Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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was positively correlated to the percentage protein

in the flour, whereas lipid and cell wall material was

negatively correlated. Starch and protein separating

efficiencies generally increase with increasing pro-

tein content. An unpublished rule of thumb has

been applied to evaluating the potential of legume

seed for air classification. If the flour is finely milled

and a 33% fines fraction yield is sought, the protein

content of that fraction is apt to be 2–2.5 times the

initial protein value of the flour. To insure uniform

products by air classification technology, it is sug-

gested that, in any particular legume being processed,

the seed falls within a narrow range of protein

content.

0012 Shifting of other components also occurs as a result

of air classification. In terms of fiber material present

in the flour, the hull fiber tends to segregate into the

coarse fraction and the cell wall fiber into the fine

fraction. Dehulling may enhance the efficiency of

the process somewhat and may be a prerequisite if

colored hulls are present. If the coarse (starch) frac-

tion is of lesser importance, dehulling need not be

practiced as it only has a small effect on the crude

fiber content of the protein fraction.

0013 Legume flours containing a-galactosides, especially

raffinose, manninotriose, stachyose, and verbascose,

generally show the protein fraction to be 40–90%

higher in these components than the initial flour,

while the starch fractions become depleted. Legumes

with trypsin inhibitors, hemagglutinating factors,

saponins, phytic acid, vicine, and convicine will

exhibit elevated concentrations of these components

in the protein fractions.

0014 The protein fraction from legumes also tends to be

enriched in ash and lipid, the latter having undesir-

able ramifications in storage of the concentrate. The

lipids likely occur as a result of the presence of a lipid-

rich coating on the protein bodies.

0015Protein concentrates prepared by air classification

may be used as emulsifiers or supplements in meat

products and patties, beverages, bread, noodles, and

spaghetti. Cowpea flour fractionated into enriched

protein and starch concentrates has been applied to

produce emulsifiers and starch jellies. There may be a

requirement to inactivate some of the antinutritional

factors. The starch fractions are increasingly being

utilized in the paper industry, especially carbonless

paper. Some use of pea starch was made in ore refin-

ing, but this has been sporadic. Nevertheless, peas

have shown the greatest potential for commercial

application of air-classification technology. There

are few major nutritional or technological problems

except for the ‘pea flavor’ and, in some cases, a darker

green color in some end products. The main obstacles

to success in classification stem back to basic agro-

nomics, the guarantee of a year-round supply at a

competitive price and of high quality, and establish-

ment of markets for the products.

Application to Cereal Grains

Wheat Flour

0016The earliest work in application of air classification

techniques to food products began in several United

States labs (Peoria, Illinois and Manhattan, Kansas)

with protein-shifting in wheat flours. Classification

has generally been less efficient than in legume flours

because of the high proportion of small starch gran-

ules that cannot be readily separated from the fine

protein particles. Approximately 81% of the granules

fall below 7.5 mm, the quantity by weight represented

tbl0001 Table 1 Yield, compositional data

, and separation efficiency of fine and coarse fractions from air classification of milled legume

flours

Legume species Starting flour

(% protein)

Fraction yield Protein content Starch content Proteinrecovery

in fines

Starchrecovery

in coarse

Fine Coarse Fine Coarse Fine Coarse

Field pea 20.4–25.3 2.3–53.0 77.7–47.0 62.5–42.6 14.5–6.5 1.4–24.7 60.0–86.2 58.1–88.8 99.8–75.3

Fababean 29.8–33.0 21.1–45.8 78.9–54.2 71.8–60.6 19.6–4.2 0–9.5 57.5–91.1 61.3–92.6 100–95.0

Navy bean 24.5–30.4 19.4–44.6 80.6–55.4 63.8–46.2 21.9–5.7 0.1–6.9 58.0–84.9 49.1–87.1 100–96.7

Lima bean 22.7–22.8 17.7–32.0 82.3–68.0 49.6–39.3 18.7–14.1 0–5.9 56.9–69.6 32.0–55.4 99.1–96.1

Great Northern

bean

22.6–24.0 22.5–42.2 77.5–57.8 53.5–45.6 15.6–6.1 0–4.2 51.5–92.3 50.0–87.0 100–96.6

Lentil 22.6–27.1 21.9–44.6 78.1–55.4 64.6–46.5 14.5–3.3 0.2–19.3 65.8–89.7 63.0–91.9 99.2–84.3

Cowpea 23.6–24.4 24.1–33.5 75.9–66.5 51.6–41.8 17.2–14.2 3.2–14.6 67.8–73.7 48.7–60.0 97.0–89.2

Mung bean 25.8–26.5 27.1–29.0 72.9–71.0 61.4–60.4 12.3–11.7 2.0–6.1 67.7–74.0 66.1 96.4–96.3

Chickpea 17.7–19.5 8.5–29.0 91.5–71.0 40.0–28.9 15.5–11.4 3.7–30.3 53.1–62.7 19.2–50.2 94.5–81.4

Soybean 52.5 72.0 28.0 54.2 50.5 74.1

On a percentage dry-weight basis.

Data are shown as a range of reported values from assorted classifier types and settings.

104 AIR CLASSIFICATION/Uses in the Food Industry

in those fractions being equal to 4, 3, and 93%,

respectively. In some studies, protein fractions having

up to 40–50% protein (but yields of only 1.5%) were

generated accompanied by a 20–30% protein frac-

tion (< 15 mm) at a yield of 11–25%. Although this

fraction could be used to enhance the protein content

of foods, commercial use has not occurred. The starch

fraction can be further processed by water extraction

to remove the protein generating purified starch for

food (e.g., puddings and thickeners) and industrial

applications.

0017 To obtain a protein-rich flour, a separation cut

point of 15 mm is usually sufficient, especially in soft

wheat. Two separations may be required, however, to

obtain a low-protein starch fraction. As an example,

10.8% protein content soft wheat flour can be frac-

tionated into three cuts: < 15, 15–35, and 35 mm, with

yields of 12, 43, and 45% having 19.5, 7.1, and

12.0% protein content, respectively. In a single oper-

ation, a 20% yield of 21–22% protein fine fraction

and an 80% yield of a coarse fraction having 7.5–8%

protein can be achieved from soft wheat flour of

around 10.5% initial protein.

0018 Using soft wheat flour, air-classification methods

have generated a very fine subfraction that is light,

fluffy, and high in protein and total dietary fiber.

Applications requiring such high-gluten flour are

found in the bagel industry.

0019 The protein-rich fractions generated from wheat

can be used as the basis for preparation of protein

isolates, or the high-protein fraction can be added to

low-protein base flours to improve the dough and

baking characteristics. This enhancement is due to

the nature of the protein components present and

the alteration of starch and ash components during

air classification. As in legumes, the soluble sugars are

also concentrated into the high protein fraction.

0020 To improve the efficiency of fine grinding and sub-

sequent air classification, it has been suggested that a

treatment of 0.1–1.0% sulfurous acid be applied for

several hours as an aid in loosening the starch protein

complex. Sorghum subjected to such treatment has

shown increased yields of protein-rich and starch-rich

fractions.

Miscellaneous Cereal Flours

0021 Barley, and especially dehulled malted barley, lends

itself to successful separation by air classification.

Dehulled malted seed with 11.5% protein can be

impact-milled and classified into a high-protein,

low-fiber fraction with 26–27% protein content and

a starch portion of about 9.5% protein. The split

ratio is in the range of 16/84. Regular dehulled barley

with 15% protein has up to 40% protein in the fine

fraction with a 17.5% yield. The benefits of such

application may be in a defibered starchy malt or

barley flour or a high-starch malt flour that may be

used in rapidly brewed malt drinks. Applications for

generating starch have shown that up to three passes

have been used in some programs, thereby yielding

fractions rich in large granule starch (77–78%

starch), which, upon subjecting to wet extraction,

produce an almost pure large-granule starch with a

higher extraction efficiency than conventional extrac-

tion processes.

0022Similarly, high-fiber byproduct streams from the

milling of rye have been impact-milled and reclassi-

fied to yield fractions that have enhanced soluble and

dietary fiber levels.

b-Glucan-enriched Flours

0023Nonconventional use of the air-classification tech-

nique has been applied to oats for the purpose of

generating a b-glucan rich bran fraction. More re-

cently, focus has also turned to barley flour. Instead

of classifying finely ground flour, oat groats have been

coarsely milled and then subjected to air classification

in order to produce a high-bran coarse fraction and a

fine flour fraction. The bran fraction can serve as an

enriched fiber source for cereal applications or as a

starting material for the isolation of b-glucan concen-

trates. Other researchers have defatted the oat flour

prior to air classification and noted that the coarse

fraction (> 30 mm) had a b-glucan content of about

17% for a 30% yield. Inactivation of enzymes would

be a prerequisite for the preparation of b-glucan

fractions by wet extraction. Preparation of barley

fractions (2–4% yield) has shown b-glucan values of

15–18%. Numerous researchers have confirmed

these results with various waxy and nonwaxy barleys.

Other Minor Uses

0024Cottonseed proteins are rarely used in edible foods

because of pigment glands containing gossypol. Air

classification has been shown to produce an edible

protein fraction if there is minimal pigment gland

damage during the milling of extracted flakes. Yields

of 35–40% protein-rich fractions with up to 65%

protein and meeting food and drug directorate stand-

ards are possible.

0025Finely ground soybean hulls have been air-classified

into fine and coarse fractions with enriched fiber.

Application of this fraction in frying batters reduces

oil absorption. Substitution of wheat flour by 20% in

cakes increases volume and acceptability. The protein

fraction from the soybean hulls may yield increased

quantities of trypsin inhibitor with anticarcinogenic

properties.

AIR CLASSIFICATION/Uses in the Food Industry 105

0026 Utilization of rapeseed and canola meals could be

enhanced if crude fiber levels could be reduced by on-

half or less. Small protein shifts of 11–17% have been

achieved in roughly 50/50 splits, but in spite of the

fiber being shifted into the coarse fraction, the two

end products are not sufficiently improved to warrant

commercialization.

0027 Although rice protein has a high nutritional value,

attempts at recovery of this protein by air classifica-

tion have failed to demonstrate any significant pro-

tein shifting. Air classification of defatted finely

ground rice bran has provided a high-protein bran

fraction of a 50% yield and 23% protein content

from an initial 19.4% protein level in the flour. Un-

fortunately, the ash content of the protein-enriched

fraction is also increased, rendering the product

unsuitable for direct food use.

0028 Potato granules produced by spray drying from a

wet milling process have been successfully classified.

Granules with about 10% protein have been classi-

fied into a 15/85 fine:coarse ratio having 38 and

3.1% protein, respectively. Assuming that the eco-

nomics of spray drying is favorable, the protein frac-

tion can provide a source of nondenatured protein

concentrate for food applications.

0029 Other miscellaneous applications of air-classifica-

tion principles to food products include the produc-

tion of fine confectioners sugar (< 5 mm) and fine

cocoa powder (< 75 mm), the latter being used to

make top-quality, smooth chocolate products. Recent

investigations have shown a possible means of gener-

ating flax lignan involving the use of hull material

removed from flaxseed by pearling then subjected to

classification yielding a fraction rich in lignan and

water-soluble fiber. The lignan can be extracted

sequentially and used in the nutraceutical industry.

0030 The combined air-classifier mills have recently

found some applications in the food industry, includ-

ing milk powder, sugar, alginates, gelatine, wheat

gluten and flour, cocoa powder, casein, and starches.

0031 Air-classification techniques applied to finely

ground flours have produced a wide range of pro-

tein/starch-rich fractions and with variable degrees

of success. The process is advantageous in that drying

and effluent disposal costs for wet plants are reduced

or excluded, a significant factor from an environmen-

tal viewpoint. There is also some flexibility for the

processor to vary the resulting flours to suit the end-

user. Increases in capital and operating costs for

the operation must be considered in light of these

benefits.

See also: Air Classification: Principles of Operation;

Barley; Cocoa: Chemistry of Processing; Production,

Products, and Use; Flour: Roller Milling Operations;

Analysis of Wheat Flours; Dietary Importance; Rice

Further Reading

Andersson AAM, Andersson R and Aman P (2000) Air

classification of barley flours. Cereal Chemistry 77:

463–467.

Gueguen J (1983) Legume seed protein extraction, process-

ing and end product characteristics. Qualitas Plan-

tarum, Plant Foods and Human Nutrition 32: 267–303.

Kadan RS, Freeman DW, Ziegler GM Jr. and Spadaro JJ

(1979) Air classification of defatted, glanded cottonseed

flours to produce edible protein product. Journal of

Food Science 44: 1522–1524.

King RD and Dietz HM (1987) Air classification of rape-

seed meal. Cereal Chemistry 64: 411–413.

Kohnhorst AL, Uebersax MA and Zabik ME (1990) Pro-

duction and functional characteristics of protein concen-

trates. Journal of American Oil Chemists’ Society 67:

285–292.

Mok C, Park D-J and Ku K-H (1995) Air classification of

barley flour. Foods and Biotechnology 4(1): 21–24.

Reichert RD (1982) Air classification of peas (Pisum sati-

vum) varying widely in protein content. Journal of Food

Science 47: 1263–1271.

Sosulski FW, Walker AF, Fedec P and Tyler RT (1987)

Comparison of air classifiers for separation of protein

and starch in pin-milled legume flours. Lebensmittel-

Wissenschaft & Technologie 20: 221–225.

Sosulski F and Youngs CG (1979) Yield and functional

properties of air-classified protein and starch fractions

from eight legume flours. Journal of American Oil

Chemists’ Society 56: 292–295.

Thomas ND (1999) The MikoPul air classifier mill. Inter-

national Sugar Journal 101: 480–481.

Tyler RT, Youngs CG and Sosulski FW (1981) Air classifi-

cation of legumes. I. Separation efficiency, yield and

composition of the starch and protein fractions. Cereal

Chemistry 58: 144–148.

Vasanthan T and Bhatty RS (1995) Starch purification after

pin milling and air classification of waxy, normal and

high amylose barleys. Cereal Chemistry 72(4): 379–384.

Vose JR, Basterrechea MJ, Gorin PAJ, Finlayson AJ and

Youngs CG (1976) Air classification of field peas and

horsebean flours: chemical studies of starch and protein

fractions. Cereal Chemistry 53: 928–936.

Wu YV and Stringfellow AC (1992) Air classification of

flours from wheats with varying hardness: protein shifts.

Cereal Chemistry 69(2): 188–191.

Wu YV and Stringfellow AC (1995) Enriched protein and

b-glucan fractions from high-protein oats by air classifi-

cation. Cereal Chemistry 72(1): 132–134.

106 AIR CLASSIFICATION/Uses in the Food Industry

Air Drying See Drying: Theory of Air-drying; Drying Using Natural Radiation; Fluidized-bed Drying; Spray

Drying; Dielectric and Osmotic Drying; Physical and Structural Changes; Chemical Changes; Hygiene; Equipment

Used in Drying Foods

ALCOHOL

Contents

Properties and Determination

Metabolism, Beneficial Effects, and Toxicology

Alcohol Consumption

Properties and Determination

A T Bakalinsky and M H Penner, Oregon State

University, Corvallis, OR, USA

Introduction

0001 The earliest written records indicate that alcohol

(ethanol or ethyl alcohol) has been enjoyed in the

human diet for thousands of years. In spite of past

and present prohibitions among some groups, an im-

pressive number and variety of alcoholic beverages

have been developed, refined, and extolled in an

extensive and rich literature. While the discovery of

alcohol was undoubtedly accidental, its microbial

origin was only established some 120 years ago

through the work of Louis Pasteur. Microbial physi-

ologists have since shown that alcohol formed

through different fermentative pathways in various

microbial species always serves the same purpose:

regeneration of an oxidized cofactor, usually the

oxidized form of nicotinamide adenine dinucleotide

(NAD

0002 Major classes of alcoholic beverages include pri-

mary products of alcoholic fermentations (beer and

wine), products of mixed alcoholic–lactic acid fer-

mentations, and since the discovery of distillation –

known to the Chinese at least 3000 years ago and

to the ancient Egyptians even earlier – distilled and

fortified beverages. Because alcohol is an excellent

extractant, exhibits low toxicity, and possesses anti-

microbial activity, it also enjoys wide use as a solvent

for food ingredients such as spices and flavors. (See

Lactic Acid Bacteria.)

Physical and Sensory Properties

0003Ethanol is a clear, colorless, flammable liquid mis-

cible with water and many organic solvents in all

ratios. It is hygroscopic and relatively nontoxic, ex-

hibiting an oral LD

in rats of 13.7 g kg

1

(grams of

ethanol per kilogram of body weight required to kill

50% of the animals). When ethanol is added to water,

a rise in temperature due to the heat of solvation

occurs with a consequent increase in volume. When

the mixture cools to the original temperature, the

volume decreases, becoming slightly less than the

sum of the initial water and ethanol volumes. Max-

imum contraction occurs at a molar ratio of eight

parts of water to one part of ethanol. At a pressure

of 101.3 kPa, a mixture of 95.6% ethanol and 4.4%

water on a mass basis forms a constant-boiling-point

mixture called an azeotrope. In practice, this means

that the concentration of ethanol cannot be increased

beyond 95.6%, by simple distillation. Some import-

ant physical properties of ethanol are listed in Table 1.

0004Ethanol has a slightly sweet taste and a character-

istic aroma. The aroma threshold determined in one

study ranged from 4 to 5 mg of ethanol per 100 ml of

tbl0001Table 1 Physical properties of ethanol

Formula CH

Molecular weight (Da) 46.07

Boiling point (



C) 78.32

Freezing point (



C) 114.1

Density, d

(g ml

1

) 0.7893

Refractive index, n

1.361

Viscosity at 20



C (cP) 1.17

Dielectric constant at 20



C 25.7

Heat of fusion (J g

1

) 104.6

Heat of vaporization at 78.32



C(Jg

1

) 839.31

ALCOHOL/Properties and Determination 107

total aqueous solution. At high concentrations, it

causes a burning sensation in the mouth. It moderates

the taste of acids, as shown by the greater tartness of

dealcoholized wines relative to the same untreated

wines. It is said to impart ‘body,’ which may be due

to the fact that at room temperature it is more viscous

than water. Addition of sugar to an ethanol–water

solution increases the threshold for ethanol, indicat-

ing that sugar masks its taste and/or aroma.

Sources of Alcohol

0005 The natural source of alcohol is fermentation which

has been defined as the oxidation of organic com-

pounds (generally carbohydrates) in the absence of

external electron acceptors. Pasteur called it ‘life

without air.’ Microorganisms ferment in order to

obtain the energy necessary for growth and reproduc-

tion and alcohol is one of several possible fermenta-

tion products. In most countries, use of alcohol in

beverages is restricted to that made by fermentation.

The vast amount of alcohol used industrially as a

solvent and substrate in chemical syntheses is made

synthetically from ethylene. The amount derived

from the fermentation of agricultural products is

largely dependent on economic considerations,

e.g., alcohol produced by fermentation is used as a

substitute for petrol in Brazil.

Alcoholic Beverages

0006 All alcoholic beverages are derived either directly or

indirectly from fermented products. As indicated in

Table 2, the major groups are fermented beverages,

products of mixed alcoholic–lactic acid fermenta-

tions, beverages made by distillation and by fortifica-

tion. Fruits containing high sugar concentrations at

maturity and nutrients at levels sufficient to support

growth of fermenting yeasts, primarily Saccharo-

myces cerevisiae, have traditionally been the raw

materials from which wines are made. Grapes are

unusual in that they contain sufficiently high levels

of sugars, nutrients, and acids to produce wines that

are microbiologically stable.

0007Beer and sake are produced from barley (other

grains are possible sources) and rice, respectively.

Unlike wines, these beverages are derived from carbo-

hydrates that are not initially fermentable. Conversion

of carbohydrates into fermentable sugars requires the

action of amylases produced by barley in the case of

beer, and by the fungus Aspergillus oryzae during

sake production. Kefir and koumiss are examples of

beverages produced by the fermentation of cows’ and

mares’ milk, respectively, by a mixture of lactic acid

bacteria and lactose-fermenting yeasts. Although

almost unknown in some western countries, kefir

and koumiss continue to enjoy great popularity in

eastern Europe. (See Beers: History and Types; Fer-

mented Milks: Other Relevant Products.)

0008Distilled beverages are derived from fermented

grains and potatoes (whiskies and vodka), sugar

cane byproducts (rum), fruits (brandies), and other

plants such as mezcal (tequila). Liqueurs are distilled

beverages that have been flavored and sweetened.

Fortified beverages require the addition of alcohol

during production, generally in the form of brandy.

Special fruit preserves are fortified to assure preserva-

tion. The intact fruits, usually cherries, are steeped in

brandy for a period of months prior to consumption.

(See Brandy and Cognac: Armagnac, Brandy, and

Cognac and their Manufacture; Rum; Whisky, Whis-

key, and Bourbon: Products and Manufacture.)

Use as a Preservative

0009Ethanol is not particularly toxic. As a sole agent of

preservation in beverages, minimum concentrations

ranging from 18 to 21% by volume are required to

assure microbiological stability. Table wines which

contain significantly less alcohol are stable because

of additional factors: their natural high acidity, low

pH, high content of phenolic compounds, and lack of

sugar. The toxicity of ethanol towards microorgan-

isms is due to a number of effects. At very high

concentrations, as in flavor extracts, ethanol acts as

a desiccant and protein denaturant. At lower concen-

trations, 10–20% by volume, the toxicity is believed

to result primarily from interactions with cell mem-

branes. In the yeast S. cerevisiae, ethanol has been

shown to inhibit several solute transport systems. In

the presence of sugar, the toxicity of ethanol is en-

hanced. This explains the stability of a class of sweet

tbl0002 Table 2 Major classes of alcoholic beverages

Fermented

Beer

Wine

Sake

Mixed alcoholic^lactic acid fermentation

Kefir

Koumiss

Distilled

Whiskies, vodka

Rum

Brandy

Tequila

Liqueurs

Fortified

Port

Sherry

Vermouth

108 ALCOHOL/Properties and Determination

dessert wines which contain several percent sugar and

only 7–10% ethanol.

Analysis

0010 Several methods are available for quantitative meas-

urement of ethanol. Some are based on ethanol-

specific chemical reactions, such as dichromate and

enzyme-catalyzed oxidation. Others are based on

selected physical properties of the product or sample

which are functions of its ethanol content, such as

boiling point or relative density. Often, separation of

the ethanol from other compounds by chromatog-

raphy, distillation, or other means is required prior

to quantification. The choice of method depends on

the need for accuracy and precision, analysis time,

potential for interfering substances, and expense.

(See Chromatography: Principles.)

0011 With respect to interfering substances, it is worth

noting that methanol is a minor but potentially toxic

constituent of distilled beverages derived from fruits.

It is formed from hydrolysis of methoxylated fruit

pectin and does not interfere with enzymatic or chro-

matographic methods of ethanol analysis.

Boiling Point Determinations (Ebulliometry)

0012 Boiling point depression is probably the most com-

monly used measure of ethanol in liquid systems. The

method is based on Raoult’s law of partial pressures.

Under ideal conditions, the law states that the total

pressure above a mixture of two miscible liquids is

equal to the sum of their partial pressures, and that

the partial pressure of each component is directly

proportional to its mole fraction in the mixture.

Stated mathematically:

¼ P

þ P

¼ P

þ P

ð1Þ

where P

is the total pressure above a solution of

liquids A and B, P

is the partial pressure of compon-

ent A, P

is the partial pressure of component B, P

the vapor pressure of component A above pure liquid

A, X

is the mole fraction of component A in the

mixture, P

is the vapor pressure of component B

above pure liquid B, and X

is the mole fraction of

component B in the mixture. Ethanol–water mixtures

have boiling points ranging from slightly less than

78.32



C (100% ethanol) to 100



C (0% ethanol) at

101.3 kPa pressure. The ethanol–water azeotropic

mixture (95.6% ethanol, w/w) boils at 78.2



C. Devi-

ations from Raoult’s law are exhibited by mixtures of

nonideal liquids, in which significant molecular inter-

actions occur between components. Ethanol–water

mixtures are examples of nonideal systems because

ethanol exhibits significant hydrophilic character.

Generally, the boiling point of pure water is measured

in addition to that of the sample and tabulated

ethanol content–boiling point data are adjusted ac-

cordingly, to account for variations in atmospheric

pressure. To prevent significant evaporative loss of

ethanol during boiling and measurement, ebulli-

ometers are fitted with a small condenser, and read-

ings are taken shortly after a constant temperature is

attained.

0013Boiling point measurements require relatively large

samples – up to 50 ml – which limits their utility.

Their accuracy is reduced in the presence of high

concentrations of other dissolved solutes. Since ebul-

liometers are simple devices, relatively inexpensive,

and sufficiently accurate for production purposes,

small wineries have used them extensively.

Relative Density Measurements (Hydrometry)

0014Relative density (specific gravity) is the ratio of the

density of a solution to the density of a reference

solution (usually water at 4



C). Relative densities of

aqueous solutions are dependent on the concentra-

tion of dissolved solutes. Two general approaches

are used to obtain relative densities.

0015The first is to measure the mass of known volumes

of sample and standard reference solutions. This

is accomplished by use of a pycnometer, a small,

relatively lightweight flask of known volume. Since

specific volume is temperature-dependent, the pycno-

meter is immersed in a water bath maintained at a

standard temperature. The mass of the sample and

reference solutions is then determined on an analyt-

ical balance and compared to tabulated standard

values. The relative density of aqueous ethanol solu-

tions reflects the concentration of ethanol, assuming

it is the only (or major) dissolved solute.

0016The second general approach is based on Archime-

des’ principle, which holds that an object immersed in

a liquid appears to lose an amount of mass equivalent

to the mass of liquid it displaces. The Westphal bal-

ance illustrates this principle. Its design is similar to

that of an equal-arm, two-pan balance. At one end, a

tube of known volume and weight is immersed in a

reference solution and the beam is balanced by add-

ition of weights to compensate for the buoyancy of

the liquid. The procedure is repeated by immersion in

the unknown liquid sample. In this manner, a relative

density is determined. A more commonly used device,

based on the same principle, is the hydrometer. Hy-

drometers are glass instruments with narrow, cali-

brated stems much like a thermometer at the top,

but with much expanded bottom halves that are

weighted with lead to insure that they float upright.

When the hydrometer is placed in a liquid sample, it

sinks to a depth that results in displacement of a

volume of liquid equal to its weight. The alcohol

ALCOHOL/Properties and Determination 109

content is read from the calibrated stem at the point

where it intersects the liquid surface. Hydrometers

designed for alcohol measurement are standardized

for use at a specific temperature and calibrated either

in percent alcohol (v/v), or



proof, where



proof ¼

2  % ethanol (v/v). Correction tables are available

for measurements taken at nonstandard tempera-

tures. Hydrometers are accurate over a limited range

of alcohol concentrations. Consequently, a set of

instruments may be required to cover the range of

interest.

0017 Since the relative density of a solution depends on

the concentration of all dissolved solutes, it is often

necessary to separate the ethanol from the other

solutes. To accomplish this, the ethanol contained in

a specified volume of product is separated by distilla-

tion. The distillate is diluted to a known volume with

water, and the relative density of the resulting solu-

tion is measured. In fermented beverages, the concen-

tration of other volatiles that codistill is significant

from a sensory perspective, but is very low compared

to that of ethanol and does not significantly affect the

relative density. In some beverages, such as bourbon

whiskey, the concentration of soluble solids other

than ethanol is sufficiently low to permit direct

measurement of relative density without need for

distillation.

0018 The international Association of Official Analyt-

ical Chemists (AOAC) has approved methods based

on specific gravity for determination of ethanol in

distilled beverages, beers, and wines. The methods

require relatively large samples, and are somewhat

tedious due to the need for distillation and strict

temperature control. However, the equipment is rela-

tively inexpensive and readily available, and precise

results can be expected from a skilled technician.

Enzymatic Determination

001 9 The enzymatic analysis of ethanol is generally based

on the reaction catalyzed by alcohol dehydrogenase.

As shown in eqn (2), the oxidation of ethanol by

NAD

yields acetaldehyde and NADH.

Ethanol

þ NAD

CHO

Acetaldehyde

þ NADH þ H

ð2Þ

The reaction is monitored photometrically at 340 nm

since NAD has no significant absorbance at this

wavelength in the oxidized form (NAD

), but has a

molar extinction coefficient of 6.23  10

lmol

1

when reduced (NADH). Primary alcohols other

than ethanol can interfere, but this is rarely a problem

in foods. Determination of ethanol concentration re-

quires that conditions be adjusted such that the equi-

librium is shifted toward acetaldehyde formation.

One approach is to trap the aldehyde as it forms by

reaction with semicarbazide. Another is to carry out

the reaction at alkaline pH. A third is to couple the

reaction with a second enzyme, aldehyde dehydro-

genase, which converts the acetaldehyde to acetic

acid. In the latter case, 2mol of NADH are formed

per mole of ethanol. (See Enzymes: Uses in Analysis.)

0020Enzyme assays have high sensitivity relative to

other methods and good accuracy due to the specifi-

city of the reaction. Often, samples require treatment

such as dilution, pH adjustment, or decolorization

prior to analysis. Material needs for this type of

assay are simple: volumetric glassware, a photometer,

and reasonable temperature control. A recurring

expense is the enzyme itself, which must be stored

and handled appropriately.

Dichromate Oxidation

0021Chemical oxidation may be used to quantify the

amount of ethanol in a sample. In the dichromate

procedure, the quantity of dichromate required to

oxidize ethanol to acetic acid is measured. In complex

beverages, the ethanol is usually separated from

potentially interfering compounds by distillation.

The ethanol in the ethanol–water distillate is then

oxidized with a known excess of dichromate in the

presence of sulfuric acid. Residual dichromate is then

reduced by ferrous ammonium sulfate in a standard

oxidation–reduction titration. A useful end-point

indicator is 1,10-o-phenanthroline-ferrous sulfate,

which turns brownish purple from blue-green. Potas-

sium dichromate is available in sufficient purity to be

used as a primary standard. Ferrous ammonium sul-

fate may be used as an approximate standard, but

solutions that are not fresh must be standardized

against dichromate.

0022The dichromate oxidation method for the measure-

ment of alcohol in wines has been approved by the

AOAC. The method is appropriate for routine

analyses only when the reagents are standardized

regularly.

Refractive Index

0023The refractive index (RI) of a medium is dependent on

its chemical composition, since under controlled con-

ditions composition dictates its electrical and mag-

netic properties. The RI of a sample is defined as the

ratio of the speed of light in a vacuum to its speed

in the sample medium. Consequently, RI values are

always greater than one. The RI of aqueous ethanol

solutions can be measured directly to indicate ethanol

concentration. When interfering compounds are pre-

sent, the RI of a distillate can be measured. Since the

RI of a liquid is temperature-sensitive, measurements

110 ALCOHOL/Properties and Determination

taken at nonstandard temperatures must be corrected

by use of conversion tables.

0024 The AOAC has approved RI-based methods for

determining ethanol in beers and wines. For wines,

the measurement is made on the distillate. Measured

RI values for beer are used in conjunction with

specific gravity measurements to calculate ethanol

content.

Chromatography

0025 Chromatography is based on the separation of

sample components due to their differing affinities

within a stationary–mobile-phase system. A success-

ful chromatographic run results in separation of the

analyte of interest from all sample components that

would otherwise interfere with its analysis. The sep-

arated analyte may then be quantified by one of sev-

eral detectors. The ethanol in most samples can be

readily separated by chromatography. Since ethanol

is volatile, direct measurement by gas chromatog-

raphy (GC) is possible. Several commercial vendors

have columns specifically designed for determining

the ethanol content of beverages. Simple, 2-m packed

columns are commonly used. Depending on the

sample, it is often beneficial to carry out a simple

pretreatment to extend the life of the column by

removing nonvolatile components. Ethanol exiting a

GC column is commonly quantified with a flame

ionization detector or by thermal conductivity. Etha-

nol separations using high-pressure liquid chroma-

tography (HPLC) are also possible. An advantage of

HPLC over GC is the possibility of simultaneous

determination of certain nonvolatile sample compon-

ents. Commercial HPLC columns for ethanol analysis

are available. Chromatographic methods, which are

expensive, are widely used in analytical laboratories

because of their specificity, increasingly simple oper-

ation, and potential for automation. (See Chromatog-

raphy: High-performance Liquid Chromatography;

Gas Chromatography.)

See also: Beers: History and Types; Brandy and Cognac:

Armagnac, Brandy, and Cognac and their Manufacture;

Chromatography: Principles; High-performance Liquid

Chromatography; Gas Chromatography; Cirrhosis and

Disorders of High Alcohol Consumption; Enzymes:

Uses in Analysis; Fermented Milks: Other Relevant

Products; Lactic Acid Bacteria; Rum; Whisky, Whiskey,

and Bourbon: Products and Manufacture

Further Reading

Amerine MA and Ough CS (1980) Methods for Analysis of

Musts and Wines. New York: Wiley.

Amerine MA and Roessler EB (1983) Wines. Their Sensory

Evaluation. New York: W.H. Freeman.

Association of Official Analytical Chemists (1990) Official

Methods of Analysis, 15th edn. Arlington, Virginia:

Association of Official Analytical Chemists.

Boulton RB, Singleton VL, Bisson LF and Kunkee RE

(1996) Principles and Practice of Winemaking. New

York: Chapman Hall.

Heath HB and Reineccius G (1986) Flavor Chemistry and

Technology. New York: Van Nostrand Reinhold.

Pederson CS (1979) Microbiology of Food Fermentations,

2nd edn. Westport, CT: AVI Publishing.

Sherman PD and Kavasmaneck PR (1980) Ethanol.

Encyclopedia of Chemical Technology, 3rd edn, vol. 9.

New York.

Sommer AE and Bu

cker R (1983) Ethanol. Encyclopedia of

Chemical Processing and Design, vol. 19. New York:

Marcel Dekker.

Zoecklein BW, Fugelsang KC, Gump BH and Nury FS

(1990) Production Wine Analysis. New York: Van

Nostrand Reinhold.

Metabolism, Beneficial Effects,

and Toxicology

C H Halsted, University of California, Davis, CA, USA

Background

0001Alcohol is both a food that provides 29.7 kJ g

1

(7.1 kcal g

1

), accounting for 5.6% of energy in

the American diet, and a drug with potential for

intoxication, addiction, and damage to several

organ systems. In the USA, alcohol is consumed to

some extent by at least two-thirds of the population,

and an estimated 5–10% consume alcohol in excess

and are at risk for addiction, liver disease, cardiomy-

opathy, pancreatic disease, and neurological defects.

The overall mortality from alcoholism in the USA is

about 100 000 deaths per year, about half due to

accidents related to intoxication, and the remainder

mainly due to chronic alcoholic liver disease and its

consequences.

Metabolism of Alcohol

0002Unlike most other drugs, alcohol is completely me-

tabolized after ingestion at a rate of 10–15 g h

1

, and

its energy content is not stored in the body. Alcohol

diffuses across all cell membrances, and after equili-

bration, its concentration is similar in all aqueous

compartments. After the human ingestion of 50 g of

alcohol, its concentration in the stomach reaches

ALCOHOL/Metabolism, Beneficial Effects, and Toxicology 111

1 mol l

1

(4.2 g dl

1

), decreasing by one-half in the

upper intestine and equilibrating with the blood alco-

hol level in the distal intestine. ‘First pass’ metabolism

is achieved in the stomach by gastric alcohol dehy-

drogenase, an enzyme located in parietal cells with

500 mM that accounts for 30% of total alcohol

metabolism in men and 10% of alcohol metabol-

ism in women. This gender difference in gastric me-

tabolism partly explains the lower tolerance of

women for alcohol. Subsequently, alcohol is metabol-

ized entirely in liver cells by two enzyme systems:

cytoplasmic alcohol dehydrogenase and a specific

and inducible microsomal cytochrome P450 enzyme,

known as CYP2E1 (Table 1). Hepatic alcohol dehy-

drogenase with K

2 mM metabolizes alcohol at

blood levels consistent with moderate drinking.

During the oxidation of alcohol by this reaction,

NAD is reduced to NADH while producing acetalde-

hyde and subsequently net energy through the pro-

duction of ATP. However, the increased ratio of

NADH to NAD is associated with increased fatty

acid and triglyceride synthesis, reduced gluconeogen-

esis, enhanced lactate production, and reduced urin-

ary excretion of uric acid. The potential clinical

consequences of these metabolic side effects include

transient fatty liver, hypertriglyceridemia, hypogly-

cemia, acidosis, and gout. On the other hand,

CYP2E1 with a K

of 20 mM accounts for the me-

tabolism of higher alcohol levels as seen with chronic

excessive alcohol use. Alcohol oxidation by CYP2E1

is energy-wasteful by utilizing NADPH at a 2:1 molar

ratio while producing greater amounts of acetalde-

hyde without generation of ATP. Increased intrahepa-

tic acetaldehyde blocks mitochondrial respiration

thereby enhancing the accumulation of fat in the

liver, increases catecholamine release while promot-

ing ketoacidosis, and, by enhancing lipid peroxida-

tion and collagen synthesis, contributes to the

development of alcoholic hepatitis and cirrhosis.

The induction of CYP2E1 by frequent alcohol con-

sumption generates a free radical that can trigger

oxidative liver injury, accounts for alcohol tolerance

in habitual users, and is associated with induction

of other microsomal enzymes that accelerate the

metabolism of drugs such as acetaminophin, bar-

biturates, and coumadin, and the catabolism of vita-

min A.

Risks Versus Benefits of Alcohol

Consumption

0003The risks or benefits of alcohol consumption are based

on quantitative estimates of the numbers of drinks per

day, where one drink of 360 ml of beer, 120 ml of

wine, or 40 ml of spirits provides 15 g of alcohol.

By these standards, ‘moderate drinking’ is defined as

one to two drinks (15–30 g of alcohol) per day or

seven to 14 drinks per week on average, and excessive

drinking that increases disease risk is defined as more

than two to three drinks (30–45 g of alcohol) per day

for men and more than one or two drinks (15–30 g of

alcohol) per day for women. Excessive drinking may

occur through habituation or addiction, and in cul-

tures where wine is consumed daily as a component

of the diet (Table 2).

0004A variety of studies from many developed countries

established a J-shaped curve for mortality risk from

alcohol consumption, wherein the lowest mortality

occurs at consumption of one or two drinks per day

compared with abstinence or to the consumption of

more than two drinks per day. Thus, the term ‘moder-

ate alcohol consumption’ is used to define consump-

tion with clinical benefit and no increased mortality

risk. Comparing all causes of mortality, the moderate

drinking level is somewhat less for women at one to

two drinks per day than in men at two to three drinks

per day. When all the causes of mortality are taken into

account, it appears that the benefit of moderate drink-

ing is confined to the prevention of coronary heart

disease and ischemic strokes, both together of which

account for the majority of deaths in the USA. Above

the level of moderation, i.e., at more than two to

three drinks per day, alcohol consumption associates

with life-threatening trauma, hemorrhagic strokes,

tbl0001 Table 1 Alcohol metabolizing enzymes

Enzyme K

(mM) Relatedeffects

Gastric cytosolic alcohol dehydrogenase 500 ‘First pass’; 30% in men, 10% in women

Liver cytosolic alcohol dehydrogenase (one to two drinks) 2 Increased NADH/NAD ratio promotes fatty acid synthesis

leading to fatty liver, preferential oxidation of ethanol over

lipid, and reduced gluconeogenesis, lactic acidosis, and

hyperuricemia

Liver microsomal CYP2E1 (more than two drinks) 20 Increased NADPH/NADP ratio consumes energy and

increases metabolism of many other drugs and vitamin A;

increased acetaldehyde inhibits mitochondrial functions

and stimulates lipid peroxidation and collagen synthesis

112 ALCOHOL/Metabolism, Beneficial Effects, and Toxicology

hypertension, esophageal, breast and colorectal

cancers, acute and chronic pancreatitis, malnutrition,

and alcoholic liver disease.

0005 Interest in the cardiovascular protective effects

of alcohol stemmed from the French report of

the paradoxical improved mortality among wine-

drinkers in the Mediterranean provinces compared

with residents of northeastern provinces, in spite of

the similar content of high-fat foods in the diet and

overall alcohol consumption. A subsequent in vitro

study at the University of California Davis demon-

strated that the oxidation of low-density lipoprotein

could be prevented by coincubation with phenolic

compounds extracted from red wine, suggesting

that the improved cardiovascular risk of French red

wine-drinkers was related to these nonalcoholic

compounds. The principal flavonoid of red wine is

catechin, which is also present in nonfermented grape

juice. The biological availability of catechin for

human subjects is similar in red wine to that in deal-

coholized red wine, but a greater amount of catechin

may be extracted from fermented grapes during

wine-making than from nonfermented grape juice. A

long-term prospective Danish study of residents of

Copenhagen concluded that the lowest mortality

rates occur in wine-drinkers, with the highest mortal-

ity in consumers of liquor. A study from Northern

California concluded that the improved mortality

in wine-drinkers related to their healthy dietary life-

style, and that red and white wine provided equal

protection.

0006 There are several explanations for the cardiopro-

tective effects of moderate alcohol consumption, in

particular the effects of red wine. Moderate consump-

tion of all types of alcoholic beverages has a posi-

tive effect on the circulating lipoprotein profile,

principally by elevating the levels of high-density lipo-

protein. Furthermore, moderate alcohol consumption

by women results in modest reductions in low-density

lipoprotein levels and increases Apo A-1 but de-

creases Apo B lipoproteins. Whereas serum triglycer-

ides increase in male alcohol consumers, they may

decrease in postmenopausal women drinkers. Moder-

ate alcohol consumption also benefits the clotting

process by increasing levels of tissue plasminogen

activator. In addition to the benefit of wine flavonoids

in reducing the oxidation of low-density lipoprotein,

they also improve the clotting risk by reducing

platelet adhesiveness.

Alcohol Toxicity

0007With the exception of traffic fatalities that may occur

after consumption of an intoxicating dose of alcohol

pending timing after the last drink, the deleterious

effects of alcohol consumption are limited to those

who consume excessive amounts of alcohol (on aver-

age, more than two to three drinks per day) on a

chronic basis. The clinical risks of alcohol toxicity

increase in proportion to increasing average daily

amount of alcohol and duration of excessive alcohol

consumption. In many instances, the development of

these clinical syndromes is modulated by nutrient

deficiency, and in the case of pancreatic insufficiency

and alcoholic liver disease, the clinical syndromes can

contribute to malnutrition.

Clinical Effects of Alcohol Toxicity

0008Excessive alcohol consumption contributes to

increased risks of alcoholic liver disease, cardiomyop-

athy, chronic pancreatitis and insufficiency, nutri-

tional anemia, certain cancers, abnormal energy

metabolism, and malnutrition that is more pro-

nounced after the development of alcoholic liver dis-

ease. Abnormal nutrient availability and metabolism

play a role in all of these clinical complications of

chronic alcoholism (Table 3).

0009Increased cancer risks Increased risk of cancer of

the oropharynx and esophagus is directly related to

the amount and duration of alcoholism, is tripled in

heavy smokers, and may be related in part to abnor-

mal carcinogen metabolism secondary to zinc and

vitamin A deficiency. The incidence of breast cancer

is increased in women who drink, in part due to

enhanced estrogen secretion, and particularly in

women who consume inadequate folate in their

diets. Colorectal cancer risk is increased in excessive

drinkers of both sexes and is related to both alcohol

toxicity and low folate status.

tbl0002 Table 2 Benefits and risks of alcohol consumption

Benefits (one to two drinks per

day)

Protection against ischemic

cardiovascular disease

Improved lipoprotein profile

Increased tissue plasminogen

activator

Wine flavonoids decrease LDL

oxidation, platelet

adhesiveness

Risks (more than three drinks

per day)

Hypertension

Cardiomyopathy

Hemorrhagic stroke

Pancreatitis

Anemia

Neurologic disorders

Cancer of esophagus, breast,

colon

Alcoholic liver disease

Malnutrition

ALCOHOL/Metabolism, Beneficial Effects, and Toxicology 113