CN117204398A - A method for evaluating the antioxidant and anti-aging effects of egg white protein polypeptides based on the Caenorhabditis elegans model - Google Patents

Abstract

The invention relates to the field of raw materials of foods and cosmetics, and discloses a method for evaluating the antioxidation and anti-aging activity of an egg white protein polypeptide based on a caenorhabditis elegans model, wherein the method for evaluating the antioxidation and anti-aging activity of the egg white protein polypeptide comprises the following steps: preparing a culture medium, culturing and synchronizing nematodes, measuring the oxidative stress life of paraquat, measuring the oxidative stress life of H2O2, measuring the activity of antioxidant enzyme, measuring the normal life, measuring the lipofuscin content, measuring the spawning quantity and the like. The method for evaluating the oxidation resistance and the aging resistance of the egg white protein polypeptide based on the caenorhabditis elegans model has the characteristics of convenient operation, low cost, short period and the like, can evaluate the effect of the egg white protein polypeptide in organisms with high efficiency, and reflects biological significance.

Description

A method to evaluate the antioxidant and anti-aging effects of egg white protein polypeptides based on the Caenorhabditis elegans model method

Technical field

The invention relates to the field of food and cosmetic raw materials, and is specifically a method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model.

Background technique

Caenorhabditis elegans, referred to as nematodes, belongs to Phylumnematoda, Seternentea, Rhabditida, Rhabditidae, and Caenorhabditis. C.elegans includes the oral cavity, pharynx, intestine, gonads, and collagenous cuticle. There are two sexes, hermaphrodites and males. Most individuals are hermaphrodites, with males accounting for only 0.05%. The germ line used in experimental research is usually hermaphrodite N2 wild-type C. elegans (C. elegans, the Bristol strain N2). C. elegans has approximately 60%-80% human homologous genes and contains at least 42% of human disease-related genes (Sugi et al., 2016) and C. elegans does not require the approval of an animal ethics committee. These advantages make C.elegans a widely used animal model, often used to study anti-aging directions.

The growth cycle of Caenorhabditis elegans is short. It only takes 3-4 days to develop from egg to adult. Under 20℃ culture conditions, the average lifespan is 2-3 weeks. A hermaphrodite adult can lay about 300 eggs. C The life cycle of .elegans includes four larval stages (L1~L4) and the adult stage. Nematode eggs will enter the L1 stage of larvae after being cultured at 20°C for 15 hours. At this time, the nematode body is only 250um, but its structure is the same as that of the adult worm. If the culture environment is suitable and the nutrition is sufficient at this time, the nematodes will grow from the L1 stage to the L4 stage in about 3 days and form adult worms. At this time, the nematode size will be 1-1.5mm. If there are not sufficient growth conditions in the L1 stage, the nematode growth will stagnate and enter dormancy. This period is called the dauer stage. The lifespan of nematodes in this stage can be as long as 6 months. If the conditions for nematode growth and adaptation are reached again, the nematodes will directly skip the L3 stage to the L4 stage and develop into adults. Taking advantage of this feature, the L1 stage larvae can be stored at -80°C for a long time and then resuscitated when used.

There is a certain basis for using Caenorhabditis elegans as a model to study the role of antioxidant stress. Oxidative stress in the usual sense means that under certain extreme circumstances, the body's tissues and organs will become imbalanced. Research shows that the increase in oxygen free radicals can lead to protein abnormalities and lipid accumulation, and cause oxidative damage to macromolecules such as DNA. Antioxidation in nematodes is closely related to oxidative stress. Studies have confirmed that Cirsium japonicum processed in different ways can improve the oxidative stress response of Caenorhabditis elegans to varying degrees. Feeding nematodes a sterol-rich plant drink improves their oxidative stress response to extreme conditions.

C. elegans is a model organism widely used in many fields such as genetic biology, gene function, aging, and drug screening. Caenorhabditis elegans has several advantages as a primary model organism for exploring various biological processes:

(1) The structure is simple and the body is transparent, making it easy to observe under a microscope; it feeds on OP50 bacteria, making it easy to culture and easy to preserve and recover.

(2) The life cycle is short, which is convenient for experimental operations; synchronization is easy, which reduces the interference of external factors on experiments.

(3) It has complex behavior, including mechanical perception and chemical perception; it is also similar to human body behavior and physiology. With age, movement slows down and oxidation products in the body increase.

(4) It is 60-80% similar to human genes, making research more meaningful.

Contents of the invention

Based on this, the present invention discloses a method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model, including medium preparation, nematode culture and synchronization, paraquat oxidative stress lifespan measurement, H2O2 oxidative stress lifespan measurement, Antioxidant enzyme activity determination, normal life span determination, lipofuscin content determination and egg laying volume determination and other steps.

Compared with the existing technology, the evaluation method of the present invention has the characteristics of easy operation, low cost, short cycle, etc., can efficiently evaluate the role of egg white protein polypeptides in the organism, and reflects the biological significance.

In order to achieve the above experimental purpose, the present invention adopts the following technical solutions

A method for evaluating the antioxidant and anti-aging effects of egg white protein polypeptides based on the Caenorhabditis elegans model mainly includes the following steps:

S1. Medium preparation: Prepare nematode growth solid medium (NGM), LB liquid medium, LB solid medium, and paraquat medium, and sterilize them for later use.

S2. Nematode culture and synchronization: streak the E.coli OP50 strain on an LB plate, pick a single colony and culture it in LB liquid medium to a certain bacterial concentration, and spread it on the NGM plate. Inoculate the frozen nematodes into NGM plates after resuscitation. After the nematodes lay eggs, wash and lyse the adult worms with M9 buffer solution and lysis solution. On the ultra-clean workbench, use a pipette to absorb an appropriate amount of nematode eggs and drop them onto the E-coated .coli OP50 NGM plate, placed in a 20°C incubator for 3 days to complete synchronized culture.

S3. Paraquat oxidative stress lifespan measurement: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. , then transfer the nematodes to 5-30mmol/L paraquat medium, record the death of plate nematodes and the number of remaining nematodes in each group every day, and pick out dead nematodes until all nematodes die.

S4, H2O2 oxidative stress lifespan measurement: Pick the nematodes obtained from S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. Dilute 10%-60% H2O2 1000 times and apply it evenly on the bacterial solution, then transfer the nematodes to the NGM medium containing H2O2, record the death of nematodes and the number of remaining nematodes every 30 minutes, and pick out the dead nematodes until All nematodes died.

S5. Antioxidant enzyme activity measurement: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. The M9 buffer was prepared into a nematode homogenate solution with a volume ratio of 5% to 30%, and the nematode tissue protein was extracted. The protein content, catalase (CAT) enzyme activity, and superoxide dismutase (Superoxide) were measured using a kit. dismutase (SOD) enzyme activity, reduced glutathione (GSH) enzyme activity, and malondialdehyde (MDA) content.

S6. Normal life span determination: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, and place them in an incubator at 20°C-25°C. The day will be regarded as day 0, and then Transfer the nematodes to new experimental and control plates every day, record the death of nematodes and the number of remaining nematodes every day, and pick out dead nematodes until all nematodes die.

S7. Determination of lipofuscin content: Pick the nematodes obtained in S2 into the plates of the experimental group and the control group, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-168h. Anesthetize the nematodes with 1%-20% NaN3, and then place them on a glass slide dripped with 3%-20% agarose pad. Observe and take photos with an inverted fluorescence microscope, and measure the fluorescence intensity with Image J software.

S8. Determination of egg laying volume: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 10-30 worms in each group, and place them in an incubator at 20℃-25℃. The day from that day is regarded as day 0. , after every 24 hours, pick the nematodes to the new experimental group and control plates until the nematodes no longer lay eggs. The oviposition plate was placed in an incubator at 20°C-25°C and incubated for 24h-72h and then the number of nematodes was recorded.

According to the above protocol, the medium used for nematode culture is described in S1.

According to the above protocol, the concentration of the E.coli OP50 bacterial solution to be inoculated as described in S2 should be between 0.1-0.8 with an OD600 of 50uL-300uL.

According to the above scheme, the number of nematodes in each group described in S3, S4, S5, S6, S7 and S8 was evenly distributed on 3-6 plates.

According to the above scheme, the criteria for judging the death of nematodes described in S3, S4 and S6 are that individual nematodes are considered dead if they do not respond to slight mechanical stimulation; nematode counting excludes nematodes hatched in the body and nematodes that escape.

According to the above scheme, the inverted fluorescence microscope observation described in S7 is to use a 10-30x objective lens to observe at an excitation wavelength of 485nm and an emission wavelength of 530nm.

To sum up, the present invention mainly has the following beneficial effects:

It has the characteristics of easy operation, low cost and short cycle. It can efficiently evaluate the role of egg white protein peptides in the body and reflect the biological significance.

Description of the drawings

Figure 1 is a comparison chart of the evaluation results of the nematodes of the present invention on paraquat stress lifespan;

Figure 2 is a comparison chart of the evaluation results of the nematodes of the present invention on hydrogen peroxide stress lifespan;

Figure 3 is a comparison chart of CAT antioxidant enzyme activity results in nematodes of the present invention;

Figure 4 is a comparison chart of SOD antioxidant enzyme activity results in nematodes of the present invention;

Figure 5 is a comparison chart of the GSH-Px antioxidant enzyme activity results in nematodes of the present invention;

Figure 6 is a comparison chart of MDA content results in the nematode of the present invention;

Figure 7 is a comparison chart of evaluation results of normal life span of nematodes of the present invention;

Figure 8 is a comparative chart of the evaluation results of lipofuscin content in nematodes according to the present invention;

Figure 9 is a comparison chart of the evaluation results of nematode egg production according to the present invention.

Detailed ways

The present invention will be further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the present invention are all commercially available.

Example 1:

Medium preparation: Prepare nematode growth solid medium (NGM), LB liquid medium, LB solid medium, and paraquat medium, and sterilize them for later use.

Nematode culture and synchronization: streak the E.coli OP50 strain on an LB plate, pick a single colony and culture it in LB liquid medium to a certain bacterial concentration, and spread it on the NGM plate. Inoculate the frozen nematodes into NGM plates after resuscitation. After the nematodes lay eggs, wash and lyse the adult worms with M9 buffer solution and lysis solution. On the ultra-clean workbench, use a pipette to absorb an appropriate amount of nematode eggs and drop them onto the E-coated .coli OP50 NGM plate, placed in a 20°C incubator for 3 days to complete synchronized culture.

Determination of paraquat oxidative stress life span: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and then transfer the nematodes to In 20mmol/L paraquat medium, the death of plate nematodes and the number of remaining nematodes in each group were recorded every day, and dead nematodes were picked out until all nematodes died.

H2O2 oxidative stress lifespan measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and dilute 50% H2O2 to 1000 After doubling, apply it evenly on the bacterial solution, then transfer the nematodes to the NGM medium containing H2O2, record the death of nematodes and the number of remaining nematodes every 30 minutes, and pick out dead nematodes until all nematodes die.

Antioxidant enzyme activity measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and use M9 buffer to adjust the volume Use a 20% nematode homogenate solution to extract nematode tissue protein, and use a kit to measure protein content, catalase (CAT) enzyme activity, superoxide dismutase (SOD) enzyme activity, and reduced form. Glutathione (GSH) enzyme activity and malondialdehyde (MDA) content.

Normal life span determination: Pick the nematodes obtained in step (2) into the experimental group and control group plates, with 180 worms in each group, and place them in an incubator at 23°C. The day is regarded as day 0, and then the nematodes are transferred every day. In the new experimental group and control plates, record the death of nematodes and the number of remaining nematodes every day, and pick out dead nematodes until all nematodes die.

Determination of lipofuscin content: Pick the nematodes obtained in step (2) into the plates of the experimental group and the control group, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and anesthetize the nematodes with 10% NaN3. Then place on a glass slide dripped with 10% agarose pad. Observe and take photos with an inverted fluorescence microscope, and measure the fluorescence intensity with Image J software.

Determination of egg laying volume: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 20 worms in each group, and place them in an incubator at 23°C. The current day is regarded as day 0, and every 24 hours Then pick the nematodes to the new experimental group and control plate and wait until the nematodes no longer lay eggs. The oviposition plate was placed in an incubator at 23°C for 24 hours and then the number of nematodes was recorded.

Example 2:

(1) Medium preparation: Prepare nematode growth solid medium (NGM), LB liquid medium, LB solid medium, and paraquat medium, and sterilize them for later use.

(2) Nematode culture and synchronization: streak the E.coli OP50 strain on an LB plate, pick a single colony and culture it in LB liquid medium to a certain bacterial concentration, and spread it on the NGM plate. Inoculate the frozen nematodes into NGM plates after resuscitation. After the nematodes lay eggs, wash and lyse the adult worms with M9 buffer solution and lysis solution. On the ultra-clean workbench, use a pipette to absorb an appropriate amount of nematode eggs and drop them onto the E-coated .coli OP50 NGM plate, placed in a 20°C incubator for 3 days to complete synchronized culture.

(3) Paraquat oxidative stress lifespan measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and then The nematodes were transferred to 20 mmol/L paraquat medium, and the death of nematodes and the number of remaining nematodes in each group were recorded every day, and dead nematodes were picked out until all nematodes died.

(4) H2O2 oxidative stress lifespan measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, and culture for 96 hours. 50% H2O2 is diluted 1000 times and spread evenly on the bacterial solution, and then the nematodes are transferred to the NGM medium containing H2O2. The death of nematodes and the number of remaining nematodes are recorded every 30 minutes, and dead nematodes are picked out until all nematodes are dead.

(5) Antioxidant enzyme activity measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and use M9 buffer Prepare a nematode homogenate solution with a volume ratio of 20%, extract nematode tissue protein, and use a kit to measure protein content, catalase (CAT) enzyme activity, and superoxide dismutase (Superoxide dismutase, SOD) enzyme activity. , reduced glutathione (Glutathione, GSH) enzyme activity, malondialdehyde (MDA) content.

(6) Normal life span measurement: Pick the nematodes obtained in step (2) into the experimental group and control group plates, with 180 worms in each group, and place them in an incubator at 23°C. The day is regarded as day 0, and then every day Transfer the nematodes to new experimental and control plates, record the death of nematodes and the number of remaining nematodes every day, and pick out dead nematodes until all nematodes die.

(7) Determination of lipofuscin content: Pick the nematodes obtained in step (2) into the experimental group and control group plates, 180 worms in each group, place them in an incubator at 23°C, culture for 96 hours, and use 10% NaN3 Nematodes were anesthetized and placed on a glass slide with a 10% agarose pad. Observe and take photos with an inverted fluorescence microscope, and measure the fluorescence intensity with Image J software.

(8) Determination of egg production: Pick the nematodes obtained in step (2) into the experimental group and control group plates, with 20 worms in each group, and place them in an incubator at 23°C. This day is regarded as day 0. After every 24 hours, pick the nematodes to a new experimental group and control group plate until the nematodes no longer lay eggs. The oviposition plate was placed in an incubator at 23°C for 24 hours and then the number of nematodes was recorded.

Activity evaluation results:

As shown in Figure 1, Figure 2, and Table 1, in the two anti-oxidative stress experiments, the growth curve of C. elegans under the action of egg white protein peptides significantly shifted to the right (p<0.01), and the average and median life spans were and maximum lifespan were increased, indicating that egg white protein peptides can alleviate oxidative stress and have antioxidant activity in nematodes. As shown in Figures 3, 4, 5, and 6, the enzyme activities of SOD, CAT, and GSH-Px in Caenorhabditis elegans were significantly increased (p<0.01) by 0.4 times and 1.25 times respectively compared with the control group. and 2 times, compared with the control group, MDA in vivo was significantly reduced (p<0.05), reduced by 28%. It shows that egg white protein peptides can activate the antioxidant enzyme activity in the body and relieve oxidative damage in the body, and have antioxidant effects. As shown in Figure 7 and Table 2, under the action of egg white protein peptide, the growth curve of nematodes significantly shifted to the right within 0-15 days; their average lifespan, median lifespan and maximum lifespan increased. It shows that egg white protein peptide has the effect of anti-aging and extending lifespan. As shown in Figure 8, the lipofuscin fluorescence in nematodes after the action of egg white protein peptides was significantly weakened, and its fluorescence intensity was reduced by 1 times (p<0.01). It shows that egg white protein peptide can reduce the accumulation of lipofuscin in the body and has antioxidant and anti-aging effects. As shown in Figure 9, there is no change in the number of eggs laid by nematodes after the action of egg white protein peptides, which has no impact on reproductive capacity and is safe and harmless.

Table 1 Evaluation results of nematodes of the present invention on paraquat stress lifespan and hydrogen peroxide stress lifespan

Table 2 Evaluation results of normal life span of nematodes of the present invention

Although embodiments of the invention have been shown and described, these specific embodiments are merely illustrative of the invention and are not intended to limit the invention. The specific features, structures, materials or characteristics described may be used in any one or more The embodiments or examples are combined in a suitable manner. After reading this description, those skilled in the art can make modifications and substitutions to the embodiments as needed without inventive contribution without departing from the principles and purposes of the present invention. and modifications, etc., as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims (6)

1. A method for evaluating the antioxidant and anti-aging effects of egg white protein polypeptides based on the Caenorhabditis elegans model, which mainly includes the following steps: S1. Medium preparation: Prepare nematode growth solid medium, LB liquid medium, LB solid medium, paraquat medium, sterilize and set aside; S2. Nematode culture and synchronization: streak the E.coli OP50 strain on an LB plate, pick a single colony and culture it in LB liquid medium to a certain bacterial concentration, and spread it on the NGM plate. Inoculate the frozen nematodes into NGM plates after resuscitation. After the nematodes lay eggs, wash and lyse the adult worms with M9 buffer solution and lysis solution. On the ultra-clean workbench, use a pipette to absorb an appropriate amount of nematode eggs and drop them onto the E-coated .coli OP50 NGM plate, placed in a 20°C incubator for 3 days to complete synchronized culture; S3. Paraquat oxidative stress lifespan measurement: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. , then transfer the nematodes to 5-30mmol/L paraquat medium, record the death of plate nematodes and the number of remaining nematodes in each group every day, and pick out dead nematodes until all nematodes die; S4, H2O2 oxidative stress lifespan measurement: Pick the nematodes obtained from S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. Dilute 10%-60% H2O2 1000 times and apply it evenly on the bacterial solution, then transfer the nematodes to the NGM medium containing H2O2, record the death of nematodes and the number of remaining nematodes every 30 minutes, and pick out the dead nematodes until All nematodes died; S5. Antioxidant enzyme activity measurement: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-120h. M9 buffer is prepared into a nematode homogenate solution with a volume ratio of 5%-30%, and the nematode tissue protein is extracted. The protein content, catalase activity, superoxide dismutase activity, and reduced glutathione are measured using a kit. Glypeptidase activity and malondialdehyde content; S6. Normal life span determination: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 150-300 worms in each group, and place them in an incubator at 20°C-25°C. The day will be regarded as day 0, and then Transfer nematodes to new experimental and control plates every day, record the death of nematodes and the number of remaining nematodes every day, and pick out dead nematodes until all nematodes die; S7. Determination of lipofuscin content: Pick the nematodes obtained in S2 into the plates of the experimental group and the control group, with 150-300 worms in each group, place them in an incubator at 20°C-25°C, and culture for 72h-168h. Anesthetize the nematodes with 1%-20% NaN3, and then place them on a glass slide dripped with 3%-20% agarose pad. Observe and take photos with an inverted fluorescence microscope, and measure the fluorescence intensity with ImageJ software; S8. Determination of egg laying volume: Pick the nematodes obtained in S2 into the experimental group and control group plates, with 10-30 worms in each group, and place them in an incubator at 20℃-25℃. The day from that day is regarded as day 0. , after every 24 hours, pick the nematodes to the new experimental group and control plates until the nematodes no longer lay eggs. The oviposition plate was placed in an incubator at 20°C-25°C and incubated for 24h-72h and then the number of nematodes was recorded. 2. A method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model according to claim 1, characterized in that: the medium described in S1 is the medium used for nematode culture. 3. A method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model according to claim 1, characterized in that: the concentration of the inoculated E.coli OP50 bacterial solution described in S2 is between 0.1-0.8 in OD600 , the nematode egg droplets applied to the E.coliOP50 NGM plate are 50uL-300uL. 4. A method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model according to claim 1, characterized in that: the average number of nematodes in each group in S3, S4, S5, S6, S7 and S8 Distributed on 3-6 plates. 5. A method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model according to claim 1, characterized in that: the nematode death judgment standard in S3, S4 and S6 is if the individual nematode is exposed to slight mechanical stress. No response to stimulation was considered dead; nematode counting excluded nematodes that hatched in the body and those that escaped. 6. A method for evaluating the anti-oxidation and anti-aging of egg white protein polypeptides based on the Caenorhabditis elegans model according to claim 1, characterized in that: the inverted fluorescence microscope observation in S7 is performed using a 10-30x objective lens at an excitation wavelength of 485nm. and observed at an emission wavelength of 530 nm.