Effects of Nisin On Staphylococcus Aureus Count and Physicochemical Properties of Minas Frescal Cheese
Effects of Nisin On Staphylococcus Aureus Count and Physicochemical Properties of Minas Frescal Cheese
Effects of Nisin On Staphylococcus Aureus Count and Physicochemical Properties of Minas Frescal Cheese
98:4364–4369
http://dx.doi.org/10.3168/jds.2015-9520
© American Dairy Science Association®, 2015.
ABSTRACT than 5.0, and that does not undergo maturation. These
features make Minas Frescal cheese highly perishable
The aim of this work was to evaluate the effects of ni- and its shelf life remarkably short, even under refrigera-
sin on in vitro and in situ Staphylococcus aureus counts. tion (Silva et al., 2003; Malheiros et al., 2012; Magenis
For in vitro experiment, milk was inoculated with 5.0 et al., 2014).
log cfu·mL−1 of S. aureus and nisin was added at con- The physicochemical characteristics of Minas Fres-
centrations of 0, 100, 200, 400, and 500 IU mL−1. The cal cheese increase the potential risk of pathogenic
main effect of the bacteriocin was lag phase extension bacteria incidence, including Salmonella spp., Listeria
from 0 h, for 0 and 100 IU·mL−1 to 8 h, when 200, 400, monocytogenes, Escherichia coli, and Staphylococcus
and 500 IU·mL−1 of nisin were used; however, log phase aureus (Carvalho et al., 2007). Staphylococcal food
was not affected. Microbial growth rate was found to poisoning is often related to cheese consumption due to
be exponential and around 0.11 log cfu·mL−1·h−1 for the presence of enterotoxins (Hennekinne et al., 2012),
all treatments. For in situ experiments, 0, 400, and 500 which in turn are probably caused by excessive human
IU·mL−1 of nisin were directly added to pasteurized milk manipulation during cheese production.
previously inoculated with 5.0 log cfu·g−1 of S. aureus. The most efficient way of reducing the initial milk
Milk, curd, and whey were analyzed to S. aureus counts. contamination and, consequently, producing a safer
Nisin at concentration of 500 IU·mL−1 was able to re- cheese is by improving the hygienic conditions during
duce S. aureus count in curd and whey, demonstrating milking and cheese production (Poli et al., 2007). How-
nisin partition between both phases. Throughout stor- ever, these practices have not been enough to ensure
age at 4°C, S. aureus count increased for all treatments, Minas Frescal cheese quality and safety because many
but the bacterial grew slower when nisin was added in studies have associated this cheese with food poisoning
both concentrations, maintaining S. aureus count about (Silva et al., 2003; Carvalho et al., 2007; André et al.,
1.5 log cycles lower than the control, despite abusive 2008; Malheiros et al., 2012; Magenis et al., 2014).
initial S. aureus count. Therefore, nisin seems to play Some studies have shown that bacteriocins can act as
an important role in reducing S. aureus initial count an additional hurdle to reduce pathogenic bacteria con-
in cheese made with highly contaminated milk. Nisin tamination in cheeses (Pinto et al., 2011; Malheiros et
showed potential to be used as an additional, important al., 2012). Bacteriocin action mechanism is particularly
hurdle to improve Minas Frescal cheese safety, without interesting when compared with traditional antibiot-
replacing good manufacturing practices. ics due to their strong antibacterial activity against a
Key words: bacteriocin, bacterial growth, high mois- broad microorganism spectrum and to the low bacterial
ture cheese, milk, food poisoning resistance rates (Malheiros et al., 2012; Tong et al.,
2014). Nisin, the most studied bacteriocin, is gener-
INTRODUCTION ally regarded as safe (GRAS) by the Joint Food and
Agriculture Organization/World Health Organization
Minas Frescal cheese is widely consumed in Brazil. (FAO/WHO) Expert Committee on Food Additives.
It is a fresh, soft white cheese produced via enzymatic Nisin is a 34 amino acid peptide produced by Lactococ-
coagulation, resulting in a preservative-free cheese hav- cus lactis ssp. lactis, and it is antimicrobially effective
ing high water activity, low salt content, and pH higher against a wide range of gram-positive bacteria (Liu et
al., 2015).
Many studies have shown that nisin is effective
Received March 2, 2015.
Accepted March 29, 2015. against Staphylococcus sp. (Hamama et al., 2002; Pinto
1
Corresponding author: [email protected] et al., 2011; Pimentel-Filho et al., 2014; Zhao et al.,
4364
NISIN EFFECTS ON MINAS CHEESE PRESERVATION 4365
2014; Liu et al., 2015). However, to the best of our Staphylococcus aureus was added to pasteurized milk
knowledge, few data are available regarding the effect to obtain an abusive initial count of 1 × 105 cfu·mL−1.
of nisin against S. aureus in Minas Frescal cheese, which Based upon in vitro tests, nisin was added at concen-
is a fresh, highly manipulated product and, thus, an trations of 400 and 500 IU·mL−1. Nisin was not added
important vehicle for the staphylococcal enterotoxin. to control milk.
Therefore, with the aim of contributing to Minas Fr- Microbiological analyses were carried out in milk,
escal cheese safety and extending its shelf life, the goal whey, and curd during cheese production as well as
of this work was to evaluate the inhibitory effect of dif- in cheese after 1, 7, 14, 21, and 30 d of storage. For
ferent nisin concentrations against S. aureus inoculated each analysis, 25-g (or 25 mL) samples were diluted in
in Minas Frescal cheese during 30 d of storage. 225 mL of 0.2% (wt/vol) peptone water. Staphylococcus
aureus was counted in Petrifilm 3M Rapid S. aureus
MATERIALS AND METHODS Count Plates (AOAC 981.15), according to the manu-
facturer’s recommendations.
Nisin Preparation
Data Analysis
For in vitro experiments, commercial nisin (Nisaplin,
Danisco Brasil Ltda, Pirapozinho, Brazil) suspensions The experiments were carried out 3 times in a com-
at 2.5% (wt/wt) in reconstituted (10% wt/vol) skim pletely randomized design. The decimal logarithms of
milk (RSM). For in situ experiments, nisin was directly S. aureus counts were evaluated by ANOVA, using the
added to milk during cheese manufacturing. Tukey test at a significance level of P < 0.05 to compare
mean values. The analyses were performed using the
Culture and Growth Conditions Software Analysis and Experimentation Group, version
8.0 (SAEG, Viçosa, Minas Gerais, Brazil).
Staphylococcus aureus (ATCC 6538) cultures were
used in this study. The stock culture was maintained
RESULTS AND DISCUSSION
at −80°C in brain heart infusion (BHI) broth (Difco,
Becton, Dickinson and Company, Franklin Lakes, NJ) Nisin In Vitro Effect Against S. aureus
containing 10% (vol/vol) of glycerol. Stock cultures
were recovered in BHI broth and subsequently subcul- Culture medium composition is known to influence
tured thrice in BHI at 37°C for 24 h. antimicrobial tests (Huys et al., 2002). Therefore, we
chose reconstituted (10% wt/wt) skim milk (RSM) to
Nisin In Vitro Antagonistic Effect Against S. aureus investigate the nisin in vitro effect on S. aureus. Recon-
stituted skim milk is rich in proteins that can interact
Staphylococcus aureus was added to nisin suspensions, with nisin, reducing its effect. Inhibition of S. aureus
prepared in RSM at 10%, at concentrations of 0, 100, inoculated in milk containing different nisin concentra-
200, 400, and 500 IU·mL−1 to obtain an initial count of tions is shown in Figure 2.
1 × 105 cfu·mL−1. Systems were incubated at 37 ± 2°C In the absence of nisin, S. aureus grew immediately
during 24 h and S. aureus was counted throughout this without a lag phase and reached a maximum popula-
period by using Petrifilm 3M Rapid S. aureus Count tion density (MPD) of 7.8 log cfu·mL−1 during 24 h
Plates (AOAC International, 2003; method 981.15). in RSM (pH = 6.5). When 100 IU·mL−1 was added
Previous studies have demonstrated that Petrifilm is to RSM, a similar behavior was observed (P > 0.05).
suitable for S. aureus count in dairy products (Viçosa Pinto et al. (2011) also did not confirm the effect of low
et al., 2010). nisin concentration (100 IU·mL−1) against S. aureus in
RSM (12% wt/wt).
Nisin Antagonistic Effect Against S. aureus Nisin concentration of 200 IU·mL−1, however, was ef-
Inoculated in Minas Frescal Cheese fective against S. aureus as lag phase was increased to 8
h. After this adaptation period, cells grew and achieved
To evaluate nisin in situ effect on S. aureus, Minas a MPD of 7.6 log cfu·mL−1 during 36 h. This result
Frescal cheese was produced with pasteurized milk demonstrates that nisin was effective both in increasing
containing 3.0% (wt/wt) of fat and added by com- the S. aureus lag phase and in reducing bacteria growth
mercial rennet and sodium chloride at 2.0% (wt/wt), in RSM even at the optimum inoculation temperature.
according to the flowchart illustrated in Figure 1. Good When nisin concentration was enhanced to 400 and
manufacturing practices were followed to avoid cheese 500 IU·mL−1, different S. aureus growth patterns were
contamination. observed if compared with lower nisin concentrations.
Initially, a reduction of 1.3 log cycles in bacteria count surviving cell regrowth after 24 h. This behavior was
occurred until 8 h of incubation, showing evidence of attributed to the presence of nisin-resistant bacteria
nisin bactericidal effect. However, after 16 h, S. aureus (Arqués et al., 2011). Grisi and Gorlach-Lira (2005)
achieved a count that compensated the initial reduc- used a nisin concentration close to that used in the
tion caused by nisin, and MPD (7.2 log cfu·mL−1) was present experiment and found that this bacteriocin was
reached until 36 h. For control and all nisin concentra- bacteriostatic up to 8 h for S. aureus growth in BHI
tions, log phase showed a similar behavior (P > 0.05) broth. However, after the lag period, the multiplication
and the exponential microbial growth rate was almost rate was similar to that in the absence of nisin, which
the same (around 0.11 log cfu·mL−1·h−1). These results is the behavior also associated with resistant cells. On
indicate that the main nisin effect is lag phase exten- the other hand, according to Gallo et al. (2007), the
sion, which is in accordance with a previous report survivors are not necessarily resistant to nisin. They
(Kiss et al., 2007). may also have escaped from nisin action due to insuf-
In another work, 10,000 IU·mL−1 of nisin was able ficient bacteriocin availability.
to reduce S. aureus counts by 4.68 log cfu·mL−1in milk Because only 400 and 500 IU·mL−1 of nisin dem-
after 4 h of incubation at 37°C, followed by bacteria onstrated potential to inhibit S. aureus mainly by in-
CONCLUSIONS
ACKNOWLEDGMENTS