The Combined Effects of Pediococcus acidilactici and Lactobacillus curvatus

On Listeria monocytogenes ATCC 43251 in Dry Fermented Sausages.

By

Frank Kofi Dogbatsey

A Research Paper Submitted in Partial Fulfillment of the

Requirements for the Master of Science Degree

With a Major m

Food & Nutritional Sciences

Approved: 6 Semester Credits

Marcia Miller-Rodeberg, PhD, Advisor.

Stephen Nold, PhD

~9.~ J Smdelar, PhD

I

The Graduate School University of Wisconsin-Stout

December, 2010.

Author:

Title:

The Graduate School University of Wisconsin-Stout

Menomonie, WI

Dogbatsey, Frank K.

The Combined Effects of P. acidilactici and L. curvatus on

L. monocytogenes ATCC 43251 in Dry Fermented Sausages

Graduate Degree/Major: MS Food and Nutritional Sciences

Research Adviser: Marcia Miller-Rodeberg, PhD

MonthrYear: May, 2011

Number of Pages: 74

Style Manual Used: American Psychological Association, 6th edition

Abstract

The use of starter cultures in meat fermentation has been known since the 15th century.

Since then, a scientific approach has been adopted to study the benefits derived from meat

cultures, including how starter cultures keep fermented meat safe for consumption through the

production of organic acids and bacteriocins that inhibit or kill pathogenic microorganisms.

2

Although not all starter cultures are capable of controlling pathogens in meat, some fermentation

cultures have been used to control the food-borne pathogens that cause disease in humans and

result in major recalls of fermented foods worldwide. The aims ofthis study were to: 1) evaluate

whether the combined effect of two common meat cultures would better control Listeria

monocytogenes in dry fermented sausage than the cultures applied individually, and 2) to

compare which of the individual cultures had the ability to most effectively control L.

monocytogenes. Additionally, this research determined whether environmental conditions

effectively control L. monocytogenes populations during fermentation and examined if the

fermentation culture conditions affected proteolysis.

3

To accomplish these tasks, L. monocytogenes (lx104 CFU/g) was inoculated into sausage

batters containing Pediococcus acidilactici, Lactobacillus curvatus, and a mixture of the two

cultures at 1x107 CFU/g. L. monocytogenes was then grown on solid Listeria selective medium

(LPM) and lactic acid bacteria were grown on MRS agar. Sausages were incubated at 3TC for

12 hours, then allowed to dry at 12°C (78% relative humidity) for 28 days. Samples were

obtained on days 0, 2, 4, 7, 14 and 28 for microbiological and environmental conditions analysis,

including water activity and pH.

Although L. monocytogenes was detected in control sausages after 28 days, when starter

cultures were used, Listeria populations were significantly reduced. Pediococcus alone inhibited

Listeria more effectively than Lactobacillus alone. A combination of starter cultures resulted in

faster decline of Listeria populations in the dry fermented sausages than the individual cultures

alone. Environmental conditions (PH and water activity) did not significantly inhibit Listeria,

since Listeria grew in uninoculated control sausages while pH and water activity declined.

Starter cultures did not impact proteolytic activities, but this could be due to limited ability to

resolved differences in protein banding patterns.

These results are important for the sausage industry. Combined starter cultures are

already used to enhance flavor, but these findings also suggest their importance in controlling

pathogenic microbes. These results also underscore the importance of biological control

methods as food industries search for better ways to produce safer meat products for consumers.

The Graduate School University of Wisconsin-Stout

Menomonie, Wisconsin

Acknowledgements

I would like to thank God for His goodness and seeing me through my years of study

here at the University of Wisconsin-Stout. I also want to thank Marcia Miller-Rodeberg for her

willingness and the advice she gave me throughout this research. I especially want to thank the

rest of my research committee members, Dr. Stephen Nold (UW-Stout), Dr. Hans Zoerb (UW-

Stout) and Dr. leffSindelar (UW-Madison). Thank you for your inspiration, thoughtful advice

and tremendous efforts to provide all the needed resources for the implementation of this

research.

In addition, I want to acknowledge the relentless efforts of lames and Nancy Burritt in

helping through my experiments and writing this paper. Their support and encouragement have

helped me come this far in my research abilities and through them, I've understood better the

saying, "Determination is truly the key to success". I also extend my appreciation to Chr.

Hansen cultures for providing me with starter cultures and valuable insight of the research.

Finally, I would like to thank my family and friends for their love, prayers, support and

encouragement throughout my academic career, without which all these would have been

impossible. Thank you all and God bless you.

4

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Table of Contents

.................................................................................................................................................... Page

Abstract ............................................................................................................................................ 2

List of Tables ................................................................................................................................... 7

List of Figures .................................................................................................................................. 8

Chapter I: Introduction ..................................................................................................................... 9

Purpose of the Study .......................................................................................................... 13

Hypotheses of the Study .................................................................................................... 13

Limitations of the Study ..................................................................................................... 14

Definition of Terms ............................................................................................................ 14

Chapter II: Literature Review ........................................................................................................ 16

History of Sausage Making ................................................................................................ 17

Traditional and Modern Methods of Sausage Manufacture .............................................. 18

Sausage Preservation and Consumer Safety ...................................................................... 18

Key Ingredients for Sausage Manufacturer ....................................................................... 20

Use of Starter Cultures in Sausage Fermentation ............................................................. .25

Starter Cultures and Control of Other Food-borne Microorganisms in Meat Products .... .29

Bacteriocins ........................................................................................................................ 32

Effects of Listeria monocytogenes on Human Health and Meat Industry ......................... 34

Chapter III: Materials and Methods .............................................................................................. .3 8

Starter Culture Strains and L. monocytogenes Inoculum Preparation ............................... 38

Genoa Salami Preparation .................................................................................................. 38

Microbial Analysis ............................................................................................................. 39

6

pH and Water Activity Determination .............................................................................. .41

Protein Separations ........................................................................................................... 42

Chapter IV: Results ........................................................................................................................ 44

Chapter V: Discussion ................................................................................................................... 55

References ...................................................................................................................................... 60

7

List of Tables

Table 1: Key Non-Meat Ingredients and Their Uses ..................................................................... 21

Table 2: Common Spices used in Sausages, Areas They are Commonly Found, and Some

Products They are Used to Produce ............................................................................... .25

Table 3: Microorganisms Used as Meat Starter Cultures ............................................................ .27

Table 4: Select Starter Culture Organisms, the Food Borne Pathogens they Control, and Meat

Products they are used to Ferment .................................................................................. 29

Table 5: Selected Bacteriocins, Their Sources, and Pathogens they Control ............................... 34

Table 6: Some Growth Conditions ofL. monocytogenes in Foods when all Other Factors

Are Optimum ............... '" ................................................................................................ 3 7

Table 7: Quantities of Various Ingredients used in Preparing Genoa Salami by Weight

and Percentage ................................................................................................................ 40

Table 8: Experimental Sample Formulations ............................................................................... .42

Table 9: Mean Population of L. monocytogenes Survival in Different Sausage Treatments

Over the 28 Day Period ................................................................................................... 46

Table 10: Mean Population of Lactic Acid in Different Sausage Treatment

Over the 28 Day Period .................................................................................................. 47

Table 11: Mean pH Measurements of Different Sausage Treatment Over the 28 Day Period ..... 50

Table 12: Mean Water Activity Measurements of Different Sausage Treatments

Over the 28 Day Period .................................................................................................. 51

8

List of Figures

Figure 1: General flow process for fermented sausage manufacture ............................................ 19

Figure 2: Flow diagram of Genoa salami preparation ................................................................. .41

Figure 3: Change in monocytogenes and lactic acid bacteria population over time ................... .48

Figure 4: Change in monocytogenes and lactic acid bacteria population over time .................... 52

Figure 5: Change in water activity and L. monocytogenes population over time ........................ 53

Figure 6: Protein bands picture from PAGE Gel-Electrophoresis for days 2 and 28 ................... 54

Chapter I: Introduction

Pork products have recently come under surveillance as a result of outbreaks of food­

borne pathogens like Listeria monocytogenes that causes listeriosis in humans. Listeria and

other pathogens are naturally found in agricultural ecosystems with slaughter houses and retail

points serving as prevalent sources (Genigeorgis, 1989). L. monocytogenes is considered an

important food-borne pathogen because of the risks it poses to immune-compromised patients,

including the elderly, children and pregnant women.

Meats used in sausage typically contain pork, beef, veal, poultry or a combination.

9

Others use elk, lamb or rabbit due to religious beliefs. Different pathogens are found in different

meat types. For example, species of Escherichia coli are sometimes found in comminuted beef

used for sausage processing while Salmonellae spp. are common in poultry. Contamination of

meat by pathogenic microorganisms is almost impossible to avoid during sausage processing due

to the nature ofthe slaughter process (Madden, 1994). Sausage manufacturers therefore seek

ways to reduce contamination to levels that do not threaten consumer health.

Contaminating meat products with L. monocytogenes often begins at the slaughter house.

Likely sources of contamination include processing utensils and animal waste products

(Stekelenburg & Kant-Muermans, 2001). It is difficult to completely eliminate L.

monocytogenes from meat production facilities as it has the ability to grow in processing

environments and can even grow in meat after packaging because is its ability to grow as in the

low-oxygen environment found in packaged meat (Buchanan & Klawitter, 1991).

Proper hygienic practice must still be followed during processing to reduce

contamination by L. monocytogenes and other pathogens. This is because the extent of

contamination and cross-contamination largely depends on the training of personnel and general

10

hygienic measures adopted by the processing facility (Yucel et aI., 2005). Methods that reduce

bacterial loads post-slaughter include the addition of inhibitory ingredients to the sausage and the

control of environmental conditions such as temperature, pH, and water during processing.

Meat preservation with saltpeter as a curing agent has been used for thousands of years to

reduce meat spoilage and disease transmission. This ancient method is still practiced today

(Jensen, 1954). During World War II, the demand for fermented meat products increased

because the military required processed meat with a longer shelf life. This led to large-scale use

of lactic acid bacterial cultures in meat fermentation and standardized methods for fermentation

to complement the use of salt in meat preservation (Caplice & Fitzgerald, 1999). Embraced by

meat processors, fermenting meats prior to consumption was also found to control food-borne

spoilage and reduce the number of pathogenic microorganisms, including Staphylococcus

aureus, Escherichia coli, and L. monocytogenes cornmon organisms in meat products (Smith et

aI., 1983a). During sausage fermentation and drying, the number of live cells of L.

monocytogenes decreases when using starter cultures, curing salts, and heat (Tolvanen et aI.,

2008).

A starter culture is an inoculum of beneficial microorganisms added to food products

such as meat, milk, and vegetables to improve food quality and safety. Starter cultures provide

rapid lactic acid development from fermentation of sugars added to the sausage (Visessanguan et

aI., 2004a) resulting in a decreased pH that retards the growth of most spoilage microorganisms

and enhances flavor and texture of the final product (Meng & Schaffner, 1997). There is a linear

relationship between numbers of lactic acid bacteria and pH (Visessanguan et aI., 2004b).

Acidity has been reported to contribute to the decline of undesirable microbes in dry fermented

sausages. This is due to the homeostatic disturbance of pathogenic and spoilage organisms

resulting from the low pH of their external environment (Leistner, 2000).

11

The use of starter culture therefore, helps to stabilize the fermentation by controlling the

microflora ofthe food (Font de Valdez et aI., 1990). Another advantage of using starter cultures

is that fermentation and ripening process are more easily controlled when culture characteristics

and starting concentrations are known. Common fermentation cultures include species of

Lactobacillus, Pediococcus, and Staphylococcus. In 1940, Jensen and Paddock (1940)

introduced Lactobacillus to dry sausage fermentation processing to reduce ripening time and

ensure retention of sausage aroma and quality. Pediococcus cerevisiae [later reclassified as P.

acidilactici (Niven, 1955)] was the first lactic acid bacteria to be developed in the United States

as a pure starter culture. Species of Staphylococcus were earlier identified as pathogenic (hence

not suitable for food fermentation) but researchers later found S. carnosus and other non­

pathogenic strains that are now used in sausage fermentation (Niinivaara et aI., 1964). The

potential use of Staphylococci in sausage fermentation was further supported by Casaburi et aI.

(2005), who evaluated the potential of S. simulans and S. carnosus stains in fermented sausages.

They found that these species possessed antioxidant properties, grew at optimal pH, temperature

and salt concentrations, and did not produce toxic compounds, allowing their use as meat starter

cultures. Unfortunately, some food-borne pathogens like Listeria, Clostridium, and Bacillus

have developed resistance mechanisms to overcome challenges posed by acidic conditions

resulting from fermenting starter cultures (Van Schaik et ai. 1999; Abee & Wouters, 1999).

Resistance mechanisms include altering cell membrane characteristics to resist changes in pH,

production of alkali to neutralize acids, and shifting metabolism to pH-stable mechanisms

(Cotter & Hill, 2003a). Since bacterial pathogens constantly develop resistance mechanisms,

12

sausage manufacturers must meet this threat by continuously developing new strategies to keep

their products safe. Adding a mixture of different starter cultures during sausage manufacture is

one way to accomplish this goal. By combining the anti-pathogen properties of different starter

cultures, we may achieve pathogen control.

One way starter cultures control other bacterial cells is by producing toxins called

bacteriocins, which are small oligopeptides commonly produced by food-grade lactic acid

bacteria (Cotter & Hill, 2003b) and are capable of inhibiting and killing a wide range of food­

borne pathogenic microorganisms (Rosenstein et aI., 2009; Ruiz-Barba et aI., 1994). Not all

lactic bacteria are capable of producing bacteriocins, so adding a mixture of starter cultures could

draw on the beneficial effects of each. Mixed starter cultures allow manufacturers to exploit

both biological (bacteriocin) and environmental properties (PH and water activity) of

fermentation cultures in controlling pathogens. Mixed cultures can target a range of pathogenic

organisms while enhancing flavors in the fermented product. In an early study, Nurmi (1966)

confirmed the ability of mixed cultures to inhibit food-borne pathogens in dry fermented

sausage. Here, Micrococcus varians and Lactobacillus plantarum were mixed to inhibit Listeria

while producing a good-flavored dry fermented sausage. Lactic cultures also achieve fat

breakdown (Collins et aI., 2003), utilize carbohydrates (Acton et al., 1977), and proteolyze meat

proteins, each contributing to sensory enhancement of the meat product. Proteolysis of meat

proteins produces free amino acids which are important for developing taste and a more tender

dry fermented sausage (Kato et al., 1994). This is a result of myofibrilar fragmentation of meat

muscle caused by commercial lactic cultures (Aksu et aI., 2002).

The present study was initiated to evaluate the impact of mixed starter cultures on

pathogen survival and meat proteolysis in dry fermented Genoa salami. The starter cultures

chosen have been shown to individually inhibit the growth and survival of undesirable

microorganisms including L. monocytogenes (Ross et aI., 2000) but to date there are no reports

of their combined effects.

Purpose of the Study

The present study aims to:

13

1. Elucidate the combined effect of Lactobacillus curvatus and Pediococcus acidilactici on

the growth and survival of Listeria monocytogenes in dry fermented sausages.

2. Understand the effects of starter cultures on meat proteolysis.

3. Compare the individual starter cultures for relative effectiveness in controlling L.

monocytogenes in dry fermented sausages.

4. Report on the effects of environmental parameters (PH and water activity) on L.

monocytogenes in sausages.

Hypotheses of the Study

1. Mixed starter cultures will decrease L. monocytogenes populations more effectively

than the individual starter cultures alone. Synergistic effects of Pediococcus and

Lactobacillus working together will decrease L. monocytogenes more rapidly than either

culture individually.

2. Environmental factors will have less effect than biological factors on the control of L.

monocytogenes in fermented sausages. While starter cultures impact sausage acidity and

water availability, these effects will be less important than biological effects such as

bacteriocin production in controlling Listeria.

Limitations of the Study

a) No sensory experiments were conducted on sausages fermented with the mixed starter

cultures to ensure their consumer acceptability.

b) Although no colonies of L. monocytogenes were detected on the LPM agar plates, there

was no further specific test to confirm the absence of the pathogen in the samples.

Definition of Terms

Amino Acids. Protein precursor molecules that share a common chemical structure

[NH2-CRH-COOH]. Amino acids can be linked through peptide bonds to form proteins.

Bacteriocins: Small, naturally occurring antimicrobial proteins produced by bacteria that are

used to inhibit the growth of competing microorganisms.

Curing. A process of preservation and flavoring, (especially fish and meat) which

involves adding of salt, sugar, nitrates or nitrite.

14

Decarboxylation. Any chemical reaction in which a carboxyl group (-COOH) is split off

from a compound, producing carbon dioxide (C02),

Entertoxins. Any of several toxins produced by intestinal bacteria.

Facultative anaerobes. Organisms that can live in the presence or absence of oxygen.

Fermentation. Food preservation processing where carbohydrates in foods are

converted to alcohols and carbon dioxide or organic acids using yeasts, or bacteria under

anaerobic conditions.

Hemolytic-uremic syndrome. Breakdown of red blood cells which results in anemia.

Hemorrhagic colitis. A syndrome (clinical) that results in bloody inflammation of the

large intestine.

Histamine. An amine released by cells that causes symptoms of an immediate allergic

reaction.

Inoculum. A small amount of bacterial culture introduced into surroundings suited to

cell growth.

15

Listeriosis. A bacterial infection caused by Listeria monocytogenes.

Methemoglobinemia. Abnormal increase in the level of hemoglobin in the blood which

affects its ability to effectively transport blood oxygen.

body.

Myoglobin. Protein in the blood that binds and stores oxygen in tissues.

Proteolysis. The breakdown of proteins into peptides and amino acids.

Purine. Natural nitrogen-containing substances present in most foods and found in the

Supernatant. The liquid remaining above the pellet after centrifugation of a suspension.

16

Chapter II: Literature Review

The primary goals of fermentation include extending shelf life of material, enhancing

flavor, and inhibiting spoilage and pathogenic microorganisms. During fermentation,

microorganisms convert carbohydrates and other sources of carbon in food material to ethanol

and carbon dioxide or organic acids. Metabolic by-products from the microorganisms involved

in fermentation preserve the food, enhance flavor and increase consumer acceptability (Rahman,

2007).

Preservation methods such as the use of irradiation, blanching, and heating are also used

in the food industry to control pathogens and spoilage microorganisms. Food fermentation may

be preferred over some of the above methods, although some processors consider irradiation and

heating methods as more effective in controlling pathogens than fermentation. One of the

reasons why fermentation is preferred is the additional flavors that fermentation products impart,

its role in developing a more nutritive and digestible product, and its role in enhancing food

preservation, as the low pH inhibits the growth of microbial pathogens (Norma & Hotchkiss,

1998). Fermentation encourages the growth of beneficial microbes and their metabolites in

foods. Fermentative bacteria, especially lactic acid bacteria, contribute to the texture and flavor

of the fermented food product. Fermentation is an inexpensive method of food preservation and

does not require sophisticated facilities. It can therefore be practiced anywhere in the world by

people with little or no training.

Fermented foods may be classified by their physical, chemical or biological properties.

Steinkraus (2002) classified fermented foods by types of organisms used in the process, the

biochemistry of the food, and the state of the food (e.g. liquid or solid). Campbell-Platt (1987)

had earlier expanded the classification to include cereal products, beverages, dairy foods,

legumes, fruits and vegetables, fish, and meat products. Based on classification, processors

identify the strains of lactic cultures that produce the results in different fermented foods. For

instance, Streptococcus acidophilus has been identified to produce best results in dairy culture

and Pediococcus acidilactici for meat products.

History of Sausage Making

17

Sausage processing evolved as an economic means of food preservation that also

converts pieces of meat into more palatable products. According to the Oxford encyclopedia of

Foods and Drinks (2004), the name sausage came from the Latin word salsus, which means

salted. This is because sausage processing began as a method of preserving meat with salt.

Records however show that Babylonians and the people of China consumed sausage as early as

1500 BC (Pederson, 1971). Meat trimmings were ground, salted, stuffed into casings, and dried

to develop the desired flavor. The evolution of cured sausage began by adding either sodium

chloride or saltpeter (potassium nitrate), and by the late 1800s scientific study showed that

saltpeter was beneficial in sausage preservation.

A variety of unique sausage fermentation techniques emerged in other parts of the world.

For example nham, a Thai fermented sausage is fermented in leaves instead of casings. The

common element for all fermentation techniques is the unique role microorganisms play in the

processing of meat products. Intensive research into the ecological and biochemical properties

of these microbes contributed to the cultivation and use of lactic bacteria in sausages.

Researchers and processors in the United States embraced the use of lactic bacteria in sausage as

early as 1930s and this later gained popularity through the work of Jensen and Paddock in 1940.

18

Traditional and Modern Methods of Sausage Manufacture

Traditionally, sausage manufacture was confined to homes and small commercial

settings. The ground meat was processed with indigenous herbs and spices. Fermentation of

meat depended on adventitious bacteria present in the butcher's premises. This means that the

quality of sausage produced at the time relied on the type and amount of beneficial

microorganism present at the time of processing. Each manufacturer followed family recipes in

preparing sausages to their own taste (American Meat Institute Foundation, 1960). Therefore,

flavor was determined by each manufacturer's location and family recipes.

Modern sausage production is independent of environmental conditions. Temperature,

humidity, moisture, and acidity are continuously and carefully controlled. Microorganisms with

specific desired properties can be grown separately and added to sausages. Recipes and

ingredients for particular types of sausages can be obtained from the literature and there are

limits to amounts of additives regarded as acceptable. Figure 1 shows the general flow process

for fermented sausage manufacture.

Sausage Preservation and Consumer Safety

Safety is a priority of meat industries all over the world. Each year millions of dollars are

spent on research for improving the safety of meat products. In the United States alone it is

estimated that food borne pathogens cause 76 million illnesses and 5000 deaths annually, many

of which result from meat borne pathogens like Listeria and Salmonella. Listeria and

Salmonella are known to be responsible for 15,000 deaths annually (Mead et aI., 1999). The

meat industry in America is therefore confronted each year with recalls of products resulting

from contamination by food borne microorganisms.

19

Pork Meat Pork fat

Frozen

Curing Salts

Cutting/Grinding Seasonings

Starter Cultures

Mixing/Filling Casing

Fermentation

Smoke

Ripening

Figure 1. General flow process for fermented sausage manufacture.

The importance of meat preservation techniques cannot be over-emphasized. As a result,

various preservation practices have emerged. Some of the practices like modified atmosphere

packaging and irradiation are beneficial in controlling undesired microorganisms without

compromising the sensory property of the meat. To combat meat-borne pathogens and still

preserve the desired sensory properties of the product, meat processors have adopted "hurdle

technology" that combines different preservation methods to inhibit spoilage and pathogenic

microorganisms without compromising the safety, nutritional, sensory, and shelflife of meat

products.

20

In hurdle technology, physical, chemical, and biological methods of preservation are

combined to preserve the food. The physical methods include addition or removal of heat,

management of water or water activity, pH, irradiation, modified atmosphere packing, the use of

microwaves, and high pressure processing. Chemical methods include the use of additives and

preservatives, curing ingredients, smoke, and antioxidants. Acid produced during fermentation

and bacteriocins produced by starter cultures are the common biopreservative methods used by

meat processors.

Key Ingredients for Sausage Manufacture

Adding ingredients to sausages, like any form of food processing, is meant to increase

consumer acceptability of the product. Adding different ingredients produces a variety of

sausage types. These ingredients not only improve the sensory properties of sausage but also

serve as preservatives and to help ensure the stability of the product. However, adding one

ingredient does not necessarily improve the sensory and safety attributes of sausages. A

combination of ingredients like curing salt, seasonings, and starter cultures have proved to be

beneficial and acceptable for producing safe and stable sausages. Some of the key ingredients

and their roles are summarized in the Table 1.

Table 1

Key Non-Meat Ingredients and Their Uses

Non Meat Ingredients

Salt

Sweeteners

Spices

Curing Agents

Starter Cultures

Uses

Preservation

Flavoring

Fermentation

Reduce Saltiness

Flavoring

Antioxidant

Flavoring

Antimicrobial Agent

Improve Color

Retards rancidity

Fermentation

Antimicrobial Agent

Examples

Sodium Chloride

Potassium Chloride

Dextrose

Sucrose

Corn Syrup

Ginger

Cloves

Black Pepper

Sodium nitrite

Sodium nitrate

Lactobacillus plantarum

Pediococcus acidilactici

Staphylococcus carnosus

21

Salts. Sodium chloride is predominantly used, though it may be replaced or combined

with potassium chloride. Combined with nitrite, sodium is considered the most effective in

inhibiting and growth of pathogenic and spoilage microorganisms. Smith and Palumbo (1973)

investigated the effect of different concentrations of sodium chloride on the survival of spoilage

bacteria in Lebanon bologna. They found that higher concentrations of sodium chloride

inhibited the growth of spoilage organisms in the absence of starter cultures. This is because salt

22

lowers water activity, making water unavailable to the microorganisms for growth. Plasmolysis

also occurs, as water flows out of the cell membrane when the osmotic potential is disrupted.

The sensory color and texture acceptability of fermented meat are also improved by the addition

of sodium chloride, potassium chloride, and calcium chloride individually or in combination.

Nitrite. Sodium nitrite is a vital ingredient for meat curing, but nitrates can sometimes

be used as a replacement. For safety and health reasons, nitrite use in dry sausage is controlled at

156 ppm as the allowable limit. Nitrites were therefore encouraged as a replacement for nitrates

even though some meat processors still use nitrates. Sodium nitrite or nitrate is added to meat

products for two reasons. First, the bright color of cured meat product is retained through a

series of reactions. Thus, nitrite is converted to nitric oxide, which then combines with

myoglobin, a pigmented protein in meat responsible for natural meat color (Gotterup et aI.,

2007). Once combined, the nitric oxide myoglobin produces the bright red color of the cured

meat. Second, sodium nitrite or nitrate is added to meat due its antimicrobial effects on

pathogens such as the ability of nitrite to inhibit formation and growth of Clostridium botulinum

spores (Dykhuizen et aI., 1996). Christiansen et aI., (1974) reported an inverse relationship

between the amount of nitrite and Clostridium cell counts during bacon manufacture. These

authors noted the reduction was dependent on both temperature and salt concentration.

Unfortunately, undesirable compounds are produced by humans who consume meats containing

nitrates and nitrites. These compounds, called nitrosamines, may induce a disorder known as

methemoglobinemia, a condition where blood oxygen is not transported efficiently because

hemoglobin is converted to methemoglobin (Hord et aI., 2009). Cassens (1995) also linked

consumption of nitrite and nitrate intake from food to cancer in young children. Several

countries therefore continue to monitor and control nitrate and nitrite levels in foods. The

American Cancer Society concluded that nitrite levels in American foods do not cause

significant cancer among Americans (American Cancer Society Dietary Guidelines A visory

Committee, 1996).

23

Sweeteners. Fresh meat contains dextrose but not in sufficient amounts for lactic acid

bacteria to produce enough acid to inhibit growth of pathogens. Fast-fermented sausages rely on

added sugar for the production of lactic acid to lower pH and prevent spoilage. Roughly 0.1-1 %

dextrose (w/w) is added to meat during sausage manufacture to yield the desired lactic acid for

good taste and safety. The added amounts vary depending on sausage type. 0.1-0.3% is added

to slow-fermented sausages while fast-fermented sausages require 0.3-1.0%. Different amounts

are added to prevent slow-fermented sausages from developing an acid taste while encouraging

fast-fermented sausage to produce acid quickly. Sugar concentrations affect the product's taste

after the meat is exposed to heat (Cassens 1994), but also reduce the harsh taste of the added

salts. Even though traditional long-dried sausage makers may not add any sugar, increasing

glucose level by 1 % in sausage decreases pH proportionally and pH is vital in the fermentation

process of sausages (Marianski & Marianski, 2008a).

Spices. Different spices and herbs have for centuries been added to foods in different

concentrations to achieve characteristic flavors. In meat processing, spices are used alone or in

combination with other ingredients like salts and sugars to give sausages and other meat products

their desired flavor, pungency and color.

Garlic, mace, rosemary, ground pepper, paprika, and ginger are spices that are most used

in sausage manufacture (Verluyten et aI., 2004). Other spices provide additional benefits when

added to sausages. Chipault et aI. (1952) was the first to report the ability of rosemary and sage

to exhibit profound antioxidant properties in meat. AI-Jay et aI. (1987) further evaluated the

24

antioxidant properties of ten spices that are mostly used in dry sausage fermentation. They

reported that clove, allspice, and black pepper exhibited antioxidant activity. This property of

clove had earlier been attributed to the presence of eugenol (Kramer, 1985). According to Gray

and Killinger (1966) the use of natural and artificial additives in foods can be use to minimize or

inhibit the growth of food-borne microbes. The inhibitory capability of some spices has been

attributed to their essential oils such as phenolic amide present in black pepper, tocopherols from

oregano, diarylheptanoid found in ginger and eugenol in clove (Kim et al., 1995). In a related

study, Blank et al. (1987) discovered that ground clove eugenol has the tendency to decrease the

rate and extent of Bacillus subtilis spore germination. Nutmeg, bay, and mace extracts added at

levels of 125 ppm were found to inhibit Clostridium botulinum toxin production in turkey

frankfurters, whereas ground cinnamon, clove, mustard, garlic, and onion added at 0.5% had

inhibitory effects on Listeria monocytogenes (Hall, 1986). In addition to the sensory qualities

spices impart to processed meat, they also serve as antioxidants and antimicrobial agents in meat

products. Table 2 below shows common spices used in sausage manufacture, their sources, and

examples of resulting products.

25

Table 2

Common Spices used in Sausages, Areas They are Commonly Found, and Some Products They

are Used to Produce

Spice Common Location Grown Use

Ginger Jamaica, West Africa Frankfurters, Pork Sausages

Garlic India, U.S.A, Italy, Mexico Polish and Smoke Sausages

Clove Brazil, Sri Lanka, Tanzania Bologna, Liver Sausage

Black Pepper Singapore, Thailand Polish Sausage, Bologna

Paprika Hungary, Ethiopia, Spain Frankfurters, Dry Sausages

Use of Starter Cultures in Sausage Fermentation

Beneficial microorganisms are vital in the manufacture of fermented sausages. They

often present naturally in meat, but can also be added in the form of starter cultures. Meat

fermentation using starter cultures ensures the presence of sufficient cell numbers to guarantee

consistent and controlled fermentation. Adding these microbes also inhibits the growth of

spoilage and pathogenic microorganisms by reducing pH, producing organic acids, and

producing inhibitory bacteriocins. Other reasons why food processors use starter cultures are

their ability to reduce variations between batches reSUlting in consistent product as well as

minimize processing time.

Some small scale manufacturers of fermented sausages still use the "back slopping

method" where meat from a previous successful fermentation is used to inoculate the new

sausage (Smith et ai., 1983b). Back slopping is still practiced in developing nations for meat,

26

dough and local beer fermentation (Holzapfel, 2002). The term starter culture was then adopted

and used for bacterial inoculation because these cells are used to initiate the fermentation process

(Cogan, 1995). Such microorganisms must be tolerant to nitrite and salts while exhibiting

growth at optimal temperatures. They must be safe for consumption, and must not produce off­

odors. Some of the genera of starter cultures include Lactobacillus, Lactococcus, and

Pediococcus. Pediococcus and Lactobacillus are used in the meat fermentation industries where

the aim is to produce fast-ripening and low acid sausages. Some ofthe important lactic acid

bacteria utilized in sausage fermentation are shown in Table 3.

Starter Cultures and Proteolysis in Sausage. During the curing process, sarcoplasmic

and myofibrillar meat proteins are hydrolysed by microbial proteases and endogenous meat

enzymes such as cathepsins. Peptides, polypeptides, and free amino acids are among the

compounds that produced through proteolysis (Diaz et aI., 1993). Desirable flavor of fermented

meat results from the production of these compounds during meat proteolysis (Hagen et aI.,

1996). Eskeland (1980) had earlier reported that the fermentation of salami with Lactobacillus

improved the taste and texture. They found that the digestibility and utilization of free amino

acids increased by the end of the fermentation process when the meat was fed to rats. The

proteolytic activities of endogenous enzymes on dry-cured ham has been studied by Toldra et aI.

(1995). Unfortunately, information regarding the contribution of fermentation microbes on

proteolysis is relatively unknown due to the lack of adequate techniques to evaluate changes

caused by fermentation microbes (Martin et aI., 2006). Roringuez et aI. (1998) studied the

activity of microorganisms isolated from meat to evaluate the proteolytic activities of moulds,

yeast and gram positive cocci in dry-cured meat. Most of the microorganisms tested showed

high proteolytic activity for myosin. They concluded that myosin hydrolysis is a suitable test for

preliminary screening of microbial proteolytic activity necessary for selecting microorganisms

that are used as meat starter cultures (Roringuez et aI., 1998).

Table 3

Microorganisms Used as Meat Starter Cultures

Microorganism Family Species Use

Lactic Acid Bacteria Pediococcus P. acidilactici Fast fermented

P. pentosaceus Acid production

Lactobacillus L. plantarum Acid production

L. pentosum Acid production

L. sakei Acid production

L. curvatus Acid production

Curing Bacteria Staphylococcus S. xylosus Flavor and Color

S. carnosus Flavor and Color

Kocuria (Micrococcus) K. varians Flavor and color

Yeast Debaryomeces D. hansenii Flavor

Candida C. Jamata Flavor

Mold Penicillium P. nalgiovense White mold

P. chrysogenum White mold

27

Fermentation microbes break down lipids in addition to proteins (Femades et aI., 1988).

Unfortunately, these bacteria require preformed B-vitamins, amino acids, purine, and pyrimidine

for growth. These bacteria therefore depend on the nutrients in meat for survival, thereby

28

reducing the nutritive content (vitamins and amino acids) during fermentation. While proteolysis

increases palatability, it may also produce toxic compounds such as biogenic amines during

fermentation (Pereira et aI., 2001). In contrast, Bover-Cid et ai. (1999) showed no correlation

between proteolysis and the production of toxic compounds. While there are numerous benefits,

it is still unclear if starter cultures also add deleterious effects on fermented meats, depending on

the type of fermentation culture and the desired product.

Starter Culture and Biogenic Amine Formation in Sausage. Biogenic amines are

nitrogen-containing compounds responsible for the essential functioning and maintenance of

living organisms. They are found in fruits, vegetables, meat, and milk and can produced in high

amounts by microorganisms through the decarboxylation of amino acids. High levels of amines

have raised health concerns due to their capacity to cause nervousness, abnormal blood pressure,

and intestinal disease (Suzzi & Gardini, 2003). Meat fermentation processes characterized by

high microbial activity can lead to high concentrations of biogenic amines in sausages. Dry

fermented sausages may have higher amounts of precursor amines compared to semi dry

sausages (Taylor et aI., 1978) resulting from the length of time required to ripen dry sausages.

For example, after meat was fermented by Pediococcus cerevisiae and dried, histamine was

found in the final product (Baumgart et aI., 1979). Eiteinmiller et ai. (1981) showed that during

fermentation of cervelat (a cooked sausage originally produced in Germany and Switzerland)

lower levels of tyrosine decarboxylase were detected when using adventitious microbes rather

than commercial starter cultures. This suggests that during fermentation with starter cultures,

biogenic amines are produced in higher levels. The consumption of high amounts of biogenic

amines such as histamine and tyramine (derived from the amino acid tyrosine) may have

toxicological effect (Edwards et aI., 1987). In general, researchers mentioned that the presence

of high levels of amines in dry fermented sausage can be reduced if the right bacteria and

fermentation process are used during sausage processing (Bover-Cid et aI., 2000).

Starter Cultures and Control of Other Food-borne Microorganisms in Meat Products

29

There are several food-borne pathogens that are of concern due to their health and

economic impacts on consumers and food processors. Some of these pathogens have resulted in

the death of individuals who have consumed contaminated foods (Donnelly, 200la). Product

recalls have cost processors fortunes (CDC, 2002a). Starter cultures play an important role in

addressing some of these concerns through their ability to control food-borne pathogens (Smith

& Palumbo, 1983a). Commercial starter cultures used to control food-borne pathogens are

presented in Table 4.

Table 4

Select Starter Culture Organisms, the Food Borne Pathogens they Control, and Meat Products

they are used to Ferment (Adapted from Smith & Palumbo, 1983)

Starter Culture

Lactobacillus plantarum

Pediococcus cerevisiae

L. plantarum+P.cerevisiae

Lactobacillus plantarum

Pediococcus cerevisiae

L. plantarum+ P. cerevisiae

Pediococcus pentosaceum

L. plantarllln+ P. cerevisiae

Pediococcus cerevisiae

Lactobacillus plantarum

Food borne Pathogen

Poliovirus

Hog Cholera Virus

Echovirus

Salmonella newport

Salmonella pul/orum

Salmonella dublin

Staphylococcus aureus

Staphylococcus aureus

Clostridium botulinum

Clostridium perfringens

Meat Product

Dry Fermented Salami

Pepperoni

Cervelat

Summer Sausage

Dry Fermented Sausage

Lebanon bologna

Genoa salami

Fermented Beef Sausage

Canned Ham

Bacon

30

Salmonella spp. Salmonella species have been implicated in the recall of meat products

in the United States with poultry being the most common. Noted among pathogenic species are

S. typhimurium, S. enteritidis, S. newport, S. virchow and S. dubline. During the fermentation of

Lebanon bologna using a mixture of Lactobacillus plantarum and Pediococcus cerevisiae,

populations of Salmonellae typhimurium and dubline were reduced more rapidly than in controls

where indigenous lactic acid bacteria were utilized (Smith & Palumbo, 1973). Similar

mechanisms were used to reduce population of S. typhimurium in both experimental and control

samples due to the low pH of the product and the drying process. The protective role of starter

cultures in sausage fermentation was also supported by Sirvio et al. (1977), who found that

Salmonellae grew in rohwurst in the absence of starter cultures at the early stages and during

drying. Smith et aI., (1983c) reported that contaminated pepperoni that undergoes a shorter

period of fermentation was found to contain lower populations of both S. typhimurium and S.

dub line irrespective of the type of fermentation process utilized. Therefore, it is still not certain

the extent that lactic acid cultures are capable of controlling Salmonellae spp.

Staphylococcus spp. Staphylococci are facultative anaerobic gram-positive cocci

bacteria existing in grape-like clusters (Kloos & Schleifer, 1975). Their cells walls are made of

peptidoglycan which is resistant to lysozymes. Some strains of Staphylococcus produce

staphylococcal enterotoxins which cause Staphylococcal food poisoning. The symptoms include

nausea, vomiting, diarrhea and dehydration. Staphylococci have been implicated in recalls of

genoa sausage contaminated by Staphylococcus enterotoxins (U.S. Centers for Disease Control,

1979). Other investigations have also reported Staphylococcus aureus contamination of

fermented sausages during processing (Smith & Palumbo, 1983b). Sameshima et al. (1998)

reported the ability of Lactobacillus starter cultures to inhibit the growth of Staphylococcus

31

aureus. They experimented on the inhibitory effect of Lactobacillus starter on different

concentrations of enterotoxin-producing Staphylococcus aureus in dry fermented sausage. Their

results showed that Lactobacillus is capable of inhibiting the population S. aureus in dry

fermented sausage, but only when the concentration of Lactobacillus is higher than S. aureus.

This supports the findings of Daly et al. (1973) who found that the inhibition and destruction of

S. aureus in sausage can be accomplished by the use of starter cultures. They also observed that

growth of S. aureus at 104 CFU/g was inhibited at 3TC when L. plantarum, P. cerevisiae or a

mixture of the two cultures were added to fermented sausages. However, when the same amount

of these starter cultures were added to the sausages and fermented at 30°C, growth of S. aureus

could not be prevented. Fermentation temperature is therefore crucial for the successful

elimination of S. aureus when starter cultures are used.

Clostridium spp. Christiansen et al. ( 1975), studied the effects of starter cultures on

Clostridium botulinum in sausages. He observed that elimination of C. botulinum and associated

toxins required the presence of nitrite and/or glucose. A mixture of cultures (L. plantarum and

P. cerevisiae) and glucose prevented C. botulinum from forming toxins during sausage

fermentation. Their findings were supported by Hutton et al. (1991), who reported that a

combination of P. acidilactici and dextrose prevented botulinum toxigenesis in chicken salad.

This was due to the catabolism of dextrose to lactic acid by P. acidilactici to reduce the pH of

chicken salad. Baran and Stevenson (1975) showed that C. perfringens does not grow when

Pediococcus cerevisiae is used as a starter culture. They observed a decrease in the number of

viable C. perfringens cells after fermentation. However, the research could not establish whether

the inhibition was a result of low pH, drying or a combination of both.

32

Escherichia coli. E. coli infection, also known as travelers' diarrhea, results from poor

sanitation causing severe abdominal cramps and bloody diarrhea. In 1984, the E. coli strain

0157:H7 was formally recognized as a pathogen that caused hemolytic-uremic syndrome and

hemorrhagic colitis. This organism has since then been implicated in numerous food-borne

outbreaks and product recalls. Teratanavat and Hooker (2003), reported that in 2002 alone the

Food Safety and Inspection Service of the USDA recorded 19 million pounds of ground beef

contaminated by E. coli 0157:H7. As a contribution to control E. coli in ground beef, Wu et ai.

(2009) experimented on the use of cranberry concentrate as antimicrobial mechanism to

eliminate E. coli from ground beef. They found cranberry concentrate was effective in reducing

the population of E. coli in ground beef. They attributed the effect to the organic and phenolic

acids produced by cranberry qmcentrates which they stated possess antimicrobial properties.

Lactic starter cultures produce lactic acid and bacteriocins that disrupt outer cell

membranes of E. coli and inhibit their growth (Alakomi et aI., 2000). Glass et ai. (1992) further

expanded these findings by investigating the fate of E. coli in commercial sausage batter. They

reported that the E. coli survived processing but did not grow during fermentation and drying

stages, regardless of the presence starter culture and curing salts. E. coli may survive and not

proliferate, but if cell counts are sufficiently high, this organism may still cause food poisoning.

Bacteriocins

In addition to organic acids from carbohydrates, most lactic acid starter cultures are

capable of producing bacteriocins. Bacteriocins have recently received attention for their roles in

inhibiting pathogenic microorganisms. These molecules are peptides produced by bacteria to

inhibit the growth of similar or closely related strains. They possess antibiotic properties, but are

not called antibiotics due to the association of this term with allergic reactions in humans

33

(Cleveland et aI., 2002a). Chemical structure and narrow specificity against strains ofthe same

or closely related species are the differences between known antibiotics and bacteriocins. (Tagg

et aI., 1976).

Lactic acid bacteria bacteriocins are antagonistic natural biopreservative and undesirable

microorganisms, that inhibit the survival of pathogens in foods, and enhance food safety

(Schillinger et aI., 1996). In 1988, the USFDA approved the use ofbacteriocins in food as

preservatives. Starter cultures remain the main source of bacteriocins and different cultures

produce different bacteriocins depending on favorable conditions for production. Nisin remains

the most important commercially available bacteriocin in use (Li et aI., 2002; Cleveland et aI.,

2002b; Cleveland et aI., 2001). Apart from nisin (produced by Lactococcus lactis (Meghrous et

aI., 1999), other bacteriocins such as curvacin A (produced by Lactobacillus curvatus), lactocin

S (produced by Lactobacillus sake L45) and pediocin PA-1 (produced by Pediococcus

acidilactici PAC 1.0) all achieve bacterial killing (Cintas et aI., 1998).

Studies have shown that lactic starter cultures added to sausage during processing have

the ability to inhibit L. monocytogenes by producing bacteriocins. These bacteriocins collapse

the cells membranes of L. monocytogenes, increasing the cell permeability and resulting in loss

of water and substances from the cell (Christensen et aI., 1992). In an experiment to evaluate the

antilisterial activity of nisin and other lactic acid bacteria bacteriocins, (Laukova et aI., 1999)

reported that enterocin CCM 4231 was effective in inhibiting the population of L.

monocytogenes in contaminated Saint Pauline cheese. They found CCM 4231 not only inhibited

growth but also killed the pathogens. There was no significant difference in pH between

experimental and control cheeses. Hence they attributed their results to the observed

bacteriocins that were produced. Murray and Richard (1997) further evaluated the capability of

34

antilisterial properties of nisin A and pediocin AcH in decontaminating pork contaminated with

Listeria. They concluded that their bacteriocins were capable of controlling Listeria, and that

nisin was more efficient than pediocin. This difference was attributed to the rapid degradation of

pediocin by proteases in the meat. Bacteriocins (Table 5) therefore playa supporting role in the

effects of starter cultures in keeping products safe and extending shelf life.

Table 5

Selected Bacteriocins, Their Sources, and Pathogens they Control

Bacteriocin

Lactocin S

Nisin

Plantaricin C

Curvacin A

Pediocin PA-1

Source

Lactobacillus sake

Lactococcus lactis

Lactobacillus plantarum

Lactobacillus curvatus

Pediococcus acidilactici

Control

Listeria, Staphylococcus,

Clostridium spp.

Listeria, Staphylococcus,

Clostridium spp.

Bacillus, Clostridium spp.

Listeria, Clostridium spp.

Listeria, Staphylococcus,

Clostridium spp.

Effects of Listeria monocytogenes on Human Health and Meat Industry

Listeria monocytogenes is a Gram-positive rod-shaped motile bacterium. It is non-spore

forming, catalase positive, and a facultative anaerobe. Colonies of L. monocytogenes appear

with a blue-green sheen in transmitted light (Henry, 1933). They are commonly found in soil,

plants, silage, sewage, and slaughterhouses (Weis & Seeliger, 1975). L. monocytogenes ferments

glucose, lactose, and rhamnose under aerobic conditions (Pine et aI., 1989), and grows well on

standard bacteriological media. Listeria utilizes fermentable glucose and therefore its growth

35

rate increases in the presence of sugar. Listeria growing on culture plates exhibits an acidic odor

that may be as a result of carboxylic acid, hydroxylic acid, and alcohol (Daneshvar et al.,1989).

L. monocytogenes is a psychotropic organism with 30-37°C as its optimum growth

temperature, but is able to grow at a wide range of temperatures from I-45°C (Gray, 1960). L.

monocytogenes is known to survive under various conditions of refrigeration, freezing, heating,

and drying, which makes it difficult for the food industries to effectively manage (Gray &

Killinger, 1966a). L. monocytogenes was recognized as a human pathogen in 1929 (Gray &

Killinger, 1966b), but the route of transmission was unclear until the 1980s when interest in the

organism grew resulting from food-borne disease outbreaks. L. monocytogenes in humans and

animals is primarily transmitted through foods (Goulet et al., 2001). It has been implicated in the

recall of numerous meat products resulting from outbreaks of food-borne listeriosis, one of

which was linked to 23 deaths and 120 illnesses in eight Northeastern States (Fugett, 2006).

Listeriosis in humans can be especially dangerous in pregnant women, the elderly, children, and

immuno-compromised adults. Human listeriosis is characterized by high mortality rates, with

clinical symptoms such as muscle aches, fever, and diarrhea (Schlech, 1996).

Several major food borne outbreaks have been documented in North America and

Europe. The incidence of L. monocytogenes in ready-to-eat foods ranges from 1-10% (Farber et

al., 1992). Apart from health impacts, food borne illnesses including listeriosis have an

economic impact of 5 billion United States dollars annually (Altekruse et al., 1997). Four major

Listeria-related outbreaks occurred in North America between 1979-1985 (Wesley & Ashton,

1991). The first was recorded in 1979, where at least 23 were hospitalized because of

consumption of L. monocytogenes- contaminated lettuce, carrots, and radishes in Boston,

Massachusetts. Two years later, in the Maritime Provinces of Canada the second outbreak

36

occurred. Forty-one cases were recorded with 17 deaths due to the consumption of contaminated

coleslaw. This was followed by another outbreak that occurred in Massachusetts in 1983 with a

case-fatality rate of 29% associated with the consumption of contaminated milk. The largest

single listeriosis outbreak occurred in 1985 in Southern California. A Mexican style cheese was

implicated as the means of infection that infected people (Donnelly, 200 1 b). A total of 86 cases

were recorded resulting in 29 deaths, 13 stillbirths and 8 neonatal deaths.

From 1998 to 1999 most of the L. monocytogenes outbreaks involved the consumption of

hotdogs with 101 cases of illness and 21 deaths (Donnelly, 2001c). In a related outbreak in

2002, twenty-five samples from a poultry processing plant (Pilgrim's Pride) in Pennsylvania

recorded L. monocytogenes contamination, resulting in a voluntarily recall of 27.4 million lbs

(7% of their annual production) of fresh and frozen ready-to-eat turkey and chicken products

(CDC, 2002b). Due to serious health consequences, L. monocytogenes has been recognized as

an important public health problem which has prompted global response from sectors including

food industries, health agencies, and government bodies aimed at detecting and controlling

infections caused by L. monocytogenes in foods (CDC, 2003).

To control the effect of L. monocytogenes on food industries and people's health, food

processers have adopted different methods of the pathogen control based on the inhibition of

growth conditions. Table 6 shows some of the conditions under which L. monocytogenes is able

to grow in foods.

37

Table 6

Some Growth Conditions oiL. monocytogenes in Foods when all Other Factors are Optimum

Growth Condition Minimum Optimum Maximum Temperature (OC) -1.5 37 45

pH 4.4 7.0 9.4

Water Activity 0.92 ~ 0.97

Sodium Chloride (%) :s 0.5 2.5 3.5

The data in Table 6 shows that when there is inconsistency in other factors L.

monocytogenes is capable of developing tolerance to inhibitory growth conditions. For instance,

L. monocytogenes was found to survive in fermented salami of water activity 0.79-0.86 held at

4°C for at least 84 days (Johnson et aI., 1988). Also the use of thermal inactivation is commonly

used to control L. monocytogenes pathogen in foods. Elevation of product temperature may

cause irreversible damage to Listeria ribosomes as well as denaturing proteins and inactivating

enzymes within the cell (Anderson et aI., 1991), L. monocytogenes can tolerate thermal

inactivation (Farber, 1989). This may be due to factors such as the strain of Listeria (El-

Shenaway et aI., 1989) and the composition and concentration of the food material (Juneja &

Eblen, 1999).

Chapter III: Materials and Methods

Starter Culture Strains and L. monocytogenes Inoculum Preparation

38

The two starter cultures used in this research were provided by Chr Hansen Cultures,

(Milwaukee, Wisconsin, USA). Pediococcus acidilactici (B-LC-20) and Lactobacillus curvatus

(B-LC-48) were supplied as powders and were stored at -20°C throughout the period. Viable

counts of starter cultures stated on the packs (lx109 CFU/g) were verified by enumeration on

MRS agar (Difco). Starter cultures were added to batches of sausages at a concentration of 107

CFU/g of meat.

Listeria monocytogenes (ATCC 43251) was acquired from Presque Isle cultures, (Erie,

Pennsylvania, USA). The culture was grown in Brian Heart Infusion (BHI, Difco) broth at 3TC

for 24 hours, then grown on lithium chloride phenyl ethanol moxalactam agar (LPM, Difco), a

Listeria selective medium, and re-cultured to obtain pure L. monocytogenes colonies. A single

colony from the LPM agar plate was inoculated into 50 ml ofBHI broth and incubated at 3TC

for 24 hours. Listeria was inoculated into the sausages at 104 CFU/g of meat. To confirm the

presence and viability of L. monocytogenes, Frazer agar plates (Difco) were streaked with the

remaining inoculum. The Frazer plates turned dark while the uninoculated plates remained

yellow in color, indicating the presence of viable Listeria.

Genoa Salami Preparation

The recipe and ingredients for preparing the salami was provided by the Meat Science

Department of the University of Wisconsin-Madison. Sausage production followed a standard

procedure for making fermented sausages described by the United States patent 3,561,977

(Mich. USA. Patent No. 3,561,977, 1971). The flow diagram of the process used in preparing

the sausages is as shown in Figure 2. Pork, ground to required specification (1/8" blade), was

39

obtained from the Marketplace Foods store in Menomonie. Fresh meat was stored at _5°C and

kept slightly frozen for 12 h prior to the experiment. The meat temperature was then brought to -

3°C for 30 min before preparing the sausage. In Table 7 are the various ingredients that were

used to prepare the batches of sausages for this experiment. They included pork trim (72% pork,

28% fat), spices, (ground black pepper, whole black pepper and garlic powder), curing agents

(curing salt, NaCI), dextrose, sodium erythobate, and starter cultures (P. acidilactici and L.

curvatus). The ingredients were mixed in a rotary mixer then stuffed into fibrous collagen

casings with a hand held stuffer. Casings were initially soaked in 80°C water to make them

pliable and reduce microbial load. Nine batches 0.45kg (1 pound) of sausages (a total of two

replicates for each treatment and control) was prepared as shown in Table 8. Sausages were

incubated at 3TC for 12 hours then allowed to ripen at 12°C and 78% relative humidity for 28

days.

Microbial Analysis

L. monocytogenes counts were enumerated by spread plating serially diluted samples.

Approximately 109 of sample was aseptically taken from sausages on days 0, 2, 4, 7, 14,28 for

pH and water activity analysis. Duplicate samples were each mixed in 90 ml of buffered peptone

water (Difco) and homogenized in a stomacher for 120 seconds. The supernatants were further

diluted serially in sterile buffered peptone water and spread on Listeria selective LPM agar for

Listeria enumeration and MRS (de Man, Rogosa and Sharpe, Difco) agar plates for lactic acid

bacteria enumeration. The plates were incubated for 48 hours at 3TC under aerobic conditions.

Colonies on plates were counted using colony counter and the populations of Listeria and lactic

acid bacteria in the samples were determined.

40

Table 7

Quantities o/Various Ingredients used in Preparing Genoa Salami by Weight and Percentage

Ingredients Weight (grams) Percentage/ppm

Meat

Total Pork Trim 450 100%

Pork 324 72%

Fat 126 28%

Curing Agents

Salt 10.125 2.25%

Dextrose 2.27 0.50%

Curing salt 1.135 156ppm

Sodium erythobate 0.248 547ppm

BHAlBHT mixture 0.027 0.01%

Spices

Ground black pepper 1.135 0.25%

Whole black pepper 0.568 0.13%

Garlic powder 0.142 0.03%

Cultures

P. addilactid 0.8 0.004%

L. curvatus 4.6 0.02%

41

I Pork (_3°C) I I Back Fat (-3T) I I

Grinding with 1/8"

blade

I I Spices

Mixing for 3 minutes I Curing Agents I

I Starter culture I Filling 1 I

Casing I I

Fermentation at 3TC (98.6°F) for 12hrs

Drying for 28days at 12°C

(54°F) and 78% R.H

Figure 2. Flow diagram of Genoa salami preparation.

pH and Water Activity Determination

Samples were homogenized in buffered peptone water. Sausage samples (1 g) were

homogenized in 9 ml sterile peptone water. pH was determined from in the homogenized

solution. Water activity of 5 g undiluted sample was measured with a water activity meter (Aqua

Lab 3TE).

42

Table 8

Experimental Sample Formulations

Formulation Batches Batch description

A - Pork A Pork

B - Uninoculated Sausage Bl Sausage

B2 Sausage + L. monocytogenes

C - P. acidilactici Cl Sausage + P. acidilactici

C2 Sausage + P. acidilactici + L. monocytogenes

D - L. curvatus Dl Sausage + L. curvatus

D2 Sausage + L. curvatus + L. monocytogenes

E - P. acidilactici + L. curvatus El Sausage + P. acidilactici + L. curvatus

Sausage + P. acidilactici + L. curvatus + L. E2 monocytogenes

Protein Separations

Experimental sausage samples were freeze-dried on days 2 and 14 for protein

determination. Homogenized sausage samples (1.5 g) were boiled for 10 min in 15 ml basic 2x

Laemmli sample loading buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol,

0.004% bromophenol blue and 0.125M Tris HCI (Laemmli 1970). Protein hydrolysis was

analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

43

with a pre-made polyacrylamide gel (Thermo Scientific, Rockford IL) and staining with

coomassie brilliant blue (Laemmli 1970). Molecular makers used ranged from 6 kDa to 200 kDa

(Thermo Scientific, Rockford IL). Samples (15 Ill) were loaded onto the gel and run at 150 V

for 1 hour. The gel was then stained for 15 minutes and then destained overnight in methanol

and acetic acid (5% acetic acid and 10% methanol).

44

Chapter IV: Results

Growth of indigenous L. monocytogenes was detected in uninoculated fresh pork and

sausages on day 14 (Figure 3, Table 9, treatments A and Bl). There was however no significant

difference in L. monocytogenes populations between uninoculated sausages compared to

sausages inoculated with Listeria (compare treatment B2 with A and Bl in Figure 3, Table 9).

By day 28, no L. monocytogenes was detected in all samples inoculated with L. monocytogenes

except sample B2 which had no starter culture added to it (Figure 3, Table 9, compare treatments

C2, D2, and E2 to B2).

Growth of indigenous lactic acid bacteria were detected in uninoculated sausage (Figure

3, Table 10 treatments A, B1 and B2), but these bacteria did not inhibit the growth of both

indigenous and inoculated L. monocytogenes. However, in the presence of lactic starter cultures,

L. monocytogenes was never detected in uninoculated sausages (Figure 3; Table 9, treatments

C1, D1, E1). There was also a significant decline of L. monocytogenes in inoculated sausages in

the presence of starter cultures (Figure 3; Table 9, treatments C2, D2, E2).

The combination of the P. acidilactici and L. curvatus resulted in rapid decline of L.

monocytogenes by seven days (Figure 3; Table 9, treatment E2). By the fourth day, there was a

significant difference between the population of L. monocytogenes in samples with mixed starter

cultures and those that have P. acidilactici (P=0.0005) or L. curvatus (P=0.0007) individually,

hence their ability to reduce population of L. monocytogenes. P. acidilactici effectively

inhibited L. monocytogenes by day 14, compared to L. curvatus which inhibited L.

monocytogenes by day 28 (compare Table 9, Figure 3 treatments C2 to D2). Lactic acid bacteria

populations either grew or remained the same until day 14 when they began to decline (Table 10,

Figure 3, CI-E2). By day 28, there was significant decline in the population of the inoculated

lactic acid bacteria (P<O.05) in all sausage samples.

45

46

Table 9

Mean Population of L. monocytogenes Survival in Different Sausage Treatments over the 28 Day Periorf

DAY 0 DAY 2 DAY 4 DAY 7 DAY 14 DAY 28

A N.D. N.D. N.D. N.D. 2.2xl04±1.3xl03 5.8xl 04±8.0xl 02

Bl N.D. N.D. N.D. N.D. 7.9xl 03±1.4xl 02 7.9xl 03±1.4xl 02

B2 1.4xl04 ±8.0xl02 1.4xl04 ±9.8xlOI 1.4xl04±9.3xlOI 1.4xl04±9.4 xl 01 3 .3x 1 04 ±4.9x 102 1.3xl 04±4.9xl 02

Cl N.D. N.D. N.D. N.D. N.D. N.D.

C2 1.4xl04 ±5.7xl02 2.8xl03±9.4xlOI 2.0xl02 ±1.4xl 01 1.9 xlOI±O.lxlOI N.D. N.D.

Dl N.D. N.D. N.D. N.D. N.D. N.D.

D2 1.2xl04 ±4.9xl 02 1.5xl 03±4.9xl 01 9. Ox 102 ± 1.1 x 102 1.0xl 03±1.5xl 02 5.0xl 02±0.8xl 01 N.D.

El N.D. N.D. N.D. N.D. N.D. N.D.

E2 1.2xl04 ±4.3xl02 7.0xl 02 ±8.0x 1 01 1.0xl 02±0.8xl 01 N.D. N.D. N.D.

'Values are mean ±95% confidence interval (s.d. *t/;Jn) A, Raw Pork; B 1, Sausage; B2, Sausage + Listeria; C 1, Sausage + Pediococcus; C2, Sausage + Pediococcus + Listeria; Dl, Sausage + Lactobacillus; D2, Sausage + Lactobacillus + Listeria; El, Sausage + Pediococcus + Lactobacillus; E2, Sausage + Pediococcus + Lactobacillus + Listeria

47

Table 10

Mean Population of Lactic Acid in Different Sausage Treatments over the 28 Day Period"

DAY 0 DAY 2 DAY 4 DAY 7 DAY 14 DAY 28

A N.D. 2.50xl06 ± 8.0xl04 8.20x107 ± 1.4xl06 5.50xl07 ± 8.0xl05 2.08xl07 ±1.2xl06 7.20xl05± 2.8xl04

Bl N.D. 8.20xI06± 2.4xl05 1.70xl07 ± 8.0xl05 8.38xl07 ± 4.9x105 5.80xl 05 ±2.4xl04 1.40xl 03 ±8.0 xl 01

B2 N.D. 4.60xl03 ± 1.4xl02 3.70xl05 ± 1.6xl04 2.53xl07 ± 2.3xl07 4.30xl05 ±1.6xl04 2.l0xl03± 2.1xl02

Cl 1.50xl07 ± 8.0x105 2.88xl07 ± 4.9x105 3.30xl07 ± 8.0xl05 2.00x107 ± 1.8xl06 1.80xl 06 ±2.7xl 05 7.50xI03± 1.1xl03

C2 1.83xl07 ± 9.4xl05 2.28x107 ± 1.7xl06 3.00x107 ± 1.6xl06 1.40xl07 ± 1.6xl06 2.1 Oxl 06 ±1.4xl 05 2.38x103 ± 2.6xl02

Dl 1.60xl07 ± 2.3x105 2.00xl07 ± 1.4xl06 4.68x107 ± 1.2xl06 2.00x107 ± 2.3xl06 2.90x 105 ±8.0x I 03 7.50xl 03 ± 6.0xl 02

D2 1.00xl07 ± 1.6xl05 1.08xl07 ± 9.4x105 4.00x107 ± 1.6xl06 6.10x107 ± 1.8xl06 4.40x105 ±2.8xl 04 1.13xI04± 2.1xl03

El 9.40xl06± 4.8x104 1.90xl07 ± 8.0xl05 9.75xI06± 1.7xl06 1.44xl07 ± 8.7xl06 4.30xl 05 ±2.4xl 04 6.70xl03± 1.6xl02

E2 2.l0x107 ± 1.8xl05 3.00x107 ± 1.4xl06 6.40x107 ± 2.8x106 7.80x107 ± 1.6xl06 3.15x105 ±3.8xI04 1.30xl04 ± 1.8xl03

aValues are mean ±95% confidence interval (s.d. *tJ~n) A, Raw Pork; B 1, Sausage; B2, Sausage + Listeria; C 1, Sausage + Pediococcus; C2, Sausage + Pediococcus + Listeria; Dl, Sausage + Lactobacillus; D2, Sausage + Lactobacillus + Listeria; E1, Sausage + Pediococcus + Lactobacillus; E2, Sausage + Pediococcus + Lactobacillus + Listeria

10' ~10'

3 10' ~ 10' :;- 105

~ 104

.~ 10' -J

10' 10' 100

10' rn 1(J8 :3 10'

S 10'

.~ 10' 10' ~

.~ 10' -J

10' 10' 100

10' ~10'

310' ~ 10' ~105

.'" * ~ 104

.~ 10' -J

10' 10' 100

10' ~ 10'

310' ~ 10' ~105 .~ ~ 104

::1 10' 10' 10' 100

1(J8 ~1(J8 0> :3 10' S1(J8

.~ 10'

~ 10' .~ 103 -J

10' 10' 100

0

48

Fig :1 Change in L. monocytogenes and Lactic Acid Bacteria Population Over Time

A. Uninoculated Pork 10' 10' 107 :§ 10' iI 105 ~ 10' to

10' ::5 10' 10' 100

81. Uninoculated sausage 10' 10' 10' &>

::::> 1(J8 u. 10' ~ 10' to

10' ::5 10' 10' 100

82. Sausage inoculated with Usteria 10' 1(J8~

~ 10' 3 10' ~ 10' ;n-

~ 10' ::5 1(J3

10' 10' 100

C1. Sausage inoculated with Pediococcus 10' 10'

~ 10' 10' U.

105 ~ to

10' ::5 103

10' 10' 100

C2. Sausage inoculated with listen's and Pediococcus 10' 10' ~ 10' 3 10' ~ 105 ;n-10' ::5 10' 10' 10' 100

5 10 15 20 25 30 Time (Days)

-*- L. monocytogenes (CFU/g) -v- Lactic acid bacteria (CFU/g)

10' 01. Sausage inoculated with Lactobacillus

10' ~ 10'

10' 10' 10' ~ 10' ~ 105 III

10' :5

IL

~ .[g .!! :3

10' 105

10' 10' 10' 10' 100

10' 10' 10'

}-~~d-__ ~ ____ ~ ______________ ~-f100

109 D2. Sausage inoculated with listeria and Lactobacillus 109

1(J8 1(J8

~ 10' 10' ~ ~ 10' 1(J8 ~ .[g 105 10' ~ .!! 10' 10' :3

10' 103

1(J3 1(J3

10' 10' 10°c-~----~----~----~----~----~~~-f 10° 1 09 E1. Saussage inoculated with Pediococcus and Lactobacillus 109

1(J8 1(J8

~ 10' 10' ~ ~ 10' 10' ~ .[g 105 105 III * 10' 10' :5 c:J

103 10' 1(J3 1(J3

10' 10' 100 10° 109 E2. Sausage inoculated with Usteria,Pediococcus, and Lactobacillus 109

1(J8 1(J8

~ 10' 10' ~ ~10' 10'~ .[g 10' 105 ;;;-

~ 10' 10' :5 1(J3 1(J3 1(J3 1(J3

1~ 1~

100 10°

o 5 10 15 20 Time (Days)

25 30

Figure 3. Change in monocytogenes and lactic acid bacteria population over time

49

The pH of the control samples declined with time (Figure 4, Table 11 treatments A and

B 1). There were differences in the rate of pH decline between the uninoculated pork and

sausage (compare Figure 4, Table 11 treatment A with B 1). pH decline, however, did not

correspond to declines in indigenous L. monocytogenes populations in uninoculated pork and

sausage (Figure 4, Table 11, treatments A and B 1). Sausages treated with L. curvatus and P.

acidilactici displayed lower pH compared with the sausage sample with no lactic starter culture

(compare Figure 4, Table 11: B2 with C2 and D2). Sausage treated with the mixed starter

culture had the lowest pH (compare Figure 4, Table 11: treatments E2 with B2, C2, and D2).

Sausage treated with mixed starter culture had the least pH of 4.6 by day 28. However, pH

decline did not correspond with the population decline of L. monocytogenes in sausage (Figure 4,

Table 11 treatment B2).

There was a general decline in water activity for control and treated sausages. Significant

decreases were detected in all samples by day 14. For example, water activity decreased in

uninoculated pork (0.96 to 0.92) and sausage (0.96 to 0.86) (Table 12 treatments A and Bl).

Water activities of treated samples also declined but the declines were lower compared to the

controls (compare Figure 5, Table 12, treatment Bl with Cl, Dl, El). Sausage treated with L.

curvatus had lower water activity compared to sausage treated with P. acidilactici (compare

Table 12, treatment C2 with D2), while sausage treated with the mixed starter culture recorded

the lowest pH among both control and treated samples (compare Figure 5, Table 12, treatment

E2 with B2, C2, and D2).

50

Table 11

Mean pH Measurements of Different Sausage Treatments over the 28 Day Periocf1

DAY 0 DAY 2 DAY 4 DAY 7 DAY 14 DAY 28

A 6.9 ± 8.0x 10-3 6.9 ± 2.8xl0-1 6.6 ± 1.4xl0-1 6.2 ± 1.7xl0-2 5.9 ± 3.4xl0-1 5.6 ± 8.0xl0-3

Bl 6.8 ± 2.3x 10-2 6.8 ± 2.8xl0-2 6.0 ± 2.8xl0-2 5.8 ± 1.7xl0-2 5.4 ± 2.5xl0-2 5.3 ± 2.4xl0-1

B2 6.8 ± 3.9xl 0-2 6.8 ± 3.0xl0-1 5.8 ± 2.1xl0-1 5.8 ± 1.4xl0-1 5.3 ± 2.5xl0-1 5.0 ± 2.lxl0-1

Cl 6.8 ± 2.8xl 0-2 6.7 ± 1.0xl0-1 5.8 ± 1.3xl0-2 5.8 ± 1.6xl0-2 4.9± l.4xl0-2 4.9 ± 8.0xl0-2

C2 6.8 ± 2.4xl0-2 6.7 ± 1.6xl0-2 5.6 ± 5.4xl0-1 5.8 ± 2.8xl0-1 5.2 ± 4.7xl0-2 4.9 ± 5.8xl0-2

Dl 6.9 ± 6.9xl0-2 6.5 ± 3.2xlO-2 6.6 ± 5.0xl0-1 5.7 ± 3.6xl0-2 5.0 ± 4.0xl0-1 4.8 ± 3.6xl0-2

D2 6.8 ± 7.2xl0-2 6.5 ± 1.6xlO-1 5.8 ± 3.9xl0-2 5.7 ± 4.3xlO-2 5.1 ± 1.4xl0-1 5.1 ± 8.0xlO-2

El 6.9 ± 3.8xl0-2 6.5 ± 3.8xl0-2 5.8± 1.6xl0-1 5.8 ± 4.2xl0-1 5.2 ± 3.3xl0-2 4.9 ± 3.6xl0-2

E2 6.8 ± 8.7xl0-2 6.5 ± 2.0xl0-2 5.8 ± 9.2xl0-2 5.8 ± 7.2xl0-2 5.0 ± 1.3xl0-1 4.6 ± 9.4xl0-2

'Values are mean ±95% confidence interval (s.d. *tI'-'n) A, Raw Pork; Bl, Sausage; B2, Sausage + Listeria; Cl, Sausage + Pediococcus; C2, Sausage + Pediococcus + Listeria; DI, Sausage + Lactobacillus; D2, Sausage + Lactobacillus + Listeria; El, Sausage + Pediococcus + Lactobacillus; E2, Sausage + Pediococcus + Lactobacillus + Listeria

Table 12

Mean Water Activity Measurements of Different Sausage Treatments over the 28 Day Periocf

DAY 0 DAY 2 DAY 4 DAY 7 DAY 14 DAY 28

A 0.960 ± 8.0 x 10-3 0.963 ± 1.1 x 10-2 0.963 ± 1.5x10-2 0.938 ± 1.6 x 10-2 0.944 ± 2.1 x10-3 0.920 ± 2.0 x 10-3

B1 0.957 ± 1.7 x 10-3 0.933 ± 8.0 x 10-4 0.916 ± 1.1 x10-2 0.920 ± 1.4 x 10-3 0.913 ± 3.8 x10-2 0.860 ± 4.0 x 10-3

B2 0.959 ± 1.9 x 10-3 0.930± 4.1 x 10-1 0.917 ± 1.6 x10-2 0.870 ± 8.0 x 10-4 0.840 ± 1.4 xl 0-3 0.834 ± 2.6 x 10-2

C1 0.958 ± 1.4 x 10-4 0.949 ± 8.0 x 10-4 0.925 ± 1.4 x10-3 0.912 ± 7.8 x 10-3 0.822 ± 1.4 xl 0-2 0.828 ± 2.5 x 10-2

C2 0.931 ± 2.0 x 10-2 0.938 ± 8.0 x 10-4 0.925 ± 3.1 x10-3 0.913 ± 1.4 x 10-3 0.810 ± 8.0 x10-4 0.803 ± 3.4 x 10-2

D1 0.980 ± 8.0 x 10-4 0.940 ± 1.4 x 10-3 0.932 ± 3.3 x10-3 0.917 ± 8.0 x 10-4 0.805 ± 1.3 x10-2 0.779 ± 6.7 x 10-3

D2 0.949 ± 1.1 x 10-3 0.941 ± 8.0 x 10-4 0.926 ± 1.3 x10-3 0.910 ± 2.0 x 10-3 0.800 ± 2.1 x10-2 0.760 ± 4.5 x 10-2

E1 0.948 ± 1.8 x 10-3 0.936 ± 8.0 x 10-4 0.921 ± 1.4 x10-3 0.912± 1.1 x 10-3 0.790 ± 8.0 x10-4 0.729 ± 2.4 x 10-2

E2 0.947 ± 8.0 x 10-4 0.937 ± 1.6 x 10-3 0.925 ± 2.1 x10-3 0.910 ± 1.8 x 10-3 0.760 ± 1.6 xlO-3 0.647 ± 1.1 x 10-2

'Values are mean ±95% confidence interval (s.d.*tI'<'n) A, Raw Pork; Bl, Sausage; B2, Sausage + Listeria; Cl, Sausage + Pediococcus; C2, Sausage + Pediococcus + Listeria; Dl, Sausage + Lactobacillus; D2, Sausage + Lactobacillus + Listeria; El, Sausage + Pediococcus + Lactobacillus; E2, Sausage + Pediococcus + Lactobacillus + Listeria

51

7.0

6.5

6.0

5.5 :c Q,

5.0

4.5 7.0

6.5

6.0 :c Q,

5.5

5.0

4.5 7.0

6.5

6.0 :c Q,

5.5

5.0

4.5 7.0

6.5

6.0 :c Q,

5.5

5.0

4.5 7.0

6.5

6.0 :c Q,

5.5

5.0

4.5

52

Fig. 4 Change in pH and L. monocytogenes Population Over Time

A. Raw Pork

10'

10' :§ 10· ::::J

LL 105 ~

~~: 1 10' -..I

10' +-~~~--*-~--__ ~ ____ ~ ____ ~ ____ ~1~

81. Uninoculated Sausage 10' 10' rn 10· ~ lOS ~ 1Q4.~

1()3~ 10' -..I

10' +-~~~--~--____ ~ ____ ~ ____ ~ ____ ~1~

10· 10' 10' :§ 10' iI lOS ~ 10' .~ lOS ~ 10' -..I

10' +-~----~--~----~----~----~----tl~

C1. Sausage inoculated with Pediococcus 10'

10':§ 10. ::::J

LL 105 ~

10' .~ 10' ~ 10'::1 10'

~~~ __ ~=w======~~========== __ ===w--rl00 C2. Sausage inoculated with Usteria and Pediococcus 10'

10':§ 10· iI 105 ~ 10' .~

10' ~ 10' -..I

10' L-~ ____ ~ ____ ~ __ ~~ ________________ ~~~100

0 5 10 15 20 25 30 Time (Days)

7.0

6.5

6.0 :c Q,

5.5

5.0

-0- pH -*- L. monocytogenes (CFU/g)

D1. Sausage inoculated with Lac/obacillus 10' 10' rn 10· ~ 105 ~ 10' .~

10' ~ 10' -..I

10'

4.5 +-................. - ...... ---.... -------....... ---t 100

7.0 02. Sausage inoculated with Usteria and Lactobacillus 108

6.5

6.0 :c Q,

5.5

5.0

107 ~ 10. ::::J

LL 105 ~

10' .~

10' ~ 10' -..I

10'

4.5 t-~----~----~----~----~----~---_r 100

7.0 E1. Sausage inoculated with Pediococcus and Lactobacillus 10'

6.5

6.0 :c Q,

5.5

5.0

107 .g> 10· ::::J

LL 10S ~

~~ j .!!!

10' -..I

10'

4.5 +-,.,...., ............. ~~--...... ~--~------........ "____/" 100 ~ 10'

6.5

6.0 :c Q,

5.5

5.0

10' 106 ~

::::J 105 ~ 10' ~

.~ 10' ~ 10' -..I

10'

4.5 -'-~ ___ ~ ... -_-._------.... -1- 100

o 5 10 15 20 25 30 Time (Days)

Figure 4. Change in pH and L. monocytogenes Population Over Time

1.00

~ 0.95

~ 0.90

~

.ill 0.85

~ 0.80

1.00

~ 0.95 ·5

~ 0.90

j 0.85

0.80

1.00

~ 0.95

·5

~ 0.90

2 ~

0.85

0.80

1.00

~ 0.95 ·5

~ 0.90

I 0.85

0.80

1.00

~ 0.95 ·5

~ 0.90 ~

fJ 0.85 S

0.80

o

Fig . .5 Change in Water Activity and L.monocytogenes Population Over Time

A Raw Pork 10" 107 ~

.Ql 10· ::J

10' 8 10· III

10' .5; 10' :3 10' 10"

B1. Uninoculated Sausage 10· 107

~ 10" ::J 10'

u.. 2-

10· .~

10' .l!! 10' ~ 10' 10"

B2. Sausage inoculated with Listeria 10· 107 ~

10· ::J u..

10' 2-10· .~ 10' !ll

10' ~ 10' 10"

C1. Sausage inoculated with Pediococcus 10· 107 :Qi 10· ~ 10' 2-10· .~ 10' .l!! 10' ~ 10' 100

C2. Sausage inoculated with Usteria and Pediococcus 10·

107 ~ 10· ~ 10' 2-10· .~ 10' .l!! 10' ~ 10' 100

5 10 15 20 25 30 Time (Days)

1.00

0.95 ~ ·5

0.90 ~ 2 0.85

~ 0.80

1.00

0.95

~ 0.90 ~ iii 0.85

~ 0.80

1.00

~0.95

:~ ~ 0.90

iii ~ 0.85

0.80

0.80

__ Water Activity

-- L monocytogenes (CFU/g)

D1. Sausage inoculated with Lactobacillus

02. Sausage inoculated with Usteria and Lactobacillus

E 1. Sausage inoculated with Pediococcus and Lactobacillus

E2. Sausage inoculated with Usteria,Pediococcus and Lactobacillus

o 5 10 15 20 25 Time (Days)

Figure 5. Change in water activity and L. monocytogenes population over time

53

54

There was no noticeable difference in the proteolytic activities of all sausage samples

(Figure 6). Protein bands for different days (Day 2 and Day 28) were similar for all samples and

showed no significant effect on protein hydrolysis. The protein bands from the PAGE Gel-

Electrophoresis in Figure 6 below also showed no significant difference between sausages with

no fermentation cultures to sausages treated with fermentation cultures (compare Figure 7, B to

C-E and G to B-1).

ABC DE F G H P:.-...., .. ,~ c;-" ~.

~ "1IIIiiIt ,'.

,,",, lI/l!l' """ I.' ..... .~ . "-" J •

~ ":.~ ,:,.l : "'.':!

: ~, ..... ~::

J K KDa

200 116 97 66

~~. '~.'~ .•. : ... ~ ;~ k!~~ ~ 45 _ _ ~ 31

'~J:';.~l~, 21

.... 17t![;;"'1 ~:

a Lanes B to E are samples from Day 2 and G to J are samples from Day 28. Lanes A, F and K, Molecular makers; Lane B

& G, Raw sausage; Lane C & H, Sausage + Pediococcus; Lane D & I, Sausage + Lactobacillus; E & J, Sausage +

Pediococcus + Lactobacillus.

Figure 6. Protein bands picture from PAGE Gel-Electrophoresis for days 2 and 28a

55

Chapter V: Discussion

The purpose of this research was to measure the effect of combined starter cultures

compared to individual cultures on the growth and survival of L. monocytogenes in the

manufacture of dry sausages. We also sought to determine the effects of environmental

parameters resulting from fermentation on the survival of L. monocytogenes, and to understand

the impact of fermentation on meat proteolysis.

We can draw three conclusions from these studies. First, mixed starter cultures greatly

impact the survival rate of L. monocytogenes. In sausages containing L. acidilactici and P.

curvatus, a rapid decline in L. monocytogenes populations was detected (1.2x 104 CFU/g on day 0

to no colonies by day 7). In contrast, L. monocytogenes populations in uninoculated controls

remained high on day 7 (1.4xl 04CFU/g) while individual starter cultures had some impact on

pathogen numbers by day 7 (1.9xlO ICFU/g with Pediococcus, lxl03CFU/g with Lactobacillus).

These results are consistent with the hypothesis that combined starter cultures controls the

growth of L. monocytogenes better than individual starter cultures.

Other researchers have reported the ability of mixed starter cultures to effectively control

food-borne pathogens. Starter cultures ferment sugar to produce lactic acid, ensure a low pH.

The lactic acid inhibits the growth and survival of unwanted food-borne pathogens. The role of

acidity as a result of mixed starter culture use is supported by Smith et al. (1975) who found that

the pathogen Salmonella typhimurium was effectively reduced when pepperoni was fermented

with a mixed culture of L. plantarum and P. cerevisia starter cultures. Christiansen et al. (1975)

also reported that low pH resulting from L. plantarum and P. cerevisia activities greatly

impacted the survival of Clostridium botulinum in summer sausage. In such ecological

situations, beneficial microorganisms may complement one another. One organism may

consume the available carbon source while the other makes use of the by-product so both

organisms can reproduce and control food-borne pathogens, making fermented meat safer for

consumption.

56

Additionally, bacteriocins produced by fermentation cultures (Soomro et ai., 2002) have

been reported to kill microbes in foods. Bacteriocins are membrane-active compounds (Jack et

ai., 1995) that bind specific receptors on their target cells and introduce pores into the cell

membrane (Fleming et ai., 1985). The bacteriocin produced by P. acidilactici has both

inhibitory and bactericidal properties (Nielsen, 1990). This study showed that P. acidilactici

more rapidly controlled L. monocytogenes than L. curvatus individually, but was less effective

than the mixed cultures. Brurberg et al. (1997) suggested that the antagonistic property of

pediocin PAl (the bacteriocin produced by Pediococcus) is higher than the activity of curvacin

A, a bacteriocin produced by Lactobacillus. This could be caused by the presence of an extra

disulfide bond in the pediocin that enhances bactericidal effects. The Pediococcus may therefore

work in tandem with the Lactobacillus, one to drop the pH, the other to kill pathogenic cells.

Lactobacillus species are slow fermenters, dropping the pH of the food material but terminating

fermentation when sugar concentrations drop below readily accessible limits (Fleming et ai.,

1984). In contrast, P. acidilactici is a fast acid fermenter due to its ability to produce lactic acid

rapidly during fermentation (Marianski & Marianski, 2008b). During this experiment, the L.

curvatus may have helped P. acidilactici ferment the available sugars, allowing the P.

acidilactici to create bacteriocins and achieve killing of L. monocytogenes.

Second, environmental conditions had less effect than biological effects on the control of

L. monocytogenes in dry sausages. pH and water activity are known to limit the growth of L.

monocytogenes (Table 6). However, populations of both indigenous and inoculated L.

57

monocytogenes increased or remained the same even though pH of sausage decreased. In the

uninoculated pork, pH decreased from 6.9 to 5.6 but the indigenous L. monocytogenes

population increased from below detection limits to 5.8xl04 CFU/g. Similar results were

recorded for control sausage and sausages fermented with the starter cultures. L. monocytogenes

requires a minimum pH of 4.4 for inhibition to occur. That pH was not attained in this study,

allowing Listeria to grow.

The pH results presented here may be spurious due to the use of buffered peptone water

as the homogenizing solvent. When sausage samples were collected they were placed in a pH

buffer that likely neutralized any acid produced by the microbes. Due to the fast fermentation of

available organic acids, pH usually drops at least 1 unit after 24 hours. The pH in these sausages

may have declined similarly over the first day of fermentation, but since they were placed in

buffer, the drop in pH was not detectable. Instead, pH declined over a much longer time frame.

The actual pH of the sausages was much likely lower than the reported values.

Given this methodological error, the declines in pH result are consistent with findings of

Zdolec et ai. (2007) who reported that a decline in the pH (5.06) may not necessarily result in the

safety of a meat product, since L. monocytogenes was reported to survive at pH as low as 4.39

(George et aI., 1988). Thus, during fermentation, low pH is a hurdle that can inhibit the growth

of undesirable food-borne pathogens, but not necessarily killing them (Vialette et aI., 2003).

Moreover, undesirable organisms exposed to pH declines may gradually adapt to the stress

develop mechanisms over time to survive the new conditions (Spyropoulou et aI., 2001). In

general, however, the lower the pH of the fermented meat product, the stronger its inhibitory

effect on undesirable microbes (Davidson, 1997). Thevenot et aI. (2005a) explains this property

58

as the ability of uncharged undissociated molecules of lactic acid to penetrate cell membranes of

targeted spoilage and pathogenic membranes.

Water availability also impacts the growth of L. monocytogenes, which requires a

minimum water activity of 0.92 to grow (Petran & Zottola, 1989). As activities of lactic acid

bacteria increase, pH declines. When the pH approaches the isoelectric point (5.2), the water

holding ability of sausage decreases and L. monocytogenes has less water available for metabolic

activity, resulting in drying and death (Thevenot et aI., 2005b). Researchers have reported 0.91-

0.93 as the minimum water activity required for the growth of L. monocytogenes (Ross et aI.,

2000). In this experiment, water activity of 0.92 was reached by day 7 in sausages fermented

with both starter cultures (Fig. 5), but this did not appear to affect the growth of L.

monocytogenes. L. monocytogenes populations continued to increase or remained the same

while water activity decreased. In the uninoculated pork and sausage controls, water activities

decreased from 0.96 on day 0 to 0.92 and 0.86 respectively by day 28. In these same samples, L.

monocytogenes populations did not experience any decline, further supporting the idea that

biological controls were more important than environmental control in this experiment.

Third, this research showed little difference in the proteolytic effects of starter cultures.

The protein banding patterns suggested the presence of myosin (200 kDa), ~-galactosidase (116

kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa), but differences between treatments

were undetectable. Protein bands did not differentiate sausages treated with starter cultures and

the controls for the different days analyzed (Fig. 6). Since the most common proteins likely

overloaded the gel's ability to analyze the more rare break-down products of proteolysis, we

failed to detect any differences between treatments. Others have found that factors such as

processing conditions or the nature of the meat flora contribute to proteolysis (Astiasaran et aI.,

59

1990). Differences in proteolytic effects between Lactobacillus curvatus and Lactobacillus sake

are reported for pork (Fadda et aI., 1998). Protein breakdown in dry fermented sausage occurs

during the ripening stage, resulting in an increase in peptides and amino acids. Lactobacillus

plantarum is capable of breaking down protein in sausage after 96 hrs of fermentation (Fadda et

aI., 2002). Although we expected to observe different patterns of protein degradation with the

addition of starter cultures, such differences may have been beyond our ability to detect these

changes via SDS-PAGE.

In conclusion, mixed starter cultures in dry sausage fermentation provided rapid killing of

the pathogen L. monocytogenes. Combining P. acidilactici and L. curvatus achieved greater

control than either culture alone, improving consumer safety. Mixed starter cultures influence

the environmental conditions of dry fermented sausage but the conditions alone were not enough

to affect L. monocytogenes populations. Chemical and biological conditions therefore act

synergistically to achieve product safety. The use of mixed starter cultures should be encouraged

in the dry fermented sausage producing industries.

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