BIOTECHNOLOGY AND BIOENGINEERING,

VOL. XVIII, PAGES 891-907 (1976)

Role of Microbial Enzymes in Flavor

Development in Foods

K. &I.
SHAHANI, R. G. ARNOLD, A. KILARA, and
B. K. DWIVEDI,* University of Nebraska,
Lincoln, Nebraska 68583

Summary
There are four main sources of enzymes in foods-these being the inherent
enzymes, enzymes from microbial contaminants, enzymes elaborated by micro-
organisms added t o foods, and specific enzymes added to foods. This study
primarily deals with the latter two sources of enzymes in food. Although both
plants and animals serve as sources of enzymes, they are not as economical or
versatile sources as are enzymes obtained from microorganisms. I n the meat
industry, proteases are used to tenderize muscle and to obtain flavor precursors.
In the preparation of cured meat products such as sausages, lipases, and proteases
from bacterial cultures are utilized. Similarly, proteases and lipases are used
in the dairy industry to develop flavor compounds. Proteases and amylases
also have applications in the baking and milling industries where they are used
to produce precursors for the nonenzymatic browning reactions. Carbohydrases
such as amylase, amyloglucosidase, and glucose isomerase have found usage in
the starch and syrup industry for the production of high dextrose and high fructose
syrups. Other enzymes such as glucose oxidase, pectinase, and naringinase are
of value to the wine and fruit juice industries. A better understanding of the
mode of action of enzymes as well as the mechanisms of development of flavor
compounds will further enhance the use of microbial enzymes to develop specific
and desired flavors in foods.

INTRODUCTION
Flavor is the result of the sensations of taste, aroma, certain
tactile responses, and physiological influences. Although taste and
tactile responses are involved in the complex attribute of a food
called flavor, odor properties or compounds have received greatest
attention in flavor investigations. Food flavors are dependent upon
odorous compounds derived singly or in combinations from more
complex precursors, including proteins, fats, carbohydrates, vitamins,
*Present address: Estee Candy Co., Parsippany, New Jersey 07054.
89 1
@ 1976 by John Wiley & Sons, Inc.
892 SHAHANI ET AL.

and essential oils. Inherent enzymes in food systems or enzymes

involved in the metabolism of food constituents by microorganisms
in foods play an important role in the development of food flavors.
Other food constituents, such as polyphenols, nucleotides, and
carotenoid pigments, also have been reported to contribute to
flavor development.
As early as 1956, Hewitt et a1.l reported that flavor lost in food
processing could be restored in a number of food products by the
addition of an appropriate enzyme preparation. They used a
blanched, dehydrated preparation of fresh watercress which was
flavorless when reconstituted. Upon addition of a tasteless, odorless
enzyme preparation from white mustard to the reconstituted product,
the typical odor and taste of watercress was regenerated within a few
minutes. This work amply demonstrated the importance of enzymes
in the flavor development of food products. Unfortunately, limited
work has been reported on mechanisms of flavor development by
enzymatic action. On the other hand, information is available on
the nature of enzymes involved in the development of important
flavor compounds. Often, purified enzymes are employed in order
to study precisely the enzymatic reactions and their intermediate
steps. Frequently, purified enzymes are used to study their speci-
ficities and concentrations using model systems. The information
thus obtained then can be applied to food systems. However, the
influence of other enzymes or their by-products and the influence of
various food ingredients upon the basic enzymic reactions need to be
evaluated prior to practical utilization of these enzymes in the food
industry.
A more complete understanding of the role of enzymes in the
development of desirable flavor in foods would aid in development of
processing methods whereby undesirable enzymes could be destroyed
selectively, while leaving the desirable enzymes relatively unaffected.
There are four main sources of enzymes in foods: a) enzymes inherent
in the food; b) enzymes contributed by microbial contaminants;
c) enzymes of desirable bacteria added to foods; and d) enzymes
added to foods. Inherent enzymes may contribute to the natural
flavor of foods, but these are often inactivated during processing.
Microbial contaminants may contribute to off flavors in foods.
The primary aim of preserving food is to reduce or eliminate these
undesirable microorganisms. Use of microorganisms in the process-
ing of foods has the advantage that microorganisms contain many
enzymes which can act in a sequential manner to produce numerous
flavor compounds. Such sequential reactions cannot be achieved
 MICROBIAL ENZYMES IN FLAVOR DEVELOPMENT 893

by a single enzyme. Additionally, it is more economical to use

microbes than a number of purified enzymes. This work will
discuss the use of enzymes and, to some extent, microorganisms for
the development of flavor in foods.

ENZYME SOURCES
Pure enzymes can be obtained from animal tissues and secretions
and plants and microorganisms by using conventional methods of
protein and enzyme purification. Plant enzymes require the growing
of a large quantity of plant material for the production of relatively
small quantities of enzymes; therefore, they do not provide an
economically feasible source. Animal enzymes are recovered from
the by-products of the meat industry and their supply is limited.
Also, the preparation of pharmaceutical products, such as insulin
from pancreas, compete with their use for enzyme extraction.
Fortunately, microbial enzymes are not subject to the limitations of
production or supply cited for plant or animal enzymes.2 Micro-
organisms, bacteria, yeasts, molds, and actinomycetes are capable
of producing a large variety of diverse enzymes. The rate and type
of enzyme production can be governed precisely through growth,
environmental factors such as nutrients, temperature, and pH,
inherent genetic adaptability, and by “genetic engineering”-a
process by which the genes of the microorganisms can be altered to
produce a specific enzyme or enzymes.

UTILIZATION OF ENZYMES FOR FOOD MODIFICATION

Enzymes offersome unique properties: a ) they are specific; b) only
catalytic amounts of enzymes are required rather than stoichiometric
amounts ; c) they generally require moderate temperatures and mild
conditions; d) they can be inactivated by appropriate changes in
temperature, pH, or a combination of the two; e) they exhibit a
narrow pH range of activity; f ) heavy metals inhibit their activity;
and g) under certain conditions some enzymes can be reactivated.
Many food products are prepared by the addition of yeasts, molds,
or bacteria to the substrate or the food. In such instances no
attempt is made to use purified or isolated enzymes or to remove the
microorganisms from the foods. Such foods include the oriental
fungus fermented soybean foods like Tempeh and cheese
including Roquefort, blue, brick, and Gorganzola, and various
sausage^.^ Uses of microbial enzymes to develop flavors in foods
are summarized in Table I.
 W
00
I+
TABLE I
Selective Use of Microbial Enzymes in Foods

Industry Application Enzyme Source

Meat, Fish Meat tenderizing Protease Fungal

Tenderizing casings Protease Fungal
Condensed fish solubles Pro t,ease Fungal
I)ry/fermented sausage Protease, lipase Bacterial
Dairy Cheese production Protease Fungal, bacterial
Cheese ripening Lipase, protease Fungal, bacterial
Cultured milk products a-Acetolactate oxidase Bacterial
Modified milk fat Lipase Fungal, bacterial
95
Baking and milling Bread making Amylase Fungal
Protease Fungal
Eggs, dried Glucose removal Glucose oxidase Fungal
Starch and syrup Corn syrup Amylase Fungal
Dextrinized corn syrup Amylase, amyloglucosidase Fungal
High fructose corn syrups Glucose isomerase Fungal
Flavor Flavor protentiators 6 p hosp hoesterases
’- Bacterial, fungal
Fruit juice Clarification Pectinase Fungal
Debittering Naringinase Fungal
Color stability, prevention of off flavors Glucose oxidase Fungal
 MICROBIAL ENZYMES I N FLAVOR DEVELOPMENT 895

Meat Industry
I n the meat industry it is customary to age the animal carcass
prior to processing. Following the slaughter of animals, the blood
circulation in the tissues ceases, which leads to the depletion of
oxygen and to anaerobic glycosis, whereby glycogen is converted to
lactic acid. The sarcoplasm within the muscle fibers contains
lysozomes, the “small packets” which contain proteolytic and
hydrolytic enzymes. These proteolytic enzymes are liberated when
the lipoprotein membranes of the lysozomes rupture a t low pH during
post-mortem aging. Proteolysis results in the liberation of free
amino acids which may contribute to or act as precursors of meat
flavor. The free amino acids also play an important role in the
development of the cooked flavor of meat. Thus, aging of meat can
be viewed from two perspectives: a) the change in texture resulting
from the resolution of rigor and b) the production of flavor com-
pounds following the hydrolysis of meat proteins. To enhance the
aging process, proteases have been used extensively. However,
microbial proteases have not been used as extensively since they
hydrolyze not only connective tissue but also the structural proteins,
causing textural defects.6 A mold Thamnidium elegans has been
used to tenderize beef steak^.^ Beef treated with this mold was
reported to be more juicy and flavorful.
Fish is another important class of muscle foods. In general, most
fish exhibit higher post-mortem pH than do warm blooded animals.
Also, post-mortem changes in fish involve the deamination of adenylic
5’-monophosphate (AMP) to inosinic 5’-monophosphate (IMP).
Bendall and Davey8 showed that during this conversion, ammonia
appeared in equimolar proportions to the disappearance of adenine
nucleotides. Fraser et al.9 observed that in cod, ATP was converted
rapidly to ADP by sarcoplasmic ATPase and the ADP was then
hydrolyzed further to AMP by myokinase. Adenylic 5’-mono-
phosphate was then further degraded leading to an accumulation of
IMP by a deaminase action. Inosinic j’-monophosphate is subse-
quently broken down to inosine and orthophosphate. Interestingly,
IMP is used commonly for flavor potentiation. Nost of the enzymic
changes occurring in red meats and fish result in the production of
flavor precursors which are converted to flavor volatiles upon cooking.
Unlike the cooked meat aroma produced from their precursors
during heat treatment, the characteristic aroma of dry or fermented
sausages is related in part to the hydrolytic and oxidative changes
occurring in the lipid fraction during ripening. The bacterial,
muscle, and adipose tissue lipases liberate fatty acids from the
896 SHAHANI ET AL

sausage lipids. The unsaturated fatty acids thus liberated con-

tribute to the flavor of the sausage directly or undergo nonenzymic
oxidation to produce lipid peroxides and carbonyl compounds.
Demeyer et al. lo reported that unsaturated fatty acids are selectively
liberated by the lipases in sausage lipids, with the rate of lipolysis
being linoleic > oleic > stearic > palmatic acid. Since pork fat is
known to have most of the linoleic and oleic acids at position-3 of
the triglyceride molecule, it has been suggested that these lipases
have specificities for position-3 of triglycerides. Volatile fatty acids,
like acetic, propionic, and butyric acids, produced by microbial
metabolism, also play a significant role in the aroma of dry sausage.
Overall, the flavor production in dry fermented sausage is de-
pendent on three principal mechanisms: a) lipolysis of fats by
microbial lipases ; b) carbohydrate metabolism via glycolysis and
Iireb’s cycle involving microbial enzymes; and c) deamination
of amino acids produced from proteins by microbial proteases. All
three mechanisms lead to the production of volatile and nonvolatile
flavor compounds.
Dairy Industry
Milk produced by normal healthy cows contains a wide variety of
enzymes. These have been catalogued by Shahani et al.” The off
flavors resulting from these inherent enzymes and enzymes con-
tributed by bacterial contaminants are beyond the scope of this
study. Therefore, only enzymes that are produced by the starter
cultures or enzymes that are used during the manufacture of dairy
products will be considered.
The contribution of microbial metabolic products to the “typical”
flavor of cultured dairy products has received considerable attention.
The volatile flavor compounds are metabolic products of Lactobacilli,
Streptococci and Leuconostoc used as starter cultures for the manu-
facture of cultured dairy products. The organisms that produce
lactic acid as the major end product of lactose metabolism are called
homofermentative organisms and utilize the Embden-hleyerhoff-
Parnas (EMP) pathway as the major route for lactose degradation.
Homofermenters, such as Streptococcus lactis, can utilize fructose,
mannose, galactose, lactose, maltose, and sucrose also as substrates
for the production of lactic acid. This is achieved by a set of
inducible enzymes.
The heterofermentative organisms, on the other hand, such as
Leuconostoc and Lactobacilli, form lactic acid in addition to ethanol,
acetate, glycerol, mannitol, and COZ. Diacetyl, which is responsible
 MICROBIAL ENZYMES I N FLAVOR DEVELOPMENT 897

for a “buttery” sweet aroma of dairy products, is thought to be

synthesized by one or two routes. One mechanism proposed in-
volves the condensation of active acetaldehyde and acetyl CoA.12
The second mechanism proposed involves the oxidative decarboxyla-
tion of a-acetolactic acid13 (Fig. 1). This second mechanism also
has been detected in yeast. The production of diacetyl via these
reactions contributes to the flavor of cultured products like cottage
cheese, sour cream, and buttermilk. However, under certain condi-
tions diacetyl can be reduced to acetoin by an enzyme called diacetyl
reductase and acetoin can further be oxidized to 2,3-butanediol. l 4
Both acetoin and 2,3-butanediol do not impart the characteristic
“buttery” flavor. While diacetyl reductase is certainly an un-
desirable enzyme in dairy products, it is highly desirable in the
brewing industry where diacetyl is regarded as an off flavor com-
ponent in beer and this enzyme helps reduce diacetyl to a nonflavor
compound.
Rennet obtained from calves’ stomachs has customarily been used
as the enzyme to coagulate milk in cheese making. Presently,
microbial rennets or proteases, derived from Endothia parasitica,
Mucor miehei, and Mucor pussilus are being used quite extensively.
Rennet does not contribute significantly to proteolysis of cheese

Acetaldehyde .l”P + Pyruvlc acid

cr-acetolactate
oxldase (?)
I
a-acetolactic acid
a-acetolactate
synthetase

+ TPP

a-acetolactate
decarboxylase

Diacetyl reductase
Co2 + Diacetyl Acetoln

2 , 3 -butaned 101
dehydrogenase

2 , 3 -butaned iol

Fig. 1. Pathway for the formation of diacetyl. Source: Wood.13

898 SHAHANI ET AL.

during ripening. However, microbial proteases do carry on pro-

teolytic action during the aging of cheese. The products of proteol-
ysis have been reported to participate in the formation of flavor
compounds in cheese.15
The flavor of cheese which develops during ripening or curing
represents a very dynamic system. l 6 The enzymes involved here
are the microbial enzymes and possibly the inherent milk enzymes
as well which act on the major components of the cheese curd. In
cheddar cheese, for example, proteases of the cheese microflora
produce amino acids and peptides which contribute to the rich
brothy background flavor. Various amines, such as tyramine,
cadaverine, putrescine, and histamine, have also been identified in
normal cheddar cheese.I7 Amino acids and amines produced by
proteolysis are precursors of a variety of aldehydes produced by
transamination and decarboxylation reactions.
The degradation of lipids is essential to cheddar cheese flavor,
with the 2- to %carbon saturated fatty acids being reported to be of
primary importance. Methyl ketones, ethanol, and 2-butanol are
also thought to be important flavor compounds in cheddar cheese.'*
Since the chemistry of mold ripened cheeses is discussed more ex-
tensively elsewhere, this presentation will deal with the role of
lipolytic enzymes in flavor development in other food products.
Hydrolysis of milk fat catalyzed by enzymes, though undesirable
in many instances, can be applied as a controlled process for the
development of desired flavors in certain products. These applica-
tions include flavor development in various simulated cheeses,
manufacture of lipolyzed milk fat products for enhancement of
butter-like flavor, and flavor development of milk chocolate. The
products conventionally flavored by the addition of lipolyzed
butterfat (Table 11) include bakery and cereal products, candy and
confectionery products, dairy products, and various other products
such as sauces, snack foods, soups, etc. There are numerous patented
methods for the use of lipase enzyme system^.^^-^^
The importance of free fatty acids to the characteristic flavors of
various dairy products gradually became apparent as technologists
studied and evaluated juxtaposedly the chemistry of food flavors.
Day30in 1966 summarized the total free fatty acid contents of various
dairy products (Table 111) and discussed the importance of these
compounds in cheese flavors. More recently, reviews of Nelson,31
D ~ i v e d iand
, ~ Arnold et al.4 have discussed the role and application
of lipolytic enzymes to the development of flavors in dairy products.
Application of lipase preparation is of great importance in Italian
 MICROBIAL ENZYMES I N FLAVOR DEVELOPMENT 899

TABLE I1
Products Conveniently Flavored by Addition of Lipolyzed Products19

Bakery/cereal products
Cake and cookie mixes
Chemically leavened bakery formulations
Sweet doughs
Cheese cake mixes
Pancake mixes
Cereals
Candy/confectionery products
Milk chocolate
Creams and cream centers
Toffee and caramel fudges
Dairy Droducts
Cheese dips
Coffee whiteners
Miscellaneous products
Filled and imitation dairy products
Margarines
Popcorn oils
Salad dressings
Sauces
Snack foods
soups

TABLE I11
Free Fatty Acids in Some Dairy Products30

Total free fatty

acids (mg/kg)

Fresh milk 415

Moderately rancid cream 1,027.
Butter 2,733
Cheddar cheese 1,793 (average,
12 samples)
Blue cheese 23,500 to 66,700
(range, 3 samples)

cheeses such as provolone and romano varieties. Data from our

laboratory32 for commercial romano cheese describe the relationship
between free fatty acids, flavor intensity, and desirability (Table IV).
Butyric acid seems to be important for flavor intensity, whereas,
desirability is related to the relative proportions of free fatty acids.
 TABLE I V
Flavor Evaluation and Free Fatty Acid Content of Five Commercial Romano Cheese Samples

Flavor (1 low, 10 high) Content of free fatty acids (mg/100 g)

LiDase
systems Desirability Intensity Comments Acetic Propionic Butyric Higher acids 3
KL 7.8 7.0 Average, typical, very good, overall good
flavor 30 1 84 1203 363
KL 7.8 6.7 Good overall flavor, clean, sharp, soapy 267 58 1142 382
3
M
KL 6.7 7.3 Slightly pungent, acidic, salty; good for c3
U.S. market 133 68 661 418 *
K 5.8 7.3 Atypical, brothy, salty, sour, lacks flavor, ?
acidic 142 59 9 13 243
K 5.2 6.7 Atypical flavor, sharp, acidic, limburger-
like 135 27 728 219

KL = mixture of kid and lamb lipases; K = kid lipase.

 MICROBIAL ENZYMES IN FLAVOR DEVELOPMENT 901

Similar data were also gathered for provolone cheese.32 The lipases
used in Italian cheeses are derived from young lambs, calves, or calf
rennet. Microbial lipases are now being investigated for the manu-
facture of simulated Italian cheeses.
Baking Industry
Enzyme modified milk fat can also be used extensively in bakery
products. Lipases used for modification of milk fat include milk
lipases, pancreatic lipase, mold lipases, bacterial lipases, and pre-
gastric esterases such as kid and lamb esterases. (Table V). When
modified milk fat was used in bread making, it was observed that
appearance and internal structure of bread were not affected by the
inclusion of modified butterfat. Trials at the American Institute
of Baking revealed that a 35 to 40% replacement of shortening
with modified butteroil products imparted a good flavor and aroma
comparable to an all butteroil control. Therefore, enzyme modified
butteroil can be of great importance commercially in the baking
industry.
The action of microbial lipases on milk fat33is dependent, of course,
on the source of the lipase as shown in Table VI. The lipase from
Achromobacter lipolyticum releases linoleic acid very selectively; the
lipase of Geotrichum candidum also selectively liberates linoleic or
oleic acid to a greater extent than does the A . lipolyticum lipase.
However, the lipase from Aspergillus niger preferentially hydrolyzes
stearic acid. Thus, the free fatty acid profile can vary significantly
with the type of lipase. Also, the pH optimum of lipase depends
upon the source of the lipase (Fig. 2). Studies in our laboratory

TABLE V
Sources of Lipases Studied for Modification of Milk Fat Emulsions for
Incorporation into Bakery Products

1. Milk lipase
2. Pancreatic lipase
3. Mold lipases
Aspergillus niger
Geotrichum candidum
Penicillium roquejorti
4. Bacterial lipases
Achromobacter lipolyticum
Pseudomonas jluorescens
5 . Pregastric esterases
Kid
Lamb
902 SHAHANI ET AL.

PH
Fig. 2. pH Optima of several microbial lipases.

have shown that P. roqueforti lipase possessed a pH optimum of

around 9, whereas A . lipolyticum lipase has an optimum pH of 8.
On the other hand, lipases from A . niger and G. candidurn have pH
optima in the acidic range. Depending upon the pH of the food
system in question, the appropriate lipase can then be chosen. It
must be recognized, however, that these studies were carried out
with particularly pure enzymes using substrates of known composi-
tions. In a food system, however, the constituents of food, such as
proteins, ash, etc., can interact with the lipid substrate and may
change some of the properties of the enzyme. That is to say,
enzyme activities determined under carefully controlled situations
may not be the same as the activities that will result upon its addition
to foods.
 MICROBIAL ENZYMES I N FLAVOR DEVELOPMENT 903

TABLE VI
Free Fatty Acids Liberated from Milk Fat by Various Microbial Lipases

Fatty acid Free fatty acids liberated by lipase action

Fatty composition
acid' of milk fat A. lipolytium G. candidum A . niger P . roqueforti

Yoof methyl ester

4 :O 4.1 trace trace trace trace
6:O 3.7 1.2 trace trace trace
8 :O 2.9 1.9 1.3 1.0 3.1
lo:o 3.1 2.2 1.1 2.1 4.4
12:o 3.4 1.0 1.9 3.2 3.2
14:O 9.5 4.2 2.4 11.0 7.6
14:l 3.6 - 1 .o 2.8 2.2
16:O 25.1 12.9 15.7 33.7 17.8
16:l 3.0 - 3.3 1.2 2.1
18:O 12.3 8.0 1.1 14.0 9.0
18:l 25.0 47.4 61 .O 28.7 47.9
18:2 1.5 6.5 5.3 1.6 2.1
18:3 0.5 8.1 5.9 0.8 1.2

-First figure refers to number of carbons, second figure refers to number of

double bonds.

Also, in the baking and milling industries, amylases and proteases

from fungal sources have been used extensively. 34 Amylases
supplement the diastatic activity of flour and a-amylases break down
starch to dextrins and maltose. Thus, higher levels of sugars
produced by enzyme action increase gas production and improve
crust color without the danger of extensive dextrinization during
baking. Table VII shows the thermostability of a-amylases from
three different sources.35 The fungal a-amylase loses much of its
activity at 80°C, whereas the bacterial a-amylase is very stable at

TABLE VII
Thermostability of ~Amylases35

yo Enzyme retained
Temp.
("C) Fungal Malted wheat flour Bacterial

80 1.6 28.0 100

85 0.0 2.1 95
90 0.0 0.0 74
95 0.0 0.0 18
904 SHAHANI ET AL.

this temperature. For this reason, fungal a-amylase is used in

baking, because after the enzyme has achieved an appropriate level
of dextrinization during dough making, it can be inactivated easily
during baking to prevent additional undesired action. Bacterial
a-amylase, on the other hand, is used in the brewing industry due
to its thermostability, since higher temperatures are involved during
the preparation of wort.
In the baking industry, proteases are used mainly to reduce
mixing times, increase extensibility of doughs, increase loaf volume,
and improve grain and texture. However, the proteolytic action
must be controlled carefully in order to avoid sticky doughs. More
importantly, sugars and amino acids produced by the microbial
amylases and proteases serve as precursors for the Maillard reaction
and the Strecker degradation, which result in nonenzymatic brown-
ing and production of volatile flavor compounds. Pyrazines, which
are formed when sugar-amine mixtures are heated, have been shown
to be important to the flavor of several baked and roasted foods.
Other Industries
In several food technological processes, enzymes are used to
prevent undesirable reactions. For example, microbial glucose
oxidase is used to remove glucose from eggs36and to remove oxygen
from soft drinks and juices.37 Removal of glucose from eggs helps
prevent undesirable browning reactions during the drying of eggs and
their subsequent storage. Here the nonenzymatic browning reac-
tions are deterred by the lack of availability of precursors, mainly
glucose. Prevention of nonenzymatic browning leads to an improve-
ment in the flavor of dried eggs. Microbial glucose oxidase is also
used to lend color stability and prevent oxidative off flavors in
fruit juices.38 As presented in Table VIII, treatment of various
citrus fruit drinks with glucose oxidase at 2 units of enzyme/oz of
juice removed 47 to 83% of the oxygen in a 20 hr period, leading to an
improvement in color stability and flavor.38 When the concentration
of enzyme was increased, the time required for oxygen depletion
was decreased.
I n the corn starch industry, carbohydrates like amylases, amylo-
glucosidases, and glucose isomerases of microbial origin are used
e x t e n ~ i v e l y . ~a-Amylases
~ convert starch to dextrins which are then
converted to glucose by amyloglucosidases and then glucose is
converted to fructose by glucose isomerase. The end product called
high fructose corn syrup is sweeter than either corn syrup or the
high dextrose corn syrup. Coincidental with the increase in sweet-
 MICROBIAL ENZYMES I N FLAVOR DEVELOPMENT 905

TABLE VIII
Oxygen Removal from Juice Drinks“

O2 Content aft.er % O2 Removed

Flavor Variab1e 30 hr (cc) in 30 hr

Orange Control 4.5 0

With enzyme’ 2.3 49
Lemon Control 3.6 0
With enzymes 1.9 47
Grapefruit Control 2.9 0
With enzymes 0.5 83

8 Two units of glucose oxidase/oz.

ness is the prevention of crystallization of sugars in such syrups.

Recently, immobilized enzyme technology has made it possible to
use these enzymes in a continuous process rather than the conven-
tional batch processes.40
Another area of use of enzymes for flavor improvement of foods
deals with a food ingredient rather than a food system. Flavor
potentiators are added to certain foods to enhance sensory responses.
Flavor potentiators can be produced by enzymic methods, solely by
enzymic reactions or in combination with chemical methods.
There are two major flavor potentiators, inosine 5’-monophosphate
(5’-IMP) and guanosine 5’-monophosphate (5’-GMP). Using
ribonucleic acid (RNA) as the substrate, bacterial 5’-phosphodi-
esterases can produce adenine 5’-monophosphate (5’-AMP), 5’-GMP,
cytidine 5’-monophosphate, and uridine 5’-monophosphate simul-
taneously. Inosine 5‘-monophosphate can be produced enzymically
or chemically from 5’-AMP. Yeast is used as a common source of
RNA and the removal of RNA from yeast increases the commerical
value of the remaining yeast as a protein source in animal feeds.41
Also, adenine produced by the chemical reaction of malonoitril and
thiourea can be converted by bacteria to adenosine and eventually
to 5’-AMP. Adenine 5’-monophosphate can be converted to 5’-IMP
enzymically or bacteria can produce inosine which can be converted
to 5’-Ih/IP by chemical methods.
I n the fruit juice industry, fungal pectinases are used widely for
the clarification of fruit juices and wines. In most cases pectinase
action affects only the clarity and color stability of the juice and
does not exert any action on flavor, the notable exception being
apple juice. Pectinase treated apple juice, when pasteurized, does
906 SHAHANI E T AL.

not develop the characteristic boiled or cooked flavor or taste nor-

mally encountered in untreated pasteurized apple juice.’ Also,
vinegar and jelly produced from depectinized juice are superior in
brilliance, color, and aroma.
Flavanoids found in a variety of citrus juices contribute heavily
to the bitterness of the juices. Naringin is one such flavanoid found
in grapefruit. Fungal enzyme naringinase breaks down naringin and
thus debitters grapefruit juice. The use of naringinase is more
common in Japan than in the U.S.42
The field of enzyme technology is making great strides, especially
with the advent of affinity chromatography and immobilization of
enzymes. These newer techniques will provide cheaper methods of
isolating enzymes and reusing them. The nature of the flavor
precursors in foods and the reactions by which these precursors are
ultimately converted to flavor impact compounds need further
investigation. If we can successfully delineate this process, then the
use of enzymes specifically to improve the flavor of foods will be
further enhanced.
This report is published as Paper No. 5047, Journal Series, Nebraska Agricul-
tural Experiment Station. The research was conducted under Project 16-17.
This study was supported by a grant from Dairy Research Inc. (DRINC).

References
1. E. J. Hewitt, D. A. M. MacKay, K. S. Konigsbacher, and T. Hasselstrom,
Food Technol., 10, 487 (1956).
2. L. A. Underkofler, R. B. Barton, and S. S. Rennert, A p p l . Microbiol., 6 ,
212 (1958).
3. W. D. Gray, The Use of Fungi as Food i n Food Processing, CRC Press,
Cleveland, Ohio, 1970.
4. R. G. Arnold, K. M. Shahani, and B. K. Dwivedi, J. Dairy Sci., 58,
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Accepted for Publication March 13, 1976