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ABSTRACT
THE EFFECT OF PROCESS PARAMETERS ON THE

FLAVOR AND METHYL KETONE CONTENT OF
QUICK RIPENED BLUE CHEESE

BY
Sharon E. Albert

Quick ripened Blue cheese was modified during
processing is an effort to improve the original method
of Hedrick, Kondrup and Williamson (1968). It was felt
that more control over the fermentation was needed in
order to assure a better, more easily reproducible
product. In an attempt to improve the cheese, the
following processing variables were employed:

1) lowering the curing temperature

2) cooking the curd to 100° F

3) direct acidification with lactic acid

4) storage of the finished product at 40° F

for 2 weeks

5) use of a white mutant strain of Penicillium

 

rogueforti.

 

All samples were analyzed for methyl ketone content and
total fat. The C5 - C13 methyl ketones were isolated
and separated using column chromatography. The cheese

was also evaluated organoleptically.

.r Sharon E. Albert
«
m0 .

(9Q; The curing temperature was lowered to 50° F in an
effort to reduce contamination by competing micro-
organisms. Cheese ripened at this temperature was cured
for 11 days and salted on days 7, 8 and 9 or 1, 9 and
10. In addition, the curd was cooked during manufacture
to prevent shattering and facilitate drainage. The two
cheeses made by this method were high in methyl ketone
content and had excellent flavor. This procedure was
suggested as a satisfactory alternative method of
manufacture.

Quick ripened Blue cheese was made by direct acidi-
fication with lactic acid in an attempt to automate the
process and reduce labor costs. Cheese made by this
method did not develop typical Blue cheese flavor and
color. Using the processing conditions described, this
cheese was considered unsatisfactory.

Quick ripened cheese was also stored at 40° F for
two weeks before analysis. The stored cheese had a much
finer flavor and higher ketone content than the original.
Such brief storage may be one way of standardizing flavor

quality of various lots of cheese.

A white mutant strain of g. rogueforti was used in

 

an attempt to produce a Blue cheese flavored product
without the blue mold color. Cheese made with this

mutant strain had a typical, although mild flavor. It was
felt that the white cheese would be preferable for
applications where the blue-green color of the mold is

objectionable or offensive.

THE EFFECT OF PROCESS PARAMETERS ON THE
FLAVOR AND METHYL KETONE CONTENT OF
QUICK RIPENED BLUE CHEESE

BY
MW":
Sharon E. Albert

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE
Department of Food Science and Human Nutrition

1974

ACKNOWLEDGMENTS

The author wishes to express sincere appreciation
to Dr. C. M. Stine for his counsel and guidance
throughout the course of this graduate program.

Appreciation and thanks are extended to members of
the guidance committee: Prof. A. L. Rippen, Department
of Food Science and Human Nutrition and Dr. W. Fields,
Department of Botany, for their advice and effort in
reading this manuscript. ,

Financial support provided by the Department of
Food Science and Human Nutrition and Dairy Research
Incorporated is gratefully acknowledged.

The author also wishes to express appreciation to
family and friends for their aid and encouragement

during this study.

ii

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O O C O O O O 0

REVIEW OF LITERATURE O O O O O O O O 0

Flavor Components of Blue Cheese and

Origin . . . . . . . . . . . . . .
Free Fatty Acids
Lipase Activity . . . . . . .
Volatile Acidity
Methyl Ketones . . . . . . . .
Other Carbonyls . . . . . . .
Proteolysis . .

Effect of Processing Variables on Flavor

Temperature . . . . . . . . .
salt 0 O 0 O O I O O O O O O O
ch-dity O O O O O O O O O O 0

Variations in the Manufacture of Blue Ch

EXPERIMENTAL METHODS . . . . . . . . .

Quantitation of Methyl Ketones . .
Solvent Purification . . . . .
Isolation of Methyl Ketones .

Fat Extraction from Cheese Sample

Formation of Dinitrophenylhydrazones

Removal of Fat from the DNPH .
Removal of Ketoglyceride DNPH

Fractionation of Monocarbonyl Derivatives

Infipfi)

Separation of Methyl Ketone DNPH into

Individual Chain Lengths . .

Determination of Individual Methyl Ketone

Concentration . . . . . .

Thin-Layer Chromatography of Methyl

Ketones . . . . . . . . . .

Determination of Percentage Recovery of th

Individual Methyl Ketones .

Production of Quick Ripened Blue Cheese

Preparation of While Mold Powder .

iii

eese

Page

Page
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 34

Sensory Evaluation of Quick Ripened Blue

Cheese . . . . . . . . . . . . . . . . . . . . 34
Quantitation of Methyl Ketones . . . . . . . . . 34
Quantitation of Free Fatty Acids . . . . . . . . 38
Methyl Ketone Content of a Commercial Blue

Cheese Sample . . . . . . . . . . . . . . . 41
Quick Ripened Blue Cheese Made by Direct
Acidification with Lactic Acid . . . . . . . . 42

Quick Ripened Blue Cheese Made Using a Mutant

White Mold . . . . . . . . . . . . . . 45
Quick Ripened Blue Cheese Made by the Original

Method and Stored at 40° F for 2 Weeks . . . . 47
Suggested Methods of Manufacture for Quick

Ripened Blue Cheese . . . . . . . . . . . . . 48

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 52
REFERENCES 0 C O I O O O O O O O O O O O O O O O C O 55

APPENDIX 0 O O O O O O O O O O O O O O O O 0 O O O O 63

iv

TABLE

LIST OF TABLES

Variations of Quick Ripened Blue Cheese
Produced . . . . . . . . . . . . . . . . . .

Sensory Evaluation of Quick Ripened Blue
Cheese 0 O O I I O O O O O O O O O O O O O 0

Concentration of Methyl Ketones in Fat
Extracted from Blue Cheese (micromoles/lO 9
Of extracted Fat) 0 O 0 O O 0 O O O O O O 0

Concentration of Free Fatty Acids in Quick
Ripened Blue Cheese (mg/Kg cheese) . . . . .

Fat Content of Blue Cheese . . . . . . . . .

Page

33

36

37

39

63

INTRODUCTION

In recent years, cheese and cheese products have
increased tremendously in popularity. The availability of
salad dressings, dips and other products utilizing Blue and
other cheeses has contributed to the increase in per capita
consumption of natural cheese. As one means of improving
production efficiency and to lower the cost of production,
Hedrick, Kondrup and Williamson (1968) devised a method of
quick ripening Blue cheese. This cheese is ripened in a
loose curd form at 62° F in an atmosphere of at least 95%
relative humidity. Less than two weeks are required for
full ripening, and significant reductions in labor costs
are realized in the process.

This project was undertaken to investigate the
possibility of modifying and improving the quick ripening
process since the flavor of quick ripened cheese was
observed by Blakely to vary and to be atypical of a com-
pletely desirable Blue cheese. Several modifications of
the quick ripening procedure were developed and this thesis
is concerned with the analysis and evaluation of cheeses

made by these procedures.

LITERATURE REVIEW

Cheese flavor is a complex phenomenon resulting from
several factors. These include the treatment of the cheese
milk, the organisms used, and the ripening conditions.
Much work has been done in the past to gain a better
understanding of the flavor of Blue mold cheeses. The
complexity of the system is evidenced by the work of Day
and Anderson (1965), where approximately 100 flavor com-
pounds were identified in the volatile fraction of Blue
cheese by gas chromatography and mass spectrometry. The
major components were methyl ketones, secondary alcohols
and esters of aliphatic acids.

The ripening products formed in cheese have been
classified into two types by Harper and Kristoffersen
(1956). Primary products (free amino acids, free fatty
acids and lactic acids) are formed by lipolysis, proteo-
lysis and glycolysis. Secondary and tertiary degradation
products are formed by the enzymatic breakdown of the
primary products. These compounds are B-keto acids,
aldehydes and ketones, acetylmethylcarbinol and diacetyl,
formed by transamination, deamination, decarboxylation and

beta oxidation reactions.

Flavor Components of Blue Cheese
and Their Origin

 

 

Free Fatty Acids

 

The degradation of milk fat forms a major source of
flavor compounds in Blue cheese. In 1914 Currie reported

that Penicillium roqueforti produced a lipase which causes

 

hydrolysis of milk triglycerides. This worker found an
increase in volatile acids during ripening of Roquefort
cheese. He attributed the "peppery" or burning flavor of
the cheese to the presence of caproic, caprylic and capric
acids. Caproic, valeric and butyric acids were found in
blue mold cheeses by Thomasow (1947). The "pungent,
peppery flavor" of Blue cheese was attributed to free fatty
acids and their oxidation products by Morris, Jezeski,
Combs and Kuramoto (1963). Harper (1959) claimed that
those fatty acids of medium to long chain length are
predominant and that free amino acids also contribute to
the flavor of Blue cheese.

Free fatty acids are important in the flavor balance
of cheese. Parmalee and Nelson (1949) observed that when
fatty acids were present in excessive amounts the cheese
displayed "an undesirable rawness of flavor", possibly due
to a change in the development of flavor. When butyric,
caproic, caprylic, capric and lauric acids were added to
Blue cheese, the resulting cheese showed a better flavor
than a control cheese. However, a "smoothness and full-

ness" of flavor was lacking. Anderson (1966) has concluded

that the overall flavor of Blue cheese does not depend on
the total volatiles present, but on the relative amounts
of the individual constituents.

Of the fatty acids present in Blue cheese, many may
still be unknown. Magidman, Herb, Barford and Reimen-
schneider (1962) conducted a study of the free fatty acids
in cows milk. At least six fatty acids were identified,
some for the first time. Utilizing gas chromatography,
Day and Anderson (1965) identified the major free fatty

acids (CZ-C ) in domestic Blue cheese. No formic,

18:3
propionic or isovaleric acid was evident in any of the
cheese samples analyzed. These workers postulate that from
75 to 85% of the acid present in cheese may exist in the
salt form. The salts of long chain acids are soaps and may
give a characteristic flavor to Blue cheese.

Blakely (1970) reported finding the same mole percent

free fatty acids in quick-ripened blue cheese as reported

in a commercial sample analyzed by Day and Anderson (1965).

Lipase Activity

 

The enzymes involved in lipid hydrolysis in cheese
originate from two sources:

1. Milk lipases

2. Microbial lipases (Stadhouders and Mulder, 1957)

The presence of a multiple lipase system in milk has
been demonstrated several times. Albrecht and Jaynes

(1955) indicated that two lipase systems exist in raw skim

milk with optima at pH 5.4 and 6.3. Two different lipase
systems were also observed by Tarassuk and Frankel (1957).
One lipase was irreversibly absorbed on the fat globule
membrane when milk was cooled, the other was associated
with casein. Harper and Gaffney (1970) concluded that
lipase is an absorbed entity on various caseins. These
workers also presented evidence for a multicomponent lipase
system in milk.

When mixed triglycerides are hydrolyzed, there is an
obvious preference for the fats containing short chain
acids (Jensen, 1964). Milk lipase preferentially releases
fatty acids from the primary positions of a triglyceride.
Since butyrate appears to be predominantly a primary ester,
an apparent preferential release of butyrate results.
Harwalker and Calbert (1961) reported that lauric and
higher acids were the major acids released at all stages
of lipolysis, but as lipolysis progressed the ratio of
these acids to butyric decreased. Very little change was
observed in the mole percentages of the other acid
fractions.

The microbial lipase present in Blue cheese originates

mainly from Penicillium roqueforti. Spores of this mold

 

are added to the cheese milk or to the curd. Morris and
Jezeski (1953) studied the lipolytic activity of a

mycelial preparation of g. roqueforti. Optimum activity

 

was obtained between pH 6.0 and 6.7 on tributyrin and pH

7.0 to 7.2 on butterfat. Reports by Inamura (1963a) and

Inamura and Kataoka (1963b, 1966) indicate two types of
lipase with optima at pH 6.5 and 7.5. Maximum lipolytic
activity on a 5% butteroil substrate was obtained at pH

8.0 and 37° C by Eitenmiller, Vakil and Shahani (1970).
Lipase activity was stimulated by MnCl2 and MgClz. The
enzyme hydrolyzed tributyrin, tricaprylin, tripropionin

and triolein in decreasing order. Wilcox, Nelson and Wood
(1955) found butyric, caproic anc caprylic acids selectively
released in reaction mixtures of butterfat and P.

roqueforti lipase.

 

Some lipolytic activity in Blue cheese may be due to
starter culture activity. Willart and Sjostrom (1959)
claimed starter organisms did not contribute to lipolysis.
However, Fryer and Reiter (1967) found a weak lipolytic
activity in seven out of fifty-six tested strains of

Streptococcus lactis.

 

Volatile Acidity

 

There may be a relationship between flavor and
volatile acidity in Blue cheese. Parmalee and Nelson
(1949) found that cheese scoring high in flavor generally
had a volatile acidity of 30 to 50 ml of 0.1 N acid per
100 g of cheese. However, no correlation was found between
fat acidities and flavor scores. Lane and Hammer (1938)
found a relationship between volatile acidities, fat
acidities and flavor scores. Cheese with high volatile

acidity and high fat acidity had more characteristic

flavor than low acid cheeses. Also, cheeses made from
homogenized milk had two to four times the volatile
acidity of cheeses made from non-homogenized milk.

The acidity of Blue cheese was studied by Coulter,
Combs and George (1938b). A minimum pH of 4.7 was
attained within 24 hours after manufacture. The pH
increased gradually to 6.5 (in approximately three months)
and then decreased to 5.7 by the ninth month of ripening.
The marked rise in pH from about 2 weeks to 10 weeks was
due to continuing proteolysis (Currie, 1914; Morris, Combs
and Coulter, 1951). Morris (1963) noted a tendency for
the rate of rise of pH to decrease as the pH approached
6.5. The lipase systems in Blue cheese were active in

the pH range occurring throughout curing.

MethylfiKetones

 

Starkle (1924) obtained heptanone-Z and nonanone-Z
by distillation of Roquefort cheese. He postulated that
these methyl ketones were formed by the metabolism of

fats by Penicillium roqueforti and that they were responsi-

 

ble for the flavor and aroma of the cheese. Later

workers (Patton, 1950; Morgan and Anderson, 1956)
identified ketones in Blue cheese using steam distillation
and chromatography. Pentanone-Z, heptanone-Z and
nonanone-2 were isolated from samples with aromas con-

sidered to be typical of Blue cheese.

The work of Coffman, Smith and Andrews (1960) showed
the major neutral components of Blue cheese to be
heptanone-Z, nonanone-Z, undecanone-Z, heptanol-Z,
nonanol-2 and an unidentified ketonic constituent.
Coffman also reported the major acids as butanoic,
pentanoic, hexanoic, octanoic and decanoic. These flavor
constitutents were isolated by vacuum distillation and
identified by infrared spectroscopy and chromatography.

Nawar and Fagerson (1962) reported finding the
following flavor constituents in Roquefort cheese:
pentanone-Z, heptanone-Z, nonanone-2, acetaldehyde,
isobutyraldehyde, butanone-Z, propanol—Z, diacetyl,
hexanone-Z, pentanol-Z, octanone-Z and heptanol-Z. Niki,
Yoshioka, and Ahiko (1966) also identified acetaldehyde,
acetone, butanone-Z, pentanone-Z, heptanone-Z and
undecanone-2 in Blue mold cheeses.

It is generally agreed that heptanone-Z is the major
ketone present in Blue cheese (Anderson, 1966; Schwartz
and Parks, 1963). Schwartz and Parks found only small
amounts of methyl ketones with chain lengths of more than
nine carbon atoms. The formations of methyl ketones did
not appear to be in a definite ratio or related to the age
of a cheese. Blakely (1970) found nonanone-2 to be the
major methyl ketone in samples of quick-ripened blue cheese.

Stokoe (1928) believed that the formation of methyl
ketones was the second step in an "abnormal" oxidation of

fatty acids. From work with Penicillium palitans and

 

Penicillium glaucum using coconut oil as a substrate, Stokoe
concluded that fatty acids had a "narcotic" effect which
caused a decarboxylation reaction. Starkle (1924)

attempted to determine the origin of methyl ketones

distilled from Roquefort cheese. Aspergillus niger, A.

 

fumigatus and P. roqueforti when grown for several weeks in

 

 

pure culture, with individual fatty acids as the carbon
source, produced methyl ketones of one less carbon atom
than the fatty acids. A mechanism advanced for the
formation of these ketones was the oxidation of the fatty
acid to the beta-keto acid followed by decarboxylation of
the beta-keto acid.

Using C14

labeled octanoate, Gehrig and Knight (1963)
confirmed the original assumption by early workers that
B-keto acids are formed from free fatty acids and then
decarboxylated to form methyl ketones. The 2-heptanone

synthesized was unlabelled and 14CO2 was produced.

Lawrence (1966) found that 2-heptanone formed from 2-14C-
octanoic acid was radioactive and that some radioactive
carbon dioxide was also present. Therefore, ketone
formation and beta-oxidation proceed simultaneously. The
2-C moiety (presumably acetate) formed by beta-oxidation
is further oxidized in the tricarboxylic acid cycle. The

pathway of fatty acid oxidation and methyl ketone formation

may be outlined by the following scheme (Hawke, 1966).

10

Pathway of Fatty_Acid Oxidation and
Methyl Ketone Formation

 

+2H

 

 

 

 

RCHZCHZCOSCOA ~** RCH=CHCOSCOA
-2H J [“20
+2H __
RCOCHZCOSCOA RCHOHCH 2COSCoA
I -2H
Deacylase CoASH
RCOCHZCOOH + COASH RCOSCOA + CH3COSCOA
Decarboxylase Further formation of CO2 + H20
CH COSCoA (via TCA
RCOCH3 + C02 3 cycle)

This mechanism provides for the formation of only methyl
ketones of one less carbon atom than the fatty acids used
as substrate.

Hawke (1966) outlined the following four steps in the
formation and subsequent metabolism of methyl ketones in
Blue cheese:

1) lipolysis of milk triglycerides to form free
fatty acids

2) formation of B-keto acids by oxidation of free
fatty acids

3) decarboxylation of B-keto acids to produce
methyl ketones

4) reduction of ketones to secondary alcohols.

11

In work with spores of P. roqueforti, Gehrig and

 

Knight (1958) claimed that spores of mycelium, formed
methyl ketones. Sporulation was necessary for the
formation of ketones and the amount of Sporulation was
directly related to ketone formation. Germinated spores
did not have the ability to form ketones. A direct
relationship was also found between the amount of oxygen
used by the spores and ketone production. No ketone was
produced under anaerobic conditions. Girolami and Knight
(1955) have shown that the oxygen uptake of spores is
related to fatty acid chain length. In later work (1963)
Gehrig and Knight stated that the spore is the active site
for the metabolism of fatty acids to ketones of one less
carbon. The ability of spores to form methyl ketones
disappeared rapidly as germination progressed. There are
conflicting reports on the ability of mycelium to produce
ketones. Contrary to the conclusions of Gehrig and Knight,
Lawrence and Hawke (1968) observed the oxidation of low
concentrations of fatty acids over a wide range of pH

values by the mycelium of P. roqueforti.

 

Lawrence (1966) identified methyl ketones produced by

washed spores of g. roqueforti in a medium containing free

 

fatty acids. The results showed that the higher the
concentration of fatty acid, the longer the time required
before the corresponding methyl ketone could be detected.
The Optimum acidity for ketone formation was between pH

5.5 and 7.0, depending on the concentration of the fatty

12

acid being oxidized. The maximum formation of heptanone-2
occurred at 27° C. Lawrence also observed that the ability
of the spores to produce ketones decreased with age. A

lag phase preceded the oxidation, which was lengthened with
decreasing spore concentration and increased fatty acid
concentration.

Girolami and Knight (1955) obtained results showing
that the restriction or stimulation of the formation of
ketones is dependent on the amount of fatty acid present.
According to Anderson (1966) the amount of methyl ketone
produced is not related directly to the amount of
corresponding fatty acid available. The conversion of
fatty acids seemed to be related to the chain length.
Octanoic acid is converted to methyl ketone most rapidly,
while butanoic and dodecanoic are the least rapid.

Dartey and Kinsella (1971) have studied the formation
of methyl ketones in Blue cheese during ripening. The
amount of the individual ketones fluctuated over the
ripening period, with the exception of heptanone-2 and
nonanone-Z.

They were observed to be the two major methyl ketones,
accounting for between 50 and 75% of the total. These
workers believe these to be the two major flavor components
of Blue cheese. During the initial days of ripening the
methyl ketone content was extremely low, but increased to
120.4 umol per 10 g of dry cheese on day 70. After day 70

there was a slow decline. No correlation was found between

13

the concentrations of individual methyl ketones. This may
be due to interconversion of methyl ketones and secondary
alcohols. Schwartz and Parks (1963) stated that the age
of a cheese does not necessarily reflect its ketone
content. Niki et 31 (1966) used a highly lipolytic

variety of g. roqueforti for the production of Blue cheese.

 

They noted that the ketone content increased rapidly after
7 weeks and reached a maximum at 21 weeks, when ketone
flavor became evident.

Dartey and Kinsella (1973) studied the oxidation of

palmitic acid by spores of P. roqueforti. Carbon dioxide

 

was formed, along with carbonyl compounds, including a
homologous series of C3 to C15 methyl ketones. The
Optimum yield of carbonyl compounds was obtained with a

spore concentration of 6.3 x 108

spores/m1 in the presence
of D-glucose, at pH 6.5 and 30° C.

The inhibition of respiration and formation of methyl
ketones by free fatty acids has been studied by several

workers. Girolami and Knight (1955) stated that fatty

acids inhibited a culture of P. roqueforti. The acids

 

became increasingly toxic with greater chain length.
Later, Gehrig and Knight (1963) found that the amount of
inhibition depends on the pH of the medium, the concen-
tration of free fatty acid and the chain length.

Dwivedi and Kinsella (1974) cultured mycelium of

P. roqueforti in lipolyzed milk fat. Their results showed

 

that low concentrations of fatty acids were metabolized

14

after a 6 hour lag period and higher concentrations
required a longer (12 hour) lag, but greater total
carbonyls were produced after 48 hours. They concluded
that the higher concentration of fatty acids have only a
temporary effect. The fatty acid concentration also
influenced the relative proportion of methyl ketones
produced. Pentanone-Z and undecanone-Z were dependent on
the concentration of fatty acids, appearing only when the

C8 and C10 fatty acids decreased.

Other Carbonyls

 

Neutral carbonyls have been identified as flavor
components of Blue cheese and subsequently studied by many
researchers. Stokoe (1928) identified secondary alcohols
among the products resulting from the action of g.

roqueforti on coconut oil. He postulated that these com-

 

pounds were the result of decarboxylation of B-hydroxy
fatty acids, but considered the possibility that they were
formed by the reduction of ketones. Pentanol-Z, heptanol-Z
and nonanol-Z were isolated by Jackson and Hussong (1958).
They claimed that these neutral volatiles were probably
formed by a reduction of the corresponding methyl ketones,
because they did not appear until considerable amounts of
ketones were produced. Day and Anderson (1965) indicated
that under anaerobic conditions this reaction is
reversible and could be responsible for the nonuniform

distribution of individual ketones. These workers found

15

that spores of P. roqueforti incubated in phosphate buffer

 

interconverted 2-pentanone and 2-pentanol. Mycelia
suspended in media which prevented Sporulation likewise
caused this type of interconversion. Somes yeasts
associated with normal Blue cheese flora may also possess
the ability to reduce methyl ketones to secondary alcohols.
This was postulated by Anderson (1966) who also observed
that the secondary alcohols appear to be present in Blue
cheese in the same ratio as methyl ketones.

Acetaldehyde has been identified in Blue cheese
(Bassett and Harper, 1958; Morgan and Anderson, 1956;
Nawar and Ferguson, 1962). PrOpionaldehyde and isobutyral-
dehyde have also been detected by Nawar and Ferguson. Day
and Anderson (1965) isolated furfural from Blue cheese.

Other compounds such as ethanol, acetylmethyl carbinol
and diacetyl were documented in Blue cheese by Bavisotto,
Rock and Lesniewski (1960), and Bassett and Harper (1958).
Day and Anderson (1965) identified methanol, ethanol,
n-pentanol, 2- and 3-methyl butanol and 2-pheny1ethanol in
Blue cheese. These workers also identified the methyl
esters of the even numbered carbon fatty acids C2 through

C The ethyl esters of the even numbered carbon fatty

12°
acids acetic through decanoic were also present.

Proteolysis

 

The protein degradation of Blue cheese varies with the

strain of P. roqueforti, the pH, and the temperature.

 

16

Niki 9i: _a_l (1966) detected an intra-cellular and an extra-
cellular protease, both having an optimum pH of 5.5. The
extra-cellular protease had a narrow pH range and contri-
buted to casein break-down only during early stages of
ripening. The intra-cellular protease, with a pH range of
5.5 to 7.0 contributed to proteolysis during the entire
ripening period. The optimum pH for proteolysis was
identified as 5.5 to 6.0 by Niskikawa (1958). An optimum
temperature of 40° C for a 0.6% casein medium was found.
Salt at a level of 2% slightly stimulated proteolysis,
while 4% caused a decrease. Inamura (1960b) found maximum
proteolysis at 42° C and pH 5.5. Starter organisms were
found to have little proteolytic activity but did stimulate

the growth of P. roqueforti.

 

High proteolytic strains of P. roqueforti showed low

 

lipase activity and vice versa, according to the work of
Niki et_al (1966). Salvadori and Salvadori (1967) also
illustrated this, as indicated by lipolysis of palmitic,
oleic, stearic, azelaic and pelargonic acids. Sato, Honda,
Yamada, Takada and Kawanami (1966) and Morris, Jezeski,
Combs and Kuramato (1955) studied the tyrosine content of
Blue cheeses. The work of Sato 33 al showed the tyrosine
content to be higher in well-ripened cheese, the content
increasing slowly during ripening. Morris et al found no
significant difference in tyrosine levels in cheeses made
from raw milk, homogenized milk, and pasteurized milk.

Modler, Brunner and Stine (1974) compared the

17

extra-cellular proteolytic activity of three strains of P.

roqueforti and isolated the enzyme from the most proteo-

 

lytic strain. The extracellular protease had a pH
optimum of 3.0 and 5.5 for bovine serum albumin and casein,

and a temperature optimum of 46° C.

Effect of Processing Variables on Flavor

 

Temperature

 

In Roquefort, France, the "cave" temperature is main-
tained at 43-46° F with a high humidity. For U.S.
manufactured Blue cheese, temperatures of 48-55° F with
90-95% relative humidity have been reported.

Peters and Nelson (1961) studied the quality of cheese
produced at different ripening temperatures. The highest
quality cheese was produced by ripening for four weeks at
50° F and then four weeks at 76° F. Cheese ripened for
eight weeks at either of these temperatures was of much
lower quality. Morris (1969) used ripening temperatures of
37 to 60° F for Blue cheese. His results showed that
higher temperatures are conducive to faster breakdown of
fat and protein but the body, color and flavor of the
cheese are of lower quality. Morris concluded that the
best combination of ripening temperatures is high tempera-
ture initially and lower temperature in the last stages of

ripening.

18

.5212

Most organisms cannot survive salt concentrations
greater than about 6 percent, and essentially none survive
a concentration of 10 percent (Morris, 1969). P.

roqueforti can grow at salt concentrations up to about 16

 

percent brine. A 10 percent brine is the concentration
maintained in Blue-veined cheeses. Therefore, a salt
concentration of approximately four percent in a cheese of
40-44 percent moisture will provide the proper brine
concentration to discourage contaminating molds, yeast
and bacteria from growing.

The effect of salt content on flavor compounds in
Blue cheese was studied by Willart and Sjostrom (1959) and
Poznanski, Jaworski and D'Obyrn (1966). Willart and
Sjostrom used l.5-4.5% NaCl in making cheese. The
greatest difference in the amount of fatty acids produced
was detected at the lower salt concentrations. The higher
the salt content used, the less higher chain length acids
were observed. However, the ratio of butyric to higher
acids remained the same regardless of salt concentration.
Poznanski gt 21 found that from salt contents of 3.0 to
3.9% the fat acidity increased with increasing salt
content. Morris and Jezeski (1953) observed that the

lipase activity of P. roqueforti decreased with increases

 

in salt concentrations.
This was also observed by Inamura (1963). The lipo-

lytic activity of P. roqueforti was decreased by greater

 

19

than 3% NaCl. The consumption of volatile fatty acids was
decreased as salt concentrations greater than 4%.
Poznanski, Jaworski and D'Obyrn (1966) reported that
acid production in Roquefort cheese diminished as the salt
content increased from 3.0 to 3.9%. It was observed by
Nelson (1970) that carbonyl production by spores of P.

roqueforti is enhanced by the addition of salt. However,

 

Dwivedi and Kinsella (1974) claimed that salt caused a
prolonged lag phase before carbonyl production by mycelium.
The relative proportion of the major methyl ketones was
not significantly different.

Lane and Hammer (1938) found that that delayed
salting to hasten mold develOpment was generally unsatis-
factory. A yellow color develoPed in the cheese which
increased with the number of days between manufacture and
salting.

Salt can also inhibit milk lipase in raw homogenized
milk and cream (Gould, 1941). Levels of 5 to 8 percent
salt were sufficient to inhibit lipolysis almost

completely.

Acidity

High acidity is required for good mold growth in
Blue-veined cheeses. Thom and Matheson (1914) and
Matheson (1921) recommended that milk be brought up to an
acidity of .23% before the addition of rennet. This was

claimed to prevent off flavor development and soft curd.

20

According to Coulter et al (1938a) the pH of Blue
cheese is influenced by the acidity of the cheese at
setting and the acidity of the whey at dipping. If the
acidity was too high during manufacture, the cheese
developed an acidy flavor, tough curd and crumbly body.
Low acid cheese has soft curd and a tendency toward off
flavors. In another paper (1938b) Coulter stated that
experimental Blue cheese reached a maximum acidity of pH
4.7 within one day after manufacture. The lowest acidity,
pH 6.5, was reached after about three months. Nesbitt
(1953) obtained results showing the maximum acidity to be
pH 5.6 after eight weeks of ripening.

Morris (1963) noted a tendency for the rate of rise
of pH to decrease as the pH approached 6.5. The lipase
systems in Blue cheese were active in the pH range
occurring throughout curing.

The oxidation of free fatty acids to methyl ketones by

g. roqueforti has also been found to be pH dependent.

 

Lawrence and Hawke (1968) studied this conversion by
mycelium and found the optimum pH to be dependent on the
concentration of the acid, varying with each fatty acid.
Mycelium grown at high pH (6.8) metabolized fatty acids
faster than mycelium grown at pH 4.0. Similar conclusions
were reached by Dwivedi and Kinsella (1974) but the effect
of pH was only temporary. Also, as long as the pH was

kept between 6 and 7 there was no significant difference in

carbonyl production. Gehrig and Knight (1963) found that

21

the maximum conversion of octanoate to heptanone-Z at 25-
30° C occurred at approximately pH 7. The maximum
oxidation of sodium palmitate into carbonyl compounds was
found to occur at pH 6.5, using a spore concentration of
approximately 6.3 x 108/m1 at 30° C (Dartey and Kinsella,
1973).

Variations in the Manufacture of Blue Cheese

Much work has been done to modify the production of
Blue cheese and Blue cheese flavorings. Knight, Mohr and

Frazier (1950) used a mutant white strain of P. roqueforti

 

to produce Blue cheese without a green-mold color. The
mutant was produced by the irradiation of spores. This
type of cheese was developed for use in the manufacture of
Blue cheese flavored spreads and dips where lack of color
is desirable. The white mold showed no significant changes
in lipolytic and proteolytic activity, but the cheese had

a milder flavor and softer, creamier body than traditional
Blue cheese.

A Blue cheese flavored salad dressing was developed by
Patton (1951). The volatile flavor components were
extracted with salad oil from the fat of melted cheese.
Other uses for the extract were suggested, including sauces,
soups, dips and spreads. Patton also prOposed the use of
a synthetic cheese flavor consisting of 1 part methyl-n-
amyl ketone to 20 parts butyric acid as a fortifying agent

for natural cheese flavor.

22

Cort and Riggs (1967) have developed a method for
producing Roquefort and Camembert type cheeses by adding

enzymes extracted from P, roqueforti and P. camemberti

 

 

during the manufacture of the cheese.
Nelson (1970) developed a 24-72 hour submerged
fermentation to produce Blue cheese flavor of a high

intensity. P. roqueforti was cultured in a sterile milk

 

base medium. The ketone profile of the flavor, as
identified by gas liquid chromatography, is similar to that
of Blue cheese.

Research has been done on direct acidification of
cheese curd, without the use of a starter culture.
Shehata and Olson (1967) developed a direct acidification
method using hydrochloric acid or lactic acid. Raw milk
at 4.4° C was acidified to pH 5.9, 5.6, 5.0, 4.85, or 4.7,
and then heated and coagulated with rennet. Curd adjusted
to pH 5.6 produced the best curd, as judged by the ease of
cutting and amount of shattering. Hydrochloric acid was
more desirable, because the lactic acid curd was too soft,
too high in moisture and difficult to handle during
draining. Singh and Kristofferson (1972) produced a cheese
flavor using a slurry prepared from directly acidified
lactic acid curd. The slurry was inoculated with 5% lactic
culture supplemented with glutathione, cobalt, riboflavin
and diacetyl. The curd, which was used for the study of
off flavor development, had physical characteristics

comparable to cheese made by conventional methods, but

23

failed to develop characteristic cheese flavor during
storage. Chanet (1972) patented a procedure for the
acidification of soft cheeses by direct acidification with
lactic acid. Whey from the coagulation stage was acidified
and heated to 100° C to precipitate proteins and mineral
salts, destroy acidifying microorganisms and inactive
enzymes. The treated whey was in amounts sufficient to
achieve the required pH.

Hedrick, Kondrup and Williamson (1968) developed a
method of quick-ripening Blue cheese which involves
ripening in a loose curd form at 62° F and greater than 95%
relative humidity for ten days. This method is more
economical than the production of conventional cheese. It
is also faster and minimizes the formation of slime.

Slime growth is reduced by the rapid mold growth. Moisture
control is important and salting is controlled by the
degree of ripening. According to Kondrup and Hedrick
(1963) judges comparing quick-ripened Blue cheese to con-
ventional Blue cheese determined that the body and flavor
of quick-ripened cheese was equal or superior to conven-
tional cheese. A modification of this method was used by
Blakely (1970). Coconut oil was used to replace milk fat.
High free fatty acid and methyl ketone contents were
evident in the flavor of the cheese, but the flavor was

considered to be atypical.

24

Lundstedt and Yau-Yee Lo (1973) have developed a
method to prepare a Blue cheese type product from soy bean
milk fortified with non-fat milk solids. This cheese also

ripens in about two weeks.

EXPERIMENTAL METHODS

Quantitation of Methyl Ketones

 

Solvent Purification

 

Benzene: Carbonyl free benzene was prepared by
refluxing reagent grade benzene with 1 gm of 2,4-dinitro-
phenylhydrazine (2,4-DNP) per 500 ml for 1-2 hours and
redistilling.

Chloroform: Refluxed over KOH for 30 minutes and

 

redistilled.

Nitromethane: Redistilled over boric acid and stored

 

in a brown bottle.

Acetonitrile: Distilled and 81-82° C fraction

 

collected.
Methanol: Refluxed over KOH for 30 minutes and
redistilled.

Hexane (Carbonyl free): High purity hexane was

 

redistilled and treated for the removal of carbonyls by

the methods of Hornstein and Crowe (1962) and Schwartz and
Parks (1961). A column was prepared using 40 m1 of concen-
trated sulfuric acid blended with 75 gm of Celite 545
(Johns Manville). A small amount of anhydrous sodium
sulfate was placed at each end of the column to serve as a
dessicant. The eluate from this column was passed over a

25

26

column of Celite, analytical filter aid (Johns Manville)
impregnated with DNP-hydrazine, phosphoric acid and water.
The hydrazones were removed by slurrying with Sea Sorb 43
(Iodine Number 80; Fisher Scientific Company, Pittsburgh)

and filtering as suggested by Blakely (1970).

Isolation of Methyl Ketones

 

The quantitative procedure for the isolation of methyl
ketones was similar to that described for fats and oils
(Schwartz, Haller and Keeney, 1963) and adapted to the
analysis of Blue cheese by Anderson (1966).

Fat Extraction from Cheese Sample: Fifteen grams of

 

Celite 545, dried for 24 hr at 150° C, and 10 g of cheese
were thoroughly ground together with a mortar and pestle.
The mixture was packed into a 25 x 500 mm chromatographic
column, fitted with a fritted glass filter. The fat was
extracted by passing 300 m1 of carbonyl free hexane through
the column and collecting the eluate. To determine the
yield of fat, a sample of the same cheese was extracted as
above and the eluate collected in a 300 m1 Erlenmeyer flask.
The hexane was evaporated on a steam bath under a stream of
nitrogen. The fat was quantitatively transferred with
ethyl ether to a pre-weighed Mojonnier fat dish. The
solvent was evaporated and the dish re-weighed.

Formation of Dinitrophenylhydrazones (DNPH): The

 

hexane-fat extract was passed through a reaction column of

Celite, analytical grade, impregnated with 2,4 DNP,

27

phosPhoric acid and water, to convert the monocarbonyls
present in the fat extract into DNPH (Schwartz, et 31,
1963). The column was first flushed with hexane until the
effluent had the same spectral properties as pure hexane
passed through a blank column. The sample was passed
through, and the column was again flushed with hexane until
the effluent had the same spectral properties as the hexane
which had been passed through the column prior to sample
addition. The hexane was then evaporated on a steam bath
under a stream of nitrogen.

Removal of Fat from the DNP-hydrazones: The lipid
material was separated from the DNPH by the procedure of
Schwartz £3 31 (1963) as modified by Anderson (1966).
Fourteen grams of Sea Sorb and 28 g Celite 545 (dried 24
hrs at 150° C) were slurried in hexane and poured into a
2.8 cm ID chromatographic column equipped with a fritted
glass filter and a 500 ml solvent reservoir. The column
was packed using 3-5 psi nitrogen pressure and the fat-
hydrazone mixture was dissolved in hexane and applied to
the column. The fat was flushed from the column by the
successive additions of 200 m1 hexane, 100 ml hexane-
benzene mixture (1:1, v/v) and 200 ml benzene. The
hydrazones were eluted with 175 m1 of a chloroform
nitromethane mixture (3:1, v/v). The solvent was removed
from the sample by evaporation on a steam bath under a

stream of nitrogen.

28

Removal of Ketoglyceride DNPH: A column of weak
alumina (Schwartz and Parts, 1961) was used to remove DNPH
of ketoglycerides from the monocarbonyl derivatives. The
sample from the preceding step was dissolved in hexane and
applied to a 7.5 9 column of partially deactivated alumina.
The monocarbonyl fraction was eluted with 100 m1 of a
benzene-hexane mixture (1:1, v/v). The solvent was
evaporated on a steam bath under a nitrogen stream.

Fractionation of Monocarbonyl Derivatives: The

 

monocarbonyl residue from the alumina column was dissolved
in hexane and applied to a 10 g Celite 545-Sea Sorb 43
(1:1, w/w) column. The Celite 545 was dried at 150° C for
24 hr. Separation of the methyl ketones from other DNPH
classes was accomplished using a modification of the
sequence of solvents suggested by Boyd, Keeney and Patton
(1965). The following sequence of solvents was used:
50 m1 quantities of 15, 25, 40, and 60 percent chloroform
in hexane, 100 ml of 80 percent chloroform in hexane, 50 ml
of 90 percent chloroform in hexane, 150 ml of chloroform,
and 50 m1 quantities of 2, 4, 6, 8 and 10 percent methanol
in chloroform. The retention volume of the classes varied
due to the variation in carbonyl content among cheese
samples.

The column eluate was monitored for absorption at 254
nm with an Isco Model UA-2 ultra-violet analyzer
(Instrumentation Specialities Co., Inc., Lincoln,

Nebraska). A strip chart recording of absorption was made

29

and each peak was collected separately. The chromato-
graphic fractions were evaporated to dryness under nitrogen,
and the residues dissolved in chloroform. A Beckman DK-ZA
ratio recording spectrophotometer was used to establish
class authenticity on the basis of the following

absorption maxima: methyl ketones, 363; saturated
aldehydes, 358; 2-3na1, 374 (Day, 1965).

Separation of Methyl Ketone DNPH into Individual

 

Chain Lengths: The methyl ketone class was separated into

 

its individual members using the liquid-liquid partition
column of Corbin, Schwartz and Keeney (1960). Twenty-
five grams of Celite, analytical filter aid (dried at
150° C for 24 hrs) were slurried with 250 ml hexane
equilibrated with acetonitrile. Thirty-three m1
acetonitrile and 0.5 ml distilled water were added and the
blended slurry was poured into a 2.8 cm ID chromatographic
column. The column was packed with nitrogen pressure
(6-8 psi). Approximately 1000 ml of hexane equilibrated
with acetonitrile was required to elute the ketones. The
column effluent was monitored for absorption at 254 nm with
an Isco Model UA-2 ultraviolet analyzer and a strip chart
recording was made. The individual members of the ketone
class were collected and the solvent evaporated under a
stream of nitrogen on a steam bath.

Determination of Individual Methyl Ketone Concentra-
tion: The concentrations of the CS- 13 methyl ketone

derivatives were determined by measuring their absorbance

30

in chloroform at 363 nm. The molar absorptivities of the
methyl ketone 2,4 DNP derivatives used for quantitation
were those reported by Day (1965). The chain length of
each methyl ketone derivative was tentatively assigned by
its retention volume on the liquid-liquid partition column.

Thin-Layer Chromatography of Methyl Ketones: Con-

 

firmation of chain length was made using the thin-layer
chromatography methods of Libbey and Day (1964), and
Edwards (1966). Prepoured plates of silica gel F-254
(Brinkman Instruments, Inc.) were used for both methods.

The method of Libbey and Day involved activating the
plates at 150° C for one hour. After cooling, they were
placed in a developing tank containing petroleum ether
with 10% silicone oil for 5-6 hr. The ether was allowed to
evaporate and the plates spotted with DNPH samples, then
developed in dioxane-HZO (65:35, v/v) for 6-8 hours.

The other method (Edwards, 1966) required a 24 hour
plate activation. The plates were developed in hexane-
ethyl acetate (4:1, v/v). To visualize the DNPH, the
plates were sprayed with 0.2% 2,7-dichlorof1uorescein in
ethanol and then with 5N NaOH. Spots were visible as deep
red-orange on an orange background.

Determination of Percentage Recoverypof the Individual

Methyl Ketones: A standard mixture of the C5'C13 methyl

 

ketones was used to determine the percentage recovery.

Concentrations used approximated those found in Blue cheese.

31

The standard ketone mixture in hexane was passed over a

2,4 DNP reaction column to convert the ketones to their
hydrazones. Three grams of butteroil were added to the
standard mixture and the sample analyzed as described for
cheese. The fat was added after hydrazone formation to
reduce the possibility of any methyl ketones present
reacting to form derivatives. Fat added in this manner did
not contribute any hydrazones to the sample.

All analyses were carried out in duplicate.

Production of Quick-Ripened Blue Cheese

Quick-ripened Blue cheese was made according to the
method of Hedrick 2E.2l (1968). Raw milk containing 3.5
percent fat was preheated to 130° F and homogenized in a two
stage homogenizer (2000 psi first stage, 500 psi second
stage). Following homogenization the milk was pasteurized
at 143° F for 30 minutes and then cooled to 84° F.

Cheese was made in 20 gallon batches using 17 9 frozen
concentrated cheese starter per batch. After the acidity
had risen, .02%, 10 m1 of decolorizer (chlorophyll and
food dye in alkaline solution), 24 g mold powder ("Midwest"
Blue Mold Powder) and 10 m1 single strength rennet were
added per 20 gallons of milk. When the curd separated
cleanly (usually one hour), it was cut with 3/8" cheese
knives. The curd was stirred thirty minutes after cutting

and again thirty minutes later. After the second stirring,

32

the whey was drained and the curd placed on a stainless
steel mesh screen in the ripening room. The depth of the
cheese curd was approximately 3 to 4 inches.

The curing room was maintained at 62° F and 95% rela-
tive humidity. The curd was stirred daily and salt was
added on the fourth, fifth and sixth days. The salting
rate was one pound for each 22 pounds of cheese. One-
fourth was added on the fourth day, one-fourth on the fifth
day, and one-half on the sixth day. The ripened Blue
cheese had a salt content of approximately 4%.

Subsequent batches of cheese were varied as to
manufacture and ripening conditions (Table 1). After
ripening 10 days the cheese usually had a flavor
characteristic of Blue cheese and was packaged in
polyethylene bags and immediately frozen at -5° F and held

at this temperature for further analysis.

Preparation of White Mold Powder

 

A slant of a white mutant of P. roqueforti was

 

obtained from Midwest Mold Company and propagated by the
method of Hussong and Hammer (1935). Whole wheat bread
was cut into 1/2" cubes and placed in wide mouth bottles.
The bottles were sterilized at 15 lbs. steam pressure for
30 minutes. After cooling, the bread was inoculated with
mold and incubated at 21° C. Approximately 11 days was
required for spore formation. The material was then

pulverized in a Waring blender and stored at 5° C.

33

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RESULTS AND DISCUSSION

The results of this study have been arranged in the
following manner. For convenience in discussing this
research the data are presented, followed by a discussion
of the effects of each processing variable. All modifi-

cations of quick ripened Blue cheese were made in duplicate.

Sensornyvaluation of Quick Ripened Blue Cheese

Each modification of quick ripened Blue cheese was
evaluated for color, flavor and texture by a panel of
experienced judges. Flavor was assigned a maximwm
possible total of 20 points, color 15 points and texture
10 points. It was felt that flavor should be given
maximum importance because the quick ripened cheese is
suitable for incorporation into other products. Color and
texture were considered to be of lesser importance. A
cheese with a score of approximately 30 out of the total
of 45 was considered acceptable. The commercial control
was a packaged, crumbled Blue cheese purchased in a local

supermarket.

Quantitation of Methyl Ketones

 

Sensory evaluation of quick ripened Blue cheese showed

that most samples were lacking in methyl ketone flavor, a

34

35

flavor which is considered typical of good Blue cheese.
During this study it was observed that the flavor score was
directly related to methyl ketone content. Starkle (1924)
and Hammer and Bryant (1937) postulated that methyl

ketones contribute to the flavor and aroma of Blue mold
cheeses. Hammer and Bryant isolated pentanone-Z,
heptanone-2 and nonanone-2, as did later workers (Patton,
1950; Morgan and Anderson, 1956). The quantity and
distribution of methyl ketones in quick ripened Blue cheese
was studied as it relates to processing parameters.

The methyl ketone content of the quick ripened cheeses
studied in this project, reported as micromoles/lo g of
extracted fat, are presented in Table 3. The processing
variations have been outlined in Table l and described in
detail in Experimental Methods. These samples were chosen
for analysis out of a larger series of different quick
ripened Blue cheeses which had been prepared in this
laboratory (Harte, 1974; Kuehler, 1974). This selection
of samples for analyses was necessary due to the length
of time required for complete separation and identification
of the methyl ketones in a single sample (approximately two
weeks per sample). With one exception, the samples were
chosen to represent the best of the cheese prepared in our
laboratory. One poor quality cheese (that prepared by
direct acidification) was also analyzed. The methyl ketone
content paralleled the sensory evaluations made on the

cheeses.

36

 

 

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37

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38

All samples contained large amounts of heptanone-2 and
nonanone-Z. Dartey and Kinsella (1971) observed that these
two methyl ketones accounted for 50 to 75% of the total
methyl ketone in Blue cheese. As shown by Blakely (1970),
nonanone-2 was found to be the major methyl ketone in quick
ripened Blue cheese. In addition to those ketones reported,

small amounts of a compound assumed to be Cl methyl

5
ketone (pentadecanone-Z) were found in all samples
analyzed. The presence of C13 and C15 methyl ketones in
Blue cheese may be due to natural breakdown of milk fat,
rather than to microbial action (Schwartz and Parks, 1963).
Results of these analyses are comparable to the findings of
Morgan and Anderson (1956), Schwartz and Parks (1963), and
Anderson and Day (1966), in that the individual ketones in

Blue cheese vary considerably, even among cheeses of good

quality.

Quantitation of Free Fatty Acids

 

These data were collected for quick ripened Blue cheese
by Harte (1974), and are presented in this thesis in Table
4. The cheese samples analyzed are identical to the
samples used in this study and are included here for
discussion because of their interrelation with methyl
ketone data and flavor properties. Free fatty acids (FFA)
were determined by the method of Iyer, Richardson, Amundson
and Boudreau (1967). Table 4 shows the FFA content of the

quick ripened Blue cheese, reported as mg acid/Kg cheese.

39

Table 4

Concentration of Free Fatty Acids n
Quick Ripened Blue Cheese (mg/Kg).

 

Cheese sample2

 

 

 

ACID

A B c D E F
Butyric 846 657 1,270 1,680 650 875
Caproic 465 228 490 485 244 401
Caprylic 523 645 424 359 314 306
Capric 2,010 1,460 824 2,240 950 2,350
Lauric 2,580 2,380 1,240 1,500 1,540 2,560
Myristic 6,720 4,630 4,090 4,660 4,630 4,520
Palmitic 9,780 10,200 12,500 12,500 13,100 12,400
Stearic 4,370 4,910 6,590 6,460 4,730 5,600
Oleic 10,930 11,100 13,900 13,000 12,900 13,000
Linoleic 1,060 936 787 750 934 1,260
Linolenic 862 844 707 651 762 914
1

From Harte, 1974.

2Quick ripened Blue cheese samples:

Lactic Acid, Direct Acidification
White Mold

Control

Control Stored for 2 Weeks at 49° F
Suggested Standard #1

Suggested Standard #2

WMDOWS’

40

Fatty acids are metabolized by the mold spore to form
B-keto acids and then decarboxylated to form methyl ketones
of one less carbon atom (Hawke, 1966).

Therefore, butyric through myristic acids are
oxidized to form the C5 through Cl3 methyl ketones
quantitated in this study. The data for the longer chain
acids are also included in Table 4 because of their con-
tribution to the flavor of Blue cheese. Anderson and Day
(1965) and others felt that the long chain fatty acids may
exist in the salt form, accounting for the slightly
”soapy" flavor characteristic of some mature Blue cheeses.

Comparison of the fatty acid and methyl ketone data
does not reveal any particular relationship of fatty acid
concentration to ultimate levels of methyl ketone. For
example, in samples C and D (a control quick ripened
cheese and the same cheese after two additional weeks
storage at 40° F) the caproic acid contents are 490 and
485 mg/Kg respectively. However there is a ten fold
increase in the corresponding methyl ketone, 2-pentanone,
in D, the stored sample, indicating a highly significant
conversion of fatty acid to ketone. The capric (C10) acid
content of the stored control increased three fold over
the initial value and the corresponding ketone, 2-nonanone,
showed a 50% increase during storage. Thus, from these
data, one cannot predict the levels of methyl ketone in a
sample of cheese and certainly not from FFA data.

Naturally, the relative concentration of the two classes of

41

compounds are quite different, the ketones being reported
in micromoles/lo g fat and the FFA as mg/Kg cheeses. If
there is some limiting factor governing the rate of con-
version of FFA to methyl ketone one could observed an
increase in total FFA without a concomitant increase in
ketone.

The amount of methyl ketone produced may be related
to the amount of fatty acid available. The results of
Girolami and Knight (1955) show that the formation of
ketones is directly dependent on the quantity of available
fatty acid. However, later work (Anderson, 1966) showed
that the amount of ketone produced is also related to the
chain length of the corresponding fatty acid. Octanoic
acid was found to convert to methyl ketone fastest, while

butanoic and dodecanoic acids were the slowest.

Methyl Ketone Content of a Commercial Blue Cheese Sample

A sample of commercial Blue cheese packed in loose
crumbled form was analyzed for methyl ketones. This cheese
was judged by sensory evaluation to be slightly superior
(Table 2) to all samples of quick ripened cheese and the
high quantities of ketone (Table 3) confirm this.
Apparently the flavor score is related to net ketone levels
in the concentrations shown here. The commercial sample
was analyzed for the purpose of comparison with the quick
ripened Blue cheeses. The results show a similar ketone

distribution, but a much higher overall quantity in the

42

commercial cheese. The quick ripened samples ranged from
28.55 micromoles/10g fat for cheese made by direct
acidification with lactic acid to 135.47 micromoles for
cheese ripened at 52° F and salted on days 1, 9 and 10.
This is in contrast to 192.79 total micromoles for the
commercial cheese.

Nonanone-Z was the most abundant ketone in this sample,
as in the quick ripened samples. Anderson and Day (1966)
found 2-heptanone to be the predominant ketone in two
samples of Roquefort and five domestic Blue cheese samples.
Two of the domestic Blue cheese samples contained equiva-
lent concentrations of 2-nonanone and 2-heptanone. In
three samples of domestic Blue cheese, Schwartz and Parks
(1963) observed 2-heptanone to be the most abundant ketone.
A Roquefort sample analyzed by Schwartz, Parks and Boyd
(1963) contained more 2-nonanone than 2-heptanone.
Blakely (1970) found 2-nonanone to be the major methyl

ketone in samples of quick ripened Blue cheese.

gpick Ripened Blue Cheese Made by Direct Acidification with

 

Lactic Acid

 

One of the more time consuming steps in the manufacture
of quick ripened Blue cheese is the ripening of the cheese
milk with starter culture. Approximately 1 to 1 1/2 hours
are required for the acidity to rise .02%. In an attempt
to shorten the procedure, quick ripened Blue cheese was

made by direct acidification with lactic acid, without the

43

use of starter culture. Lactic acid was added to the cheese
milk until the pH had been brought down to 5.70. This was
close to the pH of cheese milk ripened with starter

culture.

A Blue cheese possessing good body and texture was
produced by a direct acidification method by Shehata and
Olsen (1967). These workers obtained better results using
hydrochloric acid instead of lactic acid. They found the
lactic acid curd too soft and too high in moisture. Singh
and Kristofferson (1972) attempted to produce a Blue cheese
flavor using a slurry prepared from directly acidified
lactic acid curd. The curd had physical characteristics
comparable to normal Blue cheese, but it failed to
develop characteristic flavor during storage. Shehata
(1967) incorporated a lipolytic enzyme into milk used for
directly acidified cheese in order to encourage the
development of flavor compounds.

Quick ripened Blue cheese made in our laboratory
by this method of direct acidification failed to develop a
flavor characteristic of good Blue cheese. This observa-
tion was verified by the much lower methyl ketone content
of the directly acidified cheese compared to other quick
ripened Blue cheeses.

As shown in Table 3, the total concentration of methyl
ketones was 28.55 micromoles/log of fat compared to 64.87
for the control cheese. The levels of heptanone-2 and

nonanone-2 were almost equal and together they accounted

44

for greater than 85% of the total methyl ketones. The
other ketones are almost negligible in concentration.
This unusual ketone distribution may in part account for
the atypical flavor in this cheese sample.

The directly acidified quick ripened cheese also
failed to exhibit the characteristic drop in pH after
manufacture (Coulter, 1938b). Conventional Blue cheese
reaches a minimum pH of 4.7 within one day after manu-
facture. This increase in acidity was also characteristic
of quick ripened Blue cheese (Harte, 1974). Most quick
ripened Blue cheese samples reached a maximum acidity on
day 2 or 3. However, cheese made by this method required
four days to reach its minimum pH. Also, the pH did not
drop as far as it did with other variations. The
directly acidified curd reached a pH of 5.37, while the
pH of standard quick ripened Blue cheese fell as low as
pH 4.86 (Harte, 1974). This slow, atypical change in pH.
may be indicative of an abnormal mold growth and consequent
poor flavor production.

This cheese received the lowest rating by sensory
evaluation since it failed to develop characteristic flavor
and color. Using the processing conditions described, this
cheese was unacceptable. However, longer ripening might

have improved the cheese.

45

Quick Ripened Blue Cheese Made Using a Mutant White Mold

Cheese was made using a white mutant strain of P.
roqueforti. The standard method of Hedrick gt a; (1968)
was used, but the ripening temperature was lowered to 52° F
and the curd was cooked to 100° F after cutting. The white
mutant strain was developed by Knight gt El (1950) by the
irradiation of spores. The intent of using a white mutant
in cheesemaking is to produce a cheese possessing a
typical Blue cheese flavor without the color of the mold
being evident. This would be preferable in some applica-
tions and could conceivably increase consumer acceptance of
Blue cheese products.

The total concentration of methyl ketones in the
white cheese was 41.38 micromoles/10 g fat (Table 3).
This was fairly low compared to other samples analyzed in
this study, but still slightly higher than the values
reported by Blakely (1970) for quick ripened Blue cheese.
Blakely's values ranged from 17.7-26.0 micromoles/10 g fat.
The total FFA as outlined in Table 4 for this cheese were
also low compared to other samples of quick ripened Blue
cheese (Harte, 1974). The low ketone content is in
contrast to the results of Nelson (1970). Nelson
quantitated the total ketones in a submerged culture of

white mutants of P. roqueforti grown in homogenized milk

 

plus lipolyzed milk fat. He detected a ketone concentra—

tion 7 to 15 times that found in Blue cheese; however his

46

results were obtained in a liquid culture rather than a
ripened cheese.

The ketone profile of quick ripened Blue cheese made
with white mold was fairly similar to that of conventional
Blue cheese and other quick ripened Blue cheese samples.
The only major difference was the substantially lower
concentration of 2-heptanone (Table 4). The caprylate
level was also low as determined by Harte (1974). This
may reflect a difference in the lipase system of the
mutant white mold.

The white cheese was rated by sensory evaluation as
having a mild flavor, and again the low intensity of flavor
is reflected in a low concentration of methyl ketones.

This may be due to the fact that the ripening temperature
was only 52° F, yet the salting was done on days 4, 5 and
6. With conventional quick ripened Blue cheese salting
times were determined by the development of the blue color.
This of course was not possible with the white mutant.
Harte (1974) and Kuehler (1974) have shown that early
salting may slow down mold activity and consequently the
production of flavor compounds. Knight 25 31 (1950)
claimed Blue cheese manufactured by a traditional method
using the white mutant had normal lipolytic activity, but
a milder flavor and softer body than normal Blue cheese.
The level of flavor compounds may have been higher had
salting been delayed. Additonal research is needed in this

area.

47

Quick Ripened Blue Cheese Made by the Original Method and

Stored at 40° F for 2 Weeks

 

A control batch of cheese was made by the original
method of Hedrick 3E 21 (1968). The cheese was ripened
at 62° F for 7 days and salted on days 4, 5 and 6. Some
of this cheese was then stored for 2 weeks at 40° F. The
stored cheese was also analyzed for methyl ketones.

Sensory evaluation of both cheeses showed the stored cheese
to have a much finer flavor, although the mold color was
the same.

Both cheeses had high methyl ketone levels. The data
in Table 3 show that the total methyl ketone content of the
original cheese was 64.87 micromoles/10 g of fat, and the
stored cheese was approximately 16 micromoles higher. The
level of 2-nonanone showed the most marked increase during
storage. Undecanone-Z actually decreased in concentration
during storage, possibly due to interconversion with the
corresponding secondary alcohol. The ketone level in this
standard batch of cheese was significantly higher than that
reported by Blakely (1970) for quick ripened Blue cheese.

The FFA data (Table 4) show that the volatile (C4-C10)
fatty acids increased in storage. These data, and the
increase in methly ketones discussed above indicate that

the enzyme systems of P. roqueforti are active during

 

storage at 40° F. A cheese of quality superior to the

original standard cheese was produced by 2 weeks storage.

48

Suggested Methods of Manufacture forvguick Ripened Blue
Cheese

Many variations on the original method of Hedrick
25 31 (1968) were attempted in an effort to improve the
quick ripened Blue cheese. It was felt that more control
was needed over the fermentation in order to provide better,
more easily reproducible results. The two variations
discussed in this section are suggested as an improved
method of manufacture.

Several problems were encountered during the produc-
tion of the standard batch discussed above. Contamination
by microorganisms of the ripening curd was often seen.
Better control of the salting procedure and a lower
ripening temperature were tried in an effort to eliminate
or at least minimize this problem. Also, in an effort
to cut down the drainage time, produce a firmer curd, and
reduce curd shattering, the cheese was cooked to 100° F.

A ripening temperature of 52° F was used. One batch was
salted on days 7, 8 and 9, the other on days 1, 9 and 10.
Both were allowed to ripen for 11 days.

Cooking the cheese curd was considered preferable to
the original method. Less shattering occurred and therefore
less loss of curd fines was noted during drainage. This
was verified by weighing curd made by each method. Also,
the drainage time required was greatly reduced when
cooking was introduced, thereby reducing the amount of

labor required in the production of the cheese.

49

A lower ripening temperature was also considered to be
preferable to the original temperature of 62° F. Morris
(1969) demonstrated that higher temperatures are superior
for increased lipolysis and proteolysis, but the body,
color and flavor of the cheese are of lower quality. As
indicated in Table 2, subjective analysis of cheese made by
the standard method and by the two suggested methods showed
high scores in all areas for the suggested methods compared
to other quick ripened Blue cheeses. The increased flavor
intensity may be due to the longer ripening time. Also,
this longer ripening time may have provided for a better
balance of flavor components. The methyl ketone content
of the cheese made by the suggested methods was approxi-
mately double that of the standard method (Table 3).

The methyl ketone content of cheese made by the
second suggested method (salting on days 1, 9 and 10) was
slightly higher, although both cheeses were highly
acceptable. This variation and control of salting was
felt to be extremely important in control of flavor
development (Harte, 1974; Kuehler, 1974). Most organisms
are inhibited at the 10 percent brine concentrations
present in Blue cheese. The control of contamination in
quick ripened Blue cheese is essential because of the
rapid growth of organisms on the large area of curd surface
exposed to the atmosphere.

The enzyme systems of P. roqueforti are also affected

 

by salt concentration. Willart and Sjostrom (1959), using

50

salt concentrations of l.5-4.5%, found less higher chain
length fatty acids at the higher levels of salt. Morris
and Jezeski (1953) and Inamura (1960) reported that the

lipolytic activity of P. roqueforti decreased with

 

increasing salt concentration.

The time of salting and amount of salt were used to
control mold growth and activity in quick ripened Blue
cheese. According to the data of Harte (1974), the
addition of salt on day 1 caused a delay in the normal drop
in pH. This may be due to inhibition of the starter
culture by the salt and therefore a slower production of

lactic acid. P. roqueforti may also be inhibited by an

 

early addition of NaCl. Harte (1974) found that lipolysis
in quick ripened cheese salted on days 1, 4 and S was
retarded. However, cheese salted on days 1, 9 and 10
showed normal lipolytic activity as evidenced by the methyl
ketone and FFA data (Tables 3 and 4). The difference here
is the time span between the initial and final saltings.
This longer time interval may have allowed the enzyme

systems of P. roqueforti to overcome the initial slow

 

start. The early salting may even have been beneficial for
carbonyl production. Nelson (1970) observed that carbonyl

production by spores of P. roqueforti is enhanced by the

 

addition of salt. However, a lag phase caused by salt
addition was observed by later workers (Dwivedi and
Kinsella, 1974). These researchers noticed a prolonged

lag phase before carbonyl production by mycelium when salt

51

was added to the growth media. The salting times suggested
here (days 1, 9 and 10) may have been ideal for carbonyl
production by allowing time for the mold to overcome the
lag phase, yet enhancing total carbonyl production by

early salting. The cheese made by this second method was
described as having a clean, milk flavor. It is suggested
that salting on day l to control contaminating organisms

is a satisfactory method of production. No problems with

contamination were encountered using this procedure.

SUMMARY AND CONCLUSIONS

Quick ripened Blue cheese was prepared according to
the method of Hedrick g; E; (1968). This original proce-
dure was varied in an attempt to standardize and improve
the cheese. The following modifications were developed
and studied:

1. Direct acidification with lactic acid

2. Use of a mutant white strain of P. roqueforti

 

3. Cooking curd to 100° F during the processing of
the cheese

4. Ripening at 52° F with salting on days 1, 9 and 10

5. Ripening at 52° F with salting on days 7, 8 and 9

6. Storage of the quick ripened cheese at 40° F for
2 weeks to improve flavor quality.
All samples were subjected to sensory evaluation. The
methyl ketone content of the cheese was determined by
chromatographic separation and quantitation of 2, 4 dinitro-
phenylhydrazine derivatives of the C5'C15 carbonyls. The
cheeses were compared using a conventionally ripened
commercial Blue cheese as a control.

The commercial sample was judged superior to all
samples of quick ripened Blue cheese. Nonanone-Z was the

most abundant ketone in all of the samples analyzed.

52

53

Directly acidifying the curd with lactic acid produced
a quick ripened Blue cheese of poor quality. The methyl
ketone content was extremely low and the flavor was
atypical. The pH of such cheese during ripening was con-
sidered abnormal when compared to other quick ripened
cheeses. Due to the lack of characteristic flavor and
color, this cheese received the lowest rating by sensory
evaluation.

Cheese made with a white mutant strain of P.

roqueforti was considered satisfactory, although low in

 

flavor and methyl ketone content. The flavor was typical
of a good, milk Blue cheese although the typical blue-
green color of the mold was absent. This cheese was
considered excellent for incorporation into dips and
dressings where lack of mold color is desirable.

Quick ripened Blue cheese was shown to be improved
by storage of the ripened curd at 40° F. Cheese stored
for two weeks was judged superior in flavor. The total
methyl ketone content was also markedly increased;
nonanone-Z increased to the greatest extent, while
undecanone-2 decreased in storage.

Two modified methods of manufacture of quick ripened
cheese were developed. These modifications resulted in
improved flavor, body and texture of the cheeses. The
methyl ketone content of the cheese prepared by either

modified method was significantly higher than conventional

54

quick ripened cheeses. These variations involved cooking
of the cut cheese curd, lowering the ripening temperature

to 52° F, and salting either on days 1, 9 and 10 or on days
7, 8 and 9.

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63

APPENDIX

Fat contents of cheese samples were routinely deter-

mined and are presented below.

Table 5

Fat Content of Blue Cheese Samples

 

 

Cheese Sample % Fatl
Lactic Acid,

Direct Acidification 29.72
White Mold 28.05
Control 26.83
Control, Stored for 2 Weeks

at 40° F 25.38
Suggested Standard #1 26.61
Suggested Standard #2 27.76
Commercial Cheese 29.70

 

1Average of duplicate analyses.

"'3.

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