Antibacterial activities of secondary metabolites from Clitocybe nuda  
 
by 
 
Zheng Hou 
 
 
 
 
A thesis submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Master of Science 
 
Auburn, Alabama 
December 14, 2013 
 
 
 
Keywords: Clitocybe nuda, secondary metabolites, antibacterial activity,  
foodborne pathogen 
 
 
Copyright 2013 by Zheng Hou 
 
 
Approved by 
 
Tung-Shi Huang, Chair, Professor of Poultry Science 
Jean Weese, Professor of Poultry Science 
Thomas McCaskey, Professor of Animal Science 
 
 
 
 
ii 
 
Abstract 
 
 
The purpose of this study was to obtain a better understanding of 
the antibacterial activities of the secondary metabolites from Clitocybe 
nuda, one kind of edible mushroom. The antibacterial activity was 
determined by testing the Minimum Inhibitory Concentrations (MIC) of 50% 
log reduction of the secondary metabolites from C. nuda against four 
prevalent foodborne pathogens---Listeria monocytogenes, Salmonella 
Typhimurium, Staphylococcus aureus and Escherichia coli O157:H7, 
which were 27, 25, 23 and 40 mg/mL respectively. The thermal and pH-
stability of the antibacterial activity of the secondary metabolites from C. 
nuda were also evaluated. They were stable at 121 ?C after 30 min 
treatment and had good antibacterial activity under a broad pH range of 5-
9. The antibacterial efficacies in liquid eggs and ground beef were also 
investigated. The secondary metabolites of C. nuda were able to reduce 
about 0.2 and 0.5 log CFU/g L. monocytogenes and E. coli O157:H7 in 
ground beef, respectively. A 1 log reduction of Salmonella Typhimurium in 
liquid eggs at 50 mg/mL treatment was found.  
 
 
 
iii 
 
Acknowledgement 
 
 
First of all, I would like to sincerely thank my major advisor Dr. 
Tung-Shi Huang for his patient guidance and kind help in my master?s 
study. I am so grateful to his support and encouragement in my research. 
Next, I want to express my gratitude to my committee members, Dr. Jean 
Weese and Dr. Thomas McCaskey, who offered genuine advice and 
insightful comments on my thesis. Finally, it would be impossible for me to 
finish my graduate study without the support from my beloved family and 
friends. I deeply appreciate their encouragement during these years.  
 
 
 
 
 
 
 
 
 
 
 
 
iv 
 
Table of Contents 
 
 
Abstract ................................................................................................................. ii 
Acknowledgement ................................................................................................. iii 
List of Figures ....................................................................................................... vii 
List of Abbreviations or Symbols ......................................................................... viii 
Chapter 1 Introduction ........................................................................................... 1 
1.1 Background ................................................................................................. 1 
1.2 Purpose of study .......................................................................................... 2 
1.3 Significance of study .................................................................................... 3 
Chapter 2 Literature Review ................................................................................. 5 
2.1 Concern of food safety ................................................................................ 5 
2.2 Listeria monocytogenes ............................................................................... 7 
2.3 Escherichia coli O157:H7 ............................................................................ 9 
2.4 Salmonella ................................................................................................. 11 
2.5 Staphylococcus aureus ............................................................................. 12 
2.6 Mushrooms---Antimicrobial sources .......................................................... 13 
Chapter 3 Materials and methods ....................................................................... 16 
3.1 Preparation of the secondary metabolites from Clitocybe nuda ................ 16 
3.2 Bacterial preparation ................................................................................. 17 
3.3 Minimum inhibitory concentration (MIC) tests for the secondary metabolites 
of C. nuda ........................................................................................................ 17 
3.4 The pH effects on the antibacterial activity of the secondary metabolites of 
C. nuda ............................................................................................................ 18 
3.5 Thermal stability of the antibacterial activity of the secondary metabolites of 
C. nuda ............................................................................................................ 19 
3.6 The antibacterial efficacy of the secondary metabolites of C. nuda on 
ground beef and liquid egg samples ................................................................ 19 
3.6.1 Ground beef ........................................................................................ 20 
3.6.2 Liquid egg ........................................................................................... 20 
v 
 
3.7 Statistical analysis ..................................................................................... 21 
Chapter 4 Result and Discussion ........................................................................ 22 
4. 1 Minimum inhibitory concentration (MIC) of secondary metabolites from 
Clitocybe nuda ................................................................................................. 22 
4.2 Effects of pH on the antibacterial activities of the secondary metabolites 
from C. nuda .................................................................................................... 25 
4.2.1 Listeria monocytogenes ...................................................................... 25 
4.2.2 Salmonella Typhimurium .................................................................... 26 
4.2.3 Staphylococcus aureus ....................................................................... 27 
4.2.4 Escherichia coli O157:H7 .................................................................... 28 
4.3 Thermal stability of the secondary metabolites from C. nuda .................... 29 
4.4 Antibacterial effects of secondary metabolites from C. nuda on food 
samples ........................................................................................................... 32 
Chapter 5 Conclusion .......................................................................................... 36 
References .......................................................................................................... 38 
 
 
 
 
 
 
 
 
 
 
 
 
vi 
 
List of Tables 
 
 
Table 1-Antibacterial activity on L. monocytogenes of the secondary metabolites 
of C. nuda at various concentrations. .......................................................... 23 
Table 2-Antibacterial activity on E. coli O157:H7 of the secondary metabolites of 
C. nuda at various concentrations. .............................................................. 24 
Table 3-Antibacterial activity on Salmonella Typhimurium of the secondary 
metabolites of C. nuda at various concentrations. ..................................... 24 
Table 4-Antibacterial activity on S. aureus of the secondary metabolites of C. 
nuda at various concentrations. .................................................................... 24 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vii 
 
List of Figures 
 
 
Figure 1-pH effect on antibacterial activity of secondary metabolites from C. nuda 
against L. monocytogenes. ........................................................................... 26 
Figure 2-pH effect on the antibacterial activity of the secondary metabolites of C. 
nuda on Salmonella Typhimurium. .............................................................. 27 
Figure 3-pH effect on the antibacterial activity of the secondary metabolites of C. 
nuda on S. aureus. ......................................................................................... 28 
Figure 4-pH effect on the antibacterial activity of the secondary metabolites of C. 
nuda on E. coli O157:H7. .............................................................................. 29 
Figure 5 ?Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on L. monocytogenes. ........................................... 30 
Figure 6-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on Salmonella Typhimurium. ............................... 31 
Figure 7-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on S. aureus. .......................................................... 32 
Figure 8-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on E. coli O157:H7. ............................................... 32 
Figure 9- Anti-L. monocytogenes activity of the secondary metabolites from C. 
nuda in ground beef. ...................................................................................... 33 
Figure 10-Anti-E.coli O157:H7 activity of the secondary metabolites from C. nuda 
in ground beef. ................................................................................................ 34 
Figure 11-Anti-Salmonella Typhimurium activity of the secondary metabolites 
from C. nuda in liquid eggs. .......................................................................... 35 
 
 
 
 
 
 
 
 
viii 
 
List of Abbreviations or Symbols 
 
 
ANOVA Analysis of Variance 
BPB Butterfield?s Phosphate Buffer 
BSA Bismuth Sulfite Agar 
CDC US Centers for Disease Control and Prevention 
CFU Colony Forming Unit 
FSIS USDA Food Safety and Inspection Service 
HC Hemorrhagic Colitis 
HEA Hektoen Enteric Agar 
HUS Hemolytic-uremic Syndrome 
MOA Modified Oxford Agar 
NM Nonmotile 
OD Optical Density 
PDA Potato Dextrose Agar 
PDB Potato Dextrose Broth 
RTE Ready to Eat 
SD Standard Deviation 
SEA Staphylococcal Enterotoxin A 
SED Staphylococcal Enterotoxin D 
ix 
 
SMAC Sorbital MacConkey 
STEC Shiga Toxin-producing E. coli 
TC-SMAC Tellurite Cefixime-Sorbital MacConkey Agar 
TSA Trypticase? Soy Agar 
TSB Tryptic? Soy Broth 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 
 
Chapter 1 Introduction 
 
 
This study focuses on food safety concerns in the food processing 
industry and evaluates the potential use of the antibacterial activity of the 
secondary metabolites from Clitocybe nuda against Listeria 
monocytogenes, Escherichia coli O157:H7, Staphylococcus aureus, 
Salmonella Typhimurium.  
1.1 Background 
With the globalization of the food industry, food safety becomes a 
more important issue in the human health in the US. The Centers for 
Disease Control and Prevention (CDC) estimated that in the United States, 
31 known pathogens and more unspecified pathogens together caused 
about 47.8 million illnesses in 2011 (CDC 2011).  This data may be 
underestimated because some mild symptoms of foodborne illnesses 
were not reported.   
Among those foodborne pathogens, Listeria monocytogenes, 
Escherichia coli O157:H7, Staphylococcus aureus and Salmonella are the 
major bacteria which cause a great deal of food safety problems. A 
Listeria outbreak linked to Crave Brothers Farmstead Cheeses caused 1 
2 
 
death and 6 hospitalizations in July, 2013 (CDC 2013).  Another example 
is the multistate outbreak of E. coli O157:H7 in November, 2012 which 
involved in 33 people with 5 hospitalizations due to the consumption of 
contaminated organic spinach and spring mix blends (CDC 2012).  Many 
outbreaks of Salmonella infections are linked to live poultry. From March 
to July, 2013, more than 300 persons were ill due to infections of 
Salmonella Typhimurium linked to live poultry in backyard flocks from 37 
states all around the US (CDC 2013).     
To provide consumers with safe foods, the addition of food 
antimicrobials is one of the strategies to protect foods from foodborne 
pathogen contamination. Due to the awareness of health issues, lately, 
consumers have concerns about the use of artificial food antimicrobials. In 
addition, the prevalence of antibiotics has led to the occurrence of drug-
resistant foodborne pathogens. Thus, food scientists are looking for safe 
and effective naturally occurring food antimicrobials to replace artificial 
compounds.  
1.2 Purpose of study 
The use of food antimicrobials is an effective way to ensure the 
wholesomeness and safety of food products. In general, antimicrobials are 
divided into artificial and naturally occurring compounds (Davidson and 
others 2010). Since some consumers have concerns about the toxicity of 
synthetic chemicals added to their foods, naturally occurring antimicrobials 
3 
 
are becoming more attractive to the food industry. Naturally occurring 
antimicrobials can be isolated from animals, plants and of microbial origins 
(Ana and others 2010).   
Certain mushrooms have been found to show considerable 
antimicrobial activities. Clitocybe nuda, formerly called Lepista nuda, is 
one of them (Kiran and others 2013). This is an edible mushroom mostly 
found in Europe and North America. In previous research, the methanolic 
extract of C. nuda was found to inhibit the growth of Bacillus cereus, 
Bacillus subtilis, Staphylococcus aureus, Escherichia coli and 
Pseudomonas aeruginosa (Alves and others 2012). Chen and others 
(2012) have isolated the components from the secondary metabolites of C. 
nuda which have been known to inhibit plant pathogens, such as 
Phytophthora capsici and Xanthomonas axonopodis.  
The objectives of this study were (1) to evaluate the antibacterial 
activities of the secondary metabolites from C. nuda against Escherichia 
coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus and 
Listeria monocytogenes, (2) to test the effects of pH and temperature on 
the antimicrobial activities of the secondary metabolites from C. nuda, and 
(3) to investigate the antibacterial effects of secondary metabolites from C. 
nuda on Salmonella in liquid eggs and E. coli O157:H7 and L. 
monocytogenes in ground beef.   
1.3 Significance of study 
4 
 
Artificial food preservatives are raising issues related to the health 
concern. Some consumers expect less artificial chemicals to be added 
into their foods. It is also notable that the use of antibiotics has resulted in 
the increase of drug-resistant foodborne microorganisms (Threlfall and 
others 2000). Therefore, it becomes important for the food industry to find 
safe and effective antimicrobials from natural sources to replace artificial 
chemicals in foods. 
The antimicrobial activities of mushrooms have been investigated 
for a long time. Both mushroom extract and the secondary metabolites 
show good inhibitory effects against bacteria and fungi. However, very few 
studies of the secondary metabolites from C. nuda on their antibacterial 
activities against foodborne pathogens have been done. This study will 
obtain a better understanding of the potential use of the secondary 
metabolites from C. nuda as food antimicrobials. 
 
 
5 
 
Chapter 2 Literature Review 
 
 
2.1 Concern of food safety 
With the great expansion and globalization of the food industry, 
consumers are more concerned with food safety issues. Food safety 
describes the proper handling, preparation and storage of food products to 
prevent them from becoming hazardous to consumers. Food products can 
get contaminated physically, chemically and biologically. The presence of 
pathogenic microorganisms in foods has long been a potential threat to 
public health, especially to the immune system compromised people, 
infants, the elderly and people with chronic diseases such as diabetes, 
AIDS and others. Good hygienic practices can reduce the hazards to 
some extent, but are never enough to ensure the safety of the final 
products. Although much has been done to prevent the broadcast of most 
well-recognized pathogens, trends are changing rapidly, such as the ever-
fast growing population calls for larger and more industrialized production 
of food and the changing of eating habits. These changes result in more 
dining out. However, health conscious individuals are consuming more 
fresh produce. With the food industry trying to match the growing demand 
of food production, the use of antibiotics is increasing which may lead to 
6 
 
the emergence of gene-mutated pathogens. Yet, the globalized food 
market asks for better surveillance over imported food products (Newell 
and others 2010; Nyachuba 2010). These trends potentially increase the 
possibility of consumers coming in contact with contaminated food 
products and require stricter methods to improve food safety. 
In the US, according to the data from the Center for Disease 
Control and Prevention (CDC) records, 31 major foodborne pathogens 
and other unspecified agents are responsible for about 47.8 million of 
illnesses, 128,000 cases of hospitalization and 3,000 deaths each year 
(Scallan and others 2011; Scallan and others 2011). However, this data 
can be largely underestimated by a variety of reasons; e. g. patients with 
minor symptoms may not be aware of the infections, outbreaks without 
serious illnesses are ignored, doctors fail to link the disease with 
foodborne pathogens.  
Foodborne illnesses also cause great economic loss to the whole 
society. Robert L. Scharff (Scharff 2012) estimated that an annual cost of 
$77.7 billion was spent on the health-related losses. Therefore, it is 
beneficial to produce safe food not only for the consumers, but also for the 
food industry and the government. These combined reasons help to 
explain why the concern of food safety has always been an important 
issue to the public. 
Foodborne illness is classified into foodborne infection and 
intoxication. A foodborne agent which can invade or establish itself in the 
7 
 
host?s intestinal tract after being ingested is called the infectious pathogen. 
Examples for such pathogens are Salmonella, Campylobacter, Listeria 
monocytogenes and E. coli. The incubation period for a foodborne 
infection could range from hours to several days before the first symptom 
occurs.  
Foodborne intoxications (also known as food poisoning) occur 
when people consume food which contains toxins that have been formed 
before ingestion. S. aureus and C. botulinum are the most common toxin 
producers. A typical symptom for food poisonings is vomiting and the 
effects are generally acute.  
The growth of bacteria in food can be influenced by many factors, 
such as pH, temperature, time, water activity, oxygen, preservatives and 
microbial interactions with the food. It is essential to control foodborne 
pathogens by changing the properties of the food products. However, the 
change of the properties could affect the sensory characteristics of certain 
products. The addition of antimicrobials in food production is commonly 
used. 
2.2 Listeria monocytogenes 
Listeria monocytogenes is a group of gram-positive foodborne 
bacteria which can cause listeriosis to both humans and animals. The 
genus Listeria consists of seven species (monocytogenes, ivanovii, 
8 
 
innocuar, seeligeri, martii, welshimeri, and grayi). Among all these species, 
only L. monocytogenes and L. ivanovii are pathogenic.  
L. monocytogenes is pathogenic to both humans and animals while 
L. ivanovii cause diseases mostly to animals (V?zquez-Boland and others 
2001). In the United States, L. monocytogenes alone causes about 1,600 
cases of listeriosis infection each year, including 1,400 hospitalizations 
and 250 deaths. Listeriosis is more threatening to immune-compromized 
people, pregnant women, elder citizens, and newborns. Listeriosis 
infection can be extremely serious, especially to the populations who are 
at risk. In 2011, 72 people were infected with listeriosis after eating the 
cantaloupe grown at Jensen Farm in Colorado. In that outbreak, most of 
the sick people were older than 60, and 13 deaths were reported (CDC 
2011). Although the infectivity of L. monocytogenes is low, the fatality rate 
of listeriosis (20-30%) can be a lot higher than vibriosis and salmonellosis. 
L. monocytogenes is commonly found in the environment like soil, 
surface water, waste and food products including raw milk, dairy products 
and vegetables (Liu 2006). Plus, it has also been isolated from food-
processing plants (Autio and others 1999; Destro and others 1996; 
Miettinen and others 1999). Once L. monocytogenes presents at a plant, 
eradication planning of the bacteria must be made immediately. L. 
monocytogenes can survive in extreme environmental conditions, like acid, 
base, low temperature and salt treatments (Liu and others 2005). 
Therefore, it is difficult to be eliminated from the environment. Hot water 
9 
 
and hot steam are usually involved in the eradication plan of L. 
monocytogenes.  
L. monocytogenes can grow at refrigeration temperature which 
makes many ready-to-eat (RTE) foods vulnerable to the contamination of 
the bacteria. L. monocytogenes can re-contaminate the RTE foods by 
handling after the lethality treatment. A ?zero tolerance? policy of L. 
monocytogenes in RTE foods was established in the US to address the 
importance of removing this fatal microorganism from RTE foods such as 
deli meat and hot dogs. The Food Safety and Inspection Service (FSIS) 
provides three alternatives used to control or eliminate L. monocytogenes 
contamination in RTE food products, including post-lethality treatments 
and/or the addition of antimicrobial agents.  
2.3 Escherichia coli O157:H7 
Escherichia coli is a group of bacteria which are mostly 
nonpathogenic. However, some of the serotypes have acquired genes 
which can cause severe diseases in humans. The Shiga toxin-producing E. 
coli (STEC) have emerged as a major type of food-borne pathogens which 
can cause symptoms ranging from uncomplicated diarrhea to hemorrhagic 
colitis (HC) and the life-threatening hemolytic-uremic syndrome (HUS) 
(Hughes and others 2006). E. coli O157:H7 is the most predominant 
serotype that has been well described over the years. 
10 
 
The STEC is categorized by serotyping of O & H antigens. With 
174 O antigens and 56 H antigens in E. coli, there are many possible 
combinations of serotypes (?rskov and others 1992). The specificity of the 
O antigen is based on the polysaccharide portion of cell wall 
lipopolysaccharide and H antigen is determined by the flagella protein. 
Many of the STEC serotypes are nonmotile (NM) mutants of strains with H 
antigens (Gyles 2007). The serotyping is the basis of differentiating the 
STEC and analyzing their properties. Since E. coli O157:H7 plays the 
predominant role in all the serotypes of STEC, the STEC serotypes are 
commonly divided into O157 and non-O157.  
E. coli O157:H7 are gram-negative, rod-shaped bacterium and can 
be detected using the sorbitol MacConkey (SMAC) agar plate, because 
they have a unique phenotypic feature which is the failure of fermenting 
sorbitol within 24 h incubation.  
E. coli O157:H7 is normally transmitted through fecal-oral route. 
Humans are most likely to get infected with STEC through the 
consumption of contaminated water or food. It usually starts with the 
colonization in the intestinal tract before resulting in diseases like 
watery/bloody diarrhea, hemorrhagic colitis or even hemolytic-uremic 
syndrome.  
E. coli O157:H7 has been linked to many severe diseases and 
identified as a food borne pathogen carried frequently by cattle, as well as 
11 
 
other animals. Beef, especially ground beef, is the most frequently 
associated with E. coli O157:H7 outbreaks. Between 1982 and 2002, 
about 8,598 cases from 350 outbreaks were identified in 49 states (Rangel 
and others 2005). Among all the outbreaks, ground beef was recognized 
as the most common food vehicle which accounts for up to 41% of them. 
Other foods such as fresh produce, dairy products and drinking water 
have also been implicated in some of the outbreaks.  
2.4 Salmonella  
Salmonella is non-spore forming, gram-negative, rod-shaped 
bacterium which was discovered since the 19th century by a veterinary 
scientist named Salmon. Normally, Salmonella grow rapidly at 
temperatures between 25 and 43 ?C. However, Salmonella can still grow 
at temperatures as low as 5.3 ?C (Matches and others 1968). Therefore, it 
is important to avoid contact between raw and cooked foods even at 
refrigerated temperatures. The pH range of Salmonella is about 4.0-
9.0,with an optimal growth pH at around 7.0 (Foster 1995, Chung 1970).  
Salmonella are the cause of salmonellosis, one kind of foodborne 
disease, which causes approximately 40,000 cases of infection annually in 
the United States. Moreover, the number of the cases could be greatly 
underestimated because of the unreported minor symptoms. There are 
more than 2,400 serotypes of Salmonella (Brenner and others 2000). 
Among them, Salmonella Typhimurium is one of the most common 
12 
 
serotypes to cause infections in the United States. Salmonella is found in 
the intestinal tracts from humans, reptiles and birds. People can get 
infected after the ingestion of contaminated food products. The common 
food vehicles are eggs, poultry products, fresh produce, milk and beef. In 
2009, a large multistate outbreak of Salmonella Typhimurium sickened 
more than 700 individuals (CDC 2009). Peanut butter was identified as the 
source of the outbreak. Salmonellosis generally has three distinct 
syndromes: typhoid fever, enteric fever and gastroenteritis. However, 
gastroenteritis is the most common one.  
2.5 Staphylococcus aureus 
S. aureus is a gram-positive cocci belonging to the Micrococcaceae 
family. The most obvious characteristic of the species is its capability in 
producing golden colonies. S. aureus is ubiquitous. It is present at skin, 
respiratory tract of humans. About 30% of healthy people are the carriers 
of S. aureus and the bacteria can survive for weeks or months if they are 
colonized on mucous membranes (Archer 1998).  
Staphylococcal food poisoning occurs when individuals consume 
food products which have been contaminated by pre-formed toxins. 
Common symptoms are nausea, vomiting, abdominal cramps and 
diarrhea. The symptoms usually are rapid, even acute. It depends on the 
amount of toxins consumed and the health status of the person. Most 
patients can recover after 2-3 days. Severe cases usually occur among 
13 
 
the infants or elder people. With a toxin dose of 1.0 ?g, it is enough to 
induce intoxication. In an outbreak of staphylococcal food poisoning 
related to chocolate milk, only a total of 200 ng of staphylococcal 
enterotoxin A (SEA) was found to produce illness (Evenson and others 
1988).  
Staphylococcal enterotoxins are a group of heat stable enterotoxins 
that can even withstand the processes of canned products (Balaban and 
others 2000). SEA and SED are two of the most common toxins related to 
staphylococcal food poisoning.  
Most processed food products get contaminated through improper 
handling of the food. Since many healthy people could be the carriers of S. 
aureus, their contact with the food can be a great potential threat to the 
food safety. The proliferation of S. aureus in the food will lead to the 
accumulation of the enterotoxins which could cause disease after being 
consumed.  
2.6 Mushrooms---Antimicrobial sources 
Fungi are a group of organisms which are different from bacteria, 
plants and animals but widely distributed around the world also. The fungi 
kingdom includes yeasts, molds and mushrooms. The medicinal use of 
mushrooms has a long history in traditional Chinese medicine. To survive 
in the wild environment, mushrooms need to have the ability of producing 
bioactive compounds protecting themselves. Thus, it is possible for 
14 
 
humans to isolate those bioactive compounds for different uses. In early 
studies, mushrooms are found to have different functions, such as 
antibacterial (Hatvani 2001; Rosa and others 2003), antifungal (Ngai and 
others 2005; Wang and others 2004), antiviral (Piraino and others 1999), 
antitumor (Wang and others 1996; Wang and others 1996), antiallergic 
(Ellertsen and others 2009; Yoshino and others 2008), anti-inflammatory 
(Hirota and others 2002; Kim and others 2004), antioxidant (Cheung and 
others 2003; Song and others 2003), and hypolipidemic activities (Bobek 
and others 1991; Yang and others 2002).  
Clitocybe nuda (wood blewit) is an edible mushroom mostly found 
in Europe and North America. The color of the mushroom can range from 
lilac to purple. The percentage of the chemical components based on the 
dry matter of C. nuda has been reported to be: 59.4% crude protein, 1.8% 
lipids, 18,5% ash and 20.3% carbohydrates (Barros and others 2008). The 
mushroom is high in protein content on dry weight basis with very little 
lipid. Therefore, it can be a good dietary food source. However, there is a 
potential to cause allergies to sensitive people after eating the mushroom, 
especially when consuming the mushroom raw.  
Chen and others recently isolated three bioactive compounds from 
the 95% ethanol extract of the secondary metabolies of Clitocybe nuda: 2-
methoxy-5-methyl-6-methoxymethyl-p-benzoquinone, 6-hydroxy-2H-
pyran-3-carbaldehyde, and indole-3-carbaldehyde (Chen and others 2012). 
They also reported the antifungal ability of C. nuda in preventing the 
15 
 
Phytophthora capsici, a kind of plant pathogen which can cause severe 
disease in the earlier stage, such as peppers (Chen and others 2009).  
16 
 
Chapter 3 Materials and methods 
 
 
3.1 Preparation of the secondary metabolites from Clitocybe nuda  
The secondary metabolites from C. nuda were prepared by 
following Chen?s (Chen and others 2009) methods with modification. The 
C. nuda was grown on potato dextrose agar (PDA) (NEOGEN, Lansing, 
MI) at 25 ?C. Round C. nuda culture blocks (d=5 mm) were prepared by 
cutting the mycelium grown PDA plates with a sterilized cork-stopper borer. 
Each culture block was transferred into a 500-mL flask with 250 mL potato 
dextrose broth (PDB) (HIMEDIA, India). The inoculated PDB was 
incubated at 25 ?C for 21 days with shaking at 120 rpm. The cultured PDB 
was collected through filtration with 25 ?m filter paper (VWR filter paper 
415, VWR, Radnor, PA). The volume of the filtrate was reduced from 600 
mL to 100 mL by heating on a hot plate (CORNING, PC-420) at about 
82 ?C, and then continued to dry completely in an oven at 95 ?C 
(PRECISION, Model 16, Thermo Fisher Scientific Inc., Walham, MA). 
One liter of 95% ethanol was added to the dried sample for further 
extraction. The mixture was kept at room temperature with constantly 
stirring for 24 h and filtered through a 25 ?m filter paper followed by 
17 
 
filtering using a 0.45 ?m filter paper. The filtrate was dried at 40 ?C to 
remove ethanol. The dried extract was then re-suspended in 5 mL sterile 
water and the water solution was dried at 42 ?C in a vacuum oven 
(FISHER SCIENTIFIC, Model 282A). The dried extract was dissolved in 
sterile water to make a stock solution at 200 mg/mL. The pH of the stock 
solution was adjusted to 7 with 10 M HCl/NaOH and stored at 4 ?C. 
3.2 Bacterial preparation 
In this study, Listeria monocytogenes G3990, Escherichia coli 
O157:H7 ATCC43894, Salmonella Typhimurium ATCC13311 and 
Staphylococcus aureus ATCC12600 were used. E. coli O157:H7, 
Salmonella Typhimurium and S. aureus were cultured in Tryptic? soy 
broth (TSB) (BD, Sparks, MD) and incubated at 37 ?C with shaking at 150 
rpm for 20 h. L. monocytogenes were cultured in TSB with 0.5% yeast 
extract (AMRESCO, Solon, OH) and incubated at the same condition with 
other bacteria. The cultures were centrifuged at 4,500 rpm for 3 min and 
washed twice with Butterfield?s phosphate buffer (BPB). Then, the 
bacterial solution was diluted to an absorbance of 0.4-0.8 at O.D.640nm. 
The bacterial population was estimated based on preconstructed 
equations. The populations were adjusted to the levels for experimental 
use.  
3.3 Minimum inhibitory concentration (MIC) tests for the secondary 
metabolites of C. nuda  
18 
 
The dried extract was diluted to various concentrations and mixed 
with double strength culture medium at 1:1 ratio for the minimum inhibitory 
concentration tests against the four bacteria. The negative control was 
culture medium only. The pH of all samples was adjusted to 7.0?0.2. 
Treatments were triplicated. The inoculum of the bacteria was designated 
to 25 CFU/mL sample solution. All tested samples were incubated at 
37 ?C with shaking at 150 rpm for 24 h. After 24 h incubation, 1 mL culture 
was taken from the sample and washed twice with BPB after 
centrifugation at 4,500 rpm for 3 min. The washed cultures were properly 
made using a 10-fold serial dilution to plate out on Trypticase? soy agar 
(TSA) (BD, Sparks, MD) plates for E. coli O157:H7, Salmonella 
Typhimurium and Staphylococcus aureus and Trypticase? soy agar with 
yeast extract (TSA+YE) plates for L. monocytogenes and all plates were 
incubated at 37 ?C for 24 h. The colonies were enumerated and the 
bacterial population was calculated. The minimum inhibitory concentration 
of 50% log reduction (MIC50) was calculated by the equation from the 
linear regression of log bacterial populations vs. concentrations (mg/mL).  
All experiments were repeated. 
3.4 The pH effects on the antibacterial activity of the secondary 
metabolites of C. nuda 
The dried extract of secondary metabolites from C. nuda was 
dissolved in sterile water and concentrations were adjusted to the levels of 
2X MIC50 for each tested bacteria. The prepared extracts were mixed with 
19 
 
double strength culture media at 1:1 ratio to have growth media contained 
the extract at MIC50 levels. The negative control groups were culture 
media only without extract. The pH of samples and controls were adjusted 
to 4, 5, 6, 7, 8 and 9 with 10 M HCl/NaOH. After samples were inoculated 
with bacteria at 25 CFU/mL, they were incubated at 37 ?C with shaking at 
150 rpm. The bacterial populations of tested samples were enumerated 
using spread-plating method at 24 h incubation for further analysis. All 
experiments were repeated. 
3.5 Thermal stability of the antibacterial activity of the secondary 
metabolites of C. nuda 
The extract solution of C. nuda was placed at 4, 25, 72, 100 and 
121 ?C for 30 min. Sterile water was used as a negative control. After heat 
treatment, the secondary metabolites samples were mixed with same 
volume of double strength culture media. The pH of the samples was 
adjusted to 7 and the samples were inoculated with the test bacteria at the 
concentration of 25 CFU/mL. All inoculated samples were incubated at 
37 ?C with shaking at 150 rpm. After 24 h incubation, the bacterial 
populations were enumerated. All experiments were repeated. 
3.6 The antibacterial efficacy of the secondary metabolites of C. nuda 
on ground beef and liquid egg samples 
The antibacterial efficacies of the secondary metabolites of C. nuda 
to L. monocytogenes and E. coli O157:H7 on ground beef and to 
20 
 
Salmonella Typhimurium in liquid eggs were investigated. In this study, 
the ground beef and liquid egg were purchased from a local grocery store. 
The background of L. monocytogenes and E. coli O157:H7 on ground 
beef and Salmonella Typhimurium in liquid egg were checked.    
3.6.1 Ground beef   
One hundred and fifty grams of ground beef were weighed into a 
sterile plastic bag, and L. monocytogenes or E. coli O157:H7 were added 
at 104 CFU/ g ground beef level. Inoculated bacteria and the ground beef 
were mixed thoroughly and then the sample was put into 6 sterile bags of 
20 g each. The 6 samples were divided into two groups, 3 in each group. 
One group was used as a negative control. In the treated group, 1.5 g of 
the dried secondary metabolites of C. nuda were added into a bag. All 
samples were stored at 4 ?C. The bacterial populations of the samples 
were analyzed by using the spread-plating method on modified Oxford 
agar (MOA) plates and tellurite cefixime-sorbital MacConkey (TC-SMAC) 
agar plates on day 1, 3 and 5. All experiments were repeated. 
3.6.2 Liquid egg   
Aliquots of 6 mL liquid egg were added to 50 mL sterile centrifuge 
tubes. Nalidixic acid resistant Salmonella Typhimurium was inoculated into 
the tube at 104 CFU/ mL. The bacteria and liquid egg were mixed 
thoroughly. In the treated group, 300 mg dried secondary metabolites of C. 
nuda were added in a tube and mixed well. The control group consisted of 
21 
 
the inoculum and liquid egg only, and all samples were stored at 4 ?C for 1, 
3 and 5 days. The bacterial populations of the samples were determined 
by spread-plating on TSA+100 ppm nalidixic acid. All experiments were 
repeated. 
3.7 Statistical analysis 
All the assays were carried out in triplicate and the results were 
expressed as mean values and standard deviations (SD). The pH effects 
of the C. nuda were analyzed by using analysis of variance (ANOVA) of 
the Statistical Analysis System (Version 9.2 for Windows, SAS Institute, 
Inc., 2012) with a significance level of P = 0.05. 
22 
 
Chapter 4 Result and Discussion 
 
 
4. 1 Minimum inhibitory concentration (MIC) of secondary 
metabolites from Clitocybe nuda 
The secondary metabolites from C. nuda showed different 
antibacterial activities against Listeria monocytogenes, Salmonella 
Typhimurium, Staphylococcus aureus and Escherichia coli O157:H7. 
Various concentrations of the secondary metabolites from 0-50 mg/mL 
were tested for MIC test against L. monocytogenes and E. coli O157:H7. 
The MIC 50% log reduction was calculated using the linear regression of 
log bacterial number versus the concentration of the secondary 
metabolites after 24 h incubation. The MIC 50% log reduction for L. 
monocytogenes was 27 mg/mL (Table 1). For E. coli O157:H7, the 
inhibitory effects from 0-30 mg/mL were very low, but bacterial growth was 
completely inhibited at 50 mg/mL. The MIC 50% log reduction of E. coli 
O157:H7 was calculated from the inhibitory effects of 30 to 50 mg/mL, and 
the MIC50 was 40 mg/mL (Table 2). The concentrations of the mushroom 
secondary metabolites from 0-30 mg/mL were used for MIC50 test against 
Salmonella Typhimurium. Since the inhibitory effects from 0-15 mg/mL 
23 
 
were similar and minimal, however, and the bacterial growth was 
completely inhibited at 30 mg/mL, the MIC 50% was calculated from the 
inhibitory effects of 15-30 mg/mL. The MIC 50% log reduction of 
Salmonella Typhimurium was 25 mg/mL (Table 3). The inhibitory effects of 
the secondary metabolites against S. aureus were similar to those of 
Salmonella Typhimurium. The MIC 50% log reduction of S. aureus was 23 
mg/mL (Table 4).  
The results indicate that the secondary metabolites from C. nuda 
has better inhibitory effects against Salmonella Typhimurium and S. 
aureus than it does for L. monocytogenes and E. coli O157:H7. The 
secondary metabolites from C. nuda have been shown to have non-
selective inhibitory effects against both Gram-positive and Gram-negative 
bacteria.  
Table 1-Antibacterial activity on L. monocytogenes of the secondary 
metabolites of C. nuda at various concentrations. 
Concentration  
(mg/mL) 
Bacterial population 
(log CFU/mL) 
0 9.711?0.037 
5 9.313?0.071 
10 9.135?0.088 
15 8.250?0.167 
20 7.178?0.242 
25 5.475?0.111 
30 3.579?0.274 
40 1.709?1.480 
50 0.000?0.000 
*Linear regression was calculated from 0 to 50 mg/mL: y = -0.214x + 10.670, R? 
= 0.968; MIC50=27.19 mg/mL, n=3, x= concentration, y= log CFU/mL 
 
 
24 
 
Table 2-Antibacterial activity on E. coli O157:H7 of the secondary 
metabolites of C. nuda at various concentrations. 
Concentration 
(mg/mL) 
Bacterial population 
(log CFU/mL) 
0 9.445?0.040 
10 9.296?0.060 
20 9.249?0.011 
30 9.191?0.027 
40 4.483?0.877 
50 0.000?0.000 
*Linear regression was calculated from 30 to 50 mg/mL: y = -0.460x + 22.940, R? 
= 0.9998; MIC50=39.88 mg/mL, n=3, x= concentration, y= log CFU/mL 
 
Table 3-Antibacterial activity on Salmonella Typhimurium of the secondary 
metabolites of C. nuda at various concentrations. 
Concentration 
(mg/mL) 
Bacterial population 
(log CFU/mL) 
0 9.831?0.113 
10 9.205?0.058 
15 9.048?0.032 
20 8.831?0.073 
25 5.423?0.025 
30 0.000?0.000 
*Linear regression was calculated from 15 to 30 mg/mL: y = -0.611x + 19.574, R? 
= 0.872; MIC50=24.63 mg/mL, n=3, x= concentration, y= log CFU/mL 
 
Table 4-Antibacterial activity on S. aureus of the secondary metabolites of 
C. nuda at various concentrations. 
Concentration 
(mg/mL) 
Bacterial population 
(log CFU/mL) 
0 8.887?0.041 
5 8.880?0.106 
10 8.758?0.027 
15 8.708?0.100 
20 5.841?0.758 
30 0.000?0.000 
25 
 
*Linear regression was calculated from 15 to 30 mg/mL: y = -0.581x + 17.439, R? 
= 1; MIC50=22.52 mg/mL, n=3, x= concentration, y= log CFU/mL 
 
4.2 Effects of pH on the antibacterial activities of the secondary 
metabolites from C. nuda 
The secondary metabolites from C. nuda showed different 
antibacterial activities at different pH levels. At all pH values evaluated, the 
concentrations of the secondary metabolites of C. nuda were determined 
at MIC as previously described. The secondary metabolites were replaced 
by sterile water in the negative control groups. In general, the optimal pH 
for the growth of the four bacterial pathogens was around 7. At pH 4, all 
four pathogens could not grow in either the negative controls or in sample 
solutions. At other pH levels, the bacterial populations in all sample 
solutions were lower than those in negative controls. The results indicate 
that the secondary metabolites from C. nuda exhibit antibacterial activity 
under a wide range of pH levels.  
4.2.1 Listeria monocytogenes 
L. monocytogenes grew similarly in control samples at pH 7-9. 
Bacterial growth was inhibited and the populations were significantly lower 
at pH 5-6 than at pH 7-9. The growth of L. monocytogenes was completely 
inhibited at pH 4 and 5 with the presence of the secondary metabolites of 
C. nuda. From pH 6 to 9, the bacterial populations with the secondary 
metabolite samples were similar to each other and they were all 
26 
 
significantly lower than those in the negative control groups (Figure 1). 
The results also indicate that the anti-L. monocytogenes activity of the 
secondary metabolites from C. nuda could be enhanced in acidic 
environments.  
 
*Inoculum: 23 CFU/ mL sample 
*Concentration of treated sample: 26 mg/mL 
 
Figure 1-pH effect on antibacterial activity of secondary metabolites from 
C. nuda against L. monocytogenes. 
 
4.2.2 Salmonella Typhimurium 
In the controls, Salmonella Typhimurium populations were the 
highest in pH levels of 7 and 8, and then at pH 9, 6, and 5 in that order. In 
the samples with the secondary metabolites from C. nuda, the growth of 
Salmonella Typhimurium was inhibited and their bacterial populations 
were lower than those of the controls at various tested pH levels (Figure 2). 
Bacteria growth was completely inhibited in the samples at pH 5. In 
0
1
2
3
4
5
6
7
8
9
10
4 5 6 7 8 9
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
pH
Negative control
Secondary metabolites
treated
d 
c 
b a a a 
c? c? 
b? ab? b? a? 
27 
 
treated samples bacteria grew significantly better in alkaline conditions 
than those in neutral and acidic environments. Thus, the anti-Salmonella 
Typhimurium activity of the secondary metabolites from C. nuda was 
enhanced by acid and weakened by alkaline.  
 
 
*Inoculum: 26 CFU/mL sample 
*Concentration of treated samples: 25 mg/mL 
 
Figure 2-pH effect on the antibacterial activity of the secondary 
metabolites of C. nuda on Salmonella Typhimurium. 
 
4.2.3 Staphylococcus aureus 
For S. aureus, the peak bacterial growth in the controls was at pH 
6-8. S. aureus was able to grow in the secondary metabolites of C. nuda 
over the pH range of 5-9, and the growth was completely inhibited at pH 4. 
However, bacterial growth was significantly lower compared to the 
negative controls (Figure 3). Unlike the other three bacteria, the growth of 
S. aureus was not completely inhibited by the secondary metabolites of C. 
0
1
2
3
4
5
6
7
8
9
10
4 5 6 7 8 9
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
pH
Negative control
Secondary metabolites
treated
e 
d 
c 
a a 
b 
d? d? 
c? c? 
b? 
a? 
28 
 
nuda at pH 5. In the treated sample, the bacterial population increased 
significantly as the pH went up from 5 to 9. This indicated that the anti-S. 
aureus activity of the secondary metabolites from C. nuda was better at 
lower pH conditions.   
 
*Inoculum: 15 CFU/mL sample 
*Concentration of tested sample: 25 mg/mL 
 
Figure 3-pH effect on the antibacterial activity of the secondary 
metabolites of C. nuda on S. aureus. 
 
4.2.4 Escherichia coli O157:H7 
In the control groups, the growth of E. coli O157:H7 was the best at 
pH 7 and was completely inhibited at pH 4.  For the secondary metabolite 
treated samples, the bacterial growth was completely inhibited at pH 5. 
The bacterial populations increased dramatically from pH 6 to 9, but were 
all lower than those in the negative controls at corresponding pH values 
(Figure 4). The trends of anti-E. coli O157:H7 activity at various pH levels 
0
1
2
3
4
5
6
7
8
9
10
4 5 6 7 8 9
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
pH
Negative control
Secondary metabolites
treated
d f? 
c 
e? 
a a a 
d? c? 
b? 
a? 
b 
29 
 
were similar to that for anti-S. aureus which were as the pH levels 
decreased so did the reduction of the bacteria.   
 
 
*Inoculum: 24 CFU/mL sample 
*Concentration of tested sample: 40 mg/mL 
 
Figure 4-pH effect on the antibacterial activity of the secondary 
metabolites of C. nuda on E. coli O157:H7. 
 
4.3 Thermal stability of the secondary metabolites from C. nuda 
The secondary metabolites from C. nuda were treated at 4, 25, 72, 
100 and 121 ?C for 30 min before treatment with the various bacteria. The 
concentrations of the secondary metabolites from C. nuda were 60, 40, 50 
and 60 mg/mL for L. monocytogenes, Salmonella Typhimurium, S. aureus 
and E. coli respectively. These temperatures chosen were commonly used 
in the food industry for processing food and food products, such as 
refrigeration, pasteurization, cooking and retort heat treatment. Sterile 
0
1
2
3
4
5
6
7
8
9
10
4 5 6 7 8 9
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
pH
Negative control
Secondary
metabolites treated
d a
d 
 d d ba
d 
 d d
c
b
a
d 
 b
 d d
d? 
c?
?b
a
d 
 b
 d d
b?
a
d 
 d d
a? 
e 
e? e? f 
30 
 
water was treated at the same conditions and used in negative controls. 
The results showed that all four bacteria grew well in the control samples 
and the populations reached to around 9.0-9.5 log CFU/mL. The growth of 
the four bacteria was completely inhibited in all treated samples with the 
secondary metabolites from C. nuda at various temperatures (Figure 5, 6, 
7, 8). This indicates that the secondary metabolites from C. nuda are 
thermally stable.  
 
*Inoculum: 23 CFU/mL sample 
*Concentration of treated sample: 60 mg/mL 
 
Figure 5 ?Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on L. monocytogenes. 
0
1
2
3
4
5
6
7
8
9
10
4 25 72 100 121
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Temperature (?)
Negative control
Secondary metabolites
treated
31 
 
 
*Inoculum: 24 CFU/mL sample 
*Concentration of treated sample: 40 mg/mL 
 
Figure 6-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on Salmonella Typhimurium. 
 
*Inoculum: 12 CFU/mL sample 
*Concentration of tested sample: 50 mg/mL 
0
1
2
3
4
5
6
7
8
9
10
4 25 72 100 121
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Temperature
(?)
Negative control
Secondary metabolites
treated
0
1
2
3
4
5
6
7
8
9
10
4 25 72 100 121
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Temperature
(?C)
Negative control
Secondary metabolites
treated
32 
 
Figure 7-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on S. aureus. 
 
 
*Inoculum: 30 CFU/mL sample 
*Concentration of tested sample: 60 mg/mL 
 
Figure 8-Temperature effect on the antibacterial activity of the secondary 
metabolites of C. nuda on E. coli O157:H7. 
 
4.4 Antibacterial effects of secondary metabolites from C. nuda on 
food samples 
Approximately 104 CFU/g L. monocytogenes and E. coli O157:H7 
were inoculated into the ground beef, respectively. Liquid egg was 
inoculated with Salmonella Typhimurium at the same level. Antimicrobial 
efficiencies of the secondary metabolites from C. nuda were analyzed on 
Day 0, 1, 3 and 5.  
0
1
2
3
4
5
6
7
8
9
10
4 25 72 100 121
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Temperature
(?)
Negative control
Secondary metabolites
treated
33 
 
In the negative control group, the population of L. monocytogenes 
increased slightly during the test period, which was from 4.292 to 4.537 
log CFU/mL. The bacterial populations in the secondary metabolite treated 
samples were kept at about the same level and were lower than those in 
the control groups (Figure 9). The secondary metabolites from C. nuda 
inhibited the growth of L. monocytogenes in ground beef and resulted in 
about a 0.2 log CFU/mL reduction. However, the inhibitory efficiency of the 
secondary metabolites from C. nuda against L. monocygogenes in ground 
beef was low.  
 
Figure 9- Anti-L. monocytogenes activity of the secondary metabolites 
from C. nuda in ground beef. 
 
The bacterial populations of E. coli O157:H7 increased from 4.004 
to 4.434 log CFU/mL from day 0 to day 5 in the negative control group. 
During the first day, the bacteria grew slightly lower than from day 1 to day 
5. The bacterial population of the treated sample was reduced from 4.004 
4.0
4.1
4.2
4.3
4.4
4.5
4.6
0 1 3 5
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Time (d)
Negative control
Secondary metabolites
treated
34 
 
to 3.619 log CFU/mL after one day treatment. Then the bacterial 
population increased steadily from day 1 to day 5, reaching 4.075 log 
CFU/mL (Figure 10). The inhibitory efficiency of the secondary metabolites 
against E. coli O157:H7 in ground beef was about 0.4 log CFU/mL 
reduction, but was not as effective as in the culture medium.  
 
 
Figure 10-Anti-E.coli O157:H7 activity of the secondary metabolites from 
C. nuda in ground beef. 
 
In liquid egg, the Salmonella Typhimurium population dropped in 
both the treated samples and the negative control groups over the 5-day 
test period. The drop in the negative control samples from 3.9 to 3.7 log 
CFU/mL might be caused by the presence of the lysozyme in eggs. The 
Salmonella Typhimurium count decreased in the treated samples from 3.9 
to 3.0 log CFU/mL which might be caused by both the secondary 
metabolites and lysozyme (Figure 11). The secondary metabolites of C. 
3.6
3.7
3.8
3.9
4.0
4.1
4.2
4.3
4.4
4.5
0 1 3 5
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Time (d)
Negative control
Secondary
metabolites treated
35 
 
nuda against Salmonella Typhimurium can cause 0.7 log CFU/mL 
reduction. 
 
Figure 11-Anti-Salmonella Typhimurium activity of the secondary 
metabolites from C. nuda in liquid eggs. 
 
 
 
 
 
 
 
 
 
 
 
 
2.9
3.1
3.3
3.5
3.7
3.9
4.1
0 1 3 5
Ba
cterial
 popul
ation
(log 
CFU
/m
L)
Time (d)
Negative control
Secondary metabolites
treated
36 
 
Chapter 5 Conclusion 
 
 
In general, the secondary metabolites from Clitocybe nuda showed 
inhibitory effects against the four tested foodborne pathogens---Listeria 
monocytogenes, Salmonella Typhimurium, Staphylococcus aureus and 
Escherichia coli O157:H7.  
The secondary metabolites from C. nuda showed better inhibitory 
effects against Salmonella Typhimurium and S. aureus than L. 
monocytogenes and E. coli O157:H7. The MIC 50% log reductions were 
27, 40, 25, 23 mg/mL for L. monocytogenes, E. coli O157:H7, Salmonella 
Typhimurium and S. aureus, respectively.  
The pH effect tests, results showed that the secondary metabolites 
of C. nuda had better antibacterial activity in acidic environments. At pH 4-
5, the growth of L. monocytogenes, Salmonella Typhimurium and E. coli 
O157:H7 were completely inhibited. However, when pH increased up to 8 
and 9, the antibacterial activities decreased, except to L. monocytogenes. 
The results indicated that acid and the secondary metabolites of C. nuda 
were able to synergistically inhibit the bacterial growth.  
37 
 
The secondary metabolites of C. nuda were stable at all tested 
temperatures of 4, 25, 72, 100 and 121 ?C at pH 7. These results 
indicated that there is the potential for applying the secondary metabolites 
of C. nuda in thermal and non-thermal food processing without losing their 
antibacterial activities.  
In ground beef samples, the addition of the secondary metabolites 
of C. nuda at 75 mg/mL showed limited inhibitory efficiencies against L. 
monocytogenes and E. coli O157:H7 after 5 days of treatment. The 
complexity of ground beef matrix may provide bacteria a better 
environment from contacting with the secondary metabolites. The further 
investigation of applying the secondary metabolites to ground beef for 
controlling foodborne pathogens is needed.  
The secondary metabolites of C. nuda can inhibit the growth of 
Salmonella Typhimurium in liquid eggs and resulted in a 1 log CFU/mL 
reduction at 50 mg/mL concentration. The lysozyme in eggs appears to 
have a synergistic antibacterial activity with the metabolites. However, the 
secondary metabolites of C. nuda may cause the changes in the 
appearance and flavor of the liquid eggs. Therefore, the sensory 
evaluations of adding the secondary metabolites of C. nuda to liquid eggs 
will be necessary.  
 
 
38 
 
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