阅读说明：本技术 一种具有改善老年认知功能障碍的牡蛎活性肽及其制备方法和应用 (Oyster active peptide capable of improving senile cognitive dysfunction and preparation method and application thereof ) 是由 朱国萍 章超桦 秦小明 曹文红 卢虹玉 郑惠娜 高加龙 林海生 曾少葵 于 2020-07-16 设计创作，主要内容包括：本发明属于功能肽领域,涉及一种具有改善老年认知功能障碍牡蛎活性肽的制备方法,包括如下步骤：牡蛎经碱提酸沉提蛋白,用中性蛋白酶酶解后,经超滤膜超滤获得5-8kDa的超滤组分,即为所述牡蛎活性肽。本发明的所述牡蛎活性肽具有改善老年认知功能障碍的作用。(The invention belongs to the field of functional peptides, and relates to a preparation method of oyster active peptide capable of improving senile cognitive dysfunction, which comprises the following steps: the oyster is subjected to alkali extraction, acid precipitation and protein extraction, enzymolysis is carried out by neutral protease, and ultrafiltration is carried out by an ultrafiltration membrane to obtain an ultrafiltration component of 5-8kDa, namely the oyster active peptide. The oyster active peptide has the effect of improving senile cognitive dysfunction.)

1. A preparation method of oyster active peptide capable of improving senile cognitive dysfunction comprises the following steps: the oyster is subjected to alkali extraction, acid precipitation and protein extraction, enzymolysis is carried out by neutral protease, and ultrafiltration is carried out by an ultrafiltration membrane to obtain an ultrafiltration component of 5-8kDa, namely the oyster active peptide.

2. The production method according to claim 1, wherein the production method comprises the steps of:

s1, extracting protein by alkali extraction and acid precipitation of oyster:

homogenizing Carnis Ostreae, adding water at a ratio of 1: 1-5(w/v), homogenizing in ice bath with high speed disperser for 1-8min, adjusting pH to 10.0-14.0, stirring at low temperature for reaction, centrifuging at low temperature, collecting supernatant, adjusting pH to 4.0-6.0, centrifuging at low temperature, and collecting precipitate;

s2, enzymolysis:

adding ultrapure water into the precipitate obtained in the step S1 at a ratio of 1: 1-5(w/v), adjusting the pH to 6-8, adding neutral protease, oscillating at a constant temperature of 40-60 ℃ for 2-6h, inactivating the enzyme at a high temperature, cooling, centrifuging, and taking supernatant;

s3, ultrafiltration:

and (3) performing ultrafiltration on the oyster protein enzymolysis supernate obtained in the step S2 by using an 8kDa ultrafiltration membrane, separating the supernate penetrating through the ultrafiltration membrane by using a 5kDa ultrafiltration membrane, and obtaining the oyster active peptide as a 5-8kDa ultrafiltration component of the supernate intercepted by the 5kDa ultrafiltration membrane.

3. The production method according to claim 2, wherein step S1 includes:

homogenizing oyster meat, adding water at a ratio of 1: 2-4(w/v), homogenizing in ice bath with high speed disperser for 3-6min, adjusting pH to 11.5-12.5 with 0.08-0.12mol/L NaOH, stirring at 0-4 deg.C for 2-4h, centrifuging at 0-4 deg.C and 8000-.

4. The production method according to claim 2, wherein step S2 includes:

adding ultrapure water into the precipitate obtained in the step S1 at a ratio of 1: 2-4(w/v), adjusting the pH to 6.8-7.2 by using 0.08-0.12mol/L NaOH, adding neutral protease at a mass ratio of enzyme to substrate of 1-5: 100, carrying out constant temperature oscillation at 40-60 ℃ for 3-5h, then carrying out enzyme inactivation at 95-100 ℃ for 8-12min, cooling, then centrifuging at 10-30 ℃ and 12000r/min for 10-30min, and taking the supernatant.

5. Oyster active peptides having an effect of improving cognitive dysfunction in the elderly, prepared by the method of any one of claims 1 to 4.

6. Use of the oyster peptide according to claim 5 for the preparation of a food or a pharmaceutical product having an activity of improving cognitive dysfunction in the elderly.

Technical Field

The invention belongs to the field of functional peptides, and particularly relates to a preparation method of oyster active peptide capable of improving senile cognitive dysfunction.

Background

The oyster peptide is prepared by applying peptide molecular biotechnology to oyster processing and performing enzymolysis. The small molecular oligopeptide formed by the preparation method reserves the nutritional components such as taurine, zinc and the like which are rich in oysters, so that the oysters rich in the small molecular active peptide are absorbed more quickly and easily by the human body after being ingested than single amino acid or protein, and have more important biological activity in the aspect of human metabolism. Has higher biological value and more important physiological function than the common oyster products.

The cognitive function is the comprehensive expression of human body information acquisition, memory, calculation, expression and execution capacity and is an important component of life quality. Impaired or reduced cognitive function is an early symptom of dementia, and if the impairment is severe, daily life and care are affected. China already enters an old-aged society, and the influence of cognitive dysfunction on the health of the old is not negligible. Therefore, early intervention on influencing factors of cognitive dysfunction of the old is of practical significance and is one of important contents of the old medical science.

Disclosure of Invention

The invention aims to provide a preparation method of oyster active peptide capable of improving senile cognitive dysfunction.

In order to achieve the above object, the present invention provides a method for preparing oyster active peptide, comprising the following steps: the oyster is subjected to alkali extraction, acid precipitation and protein extraction, enzymolysis is carried out by neutral protease, and ultrafiltration is carried out by an ultrafiltration membrane to obtain an ultrafiltration component of 5-8kDa, namely the oyster active peptide.

Further, the preparation method comprises the following steps:

s1, extracting protein by alkali extraction and acid precipitation of oyster:

homogenizing Carnis Ostreae, adding water at a ratio of 1: 1-5(w/v), homogenizing in ice bath with high speed disperser for 1-8min, adjusting pH to 10.0-14.0, stirring at low temperature for reaction, centrifuging at low temperature, collecting supernatant, adjusting pH to 4.0-6.0, centrifuging at low temperature, and collecting precipitate;

s2, enzymolysis:

adding ultrapure water into the precipitate obtained in the step S1 at a ratio of 1: 1-5(w/v), adjusting the pH to 6-8, adding neutral protease, oscillating at a constant temperature of 40-60 ℃ for 2-6h, inactivating the enzyme at a high temperature, cooling, centrifuging, and taking supernatant;

s3, ultrafiltration:

and (3) performing ultrafiltration on the oyster protein enzymolysis supernate obtained in the step S2 by using an 8kDa ultrafiltration membrane, separating the supernate penetrating through the ultrafiltration membrane by using a 5kDa ultrafiltration membrane, and obtaining the oyster active peptide as a 5-8kDa ultrafiltration component of the supernate intercepted by the 5kDa ultrafiltration membrane.

Further, step S1 includes:

homogenizing oyster meat, adding water at a ratio of 1: 2-4(w/v), homogenizing in ice bath with high speed disperser for 3-6min, adjusting pH to 11.5-12.5 with 0.08-0.12mol/L NaOH, stirring at 0-4 deg.C for 2-4h, centrifuging at 0-4 deg.C and 8000-.

Further, step S2 includes:

adding ultrapure water into the precipitate obtained in the step S1 in a ratio of 1: 2-4(w/v), adjusting the pH to 6.8-7.2 by using 0.08-0.12mol/L NaOH, adding neutral protease in a mass ratio (E/S) of enzyme to substrate (E/S) of 1-5: 100, carrying out constant temperature oscillation at 40-60 ℃ for 3-5h, then carrying out enzyme inactivation at 95-100 ℃ for 8-12min, cooling, then centrifuging at 10-30 ℃ and 8000-one-drug at 12000r/min for 10-30min, and taking the supernatant.

The second aspect of the present invention provides an oyster active peptide produced by the above-mentioned method.

The invention verifies the effect of the oyster active peptide on improving the cognitive function of the elderly through a zebra fish experiment. The third aspect of the present invention provides the use of the oyster peptide for preparing a food or a pharmaceutical product having an activity of improving cognitive dysfunction in the elderly.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

Figure 1 shows the effect of oyster active peptides on the success rate of zebrafish entry into the food placement area.

Figure 2 shows the effect of oyster active peptides on zebrafish latency.

Figure 3 shows the effect of oyster bioactive peptide on the right arm travel path of zebrafish.

Figure 4 shows the effect of oyster bioactive peptide on the swimming time of the right arm of zebrafish.

Figure 5 shows the effect of oyster bioactive peptides on the BDNF content of zebrafish fish brain.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the invention, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein.

Example 1

This example illustrates the preparation of oyster active peptides according to the invention.

And (3) extracting protein by acid precipitation of oyster alkali:

homogenizing oyster meat, adding water (1 part of oyster meat and 3 parts of water) at a ratio of 1: 3(w/v), homogenizing for 5min in an ice bath by using a high-speed disperser, adjusting pH to 12.0 by using 0.1mol/L NaOH, stirring for 3h at a constant temperature of 4 ℃, centrifuging for 20min at 10000r/min at 4 ℃, taking supernatant, adjusting pH to 4.8 by using 0.1mol/L HCl, centrifuging for 20min at 4 ℃, and taking precipitate.

Enzymolysis: adding ultrapure water (1 part of protein and 3 parts of water) into the extracted protein at a ratio of 1: 3(w/v), adjusting the pH to 7.0 by using 0.1mol/L NaOH, adding neutral protease at a mass ratio of 2: 100 of enzyme to substrate, oscillating at a constant temperature of 50 ℃ for 4h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging at 20 ℃ and 10000r/min for 20min, and taking the supernatant for ultrafiltration.

And (3) ultrafiltration:

and (3) carrying out enzymolysis on the supernatant by using 8kDa ultrafiltration oyster protein, separating the supernatant which permeates through the ultrafiltration membrane by using a 5kDa ultrafiltration membrane, and obtaining a 5-8kDa ultrafiltration component which is the supernatant intercepted by the 5kDa ultrafiltration membrane, namely the oyster active peptide.

Test example 1

Influence of oyster active peptide on zebra fish ethology.

Figure 1 shows the effect of oyster active peptides on the success rate of zebrafish entry into the food placement area. Figure 2 shows the effect of oyster active peptides on the latency of zebrafish entry into the food holding area. Figure 3 shows the effect of oyster active peptide on the right arm travel path of zebrafish. Figure 4 shows the effect of oyster bioactive peptide on zebrafish right arm swimming time. Wherein YF is young fish, YF + UF2 is young fish fed with oyster active peptide; AF is old fish, and AF + UF2 is old fish fed with oyster active peptide.

As can be seen from fig. 1 to 4, the elderly fish have a lower success rate of entering the food-holding area than the young fish, and have a longer incubation period. After the oyster active peptide is fed, the success rate of the young fish and the old fish in four days is greatly improved, wherein the success rate of the young fish in the next day is obviously improved, and the success rate of the old fish in the fourth day is obviously improved. After the oyster active peptide is fed, the latency time of the young fish and the old fish entering a food placing area is reduced to different degrees in the test process of four days, wherein the young fish is significantly reduced on the third day, and the old fish is significantly reduced on the 4 th day; the moving distance of the right arm is improved in different degrees in four days, the young fish is remarkably improved in 1-3 days, and the old fish is remarkably improved in four days; the swimming time of the right arm of the old fish is obviously improved within 1-3 days, while the swimming time of the right arm of the young fish is obviously improved within 2 days and 3 days.

Test example 2

Influence of oyster active peptide on antioxidant activity of zebra fish.

Compared with the young fish, the SOD activity in the brain of the old fish is obviously reduced, the SOD activity is increased to be close to the young level after the old fish is fed with the oyster active peptide, the age has little influence on the CAT activity, but the activities of the young fish and the old fish are obviously increased after the old fish is fed with the oyster active peptide. MDA reflecting oxidative damage is greatly increased on old fish bodies, and is obviously reduced after being fed with oyster active peptide. The experimental result shows that the activity of antioxidant enzyme in vivo is reduced due to aging of the organism, the organism suffers from serious lipid peroxidation damage, and the oyster active peptide can improve the antioxidant activity of the organism and relieve the peroxidation damage of the organism.

TABLE 1 Effect of oyster active peptides on the antioxidant Activity of the brain of Zebra Fish

Note: # compares the elderly fish with the young fish; comparing fed UF2 with unfed UF 2; p < 0.05; (xi)/# # P < 0.01; p < 0.001

Test example 3

Influence of oyster active peptide on brain inflammation factors of zebra fish in young and old people.

3 proinflammatory factors IL-1 beta, TNF-alpha and INF-r are measured, wherein IL-1 beta and TNF-alpha of the aged fish are obviously increased, and the content of the aged fish fed with oyster active peptide is obviously reduced; the influence of age on INF-r is small, and the content of the oyster bioactive peptide is slightly reduced after the oyster bioactive peptide is fed; two anti-inflammatory factors IL-10 and IL-13 were determined, which were not greatly affected by age, and were slightly elevated after feeding oyster bioactive peptide, indicating chronic inflammation in brain of aging organism, and the anti-inflammatory effect of oyster bioactive peptide was produced by inhibiting the activity of proinflammatory factor rather than enhancing the activity of anti-inflammatory factor.

TABLE 2 influence of oyster active peptides on inflammatory factors of zebrafish

Note: # compares the elderly fish with the young fish; comparing fed UF2 with unfed UF 2; p < 0.05; (xi)/# # P < 0.01; p < 0.001

Test example 4

Influence of oyster active peptide on contents of zebra fish brain neurotransmitter and neurotrophic factors.

The Ach and r-GABA in three neurotransmitters are obviously reduced on the body of the aged fish, the content of DA is basically not influenced by age, and the influence on the content of DA is not large by feeding the oyster active peptide; the brain-derived neurotrophic factor BDNF reduces the content of fish brains of old fishes, and is obviously improved after being fed with oyster active peptides. Figure 5 shows the effect of oyster bioactive peptides on the BDNF content of zebrafish fish brain.

TABLE 3 Effect of oyster active peptides on the brain neurotransmitter of Zebra fish

Note: # compares the elderly fish with the young fish; comparing fed UF2 with unfed UF 2; p < 0.05; (xi)/# # P < 0.01; p < 0.001

Test example 5

Influence of oyster active peptide on zebra fish brain gene expression.

Consistent with the result of BDNF content, the gene expression level of the BDNF of the old fish is obviously reduced, and the gene expression level of the BDNF is obviously increased after the oyster active peptide is fed. TrkB and p75 are two specific receptors of BDNF, wherein the combination of TrkB and BDNF can activate a cell differentiation pathway, the combination of p75 and BDNF can activate apoptosis, the TrkB receptor of old fishes is remarkably reduced, and the TrkB receptor of old fishes is remarkably increased after being fed with oyster active peptide, which indicates that the differentiation and regeneration capability of nerve cells is reduced due to aging, and the feeding of oyster active peptide is beneficial to the growth and differentiation of the nerve cells of old fishes; age has little influence on the gene expression level of p75, and the gene expression level of p75 has little change after feeding oyster active peptide. GFAP is an astrocyte marker protein, astrocytes support and nourish neurons in the central nervous system by secreting neurotrophic factors, and play a role in neuroprotection, the gene expression level of GFAP of old fishes is reduced, which indicates that the vitality of the astrocytes is reduced due to aging, and the content of the neurotrophic factors secreted by the astrocytes is reduced, which is consistent with the measured content of the brain-derived neurotrophic factor BDNF and the result of the reduction of the gene expression level, and the gene expression level of GFAP is remarkably increased after the oyster bioactive peptide is fed, and is consistent with the measured result that the content of the BDNF and the gene expression level are increased after the oyster bioactive peptide is fed. PSD-95 (postsynaptic protein) and SYP (presynaptic protein) are synapsin marker proteins and participate in the formation and maturation of synapses and the release of neurotransmitters, the loss of the expression of PSD-95 and SYP in old fish means the loss of synapses, which indicates that the neurosynaptic plasticity is reduced due to aging, the PSD-95 is obviously increased after the oyster active peptide is fed, the SYP is improved but is not obvious, and the fact that the neurosynaptic plasticity can be improved by the oyster active peptide is shown; the expression quantity of CD11b gene related to chronic inflammation and beta-bovine lactosamine (SA beta G) gene related to aging is obviously increased on the aged fish, so that the aged fish is shown to have serious chronic brain inflammation and show obvious aging.

TABLE 4 influence of oyster active peptides on the expression of the brain genes of zebrafish

Note: # compares the elderly fish with the young fish; after feeding UF2, compared with unfed UF2

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.