Sea grape low-molecular-weight polysaccharide as well as preparation method and application thereof

Abstract

The invention aims to provide a sea grape low-molecular-weight polysaccharide as well as a preparation method and application thereof. The sea grape low-molecular-weight polysaccharide is prepared by fermenting sea grape powder with a Periconia sp.QAU-OG-Isz-15 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.21455. The molecular weight of the sea grape polysaccharide is effectively reduced through microbial degradation, the content of functional polysaccharide is increased, and the anti-AD activity is further improved. In an in-vitro experiment, compared with undegraded sea grape polysaccharide, the sea grape low-molecular-weight polysaccharide has a more obvious inhibition effect on Abeta1-42 aggregation, and in an in-vivo experiment of caenorhabditis elegans, the sea grape low-molecular-weight polysaccharide has the effects of prolonging the service life of the caenorhabditis elegans, delaying senescence and promoting the athletic ability and reproductive ability. Meanwhile, the compound has an obvious inhibition effect on Abeta1-42 in-vivo aggregation. As a natural product component, the sea grape low-molecular-weight polysaccharide has huge potential in development and application of novel anti-AD treatment drugs and functional foods.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of biological functional active components, and specifically relates to a sea grape low molecular weight polysaccharide and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, AD is characterized by global dementia, including memory impairment, aphasia, apraxia, agnosia, executive dysfunction, and personality and behavioral changes. Currently, clinical treatment of AD at home and abroad mainly relies on two types of drugs: cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. The drugs are expensive and have obvious side effects.

[0003] One theory of the cause of Alzheimer's disease is the β-amyloid protein cascade hypothesis, which believes that the abnormal secretion and deposition of β-amyloid protein (Aβ) in the brain is the core of AD. Aβ accumulates in large quantities in the brain, produces strong neurotoxicity, and causes imbalance of the brain's oxidation-antioxidation system by mediating the inflammatory cascade reaction. Therefore, effectively inhibiting the aggregation and deposition of Aβ protein has become an important means of preventing and treating AD. Summary of the invention

[0004] The purpose of the present invention is to provide a low molecular weight polysaccharide of sea grapes and a preparation method and application thereof, that is, Periconia sp.QAU-OG-Isz-15 isolated from oysters is co-fermented and degraded with sea grapes to prepare sea grape polysaccharides, and the prepared low molecular weight polysaccharide has the effect of inhibiting Aβ protein aggregation and deposition.

[0005] The present invention first provides a sea grape low molecular weight polysaccharide, which is prepared by fermenting sea grape powder with Periconia sp.QAU-OG-Isz-15 strain with a preservation number of CGMCC No.21455;

[0006] The fermentation preparation specifically includes adding sea grape powder into deionized water and fermenting with Periconia sp. QAU-OG-Isz-15 strain, centrifuging the fermentation liquid to obtain the supernatant, adding ethanol to the supernatant solution for alcohol precipitation, and the precipitate obtained by centrifugation is the sea grape low molecular weight polysaccharide.

[0007] The present invention also provides a use of the sea grape low molecular weight polysaccharide, which is the use in inhibiting the aggregation and deposition of Aβ protein;

[0008] The present invention also provides another use of the sea grape low molecular weight polysaccharide, which is the use in preparing a product for preventing and treating Alzheimer's disease;

[0009] The present invention also provides a product for preventing and treating Alzheimer's disease, which contains the sea grape low molecular weight polysaccharide in a pharmacologically effective concentration.

[0010] The present invention effectively reduces the molecular weight of sea grape polysaccharide through microbial degradation, increases the content of functional polysaccharide, and further improves the anti-AD activity. In in vitro experiments, compared with undegraded sea grape polysaccharide, sea grape low molecular weight polysaccharide has a significant effect on Aβ 1-42 In vivo experiments on Caenorhabditis elegans showed that sea grape low molecular weight polysaccharides increased the lifespan of Caenorhabditis elegans, delayed aging, and promoted athletic ability and reproductive ability. 1-42 It has a significant inhibitory effect on in vivo aggregation. This shows that sea grape low molecular weight polysaccharides, as natural product components, have great potential in the development and application of new anti-AD therapeutic drugs and functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Effects of different polysaccharides on Aβ 1-42 Relative inhibition rate plot of aggregation;

[0012] Figure 2 :The effect of different concentrations of CLPA on the paralysis rate of Caenorhabditis elegans;

[0013] Figure 3 :The effect of different concentrations of CLPA on the survival rate of Caenorhabditis elegans;

[0014] Figure 4 :The effect of different concentrations of CLPA on the motility of Caenorhabditis elegans;

[0015] Figure 5 :The effect of different concentrations of CLPA on the reproductive ability of Caenorhabditis elegans;

[0016] Figure 6 :The effect of CLPA on the antioxidant enzyme activity and MAD content in Caenorhabditis elegans;

[0017] Figure 7 :Diagram of the effect of CLPA on Aβ aggregation in Caenorhabditis elegans. DETAILED DESCRIPTION

[0018] The sea grapes used in the present invention have a scientific name of Caulerpa lentilifera, belong to the Chlorophyta Caulerpaeaceae, and are mainly grown in the South my country Sea, Southeast Asia and other regions.

[0019] The strain used in the example is the QAU-OG-Isz-15 strain (Periconia sp.) isolated from oyster, which is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration; the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is January 27, 2021; the deposit number is CGMCC No. 21455.

[0020] The plant lactobacillus and yeast in the examples are from the Functional Food Research and Development Laboratory of Qingdao Agricultural University.

[0021] The present invention is described in detail below in conjunction with embodiments and drawings.

[0022] Example 1: Preparation of sea grape polysaccharide and determination of molecular weight of the prepared polysaccharide

[0023] The sea grapes were soaked and washed with water, dried in an oven to constant weight, and crushed with a grinder to obtain sea grape powder. Four portions of 10 g sea grape powder were placed in different 1000 mL beakers, deionized water was added at a solid-liquid ratio of 1:40, and sterilized in an 80°C water bath for 30 minutes. After cooling, sterile water and activated Periconia sp. QAU-OG-Isz-15, Lactobacillus plantarum and yeast were added in an amount of 3% of the mass of the sea grape powder. The mixture was then shaken and fermented at 30°C for 48 hours, boiled for 15 minutes for sterilization, centrifuged at 10,000 r / min for 10 minutes, and the supernatant was added with three times the volume of 95% ethanol for alcohol precipitation. The mixture was allowed to stand overnight and centrifuged at 10,000 r / min for 10 minutes after sufficient sedimentation. Four groups of alcohol-precipitated polysaccharides were separated and freeze-dried to obtain sea grape polysaccharide CLP, sea grape low molecular weight polysaccharide CLPA degraded by Periconia sp. QAU-OG-Isz-15 fermentation, sea grape low molecular weight polysaccharide CLPB degraded by Lactobacillus plantarum fermentation, and sea grape low molecular weight polysaccharide CLPC degraded by yeast fermentation.

[0024] The molecular weights of the four polysaccharides were determined using an 18-angle laser light scattering instrument. First, a 1 mg / mL sample solution was prepared, passed through a 0.22 μm filter membrane, and the filtrate was tested.

[0025] Chromatographic conditions are as follows: PSS GRAM Chromatographic column; flow rate 0.5 mL / min; injection volume 100 μL; column temperature 35°C; mobile phase 0.02% sodium nitrate solution. Data were analyzed using Astra software, and the refractive index increment (dn / dc) was 0.138 mL / g.

[0026] Table 1: Molecular weight distribution data of CLP, CLPA, CLPB and CLPC

[0027]

[0028] As can be seen from Table 1, compared with CLP, the molecular weight and content of CLPA, CLPB and CLPC have changed significantly, the average molecular weight has decreased significantly, and the content of the main components of polysaccharides has increased significantly, among which CLPA has the smallest average molecular weight, indicating that the preparation of sea grape low molecular weight polysaccharides by fermentation degradation of Periconia sp. QAU-OG-Isz-15 is more effective.

[0029] Example 2: Prepared low molecular weight polysaccharide inhibits Aβ 1-42 The impact of aggregation ability

[0030] To verify the inhibitory effect of the four groups of polysaccharides on Aβ1-42 aggregation, equal amounts of Aβ1-42 freeze-dried powder were dissolved in 100 μg / mL of the four groups of polysaccharide solutions. 1-42 The final concentration of the mixture reached 10 μM. After being fully mixed with a mixer, the mixture was placed in a 37°C incubator for 24 h to promote the aggregation of Aβ1-42. ThT powder was diluted to 5 μM using PBS solution, and 180 μL of the diluted ThT solution was mixed with 20 μL of the sample solution incubated for 24 h to aggregate Aβ1-42 alone. 1-42 As a blank control, 100 μg / mL of epigallocatechin gallate (EGCG) was used as a positive control. The fluorescence intensity was measured using an ELISA reader at an excitation wavelength of λex=450 nm and an emission wavelength of λem=480 nm, and the inhibition rate of each group of polysaccharides was calculated.

[0031] Depend on Figure 1 It can be seen that CLP, CLPA, CLPB and CLPC have a significant effect on inhibiting Aβ 1-42 Aggregation has an effect, and the positive control EGCG has an effect on Aβ 1-42 The relative inhibition rate of aggregation was 60.89%, which was significantly higher than that of each sample group. 1-42 The relative inhibition rate of aggregation reached 36.58%, which was 18.15% higher than that of the CLP group. The relative inhibition rates of CLPB and CLPC were slightly lower than those of CLPA, which were 29.80% and 26.62%, respectively. This indicates that the sea grape low molecular weight polysaccharide CLPA obtained by fermentation and degradation of Periconia sp. QAU-OG-Isz-15 has a better inhibitory effect.

[0032] Example 3: CLPA against Aβ in Caenorhabditis elegans 1-42 The impact of aggregation,

[0033] 1. CLPA for Aβ in Caenorhabditis elegans 1-42 The impact of aggregation

[0034] Based on the β-amyloid cascade hypothesis, the Caenorhabditis elegans AD pathological model CL4176 was selected to upregulate human Aβ by increasing the culture temperature. 1-42 Expression in body wall muscle cells, overexpressing Aβ 1-42 Continuous accumulation produces neurotoxicity, paralyzing the nematodes and showing AD pathological characteristics.

[0035] 2. Synchronization of Caenorhabditis elegans

[0036] Inoculate E.coli OP50 into LB liquid culture medium and culture at 37°C for 14-20 hours until the absorbance of the bacterial solution at 600nm is between 0.4-0.6. Then pipette 100 μL of the bacterial solution onto an NGM plate and spread it evenly. Culture at 37°C for 12 hours to obtain sufficient E.coli OP50.

[0037] Inoculate nematodes onto a new NGM agar plate with sufficient E. coli OP50 and culture at 16°C. Transfer 10-20 adult worms with eggs onto a new NGM plate. Observe the number of eggs after 3-4 hours. Remove the adults with platinum wire when the number meets the experimental requirements. Then transfer the plate to a 16°C incubator and incubate for 30 hours to obtain synchronized larvae.

[0038] 3. Caenorhabditis elegans paralysis relief experiment

[0039] CLPA was added to E.coli OP50 culture medium to prepare CLPA-E.coliOP50 culture medium of different concentrations (0, 1, 3, 5 mg / mL), and evenly coated on the surface of NGM culture medium. After culturing at 37°C for 16 hours, it was used as a sample plate. According to the synchronization steps, synchronized eggs (about 100) were incubated on NGM sample plates containing CLPA and E.coli OP50. After culturing at 16°C for 30 hours, they were transferred to 23°C for induction. Nematodes will express Aβ at 23°C. 1-42 , along with Aβ 1-42 The accumulation of can cause paralysis symptoms in nematodes. The number of paralyzed nematodes was checked every 24 hours using a stereo microscope, and the surviving nematodes with only their heads moving were recorded as paralyzed. The date 18 hours after induction was marked as day 1, and then observed every 24 hours for a total of 5 days.

[0040] 4. Caenorhabditis elegans lifespan experiment

[0041] Sample plates and synchronized C. elegans were prepared according to the above steps. Synchronized eggs (about 100) were incubated on NGM sample plates containing CLPA and E. coli OP50, cultured at 16°C for 30 hours, and then transferred to 23°C for induction. The date after 18 hours of induction at 23°C was recorded as the first day of the lifespan experiment. Thereafter, the number of dead nematodes on the CLPA sample plates was observed every 24 hours until all died. Nematodes without movement and swallowing behavior and still without reaction after being touched by platinum wire were recorded as dead.

[0042] 5. Caenorhabditis elegans movement protection experiment

[0043] The sample plate and synchronized C. elegans were prepared according to the above steps. The synchronized eggs (about 100) were incubated on NGM sample plates containing CLPA and E. coli OP50, cultured at 16°C for 30 hours, and then transferred to 23°C for induction. The date after 18 hours of induction at 23°C was recorded as the first day of the lifespan experiment. Thereafter, the movement ability of the nematodes on the CLPA sample plate was observed every 24 hours, and the movement ability of the nematodes was tested by gently touching the body of the nematodes with platinum wire. Ten nematodes were randomly selected from each plate, and the number of head swings of the nematodes within 30 seconds was recorded. The cumulative observation was 8 days.

[0044] 6. Caenorhabditis elegans reproductive protection experiment

[0045] Prepare sample plates and synchronized C. elegans according to the above steps. Synchronized eggs (about 10) were incubated on NGM sample plates containing CLPA and E. coli OP50, cultured at 16°C for 30 hours, and then transferred to 23°C for induction. Counts were made from the first day of egg laying, and C. elegans on the old plates were transferred to fresh sample plates containing CLPA every 24 hours until the nematodes stopped laying eggs. The plates were incubated at 16°C for 24 hours, and the number of nematodes was counted before the offspring hatched and grew up.

[0046] 7. Determination of antioxidant enzyme activity and MDA content in Caenorhabditis elegans

[0047] According to the above steps, nematodes were fed with 3 mg / mL CLPA-E.coli OP50, and nematodes fed only with E.coli OP50 were used as the control group. About 200 synchronized eggs were placed on each plate. After induction at 23°C for 40 hours, the plates were removed and the nematodes were collected into 5 mL centrifuge tubes with M9 buffer. After the nematodes naturally settled to the bottom, the supernatant was discarded, and M9 buffer was added again. The washing was repeated 3 times, and then the nematodes were transferred to a 2 mL tissue grinding tube with a small amount of sterile saline, ground on ice, and the homogenate was centrifuged at 4°C and 2500r / min for 10 minutes to make a 5% homogenate. The catalase detection kit, superoxide dismutase detection kit, and malondialdehyde detection kit were used to evaluate the CAT, SOD activity, and MDA content in nematodes.

[0048] 8. Effects of CLPA on Aβ aggregation in C. elegans

[0049] To detect Aβ in nematodes 1-42 Aggregation: 40 Caenorhabditis elegans were cultured in each group according to the synchronization steps. The sample plate nematodes fed with 3 mg / mL CLPA and the blank plate without CLPA were placed at 23°C for 55 hours. The nematodes were collected using M9 buffer and washed twice with PBS (pH 7.4). The nematodes were fixed with 4% paraformaldehyde / PBS reagent at 4°C for 24 hours, and then reacted with 125mM Tris (containing 5% fresh β-mercaptoethanol, 1% Triton X-100 24 hours, pH 7.4) solution at 37°C for 24 hours. After washing twice with 1% PBS-Tween-20, they were stained with 50% ethanol containing 0.125% thioflavin T for 2 minutes in a dark environment, then washed with 50% ethanol, and finally washed with PBS. The nematodes were placed on a slide and observed and photographed using a fluorescence microscope.

[0050] The above results are summarized as follows:

[0051] 1. Effect of CLPA on the paralysis rate of Caenorhabditis elegans

[0052] As the high temperature induction time increases, excess Aβ 1-42 It will be deposited in the body of the nematode, causing the nematode to develop a paralytic phenotype and thus become paralyzed. Figure 2 As shown in the figure, with the increase of induction time, the paralysis rate of nematodes continued to increase. Compared with the blank control group (sample concentration 0 mg / mL), the degree of paralysis in the CLPA group was lighter than that in the blank control group, among which 3 mg / mL CLPA had the best protective effect on AD nematodes. The paralysis rate of nematodes fed with 3 mg / mL CLPA after induction at 23°C was significantly lower than that of the control group, and this trend became more obvious with the increase of experimental days. This shows that CLPA can reduce Aβ 1-42The toxic effect of the drug slows down the paralysis rate of nematodes.

[0053] 2. Effect of CLPA on the lifespan of Caenorhabditis elegans

[0054] Lifespan is the most direct indicator of aging in C. elegans. Figure 3 As shown in the figure, with the extension of induction time, the survival rate of nematodes gradually decreased. Compared with the blank control group, the CLPA group had an effect of prolonging the average lifespan of nematodes. On the third day of high temperature induction, the nematodes in the blank group began to die, and all the nematodes died on the 10th day; the death rate of nematodes fed with 3 mg / mL CLPA was the slowest, and death occurred on the 5th day, and almost all died on the 14th day. Compared with the blank control group, the lifespan can be extended by 4 days; feeding 1 mg / mL and 5 mg / mL CLPA also has a certain effect on prolonging the lifespan of nematodes, but the effect is lower than 3 mg / mL concentration. The above data show that CLPA can increase the lifespan of nematodes under high temperature induction, and the sample concentration is the best at 3 mg / mL. By inhibiting the aggregation of Aβ, the damage to nematode neurons is reduced.

[0055] 3. Effects of CLPA on the motility of Caenorhabditis elegans

[0056] The locomotion ability of nematodes is related to neural health. High temperature induces Aβ in nematodes. 1-42 Aggregation occurs, causing the nematode's head to become stiff and difficult to move, so the frequency of the nematode's head swing can reflect the nematode's neurological health. Figure 4 It can be seen that as the induction time increases, the frequency of head swings of nematodes decreases. On the 1st and 2nd days, the number of head swings of nematodes showed an upward trend, which may be due to the maturity of nematodes and the improvement of their movement ability. 1-42 The nematodes continued to accumulate in the body of the nematodes. Starting from the second day, the average number of swings of the head of the blank group nematodes gradually decreased from 23 times, and the average number of swings of the nematodes on the 8th day decreased to 5 times. Compared with the blank group, the 1mg / mL and 5mg / mL sample groups had a certain recovery effect on the nematode's motility. On the 8th day, the average number of swings of the nematodes increased by 6 and 4 times, respectively. 3mg / mL had the best protective effect on the nematodes. By the 8th day of the experiment, the average number of swings reached 14 times, and the rate of decline in motility was slow. This shows that CLPA can increase the motility of nematodes during the induction process.

[0057] 4. Effects of CLPA on the reproductive capacity of Caenorhabditis elegans

[0058] The amount of eggs laid by Caenorhabditis elegans is correlated with lifespan, which can be used to evaluate the aging rate of nematodes. The 23°C induction environment will damage the gonads in the nematodes, leading to a decrease in reproductive capacity. Figure 5As shown in the figure, compared with the blank control group, the egg-laying amount and total egg-laying amount of the sample groups on each day were significantly increased, among which the egg-laying amount and total egg-laying amount of the 3 mg / mL group were the highest, indicating that CLPA at a concentration of 3 mg / mL can effectively protect the gonads of nematodes and restore the egg-laying ability of nematodes. This experimental result is consistent with the paralysis, lifespan and movement experimental data. CLPA has a positive effect on Aβ 1-42 The inhibitory ability of nematodes delays paralysis and aging and enhances their movement and reproductive abilities.

[0059] 5. Effects of CLPA on antioxidant enzyme activities and MAD content in Caenorhabditis elegans

[0060] The activity of antioxidant enzymes and the content of MDA in nematodes can reflect the degree of oxidative stress. SOD, as the most important antioxidant enzyme in nematodes, is crucial to the antioxidant level in nematodes. MAD is one of the end products of lipid peroxidation and an important biomarker of oxidative stress. Oxidative stress is inseparable from Aβ toxicity. Figure 6 As shown in the figure, compared with the blank control group, the SOD enzyme activity of nematodes treated with 3 mg / mL CLPA increased by 32.95%, the CAT enzyme activity increased by 27.79%, and the MDA level decreased by 32.20%, indicating that CLPA can improve the antioxidant defense system of nematodes by regulating antioxidant enzyme activity and lipid peroxidation, thereby delaying the aging of nematodes.

[0061] 6. Effects of CLPA on Aβ deposition in C. elegans

[0062] Thioflavin T fluorescence staining is widely used in the detection of β-amyloid protein and is often used to analyze the aggregation characteristics of amyloid protein. Figure 7 As shown in A, after being induced in a 23°C environment for 55 hours, C. elegans showed obvious protein aggregation and obvious white spots. Figure 7 As shown in B, the Caenorhabditis elegans treated with 3 mg / mL CLPA had no Aβ production in vivo. 1-42 Therefore, no white spots appeared, indicating that CLPA has a positive effect on the prevention and treatment of AD.

[0063] In summary, the present invention prepares sea grape polysaccharide by co-fermentation and degradation of Periconia sp.QAU-OG-Isz-15 isolated from oysters and sea grapes, and the molecular weight of sea grape polysaccharide is effectively degraded by means of microbial fermentation; the prepared low-molecular-weight polysaccharide has better anti-AD activity.

Claims

1. A sea grape low molecular weight polysaccharide, characterized in that: The sea grape low molecular weight polysaccharide is prepared by fermenting sea grape powder with Periconia sp. QAU-OG-Isz-15 strain with a preservation number of CGMCC No.21455.

2. The sea grape low molecular weight polysaccharide according to claim 1, characterized in that The fermentation preparation comprises adding sea grape powder into deionized water and fermenting with Periconia sp. QAU-OG-Isz-15, centrifuging the fermentation liquid to obtain the supernatant, adding ethanol to the supernatant solution for alcohol precipitation, and centrifuging to obtain sea grape low molecular weight polysaccharide.

3. The sea grape low molecular weight polysaccharide according to claim 2, characterized in that: The fermentation was then carried out in a shaking incubator at 30°C for 48 hours.

4. The sea grape low molecular weight polysaccharide according to claim 2, characterized in that: The centrifugation is carried out at 10000 r / min for 10 min.

5. Use of the sea grape low molecular weight polysaccharide according to claim 1 in inhibiting Aβ protein aggregation and deposition.

6. Use of the sea grape low molecular weight polysaccharide according to claim 1 in the preparation of a product for preventing and treating Alzheimer's disease.

7. A product for preventing and treating Alzheimer's disease, characterized in that: The product contains the sea grape low molecular weight polysaccharide according to claim 1 at a pharmacologically effective concentration.

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