Biogeneration of Aromas - ACS Publications - American Chemical

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25 Generation of Flavor and Odor Compounds through Fermentation Processes L. G. Scharpf, Jr., E. W. Seitz, J. A. Morris, and M. I. Farbood Research and Development Department, International Flavors & Fragrances, Union Beach, NJ 07735 Since Neolithic times, microorganisms have been used to achieve desired flavors in foods and beverages. In recent years, a commercial demand has developed for flavoring materials derived from natural sources. Microbiologists and flavorists are exploiting the fermentative action of microorganisms to improve the flavor of alcoholic beverages, cheese, yogurt, bread, fruit, and vegetable products. Many chemicals produced by microbial processes are odorants or tastants. Categories of molecules will be reviewed as a function of substrates and microbial strains. That microorganisms are also capable of de novo synthesis of tastants and odorants from suitable substrates will be illustrated by several examples. Since the beginning of time, flavors and fragrances have played an important role in providing man with happiness, beauty and satisfaction. Up to this century many natural flavor materials were obtained from animals and higher plants. Supplies of many of these materials have dwindled due to social, economic, and political factors, conservation, wildlife protection and industrial growth. The use of microorganisms may offer an alternative method for producing natural flavor and fragrance materials. The release of odors by microbial cultures is well known by microbiologists. Many of the volatiles are produced in only trace quantities but, due to their strength, sufficient for taste and smell. Harnessing microorganisms to produce volatile flavor and odor materials in large quantity is a real technical challenge, for reasons which will be presented. Nevertheless, fermentation technology offers the potential of producing flavor and aroma substances of interest to today's consumer. 0097-6156/ 86/ 0317-0323S07.00/ 0 © 1986 American Chemical Society In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

324

BIOGENERATION OF AROMAS

Sensory Many

Properties

microbial

their

and

ars.

Primary

carbonyl


tastants

and

bitter

and

odor

sium,

volatile

Microbial A.

Diversity Many

and

depending of

the the

aerobes

processes

an

such

as

members

this

their

oxygen,

are

considerable

of

have

this

both

sympo-

focus.

mode

The

in

the

an

find

strict

switching

bread

the

acetate(l). of

molecular

which

ketones

and

their

mode,

themselves.

produce

are

produce

presence

anaerobic

anaerobes

and

fungi

reprowidely,

Examples

which

aldehydes, of

vary

oxygen.

filamentous

as

growth,

acetone,

only

to

they

their

requirements

free

capable

microorganisms -

energy

and The

carbohydrates

produce esters.

metabolic

depending

Industrial

and

also

wine

have

on

yeasts

flavors,

other

grow

as

their be

on

sole

derived Nitrogen

common

as

starch

are

requirements

in

providing microbial

for of

carbon

sugars. some

molecules For

n-alkanes

principle

blocks sources

source.

from

are

fermentation,

and

and

organic

carbon

produced

a

building

however,

other

compounds

in

most

such

versatile,

to

Carbon

components

metabolism.

2.

of

the

such

which

Source

both

can

sug-

include

class.

nutritional

are

sour,

growth:

Carbon

1.

for

butanol,

are

aerobic which

is

theme

of

and

and

example,

Clostridium,

and grow

aerobes

Saccharomyces, of

Besides for

ethanol,

in

the

organism.

genus

microorganisms

from

for

primary

These

absence

substances

environment

There

the

oxygen

the

the

metabolize

be

cells.

of

in

Among t h e

Facultative

require

bacterial

flavor/odor

will

salty,

peptides,

volatile

lactones,

terms

categories;

Requirements

of

genus

grow

include

quite

with

in

broad

acids,

terpenes.

keeping

and

two

and F r a g r a n c e M a t e r i a l s

Metabolic

chemicals,

oxygen.

the

Flavor

maintenance

and

volatile

many

of

on

metabolize

Strict

In

microorganisms

duction,

members

of

amino are

and

aroma s u b s t a n c e s

Sources

as

categories;

properties.

into

Tastants

1).

typically

two

compounds

broken

such

esters,

the

volatile

be

(Table

compounds

compounds,

taste

can

odorants

between

Metabolites

are

properties

sweet,

overlap

Microbial

metabolites

sensory

odorants

of

by

are

the

Microorganisms

cases

can

(even

hydrocarbons)

example, amino

be

forced

amino

acid

acids

auxotrophs

from Corynebacterium(2). and

incorporated

Phosphorus into

components.

They

the can

-

Both

of

structural also

become

these and

part

elements

functional of

product

are cell mole-

cules. 3.

Other Nutrients are

B.

also

Primary The

ulated very

to

small

-

required vs.

Minerals, in

Secondary

biosynthetic produce amounts

vitamins,

and t r a c e

minerals

fermentations.

Metabolites

pathways

certain by

most

of

molecules

cellular

microorganisms that

regulatory

are

can

normally

mechanisms.

be

manip-

limited

to

Industrial

In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


25. SCHARPFETAL.

Fermentation Processes

325

Table 1. Sensory Properties of Microbial Metabolites PRIMARY ODORANTS

TASTANTS & ODORANTS

PRIMARY TASTANTS

Aldehydes . acetaldehyde . phenylacetaldehyde

Amines

Amino Acids

Ketones . diacetyl . acetophenone

Fatty Acids

Peptides

Esters . ethyl butyrate

Pyrazines

Sugars

Alcohols . butanol

Lactones

Polyols

Terpenes . citronellal



326

BIOGENERATION O F A R O M A S

m i c r o b i o l o g i s t s have d e v e l o p e d t e c h n i q u e s t o s e l e c t mutant s t r a i n s in which the r e g u l a t o r y p r o c e s s is a l t e r e d in a way t h a t c e r t a i n metabolites can be over produced. Primary metabolites are s u b s t a n c e s such as sugars and amino a c i d s which are e s s e n t i a l for c e l l growth(3). Some of t h e s e s u b s t a n c e s a r e t a s t e and odor a c t i v e . C e r t a i n amino a c i d s and p e p t i d e s a r e sweet w h i l e o t h e r s a r e b i t t e r or s a l t y . Antibiotics are good examples of secondary metabolites produced by fermentation on an i n d u s t r i a l scale. Secondary m e t a b o l i t e s a r e s u b s t a n c e s which a r e n o t r e q u i r e d by t h e c e l l f o r biosynthesis. Most v o l a t i l e and o d o r - a c t i v e m a t e r i a l s f a l l into this category of metabolites, including alcohols, aldehydes, ketones, terpennoids, and l a c t o n e s . The h i g h l y odorous simple e s t e r s formed by f u n g i a r e secondary m e t a b o l i t e s . They a r e t h o u g h t t o p r o v i d e a mechanism f o r removing b o t h a c i d and a l c o h o l p r e c u r s o r s from c e l l s and t h e media; i f a l l o w e d t o accumulate t h e s e c h e m i c a l s c o u l d become toxic to the organism(5). Secondary metabolites presumably c o n t r i b u t e t o t h e m i c r o o r g a n i s m s ' s u r v i v a l by p o s s i b l y i n h i b i t i n g c o m p e t i t i v e s p e c i e s t h a t c o u l d o t h e r w i s e occupy the same e n v i r o n m e n t a l n i c h e , ( 3 ) . C o l l i n s (4) s u g g e s t s t h a t w i t h f u n g i : 1. The r e l e a s e o f v o l a t i l e s i n t o t h e i r s u r r o u n d i n g e n v i r o n ment h e l p s t o r e g u l a t e t h e i r c o m p e t i t o r s . T h i s phenomenon known in h i g h e r p l a n t s , is c a l l e d a l l e o p a t h y . 2. Some v o l a t i l e s s t i m u l a t e spore g e r m i n a t i o n . 3. Some v o l a t i l e s s e r v e as a t t r a c t a n t s . M i c r o o r g a n i s m s t h a t produce s e c o n d a r y m e t a b o l i t e s generally undergo a p e r i o d of l o g a r i t h m i c growth in which t h e s y n t h e s i s of the secondary m e t a b o l i t e is n e g l i g i b l e . When the c u l t u r e e n t e r s t h e s t a t i o n a r y phase, s e c o n d a r y m e t a b o l i t e p r o d u c t i o n is o f t e n triggered. C.

T r a d i t i o n a l Whole Food F e r m e n t a t i o n s

The most a n c i e n t example o f t h e p r o d u c t i o n o f v o l a t i l e s by m i c r o o r g a n i s m s a r e t h e t r a d i t i o n a l food f e r m e n t a t i o n s . Fermented foods a r e t e c h n o l o g i c a l p r o d u c t s which have been c o n v e r t e d by m i c r o organisms to foods which a r e more a p p e a l i n g than the raw m a t e r i a l used. One s i g n i f i c a n t outcome is the p r o d u c t i o n o f v o l a t i l e aroma and t a s t e s u b s t a n c e s . Some fermented foods a r e produced u s i n g pure cultures of microorganisms. Others make use of organisms i n d i g e n o u s to the raw m a t e r i a l . T e x t s by M a r g a l i t h ( 6 ) and R o s e ( 7 ) review the f l a v o r compounds produced by m i c r o o r g a n i s m s in fermented foods. 1. M i l k S u b s t r a t e s Cheese is perhaps t h e o l d e s t of t h e fermented f o o d s . ( F i g . 1) The b a s i c u n d e r l y i n g m i c r o b i a l t r a n s f o r m a t i o n in a l l cheese manufacture is t h e c o n v e r s i o n of l a c t o s e of m i l k i n t o l a c t i c a c i d . The m i c r o organisms in the starter culture contribute significantly to the flavor of the cheese. The secondary m i c r o b i a l f l o r a o f t h e cheese a l s o e l a b o r a t e t a s t e and odor a c t i v e s u b s t a n c e s . These organisms may be present as chance contaminants or introduced intentionally(8), and r e s u l t in d i s t i n c t i v e t y p e s of cheeses such as c h e d d a r , b l u e v e i n e d and s w i s s .



Figure 1.

Cheese odorants and tastants.



BIOGENERATION OF AROMAS U n r i p e n e d cheeses a r e made u s i n g s i m p l e l a c t i c a c i d b a c t e r i a l c u l t u r e s or streptococcal s t r a i n s which a r e facultative anaerobes. Cottage cheese is a good example in which there is bacterial p r o d u c t i o n of d i a c e t y l t h e p r i n c i p a l f l a v o r component, d e r i v e d as a by-product of pyruvate metabolism. Some c o t t a g e cheese s t a r t e r c u l t u r e s a l s o produce a c e t a l d e h y d e , which may impart a harsh f l a v o r . The s t a r t e r c u l t u r e s a l s o c o n t r i b u t e t o t h e r i p e n i n g p r o c e s s by d e g r a d i n g p r o t e i n s to primary metabolites such as p e p t i d e s , free amino a c i d s , and s u l f u r compounds, and d e g r a d i n g l i p i d s to f r e e f a t t y a c i d s , a l l of which a r e o d o r o r t a s t e a c t i v e . These transformations can be a c c o m p l i s h e d by living c e l l s , enzymes r e l e a s e d from dead c e l l s , o r r e s i d u e s of r e n n i n l e f t from the m i l k - c l o t t i n g s t e p . Cheese r i p e n i n g c o n s i s t s of a s e r i e s of b i o c h e m i c a l r e a c t i o n s , t h e most predominant ones b e i n g l a c t i c a c i d metabolism, lipolysis, proteolysis, and oxidation/reduction. The b a s i c t a s t e and t e x t u r a l s e n s a t i o n of cheese is t h o u g h t t o be due t o f a t , amino a c i d s , p r o t e i n s , p e p t i d e s , l a c t i c a c i d , and s a l t ( 9 ) . Upon t h i s f l a v o r base a r e superimposed t h e f r e e f a t t y a c i d s and a l p h a - a m i n o a c i d s , which in t u r n a r e c o n v e r t e d t o t h e important f l a v o r v o l a t i l e s , alpha-keto acids, amines, amides, esters, and k e t o n e s . F o r example, Ney, et al(10) report ~C f a t t y a c i d s as the main v o l a t i l e c o n s t i t u e n t s o f Cheddar. S m a l l amounts of b r a n c h e d C . C

2

4 and and

C^ a c i d s a l p h a - k e t o a c i d s , 2 - a l k e n o n e s , 2-aldehydes amines a r e a l s o p r e s e n t . Cheddar cheese is made w i t h the same s t r e p t o c c o c a l bacterial strains used in unripened cheese but subsequent bacterial growth of lactobacilli and pediococci can a l s o contribute to f l a v o r and aroma development. Volatile compounds from carbohydrate metabolism o f the s t a r t e r s t r e p t o c o c c i i n c l u d e d i a c e t y l , acetic acid, and a c e t a l d e h y d e . The s t a r t e r b a c t e r i a may also be i n d i r e c t l y i n v o l v e d in key f l a v o r / a r o m a compound f o r m a t i o n by c r e a t i n g e n v i r o n m e n t a l c o n d i t i o n s favorable f o r non-enzymatic r e a c t i o n s . F o r example, t h e low redox p o t e n t i a l r e s u l t i n g from s t a r t e r c u l t u r e a c t i v i t y may be i n s t r u m e n t a l in t h e p r o d u c t i o n o f s u l f u r compounds such as hydrogen s u l f i d e and m e t h a n e t h i o l , which some i n v e s t i g a t o r s b e l i e v e a r e v e r y i m p o r t a n t in Cheddar f l a v o r / a r o m a ( 8 ) . V o l a t i l e (C2-C10) f a t t y a c i d s which p l a y a r o l e in the g e n e r a l background aroma o f c h e d d a r , may be produced by l a c t i c a c i d s t a r t e r s , and lactobacilli. Mold-ripened cheeses are inoculated with mold spores which germinate and, via metabolic transformation, produce additional characteristic f l a v o r compounds. B l u e - v e i n c h e e s e s a r e good examples. In t h e s e c h e e s e s , s u r f a c e m o l d s , y e a s t s , and b a c t e r i a ( m i c r o c o c c i ) become dominant as the cheese pH d r o p s due to t h e l a c t i c f l o r a e a r l y in m a t u r a t i o n . The main



SCHARPF ET AL.

flavor

notes

and

methyl

in

blue

r o q u e f o r t i . (8) formed

from

cheeses

Methyl

fatty

ketones

acids

by

are

blue-vein

flavor.

from

to

produce

gamma-^hydroxy

of

milk often Swiss by

cheeses

to

acid

starters.

The

peptidases important

swiss

The

on

Finnish

and

workers

the

nitrogen

free

produced

in

beer,

2)

alcohols,

fermentation,

and

by

apparently

the

contain

amino

acid

investigators(13),

an great bread

and

carbon

is

Nykanen(14)

are

similar

in

cognac, of

metabolites

both

breads

shown

yeast

Several

secondary

the The

have

by

and

The

dioxide.

produced very

are

substrate.

fermentations

dough.

present

of and

fermentations the

and

whiskey,

bread

beverages

as

the

solution

are

are

of

bacteria

development,

two

sugar

ethanol

wine,

which

the

gruyère

fermentations

These

of to

of

with

sweet-tasting

components

fermentation

variet-

propionic

also

alcoholic

utilize

sugar

to

and

fermentation

some

Suomalainen

aroma

used

flavor

traditional

sugar

note

due

tastant.

function of

in

the

are

(Figure

conversion

the

two

yeast

principal

that

cheese

floral

Fermentation

by

acid

to

importance

production. based

sugars

release

other

economic

a

from other

cultures

vital

according

acids

biochemical

esters(12).

propionibacteria

which

proline,

to been

metabolites

apparently

coryneforms.

lactic

have

gamma-keto

propionibacteria;

is

initial

molds

cheeses,

distinguished

residual

subsequent

contribute

secondary

is

acids

metabolites

and

several

starter and

and

propionic

follows

as

which

of

yeasts

and

by

are

fatty

Pénicillium

also

esterified

and p h e n y l e t h y l

acid

may

well-ripened

growth

cheeses,

or

detected

different

subsequent lactic

In

be

phenylethanol ies

lactones

acids

to

by

secondary

Yeasts

glycerides

mechanisms.(8). can

due

beta-oxidation

Gamma-lactones

cheese

are

produced

decarboxylation. reported


vein

ketones

in

to

a

those

and

during

the

higher

from and

yeast

alcoholic

beverages. 2.

Grain-Derived exploits with

only

bread.

Substrates.

the a

small

Alcoholic

lactic

breads.

mentations

are

cerevisiae

and

development breads.

in

bacteria yeast

of

and During

are

formed,

a

ethanol

required and

Candida bread,

remaining

of

for

by

are

latter also

producing

contaminants to

the

Pediococcus,

and

bread

fermentation

along

with

ethanol,

of

leavening homo-

and

production used

in

bacteria.

utilis the

bacteria

belong

Streptococcus,

of

as

microorganisms

yeasts

as

manufacture

CO

combination

are

bread

occur

a

The

Certain

development

of

amount and

fermentations

quality

The

production

in

the

heteroof

high

these

fer-

Saccharomyces

involved being

organic

in

used

contribute of

bread agent,

to

rye

flavor

acids.

commercial

genera

flavor for

These baker's

Lactobacillus,

Leuconostoc. lactic

and

esters,

acetic

acids,

aldehydes,

ke-




GLUCOSE YEAST

2 C0

2 CH CH 0H 3

2

ALCOHOLIC BEVERAGES

o

BREAD

Propanol Butanol Isobutanol Pentanol Isopentanol WINE

BEER

\

Organic Acids Aldehydes Ketones Lactones Ethyl Esters Acetaldehyde Pentanal Furfural

3-Methyl Butanol Phenylethyl Alcohol Ethyl Acetate Isoamyl Acetate Organic Acids Ethyl Hexanoate Methionol

\

Organic Acids Hexanol 2-Phenyl Ethanol Octanol Isobutyraldehyde Acetaldehyde Methyl Acetate Ethyl Caproate Ethyl Caprylate Ethyl Laurate

DISTILLED SPIRITS

I

Nitrogen & Sulfur Compounds Volatile Phenols Aldehydes & Ketones Figure 2. V o l a t i l e chemicals produced in t r a d i t i o n a l yeast fermentations.


SC H A R PF ET A L.



tones, and l a c t o n e s . ( 15 ) Sour dough b r e a d s utilize Saccharomyces e x i g u u s , S. i n u s i t a t u s , and L a c t o b a c i l l u s fermentations(15, 16). Important f l a v o r a c i d s in t h i s b r e a d a r e produced by the l a c t o b a c . i l l i from maltose v i a the maltose phosphorylase pathway(17). Carboxylic a c i d s i d e n t i f i e d in s o u r dough breads i n c l u d e l a c t i c and acetic as the major o n e s , and t r a c e amounts o f the unusual 2 - h y d r o x y p r o p a n o i c , 2 - h y d r o x y , 1 , 2 , 3 - p r o p a n e t r i carboxylic, and dihydroxy butanedioic acids (15). atel, et al.,(18) suggested t h a t the conversion of a l c o h o l s t o 2 - m e t h y l ketones by the y e a s t NAD-dependent alcohol dehydrogenase is important in bread flavor formation. Additional aroma components common to yeastleavened bread are lower alcohols, acetaldehyde, p r o p a n a l , p e n t a n a l , and f u r f u r a l , and e t h y l e s t e r s , such as e t h y l a c e t a t e ( 1 9 ) . Only a few of t h e v o l a t i l e s found in f r e s h b e e r exceed t h e i r f l a v o r t h r e s h o l d v a l u e s . These i n c l u d e ethanol, 3-methylbutanol, ethyl acetate, isoamyl a c e t a t e , and e t h y l hexanoate(20) . Although the o r i g i n of these compounds is unknown, the metabolic capabilities of Saccharomyces strains could allow f o r m a t i o n o f t h e s e p r o d u c t s v i a pathways p r e v i o u s l y o u t lined. S u l f u r - c o n t a i n i n g compounds d e r i v e d from y e a s t are a l s o i m p o r t a n t in beer f l a v o r . Dimethyl s u l f i d e increases d u r i n g the c o u r s e of f e r m e n t a t i o n by e i t h e r enzymatic o r n o n - e n z y m a t i c r e a c t i o n s ( 2 1 ) . M e t h i o n o l is one of the main s u l f u r - c o n t a i n i n g aroma compounds in beer and w i n e . It is formed from m e t h i o n i n e by t h e y e a s t t h r o u g h d e a m i n a t i o n and d e c a r b o x y l a t i o n t o the aldehyde and reduction to the corresponding alcohol(21). Other yeast derived sulfur containing compounds include methionyl acetate, ethyl-3-methyl thiopropionate, and 2-methyltetrahydrothiophene-3one(21). One of the more i n t e r e s t i n g m e t a b o l i c sequences thought t o o c c u r in beer l e a d s t o l a c t o n e f o r m a t i o n . L e v u l i n i c a c i d is a d e g r a d a t i o n p r o d u c t o f g l u c o s e and 4-oxononanoic a c i d is d e r i v e d from l i n o l e i c a c i d d u r i n g malt p r e p a r a t i o n and wort b o i l i n g . I t is t h o u g h t t h a t t h e s e oxo a c i d s a r e r e d u c e d by t h e y e a s t t o the c o r r e sponding hydroxy a c i d s , which form 4 - p e n t a n o l i d e and 4nonanolide, respectively(22). 3.

Fruit Substrates. Wines a r e g e n e r a l l y described as a c i d i c a l c o h o l i c beverages which c o n t a i n a s p e c i a l b o u quet of v o l a t i l e c o n s t i t u e n t s . Factors affecting the t a s t e - a r o m a bouquet i n c l u d e s t r a i n of y e a s t u s e d , the f l o r a o f the must c o n d i t i o n s of fermentation, r e s i d u a l s u g a r , and the t y p e o f raw m a t e r i a l s . A l t h o u g h some of the f l a v o r compounds of wine a r e indigenous to the grape i t s e l f , much of the f l a v o r o f the f i n i s h e d p r o d u c t a r i s e s from the b i o c h e m i c a l a c t i o n s of the y e a s t .



BIOGENERATION OF AROMAS The products of yeast a u t o l y s i s , such as amino a c i d s may a l s o p l a y a r o l e in wine f l a v o r . The a u t o l y sate by-products may a l s o serve as substrates for secondary f e r m e n t a t i o n s . M a l i c and t a r t a r i c a c i d s , the principle organic acids in grape must and w i n e , can a l s o s e r v e as s u b s t r a t e s f o r s e c o n d a r y f e r m e n t a t i o n . L a c t o b a c i l l u s and L e u c o n o s t o c s p . a r e i n v o l v e d in t h e c o n v e r s i o n o f m a l i c a c i d t o l a c t i c a c i d and CO^ which improves flavor and mellowness of wines(16). Important wine flavor compounds derived from yeast metabolism during fermentation are: Alcohols Although there are strain specific variations, the p r i n c i p l e h i g h e r a l c o h o l s i n v o l v e d in wine aroma a r e : 1-propanol, 2-methyl-l-propanol, 1-butanol, 2phenylethanol, 2-methyl-l-butanol, 3-methyl-l-butanol, 1-hexanol, 1-octanol(23). These are likely to be derived in part, from amino a c i d s via the Erhlich pathway. O r g a n i c A c i d s p r e s e n t a r e : a c e t i c , isobutyric, isovaleric, hexanoic, and decanoic(24). Aldehydes present are: acetaldehyde, isobutyraldehyde, isovaleraldehyde, furfural(20). E s t e r s - I t is known t h a t e s t e r f o r m a t i o n in wine is m e d i a t e d by y e a s t . Only diethyl malonate and e t h y l - 2 - m e t h y l b u t y r a t e were sign i f i c a n t f o r d i f f e r e n t i a t i o n o f wine v a r i e t i e s . Ferment a t i o n d e r i v e d e s t e r s a l s o reported to c o n t r i b u t e to wine aroma a r e : methyl and e t h y l acetates, ethyl c a p r o a t e , e t h y l c a p r y l a t e , and e t h y l l a u r a t e ( 2 1 ) . Numerous r e v i e w s a r e in t h e l i t e r a t u r e on t h e f e r mentation-derived components of distilled spirits. Over 280 components have been i d e n t i f i e d in w h i s k i e s ( 2 5 ) and o v e r 400 components have been found in rum(26). The conditions of fermentation and distillation determine t h e c o n c e n t r a t i o n o f v o l a t i l e s . F o r example the concentration and composition of trace sulfur compounds in g r a i n s p i r i t s r e f l e c t s t h e manner in which the d i s t i l l a t i o n was c a r r i e d o u t . No attempt w i l l be made to compile the complete list of compounds i d e n t i f i e d to date. R a t h e r , s e v e r a l newly identified components made p o s s i b l e by the r e c e n t advances in gas chromatography/mass s p e c t r o m e t r y w i l l be r e p o r t e d . N i t r o g e n and s u l f u r c o n t a i n i n g compounds may be formed d u r i n g y e a s t f e r m e n t a t i o n in the p r o d u c t i o n o f distilled spirits(26). Newly found s u l f u r - c o n t a i n i n g compounds in rum i n c l u d e m e t h a n e t h i o l , a l k y l d i s u l f i d e s , and dimethyl s u l f o x i d e . Ethyl nicotinate, a highly odorous compound t h o u g h t t o be formed d u r i n g f e r m e n t a t i o n 25), has a l s o been found in rum. A new s u l f u r containing compound, 2-formylthiophene, is found in scotch whiskey(25). Volatile phenols can be produced by microorganisms from the d e c a r b o x y l a t i o n of p h e n o l i c acids such as f e r u l i c and c o u m a r i c , and t y r o s i n e ( 2 7 ) . Phenols may c o n t r i b u t e t o the d r y n e s s c h a r a c t e r i s t i c s in s c o t c h whiskey(25). C r e s o l s and g u i a c o l s have been r e p o r t e d t o have an e f f e c t on the aroma o f w h i s k e y s ( 2 8 ) .


25.

SCHARPFETAL.


333

Aldehydes and v i c i n a l d i k e t o n e s produced d u r i n g f e r m e n t a t i o n a l s o c o n t r i b u t e t o t h e f l a v o r p r o f i l e of d i s t i l l e d beverages. B u t y r - and v a l e r a l d e h y d e s along w i t h a l c o h o l s and e s t e r s appear t o be i n v o l v e d in t h e "core" of whiskey a r o m a ( 2 9 ) .


Pure C u l t u r e T r a n s f o r m a t i o n s on P a r t i a l l y D e f i n e d Media The r e l e a s e of o d o r - a c t i v e m a t e r i a l s by m i c r o b i a l c u l t u r e s is w e l l known by m i c r o b i o l o g i s t s . In f a c t , odor p r o p e r t i e s have been used as classification markers by m i c r o b i a l taxonomists for years. O m e l i a n s k i ( 3 0 ) was among the f i r s t i n v e s t i g a t o r s t o d e f i n e m i c r o o r ganisms on the b a s i s of the o d o r s t h e y p r o d u c e d . S i n c e t h a t t i m e , m i c r o b i o l o g i s t s have l e a r n e d t h a t t h e p r o d u c t i o n o f v o l a t i l e s v a r i e s as a f u n c t i o n o f many a d d i t i o n a l c h e m i c a l , p h y s i c a l , and b i o l o g i c a l f a c t o r s i n c l u d i n g the f o l l o w i n g : The pH of t h e media must be m o n i t o r e d a n d , in many c a s e s , h e l d c o n s t a n t . Temperatures must be c a r e f u l l y c o n t r o l l e d d u r i n g f e r m e n t a t i o n to maximize y i e l d s . The n a t u r e of the a s s i m i l a b l e c a r b o n and n i t r o g e n s o u r c e s d i r e c t l y d e t e r m i n e s the q u a l i t y o f the odor r e l e a s e d and t h e range of secondary m e t a b o l i t e s p r o d u c e d . O p t i m i z a t i o n of the production of volatiles must also take into account the physiological state of the microorganisms. For bacteria, a p p r o p r i a t e i n o c u l a may be s e v e r a l days o l d . F o r some o f t h e h i g h e r f u n g i t h e use o f 20 day i n o c u l a and 2 week c u l t u r e s to seed the p r o d u c t i o n media a r e common(31, 3 2 ) . W i t h the e x p a n s i o n of t h e f i e l d s o f m i c r o b i a l taxonomy and b i o c h e m i s t r y , a d d i t i o n a l m i c r o o r g a n i s m s have been i d e n t i f i e d which elaborate flavor and odor compounds. Table 2 compiles a few examples from t h e l i t e r a t u r e of m i c r o o r g a n i s m s which produce o d o r s in d e f i n e d m e d i a . Most o f t h e s e f e r m e n t a t i o n s have been c a r r i e d out o n l y on s m a l l l a b o r a t o r y s c a l e . In many s t u d i e s t h e volatiles p r o d u c e d are not w e l l c h a r a c t e r i z e d and l i t t l e is known about the mechanism and pathways of biosynthesis. A.

B a c t e r i a & Actinomycetes

A r o m a / t a s t e p r o d u c i n g b a c t e r i a t e n d t o u t i l i z e amino a c i d s as t h e i r p r i n c i p a l carbon source. When grown on c o a r s e wheat as a s u b s t r a t e , v o l a t i l e f a t t y a c i d s of C ~C a r e major p r o d u c t s a f t e r 72 hours(33). With b a c t e r i a even s l i g h t changes in the o x i d a t i o n s t a t e of the media may a f f e c t t h e number and t y p e of o d o r i f e r o u s metabolites. ^In g e n e r a l , o d o r s can be d e t e c t e d when b a c t e r i a l counts r e a c h 10 -10 c e l l s per gram of m e d i a ( 3 3 ) . In mixed c u l t u r e s such as those present in dairy products, bacteria modify flavor s u b s t a n c e s t h a t a r e produced by molds and y e a s t s . However, the odorants produced by b a c t e r i a are often overpowered by those produced by y e a s t s and molds due to t h e l o n g e r b i o l o g i c a l c y c l e s o f the l a t t e r s p e c i e s . Perhaps the most i m p o r t a n t b a c t e r i a in terms o f f l a v o r and odor p r o d u c t i o n a r e t h e l a c t i c s t r e p t o c o c c i d i s c u s s e d e a r l i e r . In a d d i t i o n t o a c e t a l d e h y d e , t h e s e organisms produce a wide v a r i e t y of n e u t r a l and a c i d i c c a r b o n y l compounds which r e s u l t in t h e s h a r p , buttery, f r e s h t a s t e of d a i r y p r o d u c t s . Some b a c t e r i a i n c l u d i n g


334


Table 2. Chemicals Produced by Pure Cultures of Microorganisms and Their Sensory Descriptions


BACTERIA"

ORGANISM

SENSORY DESCRIPTOR

VOLATILES PRODUCED

L a c t i c Acid Streptococci, Lactobaci H i , Leuconostoc sp Propionibacter iïïm

Sharp, b u t t e r y , f r e s h

Acetaldehyde, Diacetyl, Acetoin, L a c t i c Acid

Sour, sharp

Pseudomonads Baci1 lus sp. Corynebacterium sp.

Malty, m i l k y granary

Acetoin, dienals, Aldehydes Acetoin, 3-Methyl-l-butanal, 2-Methyl-2-hydroxy-3-keto b u t a n a l , pyrazines

Damp f o r e s t s o i l odor

2-MeO-3-isopropyl Geosmin

Aromas associated with bread & alcohol fermentation

Higher a l c o h o l s (amyl. isoamyl, p h e n y l e t h y l , e s t e r s , lactones, thio-compounds Phenylethanol and esters, terpene alcohols (linalool, citronellol, g e r a n i o l ) , short chain a l c o h o l s & e s t e r s Ethyl e s t e r s , higher a l c o h o l esters Ethyl e s t e r s , higher a l c o h o l esters Higher a l c o h o l e s t e r s Lactones

ACTINOMYCETES Streptomyces sp

Saccharomyces

sp.

Kluyveromyces sp.

F r u i t y , rose

Geotrichum sp.

F r u i t y , melon

Hanensula sp.

Floral s o i l odor Apple, pineapple Peach

Dipodascus sp. Sporobolômyces sp.

pyrazine,

MOLDS Aspergi1 lus sp. Pen i c i 11ium sp.

Fungal, musty mushroom Mushroom, blue cheese, rosey

C e r a t o c y s t i s sp.

Banana, pear, peach, plum

Trichoderma sp.

Coconut, a n i s e , cinnamon

Phe11inus sp.

F r u i t y , rose, wintergreen

Septoria sp. Lent inus" sp.

Anise or cinnamon Aromatic, f r u i t y

Unsat. a l c o h o l s 1- 0 c t e n e - 3 - o l , methyl ketones, 2- phenylethanol, thujopsene, nerolidol A l c o h o l s , e s t e r s , rnonoterpene a l c o h o l s , lactones 6-pentyl-a-pyrone Sesquiterpenes, cinnamate derivatives Methyl benzoates & s a l i c y l a t e s , benzyl a l c o h o l A l k y l & a l k o x y l pyrazines Cinnamate d e r i v a t i v e s Higher a l c o h o l s , sesquiterpenes



25.

SCHARPFETAL.


335

Bacillus, C o r y n e b a c t e r i u m and Pseudomanas s p e c i e s a r e c a p a b l e o f p r o d u c i n g d a i r y , m i l k y , g r a n a r y , and v e g e t a b l e - t y p e o d o r s when c u l t u r e d on a p p r o p r i a t e media(33) p r o b a b l y due in p a r t t o the p r e s e n c e of pyrazines(34). P y r a z i n e s a r e h e t e r o c y c l i c n i t r o g e n c o n t a i n i n g compounds w i t h unique f l a v o r p r o p e r t i e s . A l t h o u g h t h e pathways f o r s y n t h e s i s of p y r a z i n e s a r e n o t known, it has been s u g g e s t e d t h a t the mechanism f o r s y n t h e s i s in pseudononads may be s i m i l a r to t h a t i n v o l v e d in raw vegetables. In v e g e t a b l e s it has been h y p o t h e s i z e d t h a t p y r a z i n e s y n t h e s i s i n v o l v e s c o n d e n s a t i o n between a l p h a - a m i n o a c i d s and 1 , 2 dicarbonyls(35). C o r y n e b a c t e r i u m mutants have been reported to accumulate up t o 3 g of t e t r a m e t h y l p y r a z i n e per l i t e r o f c u l t u r e media a f t e r 5 days o f g r o w t h ( 3 6 ) . Actinomycetes, primarily streptomycetes, are capable of p r o d u c i n g h i g h l y odorous v o l a t i l e metabolites in low y i e l d s in submerged c u l t u r e . T h i s s u b j e c t has been reviewed in d e t a i l ( 3 7 ) . Among the more i m p o r t a n t v o l a t i l e s i d e n t i f i e d a r e : geosmin, the e a r t h y o d o r , m e t h y l i s o b o r n e o l , h a v i n g a camphor o r menthol o d o r ; 2methoxy-3-isopropyl pyrazine, w i t h a musty v e g e t a b l e odor; and m i s c e l l a n e o u s compounds such as s e s q u i t e r p e n o i d s and l a c t o n e s . B.

Yeasts

Perhaps more is known about the p r o d u c t i o n of v o l a t i l e compon e n t s in b a k e r ' s y e a s t (Saccharomyces c e r e v i s i a e ) t h a n in any o t h e r microbial species. The t y p e and q u a n t i t y of e s t e r s formed d u r i n g y e a s t f e r m e n t a t i o n s appear t o be i n f l u e n c e d by the y e a s t s t r a i n , f e r m e n t a t i o n t e m p e r a t u r e , p H , and a l c o h o l c o n c e n t r a t i o n ( 3 8 ) . W i t h b a k e r ' s y e a s t , h i g h e r amounts o f e s t e r s were found t o be t r a n s f e r r e d from the c e l l s i n t o t h e media at h i g h e r t e m p e r a t u r e s . U s i n g e t h y l c a p r y l a t e as a s u b s t r a t e , Soumalainen(38) found t h a t the e q u i l i b r i u m between s y n t h e s i s and h y d r o l y s i s depended n o t o n l y on the e s t e r c o n c e n t r a t i o n but a l s o on p H . A h i g h e r amount o f e s t e r remains in s o l u t i o n s of lower p H . The p r o d u c t i o n o f the h i g h e r a l c o h o l s , the a c e t a t e s of i s o a m y l a l c o h o l and p h e n y l e t h y l a l c o h o l , and the e t h y l e s t e r s of the C6-C10 f a t t y a c i d s has been s t u d i e d in s e m i a e r o b i c sugar f e r m e n t a t i o n s by s t r a i n s of S, c e r e v i s i a e and S. uvarum. S. c e r e v i s i a e generally produced more e s t e r s t h a n S. u va rum. Isoamyl a c e t a t e was the main e s t e r produced by S. c e r e v i s i a e , and o t h e r s , in d e c r e a s i n g o r d e r , were e t h y l c a p r y l a t e , e t h y l c a p r o a t e , e t h y l c a p r a t e and p h e n y l e t h y l acetate(39). S e v e r a l u n u s u a l t h i o compounds have been produced by Saccharomyces in model a n a e r o b i c f e r m e n t a t i o n s u s i n g amino a c i d s such as m e t h i o n i n e as the s o l e c a r b o n s o u r c e ( 2 1 ) . These model f e r m e n t a t i o n s p r o d u c e m e t h y l t h i o p r o p a n a l and t r a c e s o f o t h e r s u l f u r containing compounds, such as methionyl acetate and 2methyltetrahydrothiophene-3-one. Another i m p o r t a n t c l a s s o f t a s t e / a r o m a c h e m i c a l s p r o d u c e d by S. c e r e v i s i a e a r e l a c t o n e s . Four and f i v e oxo a c i d s a r e t r a n s f o r m e d by t h i s y e a s t i n t o o p t i c a l l y a c t i v e l a c t o n e s , o x o a c i d e t h y l e s t e r s , and c o r r e s p o n d i n g p - h y d r o x y a c i d e t h y l e s t e r s ( 2 2 ) . Volatile product accumulation kinetics in cultures of Kluyveromyces s t r a i n s i n c l u d e s s h o r t c h a i n a l c o h o l s and e s t e r s such as 2 p h e n y l e t h y l a c e t a t e . These c u l t u r e s t y p i c a l l y have a f r u i t y ,



336 rosey

odor(40).

comparable ferences

were

culture

media

of

the

amounts

the

were favored

as


of

For

the is

of in

linalool,

a l l

responses the

dif-

to

the

addition

of

derivatives

tyrosine

led

to

in

lower

strains.

of

producing

although

conditions

asparagine

of

qualitatively

quantitative

2-phenylethyl

addition

Increasing

source

was

example,

capable

culture

produced.

nitrogen

compounds

strain-dependent

derivatives

and

Changing

of

investigated,

formation

lactis

citronellol

product

the

the

2-phenylethyl

ug/L.(41).

and

whereas

Kluyveromyces such

range

strains

observed.

strains,

of

the

three

significant

phenylalanine two

Although

within

at

very

altered

temperature increased

the

and

the

mono-terpenes

low

yields

yield

of

50

and

type

concentrations

yield

of

of

citronellol

substantially. Yeasts lated

from

mashed

fruits

scribed The

from ripe

as

amino

the

and

acids of

close

to

present

in

the

aroma

been

reported

to

duce

a

floral

ethyl

esters

rosey

Diplodascus peachy two

of

C.

Molds A

(which

of

the

large In

and

media

such

tract

broth(47).

the

volatile

alcohols, common double

and

bond.

amino

Aspergillus primarily

fungi this

such

volatile and

as

of

the

to

to

strains

the

pro-

presence

intense

glucose

of

is

due

fruity

aroma

media(44).

to

the

The

presence

lactone(45,

odor

of

46).

to

alcohols

originate

the grown

on

l-octene-3-ol,

a

on

or

as

The

components

in

part

wheat

ex-

higher are

of

the

containing

from

the

one

metabolism

valine,

Among meal

the

octenols,

isoleucine,

characteristic

of

natural

malt

principal

octanols

yeasts

substrates,

similar.

pathway.

coarse

the

broth, used

to

sub-

organic

Much out

and

leucine,

Erhlich

and and

hexanols,

unsaturated

as

molds

sources.

are

of

reported

odoriferous

carried

are

fungi

The

are

of

nutrient

cereals

higher

be

been

variety

basidiomycetes

carbohydrates

carbon

butanols,

wide

mushrooms) producing

has

broth,

a

category,

utilize

and

the

such

oryzae

thought

secrete

as

principal

grains

appear

via

de-

has

m i c r o b i a l sources

metabolites.

These

phenylalanine

higher fungi

methyl

acids

were

fruits(42). be

an

liquid

Within

dextrose

fractions

fraction

isoheated

Geotrichum s t r a i n

due

release in

producing fungi

When

secondary

of

that

Hansenula

be

been

Fungi

alcohol

potato

were

odor(43).

yeast

aerobic

butanol,

volatile of

as

fruit

Another

Sporobolomyces

fleshy

on o d o r

aromas

non-heated

and gamma-cis-6-dodecene

general,

rarely

work

fresh,

to

have

fermentation

esters(43).

interesting

predominantly

culture

of

the

produce

thought

from

molds

most

stances (4). acids

melon

substances.

include

to

from

and p i n e a p p l e

and H i g h e r

number

among

are

strains

apple

in

mashed

alcohol

gamma-deca,

odor-active be

a

aroma,

aroma e m a n a t i n g

lactones,

media those the

Oospora genera,

used

substances.

release

and h i g h e r

reminiscent

and

synthetic

being

precursors

G e o t r i c h u m and

strawberries

the

and molds,

substrate

produced

powerful

mushroom

aroma(33). In

many

duce

volatiles

blue

cheese

good

only

aroma

example.

produces

cases,

molds in

the

such

generation Another

characteristic

as

spore by

example

odor

Pénicillium

and

producing stage.

only

certain is on

Trichoderma Methyl

Pénicillium

Pénicillium media

which

species

decumbens allow

pro-

ketone

and is

a

which

sporulation



25.

SCHARPFETAL.


337

to occur. T h i s o r g a n i s m produces as major odor compounds 3o c t a n o n e , p h e n y l e t h y l a l c o h o l , l - o c t e n - 3 - o l and n e r o l i d o l ( 4 ) . C e r a t o c y s t i s s p e c i e s have been s t u d i e d in some d e t a i l for t h e i r a b i l i t y t o produce f r u i t v o l a t i l e s ( 4 8 ) . These organisms grow r e a d i l y in submerged c u l t u r e and most members of t h i s genus p r o d u c e yeast-like cells. M o r e o v e r , t h e y do not r e q u i r e u n u s u a l c u l t u r e conditions(4). A v a r i e t y of d i s t i n c t i v e f r u i t l i k e aromas r a n g i n g from banana t o pear a r e produced by C e r a t o c y s t i s moniliformis depending on the time c o u r s e and c a r b o n and n i t r o g e n s o u r c e in t h e medium(49). F o r example ( T a b l e 3) b a n a n a - l i k e odor was produced when g l u c o s e and u r e a were used as c a r b o n and n i t r o g e n sources, respectively. Canned peach aroma was o b s e r v e d u s i n g g l y c e r o l and urea. C i t r u s aromas were o b s e r v e d when g a l a c t o s e and u r e a were used as substrates, and analysis revealed the presence of monoterpenes such as g e r a n i o l and c i t r o n e l l a l . A l l c u l t u r e s showed the p r e s e n c e of e t h a n o l and s h o r t - c h a i n e s t e r s such as i s o a m y l a c e t a t e , which was s u g g e s t e d t o be d e r i v e d d i r e c t l y from the amino acid leucine. F u r t h e r a n a l y s i s , s u g g e s t e d t h a t t h i s o r g a n i s m began to produce v o l a t i l e s by day 3 o r 4, r e a c h e d a p r o d u c t i o n peak at about day 6, and then stopped p r o d u c t i o n . A d d i t i o n a l studies using v a r i o u s v o l a t i l e s as s o l e c a r b o n s o u r c e s , i n d i c a t e d t h a t the e s t e r s were l o s t by e v a p o r a t i o n , but the e t h a n o l was n e u t r a l i z e d by t h e organism(49). Monoterpene p r o d u c t i o n by t h i s s p e c i e s has been found to f o l l o w t h e mevalonate pathway a f t e r depletion of the nitrogen source(50). This is t y p i c a l of secondary metabolite production. G e r a n i o l is the f i r s t compound t o a p p e a r , d e c r e a s i n g a f t e r two d a y s . Citronellol, n e r o l , g e r a n i o l , and n e r a l appear a f t e r the second d a y , and l i n a l o o l and a l p h a - t e r p i n e o l do not appear u n t i l the f o u r t h d a y . Some v o l a t i l e p r o d u c t s accumulate at s i g n i f i c a n t l e v e l s o n l y at s p e c i f i c s t a g e s in the l i f e c y c l e s of m i c r o o r g a n i s m s . T h i s may be due in p a r t t o t o x i c i t y of m e t a b o l i t e s w h i c h , when t h e y r e a c h t h r e s h o l d l e v e l s in the c u l t u r e m e d i a , i n h i b i t t h e o r g a n i s m . For example, S c h i n d l e r and Bruns(51) found t h a t C e r a t o c y s t i s v a r i o s p o r a is inhibited by higher concentrations of its own terpene metabolites. They were a b l e t o s i g n i f i c a n t l y improve y i e l d s of t h e s e p r o d u c t s by t r a p p i n g the end p r o d u c t s on r e s i n s . A fungus w i d e l y d i s t r i b u t e d in the s o i l , T r i c h o d e r m a v i r i d e , p r o d u c e s an i n t e r e s t i n g compound w i t h a c o c o n u t , peachy aroma in a n o n - a g i t a t e d l i q u i d medium c o n t a i n i n g p o t a t o e x t r a c t and s a l t s ( 5 2 ) . A f t e r t h r e e t o f o u r days c u l t i v a t i o n , the c u l t u r e s p o r u l a t e s r e l e a s i n g the m e t a b o l i t e 6 - p e n t y l - a l p h a - p y r o n e . The maximum l e v e l r e p o r t ed, however, is 0.17 g r a m / L . F u n g i a r e v e r y a d a p t a b l e t o extreme c u l t u r e c o n d i t i o n s and by c h a n g i n g the c u l t u r e c o n d i t i o n s the p r o d u c t i o n of s e c o n d a r y metabol i t e can be s t i m u l a t e d o r r e p r e s s e d . F o r example, L e n t i n u s l e p i d u s (Fr.) when c u l t u r e d on a s p a r a g i n e as the sole nitrogen source produces p r i m a r i l y c i n n a m i c a c i d d e r i v a t i v e s . A f t e r t h r e e weeks, a d d i t i o n a l compounds such as the h i g h e r a l c o h o l s , 1 - o c t a n o l and 1decanol appear. However, when c u l t u r e d on p h e n y l a l a n i n e as the s o l e n i t r o g e n s o u r c e , it p r o d u c e d a h i g h l e v e l of sesquiterpenes, in a d d i t i o n to c i n n a m a t e s , which r e s u l t e d in an a r o m a t i c - f r u i t y odor(53).




338

Table 3.

V o l a t i l e s Produced by Ceratocystis Moniliformis CHEMICALS IDENTIFIED

CARBON SOURCE

NITROGEN SOURCE

Dextrose

Urea

F r u i t y , banana

Acetate Esters, ethanol

Dextrose

Leucine

F r u i t y , o v e r - r i p e , banana

Isoamyl

Galactose

Urea

C i t r u s , g r a p e f r u i t , lemon

Monoterpenes, ethanol

Corn

Urea

Cantaloupe, banana

Dextrose

Glycine

P i n e a p p l e , lemon, sweet

Dextrose

Methionine

Weak p o t a t o

Glycerol

Urea

Canned p e a r , p e a c h

Starch

AROMA

tropical

acetate, ethanol

flower

Decalactones,

From L a n z a e t a l . (49)


ethanol

25. SCHARPFETAL.


339


The p r o d u c t i o n o f b e n z e n o i d compounds such as m e t h y l b e n z o a t e s and s a l i c y l a t e s has been n o t e d in s p e c i e s of f u n g i b e l o n g i n g t o the genus P h e l l i n u s ( 5 4 ) . P y r a z i n e s have been r e p o r t e d t o be p r e s e n t in c u l t u r e s of S e p t o r i a s p e c i e s ( 5 5 ) . S p e c i f i c M i c r o b i a l T r a n s f o r m a t i o n s on D e f i n e d M e d i a . V o l a t i l e s may be produced from p r e c u r s o r s which r e p r e s e n t the s o l e s o u r c e of c a r bon. The m i c r o o r g a n i s m s s e l e c t e d t o a c c o m p l i s h t h e s p e c i f i c b i o t r a n s f o r m a t i o n may n o t grow on such a s u b s t r a t e . A s u f f i c i e n t mass of the c e l l s may have t o be o b t a i n e d and t h e n the p r e c u r s o r i n t r o duced i n t o the culture in small q u a n t i t i e s . In t h i s way the microorganism is "forced" to carry out the "targeted" bioconversion. In general, microbial reactions offer the following supplements t o c h e m i c a l s y n t h e s e s : 1. A t t a c k on m o l e c u l a r p o s i t i o n s n o t a f f e c t e d by c h e m i c a l methods (because the p o s i t i o n s cannot be s u f f i c i e n t l y a c t i v a t e d or they r e q u i r e a number o f i n t e r m e d i a t e s t e p s b e f o r e t h e y w i l l react)(56). 2. S t e r e o s p e c i f i c i n t r o d u c t i o n o f oxygen f u n c t i o n s o r o t h e r s u b s t i t u e n t s (or a l t e r a t i o n o f such f u n c t i o n s w i t h t h e p o s s i b l e f o r m a t i o n of o p t i c a l l y a c t i v e c e n t e r s ) ( 5 6 ) . 3. C o m b i n a t i o n of m u l t i p l e r e a c t i o n s i n t o one f e r m e n t a t i o n s t e p , (can be programmed t o o c c u r in a s p e c i f i c sequence w i t h a suitable microorganism)(56). 4. Conditions for microbial reactions are mild (since many volatiles are sensitive to heat, this is a real advantage)(56). 5. L a r g e c e l l numbers and h i g h r a t e s o f growth may a l l o w c e r t a i n m i c r o o r g a n i s m s t o be f o r c e d to s u s t a i n unique r e a c t i o n s . 6. Low r e a c t i o n r a t e s can be overcome by the sheer number o f i n d i v i d u a l c e l l s and the ease of p r o b i n g m e t a b o l i c pathways by environmental manipulation. The list of known m i c r o b i a l transformations of defined chemical.substrates has grown c o n s i d e r a b l y s i n c e 1960. Kieslich (56) has d e v e l o p e d an e x t e n s i v e c o m p i l a t i o n o f t h e s e r e a c t i o n s which has become a v a l u a b l e r e f e r e n c e f o r f e r m e n t a t i o n t e c h n o l o g i s t s . The t r a n s f o r m a t i o n s have been c l a s s i f i e d by s t r u c t u r a l c l a s s by K i e s l i c h r a n g i n g from a l i c y l i c s t o p e p t i d e s . Many of the c h e m i c a l c l a s s e s a r e r e l e v a n t t o the o b j e c t i v e of p r o d u c i n g v o l a t i l e s by m i c r o b i a l conversions. M i c r o b i a l c o n v e r s i o n s from K i e s l i c h s c o m p i l a t i o n which c o u l d be u t i l i z e d in t h e p r o d u c t i o n of v o l a t i l e o d o r a n t s and t a s t a n t s are(56): 1. Oxidation. O x i d a t i o n of a r o m a t i c a l c o h o l s t o c o r r e s p o n d i n g aldehydes; Pénicillium, Pseudomonas and enzymes from Caldariomyces sp(57). have t h e a b i l i t y to convert benzyl, c i n n a m y l and o t h e r a l c o h o l s to the c o r r e s p o n d i n g a l d e h y d e s . 2. Reduction. A r o m a t i c a l d e h y d e s can be e f f e c t i v e l y r e d u c e d t o t h e i r c o r r e s p o n d i n g a l c o h o l s by m i c r o o r g a n i s m s . 3. Hydrolytic Reactions. Microorganisms can selectively hydrolyze various e s t e r s . For example, certain bacterial s t r a i n s can h y d r o l y z e s e l e c t e d 1-menthol e s t e r s such as t h e f o r m a t e s , a c e t a t e s and c a p r o a t e s . 1


340



4.

Dehydration Reactions. A l p h a - t e r p i n e o l can be formed by t h e d e h y d r a t i o n of c i s - t e r p i n h y d r a t e by B r e v i b a c t e r i u m s t r a i n s . 5. Degradation Reactions. Oxidative decarboxylations of amino a c i d s s e r v e as a good example. S c h i z o p h y l l u m commune is c a p a b l e of d e g r a d i n g p h e n y l a l a n i n e t o p h e n y l a c e t i c a c i d as w e l l as other a c i d s . 6. F o r m a t i o n o f New CC Bond. These r e a c t i o n s , such as the f o r m a t i o n of c y c l o h e x a n e r i n g s from t e r p e n e s , can be a c c o m p l i s h e d by m i c r o o r g a n i s m s . Pseudomonas and P é n i c i l l i u m sp. are c a p a b l e of f o r m i n g menthol from c i t r o n e l l a l . Over the p a s t 20 y e a r s , r e s e a r c h has uncovered many m i c r o b i a l t r a n s f o r m a t i o n s of the t e r p e n o i d s . Terpenes a r e i m p o r t a n t c o n s t i t u e n t s of f l a v o r s and f r a g r a n c e s , and can be the c e n t e r of a wide variety of microbial hydroxylations, oxidations, reductions, degradation and rearrangement reactions. See Ciegler(58), Wood(59), Collins(47), and S c h i n d l e r and Schmid(60) for separate r e v i e w s of the b i o l o g i c a l t r a n s f o r m a t i o n of n o n - s t e r o i d a l t e r p e n e s . S c a l e - U p of M i c r o b i a l Processes f o r V o l a t i l e Production M i c r o b i a l Sources. In many c a s e s , s c r e e n i n g f o r the m i c r o b i a l p r o d u c t i o n o f v o l a t i l e s can be a c h i e v e d by s e l e c t i n g pure c u l t u r e s of m i c r o o r g a n i s m s from p u b l i c c o l l e c t i o n s such as the American Type C u l t u r e C o l l e c t i o n (ATCC). The chances o f o b t a i n i n g an e f f e c t i v e culture can o f t e n be improved by s e l e c t i n g organisms known to p e r f o r m the d e s i r e d t y p e s of r e a c t i o n s . Another source for microorganisms is the isolation of appropriate strains from the environment. Here one selects organisms by e n r i c h m e n t techniques from a i r , soils, and water s o u r c e s such as l a k e s and streams and the s o i l s and s u r r o u n d i n g s o f plants which produce the substrate to be transformed. The e n v i r o n m e n t a l sample is i n c u b a t e d in a growth medium c o n t a i n i n g t h e s u b s t r a t e which s e r v e s as the s o l e c a r b o n s o u r c e . The m i c r o o r ganisms i s o l a t e d a r e t h e n "screened" by a l l o w i n g them t o grow in o r on a p p r o p r i a t e media u s i n g s t a n d a r d m i c r o b i o l o g i c a l t e c h n i q u e s . S c r e e n i n g Program. Small s c a l e fermentations or biotransformations t y p i c a l l y r a n g i n g from 10-100 ml a r e i n i t i a l l y c a r r i e d out in s m a l l f l a s k s c a l l e d "shake f l a s k s " . I n c u b a t i o n s a r e c a r r i e d out in i n c u b a t o r shakers which p r o v i d e suboptimum a e r a t i o n and p r e c i s e temperature control. Once an e f f e c t i v e m i c r o o r g a n i s m has been i d e n t i f i e d from s c r e e n i n g s t u d i e s , s c a l e - u p can be u n d e r t a k e n f i r s t in l a b o r a t o r y f e r m e n t o r s which have e f f i c i e n t s t i r r i n g and a e r a t i o n c a p a b i l i t i e s . The c a p a c i t y of t h e s e f e r m e n t o r s t y p i c a l l y range from 3-10 liters. Here one s t u d i e s a d d i t i o n a l o p e r a t i n g parameters which can a f f e c t m i c r o b i a l m e t a b o l i s m and the p r o d u c t i o n o f v o l a t i l e c h e m i c a l s . The pH o f the media must be m o n i t o r e d a n d , in many c a s e s , h e l d constant. Temperatures must be c a r e f u l l y c o n t r o l l e d d u r i n g ferment a t i o n t o maximize y i e l d s . The n a t u r e of the media, such as c a r b o n and n i t r o g e n s o u r c e s , d i r e c t l y d e t e r m i n e s the q u a l i t y of the odor r e l e a s e d and the range o f secondary m e t a b o l i t e s p r o d u c e d . O p t i m i z a t i o n of the p r o d u c t i o n of v o l a t i l e s must a l s o t a k e i n t o account t h e p h y s i o l o g i c a l s t a t e of t h e m i c r o o r g a n i s m s .


For

pilot

fermentations

would or

scale

are

Production. even

range

in

common


main

method

of

component When p u r e

aseptically. operated the

under

10,000

as

of

producing

primary

the

in

media

cultures aseptic

they

are

the

and

fermentations foreign

desired

state are

tens

volume fermenta-

now

the

most

metabolites. is

The

water.

must

sterilized

If

the

to

high

proteins.

solid

secondary

be

compounds

compared

for

plant

scale.

volatile

fermentations

must

pilot

liter

as

food

War I I

and

used,

over-run

or

replaced

these

media

for

gallons

World

conditions.

can

20-150

liters

antibiotics

fermentations after

Thus,

culture,

such

compounds,

the

fermenters to

countries

of

at

thousands

culture

western

volatile

out

scale 1,000

of

products

Submerged

of

carried

from

hundreds

fermentation tions

production

usually

Production

normally

341


SCHARPFETAL.

25.

be

and

carried

the

organisms

culture

out

fermenter contaminate

or

destroy

the

product. Selection At

the

is

of

Feasible

present

more

of

Although

the

been

variety 1.

time, an

metabolic

to

of

the

in

yields.

microorganisms the

no

can

exceed ary

from

the

the

by

slower

energy

for

Product often in

in

the

a

can

to

the

and be

and

or only

cases

the

biomass,

with

end-products can

substrates and

cannot

are

or in

of

volatile

second-

inhibitory

some

cases

finished

by

a

oxygen

large

viscous

be

product

cost

and

the

increased

organisms. (such

as

molds)

properties

broths

to

organisms

longer

may

maintain

of

require

growth

and

can the more prod-

cost. volatiles

concentration

quantity broths

recovery.

The

transfer

production of low

in

the

unwanted

and a e r a t i o n

The

producing

fungi.

microorganisms

and

increases

lost

organic organism

stripping

Highly

in very

trace

specific

some

CO^

This

greater

Some

fermentation

extraction products

the

agitation

Recovery.

only

reports

In

toxicological

higher

contamination

This

in For

Many

Many

growing

broth.

results

product

a

broth.

rheological

yields.

to

routes.

to

intrinsic

Morphology.

fermentation

been

due

energetics

volatile

toxic

Times.

time, of

produced

typically

Many

be

continuous

the

uct

can

fermentation

the

is

product.

with

are

affect

have

have

unfavorable

producing organisms.

Fermentation

possibility

reality.

production

This

media.

concentration.

Long

Organism

are

to

degraded

Toxicity.

the

fermentation

5.

of

metabolites

minimized

4.

completely

threshold on

due

culture

terpenoids

effects

microorganisms

processes

processes.

literature

be

accumulation as

few

metabolic

of

Substrate/Product such

by

commercial

volatile

Most v o l a t i l e s

difficult

substrate

3.

practiced

amounts

chemicals,

to

a

difficulties:

milligrams/liter

2.

leading

Low p r o d u c t inherently

volatiles than

microorganisms,

commercially

by

of

curiosity

events

many

technical

Routes

generation

academic

described

scaled-up

Production

of

water.

often Due

fermentor

to

of

by a

fermentation

water

Components

present their

soluble present

problems

volatility,

off-gasses.


for some

342 6.


Product Mixes. Recovered v o l a t i l e s some of which may sensory p r o p e r t i e s of the removed.


Economics of P r o d u c i n g

volatiles may be mixtures have d e l e t e r i o u s e f f e c t s on finished product and must

V o l a t i l e Chemicals

by

of the be

Fermentation

The u l t i m a t e c r i t e r i o n f o r the p r o d u c t i o n o f any s p e c i a l t y c h e m i c a l is economics. A l t h o u g h f e r m e n t a t i o n may appear t o be an a t t r a c t i v e r o u t e t o t h e p r o d u c t i o n o f v o l a t i l e s , s e v e r a l f a c t o r s can l i m i t t h e commer c i a l i z a t i o n of t h i s a p p r o a c h : 1. I n t r i n s i c a l l y low p r o d u c t y i e l d s . 2. R e l a t i v e l y low market volumes f o r p r o d u c t s which c r e a t e u n f a v o r a b l e economies of s c a l e . 3. High c a p i t a l c o s t s a s s o c i a t e d w i t h c r i t i c a l c o n t r o l & a s e p s i s requirements. IFF has been e v a l u a t i n g the p r o d u c t i o n of s e v e r a l f l a v o r mate r i a l s by f e r m e n t a t i o n . T a b l e 4 summarizes y i e l d , market p r o j e c t i o n , and c o s t s f o r two v o l a t i l e m a t e r i a l s , A & B. Based on a 5,000 L. f e r m e n t a t i o n s c a l e , t h e l o w e s t p r o j e c t e d c o s t of v o l a t i l e A is $800/kg. T h i s is due t o r e l a t i v e l y low y i e l d s (3g/L) and p r o j e c t i o n s f o r low volumes. A l t h o u g h t h e economics f o r v o l a t i l e Β are more f a v o r a b l e due t o h i g h y i e l d and g r e a t e r market demand, t h e y fall f a r s h o r t of the economics achieved f o r two other food a d d i t i v e s and f l a v o r i n g m a t e r i a l s , MSG and c i t r i c a c i d , which by c o n t r a s t , a r e o b t a i n e d at c o n s i d e r a b l y h i g h e r y i e l d s and e n j o y much greater use(60). A l t h o u g h P e n i c i l l i n G has y i e l d s comparable t o those of the volatiles, production volumes result in greater economies o f s c a l e and more f a v o r a b l e c o s t s . A l t h o u g h the economics f o r f e r m e n t a t i o n d e r i v e d v o l a t i l e s may appear t o be d i s c o u r a g i n g , c e r t a i n f a c t o r s may s t i l l make t h e e x e r c i s e a t t r a c t i v e in some c a s e s . These i n c l u d e : 1. Some fermentation-derived products may not be readily a v a i l a b l e from o t h e r b i o l o g i c a l o r c h e m i c a l s o u r c e s . 2. Some c h e m i c a l s d e r i v e d from f e r m e n t a t i o n may have h i g h f l a v o r o r odor impact w i t h s u f f i c i e n t l y low use l e v e l s t o j u s t i f y the cost. U l t i m a t e l y the f i n a l d e c i s i o n t o c o m m e r c i a l i z e w i l l be t h e r e s u l t of a t r a d e - o f f between economics, f u n c t i o n a l i t y , u n i q u e n e s s of odor and t a s t e , and consumer demands. A h i g h e r p r i c e can be c a r r i e d by a c h e m i c a l which has a unique e f f e c t on a f i n i s h e d f l a v o r or f r a g r a n c e . Large

S c a l e P r o d u c t i o n o f V o l a t i l e S u b s t a n c e s by

Fermentation

In s p i t e of u n f a v o r a b l e economics and t e c h n i c a l problems, s e v e r a l f l a v o r i n g m a t e r i a l s and i n t e r m e d i a t e s have been produced on a l a r g e s c a l e by f e r m e n t a t i o n . Fermentation d e r i v e d n a t u r a l c a r b o x y l i c a c i d s are important to t h e f l a v o r i n d u s t r y f o r use in d a i r y and sweet f l a v o r s and as sub s t r a t e s f o r the p r o d u c t i o n of n a t u r a l e s t e r s . A p r o c e s s has been developed f o r t h e p r o d u c t i o n o f b u t y r i c a c i d which u t i l i z e s the a n a e r o b i c c o n v e r s i o n o f d e x t r o s e t o b u t y r i c a c i d by the b a c t e r i u m



25. SCHARPFETAL.

343


Clostridium butyricum(62). Maximum y i e l d s o b t a i n e d were 1.2% in the medium r e p r e s e n t i n g a 40% sugar c o n v e r s i o n . B l u e cheese f l a v o r s have been p r e p a r e d v i a submerged c u l t u r e fermentations in a sterile m i l k - b a s e d medium u s i n g Pénicillium roqueforti(63). The f e r m e n t a t i o n s a r e c o n d u c t e d under p r e s s u r e w i t h low a e r a t i o n r a t e s w i t h o p t i m a l f l a v o r p r o d u c t i o n o c c u r r i n g from 2472 h o u r s . S i m i l a r l y , K o s i k o w s k i and J o l l y ( 6 4 ) p r e p a r e d b l u e cheese f l a v o r s from the f e r m e n t a t i o n of m i x t u r e s of whey, food f a t , salt and water by roqueforti. Dwivedi and K i n s e l l a ( 6 5 ) d e v e l o p e d a c o n t i n u o u s submerged f e r m e n t a t i o n o f P^ r o q u e f o r t i f o r p r o d u c t i o n of b l u e cheese f l a v o r . Among the amino a c i d s , L - g l u t a m i c a c i d can enhance o r improve the f l a v o r o f f o o d s . G l u t a m i c a c i d is p r o d u c e d v i a f e r m e n t a t i o n directly from sugar using organisms such as Corynebacterium glutamicum and B r e v i b a c t e r i u m f l a v u m . ( 6 6 ) . The s o - c a l l e d s t a r t e r d i s t i l l a t e s used by the d a i r y i n d u s t r y a r e now produced on a commercial s c a l e from l a c t i c a c i d c u l t u r e s . These d i s t i l l a t e s in which 70% of the s u b s t r a t e is converted to d i a c e t y l have been p a t e n t e d ( 6 7 ) and a r e used t o i m p a r t a b u t t e r y t a s t e to e d i b l e o i l s . They a r e m a n u f a c t u r e d by the steam d i s t i l l a t i o n of c u l t u r e s o f b a c t e r i a grown on a medium o f skim m i l k f o r t i fied w i t h 0.1% c i t r i c acid. Organisms used a r e Streptococcus lactis, S. c r e m o r i s , S. l a c t i s subsp. d i a c e t y l a c t i s , Leuconostoc c i t r o v o r u m and dextranicum. D i a c e t y l comprises 80-90% o f the f l a v o r compounds in the aqueous d i s t i l l a t e but is p r e s e n t at o n l y 10-100 ppm. Farbood and Willis(68) in a recent patent application disclosed a process f o r p r o d u c t i o n of optically active alphahydroxy decanoic acid ( gamma-decalactone) by growing Yarrowia l i p o l y t i c a on c a s t o r o i l as a s o l e s o u r c e of c a r b o n . T h i s is a good example o f a commercial a p p l i c a t i o n o f a v o l a t i l e c h e m i c a l produced by a m i c r o o r g a n i s m . Y i e l d s of up t o 6 grams p e r l i t e r c u l t u r e media were o b t a i n e d making t h i s a p r o m i s i n g i n d u s t r i a l f e r m e n t a t i o n .

Table 4.

Economics of Fermentation Products YIELD (gm/1)

PRODUCT

MARKET VOL. (kg/yr)

PRICE ($/kg)

Volatile A

3

200

800

Volatile Β

20

2,000

100

80-100

22,000

2

120-150

180,000,000

1-2

10-15

1,250,000

42

MSG

1

Citric Acid

1

Penicillin G

1

From Bartholomew & Reisman (61)


344



Conclusion The agricultural production of flavor and fragrance materials has several disadvantages, including variation in consistency and quali ty, and dependency on climatic, seasonal, geographic, and even political factors. The microbial production of flavor and fragrance materials may compliment or offer an alternative to traditional sources of these materials. Fermentation may be particularly suited to the production of unique, highly intense character impact components, i.e., substances that can potentiate the aroma and flavor of fruits, dairy and other flavors at low levels ( 100 ppm in the finished flavor). However, technical improvements in fermentation processes will have to be made to improve product costs and stimulate use. These include: 1. Better understanding of underlying microbial metabolism. 2. Increase in product yields. 3. Development of more efficient product recovery methods. 4. Search for a wider variety of microorganisms and low-cost substrates. The progress that is being made in applying fermentation tech niques to the production of volatile materials is encouraging. Several products are being actively pursued by industry and it is hoped new results will be available by the time the next symposium is held. Literature Cited 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13.

Sani, B.K., K. Das, and Ghose, T.K., Biotech. Letters 1982 4, 19-22 . Tokoro, Y., Oshima, K., Okii, Μ., Yamaguichi, Κ., Tanaka, K. and Kinoshita, S., Agr. Biol. Chem. 1970, 34, 1516. Phaff, H.I., Sci. Am. 1981, 345(3), 77-89. Collins, R.P., "The Production of Volatile Compounds by Filamentous Fungi" in Developments in Industrial Microbiology Series Vol. 20. Amer. Society of Industrial Microbiolgy 1979, p. 239. Collins, R.P. and Morgan, M.E., Phytopath. 1962, 52, 407. Margalith, P.Z., "Flavor Microbiology" Chas. C. Thomas, Springfield, Ill. 1981. Rose, A.H., In "Fermented Foods;" Rose, A.H. Ed.; Economic Microbiology Series Vol. 7, Academic Press London, 1982, p. 1. Law, B.A., Ibid. pp. 149-198. Ney, K.H., In "The Quality of Foods and Beverages" Charalambous, G., and Inglett, G., Academic Press, New York, 1981, 389. Ney, K.H., Wirotama, I. and W. Freytag, U.S. Pat. No. 3,922,365, 1977. Ney, K.H., Gordian 1973, 380. Adda, J., Roger, S. and Dumont, I., In "Flavor of Foods and Beverages;" Charalambous, G., and Inglett, G., Academic Press, New York, 1978, 65. Langsrud, T., Reinbold, G., and Hammond E., J. Dairy Sci. 1977, 60, 16.


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