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physiological parameters allow some distinction from

other food-relevant lactobacilli, it is not possible to

use a phenotypical basis to discriminate sufficiently

among L. acidophilus, L. johnsonii, L. gasseri, L.

crispatus, and L. amylovorus. All five species are

usually assigned to the L. acidophilus cluster. A dis-

tinction of these species can be facilitated by applying

genotypical techniques and methods based on DNA

homology, the molar amounts of guanine plus cyto-

sine in the DNA, or by the analysis of certain cell wall

components.

Physiological Actions of

Lactobacillus

acidophilus

0005 Because of the properties described above and its

pronounced bile salt resistance, L. acidophilus is

well adapted to the environmental conditions of the

gastrointestinal tract. Proteins in the cell wall may be

important in attaching the bacterium to the mucosal

cells of the intestine. With strain-dependent vari-

ations, L. acidophilus contributes to the inhibition

of the multiplication of pathogenic and putrefactive

bacteria in the intestine due to the production of

organic acid and trace amounts of H

. Further-

more, strain-specific inhibitory substances can be

excreted by certain strains. In this context, numerous

antagonistic peptides (bacteriocins) have been isol-

ated from certain strains of L. acidophilus. For

example, some of them were described as lactocidin,

acidophilin, acidolin, lactosin B, and lactacin B

and possess some ‘antibiotic’ potential against

salmonellae, staphylococci, Escherichia coli, and

clostridia, and partly also against other species of

lactic acid bacteria. Because of their beneficial L.

acidophilus-related properties, products containing

this bacterium have been used in the treatment of

gastrointestinal disorders and to reestablish the func-

tion of the intestine after treatment with antibiotics.

Other features of these products are the provision of

b-galactosidase to humans having an enzymatic defi-

ciency for lactose digestion or, particularly when used

in conjunction with fructooligosaccharides (oligo-

fructose), the reduction of fecal enzymes (glucuroni-

dase, nitroreductase, azoreductase) which obviously

play some role in some stages of precancerogenesis.

Since L. acidophilus produces equimolar amounts of

l(þ) and d() lactic acid, products fermented with

this bacterium offer the advantage of a reduced d()

lactate content, compared to classical yogurt. How-

ever, the acidification potential of this bacterium is

often low and varies considerably among strains.

Products with

Lactobacillus acidophilus

0006At present, a broad variety of products containing

L. acidophilus is on the market. This bacterium

has been incorporated into fermented as well as

nonfermented milks of different levels of dry matter

and fat (Figure 2). Cows’ milk is the main substrate

which is processed using the same basal technology as

applied for the manufacture of yogurt or other cul-

tured dairy products. Hence, continuous production

lines with conventional or aseptic filling systems are

used. Some of the products also contain added fruits

and flavoring agents.

0007Fermented dairy products containing L. acidoph-

ilus as a single bacterial culture are primarily of local

importance in Russia, Eastern European countries,

and Scandinavia. In contrast, in other European

regions, L. acidophilus is usually used in combination

with other microorganisms (e.g., Bifidobacterium

spp., Streptococcus thermophilus, L. delbrueckii

subsp. bulgaricus, L. casei). Milk cultured with such

multicomponent starter cultures (including L. acido-

philus) is produced in increasing numbers and

varieties and consumed frequently by many people.

Among these dairy products, a distinction can be

made between the so-called ‘mild’ yogurt products

(yogurt-related fermented milks, with or without

fruits) which are based on a fermentation with vari-

ous thermophilic bacteria (many of them are assigned

to the area of probiotics), and so-called ‘Acidophilus

milk’ products which are usually fermented by means

of mesophilic lactic acid bacteria (e.g., strains of

Lactococcus lactis or Leuconostoc cremoris or

combinations of both), in addition to L. acidophilus.

A general flow diagram for the production of such

an Acidophilus milk fermented under mesophilic

10µm

fig0001 Figure 1 Microphotograph of a Lactobacillus acidophilus culture

(deep-frozen culture concentrate cultured in MRS broth; for

detailssee Table 1).

ACIDOPHILUS

MILK

conditions is presented in Figure 3. Deep-frozen cul-

ture concentrates or freeze-dried bacteria or, very

rarely, liquid cultures are inoculated into the milk

base. The fermentation is usually performed over-

night for 15–20 h. Stirred products, with a liquid

character, are usually made, but set-style fermented

Acidophilus milk products with increased levels of

solid-nonfat are also available.

0008 Other categories of products include specially fer-

mented drinks (e.g., L. acidophilus plus yeasts, with

or without other lactic acid bacteria, resembling kefir

and named acidophilin), texturized products with a

reduced water content, which are offered in a pasty

form or cut in cubes, or powdered milk which has

been fermented with L. acidophilus before drying.

Many of these product types have a local significance

as dietary adjuncts. In Russia, these products even play

some role as therapeutic agents and have been well

recognized with regard to their medical relevance.

0009 Nonfermented milk containing L. acidophilus is

also offered by some dairies. Such products are usu-

ally produced from standardized milk which is sup-

plemented with a culture concentrate (deep-frozen

pellets or lyophylisate) of L. acidophilus under cooled

conditions, followed by stirring before filling into

cartons or beakers. Some of these products are also

fortified with fat-soluble vitamins (A, D, E), water-

soluble vitamins (thiamin), and trace elements (iron).

While a pronounced metabolic activity of the L. acid-

ophilus strains is desired for all those products which

are produced by fermentation, storage-resistant but

not fast-growing cultures (strains) are needed for the

manufacture of ‘sweet’ (nonfermented) Acidophilus

milk in order not to alter the sensory properties

during storage.

0010The sensory characteristics of nonfermented

‘sweet’ Acidophilus milk are comparable with

regular milk; those of fermented Acidophilus milk

(mesophilic varieties) are similar to those of regular

cultured or sour milks which are manufactured using

a butter flavor-producing mesophilic culture, since

almost no acetaldehyde, which is typical for yogurt,

but some diacetyl-based butter aroma is generated

during fermentation caused by citrate-fermenting

mesophilic lactic acid bacteria. Since L. acidophilus

possesses alcohol dehydrogenase activity, which is

capable of reducing acetaldehyde, only low levels of

this compound are found in the corresponding prod-

ucts. Thus, yogurt-related dairy products (thermo-

philic varieties) containing L. acidophilus often

exhibit a milder and less acidic taste than classical

yogurt, i.e., that manufactured by a cofermention of

S. thermophilus and L. delbrueckii subsp. bulgaricus.

Sensorically, this classical yogurt is dominated by

acetaldehyde which introduces some kind of astrin-

gent characteristic and typical sharpness. Moreover,

many classical yogurt cultures, in particular owing to

the Lactobacillus component of the culture, exhibit a

continued acidification activity even under cooled

conditions on the shelves of retail shops. Besides the

sensory changes, this ‘overacidification’ can also lead

to textural problems (syneresis, whey separation).

Acidophilus products

Single-culture products

Other products

'Sweet'

Acidophilus milk

Fermented

Acidophilus milk

(liquid, dried)

Acidophilus milk

cofermented with

mesophilic lactic

acid bacteria

Fermented milk

manufactured with

L. acidophilus and

other thermophilic

lactic acid bacteria

and/or

bifidobacteria

Fermented milk

manufactured with

L. acidophilus and

yeasts,

facultatively plus

meso- or

thermophilic lactic

acid bacteria

Fermented

Acidophilus paste

or cubes

(enriched with

sugar), texturized

Soymilk-based

Acidophilus milk

Multiple-culture products

fig0002 Figure 2 Survey of the diversity of food products containing Lactobacillus acidophilus.

ACIDOPHILUS

MILK 5

Obviously because of these effects, preferences of

consumers for the milder yogurts with L. acidophilus

have been observed in many countries.

0011A completely different group of Acidophilus prod-

ucts are the pharmaceutical preparations containing

L. acidophilus. Capsules, suppositories, and soluble

powders packaged in sachets are enriched with bac-

terial lyophylisates and used for therapeutic purposes.

They are marketed in pharmacies and health stores.

Bacterial Viable Count and Bacterial

Stability of Acidophilus milk

0012Although milk is a substrate containing almost a

universal array of nutrients, it does not fully meet

the growth requirements of L. acidophilus. For this

purpose, additives and growth promoters consisting

of a mixture of natural compounds which support

and enhance bacterial growth are recommended for

supplementation of the fermentation milk by most

culture suppliers. Usually, they are added to the milk

base in small amounts, before inoculation. In add-

ition, the use of multicomponent cultures offers the

advantage of inducing synergistic effects among

the bacterial microflora which may also positively

influence the propagation rate and the stability of

the bacteria.

0013According to legal aspects and to consumer expect-

ations, products labeled as Acidophilus milk or as

containing L. acidophilus necessarily have to contain

a significant number of these microorganisms. In this

context, a group of experts of the International Dairy

Federation has recommended that L. acidophilus

shall be detected in such products at a level of at

least 1 million CFU ml

1

or g, at their sell-by dates.

0014Recently, studies performed in several countries

have shown that many commercially available prod-

ucts can meet this limit, but with a considerable

number of products a decrease in the L. acidophilus

counts has been observed during a storage period of

approximately 3–5 weeks. Due to the fact that the

expression of beneficial effects is based on a high

number of active bacteria, a high viable count and

pronounced bacterial stability have become import-

ant goals in product development and optimization.

0015Viable counts of L. acidophilus-containing dairy

products are usually enumerated by culture methods

based on plate count techniques with media designed

for culturing lactic acid bacteria (e.g., MRS, Rogosa

agar, TGV agar; for details see Table 1). To enhance

the discriminatory power of these media (this is of

particular relevance in the examination of products

which contain a mixed microflora), media are modi-

fied by slight acidification and/or by supplementation

Process milk with variable dry matter

and fat contents

Dual-step homogenization

with 15 000-20 000 kPa at 65-70 8C

Heat treatment for 5-10 min

at 90-95

Cooling to fermentation temperature

(varies from 22 to 30

8C)

Inoculation with L. acidophilus and a

mesophilic starter culture

Fermentation period at

defined temperature (varies from 22 to 30

8C)

Stirring, cooling to 10-12

and filling into packaging units

(beakers, cartons, etc.)

fig0003 Figure 3 General production steps of the manufacture of

fermented Acidophilus milk using a combined fermentation with

Lactobacillus acidophilus and mesophilic lactic acid starter culture.

Data compiled after various manufacturers’ recommendations.

ACIDOPHILUS

MILK

with antibiotics (e.g., vancomycin at different levels)

or with other selective agents (cellobiose, conjugates

with chromogenic indicator dyes, esculin, etc.). In

many cases, the parallel use of different media select-

ive for each of the bacterial components is necessary

to allow the reliable microbiological monitoring of

these Acidophilus products. Moreover, microscopical

verification of isolates harvested from the different

media usually completes their routine assessment.

Although a number of media and methodologies

have been described in the literature, no official

standard method is available yet.

See also: Fermented Milks: Types of Fermented Milks;

Functional Foods; Lactic Acid Bacteria; Probiotics;

Yogurt: The Product and its Manufacture; Yogurt-based

Products; Dietary Importance

Further Reading

Fonde

n R, Mogensen G, Tanaka R and Salminen S (2000)

Effect of culture-containing dairy products on intestinal

microflora, human nutrition and health – current know-

ledge and future perspectives. In: IDF Bulletin, no. 352,

pp. 5–30, Brussels: International Dairy Federation.

Hammes WP (1995) The genus Lactobacillus. In: Wood JB

and Holzapfel WH (eds) The Genera of Lactic Acid

Bacteria, pp. 19–54. London: Blackie Academic & Pro-

fessional.

Kanbe M (1992) Uses of intestinal lactic acid bacteria and

health. In: Nakazawa Y amd Hosono A, (eds) Functions

of Fermented Milk. Challenges for the Health Sciences,

pp. 289–304. London: Elsevier Applied Science.

Kneifel W and Pacher B (1993) An X-Glu based agar

medium for the selective enumeration of Lactobacillus

acidophilus in yogurt-related milk products. Inter-

national Dairy Journal 3: 277–291.

Lee YK, Nomoto K, Salminen S and Gorbach SL (1999)

Handbook of Probiotics. New York: John Wiley.

Mital BK and Garg SK (1992) Acidophilus milk products:

manufacture and therapeutics. Food Reviews Inter-

national 8: 347–389.

Tamime AY and Robinson RK (1999) Yoghurt Science and

Technology. Cambridge: CRC, Woodhead Publishing.

ACIDS

Contents

Properties and Determination

Natural Acids and Acidulants

Properties and Determination

J D Dziezak, Dziezak & Associates, Ltd., Hoffman

Estates, IL, USA

Background

0001 In very general terms, an acid is a compound that

contains or produces hydrogen ions in aqueous solu-

tions, has a sour taste, and turns blue litmus paper

red. A more comprehensive definition, given by the

US chemist G.N. Lewis, states that acids are sub-

stances that can accept an electron pair or pairs, and

bases are substances that can donate an electron pair

or pairs. This definition, applicable to both non-

aqueous and aqueous systems, requires that an acid

be either a positive ion or a molecule with one or

more electron-deficient sites with respect to a corres-

ponding base.

0002The definition most widely used to describe acid–

base reactions in dilute solution is one that was pro-

posed independently by two scientists in 1923 – the

Danish chemist J.N. Br

Ønsted and the US chemist

T.M. Lowry. The Br

Ønsted–Lowry theory defines an

acid as a proton donor, that is, any substance (charged

or uncharged) that can release a hydrogen ion or

proton. A base is defined as a proton acceptor

or any substance that can accept a hydrogen ion or

proton.

tbl0001 Table 1 Media used for culturing Lactobacillus acidophilus

Media References

MRS agar Lactobacillus agar according to De Man JD,

Rogosa M and Sharpe ME (1960) Journal of

Applied Bacteriology 23: 130–135.

Rogosa agar Lactobacillus selective agar according to

Rogosa M Mitchell and JA, Wiseman RF (1951)

Journal of Bacteriology 62: 132–133.

TGV agar Agar medium according to Galesloot T,

Hassing F and Stadhouders J (1961)

Netherlands Milk and Dairy Journal 15: 127–150.

ACIDS/Properties and Determination 7

tbl0001 Table 1 Structure, ionization constant, pK

, and key physical and chemical properties of acidulants

Acid Structure Ionization constant(s) pK

Physical form Melting

point (



Solubility

(gper100 mlof water)

Hygroscopicity Taste characteristics

Acetic acid

COOH

1.76  10

5

at 25



C 4.76 Clear, colorless

liquid

8.5 Soluble na Tart and sour

Adipic acid

COOH

¼ 3.71  10

5

4.43 Crystalline

powder

152 1.9 g at 20



C Low level of

hygroscopicity

Smooth lingering tartness;

complements grape flavors

¼ 3.87  10

6

at 25



5.41 83 g at 90



Citric acid

COOH

HO C

¼ 7.10  10

4

3.14 Crystalline

powder

Moderately

hygroscopic

Tart; delivers a ‘burst’

of flavor

¼ 1.68  10

5

4.77

¼ 6.4  10

7

at 25



6.39

Anhydrous 153 181 g at 25



Hydrous 135–153 208 g at 25



Fumaric acid

COOH

¼ 9.30  10

4

3.03 White granules

or crystalline

powder

286 0.5 g at 20



C Nonhygroscopic Tart; has an affinity for

grape flavors

¼ 3.62  10

5

at 18



4.44 9.8 g at 100



Glucono-d-lactone

HC OH

HO CH

1.99  10

4

(for gluconic acid)

3.7 White crystalline

powder

153 59 g at 25



C Nonhygroscopic Neutral taste with acidic

aftertaste, when hydrolyzed

Lactic acid

HC OH

COOH

1.37  10

4

at 25



C 3.86 Liquid; also

available in

dry form

16.8 Very soluble na Acrid

Malic acid

COOH

HO CH

¼ 3.9  10

4

3.40 Crystalline

powder

132 62 g at 25



C Nonhygroscopic Smooth tartness

¼ 7.8  10

6

at 25



5.11

Phosphoric acid K

¼ 7.52  10

3

2.12 Liquid Very soluble

in hot water

na Acrid

¼ 6.23  10

8

7.21

¼ 2.2  10

13

12.67

and K

at 25



at 18



Tartaric acid

COOH

HO CH

HC OH

COOH

¼ 1.04  10

3

2.98 Crystalline

powder

168–170 147 g at 25



C Nonhygroscopic Extremely tart; augments

fruit flavors, especially

grape and lime

¼ 4.55  10

5

at 25



4.34

na, not applicable.

Adapted with permission from Food Technology, Acidulants: Ingredients that do more than meet the acid test. 44(1): 76–83. Institute of Food Technologists, Chicago, Illinois, USA.

0003 This article discusses the physicochemical proper-

ties of acids and describes several methods for their

analysis.

Strong Versus Weak Acids

000 4 The strength of a BrØnsted–Lowry acid depends on

how easily it releases a proton or protons. In strong

acids, owing to their weaker internal hydrogen

bonds, the protons are loosely held. As a result, in

aqueous solutions, almost all of the acid reacts with

water, leaving only a few unionized acid molecules in

the equilibrium mixture. The reaction takes place

according to eqn (1):

HA þ H

O Ð H

þ A



ð1Þ

In this equation, HA represents the undissociated

acid, H

the hydronium ion formed when a proton

combines with one molecule of water, and A



the

conjugate base of HA.

0005 Unlike strong acids, weak acids exist largely in the

undissociated state when mixed with water, since

only a small percentage of their molecules interact

with water and dissociate. Most acids found in

foods, including acetic, adipic, citric, fumaric, malic,

phosphoric and tartaric acids, and glucono-d-lactone,

are classified as weak or medium strong acids.

Physicochemical Properties

0006 Physicochemical properties, including the ionization

constant, pH, the apparent dissociation constant

(pK

) and buffering capacity, are discussed below

and are listed in Table 1.

Ionization Constant

0007 The tendency for an acid or acid group to dissociate is

defined by its ionization constant, also denoted as

. The ionization constant, given at a specified

temperature, is expressed as:

½H

½A





½HA

ð2Þ

where the brackets designate the concentration in

moles per liter. The ionization constant is a measure

of acid strength: the higher the K

value, the greater

the number of hydrogen ions liberated per mole of

acid in solution and the stronger the acid.

0008 Acids with more than one transferable hydrogen

ion per molecule are termed ‘polyprotic’ acids.

Monoprotic or monobasic acids are those that can

liberate one hydrogen ion, such as acetic acid and

lactic acid. Those containing two transferable

hydrogen ions are called diprotic or dibasic acids

and include, for example, adipic acid and fumaric

acid. Acids such as citric acid and phosphoric acid,

which have three transferable hydrogens, are called

triprotic or tribasic acids. Ionization of polyprotic

acids occurs in a stepwise manner with the transfer

of one hydrogen ion at a time. Each step is character-

ized by a different ionization constant.

0009Measurement of acidity is an important aspect of

ascertaining the safety and quality of foods. Such

measurements are given in terms of pH, which is

defined as the negative logarithm of the hydronium

ion concentration (strictly, activity):

pH ¼ log

½H



¼log

½H

:

ð3Þ

0010The lower the pH value, the higher the hydrogen

ion concentration associated with it. A pH value of

less than 7 indicates a hydrogen ion concentration

greater than 10

7

M and an acidic solution; a pH

value of more than 7 indicates a hydrogen ion concen-

tration of less than 10

7

M and a basic solution.

When the hydronium and hydroxide ions are equal

in concentration, the solution is described as neutral.

(See pH – Principles and Measurement.)

0011It is also important to note that, because the pH

scale is logarithmic, a difference of one pH unit rep-

resents a 10-fold difference in hydrogen ion concen-

tration.

0012The term pK

is defined as the negative logarithm of

the dissociation constant:

¼ log

¼log

: ð 4Þ

0013The pK

corresponds to the pH value at the mid-

point of a titration curve developed when one equiva-

lent of weak acid is titrated with base, and the pH

resulting from each incremental addition of base is

plotted against the equivalents of hydroxide ions

added.

0014The pH of a system is at the pK

when the concen-

trations of acid (HA) and conjugate base (A



) are

equal. At the pK

and, to a lesser extent, in the area

extending to within one pH unit on either side of the

, the system resists changes in pH resulting from

addition of small increments of acid or base. In other

words, at the pK

, acids and their salts function as

buffers.

0015The number of pK

s that an acid has depends on

the number of hydrogen ions it can liberate. Mono-

protic acids have a single pK

, whereas di- and tri-

protic acid have two and three pK

s, respectively.

10 ACIDS/Properties and Determination

001 6 Strong acids have low pK

values, and strong bases

have high pK

values.

Buffering Capacity

0017 A solution of a weak acid (or a weak base) and its

corresponding salt is called a buffer solution. In these

systems, the hydronium ion content is not signifi-

cantly changed when a small amount of acid or base

is added to that solution. The reason that buffer

solutions resist appreciable changes in pH can be

best illustrated by an example. If a small amount of

hydrochloric acid is added to a buffer solution com-

posed of acetic acid and sodium acetate, the protons

from the hydrochloric acid would associate with the

acetate ions to form unionized molecules of acetic

acid. As the newly formed acid molecules ionize, the

equilibrium would shift towards forming more

hydronium ions (eqn (1)). This would result in only

a very slight increase in pH.

0018 Similarly, the addition of a small amount of sodium

hydroxide to the same buffer solution would have

little effect on pH. Hydroxide ions from the sodium

hydroxide would combine with hydronium ions

in the equilibrium mixture, forming undissociated

molecules of sodium hydroxide. More of the acid

molecules would then dissociate to replace the hydro-

nium ions lost; though a new equilibrium system

would be created, it would produce only a minimal

effect on pH.

0019 The quantity of acid or base that a buffer solution is

capable of consuming before a change in pH is real-

ized is termed the ‘buffering capacity.’ The buffering

capacity is defined as the number of moles of strong

acid or base required to increase the pH by one unit in

1 l of buffer solution. The buffering capacity of a

solution is greatest at its pK

value where the concen-

trations of acid and conjugate base are equal.

Analytical Methods

0020 Quantitative determinations of acidity play an im-

portant role in ensuring food product quality and

stability. Information obtained on acid levels can

help in detecting cases of food adulteration, moni-

toring fermentation processes, and evaluating the

organoleptic properties of fermented foods. pH

determination, titratable acidity, chromatographic

methods, and capillary electrophoresis are proced-

ures commonly employed by the food industry to

determine food acids. (See Adulteration of Foods:

Detection.)

pH Determination

0021 pH can be measured by two techniques: colorimetric

and potentiometric. The colorimetric method involves

adding a suitable indicator to a solution and matching

the color of the solution to a standard solution con-

taining the same indicator. This method can estimate

pH to the nearest 0.1 pH unit.

0022A more accurate technique and the one most fre-

quently employed, the potentiometric method, uses a

pH meter to determine hydrogen ion concentration.

The two electrodes of the meter – a calomel reference

electrode and a glass indicator electrode – are im-

mersed in the solution, of known temperature,

whose pH is to be measured. The electrode potential

of the indicator electrode is linearly related to changes

in hydrogen ion concentration and therefore pH.

Titratable Acidity

0023The total concentration of acid in a solution can be

determined by titration. The titration process is per-

formed by placing in a flask a known volume of acid

solution whose concentration is unknown. To the

flask, a few drops of indicator, e.g., phenolphthalein,

which is colorless in acid solutions and pink in basic

solutions, is introduced. A base solution of known

concentration is then gradually added until the acid

is completely neutralized. This point is indicated

when the solution permanently changes color. The

concentration of acid can then be calculated from

the volume of base solution used.

0024The value obtained, called titratable acidity, is an

estimate of the total acid in the solution. It accounts

for both the free hydronium ions present in the equi-

librium mixture and the hydrogen ions released from

undissociated acid molecules. For weak acids, the

titratable acidity is different from the actual acidity

(hydrogen ion concentration), since these compounds

exist largely in the undissociated state in solution. For

strong acids, however, titratable acidity and actual

acidity are virtually the same, since strong acids and

their salts are completely ionized in solution.

Chromatographic Methods

0025Gas chromatography (GC) and high-performance

liquid chromatography (HPLC) have almost entirely

replaced paper and thin-layer chromatography as

methods for identifying and quantifying food acids.

0026Gas Chromatography GC has been used to analyze

organic acids in fruit and fruit juice. Analysis involves

preparing volatile derivatives such as methyl esters of

the organic acids, prior to their injection into the gas

chromatograph. Derivatives are chromatographed on

a nonpolar stationary phase column and detected by a

flame ionization detector.

0027By use of GC, malic acid has been shown to be a

major constituent of many fruits, including apples,

pears, grapes, peaches, and nectarines, and significant

ACIDS/Properties and Determination 11

levels of citric acid have been found in citrus fruits

such as orange, lemon, and grapefruit, and in non-

citrus fruits, including pears, nectarines, cherries,

and strawberries. (See Chromatography: Gas Chro-

matography.)

0028 High-performance Liquid Chromatography HPLC

is used more extensively than GC to determine or-

ganic acids because the technique requires little or no

chemical modification to separate these nonvolatile

compounds. Separation is usually done on either

a reversed-phase C8 or C18 column or a cation-

exchange resin column operated in the hydrogen

mode. Acids are detected by either refractive index

(RI) or ultraviolet (UV) detectors. RI detection re-

quires prior removal of any sugars present that poten-

tially can interfere with quantification; sugar removal

is not required for UV detection at 220–230 nm.

0029 Adulteration of a commercial cranberry juice drink

was detected using HPLC when the test yielded dif-

ferent results for organic acids, sugars, and anthocya-

nin pigments than those obtained for a standard juice

drink. Atypical citric and/or malic acid contents and

presence of a natural colorant, probably grape skin

extract, confirmed that the drink was adulterated.

0030 In wine-making, HPLC is used to monitor concen-

trations of tartaric, malic, succinic, citric, lactic, and

acetic acids, which contribute tartness and stability to

the finished product. A common approach involves

using a column containing a strong cation exchange

resin and eluting the sample with dilute sulfuric

acid; the eluant is then analyzed for acids by RI detec-

tion. This column has the additional advantage of

permitting the simultaneous detection and quantifica-

tion of ethanol and the monitoring of wine for adul-

teration with methanol. Organic acids in wine can

also be separated using ion chromatography with a

conductivity detector. (See Chromatography: High-

performance Liquid Chromatography.)

Capillary Electrophoresis

0031 A relatively new technique, capillary electrophoresis,

is also useful for separating and quantifying organic

acids in food systems. This technique utilizes an elec-

trical field to separate molecules on the basis of their

charge and size. Small volumes of sample, usually a

few nanoliters, are injected on to a fused silica capil-

lary tube, which is usually less than 1 m in length and

50 mm in internal diameter. The ends of the tube are

placed in electrolyte reservoirs containing electrodes.

A voltage in the range of 20–30 kV is delivered to the

electrodes by a power supply and causes the charged

molecules to move. Because organic acids are nega-

tively charged, they migrate away from more neutral

or positively charged molecules, such as sugars and

phenols, respectively. Acids are detected by a UV

detector, and the signal is sent to a data collector.

The resulting separation is graphically represented

as an electrophoregram.

Enzymatic Analysis

0032Enzyme assays provide another means of analyzing

acids. For example, an enzymatic assay of l-malic

acid uses an NAD(P)-linked malic enzyme and in-

volves spectrophotometrically measuring the absorb-

ance of NADPH, a reaction product, at 340 nm.

See also: Adulteration of Foods: Detection;

Chromatography: High-performance Liquid

Chromatography; Gas Chromatography; pH – Principles

and Measurement