Thursday, April 4, 2019
Methods of Lipid Analysis in Food
Methods of lipoid Analysis in Food lipides atomic number 18 define as the biomolecules whose solvability in water is less than that in non-polar solvents. This definition puts structur in ally distinct classes of compounds much(prenominal) as oleaginous bitters, terpenes, steroids, prostaglandins and carotenes in the same class (Carey Giuliano, 2014). early(a) definition for lipidee has also been suggested (Eoin, et al., 2005). They serve a variety of functions in living system including and non hold to structural integrity, life force storage, digestion and communication (Nelson Cox, 2005).Although lipids are essential for many body functions, not all lipids are required in same sum. Food and Agriculture Organization (FAO) recommends that total pad of the nutrition contri simplye to 20-35% of energy for adults. This should entangle 8% saturated super eke outted acid(SFA), 11% poly unsaturated buttery acid (PUFA) and less than 1% trans plunkty acid (TFA) (FAO, 2010). Chances of insulin resistance is to a greater extent in overweight person in high SFA diet (Lovejoy, et al., 2002). Similarly, uppity TFA in progress to also increases the risk of cardiovascular disease (Song, et al., 2015).Fat content also affects preference. It is proposed that fat has a unique taste Oleogustus that is dependent of chain length and is unique from five conventional tastes sweetness, sourness, bitterness, saltiness and umami (Running, Craig, Mattes, 2015). butter produced from milk with high unsaturated fatty acid (UFA) was found to be much(prenominal) spreadable, softer and less viscous (Bobe, Hammond, Freeman, Lindberg, Beitz, 2003).As the fat content and type way outs health as healthful as taste and texture, it is essential to know the fat content of food for thought. Fat take is characterized by a variety of criteria and orders. The natural selection of the criteria and method depends upon the warning type, purpose, accuracy, precision, l egal requirement as head as available funds. and then, despite having limited signifi ejectce for nutritional purpose, total lipid (TL) metrement is widely carried disclose as many food labelling regulations require the TL report. Similarly, iodine value green goddess be apply for unsatu symmetryn study when sophisticated chromatography or spectroscopy methods are unavailable (Greenfield Southgate, check of methods of digest, 2003).This review intends to compile the available methods of lipid summary of food products. Focus will be given on the type of seek required, result provided, resolving message of each criteria and method. Only brief discussion will be d wizard on the theoretical and experimental process of the method.Sample zeal is very essential part of analysis of lipid sample. A erupt section is dedicated to sample preparation. In order to analyze the lipid, suitable sample must be prepared. If the sample has lipid inside cells (such(prenominal) as meat ) the lipid should be extracted from the cells (Christie Han, Lipid Analysis, 2012). Samples must undergo nigh pretreatment before they tail end be use as test sample. Depending on the nature of sample unity or more of the following work is needed.2.1. Storage VesselPlastic container should be strictly avoided to introduce lipids. Plasticizers can leach into the sample and contaminate it. Glass vessels or Teflon coated vessels should be used. The ambiance should be argon or normality to anticipate oxidation. (Christie Han, Lipid Analysis, 2012)2.2. Protection from OxidationUnsaturated fatty acids are prone to oxidation from atmospheric group O. Once the oxidation starts, autocatalysis accelerates the process. Different products are formed during oxidation and it may follow various mechanisms. Light, heat, metals, enzyme are known to catalyze the reaction (Angelo, 1996) Oxidation interferes with lipid analysis not only by destroying the unsaturated fats but also by formati on of conjugated treble bonds which show strong ingress at UV, thus providing false positive results (Christie Han, Lipid Analysis, 2012)Elimination of oxygen is the study step in prevention of oxidation. Therefore, all steps should be done in nitrogen atmosphere as far as possible. Equipment should be flushed with nitrogen before experiments. Small add of antioxidant like 2,6-di-tert-butyl-p-cresol, which do not interfere with analytical process, may be added. Excess of these antioxidant should not be used as they can facilitate oxidation in high concentration. (Christie Han, Lipid Analysis, 2012)2.3. Drying of SampleLipid sample containing water can make analysis difficult and expertness be a tooth root of error. Solvents cannot penetrate sample with 8% moisture easily. Hygroscopic solvents like diethyl ether can absorb the moisture, decreasing its beginning efficiency. Low moisture also facilitates grinding and increases the open air area of sample (Shahidi Wanasindara , 2008). oil color spirit, the most widely used solvent requires completely dried sample. (Greenfield Southgate, Review of methods of analysis, 2003)Depending on the type of water present (free, adsorbed or water of hydration) different methods may be required for water removal (Bradley, Jr, 2010). Care should be interpreted during drying as high temperature might lead to decomposition and combination of lipids with another(prenominal) components. These associated lipids cannot be extracted by solvents. lyophilization (freeze-drying) and vacuum drying methods are favorite(a) drying methods. (Shahidi Wanasindara, 2008)2.4. Particle size reduction (Grinding)Solid food sample might need grinding. Grinding increases the surface area and decreases the length through which solvent need to penetrate the sample (Min Ellefson, 2010). Care should be taken that the particles are not too fine, too much heat is generated or too much moisture is lost. (IUPAC, 1979)2.5. HydrolysisLipids in food may be bound ionically or covalently with non-lipid components such as carbohydrate or protein. Solvents are not able to extract them efficiently. Therefore, lipid needs to be hydrolyzed with acid or alkali to turn them into free state. Significant error in lipid origin is reported when no hydrolysis is carried out. (Min Ellefson, 2010) Hydrolysis also breaks emulsified fat. (Shahidi Wanasindara, 2008)Acid hydrolysis is used for most foods overleap diary and high scratching content food which require alkaline hydrolysis. Hydrolysis is not preferred when the lipid extract needs to be unless analyzed for fatty acid components because they can cause decomposition and oxidation of the sample components. (Greenfield Southgate, Review of methods of analysis, 2003)The given sample of food might not be entirely lipid. The amount of lipid in the food sample is called total lipid concentration. It is usually expressed as fortune or per 100gm food (Moreau, 2005). Although total li pid is widely used for food labelling and regulation of food composition, it has limited value as it does not provide the type of molecule in the lipid. (Greenfield Southgate, Review of methods of analysis, 2003)There are various methods to determine total lipid in food like solvent beginning, non-solvent lineage, instrumental methods. The selection of methods depends on a number of means which is discussed below.3.1. Solvent ExtractionIn solvent extraction, the lipid component of the food is extracted by dissolving in suitable organic solvent(s). The solvent selectively dissolves the lipid while leaving the non-lipid spate undissolved. The solvent is then evaporated to leave fat residue. Total lipid is then determined gravimetrically as-(i)The preceding(prenominal) data gives the total fat. However, a significant portion of the fat includes glycerol (from triglycerides) phospholipids and other unsaponifiable matters. Thus, corrections are required so as to represent the corr ect amount of fatty acids in the sample. The correction factor is provided by FAO. (Greenfield Southgate, Appendix 5, 2003)The extracted portion of solvent extraction is highly dependent on solvent use. Hence selection of solvent is discussed in detail next.Solvent SelectionIdeal solvent should extract all lipids and lipids only. However, due to wide range of polarity of different lipid types, no single solvent can provide an ideal solution. Moreover, the solvent selected should preferably be low boiling, non-flammable, non-toxic in naiant as well as unfaltering, easily disposable after extraction, inexpensive and non- hygroscopic. It should also penetrate sample thoroughly (Min Ellefson, 2010).Petroleum ether is the most commonly used solvent for its selectivity towards lipid, cost and availability. However, diethyl ether is better solvent for lipids but its fire hazard and hygroscopic nature makes it less favorable than oil colour ether. Ethyl ether and petroleum ether is als o some metres used alternately or together for extraction. (Pomeranz Meloan, 1994) n-hexane is preferred for oil extraction. (IUPAC, 1979) medley of polar and non-polar solvents has shown to extract all the lipids from most food. However, care should be given so as to prevent extraction of unwanted portion. The lipids extracted by this method without hydrolysis is suitable for further treatment to determine fatty acid fractions. (Greenfield Southgate, Review of methods of analysis, 2003) Alcohol-ether can be used to remove fat from tissue. Water-butanol is used in cereals. Chloroform-methanol is preferred for fleshly tissue (Pomeranz Meloan, 1994).Solvent extraction is the standard method of analysis for many types of food. Hence, it is widely used and is undergoing consecutive improvement. There are different types of solvent extraction, each with its pros and cons.3.1.1. Batch ExtractionBatch extraction is a very simple, yet widely used method of extraction. The sample is mix ed with one or more solvent which along with endogenous water (if any) forms multiple layer of varying concentration. As the lipids are more soluble in non-polar solvents than in water, lipid portion goes to the layer with more solvents and non-lipid component remains in the layer with more water. The lipid part is then separated using a separating funnel. The separation is establish on partition principle hence multiple extraction of the aqueous phase is necessary to take hold most of the lipid. The weight of lipid not extracted is given by the equation below (Pomeranz Meloan, 1994)-(ii)Where, is the weight of lipid remaining, the volume of aqueous layer, volume of solvent in each extraction step, the distribution ratio of lipid in solvent, the number of extraction steps. The selection of solvent is then done using the distribution ratio of lipids in known solvents.Folch method uses chloroform-methanol extraction followed by washing with water. This extracts all lipid from tiss ue except strandin which remains with the non-lipid phase. (Folch, 1957) Folch method was improved by Bligh and Dyer to improve the speed of extraction and purify the sample at the same duration. (Bligh Dyer, 1959) Extraction using low toxicity solvents like hexane propanol has also been highly-developed. (Hara Radin, 1978)Batch extraction is usually slow and requires a large amount of solvent. When other speedy and easier methods are available, this method is not preferred. However, as no sophisticated equipment is necessary, batch extraction is very useful where the cost of equipment outweighs the usefulness of more accurate data.3.1.2. Continuous ExtractionContinuous solvent extraction recycles the solvent used so that dwarfish amount of solvent can accomplish the equivalent extraction of several steps. This process is preferred for solid samples and sample where the distribution ratio is low. These samples need multi step extraction as very little lipid is extracted to the solvent in each step. (Pomeranz Meloan, 1994)Soxhlet extractor is widely used extractor for lipid. Although first developed to cake milk fat it has developed as a standard extractor for lipid as well as other substances. (Soxhlet, 1879) It has gone various improvements since its first publication and now various modifications are commercially available.Goldfish extraction is a faster extraction system which suspends the sample in the solvent vapour. Although faster than Soxhlet based system, it might not completely extract the lipid due to channeling i.e. solvent may take a preferential path in the solid sample and may not cover firm of the sample. (Moreau, 2005)As continuous extraction is faster and uses less solvents than batch extraction it is the most widely used extraction system. Moreover, the equipment used is not very expensive and can be used for extraction of other materials. However, these processes are slow and disposal of solvent is an everyday problem.3.1.3. Pressur ized Fluid ExtractionPressurized fluid extraction (PLE) is carried out in high pressure and high temperature. In literature, this process is also called Pressurized Solvent Extraction or Accelerated Solvent Extraction. The term ASEis the registered trademark of Dionex Corporation which manufactures pressurized fluid extraction apparatus commercially. (Dean, 2009)Richter et al. studied the effect of variables like temperature, pressure, solvent volume on extraction. Their work showed that the ASE extractor could provide results comparable to Soxhlet but with trim down extraction time and solvent volume. This is attributed to decreased viscosity of solvent, weaker bond among components and increased diffusion qualification of solvent at higher temperature. Increased pressure is primarily applied to keep the solvent liquid, however, it provides the added reinforcement of forcing the solvent into pores blocked by insoluble matter. (Richter, et al., 1996)However, there is evidence th at PLE is not selective to lipids for certain foods. (Boselli, Velazco, Caboni, Lercker, 2001) Moreover, no significant difference was seen on lipid extracted from poultry meat between Folch, Soxlet or ASE method. (Toschi, Bendini, Ricci, Lercker, 2003)Pressurized solvent extraction can be highly useful in labs where routine extraction is required as it greatly reduces the extraction time and solvent use. On the other hand, the investment on the apparatus may not be economical if extraction is not carried out regularly. In all cases the stability of temperature sensitive components should be known before using PLE.3.1.4. Super censorious Fluid ExtractionSubstance in temperature above its critical temperature and pressure is called supercritical fluid. (IUPAC, 1997) Solvent property of supercritical fluid was first demonstrated in 1879. (Hannay Hogarth, 1879) They commence huge prospects in extraction because they combine the solubility power of liquid with penetration power of liquid. Moreover, their solubility can be fine-tuned by changing the pressure and temperature. Carbon dioxide and water are the most lustrous fluid for supercritical extraction due to their non-toxicity and environmentally friendly nature. (Hedrick, Mulcahey, Taylor, 1992)3.2. Non-Solvent Liquid ExtractionIn this method, the sample is treated with some liquid reagent which separates the lipid from sample and the lipid fraction is then measured. This method is for the most part used to determine milk fat. They require specialized vessels for each method and cannot determine phospholipids. (McClements, 2003)Several methods are present3.2.1. Babcock Method3.2.2. Gerber Method3.2.3. Detergent Method3.3. Instrumental MethodsDifferent instrumental methods have been developed to determine the total lipid content of the sample. They rely on some physical properties that vary consistently with lipid concentration. (McClements, 2003)Based on the property measured it is mainly of three type s3.3.1. Measuring Bulk Properties immersion concentration decreases as lipid content increases. This relation can be used to know the percentage of fat in a sample. (McClements, 2003)Electrical Conductivity Conductivity decreases as lipid content increases. Thus, it can be used as fat concentration measure. (McClements, 2003)Ultrasonic Velocity This is a fast and non-invasive method for fat content calculation. Amplitude and brightness analysis can be used to happen the fat content and also distribution. (Abdul, N, Mohd, Abu, Z, 2013) Moreover, attenuation(absorption) of ultrasound is linearly proportional to the amount of fat in the body. (Dukhin, Goetz, Travers, 2013)3.3.2. Measuring ducking of RadiationUV-VIS Fatty Acid absorb UV light proportional to its concentration. This method requires sample preparation to remove substances like proteins and hydrophobic peptides which interfere with the measurement. (Forcato, Carmine, Echeverria, Pecora, Kivatinitz, 2005) Because of t he extraction and dilution needed the process can be time consuming and labor intensive. (McClements, 2003)IR Near infrared (NIR) spectroscopy is mainly used to analyze fatty acid content in food. (Mossoba, Azizian, Kramer, 2012) Fat show strong absorbance at 5.74 mm which provides rapid and online fat composition measure. (McClements, 2003) The carbonyl absorption is the major reason for lipids NIR activity. This method requires intensive calibration with other sanction methods hence is mainly used for routine analyses of large number of similar sample. (Greenfield Southgate, Review of methods of analysis, 2003) nu wakeful magnetic resonance NMR is also a non-destructive and fast method of total fat analysis. Although it requires a calibration curve, it is better accommodate than IR or UV because it can be operated by non-experts and the calibration curve is long lasting. (Oxford Instuments molecular(a)(a) Biotools, 2010)X-Ray Fat absorbs less X-Ray than lean meat. Hence, by building a proper calibration curve, fat content in meat can be determined by X-Ray absorption (McClements, 2003)3.3.3. Measuring Scattering of RadiationLight and Ultrasonic Scattering Light as well as ultrasound waves are scattered by oil droplets present in emulsions. The linear relation between concentration of droplet and light scattering can be used to measure total fat, provided no other interfering molecules exist. (McClements, 2003)X-Ray Fat molecules show a sharp X-Ray scattering peak at 1.1 nm-1 while a water rich tissue shows a peak at 1.6nm-1. Thus, varying amount of fat can give a scattering profile which can be used for fat content determination. (Elshemey, 2011)3.4. Other Methods3.4.1. Solid Phase Extraction3.4.2. Microwave Assisted ExtractionTotal Lipid Concentration is a very simple data about food. As lipid contain diverse chemical species, in addition to total lipid, the type of lipid and their distribution also plays a major role to determine the purity, nutriti onal value, aesthetic look and taste. Hence, complete information about the type of lipids is necessary for scientist as well as legal bodies. (McClements, 2003)Sample preparation is the most important part in most method to analyses lipids. As the lipid is extracted, care should be given to prevent the change of one form of lipid to another. Hydrolysis should be rigorously prevented as it reduces triglycerides and increases free fatty acids. Extraction should cause as less oxidation as possible. (Greenfield Southgate, Review of methods of analysis, 2003) Extraction in chloroform, chloroform-methanol and hexane-isopropanol is preferred. Storage of sample in polar at -20oC is preferred. (AAFCO Lab Methods Services Committee, 2014) The various method present have their advantages and drawbacks and the preferable method depends on the type of food and the type of lipid to me examined, (Greenfield Southgate, Review of methods of analysis, 2003)4.1. ChromatographyIt is a very powerfu l tool for lipid analysis. It can give compete profile of the lipid molecules in the given sample. Chromatography separates the different components of lipid in fractions, these are then subject to spectrometric analysis which gives the molecular identity as well as relative concentration. IR, NMR and Mass Spectrometry are most commonly used. (McClements, 2003) These methods, although very reliable and cosmopolitan are very expensive due to the instrumental and reagent cost and is only carried out where complete molecular identification is required. (Greenfield Southgate, Review of methods of analysis, 2003)Three types are usedtender loving care Thin Layer Chromatography is used to find concentration of different lipid groups. The TLC eggshell is prepared with suitable adsorbent and kept in proper solvent. A drop of sample is placed on one end and let to flow. The plate after separation to different fractions is compared to standard plates to identify the lipids. The spots can be analyzed further by GC, MS, NMR. (McClements, 2003) After the advent of HPLC, TLC use has decreased considerably. However, after the availability of pre-coated plates, the use of TLC for instant result is still carried out when there are few samples only. It is cheaper than HPLC. However, care during experimentation is required. (Christie, Thin-Layer Chromatography of Lipids, 2011) This method cannot be used to separate different types of phospholipids. (Zaima, Goto-Inoue, Adachi, Mitsutoshi, 2011)HPLC High Performance Liquid Chromatography is now a preferred method for lipid analysis. This is because it is more versatile than TLC and operates at room temperature, thus can be used to analyses labile groups that cannot be done using GC. (Christie, Thin-Layer Chromatography of Lipids, 2011)GC Gas Chromatography is the preferred method for analysis of trans fatty acid. It can also be used for triglycerides and fatty acids however, methylation is necessary. (Greenfield Southgate, Rev iew of methods of analysis, 2003) Fatty acids are non-volatile, hence before carrying out GC, the lipids are saponified and methylated to give Fatty Acid Methyl Esters(FAME) which are volatile and can be used for GC. (McClements, 2003) -(2)It is now possible to convert a lipid sample of a fraction of a milligram in size to the methyl ester derivatives, separate these by gas chromatography, and have a quantitative result in under one hour. (Christie, Chapter 1 Introduction and Summary, 2011)4.2. chemical MethodsThese methods are very cheap and do not require expensive machinery. However, only crude and intermediate results are obtained.Following test gives different information on fatIodine Value It gives the fairish degree of unsaturation in the lipid. The lipid to be analyzed is titrated with ICl and the consumption of ICl gives the amount of unsaturation in lipid.Saponification Number It gives the fair(a) molecular weight of triglycerols. The triglycerols are saponified with K OH and the amount of KOH used is determined. This is the saponification number. High saponification number corresponds to low molecular weight and vice versa.Acid Value It gives the amount of free fatty acid. Here, the lipid is titrated with KOH until the solution turns alkaline. Other acids may interfere with results.(McClements, 2003)4.3. Instrumental TechniquesVarious instrumental techniques for fat analysis are present. Methods like NMR, IR, MS are usually coupled with chromatography. Measurement of density and refractive index can be used to measure change in chain length and unsaturation. (McClements, 2003)Lipids with high unsaturation undergoes aerial oxidation. This includes variety of reactions usually summarized as followsreactants primary products secondary products(unsaturated lipids and O2) (hydrogen peroxides and conjugated dienes) (ketones, aldehydes, alcohols, hydrocarbons)5.1. ChromatographyLoss of reactants as well as formation of specific products can be monit ored by using time profile.5.2. Oxygen UptakeMeasures the amount of oxygen consumed over time while maintaining constant oxygen concentration on the reaction vessel.5.3. Peroxide ValueMeasures the amount of peroxide formed by titration with iodine.5.4. Conjugated DienesMeasures the concentration of conjugated dienes by UV spectroscopy (at 233nm for diene and 268nm for trines)5.5. Thiobarbituric Acid(TBA)Measures the secondary products (aldehydes) in the sample. The sample is treated with TBA and absorbance measured at 540nm. The absorbance value corresponds with the concentration of aldehyde.5.6. Accelerated Oxidation TestsThe sample is oxidate in oxidation friendly environment and the time taken for rancidity to form is measured.These tests help to know the physicochemical characteristics corresponding to flavor, appearance, flow etc.6.1. Solid Fat Content(SFC)Measures the fraction of fat present as solid. Density measure is mostly used(3)Where is the density at given temperature and are the density if it was completely liquid or solid at the same temperature.NMR signal decay rate is also be used recently. More solid component, faster is the signal decay.Differential Scanning Calorimetry uses latent heat measure are also used to measure SFC-temperature profile.6.2. Melting PointUsed when SFC is not required but only the temperature of melting is required. Due to different components present no sharp melting height is seen. preferably different melting points are usedClear Point The temperature at which fat completely melts and becomes clearSlip point The temperature at which the fat in a capillary tube starts to slip.Wiley melting point the temperature at which a disc suspended in alcohol-water mixture turns to a sphere.6.3. Cloud PointThe temperature at which a completely melted lipid starts to develop turbidity.6.4. Smoke PointThe temperature at which the lipid starts to stinker at standard condition6.5. Flash PointThe temperature at which a flash ap pears on the surface at ignition at standard condition6.6. Fire PointThe temperature at which a continuous flame stats to form at standard condition6.7. RheologyThe measure of deformation and flow. Viscosity, elastic modulus and other pertinent flow or plasticity measure is used.(n.d.). (FOSS) Retrieved from Analytical Solutions for Food Analysis and Quality Control FOSS http//www.foss.dk//media/images/ca/soxtec8000/soxtech_extraction_sketch-jpgAAFCO Lab Methods Services Committee. (2014, January). Crude Fat Methods Considerations. Retrieved from Association of American Feed Control Officials http//www.aafco.org/Portals/0/SiteContent/ testing ground/Fat_Best_Practices_Working_Group/Crude_Fat_Methods_Considerations.pdfAbdul, H. M., N, B., Mohd, S. M., Abu, K. R., Z, M. (2013). The Use of Ultrasound As a Fat Measurement Sensor. International Conference on intellectual Instrumentation, Measurement and Applications (ICSIMA), (pp. 315-320). Kuala LAmpur. inside10.1109/ICSIMA.2013.6 717974Angelo, A. J. (1996). Lipid Oxidation in Foods. Critical Reviews in Food Science and Nutrition, 36(3), 175-224. doi10.1080/10408399609527723Bligh, E. G., Dyer, W. J. (1959). A Rapid Method of Total Lipid Extraction and Purification. Canadian Journal of Biochemistry and Physiology, 911-917.Bobe, G., Hammond, E. G., Freeman, A. E., Lindberg, G. L., Beitz, D. C. (2003, October). Texture of butter from Cows with Different Milk Fatty Acid Composition. Journal of Dairy Science, 86(10). doi10.3168/jds.S0022-0302(03)73913-7
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