Abstract: This work aimed to establish a stable method to determine monosaccharide composition of Streptococcus thermophilus exopolysaccharides by anion exchange chromatography. The gradient elution procedure was optimized with different concentration and the different deproteinization method was analyzed. The optimal chromatographic conditions were as follows: CarboPac PA20 column (3 mm × 150 mm) was used with elute rate at 0.5 mL/min, the eluent was 1.75 mmol/L NaOH for the first 20 min, followed by a linear NaAc gradient (50-200 mmol/L) with fixed 1.75 mmol/L NaOH for the next 10 min to analyze the neutral with 1.75 mmol/L NaOH. Based on the optimal method, twelve mixed monosaccharide standards were separated. The correlation coefficients of each standard were greater than 0.99, the limits of detection were 2.5-21.1 μg/L, and the recoveries ranged were 77.3%-118.5%. In addition, the optimal deproteinization method was trichloroacetic acid. The monosaccharide composition of exopolysaccharides from four S. thermophilus strains were analyzed with optimized method. The results revealed that the exopolysaccharides were mainly composed of galactosamine, rhamnose, glucosamine, galactose, glucose, mannose, galacturonic acid, and glucuronic acid. This study provides a reliable method for the monosaccharide composition analysis of exopolysaccharides from S. thermophilus.
CUI Y H,JIANG X,HAO M Y,et al.New advances in exopolysaccharides production of Streptococcus thermophilus[J].Archives of Microbiology,2017,199(6):799-809.
CHE H Q,ZHANG H,TIAN Y J,et al.Exopolysaccharide from Streptococcus thermophilus as stabilizer in fermented dairy:Binding kinetics and interactions with casein of milk[J].International Journal of Biological Macromolecules,2019,140:1 018-1 025.
PACHEKREPAPOL U,LUCEY J A,GONG Y,et al.Characterization of the chemical structures and physical properties of exopolysaccharides produced by various Streptococcus thermophilus strains[J].Journal of Dairy Science,2017,100(5):3 424-3 435.
XU Z Y,GUO Q B,ZHANG H,et al.Exopolysaccharide produced by Streptococcus thermophiles S-3:Molecular,partial structural and rheological properties[J].Carbohydrate Polymers,2018,194:132-138.
LI S Q,SHAH N P.Antioxidant and antibacterial activities of sulphated polysaccharides from Pleurotus eryngii and Streptococcus thermophilus ASCC 1275[J].Food Chemistry,2014,165:262-270.
ZHANG J,CAO Y Q,WANG J,et al.Physicochemical characteristics and bioactivities of the exopolysaccharide and its sulphated polymer from Streptococcus thermophilus GST-6[J].Carbohydrate Polymers,2016,146:368-375.
SUN N X,LIU H P,LIU S J,et al.Purification,preliminary structure and antitumor activity of exopolysaccharide produced by Streptococcus thermophilus CH9[J].Molecules,2018,23(11):e2898.
DEL PIANO M,BALZARINI M,CARMAGNOLA S,et al.Assessment of the capability of a gelling complex made of Tara gum and the exopolysaccharides produced by the microorganism Streptococcus thermophilus ST10 to prospectively restore the gut physiological barrier:A pilot study[J].Journal of Clinical Gastroenterology,2014,48:S56-S61.
SVENSSON M,WAAK E,SVENSSON U,et al.Metabolically improved exopolysaccharide production by Streptococcus thermophilus and its influence on the rheological properties of fermented milk[J].Applied and Environmental Microbiology,2005,71(10):6 398-6 400.
TANG W,LIU D,YIN J Y,et al.Consecutive and progressive purification of food-derived natural polysaccharide:Based on material,extraction process and crude polysaccharide[J].Trends in Food Science & Technology,2020,99:76-87.
李仁勇,梁立娜,牟世芬.高效阴离子交换色谱-脉冲安培检测食品中糖的应用进展[J].食品与发酵工业,2010,36(7):97-101.LI R Y,LIANG L N,MOU S F.Application advance in analysis of food carbohydrates with high performance anion exchange chromatography-pulsed amperometric detection[J].Food and Fermentation Industries,2010,36(7):97-101.
乐胜锋, 王尉,王雅萱,等.离子色谱-脉冲安培法测定芦荟多糖中7种单糖的含量[J].色谱,2019,37(3):319-324.LE S F,WANG W,WANG Y X,et al.Determination of seven monosaccharides in Aloe polysaccharide using ion chromatography with pulsed amperometric detector[J].Chinese Journal of Chromatography,2019,37(3):319-324.
FELZ S,VERMEULEN P,VAN LOOSDRECHT M C M,et al.Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS)[J].Water Research,2019,157:201-208.
MAGER D M,THOMAS A D.Extracellular polysaccharides from cyanobacterial soil crusts:A review of their role in dryland soil processes[J].Journal of Arid Environments,2011,75(2):91-97.
刘晓莹,马璐瑶,王力,等.基于高效阴离子色谱-脉冲安培法优化阿拉伯木聚糖单糖组成分析[J].分析化学,2017,45(3):416-422.LIU X Y,MA L Y,WANG L,et al.Monosaccharide composition analysis of arabinoxylan by high performance anion exchange chromatography with pulsed amperometric detection[J].Chinese Journal of Analytical Chemistry,2017,45(3):416-422.
李静, 李仁勇,梁立娜.毛细管型离子色谱-脉冲安培法检测枸杞多糖的单糖组成[J].分析化学,2012,40(9):1 415-1 420.LI J,LI R Y,LIANG L N.Determination of monosaccharide constituents in Lycium barbarum polysaccharide using capillary ion chromatography with pulsed amperometric detection[J].Chinese Journal of Analytical Chemistry,2012,40(09):1 415-1 420.
ZHANG Z Q,KHAN N M,NUNEZ K M,et al.Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection[J].Analytical Chemistry,2012,84(9):4 104-4 110.
REN W,XIA Y J,WANG G Q,et al.Bioactive exopolysaccharides from a S.thermophilus strain:Screening,purification and characterization[J].International Journal of Biological Macromolecules,2016,86:402-407.
DUBOIS M,GILLES K A,HAMILTON J K,et al.Colorimetric method for determination of sugars and related substances[J].Analytical Chemistry,1956,28(3):350-356.
BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254.
WU D T,GUO H,LIN S,et al.Review of the structural characterization,quality evaluation,and industrial application of Lycium barbarum polysaccharides[J].Trends in Food Science & Technology,2018,79:171-183.
ALYASSIN M,CAMPBELL G M,MASEY O’NEILL H,et al.Simultaneous determination of cereal monosaccharides,xylo-and arabinoxylo-oligosaccharides and uronic acids using HPAEC-PAD[J].Food Chemistry,2020,315.DOI:10.1016/j.foodchem.2020.126221.
DE SOUZA M F,PEREIRA D S,FREITAS S P,et al.Neutral sugars determination in Chlorella:Use of a one-step dilute sulfuric acid hydrolysis with reduced sample size followed by HPAEC analysis[J].Algal Research,2017,24:130-137.
XIE J H,SHEN M Y,NIE S P,et al.Analysis of monosaccharide composition of Cyclocarya paliurus polysaccharide with anion exchange chromatography[J].Carbohydrate Polymers,2013,98(1):976-981.
PINTADO A I E,FERREIRA J A,PINTADO M M E,et al.Eficiency of purification methods on the recovery of exopolysaccharides from fermentation media[J].Carbohydrate Polymers,2020,231.DOI:10.1016/j.carbpol.2019.115703.
李嘉文, 刘达,刘党生,等.乳酸菌胞外多糖产生菌的筛选与初步研究[J].中国微生态学杂志,2019,31(9):1 027-1 033.LI J W,LIU D,LIU D S,et al.Screening of and preliminary research on exopolysaccharides producing strain[J].Chinese Journal of Microecology,2019,31(9):1 027-1 033.
NIE S P,ZHANG H,LI W J,et al.Current development of polysaccharides from Ganoderma:Isolation,structure and bioactivities[J].Bioactive Carbohydrates and Dietary Fibre,2013,1(1):10-20.
ZHANG H,REN W,GUO Q B,et al.Characterization of a yogurt-quality improving exopolysaccharide from Streptococcus thermophilus AR333[J].Food Hydrocolloids,2018,81:220-228.