for preparing a small molecule protein with anti-muscle atrophy activity and its application. Background Technology

[0002] With social development and improved living standards, health issues are receiving increasing attention. In my country, sarcopenia has become a significant problem affecting the quality of life for middle-aged and elderly people. Statistics show that approximately 30% of the middle-aged and elderly population in my country suffers from varying degrees of sarcopenia. Sarcopenia not only leads to a decline in physical function but also increases the risk of falls and fractures, severely impacting patients' quality of life. Therefore, finding an effective method to combat sarcopenia has become a current research hotspot.

[0003] In numerous studies on anti-muscle atrophy, plant-derived active ingredients have received widespread attention due to their naturalness, safety, and minimal side effects. Tomatoes, a common vegetable, are rich in nutrients such as lycopene, vitamin C, and vitamin E, offering excellent health benefits. Recent research has discovered that tomato seeds contain a class of small-molecule proteins with anti-muscle atrophy activity, providing new insights for developing novel anti-muscle atrophy products.

[0004] However, research on the extraction, purification, and activity of small molecule proteins from tomato seeds is still in its early stages. Existing extraction methods for tomato seed protein mainly include water extraction, acid-base extraction, and ultrasound-assisted extraction, but these methods suffer from low extraction rates, low purity, and significant loss of active ingredients. Furthermore, existing research on the anti-muscle loss activity of small molecule proteins from tomato seeds is relatively limited, restricting their development for practical applications.

[0005] To address the aforementioned problems, this invention provides a method for preparing tomato seed small molecule protein with anti-muscle loss activity and its applications. This invention aims to provide an efficient and simple method for preparing tomato seed small molecule protein and to study its anti-muscle loss activity, providing a theoretical basis and technical support for the development of novel anti-muscle loss products. This invention has the following advantages: high extraction rate, good purity, and minimal loss of active ingredients; simple operation and easy industrialization; significant anti-muscle loss activity and broad application prospects. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a method for preparing a tomato seed-derived small molecule protein with anti-muscle attenuation activity and its application.

[0007] The objective of this invention is achieved by at least one of the following technical solutions.

[0008] This invention provides a method for preparing a small molecule protein with anti-muscle attenuation activity and its application.

[0009] A method for preparing tomato seed small molecule protein with anti-muscle atrophy effects includes the following steps: (1) Degreasing of tomato seeds: The ultra-finely pulverized tomato seed powder is degreased by steam expansion-wall breaking subcritical technology; (2) Crude extraction of tomato seed protein: Mix defatted tomato seed powder with distilled water and perform preliminary extraction in an alkaline environment, and collect the supernatant; (3) Crude protein hydrolysis of grains: The first protease and the second protease are added to the supernatant obtained in step (2) for targeted and segmented enzymatic hydrolysis, and the supernatant is collected. (4) Preparation of small molecule protein: The protein solution obtained in step (3) is concentrated under vacuum at 40~60℃, desalted, and freeze-dried under vacuum to obtain a small molecule protein with anti-muscle attenuation activity.

[0010] Furthermore, as a preferred embodiment of the small molecule protein, the active ingredient of the present invention, the small molecule protein is prepared by an alkaline method coupled with a low-temperature enzymatic hydrolysis method.

[0011] Further, the tomato seed powder mentioned in step (1) refers to the component that is dried to constant weight at 50~60℃ and has a particle size ≤ 355 μm after being pulverized.

[0012] Furthermore, the protein content of the tomato seed raw material described in step (1) is ≥ 25%.

[0013] Furthermore, the defatted tomato seed powder described in step (1) has an oil content of ≤ 5%.

[0014] Further, the alkaline environment described in step (2) is pH 9~12, the extraction time is 0.5~2 h, and the material-to-liquid ratio is 1:10~30 (g / mL).

[0015] Further, the supernatant in step (2) is obtained after centrifugation at 4000~6000 ×g for 10~30 min.

[0016] (3) Crude protein hydrolysis of seeds: The first protease and the second protease are added to the supernatant obtained in step (2) for segmented and directional enzymatic hydrolysis, and the supernatant is collected.

[0017] Furthermore, in step (3), the first protease is Bacillus licheniformis protease, and the second protease is a complex flavor protease.

[0018] Further, the segmented enzymatic hydrolysis conditions in step (3) are: hydrolysis time of 2-4 h, hydrolysis temperature of 45-65ºC, and enzyme dosage of 5000-10000 U / g.

[0019] Further, the supernatant in step (3) is obtained after centrifugation at 4000~6000 ×g for 10~30 min.

[0020] Furthermore, in step (3), the desalination refers to the removal of small molecules using membrane permeation or ultrafiltration methods, with a molecular weight limit of 1000-3000 Da.

[0021] Furthermore, the mechanism of action of the above-mentioned raw material components by protease segmentation and targeted enzymatic hydrolysis is as follows: Some common sequence features of small proteins play important roles in inflammatory responses, such as hydrophobicity. amino acids (Val, Ile, Pro) and positively charged amino acids The presence of (His, Arg, Lys). Previous studies have shown that small proteins with high levels of hydrophobic amino acids, mainly located at the N-terminus of the small protein and positively charged amino acids located at the C-terminus, exhibit higher anti-inflammatory activity.

[0022] Bacillus licheniformis protease: This is a type of alkaline protease. Because its active center contains serine, it is also known as a serine protease. It can not only hydrolyze... peptide bonds It also has hydrolysis properties. amide bond The first protease is classified as an endonuclease, which has a particularly specific cleavage effect on peptide bonds containing hydrophobic amino acid residues, and can produce large quantities of small proteins containing hydrophobic amino acids at the C-terminus or N-terminus.

[0023] Complex flavor protease: It contains two active enzymes, exo- and endo-peptide enzymes. It is mainly used to reduce or avoid the production of bitter peptides during hydrolysis, or to degrade bitter peptides into amino acids, thereby improving the taste and flavor of hydrolysates. It can also supplement the first protease, effectively breaking down the long chain structure of proteins and generating more small molecule proteins.

[0024] Furthermore, the small molecule protein has been experimentally proven to improve muscle loss.

[0025] Furthermore, the obtained tomato seed small molecule protein has a good effect on improving the decrease in swallowing frequency and motor ability during the aging process of nematodes. The effect is most significant on the 9th day of intervention, with an increase of 30% compared with the control group. At the same time, it can significantly increase the body swing frequency of nematodes, with an increase of 25% compared with the control group.

[0026] Furthermore, the obtained tomato seed small molecule protein has a positive effect on the weight recovery of sarcopenic mice and can significantly improve the decline in muscle strength in sarcopenic mice, thus improving muscle loss.

[0027] Furthermore, the present invention provides the application of the above-mentioned small molecule protein in the preparation of products that delay muscle loss.

[0028] Furthermore, the present invention provides the application of the above-mentioned small molecule protein in the preparation of articles that improve muscle performance.

[0029] As a preferred embodiment of the application described in this invention, the product includes pharmaceuticals, cosmetics, and biological products.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The method for preparing small molecule proteins provided by this invention is simple, chemically stable, and has a significant effect on improving muscle loss. It is safe, can be used for a long time, and has no side effects.

[0031] 2. The small molecule bioactive protein provided by this invention can be used in products that improve muscle loss and related symptoms, providing a new technical means for research and application in related fields. Attached Figure Description

[0032] Figure 1 The effects of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the swallowing frequency of nematodes; Figure 2 The effects of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the frequency of body swaying in nematodes; Figure 3 The effect of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on mouse body weight; Figure 4 The effects of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the tensile strength of mouse hind limbs; Figure 5 The effects of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on mouse muscle mass. Detailed Implementation

[0033] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments used, unless otherwise specified, can be obtained commercially.

[0034] Example 1 A method for preparing small molecule protein from tomato seeds: Tomato seed powder and distilled water are mixed at a material-to-water ratio of 1:10. The pH is adjusted to 12.0 using 1M NaOH, shaken at 45℃ for 2 h, and centrifuged at 5500 ×g for 10 min. The supernatant is collected. The above steps are repeated once for re-extraction. The supernatants are combined, 5000 U of the first protease is added, and the mixture is shaken in a water bath at 50℃ for 2 h. After enzyme inactivation in a boiling water bath for 10 min, 5000 U of the second protease is added for enzymatic hydrolysis once more. After enzyme inactivation, the mixture is centrifuged at 5500 ×g for 10 min, and the supernatant is collected. The mixture is concentrated under vacuum at 55℃, dialyzed for 48 h, and freeze-dried for 48 h.

[0035] Example 2 A method for preparing a small molecule protein from tomato seeds: Tomato seed powder and distilled water were mixed at a material-to-water ratio of 1:20. The pH was adjusted to 9.0 using 1M NaOH, and the mixture was shaken at 55℃ for 2 h. After centrifugation at 4000 ×g for 30 min, the supernatant was collected. The above steps were repeated once for re-extraction. The supernatants were combined, and 3000 U of the first protease was added. The mixture was shaken in a water bath at 55℃ for 3 h, and the enzyme was inactivated by boiling in a water bath for 10 min. Then, 3000 U of the second protease was added for enzymatic hydrolysis once more. After enzyme inactivation, the mixture was centrifuged at 4000 ×g for 20 min, and the supernatant was collected. The mixture was concentrated under vacuum at 40℃, dialyzed for 24 h, and then freeze-dried for 48 h.

[0036] Example 3 A method for preparing a small molecule protein from tomato seeds: Tomato seed powder and distilled water are mixed at a material-to-water ratio of 1:30. The pH is adjusted to 10.0 using 1M NaOH, and the mixture is shaken at 60℃ for 2 h. After centrifugation at 6000 ×g for 15 min, the supernatant is collected. The above steps are repeated once for re-extraction. The supernatants are combined, and 10000 U of the first protease is added. The mixture is shaken in a water bath at 60℃ for 4 h, and the enzyme is inactivated by boiling in a water bath for 10 min. Then, 10000 U of the second protease is added for enzymatic hydrolysis once more. After enzyme inactivation, the mixture is centrifuged at 6000 ×g for 15 min, and the supernatant is collected and freeze-dried for 48 h.

[0037] Comparative Example 1 Preparation of small molecule protein from tomato seed: Tomato seed powder and distilled water were mixed at a material-to-water ratio of 1:10. The pH was adjusted to 9.0 with 1M NaOH, and the mixture was shaken at 45℃ for 2 h. After centrifugation at 8000 rpm for 10 min, the supernatant was collected. The above steps were repeated once for re-extraction. The supernatants were combined, and the pH was adjusted to 4.0 with 1M HCl. After centrifugation at 8000 rpm for 10 min, the precipitate was collected. After redissolving the precipitate, 1M NaOH was added to the solution, the pH was adjusted to 7.0, and the mixture was concentrated under vacuum at 55℃ for 48 h to remove salts. Finally, it was freeze-dried for 48 h.

[0038] Comparative Example 2 Preparation of small molecule protein from tomato seed: Tomato seed powder and distilled water were mixed at a material-to-water ratio of 1:10. The pH was adjusted to 12.0 using 1M NaOH. The mixture was shaken at 45℃ for 2 h, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The above steps were repeated once for re-extraction. The supernatants were combined, and 5000 U of neutral protease was added. The mixture was shaken in a water bath at 50℃ for 2 h, and the enzyme was inactivated by boiling in a water bath for 10 min. Then, 5000 U of bromelain was added for enzymatic hydrolysis once more. After enzyme inactivation, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected and freeze-dried for 48 h.

[0039] Comparative Example 3 Preparation of small molecule protein from tomato seed: Tomato seed powder and distilled water were mixed at a material-to-water ratio of 1:10. The pH was adjusted to 10.0 using 1M NaOH. The mixture was shaken at 55℃ for 2 h, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The above steps were repeated once for re-extraction. The supernatants were combined, and 5000 U of neutral protease was added. The mixture was shaken in a water bath at 50℃ for 2 h, and the enzyme was inactivated by boiling in a water bath for 10 min. Then, 5000 U of papain was added for enzymatic hydrolysis once more. After enzyme inactivation, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected and freeze-dried for 48 h.

[0040] 1. Evaluation of the anti-muscle loss efficacy of small molecule protein in tomato seeds 1.1 Based on the nematode model Detection of nematode body twitching frequency: The anti-muscle loss effect of tomato seed protein prepared by the methods described in Examples 1, 1, 2, and 3 was analyzed by measuring the body wiggling frequency of *Caenorhabditis elegans*. The tomato seed protein prepared in Comparative Example 2 was added to the diet of synchronized nematodes. On day 8, 20 nematodes were randomly selected, and their wiggling rate was measured using a stereomicroscope. The test nematodes were placed in M9 buffer and allowed to swim freely for 30 seconds to acclimatize before the test began. One complete wiggling of the nematode's head or tail was recorded as one instance, and the time was recorded for 30 seconds.

[0041] Detection of nematode swallowing frequency: The anti-muscle attenuation effect of tomato seed protein prepared by the methods described in Examples 1, 1, 2, and 3 was analyzed by measuring the swallowing frequency of *C. elegans*. The tomato seed protein prepared in Comparative Example 2 was added to the diet of synchronized nematodes. On days 1, 5, and 9, 20 nematodes were randomly selected, and their pharyngeal swallowing frequency was measured using a stereomicroscope. One cycle of pharyngeal pump pulsation was counted as one complete pulsation, and the number of pharyngeal pump pulsations was recorded every 30 seconds.

[0042] 1.2 Based on mouse model Mouse body weight determination: The effect of tomato seed protein prepared by the methods described in Examples 1, 1, 2, and 3 on improving muscle loss in mice was analyzed by comparing changes in mouse body weight. Mice with sarcopenia were administered tomato seed protein prepared in Comparative Example 2 by gavage for 3 weeks. During the experiment, the mice's food and water intake were measured daily, and their body weight was recorded. Data differences between each group were calculated. The final body weight of the mice was recorded at the end of the experiment.

[0043] Mouse muscle mass measurement: The effect of tomato seed protein prepared by the methods described in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 on improving muscle loss in mice was analyzed by comparing changes in mouse muscle mass. Tomato seed protein prepared in Comparative Example 2 was administered to sarcopenic mice by gavage for 3 weeks. At the end of the experiment, the mice were anesthetized, and muscle tissues from the legs, including the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles, were dissected and removed. The tissues were washed in physiological saline to remove blood stains, temporarily placed on ice with filter paper to absorb moisture, and quickly weighed and recorded (accurate to 0.0001 g).

[0044] Mouse tensile strength test: The effect of tomato seed protein prepared according to the methods described in Examples 1, 1, 2, and 3 on the muscle strength of mice was analyzed by measuring the pulling force of mice using a gripping force tester. Before the formal experiment, the gripping force tester was fixed to a horizontal surface, and a piece of wire mesh was connected to the tester using a rubber band. At the start of the experiment, the mouse was placed on the platform and allowed to grip the middle of the wire mesh. Once firmly gripped, the mouse's tail was grasped, and it was pulled horizontally backward. When the animal moves backward unintentionally, it will instinctively grip the wire mesh to prevent further movement until the pulling force exceeds its maximum gripping force. Therefore, the gripping force tester automatically recorded the maximum pulling force at this point, displaying it on the screen. Each mouse was tested four times, with a 5-minute interval between each mouse, after which the mice recovered their strength. The entire experiment was performed by the same operator.

[0045] Analysis of experimental results: Figure 1 This study examines the effects of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the swallowing frequency of nematodes. In comparison, the tomato seed small molecule protein obtained by the method in Example 1 significantly increased the swallowing frequency of nematodes on days 1, 5, and 9, with the most significant effect observed on day 9, showing a 30% increase compared to the control group. As nematodes age, their swallowing frequency tends to decrease, but the tomato seed small molecule protein obtained in Example 1 significantly slowed this trend, indicating that the small molecule protein obtained by this invention has the effect of delaying muscle atrophy during the aging process.

[0046] Figure 2This study examines the effects of proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the frequency of body wriggling in nematodes. In comparison, the tomato seed small molecule protein obtained by the method in Example 1 significantly increased the body wriggling frequency of nematodes, showing a 25% increase compared to the control group. Nematode muscle mass gradually declines with aging, leading to a decrease in their motor ability. By observing and comparing the body wriggling frequency of nematodes in different intervention groups, it was found that the small molecule protein obtained in Example 1 had a more significant effect on improving the motor ability of nematodes. The results indicate that the tomato seed small molecule protein prepared by the method of this invention has the greatest impact on the body wriggling frequency of nematodes and has the potential to improve muscle loss.

[0047] Figure 3 This study investigated the effects of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on mouse body weight. In the experiment, a mouse muscle loss model was constructed using a physical waste disposal method to simulate the process of sarcopenia. One week after modeling, we observed a significant decrease in body weight and hind limb pulling strength in all groups of mice, indicating the successful establishment of the muscle loss model. To further analyze the ameliorative effect of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 on muscle loss, we administered the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 to mice via gavage for three weeks. The results showed that, compared to the model group, after gavage administration of the tomato seed small molecule protein obtained by the method of Example 1, the mouse body weight no longer decreased and gradually recovered, indicating that this small molecule protein has a positive effect on the weight recovery of sarcopenic mice. The results indicate that the tomato seed small molecule protein prepared by the method of this invention has the greatest impact on body weight and has the potential efficacy in improving muscle loss.

[0048] Figure 4 This study investigated the effects of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on the pulling force of mice. Further analysis of the effects of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 on the muscle strength of mice was conducted. Changes in the pulling force of mice were measured weekly using a dynamometer. It was observed that after gavage administration of the small molecule protein obtained by the method in Example 1, the hind limb pulling force of the mice gradually recovered, indicating that the positive control drug has a significant effect on the recovery of muscle atrophy. Meanwhile, the proteins in Comparative Examples 2 and 3 also showed a certain degree of improvement in the recovery of muscle strength in mice. The results indicate that the tomato seed small molecule protein prepared by the method of this invention has the greatest effect on the pulling force of mice, significantly improving the decline in muscle strength in sarcopenic mice and demonstrating an effect on improving muscle atrophy.

[0049] Figure 5This study investigated the effects of the proteins prepared in Example 1 and Comparative Examples 1, 2, and 3 of this invention on mouse muscle mass. In the experimental study, we performed detailed sampling and weighing analysis of mouse leg muscles to evaluate the effects of different treatment groups on the attenuation of the gastrocnemius and soleus muscles. Through precise muscle weight measurement, we found that mice treated with the tomato seed small molecule protein obtained by gavage in Example 1 showed a significant improvement in the weight of their gastrocnemius and soleus muscles compared to the model group.

[0050] Specifically, the model group mice showed a significant decrease in the weight of their gastrocnemius and soleus muscles, consistent with the pathological characteristics of sarcopenia. However, mice administered the tomato seed small molecule protein obtained by gavage in Example 1 showed a clear recovery trend in the weight of their gastrocnemius and soleus muscles. These results indicate that the tomato seed small molecule protein prepared by the method of this invention has the greatest impact on mouse muscle mass and can significantly improve muscle loss in sarcopenic mice.

[0051] In summary, the tomato seed small molecule protein prepared by the method of this invention can not only improve muscle function, such as increasing hind limb pulling strength, but also directly act on muscle tissue to promote the recovery of muscle weight and mass, thereby achieving an anti-muscle loss effect.

[0052] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing tomato seed small molecule protein with anti-muscle atrophy effects, characterized in that, Includes the following steps: (1) Degreasing of tomato seeds: The ultra-finely pulverized tomato seed powder is degreased by steam expansion-wall breaking subcritical technology; (2) Crude extraction of tomato seed protein: Mix defatted tomato seed powder with distilled water and perform preliminary extraction in an alkaline environment, and collect the supernatant; (3) Crude protein hydrolysis of grains: The first protease and the second protease are added to the supernatant obtained in step (2) for targeted and segmented enzymatic hydrolysis, and the supernatant is collected; the first protease is Bacillus licheniformis protease, and the second protease is a compound flavor protease; the segmented enzymatic hydrolysis conditions are: after the first protease hydrolysis time is 2~4 h, the second protease is added for 2~4 h of enzymatic hydrolysis, the hydrolysis temperature is 45~65ºC, and the enzyme dosage is 5000~10000 U / g; (4) Preparation of small molecule protein: The protein solution obtained in step (3) is concentrated under vacuum at 40~60℃, desalted, and freeze-dried under vacuum to obtain a small molecule protein with anti-muscle attenuation activity.

2. The method for preparing tomato seed small molecule protein with anti-muscle atrophy effect according to claim 1, characterized in that, The tomato seed powder refers to the component that has been dried to constant weight at 50~60℃ and then pulverized to a particle size ≤ 355 μm.

3. The method for preparing tomato seed small molecule protein with anti-muscle atrophy effect according to claim 1, characterized in that, The alkaline environment is pH 9-12, the extraction time is 0.5-2 h, and the material-to-liquid ratio is 1:10-30 (g / mL).

4. The method for preparing tomato seed small molecule protein with anti-muscle atrophy effect according to claim 1, characterized in that, The supernatant was obtained by centrifugation at 4000~6000 ×g for 10~30 min.

5. The method for preparing tomato seed small molecule protein with anti-muscle atrophy effect according to claim 1, characterized in that, The desalination mentioned in step (4) refers to the removal of small molecules using membrane permeation or ultrafiltration methods, with a maximum molecular weight of 1000 to 3000 Da.

6. Tomato seed small molecule protein with anti-muscle atrophy effect prepared by the preparation method according to any one of claims 1-5.

7. The application of the tomato seed small molecule protein as described in claim 6 in the preparation of a drug for delaying muscle loss.

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