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CHAPTER 2: METHODS OF FOODANALYSIS

Despite efforts over the past half-century, there is still aneed for internationally harmonized methods and data. In fact, as described inChapter 1, the development of new methods for analysing specific components ofthe energy-yielding macronutrients has increased the complexity and made thisneed greater than ever.

This chapter discusses the commonly used analytical methodsfor protein, fat and carbohydrate, and makes recommendations regarding thepreferred methods for the current state of the art and available technology.Methods that continue to be acceptable when the preferred methods cannot be usedare also noted. Analytical methods for alcohol, which can be a significantsource of energy in some diets, polyols and organic acids were not discussed,and hence no recommendations for methods are made.

2.1 ANALYTICAL METHODS FOR PROTEINS INFOODS

2.1.1 Current status

For many years, the protein content of foods has beendetermined on the basis of total nitrogen content, while the Kjeldahl (orsimilar) method has been almost universally applied to determine nitrogencontent (AOAC, 2000).Nitrogen content is then multiplied by a factorto arrive at protein content. This approach is based on two assumptions: thatdietary carbohydrates and fats do not contain nitrogen, and that nearly all ofthe nitrogen in the diet is present as amino acids in proteins. On the basis ofearly determinations, the average nitrogen (N) content of proteins was found tobe about 16 percent, which led to use of the calculation N x 6.25 (1/0.16 =6.25) to convert nitrogen content into protein content.

This use of a single factor, 6.25, is confounded by twoconsiderations. First, not all nitrogen in foods is found in proteins: it isalso contained in variable quantities of other compounds, such as free aminoacids, nucleotides, creatine and choline, where it is referred to as non-proteinnitrogen (NPN). Only a small part of NPN is available for the synthesis of(non-essential) amino acids. Second, the nitrogen content of specific aminoacids (as a percentage of weight) varies according to the molecular weight ofthe amino acid and the number of nitrogen atoms it contains (from one to four,depending on the amino acid in question). Based on these facts, and thedifferent amino acid compositions of various proteins, the nitrogen content ofproteins actually varies from about 13 to 19 percent. This would equate tonitrogen conversion factors ranging from 5.26 (1/0.19) to 7.69(1/0.13).

In response to these considerations, Jones (1941) suggestedthat N x 6.25 be abandoned and replaced by N x a factor specific for the food inquestion. These specific factors, now referred to as “Jones factors”,have been widely adopted. Jones factors for the most commonly eaten foods rangefrom 5.18 (nuts, seeds) to 6.38 (milk). It turns out, however, that most foodswith a high proportion of nitrogen as NPN contain relatively small amounts oftotal N (Merrill and Watt, 1955; and 1973).^[4] Asa result, the range of Jones factors for major sources of protein in the diet isnarrower. Jones factors for animal proteins such as meat, milk and eggs arebetween 6.25 and 6.38; those for the vegetable proteins that supply substantialquantities of protein in cereal-/legume-based diets are generally in the rangeof 5.7 to 6.25. Use of the high-end factor (6.38) relative to 6.25 increasesapparent protein content by 2 percent. Use of a specific factor of 5.7 (Sosulskiand Imafidon, 1990) rather than the general factor of 6.25 decreases theapparent protein content by 9 percent for specific foods. In practical terms,the range of differences between the general factor of 6.25 and Jones factors isnarrower than it at first appears (about 1 percent), especially for mixed diets.Table 2.1 gives examples of the Jones factors for a selection offoods.

Because proteins are made up of chains of amino acids joinedby peptide bonds, they can be hydrolysed to their component amino acids, whichcan then be measured by ion-exchange, gas-liquid or high-performance liquidchromatography. The sum of the amino acids then represents the protein content(by weight) of the food. This is sometimes referred to as a “trueprotein”. The advantage of this approach is that it requires no assumptionsabout, or knowledge of, either the NPN content of the food or the relativeproportions of specific amino acids - thus removing the two problems with theuse of total N x a conversion factor. Its disadvantage is that it requires moresophisticated equipment than the Kjeldahl method, and thus may be beyond thecapacity of many laboratories, especially those that carry out only intermittentanalyses. In addition, experience with the method is important; some amino acids(e.g. the sulphur-containing amino acids and tryptophan) are more difficult todetermine than others. Despite the complexities of amino acid analysis, ingeneral there has been reasonably good agreement among laboratories and methods(King-Brink and Sebranek, 1993).

TABLE 2.1
Specific (Jones) factors for the conversion of nitrogen content to protein content (selected foods)

Source:Adapted and modified from Merrilland Watt (1973).

2.1.2 Recommendations

1) It is recommended that protein in foods be measured as the sum of individual amino acid residues (the molecular weight of each amino acid less the molecular weight of water) plus free amino acids, whenever possible. This recommendation is made with the knowledge that there is no official Association of Analytical Communities (AOAC)^[5] method for amino acid determination in foods. Clearly, a standardized method, support for collaborative research and scientific consensus are needed in order to bring this about.

2) Related to the previous recommendation, food composition tables should reflect protein by sum of amino acids, whenever possible. Increasingly, amino acid determinations can be expected to become more widely available owing to greater capabilities within government laboratories and larger businesses in developed countries, and to the availability of external contract laboratories that are able to carry out amino acid analysis of foods at a reasonable cost for developing countries and smaller businesses.

3) To facilitate the broader use of amino acid-based values for protein by developing countries and small businesses that may lack resources, FAO and other agencies are urged to support food analysis and to disseminate updated food tables whose values for protein are based on amino acid analyses.

4) When data on amino acids analyses are not available, determination of protein based on total N content by Kjeldahl (AOAC, 2000) or similar method x a factor is considered acceptable.

5) A specific Jones factor for nitrogen content of the food being analysed should be used to convert nitrogen to protein when the specific factor is known. When the specific factor is not known, N x the general factor 6.25 should be used. Use of the general factor for individual foods that are major sources of protein in the diet introduces an error in protein content that is relative to the specific factors and ranges from -2 percent to +9 percent. Because protein contributes an average of about 15 percent of energy in most diets, the use of N x 6.25 should introduce errors of no more than about 1 percent in estimations of energy content from protein in most diets ([-2 to +9 percent] x 15).

6) It is recommended that only amino acid analysis be used to determine protein in the following:

• foods used as the sole source of nourishment, such as infant formula;
• foods/formulas designed specifically for special dietary conditions;
• novel foods.

2.2 ANALYTICAL METHODS FOR FATS INFOOD

2.2.1. Current status

There is perhaps more agreement on standardized methods ofanalysis for fat than for protein and carbohydrate. Most fat in the diet is inthe form of triglyceride (three fatty acids esterified to a glycerol moleculebackbone). There are also non-glyceride components such as sterols, e.g.cholesterol. While there is considerable interest in the roles that thesenon-glyceride components may play in metabolism, they are not important sourcesof energy in the diet (FAO, 1994).

There are accepted AOAC gravimetric methods for crude fat,which includes phospholipids and wax esters, as well as minor amounts ofnon-fatty material (AOAC, 2000). Total fat can be expressed as triglycerideequivalents determined as the sum of individual fatty acids and expressed astriglycerides (FAO, 1994). This method is satisfactory for the determination offat in a wide variety of foods.

2.2.2 Recommendations

1) For energy purposes, it is recommended thatfats be analysed as fatty acids and expressed as triglyceride equivalents, asthis approach excludes waxes and the phosphate content of phospholipids, neitherof which can be used for energy (James, Body and Smith, 1986).

2) A gravimetric method, although less desirable, isacceptable for energy evaluation purposes (AOAC, 2000).

2.3 ANALYTICAL METHODS FORCARBOHYDRATES IN FOODS

2.3.1 Current status

FAO/WHO held an expert consultation on carbohydrate in 1997.The report of this meeting (FAO, 1998) presents a detailed description of thevarious types of carbohydrates and a review of methods used for analysis, whichis summarized conceptually in the following paragraphs. Other recommendationsfrom the 1997 consultation, e.g. the nomenclature of carbohydrates, wereconsidered by the current technical workshop participants.

Total carbohydratecontent of foods has,for many years, been calculated by difference, rather than analysed directly.Under this approach, the other constituents in the food (protein, fat, water,alcohol, ash) are determined individually, summed and subtracted from the totalweight of the food. This is referred to astotal carbohydrate bydifference and is calculated by the following formula:

100 - (weight in grams [protein + fat + water +ash + alcohol] in 100 g of food)

It should be clear that carbohydrate estimated in this fashionincludes fibre, as well as some components that are not strictly speakingcarbohydrate, e.g. organic acids (Merrill and Watt, 1973).Totalcarbohydrate can also be calculated from the sum of the weights of individualcarbohydrates and fibre after each has been directly analysed.

Available carbohydrate represents that fractionof carbohydrate that can be digested by human enzymes, is absorbed and entersinto intermediary metabolism. (It does not include dietary fibre, which can be asource of energy only after fermentation - see the following subsections.)Available carbohydrate can be arrived at in two different ways: it can beestimated by difference, or analyseddirectly.^[6]To calculate availablecarbohydrate by difference, the amount of dietary fibre is analysed andsubtracted from total carbohydrate, thus:

100 - (weight in grams [protein + fat + water +ash + alcohol + dietary fibre] in 100 g of food)

This yields the estimated weight of available carbohydrate,but gives no indication of the composition of the various saccharides comprisingavailable carbohydrate. Alternatively, available carbohydrate can be derived bysumming the analysed weights of individual available carbohydrates. In eithercase, available carbohydrate can be expressed as the weight of the carbohydrateor as monosaccharide equivalents. For a summary of all these methods, see Table2.2.

Dietary fibre is a physiological and nutritionalconcept relating to those carbohydrate components of foods that are not digestedin the small intestine. Dietary fibre passes undigested from the small intestineinto the colon, where it may be fermented by bacteria (the microflora), the endresult being variable quantities of short-chain fatty acids and several gasessuch as carbon dioxide, hydrogen and methane. Short-chain fatty acids are animportant direct source of energy for the colonic mucosa; they are also absorbedand enter into intermediary metabolism (Cummings, 1981).

TABLE 2.2
Total and available carbohydrate

* May be expressed as weight (anhydrous form) oras the monosaccharide equivalents (hydrous form including water).

Chemically, dietary fibre can comprise: cellulose,hemicellulose, lignin and pectins from the walls of cells; resistant starch; andseveral other compounds (see Figure 2.1). As more has been learned about fibre,a variety of methods for analysis have been developed. Many of these measuredifferent components of fibre, and thus yield different definitions of, andvalues for, it. Three methods have had sufficient collaborative testing to begenerally accepted by such bodies as AOAC International and the BureauCommunautaire de Reference (BCR) of the European Community (EC) (FAO, 1998): theAOAC (2000) enzymatic, gravimetric method - Prosky (985.29); the enzymatic,chemical method of Englyst and Cummings (1988); and the enzymatic, chemicalmethod of Theander and Aman (1982). Monro and Burlingame (1996) have pointedout, however, that at least 15 different methods are applied for determining thedietary fibre values used in food composition tables. Their publication, and theFAO/WHO report on carbohydrates in human nutrition (FAO, 1998), discuss theseissues in more detail. The effect of having such a variety of methods fordietary fibre, each giving a somewhat different value, affects not only thevalues in food composition tables for dietary fibreper se, but alsothose for available carbohydrate by difference.

2.3.2 Recommendations

1) Available carbohydrate is a useful concept inenergy evaluation and should be retained. This recommendation is at odds withthe view of the expert consultation in 1997, which endorsed the use of the term“glycaemic carbohydrate” to mean “providing carbohydrate formetabolism” (FAO, 1998). The current group expressed concerns that“glycaemic carbohydrate” might be confused or even equated with theconcept of “glycaemic index”, which is an index that describes therelative blood glucose response to different “availablecarbohydrates”. The term “available” seems to convey adequatelythe concept of “providing carbohydrate for metabolism”, while avoidingthis confusion.

2) Carbohydrate should be analysed by a method that allowsdetermination of both available carbohydrate and dietary fibre. For energyevaluation purposes, standardized, direct analysis of available carbohydrate bysummation of individual carbohydrates (Southgate, 1976; Hicks, 1988) ispreferred to assessment of available carbohydrate by difference, i.e. totalcarbohydrate by difference minus dietary fibre. This allows the separation ofmono- and disaccharides from starches, which is useful in determination ofenergy content, as discussed in Chapter 3.

3) Determination of available carbohydrate by difference isconsidered acceptable for purposes of energy evaluation for most foods, but notfor novel foods or food for which a reduced energy content claim is to be made.In these cases, a standardized, direct analysis of available carbohydrate shouldbe carried out.

4) “Dietary fibre” is a useful concept that isfamiliar to consumers and should be retained on food labelling and in foodtables. Because the physical characteristic of solubility/insolubility does notstrictly correlate with fermentability/non-fermentability, the distinctionbetween soluble and insoluble fibre is not of value in energy evaluation, nor isit of value to the consumer.

5) The AOAC (2000) analysis - Prosky (985.29)orsimilar method should be used for dietary fibre analysis.

6) Because dietary fibre can be determined by a number ofmethods that yield different results, when the Prosky method is not used themethod used should be stated and the value should be identified by INFOODStagnames^[7](Klensinet al.,1989). In addition, the method should be identified with the tagname in foodcomposition tables.

7) Further research and scientific consensus are needed inorder to develop standardized methods of analysis of resistant starch.

Figure 2.1 - Dietary fibre:constituents and associated polysaccharide fractions

Source: Monro and Burlingame(1996).