| Bovine colostrum contains abundant oligosaccharide(OS),which have many biological activities,can application in food and the health food.It is difficult to separate the different OS due to their similar but complex structures and less sufficient research.So it is a greatly limits for the application of functional OS from bovine colostrum.This research is aims to obtain high purity of functional OS especially sialyloligosaccharid by using membrane separation technology coupling with chromatographic separation technology.Through optimizing the separation conditions to obtain high purity sialyllactose and provide a reference for the high value use of bovine colostrum.Firstly,we established a rapid and accurate method for the analysis of saccharides in bovine colostrum.Verified the method by four standards:lactose,galactose,3'sialyllactose(3 ' SL),6'sialyllactose(6' SL),which rich in bovine milk.It was found that the standard curves for lactose,galactose,3'SL,6'SL is linear from 0.03-5.00 mg/mL,0.02-5.00 mg/mL,0.05-2.00 mg/mL,0.05-2.00 mg/mL,respectively(R2>0.99).Peak area precision(coefficient of variation)were below 4%(RSD<4%),and retention time precision(coefficient of variation)were below 2%(RSD<2%).Recovery rate were from 100%-115%and LOD,LOQ were 9?g/mL,30 ?g/mL;7 ?g/mL,20 ?g/mL;125 ?g/mL,200 ?g/mL;350 ?g/mL,450 ?g/nL;respectively.Based on the method we established,we detected 25 different OS from bovine colostrum,and the concentration of the OS is about 1.93 g/L.Secondly,according to the physical and chemical characteristics of the compositions in the bovine colostrum,we chose a 10 kDa ultrafiltration membrane and a 280 Da nanofiltration membrane to separation and purification the OS in bovine colostrum.The results showed that the pressure and temperature have obvious effects on membrane flux in the process of ultrafiltration,but has no obvious effect on the recovery of protein and total carbohydrate.On the optimum condition(5 bar,40?),the retention rate of protein was 96.0%,and tatal saccharide recovery rate was 98.3%.Pressure and temperature have obvious effects on membrane flux in the process of nanofiltration.When increase the pressure the rejection rate of monosaccharide and lactose were first increased and then decreased,and when increase the temperature the rejection rate of monosaccharide and lactose is decreased.On the optimum condition(8 bar,45?)the rejection rate of monosaccharide and lactose were 41.9%and the oligosaccharides were 97.0%.Finally,we analyzed the OS composition of bovine colostrum OS crude extract and found lactose and sialyllactose(SL)were two most abundant OS.Based on the property of lactose and sialyllactose,we chose D315,QY-P001,QY-Z001,PCR642Ca,CGC100X8 as the stationary phase to select the most suitable resin by compare with the resolution,recovery rate and retention time of lactose and SL.Results showed that the cation exchange resin QY-Z001 was more suitable to separating lactose and sialyllactose,and sialyllactose was strongly retained substances,lactose was weakly retained substances on this resin.Then,we used QY-Z001 resin as the stationary phase and deionized water as mobile phase,to study how the diameter to height ratio,temperature,flow rate,sample concentration effect on the separation of lactose and sialylactose.We found that diameter to height ratio 1:35;temperature 55?;flow rate 1.0 mL/min;sample concentration 100 mg/mL was the optimal conditions for separation lactose and sialyllactose.Under the optimal condition,the resolution of lactose and sialyllactose is 0.96,the purity of sialyllactose and lactose were 84.6%,99.3%,and recovery of sialyllactose and lactose were 76.9%,100.0%respectively.Thus QY-Z001 resin is high-efficiency for the separation of sialyllactose and lactose in bovine colostrum OS crude extract.Our results provided a basis for the separation and purification of functional oligosaccharide in the bovine colostrum. |
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