| Pinctada martensii is a kind of marine pearl shellfish mainly cultured in Southern China Sea.It has been reported that the enzymatic hydrolysate of Pinctada martensii can improve the learning and memory ability of aging mice,while the relevant mechanism underlying it remain unknown.This study firstly verified that whether the enzyme hydrolysate of Pinctada martensii meat could improve learning and memory ability in an aging zebrafish model.Subsequently,Pinctada martensii meat protein was extracted by the alkaline solution and acid precipitation method.Components with different molecular weight were further obtained by ultrafiltration after enzymation,and the water extract of it was prepared by hot water extraction method.The effect of them on improving learning and memory activity were investigated in an aging zebrafish model.Possible material basis were inferred by analyzing their nutritional composition.Eventually,fractions with different taurine content were prepared from Pinctada martensii meat,and the effect of taurine on the learning and memory activity in aging zebrafish and the mechanism underlying it were investigated.This subject may provide a theoretical foundation for using Pinctada martensii meat as functional food for preventing and improving memory in the elderly generation.Main results are as following:(1)The results of composition test of the enzyme hydrolysate of Pinctada martensii showed that the enzymatic hydrolysis product contains 78.28% of crude protein,1.96%crude fat,3.88% total sugar,11.59% ash,which is rich in glutamic acid,aspartic acid,lysine,leucine and arginine,also has high contents of calcium,iron and zinc.The results of behavioral experiment showed that the enzyme hydrolysate of Pinctada martensii significantly increased the ethological success rate of aging zebrafish(P<0.05),reduced the latency period.The results of biochemical indicators showed that the enzyme hydrolysate of Pinctada martensii reduced the levels of inflammatory factors IL-1? and TNF-? in the brain tissue of aging zebrafish(P<0.05)by 11.25% and 15.25%,respectively;Meanwhile,it could increase the level of BDNF by 14.01%(P<0.05).But it had no significant effect on IL-10 content(P>0.05).(2)The protein of Pinctada martensii was isolated by the alkaline solution and acid precipitation method before enzymatically hydrolyzed.The enzymatic hydrolyzed products were fractionated by ultrafiltration technology to obtain four ultrafiltration groups > 8 K,5-8 K,3-5 K,and <3 K.The determination of the main components of each ultrafiltration components showed that the crude protein content of each ultrafiltration components is above 83%(dry basis),among which the crude protein and crude fat content of the 3-5 K component is the highest,the total sugar content increases with the molecular weight of ultrafiltration components bigger and bigger.The ash content in the <3 K component is the highest with a mass of 6.40 ± 0.04%.The total amino acid content in the <3 K component and the ratio of hydrophobic amino acids and essential amino acids to the total amino acids are the highest,respectively 80.35 g/100 g,31.54%,39.75%,each components contains 16 kinds of amino acids.The total amount of metal elements in the <3 K component is the highest,reaching 956.13mg/kg(dry basis),and the zinc content in the 3-5 K component is the highest,which is 46.81mg/kg(dry basis).In addition,the taurine content of each component increase as the molecular weight range decrease,and the highest content is 0.61 g/100g(dry basis).The results of animal experiment showed that each ultrafiltration components fails to significantly improve the behavior performance of aging zebrafish,but some or all of the components have significant effects on inflammatory factors IL-1?,TNF-?,IL-10,as well as the levels of BDNF and ?-GABA.(3)The water extracts of Pinctada martensii was prepared by the hot water extraction method.The composition analysis results showed that the composition of the extract was relatively complicated.The main components were crude protein and ash,and the mass fractions were 52.78% and 26.75%(dry basis),respectively.The content of acidic amino acids and hydrophobic amino acids is relatively high,accounting for 27.42%and 42.39% of the total amino acids respectively.In which the content of glutamic acid,glycine,and aspartic acid is the highest,which are 4.70,4.11,and 2.83 g/100g(dry basis).Taurine content is high,up to 7.42 g/100g(dry basis).The content of Ca,Fe,Zn,Mn,Cu and total content of metal elements in the water extract are much higher than any the proteolysis ultrafiltration components.The results of animal experiment showed that the success rate of the water extract group was significantly increased and the latency period decreased compared with the elderly control group(P<0.01),IL-1? and TNF-? were significantly reduced,and IL-10,BDNF and neurotransmitters(Ach and ?-GABA)increased significantly(P<0.05).(4)Hot water extraction method has been employed to prepare the water extract and residue from Pinctada martensii meat,and part of the water extract was filtered and alcohol precipitated to obtain three extracts with different taurine content.Component analysis results showed that the content of taurine and trace elements in the alcohol precipitate,water extract,residue enzymatic hydrolysate decreased successively,while the peptide content and the hydrolyzed amino acid content were the opposite.The results of animal experiment showed that the water extract and alcohol precipitation component could significantly improve the performance of behavior(P<0.05),increase the SOD,CAT enzyme activity and IL-10 content,and reduce the levels of MDA,IL-1?,and TNF-? in the brain tissue of aging zebrafish.The q PCR results showed that water extract and alcohol precipitation component could up-regulate the m RNA expression of GFAP and CD11 b,increased by 1.86 and 5.67 times,2.59 and 5.72 times respectively.It also improved the secretion of BDNF protein and the expression of BDNF in m RNA level,and up-regulated the expression of its high-affinity receptor Trk B gene by 3.32,3.81 times respectively.Meanwhile it up-regulated the expression of SYP and PSD-95,by1.87 and 1.90 times,1.84 and 4.79 times respectively.In conclusion,the enzymatic hydrolysis products of Pinctada martensii meat could improve the learning and memory ability of aging zebrafish,while the proteolytic components of Pinctada martensii didn‘t show this effect.The water extract also could improve the learning and memory ability of aging zebrafish.The effect of Pinctada martensii meat on improving learning and memory activity possiblely associated with the taurine in it.Animal experiments with taurine content from Pinctada martensii showed the effect of improving learning and memory in aging zebrafish in a dose-depended manner.The possible mechanism underlying taurine is related to its anti-oxidation,anti-inflammation property,and promoting synapse formation. |
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