Preparation Of N-acetylneuraminic Acid From Chicken Small Intestine And Its Effect On Anti-inflammatory In Vitro

N-acetylneuraminic acid(N-acetylneuraminic acid,Neu5Ac),as one of the most important forms of sialic acid,performs a variety of biological functions in the human body.N-gylcolylneuraminic acid(N-gylcolylneuraminic acid,Neu5Gc)is another form of sialic acid,which is found in the serum,cells,and tissues of cancer patients,and is closely related to the occurrence and development of cancer.Therefore,the human body should reduce the intake of Neu5Gc while supplementing Neu5Ac.Bird's nest is a natural substance with the highest content of Neu5Ac and does not contain Neu5Gc.However,due to its high price,it is not conducive to popularization.It is of great significance to find a cheap source for preparing Neu5Ac.Through literature review and high-performance liquid chromatography detection pre-experiment,it is found that the chicken small intestine is rich in Neu5Ac and does not contain Neu5Ac.Therefore,this paper uses the chicken small intestine as the raw material to study its role as N-acetylneuraminic acid.The feasibility of biological resources.Objective:To explore the best process parameters for extracting Neu5Ac from chicken small intestine,the best process parameters for the purification of Neu5Ac crude product,and to explore the anti-inflammatory effect of Neu5Ac in vitro.Provide new ideas for the development and reuse of chicken small intestine.Method:(1)Raw material processing:homogenize,freeze-dry,crush,and sieve the collected chicken intestine waste to prepare a freeze-dried powder of chicken intestine for later use.For the determination of the basic components of the freeze-dried powder of chicken small intestine waste,the protein,fat,moisture and ash content are determined with reference to the national standard method.(2)the single factor experiment is used to select the best acid in several commonly used sialic acid extracts(hydrochloric acid,sulfuric acid,citric acid,trifluoroacetic acid,acetic acid),and further the three factors like extraction acid concentration(0.01,0.05,0.10,0.15,0.20 mol/L),extraction time(2,2.5,3,3.5,4,4.5 h)and liquid with material ratio(10:1,20:1,30:1,40:1,50:1 m L/g)are determined to influence the optimal range of sialic acid extraction.Based on the single factor experiment,the Box-Behnken test design method is used to optimize the design of three factors and three levels of response surface.The effects of three factors including citric acid concentration,extraction time and the ratio of liquid to material on the sialic acid extraction are investigated to determine the optimal process parameters for sialic acid extraction from chicken intestinal waste.HPLC is used to preliminary identify the crude sialic acid extraction.(3)Ethanol precipitation method is used to remove protein from the obtained crude extraction.the four kinds of ion exchange resins like 732,D301,D201,201×7 were selected,add the amount of resin(1,2,3,4,5 g),loading concentration(4.32,8.54,17.24,34.62 mg/m L),loading flow rate(0.5,1.0,1.5,2.0,2.5,3.0m L/min),loading volume and other factors are optimized,the optimal conditions for the secondary purification of Neu5Ac by ion exchange resin are obtained.The purified Neu5Ac extract is lyophilized to determine its content.And liquid chromatography-mass spectrometry(LC-MS)is used to identify the obtained Neu5Ac.(4)The prepared Neu5Ac product is formulated to 0,0.1,10,25,50,100,200?g/m L,and the MTT method is used to detect the influence of the above-mentioned concentration intervention on the viability of RAW267.4 cells.After that,RAW264.7 cells are intervened with the above concentration,and RAW264.7 cells are stimulated to produce inflammation with lipopolysaccharide(LPS),inflammatory mediators contention like nitric oxide(NO),tumor necrosis factor-?(tumor necrosis factor-?,TNF-?),interleukin-6(interleukin-6,IL-6),interleukin-1?(interleukin-1?,IL-1?),prostaglandin E2(prostaglandin E2,PGE2)are measured.Result:(1)The results of the single factor experiment are as follows:Five kinds of commonly used acids for extracting Neu5Ac are selected,the order of the ability to release Neu5Ac is:hydrochloric acid>sulfuric acid>citric acid>trifluoroacetic acid>acetic acid.The extraction of Neu5Ac is similar,the difference was not significant(P>0.05),citric acid are chosen for follow-up experiments.When the concentration of citric acid is in the range of0.01?0.20 mol/L,the extraction rate is increased then decreased with the increase of acid concentration.When the concentration of citric acid is 0.10 mol/L,the amount of Neu5Ac extracted reaches the maximum.With the extension of the extraction time,the amount of Neu5Ac extracted first increased and then decreased.When the extraction time is 3.5 h,the amount of Neu5Ac extraction reached the peak.With the increase of the ratio of liquid to material,the amount of Neu5Ac extraction first increased and then decreased.When the ratio of liquid to material is 30:1(m L/g),the extraction rate of Neu5Ac is the largest,and the best extraction condition is 30:1(m L/g).In summary,the best acid for extracting Neu5Ac is citric acid.The best extraction conditions are citric acid concentration of 0.1 mol/L,extraction time of 3.5 h,and the ratio of liquid to material of 30:1(m L/g).(2)The response surface method is used to carry out a 3-factor 3-level optimization experiment,and the regression equation obtained is:Y(Neu5Ac extraction amount)=945.00-47.79A+99.63B+121.65C+25.05AB+108.47AC-64.43BC-47.90A~2-25.12B~2-132.62C~2.The P value of the model is less than 0.0001,indicating that the regression model of Neu5Ac extraction is extremely significant,the F value of the lack-of-fit term is 0.25(P>0.05),and the lack-of-fit term is not significant.The regression model is a good fit and can be explained well Variation in response.By comparing the F value of the three influencing factors:acid concentration,extraction time,and the ratio of liquid to material,the degree of influence of the three factors on the amount of Neu5Ac extraction are determined as follows:the ratio of liquid to material>extraction time>citric acid concentration.And there is an interaction between the concentration of citric acid and the ratio of liquid to material,and between the extraction time and the ratio of liquid to material(P<0.01).Considering the simplicity of actual operation,the results indicated that the optimized extraction conditions of Neu5Ac are as follows:citric acid concentration 0.1 mol/L,extraction time 4 h,the ratio of liquid to material(m L/g).Under this condition,the measured Neu5Ac extraction amount is(1010.39±21.87)?g/g,which ias not significantly different from the predicted value of 1025.69?g/g(P>0.05).The extract is preliminarily identified by HPLC.By comparing with the standard,according to the peak time,it can be determined that the sialic acid contained in the Neu5Ac extract is only Neu5Ac,Neu5Gc is not detected.(3)The largest adsorption rate for citric acid and the largest retention rate for Neu5Ac is D301.It is decided to use D301 resin for subsequent experiments.When the amount of resin is continuously increased,the adsorption rate of citric acid also gradually increases.When the resin addition amount reached 3 g,the upward trend of citric acid adsorption rate tended to be gentle thereafter,and a fixed resin addition amount of 3 g is decided for subsequent experiments.With the increase of citric acid concentration,the adsorption rate of citric acid showed a trend of first increasing and then decreasing.When the concentration of the loading solution is 8.54mg/m L,the adsorption rate of citric acid by the resin reaches the maximum.The initial concentration of fixed citric acid is selected as 8.54 mg/m L for subsequent separation experiments.With the gradual increase of the sample flow rate,the adsorption rate of citric acid showed a decreasing trend overall.A comprehensive comparison of the citric acid adsorption rate,combined with the separation time,a sample flow rate of 1.5 m L/min is decided to select as the sample flow rate for subsequent separation experiments.The above sample volume is the abscissa,and the citric acid concentration in the effluent is the ordinate,and the leakage curve is drawn.The experimental results show that when the effluent volume is 150 m L,citric acid leakage begins,and the dynamic adsorption penetration point of the resin is reached at this time.After that,the sample volume continue to increase,the resin's adsorption of citric acid is gradually decreased,and the citric acid concentration in the effluent is gradually increased.Therefore,The maximum sample load is 150 m L is determined.Under these conditions,the contention of the prepared Neu5Ac sample is 85.35%.The prepared sample is identified by the LC-MS method.By comparing with the Neu5Ac standard,the peak time is the same as that of the Neu5Ac standard.Theoretical relative molecular mass is consistent with Neu5Ac standard,and the sialic acid species contained in the pure Neu5Ac product identified and prepared is only Neu5Ac,Neu5Gc is not detected.(4)Study on the anti-inflammatory effect of Neu5Ac on LPS-stimulated RAW264.7 cells:After RAW264.7 was exposed to different concentrations of Neu5Ac,the results showed that compared with the blank group,the effect on the activity of RAW264.7 of the 0.1 and 10?g/m L groups is significant(P<0.05);the effect on the activity of RAW264.7 of the other 4 groups(P>0.05)is not significant.The test results of inflammatory mediators in the cell supernatant showed that:Compared with the LPS stress group,Neu5Ac of different mass concentrations could effectively inhibit the secretion of NO,the difference is significant(P<0.05),and the effect of 50?g/m L group is most obvious(P<0.05).Compared with the LPS stress group,Neu5Ac of different mass concentrations can effectively inhibit the secretion of TNF-?,and the difference is significant(P<0.05).The effect of each group of Neu5Ac is similar(P>0.05).Compared with the LPS stress group,Neu5Ac of different mass concentrations can effectively inhibit the secretion of IL-6,the difference is significant(P<0.05),and the effect of 50?g/m L group is the most obvious inhibitory(P<0.05).Compared with the LPS stress group,Neu5Ac of different mass concentrations can effectively inhibit the secretion of IL-1?,and the difference is significant(P<0.05).Among them,the best inhibitory effects on the RAW264.7 inflammatory cell secreting inflammatory factor IL-1?is 50?g/m L group(P<0.05).Compared with the LPS stress group,Neu5Ac of different mass concentrations can also inhibit the secretion of PGE2,and the difference is significant(P<0.05).Among them,the effect of 25?50?100?200?g/m L group on inhibit the secretion of inflammatory factor IL-1?by RAW264.7 inflammatory cells are similar r(P>0.05).Conclusion:(1)On the basis of single factor experiment,combined with response surface method,the extraction process conditions of Neu5Ac is optimized,the optimal process parameters of Neu5Ac extraction are:citric acid concentration 0.10 mol/L,extraction time 4 h,liquid-to-material ratio 30:1(m L/g).The obtained Neu5Ac extract is preliminarily identified by HPLC method,and it is found that the Neu5Ac species is only Neu5Ac.(2)The best conditions for the purification of Neu5Ac extraction volume are:D301 resin is 3 g,initial concentration of citric acid is 8.54 mg/m L,sample flow rate is 1.5 m L/min,maximum sample size is 150 m L.Under these purification conditions,the contention of the Neu5Ac sample obtained is 85.35%.The LC-MS method is used to further identify the Neu5Ac prepared and verified that the sialic acid contained in it is only Neu5Ac,Neu5Gc is not detected.(3)Different concentrations of Neu5Ac inhibited the secretion of NO,TNF-?,IL-6,IL-1?,and PGE2 in RAW264.7 cells stimulated by LPS,but dose-effect relationship is not good.In general,When the mass concentration of Neu5Ac is 50?g/m L,the anti-inflammatory effect is the best.