pathogenic bacteria in meat products and its interactions with … · 2.2.3 lactic acid bacteria...
TRANSCRIPT
Pathogenic bacteria in meat products and
its interactions with competitor yeasts
Ana Barbier Tello
Dissertation to obtain the Master of Science Degree in
Microbiology
Supervisors: Prof. Doutora Isabel Maria de Sá Correia Leite de Almeida
Prof. Doutor Fernando D’almeida Bernardo
Examination Committee
Chairperson: Prof. Doutor Jorge Humberto Gomes Leitão
Supervisor: Prof. Doutor Fernando D’almeida Bernardo
Member of the Committee: Prof. Doutora Maria João dos Ramos Fraqueza
May 2016
ii
iii
“The important thing is not to stop questioning. Curiosity has its own reason for existing.”
Albert Einstein
iv
Acknowledgments
Firstly, i would like to thank my advisor Professor Fernando Bernardo for all the support and
acquired knowledge. His knowledge and guidance helped me in all the time of research,
laboratory work and writing of this dissertation.
Secondly, I would like to express my sincere gratitude to Professor Maria João Fraqueza for the
time spent with me during the laboratory work and your friendship.
Besides advisor and co-advisor, I want to really thank to Engineer Maria José Fernandes and
Helena Fernandes for all the patience, friendship, support and constant motivation. The time
spend with them enriched my laboratory experience.
To Professor Isabel Sá Correia, for consenting me to develop my work in the Faculty of
Veterinary Medicine. And also, for her dedication to this course.
To Professor António Barreto, for welcoming me at the laboratory of food technology.
To Professor Luis Tavares, for welcoming me at the Faculty of Veterinary Medicine.
I thank my fellow colleagues and friends, Zé, João C., Carina, Margarida, João M., for their
friendship and company.
I thank, also, to all my new friends that I met during my stay at the laboratory of food
technology: Maria, Ellen, Nina, Raquel and Aurora.
Last but not least, I would like to express gratitude to my family and boyfriend. My parents, my
brother and sister and João Branco. Without their support I couldn’t have done it.
This study was conducted with the financial support of ‘‘Centro de Investigação Interdisciplinar
em Sanidade Animal’’ (CIISA/FMV) from Faculdade de Medicina Veterinária (Faculty of
Veterinary Medicine, University of Lisbon), by project “Portuguese traditional meat products:
strategies to improve safety and quality” (PTDC/AGRALI/119075/2010) and Project
UID/CVT/00276/2013 financed by Fundação para a Ciência e a Tecnologia (FCT).
v
Abstract
A collection of seventy-two yeast (n=72) isolates from different traditional Portuguese sausages
were tested to find out their tecnological properties and inhibitory activity to S. aureus,
Salmonella and Listeria. Around 44% of the yeast isolates presented inhibitory effect over
Staphylococcus aureus ATCC 25923; 37.5% were able to inhibit Listeria monocytogenes CECT
4300 and 26.4% of the yeasts were inhibitors of Salmonella Enteritidis CECT 4300. However,
when the extracellular compounds of yeast isolates were purified and used as inhibitory
compounds, only eight (n=8) isolates revealed inhibitory activity and Staphylococcus aureus
was the only bacteria inhibited. These isolates were identified as Candida famata, Rhodotorula
glutinis, Candida humicola, Schwanniomyces polymorphus and Candida zeylanoides. None of
these five (n=5) isolates had proteolytic activity. Candida zeylanoides, Schwanniomyces
polymorphus and Candida famata showed lipolytic activity. There is no production of gas from
glucose fermentation by any yeast tested. All the yeast isolates were catalase positive. R.
glutinis was the only one with nitrate reductase activity. Rhodotorula glutinis and Candida
humicola presented inhibitory effects over a batch of fermentative bacteria except for
Lactobacillus plantarum. Rhodotorula glutinis was inoculated in meat product matrix (in vitro
model) simultaneously with S. aureus and Salmonella in order to verify if this yeast affected the
population of these two pathogenic bacteria over time (96 h). The reduction of Salmonella
Enteritidis was not confirmed. S. aureus had a slight reduction but there was no significancy.
There was no interference in the total aerobic microorganisms at 30 ºC neither in the levels of
LAB.
Keywords: yeast, sausage, safety, starters, pathogenic bacteria, fermentative microbiota
vi
Resumo
Uma colecção de setenta e dois (n=72) isolados de leveduras com origem em diferentes
chouriços tradicionais Portugueses foram testados para averiguar as suas propriedades
tecnológicas e actividades antimicrobianas sobre S. aureus, Salmonella e Listeria. Cerca de
44% dos isolados de leveduras revelaram actividade antimicrobiana directa para
Staphylococcus aureus ATCC 25923; 37.5% foram capazes de inibir Listeria monocytogenes
CECT 4300 e 26.4% das leveduras inibiram Salmonella Enteritidis CECT 4300. Quando os
componentes extracelulares de alguns isolados de levedura foram usados como componentes
inibitórios, apenas oito (n=8) revelaram actividade inibitória e só o demonstraram sobre S.
aureus. Estes isolados foram identificados como sendo: Candida famata, Rhodotorula glutinis,
Candida humicola, Schwanniomyces polymorphus e Candida zeylanoides. Nenhum destes
cinco (n=5) isolados exibiu actividade proteolítica. C. zeylanoides, S. polymorphus e C. famata
revelaram actividade lipolítica. Nenhumas das levedura exibiu capacidade de produção de gas
a partir da fermentação da glucose. Todas eram catalase positivas. R. glutinis foi o único
isolado com actividade nitrato-reductásica. R. glutinis e C. humicola também revelaram possuir
actividade inibitória in vitro sobre um conjunto de microbiota fermentativo com excepção de
Lactobacillus plantarum. R. glutinis foi inoculada numa matrix de carne juntamente com S.
aureus e Salmonella para averiguar o comportamento destes isolados quando desafiados num
modelo in vitro ao longo do tempo (96 h). Não houve registo de redução de Salmonella
Enteritidis. O teor S. aureus sofreu uma ligeira redução mas não foi significativa. Não se
observou qualquer interferência no teor de aeróbios totais a 30 ºC nem nos de LAB.
Palavras-chave: levedura, chouriço, segurança, starter, bactérias patogénicas, microbiota
fermentativo
vii
Contents
List of Figures ................................................................................................................................ ix
List of Tables ................................................................................................................................. x
List of Abbreviations ...................................................................................................................... xi
Chapter 1 ....................................................................................................................................... 1
Introduction .................................................................................................................................... 1
1.1 Meat hygiene ........................................................................................................................... 2
1.1.1 Salmonella zoonoses ........................................................................................................... 3
1.1.2 Listeriosis.............................................................................................................................. 4
1.1.3 Food-borne outbreaks caused by Staphylococcal enterotoxins .......................................... 5
1.2 Control strategies for meat products ....................................................................................... 5
1.2.1 Legal limits for Salmonella ................................................................................................... 7
1.2.2 Legal limits for Listeria monocytogenes ............................................................................... 8
1.2.3 Guidance values or warning limits for Staphylococcus aureus ............................................ 9
1.3 Risk management of sausages ............................................................................................. 10
1.3.1 Chemical factors ................................................................................................................. 11
1.3.2 Physical factors .................................................................................................................. 12
1.3.3 Biological factors ................................................................................................................ 12
1.3.4 Characterization of yeasts .................................................................................................. 14
1.3.4.1 Antimicrobial activity of yeasts ........................................................................................ 17
Chapter 2 ..................................................................................................................................... 20
Materials and Methods ................................................................................................................ 20
2.1 Objectives .............................................................................................................................. 21
2.2 Microbial collections .............................................................................................................. 21
2.2.1 Yeast isolates ..................................................................................................................... 21
2.2.2 Pathogenic bacteria and Staphylococcus (coagulase negative)........................................ 22
2.2.3 Lactic acid bacteria (fermentative bacteria) ....................................................................... 22
2.3 Bioautography tests ............................................................................................................... 23
2.4 Evaluation of intracellular or extracellular compounds with inhibitory effect ......................... 24
2.5 Identification of indigenous yeasts ........................................................................................ 24
2.5.1 Yeasts characterization ...................................................................................................... 25
2.6 Behaviour of selected yeast in a meat product matrix .......................................................... 25
2.7 Statistical analysis ................................................................................................................. 26
Chapter 3 ..................................................................................................................................... 27
Results ......................................................................................................................................... 27
3.1 Bioautography tests ............................................................................................................... 28
3.2 Extracellular inhibitory effects................................................................................................ 28
viii
3.3 Identified yeasts .................................................................................................................... 29
3.4 Bioautography tests over LAB and bacteria from the curing process ................................... 30
3.5 Enzimatic activity ................................................................................................................... 30
3.6 Meat fermentation model ....................................................................................................... 31
Chapter 4 ..................................................................................................................................... 35
Discussion ................................................................................................................................... 35
4.1 Discussion ............................................................................................................................. 36
5. Conclusions ............................................................................................................................. 40
References .................................................................................................................................. 42
ix
List of Figures
Figure 1: Distribution of strong-evidence outbreaks by food vehicle in the EU, 2013 (adapted
from EFSA) [8]. .............................................................................................................................. 3
Figure 2: Distribution of food vehicle in strong-evidence outbreaks caused by Salmonella in the
EU, 2013 (adapted from EFSA) [8]. .............................................................................................. 4
Figure 3: General dry-fermented meat processing flow chart (adapted from Jordi Rovira and
Dorota Puszczwicz, 2015). The notes could be slightly different according to the particular/
traditional practices [47]. ............................................................................................................... 7
Figure 4: Meat starter culture bacteria: major metabolic pathways in meat fermentation (adapted
from Pier Sandro Cocconcelli and Cecilia Fontana, 2010) [27]. ................................................. 14
Figure 5: Procedure used to make the in vitro competition tests (original). ................................ 23
Figure 6: Procedure used to filter the yeast's suspensions (left figure). “Small wells” in
incorporated medium (right figure) (original). .............................................................................. 24
Figure 7:Procedure used to inoculate microorganisms in meat matrix (original). ....................... 26
Figure 8: Percentage of yeast isolates to cause inhibition in pathogenic bacteria. .................... 28
Figure 9: Yeast colony and bacteria present in direct competition (a) and filtrated compounds
tested for their inhibitory properties (b). ...................................................................................... 29
Figure 10: In vitro competition tests between yeast isolates and fermentative bacteria. ............ 30
Figure 11: Growth of Rhodotorula glutinis during 96 h at 7 ºC and 20 ºC in meat matrix model.31
Figure 12: Growth of Staphylococcus aureus during 96h at 7ºC and 20ºC in meat matrix model.
..................................................................................................................................................... 31
Figure 13: Growth of Staphylococcus during 96 h at 7 ºC and 20 ºC in meat matrix model. ..... 32
Figure 14: Growth of Salmonella Enteritidis during 96 h at 7 ºC and 20 ºC in meat matrix model.
..................................................................................................................................................... 33
Figure 15: Growth of total aerobic microorganisms (TA) during 96 h at 7 ºC and 20 ºC in meat
matrix model. ............................................................................................................................... 33
Figure 16: Growth of Lactic acid bacteria (LAB) during 96 h at 7 ºC and 20 ºC in meat matrix
model. .......................................................................................................................................... 34
x
List of Tables
Table 1 - Effective hurdles inhibitory to pathogens present in dry-fermented (adapted from
Graciela Vignolo and Silvina Fadda, 2015) [49]. ......................................................................... 11
Table 2 - Specific relationship between yeast and temperatures [51, 52]. ................................. 16
Table 3 - Yeast isolates source and their code. .......................................................................... 22
Table 4 - Gallery profiles API 32 C for each yeast isolate. C.H - Candida humicola, S.P -
Schawanniomyces polymorphus, C.F - Candida famata, R.G - Rhodotorula glutinis, C.Z -
Candida zeylanoides. (*) Esculine test that was only used when the yeast identification was
doubtfull. In this case, only Candida zeylanoides had doubfull results so the esculine test was
made. ........................................................................................................................................... 29
Table 5 - Metabolic behaviour of five selected yeasts. ............................................................... 30
Table 6 - Brief description of main substrates and products for most important muscle proteolytic
enzymes [31]. .............................................................................................................................. 36
Table 7 - Limiting aw for the growth of microorganisms (adapted from FAO, 2007) [46]. .......... 40
xi
List of Abbreviations
a.a.
Amino acids
ANOVA
Analysis of variance
Arg
Arginine
Aw
Water activity
B.A.
Biogenic Amines
BHI
Brain Heart Infusion
BPW
Buffered peptone water
CAC
“Codex Alimentarius” Comission
CCP
Critical Control Points
CDC
Center of Disease Control
CFU
Colony-forming unit
ECDC
European Centre for Disease Control
EEA
European Economic Area
EFSA
European Food Safety Authority
EU
European Union
FAO
Food and Agriculture Organization of the United Nations
FDA
Food and Drug Administration
HACCP
Hazard Analysis and Critical control points
INSA
Instituto Nacional de Saúde, Dr. Ricardo Jorge
LAB
Lactic Acid Bacteria
MAP
Modified atmosphere packaging
MRS
Man-Rogosa-Sharpe
MS
Members States
NO
Nitrosilo radical
OD
Optical Density
Orn ISO
Ornithine International Standart Organization
RH
Relative Humidity
RTE Ready to Eat
xii
SDA
Sabouraud Dextrose Agar
SDB
Sabouraud Dextrose Broth
T
Temperature
TGA
Agar Plate Count
TPP
Tripotassiumphosphate
TSA
Tryptic Soy agar
TSA
Trypticase Soy Agar
TSB
Trypticase Soy Broth
TSP
Trisodiumphosphate
USFDA VOC
United States Food and Drug Administration Volatile Organic Compounds
WHO
World Health Organization
WTO World Trade Organization
1
Chapter 1
Introduction
2
1.1 Meat hygiene
Recorrently, and all over the world, there are many reports and evidences of outbreaks illness
that incrimine microbial pathogens present in food. Food is a frequent vehicle by which many
pathogens (bacteria, viruses and parasites) can reach and colonise new hosts [1]. Worldwide,
some of the most important causes of sickness and death are related to food and water-borne
diseases. Most infectious and parasitic human diseases are shared with animals, and food of
animal origin are among the most likely to cause those diseases [2]. People having
immunocompromising conditions are living longer, but they remain particularly susceptible to
food borne disease agents. In recent decades food distribution systems have been greatly
improved, and the manufacturing of food products may occurs in a specific place and its
utilization may happen after transportation at hundreds and thousands of miles in a short lapse
of time. When a sanitary problem occurs, the amount of food involved can be astronomical.
Another important development is the global trade. Vast amounts of food are corrently shipped
from one region to another with a minimal monitoring of its microbial safety [3]. To complicate
the food safe scenarium, well-recognised food-borne pathogens, such as Salmonella spp. and
Escherichia coli, seem to be able to evolve and exploit novel opportunities and even generate
new public health risks even though food production practices change. In addition, previously
unknown foodborne pathogens, some of which are zoonotic, are constantly emerging [1].
Animals destined to produce food, whether infected or asymptomatic carriers, are sources of
spoilage and some pathogenic microorganisms. This is due to the fact that animals carry
microorganisms natural on their external surfaces, digestive and respiratory tracts which
contaminate the environment as well as food and water. Contaminations through external
animal surfaces, gastrointestinal tract, feces and environment are the most common, and these
situations may occur (i) during the slaughtering, dressing, chilling, and cutting processes of
carcasses; (ii) during processing, storage and handling of meat products; (iii) contaminating
water and other foods through manure; or (iv) through direct transference and infection from
humans [4].
Among all the meats, pork is the most widely consumed across the world and it refers to meat
obtained from the domestic pig or hog. This meat has been consumed for millennia, with pigs
being bred as far back as 7 000 years ago. There are many different forms of preparing pork
meat such as freshly cooked, cured and preserved or processed. Cured pork products have
been used traditionally as the process that greatly increases the shelf life of the meat. Today
such pork products are also extremely popular because of the flavour and distinctive taste
added through the process of curing. Sausage, ham, salami and bacon are just few examples of
preserved pork [5].
The risks of foodborne diseases associated with the consumption of untreated or not
adequately cooked pork meat are considerable because it may contain harmful microorganisms
3
and pose a risk of infection. Some pathogens that are often present in pork meat include
Listeria monocytogenes, E. coli, Salmonella, and Staphylococcus aureus. These bacteria are
mainly found in uncooked pork, however, and according to the Centers for Disease Control and
Prevention (CDC), most of these pathogens can be destroyed through adequated cooking
procedures [6]. This is why ready-to-eat (RTE) meats are of special concerns, since these may
be consumed without further cooking [7].
In 2013, “pig meat and products thereof”, represented 8% of the outbreaks by food vehicle in
EU as ilustrated in the figure 1 [8].
Figure 1: Distribution of strong-evidence outbreaks by food vehicle in the EU, 2013 (adapted from EFSA) [8].
In fact, 31% of the foodborne outbreaks in 2013 in the European Union were due to the meat
products. These results suggest that meat and meat products are an important vehicle by which
many foodborne outbreaks arise and deserve attention.
1.1.1 Salmonella zoonoses
The report of the European Food Safety Authority and the European Centre for Disease
Prevention and Control concerning to 2013 carried out in 32 European countries (28 Member
States and four non-Member States) signaled 221 cases of Salmonella contamination was
detected from a total of 27 662 tested samples of ready to eat minced meat, meat preparations
and meat products from pork.
4
Six MS tested 1 161 samples of fermented sausages at retail level, and three of them reported
that 11 samples were Salmonella-positive. Within the EU, 0.7 % units of fresh pig meat were
contaminated with Salmonella, from a total of 78 624 units [8]. The overall EU Salmonella
prevalence from the bacteriological monitoring of pigs was 8.1 %, which is higher than in 2012
(6.3 %). At the herd and slaughter batch levels, the Salmonella prevalence was 14.9 % and
30.0 %, respectively [8]. A total of 85 268 human salmonellosis cases were reported by 27 EU
MS in 2013, with 82 694 confirmed cases corresponding to a notification rate of 20.4 cases per
100 000 EU population [8].
Figure 2: Distribution of food vehicle in strong-evidence outbreaks caused by Salmonella in the EU, 2013 (adapted from EFSA) [8].
1.1.2 Listeriosis
According to Scientific Report of EFSA and ECDC, in 2013, L. monocytogenes was detected in
3.4 % of the 36 511 samples of ready-to-eat pig meat or products thereof, in 19 member states.
Among the 19 926 units tested, L. monocytogenes was found at levels above 100 CFU/g in 0.4
% of the tested samples, in 16 member states [8].
5
In 2013, 27 MS reported 1 763 confirmed human cases of listeriosis. The EU notification rate
was 0.44 cases per 100 000 population which was an 8.6 % increase compared with 2012. The
highest MS specific notification rates were observed in Finland, Spain, Sweden and Denmark
(1.12, 1.00, 0.97 and 0.91 cases per 100 000 population, respectively). The vast majority of
cases were reported to be domestically acquired [8].
In the last five years, there has been an increasing trend of listeriosis in the EU/EEA and, in
2013, the EU notification rate increased by 9.4 % compared with 2012. In 2013, seven strong-
evidence of food-borne outbreaks caused by L. monocytogenes were reported by five MS.
These outbreaks resulted in 51 cases, 11 hospitalisations and three deaths, i.e. 37.5 % of all
deaths due to strong-evidence food-borne outbreaks reported in 2013 [8].
1.1.3 Food-borne outbreaks caused by Staphylococcal
enterotoxins
Staphylococcal food poisoning is an intoxication caused by the consumption of foods containing
enterotoxins produced by certain strains of Staphylococcus aureus, a major concern for public
health. It seems that the type of foods involved differ widely among countries. Nevertheless,
ham, sausages, and other fermented meat could be associated with outbreaks [9].
In 2013, 12 MS reported 386 food-borne outbreaks caused by staphylococcal toxins. This
represents 7.4 % of all outbreaks, an increase compared with 2012 when 14 MS reported 346
outbreaks caused by staphylococcal toxins. In 2013, the overall reported rate in the EU was
0.13 per 100 000. France reported the vast majority (87 %) of the outbreaks, representing an
increase of 12 % compared with 2012 [8].
1.2 Control strategies for meat products
To place the final product in a market, the industry has to be aware of certain requisits that
prevent, not only but also, the proliferation of undesirable microorganisms. Accomplishing all
conditions and measures necessary to ensure the safety and suitability of food in all steps of
food chain (figure 3), guaranties the safest possible final product. This is ensured by the Codex
Alimentarius (“Codex Alimentarius”, 1999, 2002). The aim of the processing plant sanitation
programs is to put as many barriers as possible to avoid the entrance of contaminants into the
foodstuffs along the processing line. The four main sources that can contaminate the product
during the process are: raw material, processing environment, personnel manipulation, and
some processing steps [10].
According to the HACCP principles, in each step of the food process, all possible hazards
(physical, chemical, microbiological) are identified, their importance is evaluated and all the
6
preventive measures for their control are described (principle 1). The Critical Control Points
(CCPs) should be identified by risk assessment according to ISO 22000:2005 where monitoring
is critical for controlling the safety of the product (principle 2). For each identified CCP, critical
limits for preventive measures (principle 3) and monitoring systems (principle 4) are established.
When monitoring shows that a critical limit has not been met, corrective actions must be taken
(principle 5). Finally, procedures to verify that the system is working properly (principle 6) and
effective records to document the HACCP system are established (principle 7) [11].
The quality of the raw material used for the production of dry/cured-fermented sausages is
extremely important. Meat has to be transported at the right temperature: less than 7°C in
chilled condition or below −18°C in the case of frozen meat. In both cases, meat trimmings
should be properly packaged with plastic films inside the boxes, to avoid the contact of the meat
with any dirty surface [12]. Microbiological criteria for fresh meat recommended less than 104
CFU per cm2 of meat for total plante count and less than 100 CFU per cm2 of meat for
enterobacteriaceae counts [46]. From here, the use of correct time/temperature, preventive
equipment maintenance, use of nontoxic food-compatible, cleaning compounds, visual control
of shelf life dates according to specified —requirements, correct stuffing pressure machine,
visual check, metal detector, relative humidity, control of pH and aw of end product, control of
sealing vacuum and CO2/O2 concentration of the package are essential to the food safety of this
product [12]. The raw material must have less than 105 CFU/g of total aerobic colony and less
than 102 CFU/g of enterobacteriaceae, S. aureus and Clostridium (sulfite reducing) [46].
It is important to note that if the meat has counts of 103 of total aerobic microorganisms per
gram, it can, easly, reach spoilage levels within 2 days at 20 ºC (temperature of fermentation).
For this reason, fermentation is an important critical control point that has to be effective in
decreasing the aw, pH and time of fermentation [46].
7
Figure 3: General dry-fermented meat processing flow chart (adapted from Jordi Rovira and Dorota Puszczwicz, 2015). The notes could be slightly different according to the particular/ traditional practices [47].
1.2.1 Legal limits for Salmonella
Salmonella infection is caused by ingestion of viable cells of the bacterium. Infection occurs in
all age groups, however, host factors may increase the susceptibility to infection, for example
treatment to reduce the acidity of the stomach, are more vulnerable to infection. The infectious
dose Salmonella species is usually quite large. However, data from outbreaks has shown that
consumption of low numbers of Salmonella in food together with the mode of delivery of the
bacterium to the gastrointestinal tract can cause infection and this is particularly evident with
high fat / low water activity foods, such as chocolate, fermented meats, cheese and snacks, in
which the organism can survive for long periods of time [13].
8
According to the Regulation (EC) No 2073/2005 that have been in force since 1 January 2006
(revised by Regulations (EC) No 1441/2007 and 1086/201121), Salmonella must be absent in
relevant products when placed on the market, during their shelf-life. Absence is defined by
testing five or 30 samples of 25 g per batch, depending on the food category; however, the
definition of a batch varies widely and in official controls, often only single samples are taken to
verify compliance with the criteria [8].
1.2.2 Legal limits for Listeria monocytogenes
Listeriosis is caused by the ingestion of live cells of these bacterium. Listeria monocytogenes
bacterium occurs commonly in the environment and in raw foods, and consequently will occur in
some food production environments. Growth of this bacterium following both post-process
contaminations of cooked or processed foods or in raw foods probably represents the greatest
risk for disease transmission. L. monocytogenes can grow between 0°C to 45°C, albeit slowly at
refrigeration temperatures. The bacterium is killed by adequate cooking. Unrefrigerated foods
and those chilled for extended periods are at increased risk of allowing significant growth,
particularly if chilled temperatures are suboptimal [13].
Food safety criteria for L. monocytogenes in Regulation (EC) No. 2073/2005 (as amended) 16
are applicable to three categories of ready-to-eat foods:
In RTE products intended for infants and for special medical purposes L.
monocytogenes must not be present in 25 g of sample.
L. monocytogenes must not be present in levels exceeding 100 CFU/g during the shelf-
life of other RTE products.
In RTE foods that are able to support the growth of the bacterium, L. monocytogenes
may not be present in 25 g of sample at the time of leaving the production plant;
however, if the producer can demonstrate, to the satisfaction of the competent authority,
that the product will not exceed the limit of 100 CFU/g throughout its shelf-life, this
criterion does not apply. For many of the reported data, it was not evident whether the
RTE food tested was able to support the growth of L. monocytogenes or not. For the
non-compliance analysis of samples collected at processing, the criterion of absence in
25 g was applied, except for samples from hard cheeses and fermented sausages
(assumed to be unable to support the growth of L. monocytogenes) where the limit ≤
100 CFU/g was applied. For samples collected at retail, the limit ≤ 100 CFU/g was
applied, except for RTE products intended for infants and for special medical purposes,
where presence in L. monocytogenes must not be detected in 25 g of sample [8].
9
1.2.3 Guidance values or warning limits for Staphylococcus
aureus
Illness due to Staphylococcus aureus is caused by enterotoxins which are performed in food.
Only some S. aureus contain enterotoxin genes and therefore have the potential to cause food
poisoning. Although most cases of infection are due to S. aureus, other coagulase-positive
Staphylococcus species (e.g. S. intermedius) can also produce enterotoxins and cause
foodborne disease. Adequate cooking will kill the bacterium, however some protection is
afforded in dry, high-fat and high-salt foods. Staphylococcal enterotoxins are heat-stable and
can survive some normal cooking processes including boiling, consequently active toxin can be
present in the absence of viable organisms. Most coagulase-positive staphylococci grow
between 7°C and 48°C with no growth at refrigeration temperatures. Many people carry S.
aureus and contamination of foods after processing by food handlers can occur. Toxin
production starts at 10°C and storage of foods below this temperature should prevent its
development [13].
In foods such as ripened cheeses and fermented meat products, S. aureus levels are highest
2–3 days after initial production and may reduce significantly during storage. If levels exceed
105 CFU/g at any time during the life of a food, there is a risk of sufficient enterotoxin to cause
illness that will remain in the food product regardless of subsequent recoverable levels of this
organism.
The only food safety criterion for staphylococci in Regulation (EC) No. 2073/2005 (as amended)
is for an absence of staphylococcal enterotoxins in cheese, milk powder and whey powder in
product placed on the market during their shelf life. This Regulation has process hygiene criteria
with limits of between 10 and 105 coagulase positive staphylococci/g in cheese, milk and whey
powder during manufacture, and if values of >105 CFU/g are detected, the batch should be
tested for staphylococcal enterotoxins [13].
According to the INSA (Instituto Nacional de Saúde, Dr. Ricardo Jorge), Staphylococcus
coagulase positive has to be below 102 CFU/g to be a ready to eat satisfectory product. Above
104 CFU/g the product is considered potencial dangerous to the humans. This numbers are
values guide and not law.
10
1.3 Risk management of sausages
The transformation of raw materials to more-or-less stable foods by drying and fermentation is
well known in many ancient cultures and it has been used for many different foods [23].
Changes from raw meat to a cured meat are caused by “starter cultures” or “wild”
microorganisms, which decrease the pH. Because this is a biological system, it is influenced by
many environmental pressures that need to be controlled to produce a consistent product.
Some of these factors include a fresh, low-contaminated, consistent raw material; a consistent
inoculum; strict sanitation; control of time, temperature, and humidity during production; smoke;
and appropriate additives [15].
Today’s Europe is still the major producer and consumer of dry- cured/fermented sausages.
There are a wide variety of dry-fermented products on the European market as a consequence
of variations in the raw materials, formulations, and manufacturing processes, which come from
the habits and customs of the different countries and regions. But from a global viewpoint, two
categories can be distinguished: Northern and Mediterranean products with some specific
characteristics. In Northern products, rapid acidification by lactic acid bacteria to a final pH
below 5 and smoking ensure safety, improve shelf life, and contribute largely to the sensory
quality. Concerning to mediterranean products, acidification reaches a final pH above 5. Safety
and shelf life are mainly ensured by drying and lowered water activity [16]. Furthermore,
sausages can be classified as dry or semi-dry. Dry sausages have a water activity (aw) inferior
to 0.90, are not usually smoked or heat processed and are eaten without cooking. The aw of
semi-dry sausages ranges between 0.90-0.95 and they generally receive a heat treatment of
140-154 ºF (60-68 ºC) during smoking. Fermentation temperatures vary according to the
individual product but they are generally less than 72 ºF (22 ºC) for dry and mould-ripened
sausages and 72-79 ºF (22-26 ºC) for semi-dry varieties [17, 48]. Therefore, ingredients,
recipes, and technology result in a particular commodity with a given composition and with
given quality items as well as a product-specific microbiological composition. The potential
preservation of this type of food is based on chemical, physical or biological action leading to
cease of metabolism, or even to the destruction of the dangerous biological agent [18].
Nowadays the safety of the dry- fermented/ slightly fermented sausages is mostly maintained by
the hurdles describe in the table below (table 1) [19].
11
Table 1 - Effective hurdles inhibitory to pathogens present in dry-fermented (adapted from Graciela Vignolo and Silvina Fadda, 2015) [49].
1.3.1 Chemical factors
Basically, fresh meat contains water, protein, lipid, minerals, and vestigial amounts of
carbohydrate. Water is the major constituent, followed by proteins, but proportions change
depending on the amount of fat. So, the content of protein and water decrease when the
amount of fat increases. Proteins, lipids, and enzymes are major components of meat and fat
[20]. For this reason, chemical factors can be used for the stability of this type of food. Some
recipes make use of alcohols; aldehydes (glutare-di-aldehyde); carbohydrates, simple sugars
such as dextrose or fructose support an early drop in pH-values as they are easily broken down
by bacterial action. The breakdown of lactose is slower and takes longer. Often a mixture of
different sugars is used. Another sugar-based additive is GdL (Glucono-delta-Lactone), which
accelerates and intensifies the acidification process by reacting to glucono-acid in the presence
of water (muscle tissue water) [46]; organic acids and related compounds; inorganic acids and
related compounds as phosphorus (sodium phosphates, trisodiumphosphate TSP,
tripotassiumphosphate TPP, sodiumhypophosphite) that assist in solubilizing muscle proteins ,
sodium ascorbate (also in the U.S., sodium erythorbate) or ascorbic acid (also in the U.S.
erythorbic acid) that are used for improvement and stability of color and retardation of oxidation;
sulphur (sulfur dioxide, sodium disulfite) and anorganic nitrogen compounds such as nitrite.
Beyond antibacterial purposes, nitrite is also used for giving color to the sausage, and it works
as antioxidant. Nitrite is applied at 80 to 240 mg/kg. Nitrate and nitrite are often used in
combination, but nitrate is usually not necessary except as a reservoir for nitrite, which could be
useful in long-term meat processing.
12
1.3.2 Physical factors
The meat industry can, also, take advantage of technology to improve the safety of its products.
Physical factors can be applied like the use of different wave lengths: X-rays, ultraviolet,
microwaves, ultrasonic; the application of correct temperature that can be high or low; the use
of mechanical devices; keeping the surfaces clean; utilization of pressure and prohibition of
atmosphere (vacuum or modified atmosphere). Vacuum and modified atmosphere packaging
(MAP) are the most frequent ways for packaging. These techniques employ gas mixtures and
packaging materials technology to extend the shelf life of food. For meat products, the
atmosphere in the package is modified by pulling a vacuum and then replacing the package
atmosphere with a gas mixture of oxygen (O2) and carbon dioxide (CO2) or nitrogen (N2) and
carbon dioxide mixtures. In dry-cured meat products, the gas mixture more utilized is formed by
0% O2, 20–35% CO2, and 65–80% N2 [20].
1.3.3 Biological factors
Biological factors must also be referred and put in perspective. These factors can ensure a
better safety product and at the same time give to the sausages more and different aromas.
Consumers are increasingly demanding pathogen-free foods with minimal processing, fewer
preservatives and additives, high nutritional value, and intact sensory quality. In response to
these conflicting demands, current trends in the meat industry include the investigation of
alternatives for safer and healthier products. Biopreservation has gained increasing attention as
a means of naturally controlling the shelf life and safety of meat products. The application of
bioprotective cultures to ensure hygienic quality is a promising tool. There are tree promising
areas that had been studied. These tree areas are the use of the competing microbiota
(starters, protective cultures), the use of metabolites (bacteriocins, colicine, natamycine) and the
use of enzymes (lysozyme, lactoperoxidase).
Bacteriocins are a kind of ribosomal synthesized antimicrobial peptides produced by bacteria,
which can kill or inhibit bacterial strains closely-related or non-related to the producer bacteria,
but will not harm the bacteria themselves by specific immunity proteins. Bacteriocins become
one of the weapons against microorganisms due to the specific characteristics of large diversity
of structure and function, natural resource, and being stable to heat [20]. The Gram-positive
bacteriocins are generally divided into class I (modified peptides, lantibiotics), class II
(unmodified peptides, non-lanthionine), class III (large proteins, heat unstable) and more
recently appeared the class IV (complex peptides that contain lipid or carbohydrate moieties,
that are essentialy for activity) [29, 59]. Bacteriocins are natural food additives due to the
bacteriocin producing bacteria presence in many types of foods since ancient times, such as
cheeses, yogurts, and Portuguese fermented meat (Yang et al., 2012; Todorov et al., 2014). In
13
food technology, nisin is produced by Lactococcus lactis and was the first antibacterial peptide
found in LAB (Rogers, 1928). It is also a commercial bacteriocin used as a food preservative
against contamination by microorganisms which is marketed as Nisaplin®. It is the only
bacteriocin approved for utilization as a preservative in many foods by the U.S. Food and Drug
Administration (USFDA), and licensed as a food additive in over 45 countries (Settanni and
Corsetti, 2008). Another commercially available bacteriocin is pediocin PA-1, marketed as Alta®
2341, which inhibits the growth of Listeria monocytogenes in meat products (Settanni and
Corsetti, 2008) [20]. However, among class Ia bacteriocins, nisin has not been quite successful
in meat products because of its low solubility, uneven distribution, and lack of stability.
Lactococcal bacteriocins are not particularly adapted to sausage technology because they
display sensitivity to some ingredients and manufacture conditions [21].
Lysozyme is one of the major egg proteins that have antimicrobial and antioxidant
characteristics. Therefore, lysozyme can be used in meat processing to prevent microbial
growth and oxidative degradation in meat products during storage [22]. Egg white lysozyme
killed or prevented growth of Listeria monocytogenes in several foods but it is more active in
vegetables than in animal-derived foods that were tested, including fresh pork sausage
(bratwurst) and Camembert cheese [23].
In Europe, the starter cultures used most frequently include Lb. sakei, Lb. plantarum,
Pediococcus pentosaceus, Staphylococcus xylosus, S. carnosus, and to a lesser extent
Micrococcus spp. The advantage of a starter culture is that the same microorganisms can be
used repeatedly, which cuts down on variation of the finished product, and a larger number of
organisms can be added. These microorganisms together ensure the fermentative and curing
process. The Lactobacillus use the sugars to convert them into lactic acid which lowers the pH
releasing lots of flavour. The Staphylococcus and Microcococcus strains rapidly reduce nitrate
to nitrite and during meat processing, at about pH 5–6.5, nitrite forms NO (nitrosilo radical)
which binds to myoglobin. The NO-myoglobin or NO-hem is heat-stable and gives the meat the
red curing color. Some of these strains may also have antagonistic effects against pathogenic
bacteria. All this transformation make the dry-fermented sausages/ dry-cured sausages a place
unpleased for pathogenic bacteria [24]. However, some pathogens such as L. monocytogenes,
E. coli O157:H7, Salmonella spp., and Staphylococcus aureus have been reported to survive or
even grow in such unfavorable conditions. The ability of these pathogens to survive in acidic
foods has been recently studied [25].
In order to increase the safety of this product maybe can be interesting to test other type of
usefull microorganisms that probably will have potencial against pathogenic bacteria and
improvement of the flavour. Furthermore, very little extremely excellent product is produced,
because most starter cultures are a combination of just a few species of microorganisms and
they cannot produce as balanced a flavour as sometimes can be obtained when many species
are included [26]. Particularly in the south European countries, dry sausages are applied with
atoxinogenic yeast and fungi to produce products with specific flavor notes. This is done by
14
dipping or spraying. Mold cultures tend to suppress natural molds and, consequently, reduce
the risk of mycotoxins. Due to the extended ripening and drying for these products, the pH is
usually higher (pH >5.5), even if the pH was lower after fermentation, because molds can utilize
lactic acid and produce ammonia. This requires a final water activity lower enough for
preservation [26].
Figure 4: Meat starter culture bacteria: major metabolic pathways in meat fermentation (adapted from Pier Sandro Cocconcelli and Cecilia Fontana, 2010) [27].
In figure 4, the blue arrows correspond to Staphylococcus activity and the black arrows
correspond to LAB activity. The metabolic activities ascribed to both bacterial groups are
indicated by white arrows. Dotted - line arrows indicate action of endogenous meat enzymes.
Abbrevations: a.a., amino acids; Arg, arginine; B.A., Biogenic Amines; Orn, ornithine [27].
Microorganisms contribute to flavour compounds by the microbial degradation of amino-acids,
fatty acids and carbohydrate metabolism. Degradation reactions are transamination and further
descarboxylation of amino acids (branched, aromatic and linear) which result in their respective
aldehydes, alcohols or acids that impart aroma notes to the sausage. The fermentation of
carbohydrates is mainly performed by lactic acid bacteria that dominates the fermentation
process and produces lactic acid and other aroma compounds such as diacetyl, acetaldehyde,
ethanol, acetic and propionic acids among others. The enzymatic generation of amino acids and
free fatty acids comes from proteolysis and lipolysis reactions. In general, muscle endogenous
enzymes are responsible for these reactions especially at the beginning of the ripening process.
The further degradation of these precursors to aroma compounds is essentially mediated by
chemical and microbial reactions. All these reactions are mentioned in figure 4 [28].
1.3.4 Characterization of yeasts
Yeasts are eukaryotic and heterotrophic cells that obtain their energy (ATP) by the oxidation of
organic molecules such as glucose, fructose, galactose, mannose, maltose, sucrose, alcohols
or organic acids. These carbohydrates also act as sources for biosynthesis. Beyond carbon
15
source these microorganisms need a nitrogen source as ammonium, salt, nitrate, amino acids,
peptides, urea, purines or pyrimidines; a phosphate source; a sulfate source and lower
concentrations of potassium, magnesium, calcium, iron, zinc, and in most cases a vitamin such
as biotin, thiamine or pantothenic acid [29].
Yeasts may be physiologically classified with respect to the type of energy-generating process
involved in sugar metabolism, namely non-, facultative- or obligate fermentative. The
nonfermentative yeasts have exclusively a respiratory metabolism and are not capable of
alcoholic fermentation from glucose (e.g., Rhodotorula glutinis), while the obligate-fermentative
yeasts – “natural respiratory mutants” – are only capable of metabolizing glucose through
alcoholic fermentation (e.g., Candida slooffii). Most of the yeasts identified are facultative-
fermentative ones, and depending on the growth conditions, the type and concentration of
sugars and/or oxygen availability, may display either a fully respiratory or a fermentative
metabolism or even both in a mixed respiratory-fermentative metabolism (e.g., S. cerevisiae or
Pichia jadinii) [60]. Although the obligate fermentative species can survive under strict anaerobic
conditions they can not make it for a very long time because they need oxygen for synthesis of
certain membrane constituents, like the sterols. For this reason, sugar and oxygen are two
fundamental components for the growth of yeast species. Yeast aerobic respiration is defined
as the complete oxidation of carbon-containing molecules to CO2 and H2O by the interrelated
processes of the tricarboxylic acid (TCA) or Krebs cycle that, in eukaryotic cells, occurs in the
matrix of the mitochondria. The oxygen plays its role as the terminal electron acceptor of the
electron transport chain. In yeast anaerobic metabolism, often called “alcoholic fermentation,”
pyruvate produced by glycolysis splitin to ethanol and CO2 in a redox-neutral process [29].
Yeasts tolerate a wide range of pH and growth readily at values between 3 and 10 but, in
general, they prefer a slight acidic medium and have an optimum pH between 4.5 and 5.5. Their
temperature range (10-35 °C) is also broad (table 2), with a few species capable of growth
below or above this range, but the optimal temperature to yeasts´s growth is 26 ºC as described
in table 2. Most species can grow at a water activity (aw) of 0.85 or less, although yeasts
generally require a higher water activity [30, 31, 32].
16
Table 2 - Specific relationship between yeast and temperatures [51, 52].
The impact on growth of each ecological factor is influenced by others, e.g. at low aw the
minimum temperature for growth increases, or at low temperatures both the minimum pH and
the minimum aw permitting growth are higher. This is the basis for various combined
preservation methods. In this respect, the interactions of yeasts with other organisms in
microbial communities also play an important role. Yeasts and lactic acid bacteria occur
simultaneously in many natural habitats and food systems because they have many common
ecological determinants [29].
Yeasts are usually found in high numbers, in dry-cured meat products (up to 106 CFU/g) even if
they are not added as starter cultures in traditional methods of spontaneous curing. These high
levels could suggest that this microbial group may play an important role in the maturation
process. Yeast colonization on the exterior of dry-cured meat products could also play an
important role against pathogenic microorganisms which may cause health problems to the
consumer, such as Staphylococcus aureus and Eschericia coli [34]. Spoilage bacteria such as
Pseudomonas aeruginosa can also be controlled by yeasts. There are many evidences that
yeasts can produce extracellular proteases that have antimicrobial activity and there are very
few cases of illness in humans caused by yeasts, enabling them to be exploited as novel agents
in bio-control of food spoilage [33]. Furthermore, yeasts are considered to affect sausage colour
and flavour due to their oxygen-scavenging and lipolytic activities. They may also delay rancidity
and further catabolize products of fermentation, such as lactate produced by meat lactobacilli, to
other by-products, thereby increasing the pH and contributing to the development of less tangy
and more aromatic sausages [32]. Although yeasts are able to grow in anaerobic media, the
majority of species have a strictly aerobic metabolism. This favors yeast growth on the surface
of meat products. When yeasts arrive to processed meats as sausages, they can grow on the
outer surface, where they form a firm and white external coat. This coat controls water loss and
permits uniform dehydratation. Moreover, this coat gives to the products a peculiar and
17
characteristic appearance that is considered a criterion of quality due to the favorable whiteness
aspect that it gives to sausages [35].
Salt is the major additive in fermented meat products. It is added in levels of 2–4% (2%
minimum for desired bind, up to 3% will not retard fermentation), which will allow lactic acid
bacteria to grow and will inhibit several unwanted microorganisms [33]. The reduction of salt
and saturated fat in meat products is one key point for the meat industry due to dietetic
recommendations from the “World Health Organization” (WHO/FAO, 2003) as they may be
associated with cardiovascular diseases. The reformulation of sausages by the decrease in fat
and salt content produced important changes in the generation of aroma compounds and aroma
perception but the inoculation of D. hansenii was shown to compensate for these effects and
improve the sensory characteristics of the dry sausages. The most important contribution of D.
hansenii, in addition to the lipolysis increase and antioxidant effect, was the enhance in aroma
compounds derived from amino acid degradation and ester activity increasing the perception of
fruity and cured aroma notes [36]. Amino acid catabolism, oxidation of lipids, esterase activity
and carbohydrate catabolism are the biochemical pathways leading to the generation of most
reported VOC’s by yeasts. Some of the volatile compounds released by yeasts include aliphatic,
branched and aromatic hydrocarbons, aldehydes, ketones, alcohols, ethyl esters and sulfur
compounds. Furthermore, lipophilic alcohols and esters, like 2-methyl butanol and ethyl acetate,
respectively, revealed antifungal activity [37, 47].
The most frequently yeasts described on meat and meat products are:
Basidiomicetous yeasts represented by Candida humicola, Cryptococcus albidus, Cr. skinneri,
and Trichosporon pullulans; Ascomycetous yeasts, in both perfect and imperfect states,
represented by Debaryomyces, Candida famata, C. zeylanoides, C. guilliermondii, C.
parapsilosis, and C. kruisii; Cryptococcus; Hansenula; Hypopichia; Kluyveromyces;
Leucosporidium; Pichia; Rhodosporidium; Rhodotorula; Trichosporon; Torulopsis; and Yarrowia,
with Debaryomyces being the most commonly isolated [35].
1.3.4.1 Antimicrobial activity of yeasts
Research on killer yeasts for industry is quite recent. Killer yeasts are capable to produce toxic
compounds that cause inhibition of sensitive yeast or some bacteria. Similar to those described
for the bacteriocins, K+ (killer) phenotype of inhibitor yeast, production of these toxins in natural
environments, is an advantage in competition for food against susceptible microorganisms [38].
Antagonism of microorganisms by yeasts has been attributed primarily to:
competition for nutrients;
18
pH changes in the medium as a result of growth-coupled ion exchange or organic acid
production (heksanoat, oktanoat and dekanoat);
production of high concentrations of ethanol;
secretion of antibacterial compounds and release of antimicrobial compounds such as
killer toxins (proteins, glycoproteins, glycolipids) or “mycocins”. Yeasts can, also,
stimulate our imune response, produce proteases that degrade bacterial toxins and
inhibit the attachament of bacteria in intestinal cells [39].
Antimicrobial proteins that act as an antimicrobial agent were found in Sacharomyces cerevisiae
that produces several proteins that have little antimicrobial properties. Moreover, yeasts also
have ability in producing sulphur dioxide that could inhibit the growth of spoilage lactic acid
bacteria [39].
Candida parapsilosis has been referred as having various antimicrobial activities towards some
pathogen bacteria such as Eschericia coli, Staphylococcus aureus, and then spoilage bacteria
such as Pseudomonas aerugenes [53].
Inhibitory activity of Metschnikowia pulcherrima seems to be correlated with the iron
concentration in the media, suggesting an iron immobilization mechanism similar to pulcherrimin
formation [54].
Mycocin production occurs among many yeast genera including Saccharomyces, Candida,
Cryptococcus, Debaryomyces, Kluyveromyces, Pichia, Torulopsis, Williopsis, and
Zygosaccharomyces [38]. Mycocins are not active against bacteria and protozoa exhibiting only
fungicidal or fungistatic action. The formation of mycocins may be determined by nuclear or
plasmidic DNA [39].
Historically, the first positive indications of the antagonistic activity of yeasts published early in
the twentieth century by Hayduck (1909) and Fernbach (1909; cited in Golubev, 2006) who
reported a volatile thermolabile toxic extract from yeast probably an amine that inhibits the
growth of Escherichia coli and Staphylococci (Viljoen, 2006) [39].
Fatichenti et al. (1983) showed that the antibacterial activity of Debaryomyces hansenii against
Clostridium tyrobutyricum and Clostridium butyricum was related to its ability to produce both
extracellular and intracellular antimicrobial compounds [39].
Also, Cavalero and Cooper (2003) demonstrated that Candida bombicola produces extracellular
glycolipids called sophorosides, which have proven antibacterial activity against Staphylococcus
aureus and also inhibit Candida albicans. Having tested hundreds of dairy yeasts, Goerges et
al. (2006) reported a strain of Candida intermedia capable of reducing viable Listeria counts by
4 log10 CFU/cm2 in co-culture on agar, while three C. intermedia and one Kluyveromyces
marxianus suppressed L. monocytogenes growth by 3 log10 CFU/cm2. The same group more
recently found a strain of Pichia norvegensis (WSYC 592) able to reduce L. monocytogenes
19
counts by 7 log10 cycles, while numerous strains of Issatchenkia orientalis, Candida krusei, and
K. marxianus reduced Listeria counts by 4–5 log10 units in co-culture on agar (Goerges et al.,
2011) [39].
Assays using a Camembert curd model, the anti-listerial compounds of D. hansenii and W.
anomalus were found to reduce L. monocytogenes population by 3 log10 units during the first 9
days of ripening. The active principles are thermostable and apparently peptides and appear to
induce leakage in bacterial cells and ultimately cause bacterial lysis [39].
Many yeast genera including some Candida species produced exotoxins. These toxins in the
form of protein or glycoprotein, binding to specific receptors on the cell surface, can kill mold,
bacteria and protozoa. The toxin K28 of S. cerevisiae, lethal toxin secreted out of the cell, going
through the cytoplasmic membrane binds to target cell walls, through the golgi and endoplasmic
reticulum reaches the cytosol. An alpha unit of toxin transmitted to yeast cell nucleus and
produces toxic signal and ultimately inhibit DNA synthesis stops at the border of the celulose
[38].
For all the previously stated, in the past decades have witnessed the application of antagonistic
yeast starter cultures in various food processing industries. It is well known that final product
quality in industries, such as wine-making, sausage production, cheese ripening, bakery, and
the “fermentations” of cacao and coffee beans is affected directly by the development of
spoilage microorganisms. Antagonistic yeasts starter cultures contribute to product safety
primarily by inhibiting pathogen growth during fermentation, finishing product sensory qualities
and increasing shelf-life by inhibiting spoilage organisms [39].
20
Chapter 2
Materials and Methods
21
2.1 Objectives
The aim of this work was to identify and classify yeasts isolated from Alentejo’s sausages
presenting technological properties and inhibitory effect against pathogenic microbiota.
Antagonistic cultures added to food aiming to inhibit pathogens and/or extend the shelf life,
while ennhancing minimal changes on the sensory properties, are referred to as protective
cultures. The main objectives of bioprotection are to extend storage life, to enhance food safety,
and to improve sensory qualities [40]. To use probiotics as starter cultures for fermented
sausages, several characteristics of the strains, in addition to technological, sensory, and safety
properties, should be taken into account. The probiotic culture should be well adapted to the
conditions of fermented sausage to become dominant in the final product, competing with other
bacterial populations from meat and from starter culture. In addition, the culture should not
develop off-flavors in the product. Specific probiotic properties such as bile tolerance, resistance
to stomach acidity, and adhesion to the intestinal epithelium in addition to antimicrobial activity
will ensure strain survival in the human gastrointestinal tract. The microorganisms must be
considered GRAS (generally recognized as safe) to be use like food additives and this is
approved by FDA (Food and Drog Administration). Specification of origin, nonpathogenicity, and
antibiotic resistance characteristics of the strains should also be assessed [39].
2.2 Microbial collections
2.2.1 Yeast isolates
A collection of seventy-two (n=72) yeasts isolated from Alentejo traditional fermented sausages,
specifictly from “Linguiça (L)”,” chouriço catalão (C)”,” chourição (CH)”,” salsichão grosso
(SG)”,” chouriço de vinho (CV)”,” paio (P)”, “mixer (S1)”,” chopper (S2)”,” filling room wall (S3)”
and “filler (S4)” were kept at -80ºC in BPW (Buffered Peptone Water, Scharlau, Barcelone,
Spain) plus 20% of glycerol (w/v) until further processing. The strains were from the culture
collection of Faculty of Veterinary Medicine, University of Lisbon. To revivified them, the isolates
were inoculated in SDB (Sabouraud’s Dextrose Broth, Scharlau, Barcelone, Spain) at 25ºC for
48h. (table 3)
22
Table 3 - Yeast isolates source and their code.
After the revivification the isolates were inoculated in SDA (Sabouraud Dextrose Agar,
Scharlau, Barcelone, Spain) medium which contained per litre 3 g of peptone, 40 g of glucose,
8,5 g of NaCl and 15 g of agar. The pH used was 5,6. The isolates were kept at 25ºC during
48h.
2.2.2 Pathogenic bacteria and Staphylococcus (coagulase
negative)
Staphylococcus aureus ref.25923 ATCC, Staphylococcus xylosus ref. ATCC 8166,
Staphylococcus xylosus S4B2, Staphylococcus equorum CV2C3, Salmonella Enteritidis CECT
4300, Listeria monocytogenes CECT 937 were revivified in BHI (Brain Heart Infusion, Scharlau,
Barcelone, Spain) medium which contained 12,5 g brain extract, 5 g heart extract, 10 g
proteose peptone, 5 g sodium chloride, 2,5 g Di-sodium phosphate and 2 g dextrose, at 37ºC
for 24h. After 24h, all the samples were inoculated in TSA (Trypticase Soy Agar, Scharlau,
Barcelone, Spain) medium which contained 15 g of casein peptone, 5 g of soy peptone, 5 g of
sodium chloride and 15 g of agar per litre. The pH used was 7,3. The bacteria were kept at 37ºC
for 24h.
2.2.3 Lactic acid bacteria (fermentative bacteria)
Lactobacillus plantarum S4B6, Lactobacillus sakei L3M3, Lactobacillus sakei ref: ATCC 15323
were revived in MRS (Man, Rogosa, Sharpe, Scharlau, Barcelone, Spain) broth at 30ºC for 24h.
After 24h these lactobacillus’s samples were cultivated in MRS (Man, Rogosa, Sharpe,
23
Scharlau, Barcelone, Spain) agar which contained 10 g of peptone proteose, 8 g of meat
extract, 4 g of yeast extract, 20 g of D (+)-Glucose, 5 g of sodium acetate, 2 g of triammonium
citrate, 0,20 g of magnesium sulfate, 0,05 g manganese sulfate, 2 g of dipotassium phosphate,
1 g of polysorbate 80 and 14 g of agar. The pH used was 6,2.
2.3 Bioautography tests
To confirm which yeast isolates showed inhibitory properties, in vitro competition assays were
performed. The challengend indicators were some pathogenic bacteria and the fermentative
bacteria previously described in point 2.2 and 2.3. The microorganisms tested were all the
seventy-two yeast samples previously described in point 2.1.
For each isolate, 2 ml suspensions, with a density of 3 in McFarland scale, of the challengend
indicators were performed. Then the pathogenic bacteria suspensions and the Staphylococcus
were incorporated in 40 ml of TSA medium with 1% of glucose at 48 ºC, while the Lactobacillus
suspensions were, each one, incorporated in 40 ml of MRS agar medium at 48 ºC. The
incorporated mediums were effused in square petri dishes. The petri dishes were divided in
squares and then a different yeast was inoculated in each grid by puncture. The yeasts had
some difficulty to grow in MRS agar, so the in vitro competition tests between yeasts and
Lactobacillus were made using a second layer of Sabouraud Dextrose agar above incorporated
MRS agar, being the samples inoculated in the second layer (figure 5). This technique allowed
the growth of yeasts and also enabled the growth of Lactobacillus, by creating an anaerobic
environment. All plates were incubated at 25 ºC for 48 h.
Figure 5: Procedure used to make the in vitro competition tests (original).
24
Figure 6: Procedure used to filter the yeast's suspensions (left figure). “Small wells” in incorporated medium (right figure) (original).
2.4 Evaluation of intracellular or extracellular compounds with
inhibitory effect
In order to determine if the inhibitory compounds were excreted by the yeast samples to the
exterior, suspensions of yeast isolates, corresponding to 3 in Mcfarland scale, were grown in
TSB (Trypticase Soy Broth, Scharlau, Barcelone, Spain) medium which contained 15 g of
casein peptone, 5 g of soy peptone, 5 g of sodium chloride per litre, at 25ºC for 72h. After 72h,
the resulting suspensions went through a 0.45 µm filter (NORMAX). The different filtrated
compounds obtained were inoculated in small “wells” that were made in TSA (Trypticase Soy
Agar, Scharlau) medium incorporated with pathogenic bacteria (S. aureus, Listeria, Salmonella)
as described in point 2.4” bioautography tests” (figure 6). The incubation was performed in a
humid chamber at 25 ºC for 48 h.
2.5 Identification of indigenous yeasts
The yeast isolates were grouped based on their micro- and macro-morphological
characteristics, following the procedures described by Walt and Yarrow (1998) as the color of
the colony, the visualization of chlamydospores or not, the presence or absence of filaments
and the position and the number of gemmules [50]. The biochemical minimized test was chosen
based on the results obtained from these morphological tests. For each group, a number of
isolates that had inhibitory properties were identified using API ID 32 C kits (following the
instructions of the manufacturer, BioMerieux, Marcy-L´Etolie, France).
25
2.5.1 Yeasts characterization
Yeasts characterization was performed through proteolytic activity expression; lipolytic activity;
glucolytic activity; the presence of catalase and the presence of nitrate reductase. Proteolytic
activity was evaluated inoculating pure cultures of yeasts into tubes that contained 10 ml of a
culture medium composed by 6 g of TGA (Agar Plate Count, Merck KGaA, Darmstadt,
Germany), 25 g of gelatin (Gelatin Pancreatic Peptone, Scharlau, Barcelone, Spain), 12,5 g of
skim milk 10% (Skim Milk Powder, Oxoid, Basingstoke, Hampshire, England) per 250 ml with a
pH of 7. After 48 h of incubation at 25 ºC, the gelatin liquefaction was assessed. Lipolytic activity
was evaluated by measured the clear halos in a culture medium composed by 20 g of tributyrin
(Oxoid Unipath) per litre and 10 ml of glycerol-phosphate-calcium (Sigma, Aldrich). This last
substract allows the formation of a precipitate if yeast has lipolytic activity. After 48h of
incubation at 25 ºC the results were measured. The production of gas was tested using Duhran
tubes and Sabouraud Dextrose Broth medium in anarobic conditions. The yeast samples were
inoculated in Sabouraud Dextrose Broth on test tubes. After yeast‘s inoculation the Durhan
tubes were placed. The results were measured after 48 h of incubation at 25 ºC. The presence
of catalase was tested using a hydrogen peroxide solution 30% (w/w). Nitrate - reductasic
activity was evaluated inoculating pure cultures of yeast into tubes that contained per litre litre 5
g of peptone, 3 g of meat extract, 1 g of yeast extract, 5 g of glucose and 1 g of KNO3 until
achieved 1,7 of optic density at 600 nm. After a 48h of incubation at 25ºC two drops of NIT 1
(0,8 g of sulfanilic acid in 100 ml of acetic acid) and NIT 2 (0,6 g of N-N-dimetil-1-naphthylamina
in 100 ml of acetic acid 5N) were placed in each test tube. If solution turns red the yeast has
nitrate-reductasic activity (Van der Walt, J.P., and Yarrow, D., 1984) [10, 29, 34].
2.6 Behaviour of selected yeast in a meat product matrix
The yeast Rhodotorula glutinis was chosen for further testing. R. glutinis was chosen based on
its nitrato redutasic activity, strong inhibition zones and for it distinctive color that can be easly
detected in Sabouraud dextrose agar. The aim of this test was to evaluate the behavior of
certain microorganisms in pork meat with the adition of R. glutinis. To do this, 90 stomacher
bags were filled with 25 g of pork meat each. The meat was cut in small pieces to increase the
surface contact and the challenged microorganisms were inoculated in the meat matrix. Six
conditions were tested: 1- control, 2- control + Rhodotorula glutinis, 3- control + Salmonella, 4-
control + Rhodotorula glutinis + Salmonella, 5 - control + S. aureus, 6- control + Rhodotorula
glutinis + S. aureus. The evolution of each condition was measured five times in the course of
96 h: T0, T1 (24H), T2 (48H), T3 (72H), T4 (96H). For two days the bags stayed at 7 ºC; the last
two days the bags stayed at 20 ºC. 7 ºC represent the temperature stablish for the raw meat
until further utilization; 20 ºC represent the fermentation temperature (figure 7).
26
For each condition and time, enumeration were performed for: Salmonella, yeasts, S. aureus,
total aerobic microorganisms at 30 ºC, lactic acid bacteria (LAB), Staphylococcus. All counts
were made according with the methods proposed by ISO (International Organization for
Standardization). One ml of Rhodotorula glutinis suspension with 105 cells was inoculated in
bags 2, 4 and 6; One ml of Salmonella suspension with 104 cells was inoculated in bags 3 and
4; finally, One ml of S. aureus suspension having 104 cells was inoculated in bags 5 and 6. This
procedure was repeated 3 times for statistical relevance.
Figure 7:Procedure used to inoculate microorganisms in meat matrix (original).
2.7 Statistical analysis
For data analysis was used the Microsoft Excel 2011 program and Statistical Package for Social
Sciences (SPSS) software, version 22. An analysis of variance (ANOVA) and Tukey test using
the General Linear Model (GLM) was performed for all the microorganisms counts. All statistical
tests were performed for a significance level (P < 0.05).
27
Chapter 3
Results
28
3.1 Bioautography tests
Seven yeast isolates (n=7), corresponding to 10% of the yeast isolates, inhibit simultaneously
Listeria monocytogenes and Salmonella Enteritidis; Forth yeast samples (n=4), around 6% of
the isolates, inhibit simultaneously Listeria monocytogenes and Staphylococcus aureus; Seven
yeast samples (n=7), representing 10% of the isolates, inhibit simultaneously Salmonella
Enteritidis and Staphylococcus aureus; only one yeast sample (n=1) inhibit ,simultaneously,
Salmonella Enteritidis, Staphylococcus aureus and Listeria monocytogenes. In general,
Stapylococcus aureus was the pathogenic bacteria more sensitive to the yeast isolates with
44.4% of these causing inhibition, followed by Listeria monocytogenes with 37.5% of yeasts
causing inhibition and Salmonella Enteritidis with 26.4% of yeasts with inhibition (figure 8).
3.2 Extracellular inhibitory effects
For further testing eleven yeast isolates (n=11) were chosen to evaluate the extracellular
inhibitory effect. These eleven yeast isolates were chosen based on their capacity to inhibit
more than one pathogenic bacteria. Among these eleven yeast isolates, only eight (n=8) had
inhibitory effect through the filtrated compounds. Furthermore, these filtrated compounds only
inhibit Staphylococcus aureus.
N= 72 yeasts
37.5
26.4
44.4
Figure 8: Percentage of yeast isolates to cause inhibition in pathogenic bacteria.
29
The figure (9a) shows two distinct zones around the yeast colony, one of the halos shows a
clear intensive growth of bacteria around the yeast and the second halo shows a dark ring
corresponding to the absense of bacteria; the yeasts synergetic and antagonistic effect were
both present. In figure (9b) there is a clearly visible dark halo around the “well” that was
inoculated with the filtrated compounds; in this case only the antagonistic effect occurs.
3.3 Identified yeasts
The results of biochemical miniatured test (API 32C) identified five yeasts (n=5) (table 4) having
extracellular inhibitory effects:
Candida famata (SG3C(1));
Rhodotorula glutinis (C1C(2));
Candida humicola (CV1(2));
Schwanniomyces polymorphus (C1C(3));
Candida zeylanoides (4L1.1).
Table 4 - Gallery profiles API 32 C for each yeast isolate. C.H - Candida humicola, S.P - Schawanniomyces polymorphus, C.F - Candida famata, R.G - Rhodotorula glutinis, C.Z - Candida zeylanoides. (*) Esculine test that was only used when the yeast identification was doubtfull. In this case, only Candida zeylanoides had doubfull results so the esculine test was made.
(a) (b)
Figure 9: Yeast colony and bacteria present in direct competition (a) and filtrated compounds tested for their inhibitory properties (b).
30
3.4 Bioautography tests over LAB and bacteria from the curing
process
Only two (n=2) out of the five yeasts revealed the hability to produce inhibitory effects over all
LAB tested and bacteria from the curing process except for Lactobacillus plantarum (figure 10).
This two yeasts are: Rhodotorula glutinis and Candida humicola.
Figure 10: In vitro competition tests between yeast isolates and fermentative bacteria.
3.5 Enzimatic activity
Table 5 - Metabolic behaviour of five selected yeasts.
31
Figure 11: Growth of Rhodotorula glutinis during 96 h at 7 ºC and 20 ºC in meat matrix model.
Figure 12: Growth of Staphylococcus aureus during 96 h at 7 ºC and 20 ºC in meat matrix model.
3.6 Meat fermentation model
The initial level of Rhodotorulla glutinis at 7 ºC was similar for all three different conditions
analysed (R. glutinis, Salmonella + R. glutinis and S. aureus + R. glutinis). After 48 h, at 20 ºC,
the yeast had an exponential growth in all conditions, reaching 9 log10 CFU/g. Rhodotorula
glutinis increased 5 log10 in 48 h at 20 ºC (figure 11). Although R. glutinis was not inoculated in
the control experiment, other yeasts that naturally occur in the meat were present and detected
in SDA.
32
Figure 13: Growth of Staphylococcus during 96 h at 7 ºC and 20 ºC in meat matrix model.
The initial counts of Staphylococcus aureus at day 0 were 3 log10 CFU/g in the two conditions
analysed. After 48 h at 7 ºC, the experiment that was inoculated only with S. aureus showed a
slight decrease. At 20 ºC, one of the conditions grew up to 4-5 log10 CFU/g and another to 5-6
log10 CFU/g. The condition that was initially inoculated with S. aureus and R. glutinis showed a
slightly lower growth rate (figure 12).
The initial counts of Staphylococcus at time 0 (T0) at control and in inoculated meat with R.
glutinis were 2 log10 CFU/g. The experiment that was inoculated with S. aureus and the
experiment that was inoculated with S. aureus and R. glutinis showed initial levels of
Staphylococcus between 3-4 log10 CFU/g. From 48 h at 20 ºC until 96 h the Staphylococcus had
a steeper exponential growth in the control experiment and the experiment inoculated with R.
glutinis than the experiment inoculated with S. aureus and the experiment inoculated with S.
aureus and R. glutinis. All the experiments grew up to 5-6 log10 CFU/g until 96 h (figure 13).
33
Figure 14: Growth of Salmonella Enteritidis during 96 h at 7 ºC and 20 ºC in meat matrix model.
Figure 15: Growth of total aerobic microorganisms (TA) during 96 h at 7 ºC and 20 ºC in meat matrix model.
The growth of Salmonella in both testing conditions were very similar. The initial counts at day 0
were 3 log10 CFU/g in both cases. The counts maintained the same level until 48 h at 7 ºC.
From 48 h at 20 ºC the counts of Salmonella grew to 7-8 log10 CFU/g until 96 h (figure 14).
The initial counts of total aerobic microorganisms at 30 ºC at day 0 were 4-5 log10 CFU/g in all
conditions. The counts slightly increase until 48 h at 7 ºC. When the temperature changed to 20
ºC there was an exponential growth of TA in all the conditions. The final counts of total
microorganisms at 30 ºC at 96 h were 9-10 log10 CFU/g (figure 15). The inhibitory effect of the
R. glutinis were not observed.
34
Figure 16: Growth of Lactic acid bacteria (LAB) during 96 h at 7 ºC and 20 ºC in meat matrix model.
There was a slight difference at the initial counts of LAB in day 0 in the different conditions. The
control, the assay inoculated with Salmonella and R. glutinis, the assay inoculated only with R.
glutinis and the experiment inoculated with S. aureus had the initial counts of LAB between 2-3
log10 CFU/g. The initial counts of LAB in the experiment inoculated with Salmonella and the
experiment inoculated with S. aureus and R. glutinis were between 3-4 log10 CFU/g. When the
temperature changed to 20 ºC after 48 h there was an exponential growth in all the conditions
and the final counts after 96 h of incubation were 7-8 log10 CFU/g for all the six conditions tested
(figure 16).
35
Chapter 4
Discussion
36
4.1 Discussion
Candida famata, Rhodotorula glutinis, Candida humicola, Schwanniomyces polymorphus and
Candida zeylanoides are species frequently found in dry-fermented/ dry-cured fermented
sausages. Candida famata is the anamorphous or imperfect form of Debaryomyces hansenii.
Debaryomyces hansenii is well known in the biotechnological world due to its ability to
hydrolyze pork muscle sarcoplasmatic proteins, thereby influencing the aroma formation and
sensory quality of dry-fermented sausages [32]. This yeast is already used as a starter culture
in many procedures.
These yeasts, with exception of Candida zeylanoides, were classified as non-pathogenic by
EFSA in 2012 [37].
The strain Candida famata and the other yeasts isolates tested in this work did not show
proteolytic activity using gelatin as protein source, which does not mean that it does not have
proteolytic activity using less complex proteins. There is a large range of enzymes that
catabolize proteins:
Table 6 - Brief description of main substrates and products for most important muscle proteolytic enzymes [31].
The isolates C. famata, C. zeylanoides and S. polymorphus showed lipolytic activity. This
capacity is a way to produce lipid derived and fruity aroma compounds with high aroma impact
[36, 42]. Some papers describe Rhodotorula spp. with lipolytic activity but the strain isolated in
this work did not showed any activity. Rhodotorula has been described as being tolerant to high
concentrations of salt like Debaryomyces, Candida and Cryptococcus [34]. Furthemore,
Rhodotorula glutinis has nitrate-reductase activity that is an advantage in terms of competition
in the sausages, because of that it can become dominant in the final product.
None of the isolates showed production of gas even though C. famata has been described with
capacity to ferment glucose. Sugars are the most common substrate of fermentation, and
typical examples of fermentation products are ethanol, lactic acid, carbon dioxide, and hydrogen
gas (H2), but homolactic fermentation (producing only lactic acid) is the simplest type of
fermentation. The pyruvate from glycolysis undergoes a simple redox reaction, forming lactic
acid. It is a unique pathway, being one of the only respiration processes to not produce gas as a
37
byproduct. Overall, one molecule of glucose (or any six-carbon sugar) is converted to two
molecules of lactic acid: C6H12O6 → 2 CH3CHOHCOOH [43]. Production of carbon dioxide gas
during fermentation is highly undesirable. The result of gas production is gas bubbles in the
product or even breakage of casings if gas production is extreme [43]. R. glutinis and C.
zeylanoides have been described without glucose fermentation hability [55]. R. glutinis is strictly
aerobic so the formation of gas is inevitable. Although the formation of CO2 by aerobic
respiration this not affect the product because R. glutinis will remain in the sausage surfaces
due to its O2 uptake. In fact the levels of yeasts in the inner part of the sausage it is always
lower than the surface [56].
S. polymorphus, R. glutinis and Candida zeylanoides isolates did not show resistence to lactic
acid in biochemical miniature test (API 32C). Similar results were showed by Pereira-Dias, S.,
Potes, M.E., Marinho, A., Malfeito-Ferreira, M., Loureiro, V., 2000 [57]. Lactic acid is an
important product released by LAB during fermentation of sugars, so it is important to choose
yeast strains with potential to resist and to use lactic acid as a carbon source. Yeast strains that
metabolized lactic acid, contribute to a less tangy product. Genomic analyses are an
indispensable tool to find out genes that give explanations to the remarkable tolerance of some
yeasts to the extremely high salt, sugars and organic acid contents. For example, during wine
and beer fermentations, S. cerevisiae exhibits sequential expression and regulation of many
genes associated with carbon, nitrogen and sulfur metabolism, as well as other genes required
to tolerate stresses such as high sugar concentrations, low pH, ethanol and nutrient deficiency
[58]. The trophic relationships that occur between the prevailing sourdough LAB (Lb.
sanfranciscensis and Lb. plantarum) and yeast (S. cerevisiae) have been studied in co-culture
model systems. Bacterial growth and production of lactic and acetic acids decreased due to the
faster consumption of maltose and, especially, of glucose by S. cerevisiae when associated with
Lb. sanfranciscensis in a synthetic medium containing these carbon sources [59].
It is important to refer that in Portugal it is not traditional to add sugars to the sausages. In some
cases wine is added. For this reason, the Portuguese sausages are considered slightly
fermented with the only source of carbon being the glycogen of the pork muscle and the sugars
from the spices. The yeast isolates came from Alentejo sausages, so it is natural that these
isolates are not used to high concentrations of lactic acid.
All the isolates were catalase positive, this means that all the yeasts had antioxidative enzymes
that protect the product against the prooxidative effects by catalysing the dismutation of
hydrogen peroxide to less harmful hydroxides [31]. Oxidation of lipids and proteins is the main
cause for quality deterioration during processing and storage of meat products, causing
changes in nutritional value and sensory traits [60].
R. glutinis and C. humicola were not resistant to cicloheximide (Actdiona) according to the
results of API 32 C. Cycloheximide is an inhibitor of protein biosynthesis in eukaryotic
38
organisms. This means that there is no risk of horizontal transference of resistance genes from
any of these two microorganisms.
These five isolates of yeast showed in vitro inhibitory activity against Staphylococcus aureus ref.
25923 ATCC, Salmonella Enteritidis CECT 4300 and Listeria monocytogenes CECT 937. This
inhibition occurs at in vitro competition conditions, when the yeasts were in direct contact with
the pathogenic bacteria. In fact, the results showed a high growth rate of the bacteria around
the yeast isolates and then the inhibition occurs on a secondary halo around the yeasts. This
synergy can be explained by the death and autolysis of yeast cells that releases vitamins and
other nutrients that stimulate the growth of bacteria [58]. When the broth of yeast cultures were
filtered and their extracellular compounds were used to performed challenges, S. aureus was
the only bacteria inhibited. This means that probably these five yeasts inhibit Salmonella and
Listeria only in direct competition for nutrients or the inhibitory compounds are so volatile that
evaporate after filtration. S. aureus was probably inhibited by an extracellular protease that
remained in the filtrated compounds. The selection of the metabolite of interest relased by
yeasts could be a good solution to avoid the growth stimulation of bacteria by the yeast cell’s
death.
Rhodotorula glutinis is a natural red-pigmented yeast and its origin is related with air
contamination (Tudor and Board, 1993). This yeast is well adapted to the micro -environment
established in the dry-fermented/ dry-cured fermented sausages (assimilation of nitrate and
high concentration of salt) and showed strong inhibitory properties to pathogenic bacteria. For
this reason, this yeast was chosen for further testing in meat model challenge. Rhodotorula
species are strict aerobic yeasts with peculiarly metabolic characteristics such as the capacity to
produce glycogen during the exponential growth phase and also, great amounts of lipids and
carotenoid pigments during the stationary growth phase. R. glutinis are capable of synthesizing
β-carotene, and two other carotenoids-torularhodin and torulene. Torularhodin has strong
antimicrobial properties, and it may become a new natural antibiotic. These examples describe
the prospects for the use of carotenoids synthesized by the R. glutinis; however, it is necessary
to perform additional nutritional and toxicological tests that will allow for the introduction of
torulene and torularhodin on the commercial market [61]. Carotenoids represent a group of
valuable molecules for the pharmaceuticals, medicine, cosmetics, food and feed industries, not
only because they can act as vitamin A precursors, but also for their coloring, antioxidant, and
possible tumor-inhibiting activity, also, enhancement of the immune response leading to
protection against bacterial and fungal infections [62]. It was determined that the biomass of
these yeasts can be source of lipases as: α-l-arabinofuranosidase, invertase, pectinases, tannin
acyl hydrolase and phenylalanine ammonia lyase [61]. Basidiomycetous yeasts like R. glutinis
was also found to produce rhodotorulic acid that is the smallest of the 2,5-diketopiperazine
family of hydroxamate siderophores which is a high-affinity chelating agents for ferric iron. This
capacity of producing rhodotorulic acid improves the biological control against fungus such as
39
Penicillium expansum and iprodione-resistant Botrytis cinerea that are responsible of rot in
several foods [63, 64, 65, 66, 67, 68].
R. glutinis was added to the pork meat with S. aureus and with Salmonella Enteritidis in order to
verify if this yeast isolate actually reduces the levels of these two pathogenic bacteria over time.
Rhodotorula glutinis increased 5 log10 in 48 h at 20 ºC in all the conditions, but in fact, a
reduction of Salmonella Enteretidis was not verified. S. aureus suffered a slight reduction but it
was not significant. There was not any interference in the levels of total aerobic microorganisms
at 30 ºC (TA) neither in the number of LAB colonies with the introduction of R. glutinis. It would
be promising if the counts of TA had decreased over time with the introduction of R. glutinis.
The decreasing numbers of TA would mean that hygienicly the product would be safer to the
consumer. At the same time, it was a good result that the counts of LAB showed no difference
with or without R. glutinis. Lactic acid bacteria are essential for the success of the fermentation
process of the sausages, so it is important that yeasts and LAB were able to exist together and
take advantage from each other. For example, the efficient utilization of specific amino acids
and/or peptides generated by yeasts provides a competitive advantage and contributes to the
stability of LAB in sourdough [59]. Despite these results, the extracellular compounds that were
able to inhibit S. aureus, in the initial competition tests, deserve a deeper and longer
investigation. If these compounds were identified, purified and their concentrations optimized its
practical and technological application would become more robust.
To use R. glutinis as a starter culture in dry fermented/dry-cured fermented sausages this yeast
need to be dominant in the final product. To be dominant in the final product the yeast has to be
competitive and must not be inhibited by the spices and the additives present in the sausages
and has to be resistant to low aw and low pH. The in vitro competition chanllanges in meat
model should be repeated but with the real sausage paste: using spices and additives of
sausages. Some of these ingridients have antimicrobial activity so the results will be necessarily
different. The addition of salt will, also, reduce even more the aw so the enumeration of R.
glutinis during the ripening period of sausage would be relevant.
40
Table 7 - Limiting aw for the growth of microorganisms (adapted from FAO, 2007) [46].
If the drying process used for dry-sausages succeeds the aw will reach values under 0.90. In
such low values of aw, Salmonella spp. and Listeria spp., will struggle to survive as showed in
the table 7. The only microorganisms that are able to survive in this stressfull conditions are the
S. aureus, yeasts and moulds. Even if the Good Hygienic Practices (GHP) and the Hazard
Analysis and Critical Control Point (HACCP) scheme are correct and established in this type of
product, S. aureus remains a problem. Yeasts and moulds are the only ones capable of dealing
with this harmfull bacteria. The need for immediate action within HACCP systems excludes
microbiological control (of raw materials, semi-fabricated products, tools, equipment, and
premises) as a directly applicable control measure. Microbiological control techniques take
hours or days to obtain the results, which does not allow corrective interventions during the
usually short manufacturing period. So, if a contamination occurs during the manufacturing
period we would only know, probably, when the products would be in the market place.
Hygienically acceptable microbiological test results are, only, an indicator of the proper
functioning of the meat plant’s HACCP scheme. The use of yeast isolates as a tool of biocontrol
will probably improve the food safety with less costs to the producer [46].
5. Conclusions
There are not many studies showing the potential of yeasts and their metabolites in biocontrol of
sausages manufacturing. It is a fact that these microorganisms exist, grow and have a role
during the ripening process of this product in terms of flavour and antioxidant effects. In this
present work, isolated yeasts revealed that antimicrobial activity to pathogenic bacteria present
in meat products exist. The value of yeasts as starter culture increased and deserves a better
and longer investigation. It is crucial to understand the complex relationship between yeasts and
41
bacteria, their sinergisms and antogonisms, in order to choose the best way to take advantage
of yeast strains. Very few foodborne illnesses have been attributed to yeasts and these
microorganisms are well known in the making of wine, beer and bread having a good
acceptance in the community. Future prospects can be associated with the extraction of the
metabolites of interest as was done with bacteriocins that came from LAB. Clearly the five yeast
isolates studied in this work produced factors that can be extracted and developed. Molecular
charactherization is also an indispensable tool to identify and comprehend the genes involved in
several important characteristics of interest. Miniature API assays were always doubtful but they
are very usefull in some cases. The fermentation/ cure/ smoking process differ highly among
different countries and traditions. The time and temperature of ripening can be very distinctive
and in some cases the fermentation process is very weak or simply does not exist. It is
important to understand that a starter culture can have a good result in a Portuguese product
and not in an Italian product; the same thing can occur between a traditional or an industrial
product. Despite these differences, the low water activity (aw) is common in all the sausage
products. The yeasts and moulds can reach low aw values and could make the final product a
better and safer product decreasing the levels of Staphylococcus aureus that can, eventually,
come from the manipulation of the manufacturer. In this work Rhodotorula glutinis strain does
not decresed the levels of Salmonella Enteritidis, total aerobic microorganisms at 30 ºC and
Lactic acid bacteria in meat matrix model, S. aureus remains with some sensitivity to R. glutinis
but not significantly to be a good result. The other four yeast isolates identified in this work,
Candida famata, Candida humicola, Scwanniomyces polymorphus and Candida zeylanoides
were not tested in meat matrix model. It would be interesting to test individualy each yeast
isolate in this work in the meat matrix model and then test all these strains together in the same
conditions. This study shows that, although the yeast strains revelead antimicrobial capacity “in
vitro”, it does not mean that in food they will behave equally. The number of interactions are so
many that unpredictable results will always occur. More studies are still required to achieve the
ultimate goal. Using less additives and more natural bioprotective cultures in food may
contribute to a better and more acceptable product to the consumer. Although fermented
sausages can be produced without the use of inoculated strains, the use of starter cultures for
sausage production is increasing so as to guarantee safety and standardize product properties,
including consistent flavor and color, and to shorten the ripening period. Nevertheless, proper
hygienic and safety strains, properly identified at species and strain level, which preserve typical
characteristics, are essential in order to assure the hygienic and safety quality together with the
regional tradition for fermented sausages. A positive implantation of the starter culture used has
to be controlled and guaranteed [69]. Research mostly focuses on the pathogenic potential of
microorganisms, while neglecting their positive role. Recent scientific advances have revealed
the preponderant role of our own microbiota, our “other genome”, from the skin, gut and other
mucosa. Though this remains undoubtedly promising, one should not forget that man has not
yet finished characterizing traditional fermented foods consumed for centuries, with often
numerous isolates belonging to species with undefined roles [70].
42
References
[1] Newell, D., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Opsteegh,
M., Langelaar, M., Threfall, J., Scheutz, F., der Giessen, J. and Kruse, H. (2010). Food-borne
diseases — The challenges of 20 years ago still persist while new ones continue to emerge.
International Journal of Food Microbiology, pp.139; S3-S15.
[2] Fahrion, A., Jamir, L., Richa, K., Begum, S., Rutsa, V., Ao, S., Padmakumar, V., Deka, R.
and Grace, D. (2013). Food-Safety Hazards in the Pork Chain in Nagaland, North East India:
Implications for Human Health. International Journal of Environmental Research and Public
Health, 11(1), 403-417.
[3] Fung, D. Y. C., (2010). Chapter 28: Microbial Hazards in Foods: Food - Borne Infections and
Intoxications. In Handbook of Meat Processing, edited by Toldra, F., Ames, F., Wiley-Blackwell,
USA, pp. 566, 481-500.
[4] Skandamis, P.N., Nychas, G-J.E., Sofos, J.N, (2010). Chapter 3: Meat Decontamination. In
Handbook of Meat Processing, edited by Toldrá, F. Wiley- Blackwell, USA, pp. 566, 43-85
[5] Giuffra, E., Kijas, J.M., Amarger, V., Carlborg, O., Jeon, J.T., Andersson, L., (2000). "The
origin of the domestic pig: independent domestication and subsequent introgression". Genetics
154 (4), 1785–1791
[6] DMello, S., (2015). Health Risks of Eating Pork. Network For Health, India
[7] Syne, S., Ramsubhag, A., Adesiyun, A., (2013). Microbiological hazard analysis of ready-to-
eat meats processed at a food plant in Trinidad, West Indies. Infection Ecology & Epidemiology,
volume 3, pp.12, India
[8] EFSA (European Food Safety Authority) and ECDC (European Centre for Disease
Prevention and Control), (2015). The European Union summary report on trends and sources of
zoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSA Journal, 13(1), pp.165
[9] Pierre, C., (2015). Chapter 45: Disease Outbreaks. In Handbook of fermented Meat and
Poultry, edited by Toldrá, F., Hui, Y.H., Astiasarán, I., Sebranek, J.G., Talon R, Wiley-Blackwell,
UK, pp.555, 477-480
[10] Rovira, J., Puszczewicz, D., (2015). Chapter 47: Processing Plant Sanitation. In Handbook
of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G.,
Talon R, Wiley-Blackwell, UK, pp.555, 491-501
[11] Zorpas, A., Tzia, C., Voukali, I., Panayiotou, A., (2010). Quality and Safety Assurance
According to ISO 22000: 2005 in a Meat Delicatessen Industry of Cyprus. The Open Food
Science Journal, 4(1), 30-42.
43
[12] Fraqueza, M.J., Barreto, A.S., Ribeiro, A.M., (2015). Chapter 49: HACCP. In Handbook of
fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G., Talon
R, Wiley-Blackwell, UK, pp.555, 513-534
[13] Bolton, E., Little, C., Aird, H., Greenwood, M., McLauchlin, J., Meldrum, R., Surman-Lee,
S., Tebbutt, G., Grant, K., (2009). Section 2: Pathogens. In Guidelines for Assessing the
Microbiological Safety of Ready-to-Eat Foods Placed on the Market, edited by Healthy
Protection Agency, UK, pp.34
[14] Zeuthen P., (2015). Chapter 1: A historical perspective of meat fermentation. In Handbook
of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G.,
Talon R, Wiley-Blackwell, UK, pp.555, 3-8
[15] Ockerman, H.W., Basu, L., (2015). Chapter 2: Production and Consumption of Fermented
Meat Products. In Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H.,
Astiasarán, I., Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 9-15
[16] Fries, R., (2015). Chapter 26: International Standards: Europe. In Handbook of Fermented
Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G., Talon R, Wiley-
Blackwell, UK, pp.555, 273-284
[17] Andrés, A., Barat, J.M., Grau, R., Fito, P., (2015). Chapter 5: Principles of Drying and
Smoking. In Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H.,
Astiasarán, I., Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 37-48
[18] Yang, S., Lin, C., Sung, C. and Fang, J. (2014). Antibacterial activities of bacteriocins:
application in foods and pharmaceuticals. Front. Microbiol., 5 (241) 1-10.
[19] Vignolo, G., Fadda, S., (2015). Chapter 14: Starter Cultures: Bioprotective Cultures. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I.,
Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 147-157
[20] Rizzello, C. G., Filannino, P., Di Cagno, R., Calasso, M., & Gobbetti, M. (2014). Quorum-
sensing regulation of constitutive plantaricin by Lactobacillus plantarum strains under a model
system for vegetables and fruits. Applied and environmental microbiology, 80(2), 777-787.
[21] Vignolo, G., Fadda, S., (2015). Chapter 14: Starter Cultures: Bioprotective Cultures. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I.,
Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 147-157
[22] Abeyrathne, N.S., (2015). Use of lysozyme from chicken egg white as a nitrite replacer in
an Italian-type chicken sausage. Functional Foods in Health and Disease, 5(9), 320-330
[23] Hughey V.L., Wilger P.A., Johnson E.A., (1989). Antibacterial activity of hen egg white
lysozyme against Listeria monocytogenes Scott A in foods. Appl Environ Microbiol; 55(3), 631-8
44
[24] Kanninen, E.P., Puolanne, E., (2015). Chapter 4: Principles of Meat Fermentation. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I.,
Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 31-36
[25] Skandamis, P., Nychas, G.J.E., (2015). Chapter 42: Pathogens: Risks and Control. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I.,
Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 427-454
[26] Ockerman, H.W., Basu, L., (2015). Chapter 2: Production and Consumption of Fermented
Meat Products. In Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H.,
Astiasarán, I., Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 9-15
[27] Cocconcelli, P.S., Fontana, C., (2010). Chapter 10: Starter Cultures for Meat Fermentation.
In Handbook of meat processing, edited by Toldrá, F., Wiley-Blackwell, USA, pp.566, 199-218
[28] Flores, M., Corral, S., Cano-García, L., Salvador, A., Belloch, C., (2015). Yeast strains as
potential aroma enhancers in dry fermented sausages. International Journal of Food
Microbiology, 212, 16-24.
[29] Hatoum, R., Labrie, S., Fliss, I., (2012). Antimicrobial and Probiotic Properties of Yeasts:
From Fundamental to Novel Applications. Front. Microbio., 3, 421
[30] Loureiro, V., Malfeito-Ferreira, M., Carreira, A., (2004). Chapter 12: Detecting spoilage
yeasts. In Understanding and measuring the shelf-life of food, edited by Steele, R., Boca Raton:
CRC Press, USA, pp.407, 273-275
[31] Cano-García, L., Flores, M., Belloch, C., (2013). Molecular characterization and aromatic
potential of Debaryomyces hansenii strains isolated from naturally fermented sausages. Food
Research International, 52(1), 42-49.
[32] Selgas, M.D., Garcia, M.L., (2015). Chapter 15: Starter Cultures: Yeasts. In Handbook of
Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G.,
Talon R, Wiley-Blackwell, UK, pp.555, 159-169
[33] Ockerman, H.W., Basu, L., (2015). Chapter 2: Production and Consumption of Fermented
Meat Products. In Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H.,
Astiasarán, I., Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 9-15
[34] Corral, S., Salvador, A., Belloch, C., Flores, M., (2015). Improvement the aroma of reduced
fat and salt fermented sausages by Debaromyces hansenii inoculation. Food Control, 47, 526-
535.
[35] Purriños, L., García Fontán, M., Carballo, J., Lorenzo, J., (2013). Study of the counts,
species and characteristics of the yeast population during the manufacture of dry-cured “lacón”.
Effect of salt level. Food Microbiology, 34(1), 12-18.
45
[36] Encinas, J., López-Dıaz, T., Garcıa-López, M., Otero, A., Moreno, B., (2000). Yeast
populations on Spanish fermented sausages. Meat Science, 54(3), 203-208.
[37] Simoncini, N., Pinna, A., Toscani, T., Virgili, R., (2015). Effect of added autochthonous
yeasts on the volatile compounds of dry-cured hams. International Journal of Food
Microbiology, 212, 25-33.
[38] Bussey, H., (1991). K1 killer toxin, a pore-forming protein from yeast. Molecular
Microbiology 5 (10), 2339–43
[39] Buyuksirit, T., Kuleasan, H., (2014). Antimicrobial Agents Produced by Yeasts. International
Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering 8(10)
[40] Ozhovan I.M., Arzumanian V.G., Basnak’ian I.A., (2002). Killer toxins of clinically important
yeasts. Zh Mikrobiol Epidemiol Immunobiol.(4), 79-83
[41] Cocconcelli, P.S., (2015). Chapter 13: Starter Cultures: Bacteria. In Handbook of
Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G.,
Talon R, Wiley-Blackwell, UK, pp.555, 137-145
[42] Toldrá, F., (2015). Chapter 6: Biochemistry of Meat and Fat. In Handbook of Fermented
Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G., Talon R, Wiley-
Blackwell, UK, pp.555, 51-58
[43] Ogrydziak, D. (1993). Yeast Extracellular Proteases. Critical Reviews in Biotechnology,
13(1), pp.1-55.
[44] Thorpe, Sir Thomas Edward, Longmans, Green (1922). A dictionary of applied chemistry,
Vol. 3, UK
[45] Sebranek, J.G., (2005). Chapter 22: Semidry Fermented Sausages. In Handbook of Food
and Beverage Fermentation Technology, edited by Hui, Y.H., Meunier – Goddik, L., Hansen,
A.S., Josephsen, J., Nip, W.K., Stanfield, P.S., Toldrá, F., CRC Press, 134 pp.905, 385-396
[46] Heins, G., Hautz, P. (2016). Meat Processing Technology. [online] Fao.org. Available at:
http://www.fao.org/docrep/010/ai407e/ai407e00.htm [Accessed 30 May 2016].
[47] Rovira, J., Puszczewicz, D., (2015). Chapter 47: Processing Plant Sanitation. In Handbook
of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I., Sebranek, J.G.,
Talon R, Wiley-Blackwell, UK, pp.555, 491-501
[48] Caplice, E., (1999). Food fermentations: role of microorganisms in food production and
preservation. International Journal of Food Microbiology, 50(1-2), 131-149.
46
[49] Vignolo, G., Fadda, S., (2015). Chapter 14: Starter Cultures: Bioprotective Cultures. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F, Hui, Y.H., Astiasarán, I.,
Sebranek, J.G., Talon R, Wiley-Blackwell, UK, pp.555, 147-157
[50] Rodrigues, F., Ludovico, P., Leão, C., (2006). Chapter 6: Sugar Metabolism in Yeasts: an
Overview of Aerobic and Anaerobic Glucose Catabolism. In Biodiversity and ecophysiology of
yeasts, edited by Rosa, C. and Peter, G., Germany, pp.579, 101-121
[51] T. Deak., Steele, R., (2004). Chapter 5: Spoilage yeasts. In Understanding and Measuring
the Shelf-life of Food. Boca Raton: CRC Press.
[52] Tournas, V., Stack, M.E., Philip B., Mislivec, Herbert A., Koch, Bandler, R., (1998). Chapter
18: Yeasts, Molds and Mycotoxins. In FDA's Bacteriological Analytical Manual, Edition 8,
Revision A.
[53] Roostita, L.B., Fleet, G.H., Wendry, S.P., Apon, Z.M., Gemilang, L.U., (2011).
Determination of Yeasts Antimicrobial Activity in Milk and Meat Products. Advance Journal of
Food Science and Technology 3(6), 442-445.
[54] Csutak O., Vassu, T., Sarbu, I., Stoica, I., Cornea, P., (2013). Antagonistic Activity of Three
Newly Isolated Yeast Strains from the Surface of Fruits. Food Technol. Biotechnol. 51(1), 70–77
[55] Rodrigues, F., Ludovico, P. (2016). [online] Available at:
https://repositorium.sdum.uminho.pt/bitstream/1822/6231/1/Ch06.pdf [Accessed 30 May 2016].
[56] Mendonça, R., Gouvêa, D., Hungaro, H., Sodré, A., Querol-Simon, A. (2013). Dynamics of
the yeast flora in artisanal country style and industrial dry cured sausage (yeast in fermented
sausage). Food Control, 29(1), 143-148.
[57] Pereira-Dias, S., Potes, M., Marinho, A., Malfeito-Ferreira, M., Loureiro, V., (2000).
Characterisation of yeast flora isolated from an artesanal Portuguese ewes’ cheese.
International Journal of Food Microbiology, 60(1), 55-63.
[58] Fleet, G., (2007). Yeasts in foods and beverages: impact on product quality and safety.
Current Opinion in Biotechnology, 18(2), 170-175.
[59] Gobbetti, M., (1998). The sourdough microflora: Interactions of lactic acid bacteria and
yeasts. Trends in Food Science & Technology, 9(7), 267-274.
[60] Gallego, M., Mora, L., Aristoy, M., Toldrá, F., (2015). Evidence of peptide oxidation from
major myofibrillar proteins in dry-cured ham. Food Chemistry, 187, 230-235.
[61] Kot, A., Błażejak, S., Kurcz, A., Gientka, I., Kieliszek, M., (2016). Rhodotorula glutinis—
potential source of lipids, carotenoids, and enzymes for use in industries. Appl Microbiol
Biotechnol, 100(14), 6103-17
47
[62] Hernández-Almanza, A., Cesar Montanez, J., Aguilar-González, M., Martínez-Ávila, C.,
Rodríguez-Herrera, R. and Aguilar, C., (2014). Rhodotorula glutinis as source of pigments and
metabolites for food industry. Food Bioscience, 5, 64-72.
[63] Calvente, V., Benuzzi, D. and de Tosetti, M. (1999). Antagonistic action of siderophores
from Rhodotorula glutinis upon the postharvest pathogen Penicillium expansum. International
Biodeterioration & Biodegradation, 43(4), pp.167-172.
[64] Borthwick, A., Da costa, N., (2015). 2,5-Diketopiperazines in Food and Beverages: Taste
and Bioactivity. Critical Reviews in Food Science and Nutrition.
[65] Hider, R., Kong, X., (2010). ChemInform Abstract: Chemistry and Biology of Siderophores.
ChemInform, 41(30).
[66] Butler, Alison, (2007). Biological Inorganic Chemistry: Structure & Reactivity, pp.151-156.
[67] Van der Helm D., Winkelmann G., (1994). Hydroxamates and Polycarboxylates as Ion
Transport Agents (Siderophores). Winkelmann, G., Winge, D. Metal ions in fungi 11. Marcel
Dekker Inc, USA, pp. 39–98.
[68] Sansone, G., Rezza, I., Calvente, V., Benuzzi, D., Tosetti, M., (2005). Control of Botrytis
cinerea strains resistant to iprodione in apple with rhodotorulic acid and yeasts. Postharvest
Biology and Technology, 35(3), 245-251.
[69] Díez, J. and Patarata, L. (2013). Behavior of Salmonella spp., Listeria monocytogenes,
and Staphylococcus aureus in Chouriço de Vinho, a Dry Fermented Sausage Made from Wine-
Marinated Meat. j food prot, 76(4), 588-594.
[70] Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J., Gerds, M., Hammes, W., Harnett,
J., Huys, G., Laulund, S., Ouwehand, A., Powell, I., Prajapati, J., Seto, Y., Ter Schure, E., Van
Boven, A., Vankerckhoven, V., Zgoda, A., Tuijtelaars, S. and Hansen, E. (2012). Erratum to
“Food fermentations: microorganisms with technological beneficial use”. International Journal of
Food Microbiology, 156(3), 87-97.
48
This work was accepted in:
Book of Abstracts BioMicroWorld 2015, VI International Conference on Environmental,
Industrial and Applied Microbiology, Barcelona, Spain/ 28-30 October 2015.
Phenetic identification and characterization of the yeast
microbiota of Alentejo’s traditional dry-fermented sausage
Tello, A.B; Bernardo, F., M., Fernandes, M.H, Fernandes, Lauková A. 2, & Fraqueza, M.J1
Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine,
University of Lisbon, Av. da Universidade Técnica, Pólo Universitário, Alto da Ajuda, 1300-477
Lisbon, Portugal. [email protected]
Fermentation is one of the oldest and important methods of meat preservation. This
process is carried out by the acid lactic bacteria, the Micrococaceae and
Staphylococaceae family. The yeasts may have an important influence in flavour,
colour, texture and pathogenic bacteria protection (Selgas, M.D. & Garcia, M.L., 2015).
These microorganisms may delay the auto-oxidative process and the beginning of
rancidity and are capable of producing volatile esters compounds that influence the
flavour. The objective of this work was to identify and classify yeasts isolated from
Alentejo’s sausages presenting technological properties and inhibitory effect against
pathogenic microbiota. A collection of 72 yeasts isolated from different dry fermented
traditional Portuguese sausages (“linguiça”, “chouriço catalão”, “chourição”, “salsichão
grosso”, “chouriço de vinho” and “paio”) were evaluated. To achieve our goal, in vitro
competition tests (against pathogenic bacteria and the fermentative bacteria
Staphylococcus xylosus ATCC 8166, Lactobacillus sakei ATCC 15323,
Staphylococcus xylosus, Staphylococcus equorum, Lactobacillus plantarum and
Lactobacillus sakei that came from “linguiça” and “chouriço de vinho”) and enzymatic
activity tests (proteolytic, glycolytic, lipolytic and catalase presence) were made (Van
der Walt, J.P. & D.Yarrow, D., 1984). The evaluation of intracellular or extracellular
inhibitory effect was performed using a 0.45 µm filter. Biochemical minimized test by
API (Biomerieux, france) were used for identification. 44.4 % of the yeast isolates
presented inhibitory effect against Staphylococcus aureus ATCC 25923; 37.5% were
able to inhibit Listeria monocytogenes CECT 4300 and 26.4% of the yeasts were
inhibitors of Salmonella Enteritidis CECT 4300. Eleven yeast isolates that
simultaneously were able to inhibit these three pathogenic bacteria were chosen for
further testing to find out if the inhibitory compounds were intracellular or extracellular.
In fact, only 8 out of the 11 inhibit through extracellular compounds and they only
49
showed inhibitory properties on Staphylococcus aureus. These eight isolates were
identified as: Candida famata, Rhodotorula glutinis, Candida humicola,
Schwanniomyces polymorphus and Candida zeylanoides. None of these 8 isolates
have proteolytic or glucolytic activity, but Candida zeylanoides, Schwanniomyces
polymorphus and Candida famata show lipolytic activity. They all are catalase positive.
Rhodotorula glutinis and Candida humicola presented inhibitory properties over all
tested fermentative bacteria except for Lactobacillus plantarum. We conclude that
Candida famata, Schwanniomyces polymorphus and Candida zeylanoides are the best
choices to make a starter of yeasts that can be inoculated on sausages to improve the
flavour, with potential protection against pathogenic bacteria.
Keywords: yeast, sausage, safety, starters, pathogenic bacteria, fermentative
microbiota
References:
[1] Selgas, M.D., Garcia, M.L., (2015). Chapter 15: Starter cultures: Yeast. In
Handbook of Fermented Meat and Poultry, edited by Toldrá, F., Hui, Y.H., Astiasarán,
I., Sebranek, J.G., Talon, R., Wiley-Blackwell, UK, pp. 555, 159-169.
[2] Van der Walt, J.P., Yarrow, D., (1984). Chapter 2: Methods for the isolation,
maintenance, classification and identification of yeast. In The yeast a taxonomic study,
third revised and enlarged edition, edited by Kreger-van Rij, N.J.W., Elsevier Science
Publishers B.V., Amsterdam, pp.1082, 45-104