.

b 137 7330{:,

THE ROLE OF YEASTS D'URI

THE RIPENING OF SALAMI

by

Akua Abrafi Osei-Abunyewa

Submitted in fulfilment of the requirements

for the degree

MAGISTER SCIENTIAE

in the
Department of Microbiology and Biochemistry, Faculty of Science,
University of the Free State, Bloemfontein 9300, South Africa

Promotor: Prof. B.C. Viljoen

Co-promotor: Mr. Arno Hugo

University Free State

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March 1999 Universiteit Vrystaat

HIERDIE EKSEMPLAAR MAG ONO-ER

CJ 'EN OMSTANDIGHEDE UIT DIE

KIHLlOTEEK VERWYDER WORD NIE

 CONTENTS

Page

CHAPTER I: Literature Review 1

1.1. INTRODUmON 1
1.2. MEAT 2
1.2.1. Chemical and physical composition 2
1.2.2. Microbial spoilage of meat 3
1.2.3. Modes of meat preservation 4
1.2.3.1. Fermentation 4
1.3. HISTORICAL BACKGROUNDOF SALAMI PRODUmON 7
1.3.1. Origin of salami 7
1.3.2. Current practices 11
1.3.2.1. The use of starter cultures 11
1.4. BACTERIAL FERMENTATION 13
1.4.1. Lactic acid bacteria (LAB) 13
1.4.2. Micrococcaceae 14
1.5. YEASTS ASSOCIATED WITH MEAT 14
1.5.1. Yeast associated with fermented meats 15
1.5.2. Yeast as starter culture 15
1.5.3. Importance of yeast in fermented meats 17

CHAPTER II: The Growth and Survival ofYeasts in

Commercial Salami 18
ABSTRACT 18
2.1 INTRODumON 19
2.2 MATERIALS AND METHODS 21
2.2.1 Commercial salami formulation 21
2.2.2 Sampling methods and selection of isolates 23
2.2.3 Sampling during manufacture of salami 23
2.2.4 Physical and chemical analysis 23
 2.2.5 Yeast identification 25
2.3 RESULTS AND DISCUSSIONS 25
2.3.1 Physical and chemical analysis 25
2.3.2 Microbial enumeration 26
2.3.3 Yeasts enumeration 29

CHAPTER Ill: Key Properties for the Selection of Yeasts as

Starter Culture in the Making of Salami 37
ABSTRACT 37
3.1 INTRODUCTION 37
3.2 MATERIALS AND METHODS 40
3.2.1 Salt tolerance 40
3.2.2 Nitrate tolerance 43
3.2.3 Proteolytic activity 43
3.2.4 Lipolytic activity 43
3.2.5 Inactivation at temperatures above 50°C 43
3.3 RESULTS AND DISCUSSION 44

CHAPTER IV: The Application of Yeast Strains as Starter

Cultures in Traditional Fermented Sausage 50
ABSTRACT 51
4.1. INTRODUCTION 52
4.2. MATERIALS AND METHODS 52
4.2.1. Yeast preparation for use as starter culture 52
4.2.2. Manufacture of salami 53
4.2.3. Sampling 55
4.2.4. Chemical and physical analysis 55
4.2.5. Microbiological analysis 56
4.3. RESULTS AND DISCUSSION 56
4.3.1. Chemical and physical composition 56
4.3.2. Microbial enumeration 64
CHAPTER V: General Discussions and Conclusions 68
5.1. The growth and survival of yeast in commercial salami 68
5.2 Key properties for the selection of yeast as possible starter culture
in the making of salami 69
5.3. The application of yeast strains as starter cultures in
traditional fermented sausage 70
Future research 71

CHAPTER VI: Summary 72

References 74
Not unto us, 0 Lord, not unto us, but unto thy name give I glory, for thy
mercy, and for thy truth~ sake. Wherefore should the heathen say, where is
now thy God? But my God is in the heavens: He hath done whatsoever He
hath pleased.(ps 115,1-3)•• I will therefore sing unto my Lord, I will make a
joyful noise to the rock of my salvation. I will go before His presence with
thanksgiving and make a joyful noise unto him with psalms. The Lord is a
great God,and the greatest king above all gods.

For his pleasure he created all things - therefore I dedicate this work to him.

Receive thy praise 0 God

 ACknowledgements

I wish to thank:

Mr Osei Kwadwo Abunyewa, who encouraged and supported me,

Prof Bennie C Viljoen, whose constructive criticism and contribution made this work a
success,

Mr Arno Hugo, who despite a tight schedule helped with the manufacture of the
salami,

Ms Eileen Roodt and Mr Piet Bates, for helping with the chemical analysis,

Mrs Carol Viljoen, whose typing skills helped in producing good prints
 CHAPTER I
LITERATURE REVIEW

1.1 Introduction

Preservation by means of fermentation is one of the oldest food technologies, yet it

continues to play an important role in meat preservation in many parts of the world.
These processes can be simple, with minimal microbial involvement, or more complex,
involving defined ingredients and starter cultures with controlled environmental
conditions. Most meat fermentations rely mainly on the use of salt and spices as
ingredients, and if permitted, the addition of nitrate and nitrite. The fermentation of
meats furthermore, depends on the interaction of various intrinsic and extrinsic
environmental as well as microbiological factors including the pH, water activity, redox
potential and the presence of preservatives and naturally competitive microflora. The
final fermented product, however, granted a long and safe shelf-life of high nutritious
quality and wholesomeness.

The microbiological attributes of bacterial starter cultures have been studied in detail.
However, despite frequent occurrences of yeasts in fermented products, little
consideration has been given to the ability of yeasts to grow in salami, its contribution
to the final product or the application as possible starter cultures. Therefore, this study
reports the cell numbers and species of yeasts found in commercial salami and
examines some properties that govern the ability of yeasts to grow in salami. In
addition, selected yeast species, are incorporated individually and in association with
conventional lactic acid bacteria as starter cultures during the processing of salami.
 2

1.2. Meat

Meat is defined as 'the flesh of animals used as food, now chiefly butcher's meat,
excluding fish and poultry'. Since the predominant portion of the edible flesh of animal
carcassesconsists of muscular tissue, meat can be conveniently regarded as the post
mortem aspects of muscles (Lawrie, 1995). The principle attributes of eating quality in
meat thus depend upon the structure and chemistry of muscle.

1.2.1. Chemical composition of meat

The proximate composition of a typical, adult mammalian muscle post mortem, after
the onset of rigor mortis, but before degradative changes commence, is 75% water,
19% protein, 2.5 % lipids, 1.2% carbohydrate and 2.3% of miscellaneous non-protein
substances (Lawrie, 1995). These findings apply generally, since the basic structure of
muscles is similar between species and classes of animals. A number of factors,
however, impose variation on the relative quantities of the components of muscles,
including species, breed, sex, age, anatomical location, nutrition, and exercise. The
approximate composition of lean muscle tissues is presented in Table 1.

Table 1
Approximate composition of lean muscle tissues of meat animals (%)
Species Water Protein Fat Ash
Beef 70-73 20-22 4.8 1.0
Pork 68-70 19-20 9-11 1.4
Lamb 73 20 5-6 1.4
Chicken 73-70 20-23 4.7 1.0
Data from Fennema, O. R. (1985).
 3

Meat is a nutritious protein-rich food, which is highly perishable with a short shelf-life
unless preservation methods are incorporated (Carnpbell-Platt, 1995). Meat product
thus provides an excellent growth media for micro-organisms.

The slaughter of the live animal destroys the inherent defense mechanism in the meat
tissue that resist microbial invasions and growth. The nature of the slaughtering process
and further handling of raw product also allow spoilage and pathogenic micro-
organisms from fleece, hide (Dillon and Board, 1991) and gut to readily contaminate
the raw meat (Niven, 1961). The combination of a high water activity with a moderate
pH and the availability of a range of nutrients further encourage the rapid growth of
bacteria.

1.2.2. Microbial spoilage of meat

Raw meat may generally contain lactobacilli, micrococci, enterococci, Pseudomonas,

Escherichia, anaerobic bacteria, yeasts and moulds contaminated either endogenously

in the living animal or by subsequent post mortem contamination. Overt disease caused
by Bacillus anthracis, Mycobacterium tuberculosis or Brucella abortis would lead to
condemnation by veterinary inspectors and the meat would not reach the consumers.
However, several conditions are not readily detectable and the composition of meat
renders a good substrate for growth of spoilage bacteria such as Pseudomonas or food
poisoning bacteria including Salmonel/a, Listeria, campylobacter and Clostridium
(Leistner et al, 1989; Katsaras and Leistner, 1985; Hechelmann et al, 1988) Post
mortem contamination of meat is the major cause both of organoleptic spoilage and of
food poisoning. Apart from the availability of nutrients, the survival and growth of these
micro-organisms in meat is determined by factors like temperature, moisture
availability, pH, and the gaseous environment to which the organisms are exposed.
 4

1.2.3. Modes of meat preselVation

It is evident that the requirements of micro-organisms for survival and growth afford
the possibility of their control by storing meat under conditions which fail to provide
these requirements, or under conditions which directly inhibit the growth and
progression of the micro-organisms. Accordingly, temperature control, moisture control,
or direct microbial inhibition by means of ionizing radiation, antibiotics or various
chemicals are implemented. Fermentation as a mode of preservation, depending on the
breakdown of carbohydrates to yield various alcohols and organic acids, resulting in the
inhibition of growth of undesirable micro-organisms, was inadvertently exploited by
humans many years ago. In respect of meat, fermentation has been employed to
preserve or enhance the organoleptic attributes of comminuted products such as
salami's (Zottola, 1992).

1.2.3.1. Fermentation

Meat product safety and shelf-life are dependent upon rapid preservation techniques
instituted after-slaughter or freezing (Zottola, 1972). In general, preservation is
accomplished by synergistic effects of several methods rather than the use of a single
procedure (Zottola, 1972). Fermentation is an important preservation method, which
has evolved for meat but is rarely used alone. Preservation is usually achieved by a
combination of fermentation with the use of water activity lowering techniques
including dehydration and the addition of salt (Carnpbell-Platt, 1995). These techniques
have been the basis of traditional technologies used before the scientific basis of their
action was understood (Carnpbell-Platt, 1995).

Historically, man made the observation that the addition of salt and sugar to ground
meat followed by a holding period was conducive to preservation and resulted in an
acceptable product. As time passed various geographical areas developed unique
varieties of preserved meat products that varied in size, shape, texture and f1avor.The
 5

f1avorand texture difference were mainly attributed to variations in spicing, sugar and
salt content, meat formulation and processing characteristics. However, the stability of
these products as well as their consistency, was primarily dependent upon the
controlled conversion of sugar to lactic acid by bacteria (Deibel, 1974).

As with dead organic matter, muscles from slaughtered animal as a whole or in a

particular form, may be modified by micro-organisms during prolonged storage.
Environmental conditions and storage time greatly influence the sort and extent of
modification. With food, there can be desirable and less desirable micro-organisms,
which bring about desirable and less desirable changes. Desirable modifications are
improvement in f1avor aroma, palatability, appearance and storage characteristics.
Food is fermented if micro-organisms, or enzymes contribute to its final characteristics
(Campbell-Platt, 1987).

The main principle for fermented sausage manufacture is the function of lactic acid-
producing bacteria (Diebel et al., 1961), which reduces the pH of the meat and provides
stability against the proliferation of food pathogens and other undesirable micro-
organisms such as proteolytic and lipolytic organisms (Nurmi, 1966; Bacus and Brown
1981). Therefore, these bacteria control the ripening process and develop the required
characteristics for the manufacture of fermented meat products (Inal, 1969; Diebel et
at., 1961; Coretti, 1977). The lactic acid development inhibits undesirable micro-
organisms and allows efficient dehydration. Specific micrococci cultures also enhance
cure-meat color stability and prevent rancidity development by means of the reduction
of peroxide formation via a catalase system (Andres, 1977). Certain yeast cultures of
the Debaryomyces family have also been shown to accelerate and stabilize the color
development at the surface of dry sausages(Coretti, 1977).

Although micro-organisms have been used to enhance and preserve fermented meat
products for centuries, the respective manufactures were unaware of the technical
aspect of the process (Leistner and Rodel, 1975).. As early as 1921 researchers
 6

recognizedthe contribution of micro-organisms to meat processing. Later studies in the

1940s and 1950s further documented the role of micro-organisms and these led to the
suggested usage of microbial cultures to achieve greater consistency. Fermented meat
products have traditionally demonstrated an extended shelf-life through a combination
of reduced moisture content and pH.

The USDA recognizes sausage having a moisture/protein ratio of 3.1 or less and a pH
5.0 or less as not requiring refrigeration (USDA, 1977). Moreover shelf-stable meat
products are classified as having a pH at or below 5.2 and water activity at or below
0.95 or a pH at or below 5.0 and water activity at or below 0.91 (Leistner and Rodel,
1975). Microbial cultures have proven effective as "acidulation agents" for meat, since
the relatively slow, consistent and uniform acid release, via metabolism does not
prohibit the extraction and binding of the soluble meat proteins (Bacus, 1984). The use
of microbial cultures as a "natural preservative" is therefore appealing. Fermented dry
sausage in federally inspected meat plants is increasing by 10% per year (AMI, 1979).
Attempts to duplicate microbial action with chemical acidulants added directly to the
meat has been unsuccessful since direct rapid acidulation prohibits "binding formation"
yielding an unacceptable product texture. In addition, the organic acids, primarily lactic
acid produced by the micro-organism, are relatively "mild" and acceptable to the palate.

Fermentation as a means of meat preservation is becoming more significant with

increasing energy cost for refrigeration, freezing and/or dehydration. Lowering meat
pH provides an economic method to enhance product stability while preserving the
nutritive and quality characteristics (Bacus, 1984). Although the direct contributions of
micro-organisms to the nutritive value of meat products have not been studied
extensively, the prolonged stability of fermented meats allows for greater consumption
of perishable raw material. This natural preservation system also precludes alternative
means of preservations such as extreme heat and chemicals that may reduce nutritive
value (Bacus and Brown, 1981). The high protein value of most fermented meats
(Kiernat et al, 1964) generally results from the drying process that is consistently
 7

achieved through controlled fermentation. Fermented products will certainly play a key
role in the increasing meat product market development since these products have
good stability without refrigeration and the initial nutritive value is maintained.
According to the FAOthe production and consumption of meat products will increase in
future. Growth is expected in industrialized and especially in developing countries
(Bacus, 1984).

Over the years, producers of fermented meats and other fermented foods have been
bothered by the long production time involved with the consequent relatively high
prices of the finished products. Therefore, developing work has been aimed at reducing
the time of manufacture. As far as fermented meat products are concerned we are
most likely at the beginning of a new era of class of foods and food ingredients.

1.3. Historical background of salami production

1.3.1. Origin of salami

A significant advance in man's history was the transition from food gathering to food
production. Man learned that the proper handling and storage of many perishable food
. stuffs brought about changes in their physical, chemical, and organoleptic
characteristics that proved desirable and yielded greater product stability. Meats could
be grounded, mixed with salt and spices and held at cool temperatures to provide a
wide variety of sausage products that were both safe for consumption and were
acceptable (Bacus, 1984).

Sausageis one of the oldest forms of processed food and was consumed by the ancient
Babylonians, Romans and Greeksduring their military campaigns. Preserved sausages,
as a meat supply, were credited as one of the main factors in the success of Caesar's
legions (Pederson, 1979). The origin of meat processing probably occurred when
primitive man first realized that he must either rapidly consume the fresh meat after
 8

slaughter, or it would spoil and be unfit for consumption. Egyptians recorded the
preservation of meat by salting and sun drying. The early Romans are credited with
first using ice and snow to preserve food. The preparation of sausage by cutting or
grinding the meat, seasoning it with salt and spices, and drying it in rolls became an
effective means to preserve fresh meat (Bacus, 1984).

Man's oldest method of cooking was the open fire. Therefore the use of heat and
smoke would have been recognized early as useful methods for preparing and
preserving meat. The use of drying, whether by air, sun or fire was also known long
before recorded history (Smith, 1987). Most reviews on fermented meats pointed out
that drying and fermentation are probably the oldest form of preservation (Bacus, 1984;
Smith, 1987; Roca and Incze, 1990). These authors claimed that these preservation
methods are several thousands of years old. Smith (1987) made reference to Homer's
Odyssey, ca 900 BC and sausages of the old Roman empire. Leistner (1986a) citing
Lissner (1939), mentioned that "sausage" as such is an ancient word in many
languages. Thus Wurst is an Indo- German word meaning "to turn" or "to twist"
probably derived from Latin. Sausage is also well known as kolbasa in Slavic, derived
from Hebrew, meaning "all kinds of meat". The origin of the name "salami" seem
uncertain. Most authors such as Leistner (1986a) reported that it is derived from Latin,
simply meaning "salt", whereas Bacus (1984) claimed that it is derived from the name
of the city Salamis on Cyprus. Adams (1986) claimed that the production of fermented
sausages is thought to have originated in the countries surrounding the Mediterranean
Sea (Zeuthen, 1995).

It seems the art of sausage production, in most cases can be traced back to southern
Europe, from where it spread to other European countries. Leistner (1986a) thus
mentioned that the most well known cured and fermented German sausage probably
first produced by Italians, are only ca.250 years old and the Hungarian salami is not
more than 150 years old. Adams (1986) wrote that the European emigrants established
production both in the USA, South America and Australia and that knowledge about
 9

fermented sausages in the Seychelles,the Philippines and Papua New Guinea is largely
a result of European influence. Climatic variation during the year has influenced the
names of fermented sausagesin some countries. Hungarian salami, the "winter salami"
according to Incze (1986) originated in Italy where the climate in northern Italy was far
better suited for the production of fermented sausages. Although the conditions were
less optimal in Hungary, it turned out to be possible to dry fermented sausages in
Hungary during winter months without difficulty hence the name "winter salami".
Similarly the name "summer sausage" was given because the sausage was
manufactured mainly during the summer, where for safety and shelf-life reasons
microbial growth was stopped by heating the sausage as the last processing step
(Zeuthen, 1995).

With a less lavish supply of red meat throughout Europe and the Mediterranean area
through recorded history, evolved the need and opportunity for people of these regions
to combine all potentially edible by-products with muscle meats and spices.
Consequently, a significant proportion of traditional European sausagesconsist basically
of a cured (i.e. nitrite-containing) red meat emulsion of widely varying texture and
moderately varying fat content in which is suspended fat and/or one or more edible
offals in some preferred form (e.g. chopped, diced, minced or strips). A very large
proportion of the meat consumed in European countries is in the form of sausage
products most of which are seen as high quality items equivalent in value to fresh meat
(Smith, 1987). The drying of meat became very common along the shores of the
Mediterranean when artificial refrigeration was not yet available as a means of food
preservation. The early Roman butchers cut beef and pork in small pieces, added salt
and spices, stuffed them into skins, or washed animal intestine and placed them in
special rooms to dry.

Preparations and spicing of various sausages became a culinary art in these

Mediterranean countries and later in upper Europe. These meat-processing operations
grew rapidly and have lead to the development of our current dry and semi-dry
 10

sausages. The manufacturing practices are still considered to be more of an art than a
science.

The dry and semi-dry sausages were developed to maintain stability under the
prevailing conditions of each area. The basis for processing the meat was preservation
by inhibiting or deterring microbial decomposition (Bacus, 1984). To direct the sausage
fermentation, "back slopping" was used originally, in which some meat from a previous
fermentation was added to encourage establishment of desired microflora. Presently
manufacturers use starter cultures (Smith and Palumbo, 1973). Fermentation as an
effective method of both preservation and flavor development appears to date from at
least several centuries BC (Smith, 1987). Coretti, (1971) defined fermentation as the
production of lactic acid, but he used the term "ripening" to refer to all the chemical,
physical, microbiological and enzymatic changes taking place in the sausage and which
are temperature and humidity controlled.

Whilst the use of fermentation was fortuitous, and not properly understood until recent
times it has been used for well over 2000 years thereby establishing a distinctive
category of sausage products. The process of salting, curing, smoking, drying and
fermenting all contribute greatly to the early development of sausage through their
preservation effects on the meat products. The addition of salt reduces the availability
of water to inhibit bacterial growth, whilst the addition of nitrite prevents the growth of
Clostridium organisms. Many of the phenolic compounds deposited by smoking makes
the surface of sausage much less suitable for microbial activity. Drying, like salting,
also reduces the availability of water whereas fermentation increases the population of
desirable bacteria, thereby making it difficult for undesirable bacteria to become
established (Smith,1987).

The efficacy of the process could only have been established by trial and error over a
long period of time. This gives rise to a deal of mythology surrounding traditional
sausage products to this day. In this, only back slopping is stuck to. The fact that
 11

without elaborate microbiological testing, one cannot be sure early enough that
fermentation in that preceding batch was complete, is ignored by the traditionalist.
(Smith, 1987).

The traditional process in the pre-1940 period relied on "natural fermentation" which
was governed by the controls inherent in the process. Some manufacturers observed
that, better consistency and stability were achieved by "back inoculating" portions of
the recently fermented meat into a freshly prepared batch. Both this "back slopping"
technique and the traditional process of relying on the natural fermentation by the
indigenous bacteria are still commonly practiced in modern times (Daly et al, 1973;
USDA, 1977).

1.3.2. Current practices

1.3.2.1. The use of starter cultures

It is now known that the processing of sausages involves a biological fermentation

process whereby specific bacteria and/or yeast, transform sugars to a variety of acids
and/or alcohol that inhibit undesirable micro-organisms including food pathogens.
These fermented meat owe their production, flavor, texture, nutritional, stability and/or
other characteristics to the activities of the beneficial micro-organisms (Bacus, 1984).

According to Lucke (1985), intensive research in sausage fermentation was only

initiated when the traditional empirical methods of manufactures no longer met the
requirement of large-scale, low-cost industrial production with short ripening and highly
standardized products (in the USA) in the 1930s. In Europe it was in the 1950s when
the first systematic studies on the microbiology and production of fermented sausage
were first published (Zeuthen, 1995). In 1940, following the successful use of starter
cultures in cheese fermentation, attempts were made to develop prepared starter
cultures for meat fermentation (Bacus and Brown, 1981). The concept of using an
 12

inoculum of a defined microbial culture originated in the United States with a series of
patents issued in the period 1920-1940 (Everson etaI., 1970; Kurk,U.S. Patent 1921).

However, the majority of these bacteria strains were of dairy origin and did not
proliferate in meat mixtures, presumably because the lack of tolerance to salt and/or
nitrite (Jensen and Paddock, 1940). Subsequent attempts utilized lactobacilli, the
predominant microflora of the marketed meat products, but these strains did not readily
survive lyophilization, which was the main method of distributing dairy starters.
Pediococcus cerevisiae was introduced as the first commercially available meat starter
culture, since it survived lyophilization (Deibel et aI1961).

The desirable micro-organisms have been isolated, identified, propagated separately in

the laboratory, and subsequently reintroduced as starter cultures. The starter cultures
are added to achieve a controlled fermentation that ensures and enhances the
production of the desired end products (Bacus, 1984). Presently, the primary bacterial
genera which are successfully utilized as meat starter cultures, are Micrococcus
(Niinivaara, 1955; Nurmi, 1966), Lactobacillus (Nurmi 1966; Everson et al., 1970), and
Pediococcus (Deibel and Niven, 1957).

In Europe, meat starter cultures consisting of specific strains of molds and yeasts are
also utilized for unique flavor development and prolonged shelf-life (Eilberg and Liepe,
1977; Coretti, 1977). New cultures are designed to yield unique f1avor attributes,
function as more effective preservatives of color and f1avor, and/or to retard rancidity
development and proliferation of undesirable micro-organisms. Consistent acid
production will be maintained through the utilization of culture blends, whereby the
specialized cultures will be combined with proficient acid producers (Bacus, 1984). The
emergence of genetic manipulating techniques probably will contribute to a greater
degree of control of microbial characteristics and improve production yields (Lee et al.,
1971).
 13

Basedon the large number of different sausage varieties recognized, it is not difficult to
account for this phenomenon. There are many possible variations associated with the
formulation, flavoring and processing of sausages. It requires only a relatively small
change in anyone of these, to make a substantial difference to the final product.
(Smith, 1987).

1.4. Bacterial fermentation

1.4.1. Lactic acid bacteria (LAB)

Micro-organisms important for the normal raw sausage aging/curing belong to the lactic
acid bacteria lactobacillus, Pediococcus and Micrococcaceae (Leistner, 1991). Lactic
acid bacteria as starter culture, cause the decline in pH by the fermentation of sugars
under which nitrite degradation is supported, which improves colour formation. They
also produce bacteriocin which in combination with reduced pH are capable of inhibiting
growth of undesirable micro-organisms. Reduced pH consequently produces a firm
product.

In meat fermentation two important types of microbial contaminants are required. One
is needed to reduce added nitrate to nitrite thus producing cured meat calor in sausage.
The second is needed to ferment the added sugar and produce the tangy flavor, which
characterizes these sausages (Deibel et al., 1961; Nurmi, 1966; Coretti, 1977). Lactic
acid bacteria and micrococcaceae starter cultures in perfect conditions are added to
sausage emulsion at levels of 106-107 org/g of meat. Yeasts are added at levels of
about 106 (Lucke and Hechelmann, 1987), the starter cultures grow to a high number
of about 108 and then stabilize during late ripening. Samelis et al. (1994), yeast
numbers were lower not exceeding 106cfu/g throughout processing. Lactic acid
bacteria produce lactic acid from the fermentation of sugar thereby decreasing the pH
and producing a sour or tangy taste.
 14

1.4.2. Micrococcaceae

Unlike lactic acid bacteria members of the family Micrococaceae are acid-sensitive.
Strain of the genera Micrococcus and Staphylococci by enzymatic actions reduce nitrate
to nitrite, and produce calatase capable of breaking down peroxides (Leisner, 1991;
Lucke and Hechelmann, 1987).

Micrococci capable of anaerobic growth ferment glucose (Jessen, 1995) Catalase

formation by Micrococcaceae and yeasts reduce peroxides consequently delaying the
onset of rancidity and giving flavour to the meat. (Leistner, 1991). Coagulase negative
nonpathogenic strains of Staphylococcus carnosus and Staphylococcus xylosus are often
used as starter cultures in dry sausage fermentation. Gokalp and Okerman (1985) and
Sanz et al. (1988) noticed a high numbers (108dujg) of lactic acid bacteria and
107dujg of Micrococcaceaeafter four days fermentation.

1.5. Yeasts associated with meat

Like maids, yeasts are usually present in low numbers on fresh meat but can compete
with bacteria if the surface of the meat becomes dry or competition with bacteria is
reduce due to the presence of sulphite (Dillon and Board, 1991). Jay and Margitic
(1981) reported yeast counts of 200 to 6.2 X 104jg on fresh ground beef. During low
temperature storage of meat, yeast counts may increase and eventually dominate the
microflora. Lowry and Gill (1984) observed that yeast counts on the loins of lamb
packaged in gas-permeable plastic film increased from 10jcm2 to 106jcm2 after storage
for 20 weeks at -5°C suggesting successful competition with psychrotrophic bacteria
flora (Cook, 1995).

In review by Jay (1978) species of Candida, Debaryomyces, and Torulopsis were listed
as the most frequently isolated genera from meats. Other genera associated with fresh
 15

meat include Bul/era, Cryptococcus, Pichia, Saccharomyces, Schizosaccharomyces,

Torulaspora, Trichosporon and Wil/iopsis. On processed meat such as sausages,
burgers, luncheon meat and smoked ham, Debaryomyces hansenii, Trichosporon spp.
and candida spp. such as Candida zeylanoides may form a significant component (Jay,
1978; McCarthy and Damoglou, 1993; Viljoen et aI., 1993). Although numbers of yeasts
on meat are generally lower than the numbers of spoilage bacteria, they can
occasionally proliferate to high numbers forming a visible surface slime, particularly on
some types of sausages. Dry cured meat products such as sausage and ham are
frequently contaminated with yeasts (Leistner and Bem, 1970) being more typical on
the surface than inside the product. The most common genera found included
Debaryomyces and Candida. On Spanish dry ham yeasts have been demonstrated in
high numbers throughout the ripening period (Casado et al., 1991).

1.5.1. Yeasts associated with fermented meats

Most studies on yeast in fermented meats have been focused on fermented sausages
rather than hams (Deak and Beuchat, 1987). Work performed by Leistner and Bem
(1970) and Monte et al. (1986) showed Debaryomyces hansenii as the most frequent
yeast on fermented meats despite the occurrence of Candida rugo5a, Candida cutenula
and Yarrowia lipolytica. Species of candida, Cryptococcus, Debaryomyces, Pichia,
Rhodotorula and Trichosporon have been isolated by Hadlock et al. (1976). Studies on
yeasts microflora of dry-cured Spanish hams showed numbers of 103jg after the
addition of salt rising to to 106jg in the middle of fermentation and stabilizing at 104at
the end of the curing process. (Huerta et al., 1988).

1.5.2. Yeasts as starter cultures

The intrinsic factors of sausage mix possess a natural selectivity for promoting the
development of the desired microflora. A traditional means of ensuring that the proper
microbial flora were present, was to add up to 5% of a previous mix to a fresh batch,
 16

the so called "back slopping" method, which has been known for centuries in bread
manufacturing. However, due to the increasing use of starter cultures, which are
applied to great effect in the dairy industries, attempts were made to develop starter
cultures for other foods such as meats (Jessen, 1995).

The starter cultures provide the sausage maker an additional control mechanism to
ensure that the product is what is desired. Micro-organisms are considered desirable as
natural food preservatives which have an extensive record of effectiveness and safety in
a wide range of food systems. The use of cultures can often preclude the necessity for
other food preservatives (Bacus, 1984). Although lactic acid bacteria and micrococci
are the predominant micro-organisms associated with fermented meats, yeasts have
also been incorporated. The yeasts used, have often been isolated from cured
fermented meats (Zeuthen, 1995). When added directly to meat mix at an inoculation
level of 106-107/g, the activity of the yeasts is mainly observed in the periphery, and the
oxygen consumption accelerates the exterior color formation. The addition of yeasts
result in a characteristic f1avor, particularly desirable for Italian types of sausages
(Leistner and Bern, 1970; Rossmanith et al., 1972; Coretti, 1977).

Although little is known about the growth and kinetics of yeasts in fermented meats,
the yeasts can tolerate the reduced water activity, high salt concentrations, low pH, and
may contribute to the organoleptic properties of salami. Debaryomyces and Candida
spp. growing on the surface, consume oxygen, degrade peroxide, show lipolytic and
proteolytic activity, reduce moisture loss during curing and protect the meat from light.
Both f1avor and colour, have been reported to be improved by the addition of
Debaryomyces hansenii (Rossmanith et al., 1972; Coretti, 1977).

Some starter cultures contain yeasts. Early workers such as Rossmanith et al (1972)
and Lucke and Hechelmann (1987) reported that Debaryomyces hansenii, identified as
D. kloeckeri, was found to give the best performance in dry sausage ripening. Coretti
(1973) in his review on the microbiology of fermented sausages, reported that rapid
 17

and stable development of red calor and acceptable aroma could be obtained with D.
kloeckeri, D. canterelli or D. pfaffi as well as with a mixture of micrococci, lactic acid

bacteria and D. hansenii .

Yeasts have also been used as starter cultures by Miteva et al (1986), who reported on
the use of Candida utilis. Similarly, Gehlen et al (1991) reported on the influence of
Debaryomyces hansenii in association with lactic acid bacteria and micrococci. In two
reports on soujouk, a fermented Turkish sausage made from beef and mutton, Gokalp
(1985, 1986) stressed how the use of various mixtures of cultures can be improved by
adding D. hansenii.

1.5.3. Importance of yeasts in fermented meat

In commercial starter culture preparations, a yeast strain is offered for exterior and
interior use (Rudolf Muller; Rhone-PoulencTexel; Laboratories Roger). The strain used
is classified as Debaryomyces hansenii. The species is characterized by high salt
tolerance, no nitrate decomposition and high oxygen demands (Jensen, 1995). When
added to the raw sausage mix as a starter culture (D. hanseniJ), it utilizes oxygen,
causing the sausage to turn red rapidly.

By breaking down fat and protein, forming specific metallic products, yeasts can
improve the aroma of fermented sausages (Metiva et et., 1986) and, by the formation
of catalase delay the onset of rancidity. In France consumers prefer sausages with a
fine white coating (sausage bloom), and accordingly yeasts are used to inoculate the
surface (Leisner, 1995).

In yeast and maid-ripened products, free fatty acids react with air oxygen, producing
first hydroperoxides (Cerise et al, 1973) and then aldehydes and ketones and volatile
fatty acids. These substances have a very strong aroma and are to be found especially
in high quality dry sausagesthat have been ripen for a very long time (Langner, 1972).
 18

CHAPTER II

THE GROWTH AND SURVIVAL OF YEASTS IN

COMMERCIAL SALAMI

A.A. Osei Abunyewa; B.C. Viljoen; *A. Hugo.

Department of Microbiology and Biochemistry; *Department of Food Science, U.O.F.S.,
P.O. Box 399, Bloemfontein 9300, South Africa.

Abstract

The survival of yeast during the production of commercial salami was investigated. 108
distinctive yeast strains were isolated and identified during the processing of the salami.
Initially, the number of yeasts remained below 103 duig, but their numbers increased
after the 12th day of maturation reaching a maximum of 2.0x 105duig at day 20.
During maturation, the pH declined from 5.72 to 4.36, water content from 58% to 43%
while the salt content increased by 1%. The number of lactic acid bacteria remained
above 105 duig throughout processing and maturation. Of the 108 yeast strains
isolated, 22 strains were identified as members of the species Debaryomyces hansenii
being present in all samples taken. Rhodotoru/a muci/aginosa, Bul/era variabi/is and
Cryptococcus a/bidus, in that order, were also frequently isolated during processing and
maturation.
 19

2.1. Introduction

Life is characterized by constant interactions between organisms; this obviously applies

to plants and animals but it certainly also applies to microbes. The functional ecological
co-existence of microbes within a limited biotope is referred to as bioecoenosis. The
species involved may either exert a positive effect on each other (synergism) or may
suppress each others growth and development (antagonism) (Liepe, 1981). With the
application of synergism and antagonism, starter cultures have been developed. The
use of a starter culture provides sufficient microbial numbers to ensure the numerical
dominance over the natural contaminating flora (as meat is an excellent growth
medium for the growth of microorganisms) which include pathogens. The use of
starter cultures in combination with the proper processing controls, therefore guarantee
the safety and quality of the final product (Bacus and Brown, 1981).

Systematic inoculation with microorganisms, as it has been in practice in other

nutritional sectors such as the brewing and dairy industries, was introduced to meat
technology only recently (as compared to when it was applied to nutritional sectors
mentioned). Nevertheless, it has been several decades that the fermentation
technology for meat products has been available for use of microorganisms as starter
cultures. The starter culture consists of single or mixed cultures of assorted non-
hazardous strains of microorganisms. The selected strains may induce specific
enzymatic activities to yield specific modification of the substrate under controlled
conditions (Liepe, 1978d). Under controlled conditions, it was possible to eliminate
potentially harmful salmonellae, staphylococci and clostridia. Consequently quality
requirements in food sanitation could be met (Barber and Diebel 1972; Halnes and
Harmon, 1973; Liebetrau and Grossmann, 1976; Niskanen and Nurmi, 1976; Sirvio et
al.,1977; Masters, 1979; Tanaka et al.,1980). Medical research on substitution therapy
and biotechnological investigations on starter cultures used in food production have
revealed a multitude of bacterial interactions in such biotopes as the intestinal tract
 20

(Reuter, 1965; Tramer, 1966; Dahiya and Speek, 1967; Bungay and Bungay, 1968;
Reuter, 1969; Daly et al., 1971; Reuter, 1972a-c and Rantala and Nurmi, 1973). In this
context, fermented sausages may act similarly as the intestines.

The inclusion of bacteria in the development of starter cultures has been frequently
investigated, while little attention is given to the role of yeasts in the fermentation of
sausages. Pioneer work on the yeast flora present in fermented sausages was
conducted by Cesari (1919) and Cesari and Guilliermond (1920) who established the
importance of the "fleur du saucisson"and recommended the use of pure yeast cultures
for flavouring in fermented sausages (Liepe, 1981). Work performed by Jay and
Margitic (1981) showed that on untreated meat like fresh ground beef, low counts of
yeasts (2x101-6.2x104 du/g) existed, which corresponded with similar findings of Dowell
and Board (1968) and Dyett and Shelley (1966) who stated that yeasts are common
contaminants of sausages. Several studies have since dealt with the yeasts supposed to
participate in the maturing of various dried meat products, contributing to the
organoleptic characteristics of the products (Arnau et al., 1987; Comi and Cantoni,
1983; Inigo et al., 1970; Smith and Palumbo, 1973). Strains of the genus
Debaryomyces predominated on dried sausagesdue to their exceptional high tolerance
of salt (Leistner and Bern 1970; Comi and Cantoni, 1980).

Rossmanith et al (1972) reported that curing color and flavour of sausages could be
improved by the addition of selected Debaryomyces strains as part of the starter
culture. Correti (1977) supported the findings and stressed that a combination of D.
hansenii, lactobacilii and micrococci resulted in better flavour and taste development of
sausages. It is evident from literature that yeasts are widely distributed on/in plants,
air, water, soil and animals (Walker, 1977). The live animal and particular its hide, hair
or fleece, obviously contribute substantially to the microbial contamination in the
abattoir (Empey and Scott, 1939; Ayres, 1955) and since meat is an ideal growth
medium for many microorganisms, yeast contamination may cause spoilage especially
when bacterial loads are inhibited.
 21

This study was therefore conducted to determine the growth and survival of the natural
contaminating yeasts present during the manufacture of commercial salami.

2.2. Material and Methods

2.2.1 Commercial salami manufacture

Three 10kg batches of salami were prepared. From each batch a total of 25 individual
salamis weighing 350g-450g were made according to the protocol indicated in Table
2.1. All meat portions were frozen at a temperature of -15°e to avoid "smearing" of the
fat on the lean meat surface, which may cause problems during sausage dehydration.
The fat used was from good quality pork lard. Meat portions and the ingredients as
given in Table 2.1 were mixed sequentially in a bowl cutter. Beef was chopped into
10mm particle size. Starter culture and spices were evenly sprinkled on the beef and
chopped to fine particles. Pork was then added and chopped to 20mm particle size
followed by the addition of lard and curing salt. Finally, all were chopped to particle
size of 4.5mm making sure that the entire batch was thoroughly mixed. The mixture
was carefully packed into ealpak Fibrous Bak 65/50 to avoid trapping air and both ends
were tied with strings. Three samples from each batch were weighed and marked for
weight loss determination. On sampling occasions, they were weighed to determine the
weight loss of the salami. The prepared salami samples were hanged on racks in a
fermentation chamber at a temperature of 22°e and relative humidity of 90% for 2days.
After 2days of fermentation, a 10 min smoke treatment at 18°e was carried out. Finally
the sausages were transferred to a drying room at a temperature of 12°e and 75-80%
RH until the product lost 20% and more weight.
Table 2.1: Composition of commercial salami

Ingredients Percentage by weight (%)

Beef 40.000

Pork 34.730

Pork back fat 20.000

Spices 2.176

Curing salt 3.046

Starter culture (freeze dried) o 11/26/98.500

Staphylococcus carnosus & Lactobacillus penttosus 2:1

22
 23

2.2.2 Sampling methods and selection of isolates

Samplesof the salami were taken from each batch on a 6hr basis during fermentation
for a 2 day period. After 2 days, sampling was performed consecutively every 48hrs.
One salami from every batch was transferred to the laboratory in a cooler box and
analyzed in duplicate on every sampling occasion. Samples of the fresh meat and
frozen meat were also taken.

2.2.3 Sampling during manufacture of salami

Salami samples were prepared for microbiological analysis by cleaning the surface of
the salami with 70% ethanol. A cut was made through the salami using a sterile knife
and the casing removed aseptically. 109 sample was cut using a sterile knife and
transferred into a Mason jar (Metaxopoulos et al. 1981b) containing 90ml of sterile
peptone water and blended for 3minutes Appropriate serial dilutions were made from
the slurry and plated on MRS, PCA, and YGC agar by the spread plate method and
incubated at temperatures as indicated in Table 2.2

2.2.4 Physical and Chemical Analysis

On every sampling occasion the aw, pH, °10 fat content, % protein content % salt
content and Oio moisture content were measured. The awof the salami was determined
on a TH 200 Novasina Thermoconstanter. With a Janke and Kunkei Ultra Turrax T.25,
109 of the samples were homogenized in 100mlof distilled water and the pH was
measured at 24°( with a HI 9321 Microprocessor pH meter (Hanna instruments). Salt,
protein, fat and moisture contents were determined by the methods proposed by the
Association of Official Analytical Chemist (AOAC) (1990).
Table 2.2: Culture media, temperature and times of incubation used for microbial analysis of

commercial salami

Incubation time (h) Growth medium

Yeast count, YGC 96 Yeast extract; glucose; chloramphenicol agar (Oxoid CM 139)

Yeast count, YM 96 Yeast extract malt extract agar (pH 3.5)

Total count, PCA 48 Standard plate count agar (Oxoid CM 361)

Lactobacilli, MRS 48 De Man Rosoga and Sharpe (MRS) (Oxoid CM 361)

24
 25

2.2.5 Yeast Identification

Representative yeast isolates obtained from the highest dilution on YGC plates were
purified by streaking on Yeast extract malt extract agar (YM) (Wickerham, 1951) and
maintained at 4°C on the same media. Strains were identified to the species level
according to the conventional methods of Barnett et al., (1983) and Kreger-van Rij
(1984).

2.3 Results and discussion

2.3.1 Physical and Chemical analysis

The pH in dry sausages as indicated by Demeyer et al. (1981) is principally determined

by lactate, ammonia and water content interacting with proteins resulting in the
variation of pH. The pH of the salami (Table 2.3) during the fermentation period of
48hrs increased from 5.72 to 5.88 within the first 18hrs ending at 5.78 after 48hrs.
During the ripening period, pH decreased gradually reaching a lowest value of 4.36 at
the end after 696hrs. The decrease in pH is attributed to the utilization of available
sugars by the lactic acid bacteria resulting in the production of organic acids. There
have been many debates regarding the precise pH and lor aw levels require for a shelf-
stable meat product that has not undergone heat processing. Many published criteria
suggested different threshold levels of pH and/or aw to secure the safety of the product.
(Lee and Styliadis 1996). Ledward (1985) suggested an aw < or = 0.85 or pH < 5 as
the criteria, while Canadian Federal Guideline suggested an aw< 0.92 and pH < 5.3.
Meisel et al (1989) studied the survival of salmonella in salami and indicated that
salmonella did not survive when the aw was < 0.96 and pH< 4.84.

According to Roca and Incze (1990) to secure the expected stability and safety of meat
that are not heated during processing and are consumed raw, the aw has to be in the
range of 0.80-0.94 depending on the drying process used and the actual pH value. The
 26

pH and awvalues reported in this study were less than the ranges given (fable 2.3). A
pH level less than 5.30 was obtained after 288 hrs while a aw level less than 0.92 was
reached after 432hrs. At the same time, the salami had lost 21.41% of its initial weight
(fable 2.3). Spoilage organisms and pathogens are not resistant to low moisture
content which corresponded with low aw values and furthermore the low pH enhances
the product shelf life. The final weight loss of the salami, which is a function of
moisture loss, was 26.66% when the water activity reached a lowest value of 0.909.
The moisture content decreased from 58% to 43.22% (fable 2.3) during the total time
of processing. The gradual moisture loss of the salami reflected the rate and extent of
increase in protein content of the salami from 16.834% to 22.711%, fat content from
19.659% to 26.487% and salt content from 3.070% to 4.131% during the processing
period (fable 2.3).

2.3.2 Microbial Enumeration

Table 2.4 shows the comparative analysis of microbial growth. Lactic acid bacteria
counts were obtained on De Man, Rogosaand Sharpe agar (MRS), total bacteria counts
were determined using standard plate count agar (PCA) and yeast counts on yeast
glucose chloramphenicol agar (YGC). Lactic acid bacteria numbers increased gradually
from log 5.36/g from the beginning of processing and reached a maximum of log
6.52/g after 240 hrs during the ripening stage. The total bacteria and lactic acid
bacteria counts were similar during fermentation and maturation with the exception of
the time lapse between 42 and 96 hrs. The similar counts clearly indicated that the
processing of salami is mainly governed by the added starter culture and the possible
inhibition of normal contaminating bacteria, which corresponded with the decline in pH
as a result of the production of lactic acid bacteria. The total bacteria numbers
increased from log 4.99/g to log 6.89/g during the fermentation stage. The high
bacterial numbers during the fermentation stage may contribute to the reduction of
nitrate to nitrite thereby influencing the appearance, odour, flavour and safety of the
product (Adams, 1986). Bacterial numbers continued to increase (Coretti, 1956) during
Table 2.3: The changes in chemical and physical analysis during the fermentation of salami over a period of 700hrs

Salt content ("lo) pH Wt Loss ("lo) aw

Protein content (%) Moisture content ("lo) Fat content ("lo)
Time (h)
--

Fermentation
3.0707 5.72 0 0.950
16.834 57.986 19.659
0
3.0983 5.72 0.89 0.949
16.964 57.008 19.832
6
3.1474 5.80 2.42 0.900
17.255 56.943 20.135
12
3.179 5.88 3.39 0.947
17.427 56.504 20.337
18
3.2196 5.83 4.60 0.947
17.649 55.945 20.812
24
3.2541 5.80 5.60 0.947
55.475 20.812
so 17.838
3.2943 5.00 6.91 0.946
18.057 54.918 21.070
36
3.3272 5.79 7.91 0.944
18.282 54.325 21.342
42
3.3373 5.78 8.87 0.944
18.484 53.848 21.563
48

Wt loss = Weight loss aw = Water activity

Table 2.3 continued

pH Wt Loss (%) aw
Moisture content (%) Fat content (%) Salt content (%)
Time (h) Protein content (%)
-

Ripening
5.64 11.(15 0.936
22.084 3.4551
96 18.940 52.644
5.58 12.58 0.938
22.467 3.5149
144 19.270 51.917
5.55 13.80 0.937
22.806 3.5675
192 19.558 51.200
5.45 15.23 0.930
23.168 3.6233
240 19.864 50.436
5.31 16.90 0.929
23.640 3.6960
288 20.263 49.441
4.96 18.41 0.923
24.079 3.7640
336 20.636 48.511
4.60 19.95 0.922
24.546 3.8367
384 21.035 47.523
4.88 21.41 0.922
24.800 3.8893
432 21.229 46.908
4.77 22.21 0.919
25.072 3.9185
480 21484 46.401
4.72 23.00 0.919
25.393 3.9666
528 21.746 45.735
4.53 24.09 0.921
25.719 4.0149
576 22.010 45.058
4.52 25.35 0.917
26.104 4.1063
624 22.326 44.251
4.36 26.66 0.909
26.488 4.1312
696 22.711 43.220

wt loss = Weight loss aw = Water activity

 29

the first part of the maturation stage probably due to the higher pH values and water
content present at the time. After 240hrs, however, the bacterial numbers remained
constant.

Although no yeasts were added to the salami formulation an increase in yeast numbers
from log 3.66/g to Log 5.36/g during ripening was noticed. The high yeast numbers is
attributed to natural contaminating yeast present in the meat (Dillon and Board, 1991),
processing equipment, and workers' hands and aprons. Yeast counts remained low
during the fermentation stages when bacterial numbers progressed but their numbers
rapidly increased during the maturation stage. A significant increase (log 2/g) in yeast
numbers, was observed during the maturation stages when the bacterial numbers
stabilized (Table 2.4). The highest yeast count of log 5.36/g was observed after 528
hrs. The high number of yeasts observed during the later stages of maturation suggests
that the yeasts may have played an important role in the ripening of the salami.

The progressive growth of lactic acid bacteria during the fermentation stages, and
yeasts during the ripening stages may indicate a competition between the
microorganisms for available substrates. However, the interaction between the yeasts
and lactic acid bacteria at the later stages appears to be synergistic since both
populations continued to survive at high numbers with none being inhibited by the
other.

2.3.3 Yeast enumeration

Of the yeasts isolated, 12 species from 7 different genera were isolated from the raw
meat (Table 2.5), Candida, Bul/era, Trichosporon, Cryptococcus, Rhodotorula,
Debaryomyces and Lipomyces. Dillon and Board (1991) have confirmed most of these
yeasts as natural flora present in the hide, fleece, and carcasses of field animals. In
addition to the above genera, six other genera were isolated from the salami during
processing, (Schwanniomyces, Galactomyces, Sterigmatomyces, Pichia, Torulaspora and
Table 2.4: Log counts s" during the production of salami over a period of 700hrs

log counts g-1

Time (hrs) PCA MRS YGC

Fermentation

0 4.00 5.36 3.90

6 5.36 5.88 3.51

12 5.61 5.71 3.64

18 5.45 5.63 3.00

24 5.45 5.41 3.91

eo 5.43 5.78 3.90

36 5.64 5.91 3.72

42 6.63 6.03 3.49

48 6.89 6.08 3.57

Ripening

96 6.27 6.11 3.66

144 6.26 6.21 4.24

192 6.47 6.39 4.39

240 6.58 6.52 4.52

288 6.48 6.33 4.43

336 6.49 6.42 4.24

384 6.42 6.34 5.08

432 6.33 6.31 5.31

480 6.43 6.43 5.25

528 6.57 6.14 5.36

576 6.53 6.48 5.17

624 6.23 6.14 5.24

696 6.35 6.39 5.18

PCA= Total bacteria counts MRS= Lactic acid bacteria counts YGC= Yeast counts

30
Table 2.5: Yeasts associated with salami processing

Source of contamination

Fresh meat Frozen meat

Isolates Beef Pork Lard Beef Pork Lard

Bul/era variabilis + + +

Candida haemu/onii +
Candida vinaria +

Candida zey/anoides + + + +

Cryptococcus a/bidus + +

Cryptococccus hungaricus +

Cryptococcus /aurentii +

Debaryomycse hansenii + + + + +

Lipomyces tetrasporus +

Rhodotoru/a minuta + + +

Rhodotoru/a muci/aginosa + + +

Trichosporon beigeIii + + + + +
 32

Sporobolomyces). candida, Cryptococcus, Debaryomyces, Galadomyces, Rhodotorula,

Pichia, Trichosporon and Torulaspora were frequently isolated from raw and fermented

meats (Leistner and Ayres, 1968; Hadlock et al, 1976; Smith and Hadlock, 1976; Jay,
1978; Comi and cantoni, 1983; Williams, 1990; Mccarthy and Damoglou, 1993). The
other yeast species being present might have originated from the meat, hands of those
who prepared the salami, processing equipment or the air.

Although the initial yeast flora present in the sausage emulsion and raw meat was
extremely variable, D. hansenii strains were isolated frequently during the fermentation
and ripening stages. The species appeared to be the most abundant yeast species
associated with the processing of salami (Table 2.6) representing 20.37% of the total
number of yeast strains isolated. The frequent presence of the species corresponded
with results obtained by various authors (Hammes et al, 1985; Lucke and Hechelmann,
1987; Samelis et al., 1994). Rhodotorula mucilaginosa, a typical air contaminant strain,
described by Deak (1993) as a frequent food isolate followed with 14.81%. Bul/era
variabilis, Cryptococcus albidus, and Trichosporon beigeIii were isolated at percentages

of 13.89%, 10.18% and 9.26% respectively while the same number of Candida
zeylanoides and Schwanniomyces occidentalis strains were isolated, 5.5%. Each of the
remaining species represented less than 5% of the total number of yeast isolated.
Torulaspora delbruecki, Rhodotorula mucilagenosa, R. minuta, Cryptococcus albidus, C
laurentii, Candida zeylanoides and Galactomyces geotrichum are reported to be
frequently isolated from meats and meat products. Deak and Beauchat (1996)

The incidence of yeast isolated throughout the salami processing is shown in Table 2.7.
C gropengiesseri, P. philogaea, P. farinosa, R. minuta, S. halophilus and Sp roseus
were isolated only during the fermentation stage which may be an indication that these
yeast were inhibited by the reducing water activity. Despite the frequent occurrence of
T. beigeIii strains during the fermentation stage, no strains appeared during the
ripening stages. T. delbruecki, D. polymorphous, S. occidentalis and C haemuloni/were
not frequently isolated during processing which may be an indication that these yeast
Table 2.5: Yeasts associated with salami processing

Source of contamination

Fresh meat Frozen meat

Isolates Beef Pork Lard Beef Pork Lard

Bullera variabilis + + +

Candida haemulonii +

Candida vinaria +

Candida zeylanoides + + + +
Cryptococcus albidus + +

Cryptococccus hungaricus +

Cryptococcus laurentii +

Debaryomycse hansenii + + + + +

Lipomyces tetrasporus +

Rhodotorula minuta + + +

Rhodotorula mucilaginosa + + +

Trichosporon beigeiii + + + + +
------- - -
Table 2.6: Frequency of occurrence of yeast isolates during salami processing over a period of

696 hrs
Number of Strains %
Isolates

15 13.89
Bul/era variabilis
5 4.29
Candida haemu/onii
0.93
Candida gropengiesseri
6 5.55
Candida zey/anoides
11 10.18
Cryptococcus a/bidus
22 20.37
Debaryomyces hansenii
1 0.93
Debaryomyces po/ymorphus
5 4.29
Debaryomyces vanrijiae
0.93
Galactomyces geotrichum
0.93
Pichia farinosa
1 0.93
Pichia philogaea
2 1.85
Rhodotoru/a minuta
16 14.81
Rhodotoru/a mucilaginosa
1 0.93
Sterigmatomyces ha/ophilus
6 5.55
Schwanniomyces occidentalis
0.93
Sporob%myces roseus
10 9.26
Trichosporonn beige/ii
0.93
To/u/opsis de/bruecki

34
Table 2.7: The incidence of yeast during salami processing over a period of 696 hrs

Time (hrs)

Maturation/Ripening
Fermentation
12 18 24 3:) 36 42 48 96 144
Yeast Strain 0 6

+ + + + + +
B. variabilis + +

C. gropengiensseri +
+ +
C. haemu/onii
+ + + +
C. zey/anoides
+ +
Cry. a/bidus
+ + + + + + + +
Dhansenii + + +
+ +
D. vanrijiae
G. geotrichum +

P. tarinosa +
+
P.philogaea
+ +
R. minuta
+ + + + +
R. mucilaginosa + +
+ +
S. ha/opilus
+ +
S. occidentalis +
+
Sp. roseus
+ + + + + +
T. beigeIIi + + +
Table 2.7: continued

Time (hrs)

Maturation/Ripening
Yeast strain 192 240 288 336 384 432 480 528 576 624 696

B. variabilis + + + + + + +

C. haemulonii + +
C. zeylanoides + +

Cry. albidus + + + + + + + + +

D.hansenii + + + + + + + + + + +

D. polymorphus +

D. vanrijiae + + +

G. geotrichum +

R. mucilaginosa + + + + + + + + +

Sw. occidentalis + + +

T. beigeIii +
To. delbruecki +

B.= Bul/era C.= Candida Cry.= Cryptococcus D.= Debaryomyces G.= Galactomyces P.= Pichia R.= Rhodotorula S.= Sporoblomyces St.= Sterigmatomyces SW.= Schwanniomyces

T.= Trichosporon To.= Torulaspora

 36

strains are not part of the yeast community of the salami that developed during salami
processing. Since small portions (lOg) of the salami were taken for microbial analysis
they might have been missed out during sampling. Deak and Beuchat (1996) indicated
that micro-organisms favoured in foods are those that posses the necessary
physiological attributes to respond to ecological determinants. The frequent
occurrences of Calbidus, 8. variabilis, R. mucilaginosa, C zeylanoides and D. hansenii
can therefore be attributed to their tolerance of low temperatures, high salt
concentrations low pH levels and resistance against their environment. C albidus an
anamorphic yeast with basidiomycetous affinity exhibits proteolytic activity (Huerta et
al., 1988) and may cause spoilage by hydrolysation of the proteins in the salami. D.

hansenii and C zeylanoides are able to reduce the fat rancidity of the salami by
hydrolyzing lipids through lipolytic activity (Metiva et al, 1986). Furthermore, most of
these species have the ability to utilize organic acids produced by the lactic acid
bacteria (Fleet, 1990; Besancon et al. 1992; Roostita and Fleet, 1996) which result in an
increase in pH. An increase in pH due to excessive growth of contaminating yeasts,
might result in a decline in its preservation action (Fleet, 1992) making the salami
susceptible to microbial spoilage of pathogens and undesired bacteria. Strong growth
in the presence of salt, growth at low temperatures and the ability to utilize organic
acids are considered as key determinant that encouraged the presence of
Debaryomyces hansenii (Guerzoni, 1993b; Van Eck et al. 1993). Considering that this
species was the most resistant and proliferating yeast found in this study, further
research on its effect on organoleptic characteristics of salami and interaction with the
lactic acid bacteria seems promising.
 37

CHAPTER III

KEY PROPERTIES FOR THE SELECTION OF YEASTS AS

POSSIBLE STARTER CULTURES
IN THE MAKING OF SALAMI

Abstract

Practices in the manufacture of raw, dry sausage have been of much public concern
since the finished products are not cooked before eating. The use of starter cultures
assures better quality products with shorter production time, and a longer shelf-life.
Care must be taken, however, not to put the consumers' health at risk since raw meat
is an ideal habitat for the growth of pathogenic micro-organisms. Nineteen yeast
species isolated from the natural microflora of commercial salami during production
were examined based on relevant key properties proposed for selecting good starter
cultures. All the yeasts survived NaCl concentrations at 4-8%, 80-240ppm nitrite
concentrations and lacked proteolytic activity except for Trichosporon beige/ii. Lipolytic
activity proved to be variable. Selected lipolytic positive Debaryomyces hansenii and D.
po/ymorpus strains were inactivated at temperatures above 50°C.

3.1. Introduction

The application of starter cultures in the fermentation of food products is derived from
the isolation and identification of micro-organisms responsible for the desired effects
and its addition to fermented food at the appropriate stage of processing (Bacus,
 38

1984). This resulted in the selection of lactic acid bacteria, Micrococcaceae (Jensen and
Paddock, 1940), and yeast strains (Coretti, 1977) for use as starter culture in sausage
fermentation.

Flavour development in raw sausages is attributed to the action of microbial enzymes

(Coretti, 1965) while lowering the pH and, the excretion of anti-microbial compounds
ensure a safe and long shelf-life stable product (Lucke and Hechelmann, 1987). The
first starter cultures were commercially available in the 60's, although suggestions and
patent registration had long been made in 1919, for the use of various micro-organisms
in the preparation of dry sausages (Coretti, 1977; Bacus and Brown, 1981; Liepe 1983).
In the USA, Deibel (1956), Deibel and Niven (1957) and Deibel et al. (1961)
recommended Pediococcuss cerevisiae to be used in meat fermentation as starter
culture. Yet the first starter culture used on a large scale consisted of Pediococcus
acidulatici, whereas Micrococcus ("M53") proposed by Niinivaara (1955), was used in
Europe.

Many patent publications proposed the use of Pediococcus and Lactobacillus plantarum
(Everson et al., 1974), as well as mixed cultures (Gryczka, 1977; Gryczka and Shah,
1979). Bacterial starter cultures in Europe therefore, comprised of staphylococci in
combination with lactobacilii or pediococci (Coretti, 1977; Liepe, 1978d; Bacus and
Brown, 1981). A changeover to mixed cultures consisting of lactic acid bacteria used in
combination with Micrococcaceae was established a few years later in Central Europe
(Lucke and Hechelmann, 1987). Although mould ripened sausagesare not so common,
patented procedures for the covering of fermented sausages with mycelia were
established in the sixties. Racovita and Racovita (1968) later described the method of
spraying sausages with spores of certain species of Penicillium. Research on the
improvement of fermented sausages resulted in the inclusion of yeasts as potential
starter cultures for meat fermentation as eventually applied in the fermentation of
Italian salami (Liestner and Bem, 1970; Coretti, 1977).
 39

To be accepted as starter culture, it is expected that the micro-organisms used be more

reliable and faster growing than the natural microflora, without endangering the quality
of the product or health of the consumers (Lucke and Hechelmann, 1987). Lactic acid
bacteria involved in meat fermentation produce organic acids, which inhibit the growth
of undesirable micro-organisms and contribute to the development and stability of
colour and aroma. Micrococcaceae strains reduce nitrate to nitrite and form catalase
which breaks down peroxide (Lucke, 1985). Moulds protect the surface of the dry
sausage from sunlight and oxygen, and prevent colonization of other pathogenic
moulds. Stiebing and Rhodel (1988) reported that Penicillium nalgiovence contributed to
better aroma. Yeasts contribute to the safety by breaking down peroxides within the
sausage and utilize oxygen, which might be available for the growth of undesirable
micro-organisms.

During the production of commercial salami, Debaryomyces hansenii strains were most
frequently isolated. Coretti (1973) reported rapid and stable red meat colour
development and acceptable aroma with the application of Debaryomyces kloeckeri, D.
cantarelli or D. pfaffi as well as with a mixture of micrococci, lactic acid bacteria and D.
hansenii. Huerta et al (1988) found Debaryomyces spp. to be the dominant yeast in
ham. Debaryomyces hansenii has been indicated by Leistner and Bem (1970), Jay
(1978), Monte et al (1986), Mccarthy and Damouglou (1993) and Viljoen et al (1993)
as the most significant yeast in processed meat. Good performance of D. hansenii in
sausage ripening was reported by Rossmanith et aI., (1972) and Gokalp (1986). D.
hansenii, a psychrotrophic yeast species can tolerate low pH values, low water activity

and high salt content, (Besancon et al., 1992; Roostita and Fleet, 1996) Besancon et al
(1992) demonstrated that some strains of D. hansenii could tolerate up to 20% NaCI.
Based on these properties, Vayssier (1979) suggested the use of D. hansenii as starter
culture alone or in combination with Penicillium nalgeovense.
In a previous study, two Debaryomyces species identified as Debaryomyces hansenii
and Debaryomyces polymorphus were frequently isolated during the processing and
ripening of commercial salami indicating that they were able to survive at low pH levels,
 40

water activity and high salt concentrations. The two species are therefore selected for
further examination and compared with other yeasts naturally isolated during salami
making. The yeasts are selected based on some of the key properties (Deibel, 1974)
proposed to select a good starter culture.

Characteristics of a good starter culture as suggested by (Deibel, 1974) are the

following:
1. Should be salt tolerant
2. Have the ability to grow well in the presence of 80-100ppm nitrite
3. Have an optimum growth at 32.2°C with series of 26.6-43°C
4. Be non-proteolytic
5. Be non-lipolytic
6. Must not produce off flavors as by-product of fermentation
7. Be non pathogenic
8. Be inactive at temperatures above 50°C
9. Have a rapid growth in 6% pickling solution.

3.2. Materials and methods

Yeasts isolated and identified according to the conventional methods of Barnett et al

(1983) from a previous study (Table 3.1), were maintained on YM (yeast extract malt
extract, Wickerham, 1951) agar stored at 4°(.

3.2.1. Salt tolerance

The isolated yeasts were inoculated into sterile test tubes containing YM broth medium
(Table 3.2) containing 4%, 6%, and 8% NaCland incubated at 25°C for 5 days. Media
appeared cloudy with positive results and remained clear with negative results.
Table 3.1 List of yeast isolates identified previously

l. Bul/era variabilis 16. Sterigmatomyces roseus

2. Candida haemulonii 17. Schwanniomyces occidentalis

3. Candida gropengiesseri 18. Trichosporon beigelii

4. Cryptococcus albidus 19. Torulaspora delbruecki

5. Cryptococcus laurentii

6. Candida zeylanoides

7. Debaryomyces hansenii

8. Debaryomyces polymorphus

9. Debaryomyces vanrijiae

10. Galactomyces geotrichum

Il. Pichia farinosa

12. Pichia philogaea

13. Rhodotorula minuta

14. Rhodotorula mucilaginosa

15. Sporobolomyces halophilus

Table 3.2: Media formulation, sterilizing temperature and time

Media Ingredients Quantlty(g/ml) Sterilizing temperature (OC) Time (mln)

YM broth Yeast extract 3g 121 15

Malt extract 3g
Peptone Sg
Glucose 10g
Distilled water 1000ml

Casein Digestion Skim milk powder 50g 121 15

Bacteriological agar 25g

Distilled water 1500ml

Tributyrin Peptone Sg 121 15

Yeast extract 3g
Glycerol tributyrate 10g
Bacteriological agar 20g
Distilled water 1000ml

Rhodamine B Nutrient broth 8g 121 15

NaCI 4g

Bacteriologocal agar 16g

Olive oil 32.25ml
Distilled water 1000 ml
 43

3.2.2. Nitrite tolerance

Yeast isolates (Table 3.1) were streaked on sterile Petri dishes with YM agar containing
80ppm, 160ppm and 240ppm nitrite and incubated at 25°C for Sdays (Table 3.2).

3.2.3. Proteolytic activity

Yeast isolates (Table 3.1) were streaked on Petri dishes with casein digestion agar
(Ahearn et al., 1968), and incubated at 25°C for 5 days (Table 3.2). Clear transparent
zone around the streaked area indicated a positive result

3.2.4. Lipolytic activity

Isolated yeasts (Table 3.1) were streaked on sterile petri dishes with olive oil and
Rhodamine B agar (Kouker and Jaeger, 1987) and Tributyrin (glycerol tirbutyrate)
(Fryer et al., 1966) agar, and the plates were incubated at 30°C for 3 to 6 days.

Rhodamine B (Table 3.2) was emulsified with a Brason Sonifer Cell Disrupter B-30 and
the final pH of the solution adjusted to 7.0 before sterilizing. Media were cooled to
70°C and 10mlof filter sterilized Rhodamine B solution [(0.1% wjv) final concentration]
added. The mixture was shaken and allowed standing for 10min to reduce foaming
before pouring into sterile Petri dishes. Flourescence of the streaked area under a
flourescent light in the dark gave positive results

3.2.5. Rate of inactivation at temperatures above so: C

Yeast isolates (Table 3.1) were seeded in triplicate in YM broth (Table 3.2) (200ml) in
1000ml Elernmeyer flasks and incubated at 50°C, 55°C and 60°C with vigorous agitation
on rotary a shaker (180rpm) through 1S0min. From each flask 10ml was taken for
microbial enumeration at consecutive intervals of 6 hrs for a period of 60hrs. Serial
 44

dilutions were prepared in buffered peptone water and spread plated on YGC (yeast
glucose chloramphenicol) agar. Highest serial dilutions which represented colonies
between 30-300 colonies per plate were counted.

3.3. Results and discussion

Resultsobtained are indicated in Table 3.3 indicating the ability of all the isolated yeasts
to grow in the presence of 4-8% NaCl and 80-240ppm nitrite, proteolytic and lipolytic
activities are also represented in Table 3.3. Several reports referred to the isolation of
yeasts from dry cured meats with reduced product aw by the addition of salts (Comi et
al., 1982; Monte et al., 1986; Huerta et al., 1988; Molina et al., 1990; Gimenez, 1992).
In a review by Jay (1978), candida, Debaryomyces and Torulopsis (now regarded as
Candida) were listed the most frequently isolated genera from meats. Other genera
associated with fresh meat include Bul/era, Cryptococcus, Pichia, Saccharomyces,

Schizosaccharomyces, Torulaspora, Trichosporon and Wil/iopsis. McCarthy and

Damouglou (1993) and Viljoen et al. (1993) reported on the predominance of Candida
spp., Trichosporon spp. and Debaryomyces hansenii in sausage, luncheon meat and
smoked ham and considered these species as part of the established yeast community.
Work performed by Saldanha-da Gama et al. (1997) showed the predominating yeasts
in Portuguese pork based products to be Debaryomyces hansenii, Cryptococcus
laurentii, Cryptocuecus humicolus, D. polymorphus and Pichia spp. These authors noted

that all yeast isolated from ham were able to grow in the presence of 5-8% NaCI.
Some D. hansenii strains have been demonstrated to tolerate up to 20% NaCl
(Besancon et al., 1992).

Yeasts grow at lower aw levels in a sugar medium than compared to growth in a salt
medium (Van Eck et al., 1993). Tokuaka et al. (1985) listed 30 yeast strains isolated
from high sugar foods, which showed exceptional tolerance against high sugar
concentrations capable of growth at a aw range of 0.912-0.876. Torulaspora delbrueckii
and D. hansenii are typical osmotolerant species isolated from high sugar foods..
Table 3.3: Key properties of a good starter culture

Nitrite tolerance Salt tolerance Lipolytic acivity Casein digestion

Concentration (ppm) Concentration (%)

Isolates 80 160 240 2 4 6 Tributyrin Rhodamine B

1. Bul/era variabilis + + + + + + + +
2. C. haemulonii + + + + + + + +
3. C. gropengiesseri + + + + + + + +
4. Cry. albid':ls + + + + + + + +
5. Cry. laurenti + + + + + + +
6. Cry. zeylanoides + + + + + + + +
7. D. hansenii + + + + + + + +
7. D. hansenii + + + + + + +
8. D. polymorphus + + + + + + + +
9. D. vanrijiae + + + + + + +
9. D. vanrijiae + + + + + + + +
10. G. geotrichum + + + + + + + +
11. P. farinosa + + + + + + + +

C.= Candida Cry.= Cryptococcus D.= Debaryomyces G.= Galactomyces P.= Pichia
ppm = Parts per million % = percentage
Table 3.3: continued

Nitrite tolerance Salt tolerance Lipolytic acivity Casein digestion

Concentration (ppm) Concentration (%)

Isolates 80 160 240 2 4 6 Tributyrin Rhodamine B
-

12. P. philogaea + + + + + + + +
13. R. minuta + + + + + + +

14. R. muci/agenosa + + + + + + +
15. S. ha/ophilus + + + + + + + +
16. St. roseus + + + + + + + +

17. Sw. occidenta/is + + + + + + + +

18. T. beige/ii + + + + + + + + +
19. To. de/brueckii + + + + + + + +

P.= Pichia R.= Rhodotorula S.= Sporoblomyces St.= Sterigmatomyces Sw.= Schwanniomyces T.= Trichosporon To.= Torulaspora
ppm =Parts per million %= percentage
 47

Touaka et al (1991) indicated the minimum aw for growth in NaCI for D. hansenii, T.
delbruecki and Rodotorula mucilaginosa are 0.84, 0.90 and 0.90 respectively.
According to Van Eck et al. (1993) the minimum aw for growth in NaCl is 0.93 for
Schwanniomyces occidentalis, 0.90 for Pichia farinosa and 0.88 for D. hansenii. The
spoilage of yeasts in meat products due to tolerance of low aw, values, however, is not
considered to be of any great importance (Deak, 1991), the spoilage appears to be
related to the yeasts' proteolytic and lipolytic activity which also include desirable
effects.

Table 3.3 shows the lipolytic and proteolytic activities of the yeasts. All the species
exhibited no proteolytic activity, except Trichosporon beigeIii. Huerta et al. (1988)
found that Trichosporon spp. were the only yeast species with proteolytic activity in
ham. All the yeast isolates included in this study proved to be lipolytically positive with
the exception of Rhodotorula species and Cryptococcus laurentii. Debaryomyces spp.
showed variable lipolytic results. The high frequency of yeasts presenting lipolytic
active results is in agreement with report by of Saldanha-da-Gama et aI., (1997).

Despite several good characteristics presented by most of the isolated yeasts, two
strains from the genus Debaryomyces were selected for use as starter cultures in the
production of commercial salami as they appeared to remain stable in meat related
environments, reaching high population cells when grown in meat (DoweIl and Board,
1968; Samelis et al, 1994). Work done on yeasts in meats and meat products depicted
the promising use of Debaryomyces, especially D. hansenii as a good starter culture for
sausage production. (Coretti, 1977; Hammes et aI., 1985; Lucke and Hechelmann,
1987; Gehlen et aI., 1991). Yeasts are generally not considered to be of any
importance in the spoilage of refrigerated, cured and vacuum packed meat, and related
meat products (Jay, 1987; Dillon and Board, 1991).

The positive contributions of yeasts to humankind are known and their public health
significance in foods and beverages has been considered negligible. Debaryomyces
 48

hansenii is regarded generally as safe and obtained GRAS status (Fleet, 1992)

Lipolytically active Debaryomyces hansenii and D polymorphus strains were selected

and their survival rate at high temperatures determined. Both species were inactivated
within 60 hrs at temperatures above 50°C (Figs 1, 2 and 3). Yeasts are generally killed
within minutes at temperatures between 55°C and 65°C however, the composition of
food affects the rate of in-activation (Lund, 1951; Put and De Jong, 1982b; Su and
Beauchat 1984; Engel et aI.1994). Debaryomyces spp. are capable of surviving a wide
range of temperatures with an optimum growth temperature of 32-37°C (Vidal-Liera et
al., 1979).

However, species were isolated from sugar cane mill capable of growth at 40°C (Stokes,
1971). The minimum growth temperature for most yeast is Doe, although obligate
pychrophlic strains can grow at -7°C. D. hansenii is frequently isolated from chilled
foods (Guerzoni et aI., 1993b).

Despite the less obvious but detrimental effects, including production of off-flavours and
taints, the production of metabolites such as acetaldehyde that can neutralize the
preservative effects of sulphur dioxide and the utilization of sodium nitrite added as
curing agent and preservative, (Dalton et aI., 1984; Dillon and Board, 1990), D.
hansenii contributes positively to product quality in Italian salamis (Grazia et aI., 1989).
Based on the results obtained in this study, and the proposed key characteristics to
assure a stable starter culture (Deibel, 1974). Debaryomyces spp proved to be
accepted as a potential starter culture in salami processing.
 SUvivaI at 5O"C

8
7

:ês
::J

~4
8'3
..J

1
0
0 20 40 00 00

llme

Fig. 1 The survival of D. hansenii and D. polymorphus at 500e

Survival at 55°C

--<>-0. hansenii
--r.J--D. poJymorphus

10 15 20
time (hrs)

Fig. 2 The survival of D. hansenii and D. polymorphus at 55°e

10 rs 20
Time (hrs)

Fig. 3 The survival of D. hanserui and D. polymorphus at 60

0
e
 50

CHAPTER IV

THE' APPLICATION OF YEAST STRAINS AS STARTER

CULTURES IN TRADITIONAL FERMENTED SAUSAGE

A. A. Osei-Abunyewa, B. C. Viljoen, *A Hugo.

Department of Microbiology and Biochemistry, *Department of Food Science U.F.S.,

Bloemfontein, 9300,South Africa

Abstract

Natural yeast strains isolated from traditional salami identified as Debaryomyces

hansenii and Debaryomyces polymorphus were incorporated as starter cultures in the
production of salami. Debaryomyces hansenii in combination with lactic acid bacteria
were used as starter culture in the first batch (DhS). In the second batch (Dh),
Debaryomyces hansenii as a single strain was used. The third batch (DhDp) contained
Debaryomyces hansenii in combination with Debaryomyces polymorphous as starter
culture, a fourth batch (S) serving as control contained lactic acid bacteria as starter
culture. After a maturation time of 624 hours, all the batches attained a 20% moisture
loss require for maturity with the exception of batch 4 which had obtained a moisture
loss of 19.05°/0. DhDp obtained 21.0659% moisture loss, which was an equivalent of
the weight loss; DhS attained 20.7834% and Dh, 20.3484%. The results obtained for
physical and chemical analysis between the different batches were similar. The salamis
appeared reddish brown after 48hrs but the batch containing yeast and lactic acid
 51

bacteria as starter cultures was slightly firmer than the others were. The pH values of
the salamis at the end of maturation were higher for batch 2 using a yeast isolate as a
single starter culture, while the highest pH values were obtained when two yeast strains
were incorporated.

4.1. Introduction

Ready-to-eat dried meat products, which mayor may not be fermented, present a
unique concern because they are prepared from raw meat which has not been cooked
(Lee and Styliads., 1996). No matter how hygienic raw meat may appear, it contains
numerous micro-organisms, which may potentially be harmful. Jay (1996) established
that the typical contaminating microflora in fresh meat and meat products
predominantly consist of bacteria. Yeast, however, despite limited references, also play
a substantial role in the spoilage of meat and meat products (Deak 1991, Fleet 1992).
Nevertheless, occasionally the role of yeasts in meat has been described (Dalton and
Board, 1984; Bank and Board, 1987; Viljoen et al., 1993).

Bacus (1986) and Lucke (1988) reported that various microbial groups are involved in
the ripening process of dry fermented sausage ie. lactic acid bacteria, micrococcaceae,
yeasts and fungi. According to Metaxopoulos et al. (1996) the microorganisms
suppress the growth of pathogenic organisms due to their enzymatic activities while
yeast play a dual role by either contributing to the final product or being detrimental in
causing spoilage. Most sausage industries ignore the importance of yeast strains as
starter cultures (Lucke and Hechel-mann, 1987) which may be a preventing strategy
since improper usage of yeast strains as starter culture may lead to severe defects
(Meisel et al., 1989). However, the meat industry remains interested in the addition of
yeasts as starter culture mainly because of their safety record (Nagodawithama, 1992).
Yeasts furthermore contribute to sausage flavour by attacking lipids and proteins and
 52

exert certain anti-oxidant effects by destroying peroxide and depleting oxygen from the
surface of the product (Nagodawithama, 1992).

Gehlen et al (1991) reported strains of Debaryomyces hansenii have a strong influence

on the microbiology and chemical composition of dry sausages due to its reducing
ability of fortuitous staphylococci and their contribution to the improvement of taste and
surface color in dried sausages. Meisel et al (1989) however, warned that care must be
taken to control the reduction of anti-microbial properties of organic acids and to limit
high residual values of nitrate in fermented sausages by the addition of yeast cultures.
Consequently the meat industry continues to seek a proper yeast species that has the
ability to impart the expected rich meaty flavor.

Debaryomyces hansenii, the proven predominant yeast species in traditional salami,

requires oxygen for reproduction (Metaxopoulos et al., 1996), destroys peroxides
(Coretti 1973), is salt tolerant, does not reduce nitrate, and occurs frequently in cured
meat products (Leistner and Bern, 1970). Even though Debaryomyces hansenii strains
do not reduce nitrate, some starter cultures on the German market contain
Debaryomyces hansenii as a single strain or in combination with lactic acid bacteria and
staphylococci (Hammes et al., 1985). Therefore, in this study we endeavoured to
determine the performance of Debaryomyces hansenii as a single starter culture, in
combination with another Debaryomyces species, and when used in combination with
traditional lactic acid bacterial starter culture in the manufacture of salami.

4.2. Materials and methods

4.2.1. Yeast preparation for use as sterter culture

Debaryomyces hansenii and Debaryomyces polymorphous isolated during a previous

study were selected as yeast representatives for usage as starter cultures.
Debaryomyces hansenii proved to be the predominant yeast isolate during processing
 53

of salami while Debaryomyces polymorphous was isolated during the period of

maturation. The yeast cells were prepared by seeding in glucose-yeast nitrogen base
(YNB, Difco, Detroit, Michigan, U.S.A.) medium (400ml) in 1000ml Elernmeyer flask and
cultured with vigorous agitation on a rotary shaker (180rpm) at 30°C until late
exponential phase. The cells were harvested by centrifugation for 5 minutes using a
BeckmanJ2-21 centrifuge and a JA-14 rotor spinning at 11000rpm.

4.2.2. Manufacture of salami

Four batches of 5kg of salami were prepared. Individual salamis weighing 250g~300g
were prepared from the different batches. The formulation for salami processing is
according to the protocol indicated in Table 4.1. For Batch 1 (OhS), the starter
composition used was FloraCarn (Chr. Hansen, Denmark) consisting of Staphylococcus
carnosus and Lactobacillus pentosus in the ratio of 2: 1 and Debaryomyces hansenii.
The ratio of Floracarn to Debaryomyces hansenii was 1:1. For Batch 2 (Dh),
Debaryomyces hansenii was used as the single starter organism and for Batch 3 (DhDp)
Debaryomyces hansenii and Debaryomyces polymorphous were mixed in the ratio of
1:1. Batch 4 (S), served as a control used for comparable reasons to establish the
maturation time. The only analysis which was done on batch 4 was the rate of weight
loss. Batch 4 comprised of only FloraCarnas starter culture.

All meat portions were frozen at a temperature of -15°C to avoid "smearing" of the fat
on the lean meat surface, which may cause problems during sausage dehydration. The
fat used was from good quality pork lard. Meat portions and the ingredients were mixed
sequentially in a bowl cutter. Beef was chopped into 10mm particle size. Starter culture
and spices were evenly sprinkled on the beef and chopped to fine particles. Pork was
then added and chopped to 20mm particle size after the lard and curing salt were
added. Finally, the mixture was chopped to a particle size of 4.5mm making sure that
the entire batch was thoroughly mixed. The mixture was carefully packed into Calpak
Fibrous Bak 65/50 to avoid trapping air and both ends were tied with strings. Three
Table 4.1: Composition of commercial salami

Ingredients Percentage by weight (%)

Beef 40.000

Pork 34.730

Pork back fat 20.000

Spices 2.176

Curing salt 3.046

Starter culture (freeze dried) o 11/26/98.500

Staphylococcus carnosus & Lactobacillus penttosus 2:1

54
 55

samples from each batch were weighed and marked for weight loss determination. On
sampling occasions, the marked samples were weighed to determine the weight loss of
the salami. The prepared salami samples were hanged on racks and placed in a
fermentation chamber with a temperature of 22°C and relative humidity of 90% for
48hrs. After 48hrs of fermentation, a 10 min smoke treatment at 18°C was carried out.
Finally the sausages were transferred to a drying chamber with a temperature of 12°C
and 75-80% RH until the product lost 20% and more weight.

4.2.3. Sampling

Sampling was done 6 hourly for the first 2 days of fermentation starting directly after
processing. Consecutive sampling was performed every 4 days. On every sampling
occasion, one salami from each batch was transferred to the lab, in a cooler box for
microbial and chemical analysis.

4.2.4. Chemical and Physical Analysis

On every sampling occasion a 109 sample from each batch of salami was homogenized
using a Janke & Kunkei Ultra-Turrax T 25 for 60 s in 20ml of distilled water and made
up to 100ml with distilled water. The pH was measured with a HI 9321 Microprocessor
pH meter (Hanna Instrument). Another 109 sample from each salami was taken,
homogenized with 12ml distilled water and centrifuged for 5min at 11000rpm with a
Beckman J2-21centrifuge. The supernatant was extracted and used for lactic acid
determination by liquid chromatography (Waters Lambda-Max 480) with a high
performance (Bio-Rad. Aninex APX-87H) exclusion column and O.OlN sulphuric acid as
eluent. The flow rate was O.5ml!min and the detection wavelength 210nm. Samples
from the same salami were also taken for water activity determination using the
NovasinaThermoconstanter TH200. Salt, protein, fat and moisture were determined by
the methods proposed by the Association of Official Analytical Chemist (AOAC, 1990).
 56

4.2.5 Microbiological analysis

The salami samples were prepared for microbial analysis by cleaning the salami surface
with 70% ethanol. A cut was made through the salami casing with a sterile knife and
the casing removed aseptically. For each sample, 109 portions of the meat were taken
with a sterile knife and homogenized in a stomacher (Janke & Kunkei Ultra T2S) for 60
s in 90ml sterile bactopeptone. Further decimal dilutions of the suspensions were
carried out as required for microbiological assay in 9ml sterile bactopeptone. Plating
was done in duplicate by the spread plate method. The growth media used and
incubation periods are indicated in Table 4.2.

4.3. Results and discussion

4.3.1. Chemical and physical composition

The changes in chemical and physical analysis of the three batches of salami are
indicated in Tables 4.3-4.6. As frozen raw material was the main ingredient for
processing, the temperature of the sausage after filling was about O°e. When sausages
at this temperature were transferred immediately into an air-conditioned chamber at a
temperature of 22°( and 90% humidity, a microclimate with a temperature
approximately that of the sausage was established. The saturation vapour pressure of
the chamber declined, leading to a less water vapour absorption by the air. Surplus
humidity was deposited on the surface of the salami as condensate (Stiebing and
Rhodel, 1988) resulting in an increase in the initial weight of all the salamis with the
exception of batch 1 (DhS, containing bacteria mixed with yeast as starters).
Dehydration rate for all the salamis was therefore generally poor during fermentation,
which resulted in an extended maturation period. Moulds grew on the surface of all the
salamis sporadically during fermentation and maturation. Frequent cleaning with 5%
vinegar solved this problem.
Table 4.2: Culture media, temperature and times of incubation used for microbial analysis of

commercial salami

Incubation time (h) Growth medium

Yeast count, YGC 96 Yeast extract; glucose; chloramphenicol agar (Oxoid CM

139)

Yeast count, YM 96 Yeast extract malt extract agar (pH 3.5)

Total cou nt, PCA 48 Standard plate count agar (Oxoid CM 361)

Lactobacilli, MRS 48 De Man Rosoga and Sharpe (MRS) (Oxoid CM 361)

57
Table 4.3: Comparative chemical and physical changes during the processing of salami over a period of 624 hours

Weight loss (%) Moisture content (%)

Time Batch 1 Batch 2 Batch 3 Batch 4 Batch 1 Batch 2 Batch 3

Fermentation
0 0 0 0 0 57.0714 56.7393 56.4988
6 0.1467 -0.1215 -0.5129 -0.2501 57.0083 56.7913 56.7208
18 1.4559 1.1991 1.4065 0.8531 56.4372 56.2143 55.8782
24 1.2914 0.8791 0.6852 0.5219 56.5098 56.3556 56.1987
30 1.3772 0.8143 0.6051 0.3161 56.4719 56.3842 56.2340
42 0.8621 0.2107 -0.1563 -0.1630 56.6981 56.6480 56.5667
48 0.8013 0.1215 -0.3045 -0.3745

Ripening
144 3.799 3.6014 3.4783 1.8500 55.3762 55.1231 54.9312
240 7.3761 7.2271 7.8141 5.2112 53.6528 53.3693 52.8115
336 9.2899 8.9569 9.1324 7.4133 52.6749 52.4833 52.1268
432 13.7028 13.3482 13.6905 10.6411 50.2549 50.0753 49.5986
528 15.3139 15.0334 15.3997 15.0251 49.3086 49.0850 48.5803
624 20.7834 20.3484 21.0659 19.0509

Batch 1= Debaryomyces hansenii and conventional starters; Batch 2= D. hansenii as starter culture; Batch 3= D. hansenii and D. polymorph us strains as starter

cultures; Batch 4= conventional starter culture.

Table 4.4: Comparative chemical and physical analysis during the processing of salami over a period of 624 hours

Aw Lactic acid content (mglg)

Batch 3 Batch 1 Batch 2 Batch 3

Time Batch 1 Batch 2

Fermentation
0.169487 0.128554 0.103405
0 0.950 0.947 0.947
0.374401 0.142380 0.118556
6 0.946 0.944 0.946
0.163500 0.132979 0.135425
18 0.944 0.948 0.950
0.174230 0.142737 0.150471
24 0.946 0.947 0.948
0.244217 0.140796 0.158247
30 0.945 0.946 0.948
0.158174 0.184229 0.149979
42 0.945 0.949 0.948
0.947 0.149227 0.177125
48 0.947 0.947

Ripening
0.241101 0.215974 0.222855
144 0.945 0.944 0.945
0.343361 0.250833 0.265239
240 0.943 0.942 0.941
0.329300 0.180192 0.294203
336 0.942 0.941 0.942
0.343087 0.313001 0.219847
432 0.937 0.937 0.937
0.358796 0.309592 0.335808
528 0.936 0.935 0.936
0.409496 0.346934 0.304665
624 0.930 0.928 0.929

Batch 1= Debaryomyces hansenii and conventional starters; Batch 2= D. hansenii as starter culture; Batch 3 = D. hansenii and D. polymorphus stains as starter cultures
Table 4.5: Comparative chemical and physical analysis during the processing of salami over a period of 624 hrs

pH
Salt content (%)

Batch 1 Batch 2 Batch 3

Batch 1 Batch 2 Batch 3
Time

Fermentation 5.15
5.26 5.16
3.0250 3.0250 3.0250
0 5.10
5.46 4.96
3.0294 3.0213 3.0096
6 5.23
5.33 5.20
3.0697 3.0617 3.0682
18 5.22
5.26 5.24
3.0646 3.0518 3.0549
24 5.24
5.36 5.24
3.0672 3.0498 3.0434
30 5.43
5.39 5.27
3.0513 3.0314 3.0203
42 5.40
5.45 5.30
3.0494 3.0287 3.0158
48

Ripening 5.29
5.10 5.14
3.1445 3.1380 3.0340
144 5.04
4.82 4.92
3.2659 3.2606 3.2814
240 4.83
4.75 4.72
3.3348 3.3226 3.3290
336 5.00
4.92 4.86
3.5053 3.4910 3.5048
432 4.94
4.85 5.04
3.5720 3.5602 3.5756
528 4.85
4.66 4.87
3.8186 3.7978 3.8328
624

Batch 1= Debaryomyces hansenii and conventional starters; Batch 2= D. hansenii as starter culture; Batch 3= D. hansenii and D. po/ymorphus strains as starter

cultures
Table 4.6: Comparative chemical and physical analysis during the processing of salami over a period of 624 hrs

Fat content (%) Protei n content (%)

Time Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3

Fermentation
0 20.3152 20.9494 20.3230 16.5251 16.6339 15.5188
6 20.345 20.9240 20.9250 16.5494 16.6137 15.4396
18 20.6153 21.2037 21.3323 16.7692 16.8358 15.7402
24 20.5810 21.1352 21.1774 16.7414 16.7814 15.6295
30 20.5989 21.1214 21.1666 16.7559 16.7709 15.6133
42 20.4919 20.9936 20.9995 16.6688 16.6690 15.4946
48 20.4793 20.9749 20.9684 16.6586 16.6541 15.4717

Ripening
144 21.1174 21.7321 21.7902 17.1777 17.2553 16.0780
240 21.9330 22.5814 22.8151 17.8411 17.9299 16.8342
336 22.3957 23.0104 23.1461 18.2175 18.2703 17.0780
432 23.5410 24.1765 24.3685 19.1962 19.1962 17.9804

528 23.9888 24.6560 24.8608 19.5770 19.5770 18.3437

624 25.6451 26.3013 26.6454 20.8833 20.8833 19.6605

Batch 1= Debaryomyces hansenii and conventional starters; Batch 2= D. hansenii as starter culture; Batch 3= D. hansenii and D. polymorph us
strains as starter cultures
 62

Within 6hrs of fermentation the prepared salamis from batch 2 (Oh, with Debaryomyces
hansenii as starter) exhibited an increase in weight of 0.1215%, batch 3 (with a mixture
of two strains of yeast added as starter culture) 0.5129% and batch 4 (control)
0.2501% (Table 4.3). Weight loss determination is accepted as a method to determine
the ripening pattern in dry sausages (Stiebing and Rhodel, 1988). The weight loss
increased during ripening when the R.H. was reduced from 90 to about 75%. After
624hrs, all the salamis containing yeast starters attained a percentage weight loss
above 20% (OhS, 20.78%; Oh, 20.35%; DhDp, 21.07). Batch 4 that comprised of the
conventional bacteria starter cultures had lost only 19.05°/0. The weight loss which, is a
function of the weight loss reflected a decline in the moisture content. The trend of
moisture loss for all three batches was similar. The initial moisture contents of batches
1, 2 and 3 were 57.0714%, 56.7393°/0 and 56.4988% respectively but at the end of
ripening, batch 1 had lost 11.2628%, batch 2, 11.0517% and batch 3, 11.6096%.

The water activity of foods influences the growth and metabolic activity (including toxin
production) of micro-organisms as well as their survival and resistance (TroIIer and
Christian, 1978). Debaryomyces hansenii is very resistant to low levels of water
activity, capable of growth in water activity levels as low as 0.88 (Van Eck et aI., 1993).
The water activity and lactic acid content of the three batches are presented in Table
4.4. From initial water activity levels of 0.950, 0.947 and 0.947 for batches 1, 2 and 3
the water activity levels gradually declined to 0.930, 0.928 and 0.929 respectively after
624 hrs. Despite continued disagreements on the water activity requirements of all
micro-organisms (Leistner et aI., 1976), it is accepted that enteropathogenic Escherichia
coli strains are inhibited at water activity levels below 0.96 (Tomtov et aI., 1974).

During fermentation, the lactic acid content for batch 1 which contained lactic acid
bacteria and yeast as starter cultures, increased rapidly within six hrs followed by a
rapid decline in the next 12 hrs. Thereafter, a gradual increase was recorded until the
end of maturation when a maximum of 0.409mg/g, which was the highest of the three
batches, was obtained. Lactic acid content for batches without lactic acid bacteria
 63

starter cultures (batches 2 and 3) fluctuated but both showed a gradual increase from
the beginning to the end of production. Batch 2 exhibited an initial lactic acid content
of 0.128mg/g and batch 3, 0.103mg/g. At the end of ripening, lactic acid content for
batch 2, was 0.347mg/g and batch 3 a content of 0.304mg/g. An interesting feature is
the similar increases in lactic acid content in batches 2 and 3 compared to batch 1,
although lower final contents after 624hrs were obtained, despite the absence of lactic
acid bacteria as part of the starter culture. However, high numbers of contaminating
lactic acid bacteria originating from processing equipment, hands and aprons of workers
or the meat might have contributed to the high lactic acid content.

Salt content and pH levels for all three batches are shown in Table 4.5. Salt content
remained similar for all three batches exhibiting an increase less than 1% (DhS, 3.025-
3.8186%; Dh, 3.025-3.7978% and DhDp, 3.025%-3.8328%). The total decline in pH
during processing was minimal (less than 1 pH unit) as compared to Belgian, German
\

and Spanish dry salamis which obtained pH values of 4.6-5 after fermentation and
continued to decline during ripening (De Ketelaere et al., 1974; Palumbo et al, 1976;
Lucke, 1985 and Sanz et al., 1988). The slight decrease in pH in this study correspond
with results obtained with Italian and Hungarian salamis which exhibit a pH decrease of
about 0.5 units (Komendy and Gantner, 1962; Baldini et al., 1983). The slight pH
increase observed after 432hrs might be due to the utilization of lactic acid (Raimahone
et al., 1988) and/or the production of amine and ammonia by the high number of yeast
present at the time (Lucke, 1988) which could favour the growth of spoilage bacteria
(Walker, 1977). Despite the detrimental effect of pH increase caused by yeasts, the
strong synergistic inhibitory effect on fortuitous staphylococci by Debaryomyces
hansenii and Lactobacillus curvstus has been demonstrated (Sorrels and Speck, 1970;
Meisel et al. 1989). The individual inhibitory effect is attributed to nitrite production by
L. auvetus and oxygen depletion by D. hansenii. Fat content represented in Table 4.6

shows an increase of 5.6%, 5.4% and 5.3% fat content for batches 3, 2 and 1
respectively; an increase of approximately 5.6% protein content for all the batches is
 64

indicated in Table 6. Batch 2 and 1 had the highest protein contents (20.8833%) after
maturation.

4.3.2 Microbiological enumeration

The progression of the lactic acid bacteria present similar growth patterns for all three
batches, the highest microbial counts recorded were obtained during ripening after
144hrs for batches 1 and 3 (Log 6.717/g and Log 6.647/g respectively) and after
240hrs for batch 2 [(Log 6.723/g) (Table 4.7)]. Similar growth patterns were reported
by Lucke (1985).

The processing of the meat into raw sausages that reduced the aw and rapidly
consumed the oxygen present within the mixture (Lucke, 1985) might have eliminated
pseudomonas (Hechelmannet al. 1977) and Enterobacteriaceae (Barth, 1960; Grau,
1981; Gill. 1982). Consequently, a shift in predominating microflora composition
present in the salami towards the lactic acid bacteria, micrococci (Niinivaara and Pohja,
1956; Ten Cate, 1960; Incze, 1965; Reuter, 1967; Ayroulet and Fournaud, 1976;
Hofmann and Scharner, 1980) and yeasts resulted. The availability of sugars, low acid
production and relatively high water activity further enhanced the rapid growth of lactic
acid bacteria during fermentation. The rapid growth and progression during
fermentation corresponded with results reported by Lucke (1985), indicating that the
activity of Micrococcaceae and Gram-negative bacteria under these conditions outgrow
competing micro- organisms. Lactic acid bacteria proved to be the major microbial
component present in the meat and during salami processing, confirming previous
reports on the predominance of lactic acid bacteria in dry fermented sausages whether
or not lactic acid bacteria starter cultures were added (Smith and Palumbo, 1973;
Metaxopoulos et al., 1981a,b; Gokalp and Okerman, 1985; Cabalan and Genigeorgis,
1986). Samelis et al. (1996) reported on the elimination of streptobacteria II,
betabacteria and streptococci in Greek dry salami during fermentation even if significant
numbers were present during early fermentation.
Table 4.7: Comparative microbial counts during the processing of salami over a period of624 hrs

Log counts g-I

MRS YGC
Time Batch 1 Batch 2 Batch 3 Batch 1 Batch 2 Batch 3

Fermentation
0 5155 5.037 5.418 4.771 5.152 5.415
6 5.037 4.929 5.143 4.832 5.037 5.190
18 5.497 5.301 5.324 5.223 5.283 5.152
24 5.924 5.021 5.398 5.243 5.471 5.356
30 6.041 5.881 5.875 5.021 5.486 5.326
42 6.324 6.149 6.093 5.272 5.885 5.179
48 6.576 6.134 6.310 5.093 5.378

Ripening
144 6.717 6.620 6.647 5.545 5.535 5.354
240 6.556 6.723 6.635 6.057 5.939 5.505
336 6.577 6.560 6.536 4.732 5.143 4.903
432 6.600 6.577 6.468 4.531 5.093 4.845
528 6.522 6.375 6.635 4.544 4.785 4.991
624 6.539 6.240 6.417 4.505 5.090 4.653

MRS =Lactic acid bacteria count YGC = yeast counts

Batch 1= Debaryomyces hansenii and conventional starters; Batch 2= D. hansenii as starter culture; Batch 3= D. hansenii and D. polymorphus strains as starter cultures
 66

The bacteria being not capable of resisting the low water activity, low pH and high salt
content were inhibited leaving the competing lactic acid bacteria to establish itself in the
stressful environment which consequently resulted in the stabilization of bacterial
numbers. According to Reuter (1972), Micrococcaceae strains showed little or no
growth during ripening. Hammes et al (1985) confirmed the poor growth and survival
of Micrococcaceae during sausage ripening despite being present. Yeasts, however,
competed very well under the environmental stresses exerted (high salt, low water
activity and low pH value) (Leistner and Bern, 1970; Jay, 1979; Beuchat, 1983). During
fermentation, the bacterial numbers increased rapidly while yeast numbers remained
stable due to the inability of the yeasts to proliferate when bacterial numbers competing
for the same nutrients are high (Walker and Ayres, 1970; Walker, 1977). In all
batches, highest counts of yeast numbers were recorded after 240hrs during ripening.
At that time bacterial numbers remained relatively high but yeast competed better
probably due to the availability of higher amounts of organic acids. Surprisingly, batch
1 with a mixture of bacteria and yeast as starter cultures, exhibited the highest yeast
count (Log 6.057). The higher proportions of organic acids produced by the lactic acid
bacteria starter cultures resulting in the progression of D. hansenii strains has been
reported (Raimihone et al 1988). The lower numbers of lactic acid bacteria present
during the early stages of maturation also enhanced the proliferation of the yeasts
(Miller 1979, Walker and Aryes, 1970; Walker, 1977).

The decline in yeasts' numbers during ripening corresponded with result by Gehlen et
al (1991) who indicated on the inhibition of yeast species by bacteria Lactobacillus

curvatus and Micrococcus varians in the later stages of ripening.

In this study the production time for batch 3 with yeasts as starter culture was reduced
by 48hrs as compared to batch 4, which contained the standard starter cluture. All the
salamis exhibited a substantial red surface colour which had already occurred after
48hrs of fermentation, a situation noticed by Gehlen et al (1991) in sausage fermented
with and without conventional starter culture indicating all batches were fully cured.

•
 67

The batches prepared without lactic acid bacteria starter cultures were judged better
with smooth "sweet" taste according to an unprofessional taste panel. The taste panel
all agreed on a less firm structure for the yeast fermented salamis compared to the
salamis with lactic acid bacteria starter culture, an observation confirmed by Lucke and
Hechelmann (1987).
 68

CHAPTER V

GENERAL DISCUSSION AND CONCLUSIONS

The traditional approach to salami fermentation has been well documented. Although
there have been numerous reports on the role of lactic acid starter bacteria in salami,
little consideration has been given to the role of yeasts in fermented meats. Reports
usually referred to the presence of yeasts in meat, but few have attempted to quantify
the yeasts present in fermented meat, to examine the ability of yeasts to grow in
fermented meats or to seek biochemical explanations of such growth. The concept that
fermented meats, as a group, may represent a specialized ecological environment for
the selective occurrence and growth of certain yeast species, is also neglected.

5.1 The growth and survival of yeasts in commercial salami

The results obtained in this study, obtained from South African commercial salami, were
in agreement with results reported in literature. A decrease in the pH values, was
observed as expected, since the fermentation process was mainly governed by the
activities of lactic acid bacteria, which ferment simple sugars forming organic acids.
The decrease in aw levels was due to high salt concentrations, which consequently
resulted in reduced weight of the salami's as a function of moisture loss. The moisture
loss corresponded with an increase in salt, fat and protein contents.

An increase in both yeasts, and bacterial numbers confirmed the synergistic relationship
that exists between yeasts and bacteria in restricted environments. Lactic acid bacteria
 69

predominated in all salami's processed, despite the rapid progression of yeasts during
ripening. This situation controlled the proliferation of yeasts by preventing the
progression of numbers high enough to cause spoilage.

Based on the results, Bul/era variabi/is, candida haemu/onii, candida gropengiesseri,

Crytopcoccus a/bidus, Cryptococcus /aurentii, Candida zey/anoides, Debaryomyces
hansenii, Debaryomyces po/ymorphus, Debaryomyces vanrijiae, Ga/actomyces
geotrichum, Pichia farinosa, Pichia phi/ogaea, Rhodotoru/a minuta, Rhodotoru/a,
muci/aginosa, Sporob%myces haophi/us, Sterigmatomyces roseus, Schwanniomyces
occidenta/is, Toru/aspora de/bruecki and Trichosporon beige/ii were established as
typical contaminants associated with meat and salami. Except for Bul/era,
Schwanniomyces, Sporob%myces and Sterigmatomyces, all the above mentioned
genera were previously isolated from meats and meat products. The sources of
contamination mainly included the raw meat, air equipment and from animals' skin and
hide.

D. hanseni/~ R. muci/aginosa, B. variabi/is, C a/bidus and C zey/anoides were

considered the dominant yeast community associated with commercial salami, being
present at frequent intervals. D. hansenii strains were the most frequently isolated, and
clearly predominated in salami processing. The substantial role played by D. hansenii
during the ripening stages of the salami, can therefore not be ignored.

5.2 Key properties for the selection of yeasts as possible starter cultures in
salami production.

Results obtained, indicated that all the yeast species isolated from commercial salami
are tolerant to 4-8% Nael and 80-240ppm nitrite. Of the nineteen species investigated,
only Trichosporon beige/ii exhibited proteolytic activity. Debaryomyces hansenii and D.
vanrijiae showed variable lipolytic activity. All Rhodotoru/a species showed no lipolytic
activity, whilst the rest were alllipolytically active.
 70

According to literature, Debaryomyces hanseriii has attained GRASstatus. The species,

furthermore, is capable of growth at low temperatures, low aw in a salty medium, low
pH and generally is frequently associated with fermented meat products. Based on
these properties, D. hansenii and D. polymorphus were selected, both of which were
isolated on the last sampling occasion of commercial salami production. The two
species, on examination for inactivation temperatures, proved to be inactivated at
temperatures above 50°Cwithin a maximum of 60hrs.

5.3 The application of yeast strains as starter culture in traditional

fermented sausage

Despite the use of Debaryomyces hansenii as a single strain, or mixed with

Debaryomyces polymorphus or conventional starter cultures as starter cultures in the
formulation of traditional salami, there was a decline in pH values at the end of
production. This is confirmed in literature indicating that the process of fermentation in
salami making is governed by the activities of lactic acid bacteria. The decline in pH
was restricted due to the activities of yeasts in the utilization of lactic acid and the
production of amine and ammonia.

Water activity values declined in correspondence with decreases in the weight,

moisture, fat, protein and salt contents. The implementation of yeasts as starter
culture did not inhibit the reddish-brown colour formation in the meat. Lactic acid
bacteria in this study were the most predominant microflora found in the salami. The
proliferation of the yeasts to numbers higher than the bacterial numbers would have
left the salami susceptible to spoilage by undesirable bacteria.

Debaryomyces hansenii and Debaryomyces polymorphus added to the salami

formulation without the conventional starter culture resulted in a less sour and tastier,
 71

product. Moreover, ripening time was reduced but the consistency was less firm. The
softness of the salami obtained with the inclusion of only yeasts as starter cultures, was
a result of the higher pH according to literature. The application of yeasts as starter
cultures in this study, has been successful, resulting in a product comparable with
products prepared with the conventional starter culture and without any major defect.

The positive results obtained in this study is a clear indication that Debaryomyces
hansenii and Debaryomyces polymorphus could be used in the future as starter culture
in the formulation of salami leading to a better taste and shorter ripening time than
salami produced with the present traditional bacteria starter cultures.

Future Research: Recommendation is therefore made for future research to include,

A. The survival levels of Debaryomyces hansenii and Debaryomyces

polymorphus after lyophilization.
B. The contribution of strains to the organoleptic characteristics of salami.
C. The type of possibleantibiotics excreted by the isolated yeast strains.
D. The possible antagonistic effect of the isolated strains against fungi and
bacterial contamination.
 72

CHAPTER VI
•

SUMMARY

Yeasts play a substantial role in the processing of salami, being present at high
numbers during the ripening stages contributing to aroma and flavour development.
Accordingly, the yeasts occurring as natural microflora in commercial salami were
quantified, isolated and identified according to conventional identification and
enumeration techniques. Depending on conditions, the yeasts grew to maximum
populations of 107duIg during the ripening stages.

Characterization of the naturally contaminating yeasts of commercial salami revealed

108 yeast species, belonging to 12 genera. The yeasts most frequently isolated were
from the genera Debaryomyces, Rhodotorula, Bul/era, Candida, Cryptococcus,
Trichosporon and Schwanniomyces in that order. Low appearances of Pichia,
Galactomyces, Sporobolomyces, Sterigmatomyces and Torulaspora were observed.

Debaryomyces hansenii, Rhodotorula mucilaginosa, Bul/era variabilis and Cryptococcus

albidus were established as representing the typical yeast community associated with
salami. These species were frequently isolated from the meat, during processing and
maturation. The most frequently isolated yeast species, was D. hansenii, isolated on
every sampling occasion.

The isolated yeasts were examined based on relevant key properties that governed
their growth and survival in fermented meat, and proposed as representative selective
characteristics to determine a good starter culture. All the yeasts were tolerant to 4-8%
 73

NaCl and 80-240ppm nitrite. All the yeasts proved to be non-proteolyic with the
exception of Trichosporon beigeli~ whereas lipolitic activity appeared variable.

Two predominating yeast isolates, Debaryomyces hansenii and Debaryomyces

polymorphus, exhibited relevant qualities regarding fermentation of meat.

Consequently, they were applied in the formulation as starter cultures in an attempt to
prepare a salami based on yeast fermentation. The incorporation of D. hansenii,
individually, or in association with conventional starter cultures, resulted in a product
with improved taste and reduced maturation time.
 74

REFERENCES

Adams, M.R., (1986) Fermented flesh foods. Progr. Indust. Microbiol., 23, 159-198.

Ahearn, D. G., Meyers, S.P. and Nichois, R.A. (1968) Extracellular proteinase ofyeasts-
like and fungi. Appli. Microbiol. 16, 1370-1374.

AMI, (1979) "Meat Facts: A Statistical Summary about America's Largest Food
Industry Am. Meat Inst., Washington, D.C.

Anderson, P.l, MacNeil, K. and Watson, K.A., (1986) High-efficiency carbohydrate

fermentation to ethalate temperatures above 40°C by Kluyveromyces marxianus
var marxianus isolated from sugar mills Appli. Environ. Bacteriol. 51, 1314-
1320.

Andres, C., (1977) Starter cultures for sausages has two micro-organisms for better
performance. Food Proc., 38(1), 132.

AOAC., (1990). Association of Official Analytical Chemist

Arnau, L, Maneja, E., yMontfort, J. M., (1987). Factores que influyen en la apricon de
precipitados de tirosina. Carnica 2000, 48. 106-110.

Ayres, le., (1955). Microbiological implications in the handling slaughtering and

dressing of meat animals. Advances in Food Research 6,106-161.

Ayroulet, M., and Fournaud, J. (1976) Mikrobiolgische Wechselwirkungen bei der

Rohwurstherstellung. Fleischwirtschaft 56, 1331-4.

Bacus,1., (1984) Utilization of micro-organisms in meat processing. A handbook for

meat. Plant operators. John Wiley, New York.
 75

Bacus, J. N. (1986). Fermented meat and poultry products. In Advances in Meat

Research: ''Meat and Poultry Microbiology" Pearson, A. M. and Dutson, T.R
(Eds) V. Z. P 123. Macmillan.

Bacus, IN. and Brown, W.L., (1981) Use of microbial cultures: meat products. Food
Technol., 35, 74-78.

Baldini, P., Mamia, F. and Raczynski, R G. (1983) Influence of the chemical and
physical-chemical properties of the formulations on pH lowering and weight loss
in the ripening of Italian salami. Proceedings of the 29thEuropean Congress of
Meat Research Workers Salsomaggiore, pp 189-97.

Banks, lG. &Board RG. (1987) Some factors influencing the recovery of yeast and
mould from chilled foods. Int. J. Food Microbiol. 4. 197-206.

Barber, L. E. and Diebel, R. H. (1972). The effect of pH and oxygen tension on

staphylococcal growth and enterotoxin production in fermented sausage.
Appli. Microbiol. 24,891.

Bamett, lA., Payne, R.W., and Yarrow, D., (1983) Yeast: Characteristics and
identification of yeasts. Cambridge. Cambridge University Press.

Barth, W. (1960) Welchen Einfluss haben pH-Wert, Kochsalz und Nitrit auf die
Bakterienflora des Rohwurstbrates/ Dissertation, Veterinarmedizinische
Fakultat, Freie Universitat Berlin, 50 pp.

Besancon, x., Smet, C., Chabalier, C., Rivemale, M., Reverbel, lP., Ratomahenina,
R., and Galzy P. ( 1992) Study of surface yeast flora of Roquefort cheese. Int. J.
Food microbiol. 17, 9-18.
 76

Beuchat, L. R. (1982) Thermal inactivation of yeasts in fluit juices supplemented with

food preservatives and sucrose Journal of food science 46, 771-777.

Bungay,H.R and, Bungay, M.L., (1968). "Microbial interactions in continuous culture.

Adv. Appl. Microbiol. 10,269-290.

Cahalan, D. L. and Genigeorgis, C. (1986) Study of the microflora during the natural
commercial fermentation of Italian-dry ansalami and identification of species
suitable as starters. In Proceedings of the 32nd European meeting of meat research
workers 5,263-266.

Campbell-Platt, G., (1987). Fermented foods of the world: A dictionary and guide.
314pp. Butterworths, London.

Campbell-Platt, G., (1995) Fermented Meats -A world perspective. Blackie Academic.

and Professional. London. pp 39-51.

Casado, M.-J.M, Borras, M.-A D. and Agnilar, RV., (1991) Fungal flora present on the
surface of cured Spanish ham. Fleischwirtschaft, 71(11). 1300-l302.

Cerise, L., Bracco, u., Horman, I., Sozzi, T. and Wuhrmann, T. J. (1973) Veranderung
des Lipidanteils wahrend des Reifungsprozesses von salami aus reinem

Scheweinefleisch. Fleischwirtschaft, 53, 223-225.

Cesari, E. -P., (1919). "La maturation du saucisson" Academie des Sciences Paris. 168,
802-803 .

Cesari, E.P. and Guiliermond, A, (1920). Les levures de saucissons" Ann Inst. Pasteur
34, 229-248.
 77

Comi, G. and Cantoni, C., (1980). Yeast in matured raw sausage. Industrie Alimentari
19, 850-60.

Comi, G., Cantoni, c., and Traldi, c., (1982) Conservazione di sciruppi con aife
Conzentrzioni di succheri. Ind. Bevande 11(59), 169-171.

Comi, G. and Cantoni, c., (1983). Presenza di lieviti nei pros ciulti crudi stagionati. Ind.
Alimentar, 22, 102-104.

Cook, P.E., (1995) Fungal ripen meats and meat products. In Fermented meats.eds G.
Campbell-Platt, P.E. Cook, Blackie Academic and Professional. London. pp 110-
125

Coretti, (1956). Fleischwirtschaft, 8, 197.

Coretti, K., (1965) Vorkommen und Bedeutung lipolytischer Mikroorganismen in

Dauerwursten. Fleischwirtschaft, 4521-23.

Coretti, K., (1971) Rhowurstreifung und Fehlerzeugnisse bei der

Rhowurstherstelliung.Verlag Rheinhessische Druckwerkstatte, Alzey (fed. Rep.
Germany.

Coretti, K. (1973) Murum interessiert den Praktiker die Microbiologie der Rohwurstrei
Feischwirtschaft 53, 907-903.

Coretti, K., (1977) Starterkulturen in der Fleischwirtschaft. Fleischwirtschaft, 57,386-

394.

Dahiya, R. S. and Speck, M.L., (1967). Hydrogen peroxide formation by lactobacilli and
its effect on Staphylococcus aureus. J. Dairy Sci. 51, 1568
 78

Dalton, H.K. and Board, RG., (1984) The yeast of fresh British sausage and minced
Beef J. Microbiol. Serl. Antonie van Leeuwenhoek, 50,227.

Daly, C., Sandine, W.E. and Elliker, P.R., (1971). "Interactions of food-starter cultures
and food-borne pathogens" J. Milk Food Technol. 35, (6) 349-357.

Daly, e., Chance, N., Sandine, W.E. and Elliker, P.R, (1973) Control of
Staphylococcus aureus in sausage by starter culture and chemical acidulation. J.
Food Sci. 38: 426

Deak, T., (1991) Food bome yeast. Adv. Appli. Microbiol. 36,179-279.

Deak, T., (1993) Simplified techniques for identifying food borne yeasts.
International Journal of Food Microbiology 19 (1) 15-26

Deak, T. and Beuchat, L. R (1987) Identification of food borne yeasts J. Food Protect
50, 243-264.

Deak, T. and Beuchat. L.R, (1996) Handbook of Food Spoilage. Yeasts. CRC press,
New York.

Deibel, R.H., (1956) Bacteriological aspects of pure starter culture in the manufacture of
sausage. American meat Institute Foundation, Circular No. 20, p. 14.

Deibel, RH., (1974). Technology of fermented semi-dried and dried sausage. In Proc.
Meat Ind. Res. Conf, P.57. Am Meat Inst. Found., Washington D.e.

Deibel, RH. and Niven, e.F., (1957) Pediococcus cerevisiae: A starter culture for
summer sausage. Bacteriol. Proc. 1957: 14.

Deibel, RH., Niven, e.F. and Wilson, G.D., (1961a) Appl. Micobiol. 9,156-161.
 79

Deibel, RH., Wilson, G.A., and Niven Jr., C.F., (1961b) Microbiology of meat curing.
IV. A Iyophilyzed Pediococcus cerevisiae starter culture for fermented sausage.
Appli. Microbil. 9,239-243.

De Ketelaere, A,Demeyer D., Vandekerckhove, P. and Vervaeke, J. (1974) Food Sci.,

39,297

Demeyer, D. I.,Vandekerckove, L. and Moerman, R, (1979). Meat Sci. 161

Demeyer, D. I., Vandekerckove, L., Vermeulen, L.and Moerman R, (1981) Compounds

determining pH in dry sausages, Europ. Meet. Of Meat Res. Worker, Kulmbach,
CA.

Dillon, V.M. and Board, R G., (1990) A study of sulfite tolerant yeast from comminuted
lamb products Biotech. Appli. Biochem., 12,99.

Dillon, V.M. and Board,RG., (1991). Yeast associated with red meats (A review),
Journal of Applied Bacteriology, 71,93-108.

Diara, H. A., (1992) The indigenous fermented foods of Sudan. In: Applications of
Biotechnology in food processing in Africa. Pp. 23-40. UNDO Veinna.

Dowdell, M.l and Board, RG., (1968). A microbiological survey of British fresh
sausage. Journal of applied bacteriology 31,378-396.

Dyett, E.land Shelley, D., (1962). The microiology of British fermented sausage. 1st
Int. Congo Food Sci. and technol. London.

Dyett, E.I. and Shelley, D., (1966). The effect of sulphite preservative in British J. Appl.
Bact. 29,439. Fresh sausage
 80

Eilberg, B.L. and Lieip, H., (1977) Possible improvement in dry sausage technology by
adding Streptomyces as a starter culture. Die Fleischwirtschaft 9 1698.

Empey, W.A. and Scott, W.J., (1939). Investigations on chilled beef Partl Microbial
contamination acquired in the meatworks. Australian Council for Scientific and
Industrial Research Bulletin.

Engel, G., Rosch, N.and Heller, K.J., (1994) Heat tolerance of heat ofyeasts. Kieier
Milchwietsch. Forschungsber, 46, (1),81-90.

Everson C.W. Danner, W.E. and Hammes, P.A. (1970) 1. Agr. Food Chem., 18 (4) pp
570-571.
Everson, C.W., Danner, WE. and Hammes, P.A., (1974) Process for curing dry and
semi-dry sausages. U.S. Pat. 3814817.

Fennema,O.R. (1985) Food chemistry 2nd ed. Rev and expanded, N.Y., Dekkar.

Fleet, G.H. (1990) Cell walls, In The Yeast. Vol. 4, 2nd ed. Rose A. H. and Harisson 1.
S. Eds. Acadamic Press, London, 200.

Fleet, G. (1992) Spoilage Yeast. Crit Rev. Biotechnol. 12, 1-14.

Fryer, T. F., Lawrence, R. C. and Reiter, B., (1966). Methods for the isolation and
enumeration oflipolytic oranisms. 1. Dairy Sci. 50,477-484.

Galzy, P., (1992) Study of yeast flora ofroquefort cheese. Int. 1. Food Microbiol. 17,9-
18.
 81

Gehlen, K-H.; Meisel C.;Fisher, A; Hammes W.P (1991) Influences of the yeast
Debaryomyces hansenii on dry sausage fermentation. Proc. 37th int.
Congr. Meat Sci. Technol. 2 pp871-876.

Genigeorgis, C. A (1976) Quality control of fermented meats. Javma 169,1220-1228.

Gill, C. O. (1982) Microbial interaction with meats. In meae Microbiology, M. H.

Brown, Ed. Applied Science Publishers, London, pp. 225-64.

Gimenez, E.H., (1992) Evolution of the microbiological parameters in the production of

Dry-cured hams (in Spanish) Proc. of the 8th Scientific Meeting of the Food
Microbiol. Group.

Gokalp, H. Y. and Ockerman, H. W. (1985) Turkish-styled fermented sausages

(soudjouk) manufactured by adding different starter cultures and using different
ripening temperatures. Fleishwirtsch. 65, 1235-1239

Gokalp, H. Y., (1986) Residual nitrate, nitrite, carbonyl and TBA values of Turkish
soujouk manufactured by adding different starter culture and using different
ripening temperature. l Food Technol.21, 615-625.

Grau, F. H. (1981) Role of pH, lactate, and aerobosis in controlling the growth of some
fermentative gram negative bacteria on beef Appl. Environ. Microbiol.
42, 1034-50.

Grazia, I., Suzzi, G., Romanio, P. and Giudici, P., (1989) The yeasts in meat products,
5, s495.

Gryczka, Al, (1977) Bacterial composition and their use in fermented meats. U.S. Pat.
4013797
 82

Gryczka, A.l and Shah, RB., (1979) Process for the treatment of meat with
composition including Micrococcus varians and lactic acid producing bacteria.
U.S. Pat. 4147807.

Guerzoni, M.E., Sinigaglia, M. and Gardini, F., (1993) Interaction between isolation
source, cellular fatty acid composition and stress tolerant Saccharomyces
cerevisiae and its subspecies. J. Appli. Bacteriol. 75: 588-294.

Hadlock, D., Samson RA. and Schinorr, B., (1975). Moulds and meat: The genus
Penicillium. Feischwirtschaft, 55, 969-983.

Hadloek D, Samsom RA. and Schipper M.A.A. (1976). Schimmelpilze und Fleish
Kontaminationflora. Fleischwirtschaft 56, 372-376.

Haines, N.C. and Harmon L.G., (1973). Effect of lactic acid bacteria on growth of
Staphylococcus aureus and production of enterotoxin. Appli. Microbiol. 25,436.

Hammes, W.P., Rolz, I. and Blantleon, A., (1985). Microbiologische Untersuchung der
auf dem deutschen Markt vorhandenen Starterkulturpraparate fur die
Rohwurstbereitung. Fleischwirtsch. 65, 1235-1239.

Hechelmann, H., Holzapfel, W. and Bern, Z. (1977) Vorkmmen und Bedeutung von
Pseudomonadaceae bei der Rohwurstreifung Mitteilungsblatt der Bundesanstalt
fur Fleischforschung Kulmbach Paryt 55. 2963-5.

Hechelmann, H, Lucke, F.-K. and Schillinger, u., (1988) Ursachen und Vermeidung
von Staphylococcus aureus-Intoxikationen nach Verzehr von Rohwurst
und Rohschinken. Mitteilungsbl. Bundesanstalt Fleischforsch. N. 100, 7956-
7964.
 83

Hofmann, H-B. and Schamer, E. (1980) Microbiologische und sensorische

Untersuchungen zur Reifungadynamik von Rohwursten, ausgereift. I. Mitt.:
Veranderung der Mikrflora wahrend der Reifung Nahrung 24,285-93.

Huerta, T., Querol, A. and Hemandez-Haba, J, (1988). Yeast of dry cured hams.
Qualitative and quantitative aspects. Microbiologie, aliments, nutrition 5,247-
252.

Inal, T., (1969) Fleicherei 1, 6-8.

Incze, K. (1965) Die mikrobiologische Veranderungen des Reifungsprozesses. 1.

Gyulaer wurst. Husipar 14,208-12 (In Hungarian)

Incze, K., (1986) Technologie und Mikrobiologie der ungarischen Salami.

Fleischwirtschaft, 66, 1305-1311.

Inigo, B., Arroyo, V. and Somavilla, J, (1970). Estudio microbiologico del proceso de
fabricacion de embutidos. Identificacion de lavaduras. Rev. Agroquim.
Technol. Aliment. 10,406-410.

Jay, JM., (I 978a). Meat and seafood. In: Food and Beverages Mycology. (ed. L.R.
Beuchat) pp. 155-173 Avi Publishing.

Jay, JM., (l978b) Meats, poultry and seafoods. In: Foods and Beverage Mycology (ed.
Beuchat, L.R.) AVI Publishing Westport, Ct. pp 129-144.

Jay, JM., (I987) Meats, poultry and seafoods. In: Foods and Beverage Mycology, 2nd
ed. (ed. Beucht, L. R.) Van Nostrand Reinhold, New York.
 84

Jay, J. M. (1979) Occurrence and significance of specific yeasts and moulds in meats,
poultry and seafoods. In food Mycology ed. Rhodes, M. E. pp. 44-55. Boston: G.
K. Hall.

Jay, J. M. (1996) Modem Food Mirobiology, 5th ed Chapman and Hall, New York

Jay, J.M. and Margitic S., (1981) Incidence of yeast in fresh ground beef and their ratios
to bacteria Journal offood Science 46,648-649.

Jensen, LB., (1935) 'Meat treating method' U.S. Pat. 2002146

Jensen, L.B., Paddock, L.S., (1940) US Patent 2,225-783

Jessen, B. (1995) Starter culture for meat fermentation In Fermented Meats eds G.
Campbell-Platt and P.E. Cook, Blackie Academic and Professional.
London. pp 130-154

Katsaras, K. and Leistner L. (1985) Staphylococcus aureus und Clostridium botulinum.

Bedeutung bel Rohwurst und Rohschinken. In Mikrobiologie und Qualtat von
Rohwurst und Rohschinken. Herausgegeben vom Institut fur Mikrobiologie
Toxikologie und Histologie der Bundesanstalt fur Fleischforschung,Kulmbach,
pp. 152-172.

Kiernat, B.H., Johnson, J. and Siedler, A., (1964) A summary of the nutrient content of
meat. Bull 57. Am. Meat Inst. Found. Chicago III

Kormendy, L. and Gantner G. (1962) Uber freie Aminosauren bei der Rohwurstreifung
Fleischwirtschaft 12, 774-80.

Kouker, G. and Jaeger, K-K., (1987). Specific and sensitive plate assay for bacterial
lipases. Appli. Environ. Microbiol. 53,211-213.
 85

Kreger-Van Rij, N. J. W. (ed) (1984). The yeast - a taxanomic study. Amsterdam;

Elsevier sceince.

Kurk, U.S. Patent 1,380,070, (1921)

Langner, H.l, (1972) Aromastoffe in der Rohwurst. Fleischwirtschaft 52, 1299-1306.

Lawrie, R. (1995) The structure, composition and preservation of meat In Fermented

meats eds G. Campbell-Platt and P. E. Cook Blackie Academic and Professional.
London. pp 1-33.

Lee, W.H., Reimann, H.P. and Al-Mashat, A.I., (1971) Controlled fermentation and
prevention of undesirable bacteria growth in food. U.S. Patent 3,794,739.

Lee M. B. and Styliadis S. (1996) A survey of pH and water activity levels in processed
salami and sausage in Metro Toronto.

Ledward, D.A. (1985). Novel intermediate moisture meat products pp. 447-450. In

Leisner, L., (1991) Fleischerei 42, (3) ppl-5; (4)3-5

Leistner, L., (1995) Stable and safe fermented sausages world-wide. In Fremented
Meats. (Ed Campbell-Platt)

Leistner L. and Ayres lC. (1968). Moulds in Meat. Fleishwirtschaft 48,62-65.

Leistner I. und Bern, Z.(1970) Vorkommen und Bedeutung von Helfen bei
Pokeelfleischwaren Fleischwirtschaft 50, 350-351.
 86

Leistner, L.and Rodel, W., (1975) The significant of water activity for micro-organisms
in meats. In water relation of foods (Edited by R.B.Duckworth); P.309.
Academic Press, New York.

Leistner, L. and Rhodel, W. (1976) Inhibition and Inactivation of Vegetative Microbes.

(F. A. Skinner and W. B. Hugo, eds.) Academic Press, New York.

Leistner, L., (1986a) AlIgemeines uber Rohwurst. Fleischwirtschaft, 66 290-300.

Leistner, L., Schmidt, V. and Kaya, M., (1989) Bedeutung des Vorkommens von
Listerien bei Fleisch und Fleischerzeugnissen. Mitteilungsbl. Bundesanstalt
Fleischforschung, 28, 440-445.

Lerche, M., (1957). Bert. Munch. Tierarztl. Wschr. 70, 13.

a. Fleischwirtschaft 56(2), 178, 180-183.

Liebetrau, B. and Grossman, G. (1976). Die bedeutung von Starterkulturen zur

Rohwurstreifung m Rahmen der Lebensmittel-und Emahrungshygiene. Nature
20(5),489-493.

Liepe, H.-v., (1978a) In La Charcuterie Crue et Les Produits Saumures. (Migaud-

Frentz, ed.) Editions, Soussana, S.A., Orly (France).

Liepe, H-V., (1978b) Herstelung eines lnokulationmaterials zur Erzeugung emse

dichten Schimmelrasens. German Pat. 2614147.

Liepe, H.-v., (1978c) L'utilisation des bacteries dan la technologie du saucisson cru.
RTVA (Revue Technique Veterinaire de I'Alimentation) 138, 3, 5-15.

Liepe,H-U (1978d). Starterkulturen fur die Lebensmitteltechnik 4,26-30.

 87

Liepe, H-U (1981) . Starter culture in meat production, Biotechnology. 5,399-424.

Liepe, H-U., (1983) Daten-Taschenbuch fur die Rohpokelwaren-Technologie. Rudolf

Muller and Co., Pohlheim 1. Druckerei Gebhard, Offenbach (Fed. Rep.
Germany).

Lissner, E., (1939) Wurstologia oder es geht urn die Wurst. Hauserpresse, Hans,
Schaefer, Frankfurt am Main.

Lowry, P.D. and Gill, C.O., (1984) Temperature and water activity minima for growth
of spoilage mold from meat. 1. Appli. Bact. 56 193-199.

Lucke, F-K, (1985) Fermented sausages. In Microbiology of Fermented foods. Vo12.

(Ed. B.1.B. Wood), pp. 41-83. Elsevier Applied Science. London.

Lucke, F-K., (1985) Mikrobiologische Vorgange bei der Herstellung von Rohwurst und
Rohschinken. In: Mikrobiologie und Qualitat von Rohwurst und Rohschinken.
Her ausgegeben vom Institut fur Mikrobiloge, Toxikologie und Histokologie der
Bundesanstalt fur Fleischforschung, Kulmbach. 85-102.

Lucke F-K. (1988) Microbiological processes in the manufacture of dry sausages and raw
ham. Fleischwirtsc. Inter. 2: 3.

Lucke, F-K. and Hechelmann, H., (1987) Starter cultures for dry suasage and raw ham.
Composition and effect. Fleischwirtschaft. Composition and effect.
Fleischwirtschaft. 67,307-314.

Lund, A, (1951) Some beer spoilage yeast and their heat resistance. 1. Inst. Brew.
57, 36-41.
 88

Masters, B.D. (1979). Fate of Salmonella inoculated into fermented sausage. M.S.
Thesis, University_of Florida, Gainsville.

McCarthy, 1.A. and Damoglou, A.P., (1993) The effect of low-dose gamma irradiation
on yeast of British fresh sausage. Food Mocrobiol. 10,439-446.

Meisel, C., Gehlen, K.H. ,Fischer A. and Hammes W.P. (1989) Inhibition of the growth
of Staphylococcus aureus in dry sausage by Lactobacillus curvatus, Micrococcus
varians and Debaryomyces hansenii. Food biotechnol3: 145

Metaxopoulos, 1.,Genigeorgis, c., Fanelli, M .. 1., Franti, C. and Cosma, E. (1981a)

Production of Italian dry salami. I Initiation of staphylococcal growth in salami
under commercial manufacturing conditions. 1. Food Protect. 44, 347-352.

Metaxopoulos, 1., Genigeorgis, c., Fanelli, M. 1. Franti, C. and Cosma, E. (1981 b)

Production of Italian dry salami. Effect of starter culture and chemical acidulation
on Staphylococcus aureus growth in salami under commercial manufacturing
conditions Appli. Environ. Microbiol. 42, 863-87l.

Metaxopoulos, J.; Stavropoulos, S.;Kakouri, A. and Samelis 1. (1996) Yeast isolated from
traditional Greek dry salami. 1. Food Sci. 8 (1) pg 25-32.

Metiva, E., Kirova, E.,Gadjeva, D. and Radeva, M., (1986) Sensory aroma and taste
profiles of raw dried sausages manufactured with a lipolytically active yeast
culture. Nahurung 829-832.

Miller, M. W. (1979) Yeasts in food spoilage. An update, Food Technol., 33, 76.

Molina, I., Silla, H., and Flores, 1., (1990) Study of the microbial flora in dry ham 4.
Yeasts. Fleischwirtschaft 70, 74-76.
 89

Monte, E., Villanueva, JR., and Dominguez, A., (1986) Fungal profiles of Spanish
country cured hams. Int. J Food Microbio1.3, 355-359.

Nagodawithama, T. (1992) Yeast derived flavours and flavour enhancers and their
probable mode of action. Food Technology 46 (11) pgs 138, 140-142. 144.

Niinivaara, F. P. and Pohja. M. S. (1956) Erfahrungen uber die Herstellung von

Rohwurst mittels einer Bakterienreinkultur. Fleischwirtschaft, 9, 264-286.

Niinivaara, F.P., (1955) Uber den Einfluss von Bacterienkulturen auf die Reifung und.
Umrotung der Rohwurst. Acta Agralia Fennica, 85 1-128.

Niinivaara, F.P., (1955). The influence of pure bacteria cultures on ageing and changes
of the red color of dry sausage. Suomen Maataloustieteellisen seuran Julkaisuja
No 84, Acta Agralis Fennica (Helsinki).

Niskanen, A. and Nurmi, E, . (1976). Effects of starter culture on staphylococcal

enterotoxin and thermonuclease production in dry sausage. Appl. Micobol. 34,
(1), Il.

Niven, C.F., (1957) Lactobacillus viridescens nov. spec., a heterofermentative species

that produces a green discolouration of cured meat pigments. J Bact., 73, 758-59

Niven, C.F., (1961). Microbiol. of meats. Circular 68. Am Meat Inst. Found.
Washington D. C.

Nurmi, E., (1966) Acta. agr. Fenn. No 8.1-77

Pal umbo, S. A., Zaika, L. L., Kissinger, J C. and Smith, J L. (1976) Microbiology and
Technology of the pepperoni process. 1. Food Sci. 41, 1270- 1272.
 90

Pederson, G.S., (1979). Microbiology of food fermentations (2nd ed.). AVI Publishing
Co., Westport, COnD.

Put, HM.C. and De Jong, 1., (1982b) The heat resistance of ascospore of four
Saccharomyces spp isolated from spoiled heat processed soft drinks and fruit
products. 1. Appli. Bacteriol. 40,135-152.

Racovita, M., and Racovita, A., (1968) Die Reimpfung von Dauerwurst mit
Schimmelpilzen .. Fleischwittschaft 48(7), 893-987.

Ramihone, M., Sirami, 1., Larpent, 1. P. and Girard, 1. P. (1988) Gout acide des
saucissons secs. Viandes et Produits Cames, 9 (6),291-298.

Rantala, M. and Nurmi, E (1973) Prevention of growth of Salmonella irfamis in chicks

by the flora of the alimentary tract of chicken. Br. Poult. Sci. 14, 624-630.

Reuter, G., (1965). "Untersuchungen uber die Zusammensetzung und die

BeeintluBbarkeit der menschichen Magen-undd Damtlora unter besonderer
Berucksichtigung der Laktobazillen. Emanrungsforschung X, 429-435.

Reuter, G. (1967) Atypische Steptobakterien als dominierende Flora 10 reifer und

gelagerter Rowurst. Fleischwitschaft 47, 397-402.

Reuter, G. (1969) . Zusammensetzung und Anwendung von Bakterienkulturen fur

therapeutische Zwecke. Arzneimittelforschung 19 (1), 103-109

Reuter, G., (1972a). Reifung von Rohwursten vom Typ der Deutschen Salami mit
Laktobazillen- und Mikrokokken-Starterkulturen. Proc. is" Meeting Meat
Research Workers, Guelph (Canada), pp. 666-669
 91

Reuter, G. (1972b). Versuche zur Rohwurstreifung mit Laktobazillen- und Mikrokokken-

Starterkulturen. Fleischwirtschaft 52 (4), 465-473.

Reuter, G. (1972c). Untersuchungen zur antagonistischen Wirkung von

Milchsaurebakterien auf andere Keimgruppen der Lebensmittelflora. Zentralbl.
Vet. Med. B. 19,320-334.

Reuter, G. (1972). Versuche zur Rohwurstreifung mit Laktobazillen- und Mikrokokken-

Starterkulturen. Fleischwirtschaft 52, 465-468, 471-473.

Roca, M. and Incze, K. (1990) Fermented sausages. Food Reviews International, 6(1),
91-118. Rudolf Muller data sheets, Giessener Strasse 94, D-35415, Pohleim.
Germany.

Roostita, R., Fleet G. H. (1996) The occurrence and growth of yeast in camembert and
blue-veined cheeses International Journal of Food Microbiology 28 (3) 393-404

Rossmanith, E., Mantzlaff, H-j., Streng, B., Christ, Wand Leistner, L., (1972) Hefen
als Starterkulturen fur Rohwurste. Jahresbericht der BAFF, Kulmbach, I, 47-48.

Saldanha-da-Gamma, A., Malfeito-Ferreira, M., and Loureiro, v., (1997)

Characterization of yeasts associated with Portuguese pork-based products. Int. 1.
of Food Microbiol., 37,201-207.

Samelis, 1., Stavropoulos, Kakouri, A., Metaxopoulos (1994). 1. Food Microbiology,

11,447-460.

Samelis, J., Tsakalidou, E., Metaxopoulos, I. and Kalantzopoulos, G. (1996).

Differentiation of Lactobacillus sake and Lactobacillus curvalons isolated from
 92

naturally fermented Greek dry salami by SOS-PAGE of whole cell proteins. J.

Applied Bacteriology, 78 (2), 157-163.

Sanz, B., Selgas, D.,Parejo, I. and Ordonez, 1. (1988) Characteristics of lactobacilli

isolated from dry fermented sausages. Int. 1. Food Microbiol. 6,199-205.

Simatos, D. and Multon, J.L., (ed) Properties of water in foods. Martinus Nijhoff
Publishers, Dordrecht, Netherland.

Sirvio, P., Nurmi, E., Poulanne, E. and Niinivaara, F. P., (1977) Der EinfluB uon
Starterkulturen und verschiedenen Zusatzstoffen auf das Wachstum von
Salmonella Senftenberg in Rohwurst Fleischwirtschaft 56 (5), 1007-1012.

Smith,D.R., (1987) Sausage a food of myth, mystery and marvel. CSIRO. Food Res
Quart.,47, 1-8.

Smith, G. and Palumbo, S., Microbiology of Lebanon Bolonia. Appli. Microbiol.. (1973)
26, 489-496.

Smith, M. TH. and Hadlok, R., (1976) Hefen und feisch: vorkommer, systematik und
differenzierung. Feischwirtschaft 56,379-384

Smith, J.L. and Palumbo, S.A., (1983) Use of starter cultures in meats. J. Food Protect;
46, 997-1006.

Sorrels, K. M and Speck, (1970) Diary Sci., 53, 239.

Stiebing, A. and Rhodel, W., (1988) Influence of relative humidity on the ripening of
dry sausage. Fleischwirtschaft 68 (10), 1287-129l.
 ..

93

Stokes,1.L., (1971) Influence of temperature on the growth and metabolism ofyeasts. In

The Yeast. Vol. 2 (ed Rose, A. H. and Harrison, 1. S.) Academic Press New

York.

Su, W. and Beuchat, L.R, (1984) Combined effect of growth medium, age and phase of
sporulation on heat resistance and recovery of Hansenula anomala. ycopathlogia

87, 129-134.

Tanaka, N., Traisman, E., Lee, M.H., Casseus, RG.and Foster, E.M., (1980) "Inhibition
of botulinum toxin formation in bacon"

Ten Cate, L., (1960) Fleischwirtschaft 40.

Tomtov, D., Bern, Z. and Leistner, L. (1974) RIM, 6, 3.

Tokuaka, K., Ishitani, T., Goto, S., and Komagata, K., (1985) Identification of yeast
isolated from high sugar foods. 1. Gen. Appli. Microbiol. 31,411-427.

Touaka, K., and Ishitani, T., (1991) The minimum water activity for the growth of yeast
isolated from high sugar foods. 1. Gen. Appli. Microbil. 37, 111-119.

Tramer, 1. (1966) Inhibitory effect of Lactobacillus acidophiluus. Nature (London) 211,

204-205.

Troll er, 1. A. and Christian, 1. H. B. (1978) Water Activity and Food. New York:
Academic Press.

USDA (1977[) The staphylococcal enterotoxin problem in fermented sausage. Task

Force Report. F.S.Q.S., Washington D.e.
 94

Van Eck, 1. H., Prior, B.A and Brandt, E. V. (1993) Journal of General Microbiology
139, 1047-1054.

Vayssier, Y. (1979) New mould culture for surface ripening of dry sausage. RTV A, 18,
50.

Vidal-Leira, M., Buckley, H. and Uden, N., (1979) Distribution of the maximum
temperature for growth among yeasts. Micologia 71, 493-50l.

Viljoen, B.C., Dykes G.A, Callis M., von Holy, A, (1993) Yeast associated with
vienna sausage packaging. Int. 1. Food Microbiol. 37,201-207.

Walker, H.W., (1977) Spoilage of food by yeast. Food Technology 31, 57-61, 65.

Walker, H. W. and Ayres, 1. C. (1970) Yeasts as spoilage organisms. In Yeasts. Yeast

Technology, Vol. 3 (eds Rose, A H. and Harrison, 1. S.) Academic Press,
London. pp. 463-527.

Wickerham, L. 1., (1951) Taxanomy of Yeast. Technical Bulletin 1029, US

Department of Agriculture, Washington, DC.

Williams, W. P (1990) Penicilli un and Aspergillus in the food microbiology laboratory.

In Modern Concepts in Penicillium and Aspergillus Classification. Eds R. A
Sampson and 1. 1.1. Pitt, pp. 121-137, Plenum Press, New York.

Zeuthen, P., (1995) Historical aspect of meat fermentation. In Fermented Meats (Ed
Campbell-Platt)

Zottola, E.A, (1972) Introduction to meat microbiology, AMI. Center for continuing
education, Am. Meat Inst., Washington, DC.