KR101342025B1 - Compositions for improvement of recognition and manufacturing method for the same - Google Patents

Abstract

The present invention relates to a composition for improving cognitive ability and a method of manufacturing the same. More specifically, the present invention relates to a composition for improving cognitive abilities and a method for producing the same, comprising a gelatin-derived enzyme as an active ingredient. According to the present invention, it is possible to develop an anti-dementia substance in the pork shell, which is a food material without side effects, and to improve the national health and health through the intake of safe natural products and achieve high value of the pork shell, which is a livestock by-product.

Description

Composition for improving cognition ability and a method for manufacturing the same {Compositions for improvement of recognition and manufacturing method for the same}

The present invention relates to a composition for improving cognitive ability and a method of manufacturing the same. More specifically, the present invention relates to a composition for improving cognitive abilities and a method for producing the same, comprising a gelatin-derived enzyme as an active ingredient.

The age distribution of dementia patients in Korea consists of 5-8% for people over 64 years old, 15-20% for people over 75 years old and 25-50% for people over 85 years old. Neurodegenerative disorders, such as Alzheimer's disease (AD), are chronic diseases characterized by loss of memory and cognition. The loss of cholinergic neurons in AD patients leads to a decrease in acetylcholinetransase (ChAT) and acetylcholinesterase (AChE) activity in the cerebral cortex and hippocampus. Has reported a link between the progression of dementia and the severity of neuropathological features of AD (Hugo et al., 1996; Chung et al., 2001). Thus, the recent cholinergic system transmits electrical signals in the human body. Increasing levels of AChE at synaptic sites through cholinergic agonists or AChE inhibitors are known to improve acetylcholin (ACh), a neurotransmitter. Animal hair with cognitive deficits, such as learning and memory, by anticholinergic drugs such as scopolamine Study on also in progress (Chung et al, 2001;.. Papandreou et al, 2009). As a number of side effects have been reported from AChE inhibitors, which are currently being used to improve the cognitive function of AD patients, research and development of natural-derived AChE inhibitors that can replace existing AChE inhibitors are emerging.

Under such technical background, the present inventors have made intensive efforts to develop a composition for improving cognitive ability, and have thus completed the present invention.

After all, the technical problem to be achieved by the present invention is to provide a composition for improving cognitive ability, characterized in that it contains a gelatin-derived enzyme degradation product as an active ingredient.

Another technical problem to be achieved by the present invention is to provide a method for preparing a composition for improving cognition ability, comprising a gelatin-derived enzyme degradation product as an active ingredient.

According to an aspect of the present invention, a composition for improving cognitive ability, comprising a gelatin-derived enzymatic degradation product as an active ingredient may be provided.

In one embodiment, the enzymatic digest may be a fraction of a gelatin derived enzymatic digest with a molecular weight of 200 to 50,000 Da.

In one embodiment, the enzymatic digest may be a fraction of a gelatin derived enzymatic digest with a molecular weight of 200 to 3,000 Da.

In one embodiment, the gelatin may be derived from pig skin.

In one embodiment, the enzyme may be one or more selected from the group consisting of alcalase, protamex and flavorzyme.

According to another aspect of the invention, the step of hydrolyzing the gelatin to obtain a gelatin solution; Enzymatically degrading the gelatin solution at 30 to 60 ° C. for 2 to 24 hours after adding the enzyme under the condition of pH 6 to 9; And desalination of the decomposition product may be provided a method for producing a composition for improving cognitive ability comprising the step of obtaining a fraction for each molecular weight.

In one embodiment, the enzyme may be at least one selected from alcalase, protamex, and flavorzyme.

In one embodiment, the enzyme and gelatin may be reacted at a weight ratio of 1:50 to 500.

According to the present invention, it is possible to develop an anti-dementia substance in the pork shell, which is a food material without side effects, and to improve the national health and health through the intake of safe natural products and achieve high value of the pork shell, which is a livestock by-product.

Figure 1 shows the inhibitory effect (%) of acetylcholine esterase of gelatin degrading enzyme.
Figure 2 shows the inhibitory effect (%) in the brain acetylcholinesterase after 16 weeks of gelatin enzyme digestion.
Figure 3 shows the results of the Y-maze test (%) after gelatin enzyme digestion.
Figure 4 shows the results of passive avoidance (sec) test after feeding gelatin enzyme digest.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, a composition for improving cognitive ability, comprising a gelatin-derived enzymatic degradation product as an active ingredient may be provided.

In this case, the enzymatic digest may be a fraction of a gelatin-derived enzymatic digest with a molecular weight of 200 to 50,000 Da.

According to an embodiment, the enzymatic digest may be a fraction of a gelatin derived enzymatic digest with a molecular weight of 200 to 3,000 Da.

According to one embodiment, the gelatin may be derived from pig skin.

According to one embodiment, the enzyme may be one or more selected from alcalase, protamex and flavorzyme.

According to another aspect of the invention, the step of hydrolyzing gelatin to obtain a gelatin solution; Enzymatically degrading the gelatin solution at 30 to 60 ° C. for 2 to 24 hours after adding the enzyme under the condition of pH 6 to 9; And desalination of the decomposition product may be provided a method for producing a composition for improving cognitive ability comprising the step of obtaining a fraction for each molecular weight.

According to one embodiment, the enzyme may be at least one selected from alcalase, protamex and flavorzyme.

According to one embodiment, the enzyme and gelatin can be reacted in a weight ratio of 1: 50 ~ 500.

In the present invention, the proteolytic enzyme used for the enzymatic digestion of gelatin may be used at least one selected from alcalase, protamex and flavorzyme.

Protease and gelatin are preferably used in a weight ratio of 1: 50 to 500. The reaction time, temperature, and pH of the enzyme may be different depending on the enzymes used, but it is preferable to carry out under conditions of 2 to 24 hours, 30 to 60 ° C, and pH 6.0 to 9.0, respectively. After desalting the obtained enzymatically decomposed product appropriately, it is preferable to separate and purify by ultrafiltration, gel filtration and various chromatography, membrane filter, and method using isoelectric point.

The gelatin hydrolyzate separated by the above method was found to have a molecular weight distribution of 200 to 50,000 Da, preferably 200 to 3,000 Da, by gel filtration and membrane filter.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1.gelatin Enzyme  Produce

(1) Hydrolysis of Gelatin Fractions

As shown in FIG. 1, 5 times of water was added to donpi gelatin (Note, Geltec) and left for 10 minutes, followed by hydration by stirring at 80 ° C. at a speed of 200 rpm for 1 hour. After that, the white foam was rolled out to obtain a yellow gelatin solution.

(2) Preparation of Gelatin Enzyme Degradation Products

The pH of the gelatine solution obtained above was adjusted to 7.0, and 1 g of AP (Alcalase + Protamex), PF (Protamex + Flavorzyme), FA (Flavorzyme + Alcalse), APF (Alcalase + Protamex + Flavorzyme Bosa NISA]) was added to decompose at 50 ° C. for 4, 12 and 24 hours, and then the salts were removed and the gelatine enzymatic products were 50 kDa or less, 50 kDa to 3 kDa, 3 kDa or less using a membrane filter. It was separated into a decomposition product of. 36 digests were prepared by dividing the enzyme digestion by digestion time, treatment enzyme and molecular weight size, and gelatin was prepared before enzymatic digestion as a positive control. Descriptions of these samples are shown in Table 1 below.

Sample number Molecular Weight (Da) Decomposition time Enzyme combination Sample number Molecular Weight (Da) Time Enzyme combination One





= 50,000






4
One 20

50,000-3,000

12 4 2 2 21

24
One 3 3 22 2 4 4 23 3 5
12
One 24 4 6 2 2525





= 3,000




4
One 7 3 26 2 8 4 27 3 9
24

One 28 4 10 2 29
12

One 11 3 30 2 12 4 31 3 13


50,000-3,000



4
One 32 4 14 2 33
24

One 15 3 34 2 16 4 35 3 17
12
One 36 4 18 2 37 gelatin - - 19 3

In Table 1, the enzyme combination is 1: alcalase + protamex, 2: protamex + flavorzyme, 3: flavorzyme + alcalase, 4: alkalase + pro Tamex + flavorzyme.

Example  2. Acetylcholinease Activity Inhibition  Measure

Acetylcholinesterase (AChE) inhibitory activity was measured using acetylcholine iodide as a substrate. The enzyme was homogenized using 12.5 mM sodium phosphate (pH 7.0) buffer in the brain of ICR mice, and centrifuged to extract the supernatant and used as an enzyme source. All extraction processes were performed at 4, and BCA Protein Assay kit (Sigma Co., USA) was used to measure the protein content of the extracted enzyme solution. Take 30μL of 50mM sodium phosphate biffer (pH8.0), add 10μL of extract and mix for 5 seconds. Add 10 μL of brain enzyme and 50 μL of Ellman's reaction mixture [0.5 mM acetylthiocholine, 1 mM 5,5'dithio-bis (2-nitrobenzoic acid)] and mix for 5 seconds. After incubation at 37 ° C. for 30 minutes, absorbance was measured every 2 minutes at 450 nm (Ellmans et al., 1961).

Example  3. Manual evasion experiment Passive avoidance test )

Passive avoidance experiments used a shuttle box consisting of two rooms, a dark room and a bright room. The two rooms have access to the guillotine doors, the bright ones are equipped with 100W bulbs, and the dark ones are connected to an electrical stimulator to allow electricity to flow along the grid. The experiment was conducted for two days. The first day was a study period, and 30 minutes before the lab, scopolamine was intraperitoneally administered at a dose of 1.0 mg / kg. Put the mouse in a bright room, after 10 seconds of search time, the bulb is turned on and the guillotine door is opened, the mouse moves to the dark room, and after the guillotine door is opened, the time is repeated twice until the mouse enters the dark place. Only migrated mice were selected and used. When the mouse entered the dark room, the guillotine door was closed and a 0.5 mA electric shock was applied for 5 seconds. The guillotine door was opened to move to a bright room with no electricity, and the study was completed for 120 seconds. After 24 hours the next day, the mouse was placed in the bright compartment again and after 10 seconds of search time the guillotine door was opened to allow access to the dark compartment. If you form a memory that experiences an electric shock when you enter a dark place from a bright place that the mouse hates, the memory test does not go into the dark place but stays in the bright place for a long time, so it takes 300 seconds for all four feet to enter the dark place. Measured to confirm the effect of improving memory.

Example  4. Y-Maze Experiment

In the Y-maze experiment, 16 weeks of gelatin enzyme digestion was administered, and then the scopolamine was intraperitoneally administered at the concentration of 1.0 mg / kg on the last day. In the Y-shaped labyrinth made of black acrylic plates according to the standard, each of the three papers was set to A, B, and C, and the mouse was placed on one branch and allowed to move freely for 8 minutes. It continued when the tail had been completely entered and also recorded when the branch had entered once again. If three different branches were entered in turn, one point was awarded. If no entry was made in a row, no points were awarded. Spontaneous alteration is expressed as the probability of dividing the measured score by 2 minus the total number of crossings.

Change (Cross) Behavior (%) = (A, BC Number of Crosses / Total Number of Crosses-2) * 100

In the present invention, based on the hypothesis that various cognitive disorders such as memory loss and learning loss caused by Alzheimer's dementia are mainly caused by damage of AChE neurons at the base of cerebral base (Chung et al., 2001). This study was conducted to investigate the preventive effect on AD by investigating AChE inhibitory activity.

1) Inhibition of Acetylcholine Degradation

 Increasing acetylcholine is known to improve memory and cognition. Acetylcholine in the brain is produced in the brain neurons by an enzyme called choline acetyltrasferase, released into the synaptic cleft, and then attached to the acetylcholine receptors in postsynaptic neurons to cholinergic neurotransmission. (cholinergic neurotransmission), which is regulated by acetylcholine degrading enzymes in the synaptic cleft. Inhibiting the action of acetylcholinesterase (acetylcholinesterase) prevents the breakdown of acetylcholine (acetylcholine) to increase the amount of acetylcholine available (maximum) and cholinergic neurotransmission increases to improve cognition, such as memory. This is the same effect as Aricept, which is currently used as a treatment for mild to moderate dementia in Alzheimer's. (Note: Aricept; Donepezil-HCl, an Alzheimer's dementia drug, reversibly inhibits Acetylcholinesterase, enhancing cholinergic functions related to cognitive function)

Gelatin enzyme digest was obtained from 37 samples of gelatin obtained from pig skin. The blank group refers to the group not treated with anything, and the control group refers to the group treated with only the enzyme and the substrate without processing the sample. When all the values were converted to 100% of the control group, the value of the Blank group was 14.80 ± 0.15%. In the group treated with # 17 [50k> Da> 3k 12 hrs (1)], acetylcholinesterase inhibitory effect was shown to be the best, and the # 17 treated group showed 44.92 ± 1.39% of activity. For comparison, 50KDa gelatin without enzyme treatment showed AChE activity of 55.86 ± 4.35% (FIG. 1).

Inhibition of acetylcholinesterase in the brain after 16 weeks of feeding gelatin enzyme degradants at 1.0, 2.0, and 4.0% showed a significant inhibitory effect of 48.93 ± 1.83, 47.79 ± 0.68 and 52.08 ± 1.16%, respectively. It was shown that the effect of improving the cognitive ability (Fig. 2). In Figure 2, the control: control, 1: gelatin lysate 1% feed group, 2: gelatin lysate 2% feed group, 3: gelatin lysate means 4% feed group.

2) Y-type maze

In order to determine the influence on the spatial cognition of gelatin enzyme digestion was measured using the Y-maze experiment (Fig. 3). The control group showed 38.99%, whereas the cognitive improvement was found to be 50.73 ± 1.72, 52.53 ± 2.56, 43.92 ± 4.92% when feeding 1.0, 2.0 and 4.0% of the gelatin enzyme digest, respectively, and significantly higher than the control when feeding 1.0 and 2.0%. As a result, cognitive ability was improved (p <0.05). In Figure 3, the control: control, 1: gelatin lysate 1% feed group, 2: gelatin lysate 2% feed group, 3: gelatin lysate means 4% feed group.

3) Manual evasion ability

As a result of passive avoidance experiment using forgetful-induced mice generated by scopolamine administration, the experimental group administered scopolamine showed a latency time of 38.33 ± 0.88 seconds (FIG. 4). On the other hand, when fed 1.0, 2.0, and 4.0% of gelatin degradate, 38.00 ± 7.56, 46.33 ± 5.36, and 48.80 ± 4.28 seconds, respectively, tended to increase, but there was no significant difference.

In Figure 4, the control: control, 1: gelatin lysate 1% feed group, 2: gelatin lysate 2% feed group, 3: gelatin lysate means 4% feed group.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

Fractions of gelatin-derived enzymes with molecular weights of 200 to 3,000 Da, digested with one or more enzymes selected from the group consisting of alcalase, protamex and flavorzyme, are active ingredients. A composition for improving cognitive ability, containing as acetylcholinesterase inhibitory activity.
delete delete The method of claim 1, wherein the gelatin is a composition for improving cognitive ability, characterized in that derived from pig skin.
delete delete delete delete

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