Composition,Structure And The In Vitro Digestibility Characteristics Of Sheep Milk Triacylglycerols

Sheep milk has a high nutritional value and enormous potential application in the development of new infant formula.Sheep milk fat is an important energy source,however,studies about the characteristics of composition,structure,digestibility and absorption of sheep milk fat are very limited.The basic properties of sheep milk,composition and structure of sheep milk triacylglycerols（TAG）were systematically investigated in this study.Additonally,the relationship of sheep milk TAG structure and its digestibility was studied by using the in vitro gastrointestinal model,and the digestibility characteristics of TAG in homogenized sheep milk and sheep milk powder were also studied.Firstly,a GC-FID method for simultaneously analysis of various milk fatty acids was developed.SP-2560 column（100 m×0.25 mm,i.d.,0.20μm）was applied.The split ratio was set at 30:1,the gas flow rate was set at 1.0 m L/min,the injector temperature was set at 260°C,and the detector temperature was set at 280°C.The oven temperature was programmed from 140 to 220°C at a rate of 2°C/min within65 min.This new method is accurate,precise,and is capable to quantify up to 59 milk fatty acids.The determination methods for TAG,diacylgylcerols（DAG）and monoacylgylcerols（sn-2 MAG）in milk by HPLC-ESI-MS/MS were developed respectively.Coreshell C18 column（150 mm×4.6 mm,i.d.,2.6μm）was applied.Column temperature was set at 50℃.Binary gradient elution was applied with the mixture of methanol/water（1:1,v/v,0.1%formic acid,10 mmol/L ammonium formate,8μmol/L phosphoric acid）used as solvent A,and isopropanol（0.1%formic acid,8μmol/L phosphoric acid）used as solvent B.Full scans of TAG（m/z 300-1000）,DAG（m/z 150-1000）and MAG（m/z 150-1000）in the ESI positive mode was applied for MS¹,and the precursor ions[M+NH₄]⁺were obtained.Multiple reaction monitoring scans（MRM）was applied for MS² to monitor the product ions of[M+NH₄]⁺.The product ions of[M-R_nCOO]⁺,[MAG（R_nCO）]⁺and[R_nCO]⁺were selected respectively for the quantification of TAG,DAG and sn-2 MAG.Precision and accrucy of the three methods met the requirement of GB/T 6379.1-2004.The methods can differentiate the fatty acid positions in TAG molecular,and are able to semi-quantify TAG,DAG and sn-2 MAG in milk.Characteristics of composition and structures of TAG in sheep milk were investigated by comparing with cow milk TAG using the developed HPLC-ESI-MS/MS method.Sheep milk contained 101 TAG molecular species,while cow milk contained 105 molecular species of TAG.There were 89 species in two milks which had the same structures,12 unique species in sheep milk and 16 unique species in cow milk.The 12 unique TAG molecular species in sheep milk accounted for 17.1%of total TAG.Among them,6 species were MLM type（medium chain-medium/long chain-medium/short chain）of TAG（accounting for 5.8%of total TAG）,5 species were long-chain TAG（accounting for 9.5%of total TAG）which contained unsaturated fatty acids on the sn-2 position.Sheep milk had higher proportions of MLM type of TAG（13.6%）and polyunsaturated TAG（21.1%）,which were 2-folds and 1.5-folds of that in cow milk,respectively.Digestibility of TAG in sheep milk were investigated by comparing with cow milk TAG using the in vitro gastrointestinal digestion model.In the gastric phase,gastric lipase can pass through the milk fat globule membrane and hydrolyze TAG generating sn-1,3 DAG and fatty acid.Sn-1,3 DAG can be hydrolyzed further by gastric lipase producing sn-2 MAG and fatty acid.The digestibility of TAG in sheep milk was highly correlated to the total carbon number and the fatty acid position,while was poorly related to the total double bonds.MLM type of TAG was preferentially hydrolyzed by gastric lipase,their digesbilities ranged from 23.1%to31.2%.The digestibility of other TAG ranged from 9.4%to 21.1%.The digestibility of sheep milk TAG in total（16.1%）was significantly higher than cow milk TAG（12.4%）.In the intestinal phase,the digestion rate of sheep milk gastric digesta was slower than cow milk digesta.The intestinal digestion of sheep milk gastric digesta reached equilibrium after 30 min,while the equilibrium time of the intestinal digestion of cow milk gastric digesta was 10 min.Only～75%of total TAG was digested in the first 15 min of intestinal digestion of sheep milk gastric digesta.This was due to the accumulation of sn-1,3 DAG,sn-2 MAG and fatty acids which generated in gastric phase on the surface of milk fat globule membrane,these surface-active compounds inhibited the absorption of pancreatic lipase on the milk fat globule membrane.Therefore,the digestion rate of sheep milk gastric digesta was slow in the beginning of the intestinal phase.This inhibition decreased along with the intestinal digestion progress,the final digestion extent of sheep milk TAG was 89.8%,which was slightly lower than that of cow milk TAG（93.3%）.The final gastrointestinal digesta of sheep milk contained high amounts of unsaturated fatty acids and unsaturated sn-2 MAG,which are highly absorbable and nutritious compounds.Homogenization greatly decreased the milk fat globule size,changed the composition and structure of milk fat globule membrane,however,it did not change the composition and molecular structure of sheep milk TAG（H-SM）.The unsaturated TAG was unstable,hydorlyzed and oxidized during the spary-drying processing.Therefore,the percentage of unsaturated TAG in sheep milk powder（P-SM）decreased.In the gastric phase,the digestibility of total TAG in P-SM（26.5%）was higher than that of H-SM（24.1%）,and both were～0.5-folds higher than that of raw milk.In the intestinal phase,the digestion rate of TAG in H-SM and P-SM increased significantly,the equilibrium time of the intestinal digestion of TAG in H-SM and P-SM were shortened to 10 min and 5 min,respectively.Homogenization and spray-drying were able to increase the digestibility of sheep milk TAG in the gastrointestinal phase,and improved the low digestion rate of sheep milk TAG in the intestinal phase.Therefore,composition,structure of TAG as well as industrial process can affect the digestibility and absorption of sheep milk fat.