Neris, D. J. G. et. al., 176
Vol. 4, N.3: pp. 176-183, August, 2013 ISSN: 2179-4804
Journal of Biotechnology and Biodiversity
Nisin in the biopreservation of Bordô (Ives) and Niágara table wines from Santa Catarina, Brazil
Daiana Jaqueline Gatti Neris1, Sidnei Emilio Bordignon-Junior2*, César Milton Baratto3 , Jane Mary Lafayette Neves Gelinski 3
ABSTRACT
The searches for new antimicrobial compounds to control the lactic acid bacteria in winemaking are based in the increasing global concern about the chemical preservatives restrictions and the development of super-resistant strains continuously exposed to sulfites in wineries for decades. The antimicrobial activity of the nisin as a biopreservative was studied for Bordô (Ives) and Niágara grape wines from Santa Catarina, Brazil. Measurements of antimicrobial activity were made by well-diffusion assays. From the eleven previously assessed strains of nisin-
susceptible lactic bacteria (nisin concentration 1000 IU ml-1), four were selected for definitive assays with nisin (100 IU ml-1) dissolved in the wines. Positive results for inhibition were obtained for the four strains selected. Next,
the direct inhibitory action was assessed in wines artificially inoculated and then treated with nisin. After 60 days of storage, there was reduction in the total bacterial population as compared to control, especially in Bordô (Ives) wine, while the physic-chemical parameters were not influenced by the nisin treatment. The inhibitory activity of nisin was not affected when it was dissolved in wine in the antimicrobial assays, and its potential utilization as biopreservative should be able to aid on the control of autochthonous microbiota, but further studies are required to conclude more precisely the nisin effects at long term in wines.
Keywords: Antimicrobial, lactic acid bacteria, malolactic fermentation, vinification, bacteriocin.
Nisina na bioconservação de vinhos de mesa Bordô (Ives) e Niágara de Santa Catarina, Brasil
RESUMO
As buscas por novos compostos antimicrobianos capazes de inibir a proliferação de bactérias ácido-lácticas na vinificação são motivadas pela crescente preocupação global com as restrições do uso de conservantes químicos, e ainda pelo risco de desenvolvimento de linhagens super-resistentes continuadamente expostas aos sulfitos durante
décadas nas vinícolas. A atividade antimicrobiana da nisina como bioconservante foi estudada para vinhos Bordô e
Niágara de Santa Catarina. Os ensaios de atividade inibitória foram realizados pela técnica de difusão em poços, inicialmente para 11 bactérias lácticas que se mostraram susceptíveis sob a concentração de 1000 UI mL-1. Dentre
estas, quatro foram selecionadas para os ensaios definitivos de nisina dissolvida em vinho sob a concentração de 100 UI mL-1. Foram obtidos resultados positivos para as quatro linhagens. Em seguida, a ação inibitória foi diretamente
avaliada em vinhos artificialmente inoculados com as mesmas quatro linhagens e depois tratados com nisina. Depois de 60 dias de estocagem, houve uma redução na população bacteriana total quando comparada ao controle, especialmente para amostra de vinho Bordô (Ives), ao mesmo tempo, os parâmetros físico-químicos do vinho não
foram alterados pelo tratamento com nisina. O potencial inibitório da bacteriocina não foi perdido quando dissolvido em vinho, durante os ensaios antimicrobianos em ágar, e seu potencial de utilização como bioconservante poderia contribuir no controle da microbiota autóctone, sendo necessários outros estudos para concluir precisamente os efeitos da nisina nos vinhos em longo prazo.
Palavras-chave: Antimicrobiano, bactéria ácido-láctica, fermentação maloláctica, vinificação, bacteriocina. *Corresponding author
1MazonWinery, Genésio Mazon Road, Urussanga, 88840-000, Santa Catarina, Brazil
2,*Department of Food Engineering and Science, São Paulo State University (UNESP), Cristóvão Colombo street, 15054-000, São José do Rio Preto, São Paulo, Brazil, bordig@gmail.com
3Nucleus of Biotechnology, University of the West of Santa Catarina (UNOESC), 198 Paese street, 89560-000, Videira, Santa Catarina, Brazil
J. Biotec. Biodivers. v. 4, N.3: pp. 176-183, Aug. 2013
https://doi.org/10.20873/jbb.uft.cemaf.v4n3.neris
Neris, D. J. G. et. al., 177
INTRODUCTION
The wine’s microbiology is composed, besides of
ethanologenic yeasts, for lactic acid bacteria (LAB) responsible by the secondary fermentation, which is an important step to
improve the quality and guaranteed the wine stabilization. The main source from these bacteria
is the surface of grapes and they remain in the must after the milling step as negligible populations during the first stage of vinification
(tumultuous fermentation by yeasts). The malolactic fermentation (MLF) occurs precisely at the moment when the LAB population
overcomes the adaptation phase and shows an intense proliferation, which may be facilitated by adding starter cultures (selected LAB strains), a common practice in the winemaking worldwide. Despite the extension of the LAB group, only four genera are acknowledged to be able to develop during the MLF in such limiting
conditions, characterized by an acidified pH, high
ethanol concentration and the presence of sulfur dioxide, widely used as a preservative in the process. Oenococcus oeni is the best adapted
species and it is recognized for its beneficial
effects on the sensory evolution of the wine after the MLF (Fugelsang & Edwards, 2007). Due to
its physiological properties, O. oeni has high er tolerance to the sulfites than other LAB, thereby
ensuring its prevalence in the winemaking environment. Different tolerance mechanisms of
O. oeni to sulfites are discussed by several
authors (Guzzo et al., 1998; Rojo-Bezares et al., 2007). Others LAB species related to
winemaking are the Lactobacillus (Stratiotis & Dicks, 2002) and, less frequently, the
Pediococcus strains (Rhodes et al., 2003; Du Plessis et al., 2004). Among lactobacilli, the
heterofermentative species are prevalent ,
especially L. delbrueckii (Costantini et al., 2009). Leuconostoc mesenteroides has been isolated in wine samples (Du Plessis et al., 2004), however
its low incidence is probably due the greater sensitivity to alcohol compared with other genera
(Oliva-Neto & Yokoya, 2001).
During advanced stages of winemaking, some
populations of the LAB that are best adapted to the medium may remain in the wine during
storage and aging. Although they multiply slowly
during these stages, the presence of such bacteria after the MLF is undesirable due to the risk of
formation of residual compounds which are deteriorative to the sensorial quality of the wine,
generating an increase in volatile acidity, excessive rancidity, the presence of bitterness, among other well-known aromatic defects such
as mannitol taint, mousiness, ropyness and
geranium off-flavor (Fugelsang & Edwards, 2007; Costantini et al., 2009).
Traditionally the bacterial control in winemaking is performed by the use of chemical compounds
derived from sulfur, which are incorporated since
the beginning process - into grape must – until the advanced stages, as salts in aqueous solution,
or as sulfur dioxide gas (SO2). Nowadays the use of sulfites is considered a compulsory treatment in vinification processes worldwide due to its
antioxidant, antioxidase and antimicrobial effects, especially against native microorganisms . However it is known that excessive SO 2
concentrations can preclude completely the proliferation of LAB and generate depreciative
residual compounds, as hydrosulfates and mercaptans, which have negative effects on the
olfactory quality of the wine. Sulfites should be used cautiously due also to legal determinations, for instance quantity limits set for industrialized foods and beverages, as well as, in some countries, specific labeling for products containing sulfites. There is a strong global trend
to reduce the concentration of chemical preservatives in winemaking (Constantini et al. , 2009).
Bacteriocins have been investigated as an alternative for the microbial control of undesirable LAB. In the context of vinification, two decades ago the pioneering works of Radler
(1990a, 1990b) and Daeschel et al. (1991)
already suggested that the use of sulfites should be reduced by incorporating nisin into the process. Nisin is produced by strains of
Lactococcus lactis subsp. lactis and it has been known since 1928 (Rogers & Whittien, England). It has had GRAS (Generally Regarded as Safe)
status since 1988 and it is used in the food processing in many countries (Arauz et al., 2009;
Zacharaf & Lovitt, 2012), including in Brazil through of MERCOSUL Resolutions n. 79/1994
and 82/1996.
Nowadays, it is known that nisin is stable to the conditions of vinification and does not affect
yeast cell growth or the sensory profile of wine (Knoll et al. 2008), although authorization for
industrial use in winemaking has been difficult to obtain. This undefined situation is strengthened
by contradictory studies, as the results about the
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Neris, D. J. G. et. al., 178
nisin and phenolic compounds interactions (Daeschel & Bower, 1993-1994; Knoll et al. , 2008), by the immense heterogeneity of the wine
profiles (physicochemical properties influenced
by grape variety and the type of vinification that is carried out) that are produced in different
regions and, besides these, the strong resistance of wine producers, which are one of the most
traditional industrial sector. All these constraints
tend to hinder to take general conclusions about new alternatives of wine preservation. Thus ,
there is a need for deeper and specific studies for each wine producer region. In this study, nisin
was evaluated as a possible microbial control agent for the wine varieties that are typical of the
state of Santa Catarina, Brazil, which are made from Bordô (Ives) and Niágara grapes .
MATERIAL AND METHODS Microorganisms and culture conditions
The LAB strains used in this study were obtained from the culture collection of the Laboratory of
Food Microbiology of the University of the West
of Santa Catarina (UNOESC), Videira campus, and also isolated from the wine microbiota kindly
provided by the Laboratory of Microbiology of
the EPAGRI Experimental Station in Videira, Santa Catarina, Brazil. All LAB were reactivated
from stock strains which were kept under freeze
(-20 °C) with 10% glycerol. For reactivation they were cultivated in De Man, Rogosa & Sharpe (MRS) broth, except those of the genus
Enterococcus, grown in Brain Heart Infusion
broth (BHI). Incubation was performed in microaerophilic condition and at 35 °C, except
for Oenococcus oeni, Lactobacillus casei and L. brevis which were incubated at 30 °C.
Preliminary screening for nisin sensitivity Eleven strains (Table 1) among the genera
Lactobacillus, Oenococcus, Enterococcus,
Leuconostoc and Pediococcus were evaluated for
the inhibitory potential of commercial nisin (Nisaplin®, Aplin & Barrett). The evaluation was
carried out by well diffusion assay (Ammor et al., 2006) inoculating each indicator microorganism from an overnight-enriched culture into plates that received the MRS or BHI media (pour
plate)with pH previously adjusted to 5.0. After solidification, the agars were perforated in two
places - one for the addition of 50 µl of nisin
solution diluted in HCl 0.02 M and containing a final concentration of 1,000 IU ml-1, and an other
for the addition of 50 µl of sterile distilled water (negative control). All the plates were incubated
at 30 °C or 35 °C/24 hours in according each indicator microorganism. The results were
determined by the presence or absence of an inhibition halo on the indicator lactic culture at
the end of the assay.
Confirmation of inhibitory activity
Four strains (Lactobacillus brevis, L. delbrueckii subsp. lactis, Pediococcus acidilactici and
Oenococcus oeni) were selected for definitive
inhibition assays using nisin solution at a final concentration of 100 IU ml-1, and sulfur dioxide
(SO2) dissolved in a sample of red Bordô wine or
of white Niágara wine to a initial concentration of 32 mg l-1 of free SO2. In this assay, the MRS agar was supplemented with 2% sterilized
Niágara table wine when Lactobacillus spp. and Pediococcus sp. were used as target strain. In the Oenococcus oeni assay, the malolactic culture medium (MLO) was used and supplemented with
3% tomato extract, as described by Rojo- Bezares et al. (2007), with modifications (original tomato extract concentration as 10%). Four holes were
perforated on the agar plates for the addition of
one of the following substances: 50 µl of nisin (100 IU ml-1), 50 µl of sterile distilled water (control), 50 µl of Bordô (Ives) wine containing SO2 (32 mg l-1), and 50 µl of Niágara wine containing SO2 (32 mg l-1). The agar plates were incubated at 30°C in microaerophilic condition
for 48 hours (to MRS medium) or for 4 days (to
MLO medium). All evaluations were performed in triplicate and in two repetitions. At the end of each incubation period the inhibition halo
diameter obtained on the indicator microorganisms previously inoculated (pour plate) on agar plates was measured in millimeters
(mm) .
Activity of nisin in wine
A 10 ml sample of Niágara wine containing 32 mgl-1 of SO2 was supplemented with 1.0 mg of commercial nisin (106 IU g-1 initial concentration)
diluted directly into the wine to obtain a final nisin concentration of 100 IU ml-1. The
antimicrobial potential of this sample was
evaluated through of 50 µl aliquots in well diffusion assay, according to describe above, for
each one of the four indicator microorganisms in
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Neris, D. J. G. et. al., 179
the same test conditions. Comparatively, 50µl of the same wine containing SO2 was used in each assay as control (without nisin addition) .
Viability assessment of bacteria and
physicochemical evaluation of wine with nisin A sample of 720 ml Bordô (Ives) wine an d
Niágara wine received inocula of lactic acid bacteria (104 CFU ml-1 each) to form a mix from
Lactobacillus brevis, L. delbrueckii subsp. lactis, Pediococcus acidilactici and Oenococcus oeni .
These inocula were obtained by centrifugation (1,300 xg, 5 minutes) from an initial culture as
previously described, and inoculated in each wine sample at time zero (t=0). In parallel others two
samples of 720 ml (one Bordô wine, one Niágara
wine) received commercial nisin ( final concentration of 100 UI ml-1), besides of the
inocula. A third sample of each wine was kept
under the same conditions without inoculum or nisin to further comparisons (original sample) .
The microbial monitoring of the LAB added to the wines was made at time zero (t0=0) and at the end of the storage period (tf=60 days) for all 720 ml samples of wine, which were kept under environmental conditions of low humidity and
the absence of light. For the quantification of LAB (CFU ml-1) after the incubation period, 1 ml
aliquots of each sample were plated on MRS agar (pour plate) and incubated at 30 °C under microaerophilic conditions for 7 days. All
analyses were performed in duplicate.
Physicochemical analyses were performed to monitor the parameters of density (mg l-1 ), alcoholic degree (% v/v), total acidity (meq l-1 )
and free sulfur dioxide - SO2 (mg l-1), accord to
standard methods described by Brazilian Ministry
of Agriculture, Livestock and Supply (BRASIL, 2006), in triplicate for all samples in t0 and tf. All
the data obtained from the microbial and
physicochemical parameters were statistically evaluated by variance analysis (ANOVA).
Table 1. Qualitative assessment of the spectrum of action of nisin on lactic acid bacteria commonly found in wine microbiota
Target microorganism Origin Susceptibility
Enterococcus faecium Culture collection - ATCC 6569 +
Enterococcus faecalis Culture collection - ATCC 19433 +
Oenococcus oeni
Commercial strain (Biolact Acclimaté PB1025, AEB Group® )
+
Leuconostoc mesenteroides subsp. cremoris
Isolated from meat*
+
Lactobacillus delbrueckii subsp. lactis Culture collection - ATCC 7830 ++
Lactobacillus fermentum Culture collection - ATCC 9338 ++
Lactobacillus plantarum Culture collection - ATCC 8014 +
Lactobacillus casei Isolated from wine** ++
Lactobacillus brevis Isolated from wine** +
Pediococcus pentosaceus Culture collection - ATCC 33314 ++
Pediococcus acidilactici Culture collection - ATCC 8042 ++
* UNOESC Collection (Videira, SC, Brazil)
** EPAGRI Experimental Station (Videira, SC, Brazil)
Key: (+) weak inhibition halo, (++) well-defined inhibition halo.
RESULTS AND DISCUSSION Inhibitory activity assays
In the preliminary screening for the antimicrobial
activity, all 11 LAB strains were observed to be sensitive to 1000 UI ml-1of the bacteriocin.
Among the species tested, Pediococcus spp. and Lactobacillus spp. (except L. plantarum) showed greater susceptibility when compared to
Enterococcus spp., Oenococcus oeni and
Leuconostoc mesenteroides, as shown in Table 1. The records for the inhibition halos obtained in
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Neris, D. J. G. et. al., 180
the second assay with nisin are shown in Figure 1 for the four lactic acid bacteria selected at this stage. The following measures of inhibition
halos, excluding the diameter of the well, were
obtained for each microorganism: (a) Lactobacillus brevis 10.6mm, (b) Lactobacillus
delbrueckii subsp. lactis 8.4mm, (c) Pediococcus acidilactici 4.0mm, and (d) Oenococcus oeni
7.8mm. It can be drawn from the well- defined
inhibition halos on the surface of each agar that the effectiveness of the bacteriocin remained
satisfactory even at a concentration 10 times lower than that used in the first assay (Table 1).
Lactobacillus spp. was again the most sensitive. O. oeni proved to be more susceptible than P.
acidilactici in this assessment. As expected, no
inhibition halo was observed on the wine samples containing SO2. In addition, similarly to the
studies of Rojo-Bezares et al. (2007), this study
has revealed an effective antimicrobial action against wine isolates (Lactobacillus casei and L.
brevis ).
Figure 1. Antimicrobial action of nisin against:
(a) Lactobacillus brevis, (b) Lactobacillus delbrueckii subsp. lactis, (c) Pediococcus acidilactici, (d) Oenococcus oeni .
Key: N = nisin; A = sterilized water; VT = Bordô wine control; VB = Niágara wine control.
These results are interesting because currently
there is a concern over the development of super - resistant strains in wineries where cultures are routinely exposed to sulfur compounds, thus the
susceptibility of LAB to other inhibitory
compounds is appreciated. The survival of a viable population in the bottled product is the
most worrying contamination, responsible by the known "second growth" which can use of
residual L-malate as carbon source (Fugelsang & Edwards, 2007).
An effective control of O. oeni by alternative antimicrobial compounds is really needed if we
consider that it can survive in a concentration of 100 mg l-1 of free SO2 (Lafon-Lafourcade et al. ,
1983). Rojo-Bezares et al. (2007) reported that O. oeni has low resistance to nisin, which was
ascertained from the fact that the minimal
inhibitory concentration (MIC) for the Oenococcus group was always much lower
compared to other LAB composed of strains of Lactobacillus spp., Pediococcus spp. and
Leuconostoc mesenteroides for the same treatments with nisin, nisin + ethanol and nisin +
metabisulfite. In our assays (with nisin diluted on
HCl), the halos between L. delbrueckii subsp. lactis and O. oeni are very close, but when nisin was dissolved directly in the wine (next assays),
some differences are observed about the halos diameter in the same four target bacteria, as
presented below.
Figure 2 shows the antimicrobial activity of nisin dissolved in wine, which has not lost its activity. This is evidenced by the formation of well - defined inhibition halos against the four LAB evaluated. These inhibition halos had the
following measures of diameters: Lactobacillus brevis: 6.4 mm, Lactobacillus delbrueckii subsp . lactis: 4.7 mm, Pediococcus acidilactici: 4.3 mm , and Oenococcus oeni: 9.0 mm. The activities against Lactobacillus strains were partly reduced (to 61% and 54% in L. brevis and L. delbrueckii, respectively), whereas the O. oeni halo increased
15%, in comparison to the first inhibitory assay with 100 IU ml-1 of nisin. In this case, our result s
are in agreement with Rojo-Bezares et al. (2007) about the higher susceptibility of the O. oeni than
Lactobacillus spp. in nisin assays.
Bacterial viability in treated wines
It was found that the Niágara wine samples stored for 60 days showed a reduction in bacterial
population when compared to the initial count (control), both in the absence and in the presence
of nisin, with a reduction of approximately 2.1
and 2.3 log cycles respectively. However there was no effect of nisin compared to the group
without nisin after 60 days. Therefore, the effect of the nisin was not significant in reducing of
countable lactic acid bacteria in Niágara wine (Figure 3). In the Bordô (Ives) wine samples
there was a reduction of 1.5 log cycles for the
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Neris, D. J. G. et. al., 181
treatment with inoculum only in comparison with control, and a reduction of approximately 2.4 log cycles for the treatment with inoculum
and nisin after 60 days, in comparison with control (with inoculum at day zero). This
other evaluations at different storage times and wine profiles are required to conclude more precisely the nisin effects at long term.
suggests a possible antimicrobial effect by nisin,
although no significant difference was observed considering the standard deviation. In the original
samples (not shown in the graph), which did not
receive neither inoculum nor nisin, LAB populations were also detected in the order of 10 3
UFC ml-1 in all samples (already considered to be in the control group ).
10
9
8
7
6
5
4
3
2
1
0
7.40
7.90
Niágara wine 6.38
5.56
Bordô wine 5.38 5.49
Control (wine + inoculum) Time 0
wine + inoculum after 60 days
wine +inoculum + nisin after 60 days
Figure 2. Antimicrobial action of nisin (100 IU ml-1) diluted in Niágara wine, where: (a) Lactobacillus brevis, (b) Lactobacillus delbrueckii subsp. lactis, (c) Pediococcus acidilactici, (d) Oenococcus oeni .
Key: V+N = Niágara wine + nisin; V = control, only wine.
Physicochemical evaluations in treated wines According to Table 2, the presence of LAB
and/or nisin during the 60 days of (white and red) wine storage did not promote any relevant changes in the physicochemical properties tested,
despite of the parameter free SO2 which decreased with time in all samples. This is a
normal tendency which always occurs in the any
wine storage due to its volatility and, thus, it is not assigned to nisin presence. In respect to total
acidity, slight differences were observed and the
values at the end of storage time were equal or less than the initial total acidity. This is a favorable situation, because if a microbiologic
contamination occurs normally the acidity is
increased by organic acid formation, especially by lactic acid bacteria. Preliminarily, the nisin
seems not depreciative to the wine quality, but
Treatments
Figure 3. Cell viability of mix of lactic acid bacteria in Niágara and Bordô (Ives) wine in the presence or absence of nisin after 60 days of storage.
*Error bars respect the standard deviation.
CONCLUSION
The results of this study contribute to extend the
well known antimicrobial action of nisin on general lactic acid bacteria against wine isolated
bacteria also. The utilization of nisin as a complementary preservative in the Niágara and Bordô (Ives) winemaking would be able to aid on
the control of autochthonous microbiota responsible for microbiological diseases, and could reduce the sulfite concentration required
currently. Further studies might also examine the joint use of sulfite + nisin in industrial processes, which may certainly improve the preservative
effect. Overall the experimental microbial contamination did not affect the physicochemical parameters during the study period, but studies involving longer periods should be carried out to
assess the interference both of lactic acid bacteria as nisin on the analytical and sensory quality of
the wine.
ACKNOWLEDGEMENTS
The authors are grateful to the government of the State of Santa Catarina, Brazil, for the research
grant received from the Ministry of Education.
Neris, D. J. G. et. al., 182
Table 2. Physicochemical evaluations of Niágara and Bordô (Ives) wine during storage
Physico chemical parameters
Sample
T = 0 Control
Wine after storage
With inoculum only With inoculum + nisin
Relative Density (mg l1 )
Niágara Bordô
0.99 a
0.99 a
0.99 a
0.99 a
0.99 a
0.99 a
Alcohol (% v/v)
Niágara Bordô
10.30 a
10.50 a
10.50 a
10.30 a
10.50 a
10.50 a
Free SO2 (mg l-1) Niágara Bordô
32.00 a
32.00 a
22.40 b
19.20 b
25.60 c
19.20 b
Total Acidity (meq l-1 )
Niágara Bordô
100.00 a
103.00 a
96.00 b
99.00 b
98.00 b
103.00 a
*For each treatment: different letters represent significant statistical differences when p = 0.05 .
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