WEEK 8 FAT ANALYSIS

FST 314

INTRODUCTION
Lipids, proteins, and carbohydrates constitute the principal structural components of foods Lipids are a group of substances that in general are soluble in ether, chloroform, or other organic solvents but are sparingly soluble in water The most widely accepted definition is based on solubility Lipids comprise a broad group of substances that have some common properties and compositional similarities

INTRODUCTION
Triacylglycerols are fats and oils that represent the most prevalent category of the group of compounds known as lipids The terms lipids, fats, and oils are often used interchangely The term lipid commonly refers to the broad, total collection of food molecules that meet the definition based on solubility While there is no exact scientific definition, the US FDA has established a regulatory definition for nutrition labeling purposes The FDA has defined total fat as the sum of fatty acids from C4 C24, calculated as triglycerides

INTRODUCTION
General classification of lipids is useful to differentiate lipids in foods: Simple Lipids:

Fats: Esters of fatty acids with glycerol triacylglycerols Waxes: Esters of fatty acids with long-chain alcohols other than glycerols

Compound Lipids:
Phospholipids: Glycerol esters of fatty acids, phosphoric acids, and other groups containing nitrogen Cerebrosides: Compounds containing fatty acids, a carbohydrate, and a nitrogen Sphingolipids: Compounds containing fatty acids, a nitrogen, and a phosphoryl group

IMPORTANCE OF ANALYSIS

An accurate and precise quantitative and qualitative analysis of lipids in foods is important for:
Accurate nutritional labelling Determination of whether the food meets the standard of identity To ensure that the product meets manufacturing specifications

Inaccuracies in analysis may prove costly for manufacturers and could result in a product of undesirable quality and functionality
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SAMPLE SELECTION AND PRESERVATION

Validity of the results depends on proper sampling and preservation of the sample prior to analysis Composition of the sample analyzed should represent as closely as possible that of the food from which it was taken. Sample preparation required in lipid analysis depends on:

Type of food being analyzed (e.g. meat, milk, margarine, cookie, dairy cream) Nature of the lipid component (e.g. volatility, susceptibility to oxidation, physical state) Type of analytical procedure used (e.g. solvent extraction, non-solvent extraction or instrumental)

SAMPLE SELECTION AND PRESERVATION

In order to decide the most appropriate sample preparation procedure, it is necessary to have knowledge of the physical structure and location of the principal lipids present in the food. Since each food is different it is necessary to use different procedures for each one. Official methods have been developed for specific types of foods that stipulate the precise sample preparation procedure that should be followed.

SAMPLE SELECTION AND PRESERVATION

Sample preparation should be carried out using an environment that minimizes any changes in the properties of the lipid fraction. If lipid oxidation is a problem it is important to preserve the sample using either a nitrogen atmosphere, cold temperature, low light or adding antioxidants. If the solid fat content or crystal structure is important it may be necessary to carefully control the temperature and handling of the sample.

DETERMINATION TOTAL LIPID CONCENTRATION

It is important to be able to accurately determine the total fat content of foods for a number of reasons:

Economic (not to give away expensive ingredients) Legal Health (development of low fat foods) Quality (food properties depend on the total lipid content) Processing (processing conditions depend on the total lipid content)
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DETERMINATION TOTAL LIPID CONCENTRATION

The principle physicochemical characteristics of lipids (the "analyte") used to distinguish them from the other components in foods (the "matrix") are:
Solubility in organic solvents Immiscibility with water Physical characteristics (e.g., relatively low density) and spectroscopic properties.

Analytical techniques based on these principles can be categorized into three types:
1) 2) 3)

solvent extraction non-solvent extraction instrumental methods.

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SOLVENT EXTRACTION

Because lipids are soluble in organic solvents, but insoluble in water, the food analyst can separate the lipid components in foods from water soluble components, such as proteins, carbohydrates and minerals. Solvent extraction techniques are one of the most commonly used methods of isolating lipids from foods and of determining the total lipid content of foods. Sample Preparation The preparation of a sample for solvent extraction usually involves a number of steps: Drying sample. It is often necessary to dry samples prior to solvent extraction, because many organic solvents cannot easily penetrate into foods containing water, and therefore extraction would be inefficient.

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SOLVENT EXTRACTION

Sample Preparation Particle size reduction. Dried samples are usually finely ground prior to solvent extraction to produce a more homogeneous sample and to increase the surface area of lipid exposed to the solvent. Acid hydrolysis. Some foods contain lipids that are bound to proteins (lipoproteins) or polysaccharides (glycolipids). To determine the concentration of these components it is necessary to break the bonds which hold the lipid and non-lipid components together prior to solvent extraction. Acid hydrolysis is commonly used to release bound lipids into easily extractable forms, e.g. a sample is digested by heating it for 1 hour in the presence of 3N HCl acid

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SOLVENT EXTRACTION
Solvent Selection Ideal solvents for fat extraction should have a high solvent power for lipids and low or no solvent power for proteins, amino acids, and carbohydrates They should evaporate readily and leave no residue Have relatively low boiling points Be non flammable and nontoxic in both liquid and vapor states

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SOLVENT EXTRACTION
Solvent Selection Should penetrate sample particles readily It is difficult to find a solvent that meets all the requirements, ethyl ether and petroleum ether are the most commonly used solvents Pentane and hexane are also used to extract oil from soybeans A combination of two or three solvents is frequently used

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SOLVENT EXTRACTION
Solvent Selection (continued) Ethyl ether:
Boiling point of 34.6 C Better solvent for fat than petroleum ether Generally expensive compared to other solvents Has a greater danger of explosion and fire

Petroleum ether:

Low boiling point fraction of petroleum and is composed mainly of pentane and hexane Boiling point of 35-38 C More hydrophobic than ethyl ether Selective for more hydrophobic lipids, cheaper and less flammable than ethyl ether Detailed properties of petroleum ether for extraction are described in AOAC method 945.16

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CONTINUOUS SOLVENT EXTRACTION METHOD: GOLDFISH METHOD

For continuous solvent extraction, solvent from a boiling flask continuously flows over the sample held in a ceramic thimble Fat content is measured by weight loss of the sample or by weight of the fat removed Continuous methods give faster and more efficient extraction than semicontiusous extraction methods However they may cause channeling which results in incomplete extraction The Goldfish, as well as the Wiley and Underwriters tests are examples of continuous lipid extraction methods

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SEMICONTINUOUS SOLVENT EXTRACTION METHOD: SOXHLET METHOD

For semicontinuous solvent extraction, the solvent builds up in the extraction chamber for 5-10min and completely surrounds the sample and then siphons back to the boiling flask Fat content is measured by weight loss of the sample or by weight of the fat removed This method provides a soaking effect of the sample and does not cause channeling However, this method requires more time than the continuous method Instrumentation for a more rapid and automated version of the Soxhlet method is available (e.g.. Soxtec, FOSS, Eden Prairie) and is used in QC applications

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SEMICONTINUOUS SOLVENT EXTRACTION METHOD: SOXHLET METHOD

http://www.youtube.com/watch?v=2 L6MDOW88zc

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DISCONTINUOUS SOLVENT EXTRACTION METHOD: MOJONNIER METHOD

Fat is extracted with a mixture of ethyl ether and petroleum ether in a Mojonnier flask, and the extracted fat is dried to a constant weight and expressed as percent fat by weight Does not require removal of moisture from the sample It can be applied to both liquid and solid samples Petroleum ether is used to purify the extracted fat Was developed for and is applied primarily to dairy foods, but is applicable to other foods, specifically fat from flour and pet food Both involve an acid hydrolysis with HCl, followed by extraction with a combination of ethyl ether and petroleum ether (AOAC 989.05)

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DISCONTINUOUS SOLVENT EXTRACTION METHOD: MOJONNIER METHOD

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TOTAL FAT BY GC FOR NUTRITION LABELING

AOAC Method 996.06 Fat and fatty acids are extracted from food by hydrolytic methods (acidic hydrolysis for most products, alkaline hydrolysis for dairy products, and combination for cheese) Pyrogallic acid is added to minimize oxidative degradation of fatty acids during analysis Triglyceride, triundecanoin (C11:0) is added as internal standard

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TOTAL FAT BY GC FOR NUTRITION LABELING

Fat is extracted into ether, then methylated to fatty acid methyl esters (FAMEs) using BF3 (boron trifluoride) in methanol FAMEs are quantitatively measured by capillary GC against (C11:0) internal standard Total fat is calculated as sum of individual fatty acids expressed as triglyceride equivalents Saturated and monounsaturated fats are calculated as sum of respective fatty acids

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NONSOLVENT WET EXTRACTION METHODS: BABCOCK METHOD FOR MILK FAT

The Babcock method, H2SO4 is added to a known amount of milk in the babcock bottle The sulfuric acid digests protein, generates heat, and releases the fat Centrifugation and hot water addition isolate fat for quantification in the graduated portion of the test bottle The fat is measured volumetrically, but the result is expressed as a percent fat by weight

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NONSOLVENT WET EXTRACTION METHODS: BABCOCK METHOD FOR MILK FAT

Is a common official method for the determination of fat in milk It takes about 45 min and duplicate tests should agree within 0.1% It does not determine the phospholipids in the milk products It is not applicable to products containing chocolate or added sugar without modification because of charring of chocolate and sugars by sulfuric acid

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NONSOLVENT WET EXTRACTION METHODS: GERBER METHOD FOR MILK FAT

The principle of the Gerber method is similar to that of the Babcock method It uses sulfuric acid and amyl alcohol The sulfuric acid digests proteins and carbohydrates, releases fat, and maintains the fat in a liquid state by generating heat Simpler and faster and has wider application to a variety of dairy products than the Babcock method The isoamyl alcohol generally prevents the charring of sugar found with the regular Babcock method This test is more popular in Europe than in North America

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INSTRUMENTAL METHODS
Instrumental methods offer numerous attractive features compared with the previously described extraction methods In general, they are rapid, non-destructive, and require minimal sample preparation and chemical consumption However, the equipment can be expensive and measurements often require the establishment of calibration curves specific to various compositions Despite these drawbacks, several of the following instrument methods are very widely used in QC as well as R&D

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INSTRUMENTAL METHODS

Infrared Method Based on absorption of IR energy by fat at a wavelength of 5.73um The more energy absorption, the higher the fat content Mid-IR spectroscopy is used in Infrared Milk Analyzers to determine milk fat content (AOAC 972.16) Near-IR spectroscopy has been used to measure fat content of commodities such as meats, cereals, and oilseeds
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INSTRUMENTAL METHODS

Specific Gravity (Foss-Let Method)

Determined as a function of the specific gravity of a sample solvent extract A sample of known weight is extracted for 1.5-2min in a vibration-reaction chamber with perchloroethylene The extract is filtered and using a thermostatically controlled device with a digital readout, its specific gravity is determined The reading can then be converted to oil or fat percentage using a conversion chart

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INSTRUMENTAL METHODS

Nuclear Magnetic Resonance

NMR can be used to measure lipids in food materials in a non-destructive way One of the most popular methods for use in determining lipid melting curves to measure solid fat content With more affordable instruments is becoming more popular for measuring total fat content Total fat content can be measured using low-resolution pulsed NMR NMR is very rapid and accurate Although the principles of NMR are complex, the use is simple, especially due to the high degree of automation and computer control

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COMPARISON OF METHODS
Soxhlet extraction is a common crude fat determination method in many foods However, this method requires a dried sample for the hydroscopic ethyl ether extraction If the samples are moist or liquid foods, the Mojonnier method is generally applicable to determination of fat content Acid hydrolysis or alkaline hydrolysis is widely used on many finished foods Instrumental methods such as IR and NMR are very simple, reproducible, and fast, but are available only for fat determination for specific foods, and require a standard curve

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SUMMARY
Lipids are generally defined by their solubility characteristics rather than by some common structural feature Lipids in foods can be classified as simple, compound, or derived lipids The lipid content of foods varies widely, but quantitation is important because of regulatory requirements, nutritive value, and functional properties

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SUMMARY

To analyze fat content accurately and precisely, it is essential to have knowledge of the:
General composition of the lipids in the foods Physical and chemical properties of the lipids in the foods The principles of fat determination

There is no single standard method for the determination of fats in different foods

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SUMMARY
The validity of any fat analysis depends on proper sampling and preservation of the sample prior to analysis Predyring of the sample, particle size reduction, and acid hydrolysis prior to analysis also bay be necessary Total lipid content of foods is commonly determined by organic solvent extraction methods, which can be classified as continuous, semicontinuous, discontinuous, or by GC analysis for nutrition labelling

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SUMMARY
Nonsolvent wet extraction methods such as Babcock or Gerber, are commonly used for certain types of food products Instrumental methods such as NMR infrared and Foss-Let are also available for fat determination of specific foods These methods are rapid and so may be useful for QC but generally require correlation to a standard solvent extraction method

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REFERENCES

Textbook: Neilson et. al. (2010). Food Analysis, 4th Edition. Springer Publishing

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