Quantitative Determination Of Bovine Lactoferrin And Lactulose In Dairy Products

Dairy products are made from fresh milk,such as raw cow milk or raw goat milk,being added with appropriate amounts of vitamins,minerals and other nutrients.It is an excellent source of nutritional contents,which meets human health needs,and contains all the nutrients needed for the healthy growth of infants.With the development of people's life,the requirements for foods,especially dairy products with high nutritional value,are getting higher and higher.Hence,currently,more comprehensive laws and regulations have been formulated for dairy products from farms to shelves,and established more complete detection technology in the world.Functional nutrients in dairy products are becoming increasingly popular with consumers.Milk quality and potential risks are closely related to their processing methods.Bovine lactoferrin and lactulose,two reliable indexes,are concerned,but they are not yet simple and applicable detection platform.It is of great significance to establish a fast,sensitive and reliable detection approach for bovine lactoferrin and lactulose in dairy products to protect consumers'health.This paper applyed liquid chromatography-tandem mass spectrometry(LC-MS/MS)and liquid chromatography-evaporative light scattering detection(LC-ELSD)to detect bovine lactoferrin and lactulose in dairy products.The main research contents and results are as follows:1.A liquid chromatography-tandem mass spectrometry was established for determination of bovine lactoferrin in dairy products.Sample were treated by degreasing and tryptic hydrolysis.Peptides of bovine lactoferrin were identified using ultra-performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry(UPLC-Q/Exactive-HRMS)system,in data-dependent acquisition(Date-dependent MS~2)mode,and analyzed by Protein pilot.Eight species-specific marker peptides of bovine lactoferrin were identified by sequence alignment.Three marker peptides with high response strength and stability were chosen for further quantitative research by high-performance liquid chromatography-triple quadrupole mass spectrometry(HPLC-Qq Q-MS).The sample was performed on an Aeris PEPTIDE XB-C18 reversed-phase chromatography column(100×2.1 mm,1.7?m)with the mobile phases of 0.1%(v/v)formic acid aqueous solution and acetonitrile.The determination performed by MS/MS in electrospray positive ion mode(ESI~+)and established a multiple reaction monitoring(MRM)detection method for bovine lactoferrin.The external standard method with goat milk powder as the blank matrix and the internal standard method with internal standard peptides added were used to quantify the content of bovine lactoferrin in the milk powder samples.The results showed that the external standard quantitative results of the three peptides and the internal standard method were not significant.difference.The method showed a good linear relationship in the range of 0.1-75 mg/kg.The limits of detections and quantitation were in the range of 0.019-0.041 mg/kg and 0.077-0.137 mg/kg,respectively.The average recovery was93.8%-104.6%.The Intra-day and Inter-day RSDs were lower than 8.8%and 10.9%,respectively.This method presents various advantages such as simple pretreatment,environmental protection,strong anti-interference,high sensitivity and reproducible,suitable for the quantitative of bovine lactoferrin in dairy products such as milk powder,yogurt and liquid milk.2.The methods for the determination of lactulose in liquid milk based on LC-ELSD and LC-MS/MS were studied,respectively.The sample was precipitated the protein by ammonium acetate,and the supernatant was detected after centrifugation.Lactulose was determined based on the LC-ELSD,separated by an Exsil 100 HAAX column(100×2 mm,1.5?m),with a column flow rate of 0.8 m L/min,and an isocratic elution of acetonitrile-water.We set evaporating gas temperature as 60?,evaporating gas temperature as 70?,gas flow rate as 1.4 m L/min to obtain better lactose-lactulose resolution.Using lactose-free milk as a blank matrix for matrix matching,an external standard quantitative analysis method was established to investigate matrix effects and methodological verification.The results showed that the matrix was enhanced,and the average recovery rate of the method was 83-99%,the intra-day and inter-day RSDs were 1.13-5.24%and1.38-7.08%.The limit of detection and quantification of lactulose were 0.3 mg/kg and 1 mg/kg,respectively.The results demonstrated a good linear relationship in the range of 1.0-100 mg/kg.This method has short detection time and low cost,and is suitable for quantitatively detecting the content of lactulose in liquid milk such as UHT milk,and identifying whether it contains reconstituted milk.Based on the LC-MS/MS method for determination of lactulose in liquid milk,Waters XBridge BEH Amide column(100×2.1 mm,1.7?m)was used to separate lactulose-lactose in the sample,and gradient washing was established with acetonitrile-aqueous ammonia as mobile phase.The matrix-matched external standard method was used for quantification in the mode of ESI~-.The results showed a good linear relationship within 0.05-50mg/kg.This method's limit of quantification was 0.05 mg/kg,and the average recovery was92.0-111.9%.The intra-day and inter-day RSDs were less than 7.73%and 12.70%.This method can effectively separate lactulose and lactose,with simple pretreatment,time-saving,low quantification limit,but the detection time is longer than that of LC-ELSD.It can meet the requirements of determination of lactulose content in liquid milk with low lactulose content such as pasteurized milk.