Antimicrobial action of lactoferrin on Staphylococcus aureus
inoculated in Minas frescal cheese
SUMMARY. The effect of the addition of lactoferrin
on Staphylococcus aureus to Minas frescal cheese
was evaluated. Three cheeses were made:
control (S. aureus), Lf-2% (2% lactoferrin + S. aureus)
and Lf-4% (4% lactoferrin + S. aureus). Cheeses
were packaged and S. aureus populations were
determined on days 1, 8, 15, 22 and 29 of storage at
6°C. The experiment was repeated three times. S.
aureus population increased 1.3 logarithmic cycles
in the control cheese during storage, while it decreased
to numbers below the detection limit in cheeses
containing lactoferrin, over the same period. Moreover,
antimicrobial effect showed to be dose-dependent.
Key words: Pathogenic microorganisms, antimicrobial
activity, bioactive compounds.
RESUMO. Ação antimicrobiana de lactoferrina sobre Staphylococcus
aureus inoculado em queijo Minas frescal. O efeito da
adição de lactoferrina sobre a viabilidade de Staphylococcus aureus
inoculado em queijo Minas frescal foi avaliado. Três queijos foram
produzidos: controle (S. aureus), Lf-2% (2% lactoferrina + S. aureus)
e Lf-4% (4% lactoferrina + S. aureus). Os queijos foram armazenados
e as populações de S. aureus foram determinadas
durante o armazenamento refrigerado a 6°C nos dias 1, 8, 15, 22 e
29. Os experimentos foram realizados em triplicata. As populações
de S. aureus aumentaram 1,3 ciclos logarítmicos nos queijos controle
durante o armazenamento, enquanto que essas populações
foram reduzidas abaixo do limite de detecção nos queijos contendo
lactoferrina no mesmo período. Além disso, observou-se que o
efeito antimicrobiano foi dose-dependente.
Palavras-chave: Microrganismos patogênicos, atividade antimicrobiana,
Minas frescal is a popular Brazilian unripened cheese
made from cow’s milk. Due to its high moisture
content, low acidity and soft texture, it is susceptible to
the development of bacteria that, in addition to limiting
the shelf life of the product, may impose a health hazard
to consumers, since pathogenic bacteria frequently associated
with outbreaks of foodborne diseases, such as
those caused by Staphylococcus aureus, which can easily
contaminate and develop in this cheese (1).
Contamination of cheeses by S. aureus is mainly
attributed to the use of raw milk in its manufacturing,
or to defects and flaws during processing, principally
during pasteurization, post-processing handling or
along the distribution chain of the product (2). This
microorganism, a ubiquitous pathogen, can cause
human diseases like mastitis, toxic shock syndrome
and staphylococcal food poisoning (3). In addition, resistant
and multi-drug-resistant staphylococcal strains
have been isolated from humans and animals (4).
S. aureus in foods has become a major health threat
to people all over the world due to the its high transmissibility
and virulence, increasing predominance
over antibiotic-resistant bacteria, limited therapeutic
options and wide distribution in both hospital and
community settings (5). For that reason, alternatives
must be developed by the industries to ensure the microbiological
safety of the foods, and consequently,
the health of the consumer.
Lactoferrin and its peptides are a promising class of
antimicrobial compounds in the fight against pathogenic
microorganisms, including S. aureus. Lactoferrin
is a multifunctional iron-binding protein belonging to
the transferrin family and is found in mammals, produced
by the mammary glands, glandular tissue on mucosal
surfaces and is also present in secretory granules
of polymorphonuclear leukocytes (6).
Lactoferrin may have beneficial preventive and
therapeutic effects on infections, inflammations, and
Alisson Santana Da Silva, Edson Renato Honjoya, Sibele Camilo Cardoso, Cínthia Hoch Batista De Souza,
Marcela De Rezende Costa, Elsa Helena Walter De Santana, Lina Casale Aragon-Alegro
Universidade Norte do Paraná, UNOPAR, PR, Brasil. Universidade Federal de Mato Grosso do Sul
(UFMS), Faculdade de Medicina Veterinária e Zootecnia, Campo Grande, MS, Brasil.
ARCHIVOS LATINOAMERICANOS DE NUTRICIÓN
Órgano Oficial de la Sociedad Latinoamericana de Nutrición
Vol. 62 Nº 1, 2012
ANTIMICROBIAL ACTION OF LACTOFERRIN ON STAPHYLOCOCCUS AUREUS 69
cancer, in addition to enhancing the iron status and the
growth performance of vulnerable groups (7).
The bactericidal function of bovine lactoferrin is
partially the result of the direct interaction between the
positive charged regions with anionic molecules present
on the surface of some microorganisms, which
causes an increase in the membrane’s permeability, inflicting
damage to the bacteria (8).
According to Shimazaki (9), there are three models
to explain the multi-functionality of lactoferrin. The
first suggests that specific sites or fragments of the lactoferrin
molecule constitute binding or active sites that
enable lactoferrin to function. The second model involves
lactoferrin binding and/or transferring various
biological substances and behaving in a cooperative
or competitive manner. And the last model suggests
that lactoferrin induces or suppresses the production
of chemical mediators to control immune cells on the
intestinal epithelium, thereby affecting tissues or organs
apart from the digestive tract.
Thus, the objective of this study was to evaluate
the action of two different concentrations of lactoferrin
on Staphylococcus aureus added to Minas frescal cheese,
with the aim of exploring the protective potential
of lactoferrin - a natural component of milk - to inhibit
the growth of pathogenic microorganisms in foods.
MATERIALS AND METHODS
Preparation of microbial culture
The Staphylococcus aureus strain, obtained from the
Food Microbiology Lab of the Faculty of Pharmaceutical
Sciences, University of Sao Paulo, Brazil, was
grown in brain heart infusion broth (BHI) and incubated
at 37o C. After 24 h, the broth was centrifuged at 1050
x g for 15 minutes at 4oC, washed with 0,85% (w/v)
saline solution, aliquoted in 1 mL portions and kept frozen
at -80oC with 25% (v/v) glycerol. Each 1 mL portion
contained approximately 5 x 102 cfu.mL-1.
Manufacturing of Minas frescal cheeses
The fat content of whole pasteurized milk was standardized
to 3% by adding pasteurized skimmed milk,
subsequently heated to 35°C and added with 50% calcium
chloride (0.25 mL L-1), lactic acid 85% (0.25 mL
L-1) and rennet (1:10 dilution in distilled water). After
30 minutes, curdling was verified and the clot was cut
and left to
|| stand for five minutes. Next, part of the
whey was drained off, followed by the addition of salt
and (10 g L-1) and an additional holding time of two
minutes. After another additional round of draining off
part of the whey, the cut curd was inoculated with a
population of 5 × 102 CFU mL-1 Staphylococcus
aureus and divided into three equal parts, with lactoferrin
(0.2 g/100 g curd and 0.4 g/100 g curd) being
added to two of these parts. After molding, the cheese
curds turned and stacked three times at 15 minute-intervals.
After a period of 12 hours cold storage at 6°C,
the cheeses were released from the molds and cut up
into 25 g-portions, which were immediately vacuumpacked
into polyethylene packages and stored at 6°C.
The entire experiment was repeated three times.
Enumeration of Staphylococcus aureus
On days 1, 8, 15, 22 and 29 of storage, the
Staphylococcus aureus populations contained in the
Minas frescal cheeses were enumerated according to
official methods (10). On each day of analysis, sample
portions each weighing 25 g were homogenized with
225 mL of a sterile saline solution (0.85%) in appropriate
plastic bags. From this initial dilution, a series
of decimal dilutions was prepared using the same diluent.
Aliquots of 0.1 mL of the dilutions were seeded
onto Petri plates containing Baird-Parker Agar supplemented
with potassium tellurite and an egg yolk solution
and subsequently incubated at 35°C. After 48
hours, typical colonies (black-colored, with or without
a halo) were counted and the population calculated and
expressed as Colony Forming Units (CFU) per gram
of cheese. A total of five suspected colonies from each
plate was subjected to the catalase and coagulase tests
After 1, 8, 15, 22 and 29 days of storage, the pH of
the cheeses was determined according to the procedures
of the American Association of Official Agricultural
The effect of the addition of lactoferrin the pH and
S. aureus population of Minas frescal cheese throughout
the shelf life investigated was evaluated by analysis
of variance (ANOVA) using the Statistica software
The initial mean pH values of the cheeses were
6.44, for the control cheese, 6.42 for the cheese added
70 SANTANA DA SILVA et al.
with 2% lactoferrin, and 6.40 for the cheese containing
4% lactoferrin (Figure 1).
It was found that in the control cheese, the S.
aureus populations remained constant up to 15 days
of storage, verifying from that point in time onwards
a statistically significant increase (p<0.05) in the
counts, greater than one logarithmic cycle (Figure 2).
When added with lactoferrin, it was observed that this
protein did not allow the multiplication of S. aureus
during the storage period of the cheeses (Figure 2). At
the concentration of 2%, it was verified that the population
of S. aureus remained unchanged for 15 days
(p>0.05), declining significantly (p<0.05) from that
point in time onwards until it was no further detected
by the method utilized up to the end of the storage period.
When 4% lactoferrin was added, the S. aureus
populations started to decline already after the 8th day.
According to Furtado (14), the initial pH of Minas
frescal cheese produced with lactic acid varies between
6.3 and 6.5. These values may fall to pH 5.2 –
5.4 during shelf life, as a function of the degree of contamination
and the overall conditions of the points of
sale. It can be observed from Figure 1 that the pH of
the control cheese and those treated with lactoferrin
declined to the same extent and with a similar time
course throughout the 29 days of refrigerated storage
investigated (p<0.05). The reduction was 0.09 for the
control sample and 0.08 for the lactoferrin-added samples,
with the pH values never falling to levels lower
than 6.34. This drop in pH is probably due to the production
of lactic acid during fermentation of lactose
by bacteria (15). However, these values are not considered
a limiting factor for the proliferation of S.
aureus. According to Beresford et al (16), the optimal
pH for the multiplication of most of the bacteria is
close to neutral and their multiplication is impaired at
values lower than 5.0.
Other authors found pH values similar to those observed
in the present study when evaluating Minas
frescal cheeses produced by direct acidification (17,
18). Cheeses acidified with lactic acid have higher pH
values than those acidified with lactic starter culture.
In Brazil, there is a trend towards partially or totally
replacing lactic starter cultures by lactic acid in cheeses,
although it is known that only the addition of lactic
cultures ensures the permanent production of lactic
acid and, consequently, lower pH values of the pro-
Mean values of the S. aureus populations of the
Minas frescal control cheese and the cheeses treated
with lactoferrin, kept under cold storage for 29 days.
C = control cheese; Lf-2% = cheese with 2%
added lactoferrin; Lf-4% = cheese
with 4% added lactoferrin.
Mean pH values observed in Minas frescal control
cheese and cheeses treated with lactoferrin,
during the 29 days of storage under refrigeration.
C = control cheese; Lf-2% = cheese with 2% added
lactoferrin; Lf-4% = cheese with
4% added lactoferrin.
The mean counts of the S. aureus populations of
the control cheese and the lactoferrin treatments in the
course of the 29-day storage period investigated are
presented in Figure 2. The initial population - approximately
2.5 log CFU g-1 (p>0.05) - was the same for
all the formulations, which is the maximum population
of coagulase-positive Staphylococcus per gram of very
high moisture cheese allowed by the Brazilian food regulations
currently in force (13).
ANTIMICROBIAL ACTION OF LACTOFERRIN ON STAPHYLOCOCCUS AUREUS 71
ducts during storage (19), which would help to control
the development of spoilage and pathogenic bacteria
that may be present in this food.
Regarding S. aureus populations, if we consider that
the freshness or "best before" date estimated by the manufacturers
of Minas frescal covers a period varying
from 21 to 30 days, cheeses containing populations
close to 2.5 log UFC g-1 when distributed to the retail
establishments and which were released for sale by legislation
may become a serious public health hazard.
In Brazil, the presence of S. aureus is relatively
common in fresh, unripened cheeses, with populations
well above the legally permitted levels.
Several authors have reported on in vitro antimicrobial
||ity of lactoferrin against pathogens (20,
21, 22, 23), but only a few evaluated this activity
against S. aureus (5, 24).
At present, several natural antimicrobial compounds
that prevent the development of spoilage and
pathogenic microorganisms have been studied by the
food industry, such as is the case of lactoferrin. One
of the reasons for these investigations lies in the fact
that the digestion of this protein by pepsin generates
two fragments - lactoferricin and lactoferrampin -
which are even more potent against bacteria, compared
to the intact protein (5).
Furthermore, some S. aureus strains are known to
be resistant to antimicrobials and the possibility to use
lactoferrin to control them, by sequestering iron and
limiting the use of this nutrient by pathogen (25) in
food is interesting.
Murdock e Matthews (26) evaluated the antimicrobial
activity of bovine lactoferrin hydrolysate with pepsin
against Escherichia coli and Listeria monocytogenes
in peptone yeast extract glucose broth and ultra-high
temperature milk. The authors concluded that, under
conditions of low pH and refrigeration temperatures,
lactoferrin hydrolysate can limit or reduce the populations
of pathogenic bacteria in a dairy product.
Lactoferrin prevented the increase of the S. aureus
population in the cheeses, at the two concentrations
tested, acting initially as a bacteriostatic when the cheeses
were inoculated with a population of 5 × 102 UFC
g-1 S. aureus. Furthermore, bactericidal action was observed
after the 15th day of storage when lactoferrin
was added at a concentration of 2%, and after the 8th
day at the concentration of 4%, which is interesting in
view of the fact that this type of cheese has a short
We would like to thank the Universidade Norte do
Paraná (UNOPAR) and the Fundação Nacional de
Desenvolvimento do Ensino Superior Particular (FUNADESP),
for supplying the financial support that
made this research project possible.
1. Nascimento MS, Moreno I, Kuaye AY. Applicability of
bacteriocin-producing Lactobacillus plantarum, Enterococcus
faecium and Lactococcus lactis ssp. lactis as
adjunct starter in Minas Frescal cheesemaking. Int J
Dairy Technol. 2008; 61: 352-57.
2. Borges MF, Arcuri EF, Pereira JL, Feitosa T, Kuaye AY.
Staphylococcus enterotoxigênicos em leite e produtos
lácteos, suas enterotoxinas e genes associados: revisão.
Boletim CEPPA. 2008; 26: 71-86.
3. Le Loir Y, Baron F, Gautier M. Staphylococcus aureus
and food poisoning. Genet Mol Res. 2003; 2: 63-76.
4. Miller LG, Kaplan SL. Staphylococcus aureus: a
community pathogen. Infect Dis Clin N Am. 2009;
5. Flores-Villaseñor H, Canizalez-Román A, Reyes-
Lopez, KN, Garza M, Zazueta-Beltrán J, León-Sicairos
N, Bolscher JGM. Bactericidal effect of bovine lactoferrin,
LFcin, LFampin and LFchimera on antibioticresistant
Staphylococcus aureus and Escherichia coli.
Biometals. 2010; 23: 569-78.
6. Lonnerdal B, Iyer S. Lactoferrin: molecular structure and
biological function. Annu Rev Nutr. 1995; 15: 93-110.
7. Lonnerdal B. Nutritional roles of lactoferrin. Curr Opin
Clin Nutr Metab Care. 2009; 12: 293-97.
8. Haversen L, Kondor N, Baltzer L, Hanson LA, Dolphin
GT, Duner K, Mattsby-Baltzer I. Structure-microbicidal
activity relationship of synthetic fragments derived
from the antibacterial alpha-helix of human lactoferrin.
Antimicrob Agents Ch. 2010; 54: 418-25.
9. Shimazaki KI, Kushida T. A preliminary approach to
creating an overview of lactoferrin multi-functionality
utilizing a text mining method. Biometals. 2010; 23:
10. Lancette G, Bennett RW. Staphylococcus aureus and
Staphylococcal Enterotoxins. In: Compendium of methods
for the microbiological examination of foods.
72 SANTANA DA SILVA et al.
American Public Health Association (APHA); 2001. p.
11. Association of Official Analytical Chemists. Official
Methods of Analysis. 17th ed. Washington, DC:
12. Statsoft Inc. STATISTICA for Windows [Computer
program manual], Tulsa, OK: StatSoft, Inc., 2000.
13. Brasil. Agência Nacional de Vigilância Sanitária – ANVISA.
Resolução RDC n° 12, de 02 de janeiro de 2001,
14. Furtado MM. Principais problemas dos queijos: causas
e prevenção. São Paulo: Fonte Comunicações e Editora;
15. Eskin MNA. Biochemistry of foods. London: Academic
16. Beresford TP, Fitzsimons NA, Brennan L, Cogan TM.
Recent advances in cheese microbiology. Int Dairy J.
2001; 11: 259-74.
17. Souza CHB, Saad SMI. Viability of Lactobacillus
acidophilus La-5 added solely or in co-culture with a
yoghurt starter culture and implications on physicochemical
and related properties of Minas fresh cheese
during storage. Food Sci Technol. 2009; 42: 633-40.
18. Alves CCC. Comportamento da Escherichia coli em
queijo minas Frescal elaborado com utilização de
Lactobacillus acidophilus e de acidificação direta com
ácido lático. [Dissertação]. Rio de Janeiro: Universidade
Federal Fluminense; 2010.
19. Rocha JS, Buriti FCA, Saad SMI. Condições de processamento
e comercialização de queijo-de-minas frescal.
Arq Bras Med Vet Zootec. 2006; 58: 263-72.
20. Oliveira IR, Araujo AN, Bao SN, Giugliano LG. Binding
of lactoferrin and free secretory component to enterotoxigenic
Escherichia coli. FEMS Microbiol Lett.
2001; 203: 29-33.
21. Di Biase AM, Tinari A, Pietrantoni A, Antonini G, Valenti
P, Conte MP, Superti F. Effect of bovine lactoferricin
on enteropathogenic Yersinia adhesion and
invasion in HEp-2 cells. J Med Microbiol. 2004; 53:
22. Superti F, Pietrantoni A, Di Biase AM, Longhi C, Valenti
P, Tinari A. Inv-mediated apoptosis of epithelial
cells infected with enteropathogenic Yersinia: a protective
effect of lactoferrin. Res Microbiol. 2005; 156:
23. Murdock CA, Cleveland J, Matthews KR, Chikindas
ML. The synergistic effect of nisin and lactoferrin on
the inhibition of Listeria monocytogenes and Escherichia
coli O157:H7. Lett Appl Microbiol. 2007; 44:
24. D’Almeida WK. Efeito da lactoferrina na conservação
do queijo Minas frescal. [Dissertação]. Londrina: Universidade
Norte do Paraná, 2009.
25. Bhimani RS, Vendrov Y, Furmanski P. Influence of lactoferrin
feeding and injection against systemic staphylococcal
infections in mice. J. Appl. Microbiol. 1999;
26. Murdock CA, Matthews KR. Antibacterial activity of
pepsin-digested lactoferrin on foodborne pathogens in
buffered broth systems and ultra-high temperature milk
with EDTA. J Appl Microbiol. 2002; 93: 850-56.