Chromatographic and Mass Spectrometric Determination of Molecular Species of Short-Chain Triacylglycerols in Butterfat

Short-chain triacylglycerols (SC-TAGs) comprise at least one esterified fatty acid with two to six carbon atoms. A high molar proportion of SC-TAGs in bovine milk fat (MF) is a unique feature among edible fats and oils and results in characteristic biological, chemical, physical, and technological properties of MF. Further, high content of SC-TAGs induces specific requirements for TAG analysis. The main objective of the present study was to develop chromatographic (gas chromatography, GC; normal-phase high-performance liquid chromatography, np-HPLC) and mass spectrometric (MS) (electron ionization MS, EI–MS; electrospray ionization MS, ESI–MS/ESI–MS/MS) methods to separate, identify, and quantify regio- and acyl chain isomers of SC-TAG species in model TAG mixtures and in butterfat (BF). An improved method was developed to fractionate BF TAGs into saturated, monoene, and polyene TAGs on a solid-phase extraction column in silver ion mode prior to GC and GC–EI–MS. This method enabled quantification of otherwise coeluting regio- and acyl chain isomers of SC-TAG species with the same number of acyl carbons but different degree of unsaturation. The elution order of the acyl chain isomers of isobaric TAG species and the regioisomers of SC-TAGs on a polarizable phenyl(65%)methylsilicone GC-column was empirically established using retention indices calculated for a high number (112) of synthesized TAG species and isomers. Separation power of the column proved to be high enough to resolve the regioisomers of mono-SC-TAGs. Correspondingly, np-HPLC was shown to resolve the regioisomers of mono- and dibutyryl TAGs to the baseline, monocaproyl TAGs close to the baseline, and dicaproyl TAGs partially. Even few regioisomer pairs of long-chain TAGs containing fatty acyl 20:0 were resolved as well. The present study established that in ESI–MS/MS, the cleavage of SC-acyls (4:0, 6:0) from the sn-1(3) positions of mono-SC-TAGs was at least 2.3 times higher than that from the sn-2 position, hence enabling clear-cut differentiation between regioisomers. Due to intricate TAG composition of BF specific molar correction factors (MCFs) were used in quantification of TAGs by GC, GC–EI–MS, and np-HPLC–ESI–MS. In both MS methods, extensive variation in MCFs was observed due to different molecular size, degree of unsaturation, and regio- and acyl chain isomerism of TAGs, but substantial number (>100) of empirically determined MCFs enabled extrapolation of MCFs for more uncommon TAGs. Both MS methods provided new information that emphasized the need to use specific MCFs for the sn-1(3) and sn-2 TAG isomers. All quantification methods yielded relatively unsurprising and similar general distribution of TAG classes and selection of the most common TAG species in BF. However, 64 minor TAGs of 336 even-numbered TAG species that were determined by np-HPLC–ESI–MS were confirmed for the first time to exist in BF. A high number of them were SC-TAGs including monoacetyl TAGs, rare sn-2 isomers of mono-SC-TAGs, and di- and tri-SC-TAGs. The present study suggested that the developed methods were reliable choices to determine both the most common TAGs and more rare TAGs in BF. All methods enabled quantification of the regioisomers of SC-TAGs in BF. The methods can be adapted to detect adulteration of MF, to elucidate biological or technological function of MF, and to develop effective procedures of MF modification. They can be applied to investigation of structured TAGs with short-chain fatty acyls.Lyhytketjuisiin triasyyliglyseroleihin (SC-TAG) on esteröityneenä vähintään yksi lyhytketjuinen rasvahappo, joka sisältää 2–6 hiiliatomia. Maitorasvan (MF) TAG-koostumus eroaa muista yleisistä ravintorasvoista ja -öljyistä siinä, että SC-TAG:ien osuus (mol-%) on MF:ssa poikkeuksellisen suuri, mikä vaikuttaa MF:n biologisiin, kemiallisiin, fysikaalisiin ja teknologisiin ominaisuuksiin ja tekee MF:n TAG:ien analysoinnista haasteellista. Tämän tutkimuksen tavoitteena oli kehittää kromatografisia (kaasukromatografisia, GC; normaalifaasinestekromatografisia, np-HPLC) ja massaspektrometrisiä (MS) (elektroni-ionisaatio-MS, EI–MS; sähkösumutusionisaatio-MS, ESI–MS / ESI–MS/MS) menetelmiä erottamaan, tunnistamaan ja kvantitoimaan voirasvan (BF) SC TAG:ien asyyliketju- ja sn 1(3)/sn 2-paikkaisomeereja. Tutkimuksessa kehitettiin yksinkertainen hopeaioni-kiinteäfaasiuuttomenetelmä (Ag-SPE), jota käytettiin fraktioimaan BF:n TAG:t tyydyttyneisiin ja kerta- ja monityydyttymättömiin TAG:eihin. Ag-SPE:n käyttö ennen GC- ja GC–EI–MS-analyysejä mahdollisti samanaikaisesti eluoituvien, mutta eri tyydyttyneisyysasteisten SC-TAG:ien asyyliketju- ja sn 1(3)/sn 2-paikkaisomeerien kvantitoinnin GC:sti ja GC–EI–MS:sti. TAG:ien ja TAG-isomeerien eluointijärjestyksen määrittämiseksi 112 erilaista TAG:a ja TAG-isomeeria syntetisoitiin kemiallisella vaihtoesteröinnillä ja niille määritettiin retentioindeksit (RI) polarisoituvalla fenyyli(65%)metyylisilikoni-GC-kolonnilla. RI-vertailu osoitti, että kolonnin erotuskyky oli riittävä erottamaan kaikkien mono-SC-TAG:ien sn 1(3)/sn 2-paikkaisomeerit toisistaan. Vastaavasti havaittiin, että peräkkäin kytketyt np-HPLC-kolonnit (3 kpl) erottivat täydellisesti kaikkien mono- ja dibutyryyli-TAG:ien ja lähes täydellisesti monokaproyyli-TAG:ien paikkaisomeerit toisistaan sekä dikaproyyli-TAG:ien isomeerit osittain. Yllättäen myös sellaisten pitkäketjuisten-TAG:ien, joihin oli esteröityneenä C20:0-rasvahappo, sn 1(3)/sn 2-paikkaisomeerit erottuivat toisistaan. Tutkimus osoitti lisäksi, että ESI–MS/MS:ssa lyhytketjuiset asyylit (C4:0, C6:0) lohkeavat vähintään 2,3 kertaa helpommin SC-TAG:ien sn-1(3)- asemista kuin sn-2-asemasta, mikä mahdollistaa vastaavien paikkaisomeerien erottamisen tällä menetelmällä. Kaikissa kvantitointimenetelmissä (GC, GC–EI–MS ja np-HPLC–ESI–MS) käytettiin spesifisiä, molaarisia korjauskertoimia (MCF) TAG:eille. Molemmissa MS-menetelmissä todettiin TAG:ien molekyylikoon, tyydyttyneisyyden ja asyyliketju- ja sn 1(3)/sn 2-paikkaisomerian vaikuttavan MCF:iin, minkä vuoksi oli välttämätöntä käyttää spesifisiä MCF:a myös SC-TAG:ien sn 1(3)/sn 2 paikkaisomeerien kvantitoinnissa. Kokeellisesti määritettyjen MCF:ien verraten suuri lukumäärä (>100) mahdollisti harvinaisempien TAG:ien MCF:ien päättelemisen. Kaikilla käytetyillä menetelmillä pystyttiin luotettavasti määrittämään TAG-jakautuma ja yleisimmät TAG:t BF:ssa. BF:sta määritettiin np-HPLC–ESI–MS:sti kaikkiaan 336 erilaista parillista TAG:a, joista 64:ä ei ole aiemmin kokeellisesti todettu BF:ssa. Suuri osa uusista TAG:eista oli monoasetyyli-TAG:ja, harvinaisia mono-SC-TAG:ien sn-2-isomeereja ja di/tri-SC-TAG:ja. Tässä tutkimuksessa käytetyillä menetelmillä pystyttiin kvantitoimaan BF:n SC-TAG:t ja niiden sn 1(3)/sn 2-paikkaisomeerit. Käytetyt menetelmät ovat sovellettavissa MF-väärennysten havaitsemiseen, MF:n biologisten ja teknologisten ominaisuuksien tutkimiseen ja MF:n muokkauksen seurantaan. Ne soveltuvat myös räätälöityjen SC-TAG:ien analysointiin

Effects of milk fat, unhydrogenated and partially hydrogenated vegetable oils on fat metabolism of growing pigs: I. Growth, feed utilization and carcass quality in pigs fed different fats and oils

Two trials were conducted to study the effects of different dietary fats on the performance, carcass quality and meat quality of 75 crossbred growing pigs. The experimental diets contained 14.3% butter oil (BO), low erucic acid rapeseed oil (RO), sunflower oil (SO) or partially hydrogenated sunflower oil (HSO). The cream (CR) content of the diets was 29.4%. The dietary fat addition comprised about 36% of the net energy content of the diets. The fatty acid composition of the dietary fats had a clear influence on the fatty acid composition of the adipose tissue of the pigs. Vegetable oils (RO and SO) increased the unsaturated fatty acid content of the adipose tissue and decreased the firmness of the backfat compared to the effects by milk fat (80, CR) (

Modification of butterfat by selective hydrolysis and interesterification by lipase: Process and product characterization

Butterfat was chemically modified via combined hydrolysis and interesterification, catalyzed by a commercial lipase immobilized onto a bundle of hydrophobic hollow fibers. The main goal of this research effort was to engineer butterfat with improved nutritional properties by taking advantage of the sn-1,3 specificity and fatty acid specificity of a lipase in hydrolysis and ester interchange reactions, and concomitantly decrease its level of long-chain saturated fatty acid residues (viz., lauric, myristic, and palmitic acids) and change its melting properties. All reactions were carried out at 40°C in a solvent free system under controlled water activity, and their extent was monitored via chromatographic assays for free fatty acids, esterified fatty acid moieties, and triacylglycerols; the thermal behavior of the modified butterfat was also assessed via calorimetry. Lipase-modified butterfat possesses a wider melting temperature range than regular butterfat. The total saturated triacylglycerols decreased by 2.2%, whereas triacylglycerols with 28–46 acyl carbons (which contained two or three lauric, myristic, or palmitic acid moieties) decreased by 13%. The total monoene triacylglycerols increased by 5.4%, whereas polyene triacylglycerols decreased by 2.9%. The triacylglycerols of interesterified butterfat had ca. 10.9% less lauric, 10.7% less myristic, and 13.6% less palmitic acid residues than those of the original butterfat

Tammanmaidon perus- ja rasvahappokoostumus

vo

Effects of milk fat, unhydrogenated and partially hydrogenated vegetable oils on fat metabolism of growing pigs : I.Growth, feed utilization and carcass quality in pigs fed different fats and oils

Tutkimus kuului yhteistutkimukseen "Ravintorasvojen vaikutus sydän - ja verisuonitautien riskitekijöihin". Tämän osatutkimuksen tarkoituksena oli selvittää erilaisten ravintorasvojen; voi -, rypsi -, auringonkukka - ja kovetetun auringonkukkaöljyn sekä kerman vaikutus sikojen kasvuun, rehun hyväksikäyttöön, teuraslaatuun ja lihan laatuun. Veren kolesterolitutkimuksen tulokset julkaistaan toisaalla tässä lehdessä (KEMPPINEN et al. 1993). Voi-, rypsi-, auringonkukka- ja kovetetun auringonkukkaöljyn osuus rehusta oli 14,3 %. Kerman määrä rehuseoksessa oli 29,4 %. Perusrehu, johon rasvat lisättiin, koostui vähärasvaisista ja kuitupitoisista raaka-aineista. Rasvan lisäystaso vastasi noin 36 % rehun nettoenergiasta. Siat ruokittiin kaksi kertaa päivässä rajoitetuin normein (1,2-2,8 ry/el/pv). Ravintorasvojen rasvahappokoostumuksella oli selvä vaikutus sikojen silavan rasvahappokoostumukseen. Kasviöljyjen monityydyttymättömät rasvahapot, erityisesti linolihappo, varastoituivat suoraan silavaan ja heikensivät merkitsevästi silavan kiinteyttä. Kovetettu auringonkukkaöljy nosti transrasvahappojen pitoisuutta silavassa. Silavan linolihappopitoisuuden ja kiinteyden välillä oli voimakas negatiivinen korrelaatio (p<0.001). Kasviöljyjen ja maitorasvan välillä ei ollut muita eroja sikojen tuotantotuloksissa kuin maitorasvalla saatu hivenen heikompi rehun hyväksikäyttö. Öljyn kovettaminen heikensi sen energia-arvoa. Tulokset osoittivat, että kasviöljy ja voirasva soveltuvat lihasikojen rehuksi. Kokeessa käytetty kasviöljyn lisäystaso oli kuitenkin liian korkea, koska se heikensi selvästi silavan kiinteyttä.Two trials were conducted to study the effects of different dietary fats on the performance, carcass quality and meat quality of 75 crossbred growing pigs. The experimental diets contained 14.3% butter oil (BO), low erucic acid rapeseed oil (R0), sunflower oil (SO) or partially hydrogenated sunflower oil (HSO). The cream (CR) content of the diets was 29.4%. The dietary fat addition comprised about 36% of the net energy content of the diets. The fatty acid composition of the dietary fats had a clear influence on the fatty acid composition of the adipose tissue of the pigs. Vegetable oils (RO and SO) increased the unsaturated fatty acid content of the adipose tissue and decreased the firmness of the backfat compared to the effects by milk fat (BO, CR) (p<0.001). The correlation between linoleic acid content and firmness of back fat was significant (r= -0.83) (p<0.001). The trans fatty acid (C18:1) found in HSO seems to be incorporated intact into the fatty tissue of the pig to a large extent and a significant difference was found in the contents of trans fatty acid C18:1 between the SO and HSO treatments (P<0.001). Significant differences were found in feed conversion efficiency, kg DM/kg gain, (p<0.05) and in the firmness of back fat (p<0.001) between the present vegetable oils (RO, SO, HSO) and the milk fat (BO, CR) dietary groups. Hydrogenated sunflower oil (HSO) increased the firmness of back fat compared to unhydrogenated sunflower oil (SO) (p<0.001), but in contrast, it significantly reduced daily gain and feed conversion efficiency (p<0.05). A vegetable oil addition of the level used in the present study is not recomended for pig diets because of the undesirable consistency of back fat.VokIrtonumeroiden myynti MTT:n kirjasto, Vuosikertatilauksia välittävät mm. kirjakaupatRavintorasvat lihasikojen ruokinnass

Ravintorasvat lihasikojen ruokinnassa

Two trials were conducted to study the effects of different dietary fats on the performance, carcass quality and meat quality of 75 crossbred growing pigs. The experimental diets contained 14.3% butter oil (BO), low erucic acid rapeseed oil (RO), sunflower oil (SO) or partially hydrogenated sunflower oil (HSO). The cream (CR) content of the diets was 29.4%. The dietary fat addition comprised about 36% of the net energy content of the diets. The fatty acid composition of the dietary fats had a clear influence on the fatty acid composition of the adipose tissue of the pigs. Vegetable oils (RO and SO) increased the unsaturated fatty acid content of the adipose tissue and decreased the firmness of the backfat compared to the effects by milk fat (80, CR) (pTutkimus kuului yhteistutkimukseen "Ravintorasvojen vaikutus sydän - ja verisuonitautienriskitekijöihin". Tämän osatutkimuksen tarkoituksena oli selvittää erilaisten ravintorasvojen; voi -, rypsi -, auringonkukka - jakovetetun auringonkukkaöljyn sekä kerman vaikutus sikojen kasvuun, rehun hyväksikäyttöön, teuraslaatuun ja lihan laatuun. Veren kolesterolitutkimuksen tulokset julkaistaan toisaalla tässä lehdessä (Kemppinen et al. 1993). Voi-, rypsi-, auringonkukka- ja kovetetun auringonkukkaöljyn osuus rehusta oli 14,3 %. Kerman määrä rehuseoksessa oli 29,4 %. Perusrehu, johon rasvat lisättiin, koostui vähärasvaisista ja kuitupitoisista raaka-aineista. Rasvan lisäystaso vastasi noin 36 % rehun nettoenergiasta. Siat ruokittiin kaksi kertaa päivässä rajoitetuin normein (1,2-2,8 ry/el/pv). Ravintorasvojen rasvahappokoostumuksella oli selvä vaikutus sikojen silavan rasvahappokoostumukseen. Kasviöljyjen monityydyttymättömät rasvahapot, erityisesti linolihappo, varastoituivat suoraan silavaan ja heikensivät merkitsevästi silavan kiinteyttä. Kovetettu auringonkukkaöljy nosti transrasvahappojen pitoisuutta silavassa. Silavan linolihappopitoisuuden ja kiinteyden välillä oli voimakas negatiivinen korrelaatio (

Lipase-catalyzed acidolysis of butterfat with oleic acid: characterization of process and product

The modification of anhydrous butterfat via interesterification reactions with oleic acid catalyzed by a lipase from Mucor circinelloides immobilized by adsorption onto hydrophobic hollow fibers is described. A reasonable degree of incorporation of free (externally added) oleic acid into the triacylglycerols of butterfat has been achieved while short-chain fatty acid residues remained virtually unaffected. Total saturated triacylglycerols decreased by 27%, and triacylglycerols with 32–44 acyl carbons (which contained two or three lauric, myristic, or palmitic acid residues) decreased by 33%. Total monoene and polyene triacylglycerols increased by 21% and 17%, respectively. The triacylglycerols (TAG) of interesterified butterfat had approximately 27% more oleic acid residues and approximately 8% less lauric, 6% less myristic, and 6% less palmitic acid residues than those of the original butterfat; the fraction of low-melting TAG peak increased by 19% whereas that of high-melting TAG decreased by 83%. Although a certain degree of butterfat hydrolysis was observed, enzymatic acidolysis was technically feasible and able to produce a modified butterfat with a stronger nutraceutical character

Bioaccessibility of vitamin B12 synthesized by Propionibacterium freudenreichii and from products made with fermented wheat bran extract

The bioaccessibility of vitamin B12 (B12) in plant-based products fortified using wheat bran extract fermented with B12-producing food-grade Propionibacterium freudenreichii was studied by applying a standard static in vitro model. At first, a culture of P. freudenreichii, fresh or heat-treated, was subjected to in vitro assays. Then, food ingredients or products were evaluated for their in vitro bioaccessibility: spray-dried wheat bran extract powder, pasta made with an extruder using fermented bran extract and breads made with spray-dried powder or with added cyanocobalamin. B12 bioaccessibility from the fresh P. freudenreichii culture was only ca. 53%, which, when heated, increased to 73%. The bioaccessibility of B12 from the food products varied from 75% (spray-dried powder) to 95% (breads). B12 from the fortified bread was as bioaccessible as from the bread made with added cyanocobalamin (99%). The in vitro results suggest that B12 synthesized by P. freudenreichii, when fortified in the studied cereal-based products, is largely bioaccessible and could be available for absorption. Plant-based products fortified using fermentation with P. freudenreichii could thus be considered excellent sources of bioaccessible B12.Peer reviewe

Effects of milk fat, unhydrogenated and partially hydrogenated vegetable oils on serum lipoproteins in growing pigs

1. Crossbred Yorkshire (Yorkshire*Landrace) pigs were fed butter oil, cream, low erucic acid rapeseed oil, sunflower oil and partially hydrogenated sunflower oil in amounts representing 30% of energy for periods of up to 13 weeks. 2. After 13 wk of feeding serum total cholesterol levels of pigs fed milk fat were significantly higher than of pigs fed vegetable oils. 3. The difference in cholesterol was mainly due to an increase in the density range of 1.063-1.125 g/ml containing pig LDL2 and some HDL. 4. A shift towards smaller LDL paricle size was apparent in pigs fed milk fat. 5. The effects of dietary trans fatty acids did not differ from cis polyunsaturated or monounsaturated fatty acids.vo

Effects of milk fat, unhydrogenated and partially hydrogenated vegetable oils on fat metabolism of growing pigs: II. Changes in serum cholesterol and triglyceride levels

Fifty mainly crossbred growing pigs were fed diets composed of a basal feed (2% fat) and supplemented 30% gross energy in the form of butter oil, cream, low erucic acid rapeseed oil (LEAR), sunflower oil or partially hydrogenated sunflower oil for 88 days in order to evaluate the effects of different food fats on their serum cholesterol and triglyceride levels. The levels of serum total cholesterol in the animals fed butter oil (3.47 mmol/l) and cream (3.48 mmol/l) were significantly (