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Department of Food Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch 7602, South Africa
DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health
Sciences, Stellenbosch University, PO Box 19063, Tygerberg 7505, South Africa
c
Institute for Infectious Diseases and Zoonosis, Department of Veterinary Science, LMU, Munich, Germany
d
Department of Microbial-, Biochemical- and Food Biotechnology, University of the Free State. PO Box/Posbus 339, Bloemfontein 9300, South Africa
b
a r t i c l e
i n f o
Article history:
Received 19 September 2013
Received in revised form 21 October 2015
Accepted 23 October 2015
Available online 25 October 2015
Keywords:
Mycobacterium bovis BCG
Survival
Milk fermentation
Africa
a b s t r a c t
Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption
of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular
traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage
bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to
determine the effect of the fermentation process on the survival of M. bovis BCG in milk.
M. bovis BCG at concentrations of 6 log CFU/ml was added to products of ker fermentation. The survival of
M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar
plates enriched with 2% BD BACTEC PANTA. M. bovis BCG was increasingly reduced in sterile ker that was
fermented for a period of 24 h and longer. In the milk fermented with ker grains, Lactobacillus paracasei
subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24 h and by
2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation.
Results from this study show that long term fermentation under certain conditions may have the potential to
inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation
should be combined with other hurdle technologies such as boiling and milk pasteurisation.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Mycobacterium bovis is the main causative agent of tuberculosis (TB)
in cattle. This bacterium is closely related to Mycobacterium tuberculosis,
the causal agent of TB in humans. These two bacterial species belong to
the same species complex, namely the M. tuberculosis complex (Neill
et al., 2005; Smith et al., 2006). M. bovis is pathogenic to various animal
species and can spread from livestock to humans. The consumption of
raw milk and raw milk products contaminated with these bacilli due
to shedding of viable M. bovis cells into the milk is the most common
transmission route of M. bovis to humans (de la Rua-Domenech, 2006;
Michel et al., 2010; Rowe and Donaghy, 2008; Thoen et al., 2006). An
aggravating factor in the occurrence of zoonotic TB in Africa is the prevalence of the human immunodeciency virus (HIV) and malnutrition
which contributes to the increased risk of susceptibility of humans to
M. bovis (Michel et al., 2010).
Corresponding author.
E-mail addresses: 16392574@sun.ac.za (C.L.S. Macuamule), iw@sun.ac.za (I.J. Wiid),
pvh@sun.ac.za (P.D. van Helden), Manfred.Tanner@micro.vetmed.uni-muenchen.de
(M. Tanner), witthuhnrc@ufs.ac.za (R.C. Witthuhn).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.10.024
0168-1605/ 2015 Elsevier B.V. All rights reserved.
C.L.S. Macuamule et al. / International Journal of Food Microbiology 217 (2016) 170176
171
of mesophilic LAB, yeast and moulds that characterise ker justify the
selection of this product as a model of milk fermentation.
2. Materials and methods
2.1. Microorganisms and culture conditions
Mycobacterium bovis Bacillus Calmette-Gurin (BCG) Pasteur (ATCC,
35734) is an attenuated strain obtained from the Division of Molecular
Biology and Human Genetics (Stellenbosch University, South Africa)
and was used in all experiments. M. bovis BCG shares characteristics
with M. bovis and M. tuberculosis as all of them are slow growing
mycobacteria within the M. tuberculosis complex (Pittius et al., 2012).
It was cultured on Middlebrook 7H9 broth (Difco Becton Dickinson,
USA) at 37 C for 7 days to an A600 nm of 0.5 to 0.6 as measured by a
DU530 spectrophotometer (Beckman Coulter). Colony forming
units (CFU's) were determined on Middlebrook 7H10 agar plates
supplemented with PANTA Plus when needed. Actively growing
mycobacteria at a concentration of approximately 6 log CFU/ml were
used to inoculate the milk and fermented milk product samples.
The ker grains used as starter cultures for fermentation were obtained from the Department of Food Science (Stellenbosch University,
South Africa) and added to and incubated at 25 C for 24 h. The grains
were retrieved by sieving, the grains inoculated into fresh double
pasteurised milk, and incubated at 25 C for 24 h. This procedure was
repeated for six subsequent days before the grains were considered
active and used as starter inoculum with LAB counts in MRS agar of
approximately 2.5 105 CFU/ml.
Pure cultures of Lactobacillus paracasei subsp. paracasei CHB 2121,
Lactobacillus casei NWL63 and Lactococcus lactis subsp. lactis NM161-4,
strains from the strain bank of the Department of Food Science (Stellenbosch University) were used as single-strain starters. These strains were
previously isolated from 5 indigenous fermented milks from subSaharan African countries, namely Omashikwa from Namibia, Masse
from Mozambique and Ehekapmkaika from Uganda and two commercially fermented milks, namely Chambiko from Malawi and Omaere
from Namibia. Prevalent microbial species were selected for this study
(Schutte, 2013). These single-strain starters were activated by inoculating 1 ml of each stock culture into 20 ml MRS broth (Merck) followed by
incubation for 24 h at 30 C to an A600 nm density of 0.7 to 0.8 as
previously described.
2.2. Milk fermentation
To prepare double pasteurised milk, fresh pasteurised full-cream
milk purchased at a local supermarket was heat treated at 80 C for
15 min. Ker was manufactured by adding 9.0 g of the activated ker
grains into 500 ml of double-pasteurised milk in sterile screw-capped
bottles followed by incubation at 25 C for 72 h.
Milk was also fermented with the pure LAB cultures, namely Lb.
paracasei subsp. paracasei CHB 2121(A), LAB2 Lactobacillus casei
NWL63 (B) and LAB3 Lc. lactis subsp. lactis NM161-4 (C) by adding
aseptically 5 ml of activated cultures into 500 ml of double-pasteurised
milk in sterile screw-capped bottles followed by gentle agitation for
30 s and incubation at 30 C for 72 h.
2.3. Challenge assays
To assess the behaviour of M. bovis BCG when exposed to a mixture
of undetermined metabolites during different stages of ker fermentation and to eliminate the interference of actively growing LAB in the
M. bovis BCG enumerations, the fermenting ker was sterilised either
by microltration or by autoclaving.
50 ml of fermenting ker samples were taken at time intervals (18,
24, 36, 48 and 72 h) and centrifuged (13,000 g for 30 min at 4 C)
and the supernatant collected, centrifuged again and ltered through
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Fig. 1. Viability of M. bovis BCG in ker supernatant (A) and heat-sterilised ker (B) when fermentation was stopped after 6 h (), 12 h (), 18 h (), 24 h (), 36 h (),
48 h () and in control ( *- -). Data is presented as mean and the indicated standard deviations of three replicates.
log after 24 h, whereas the maximum growth of about 8.5 log CFU/ml
and a 1.6% lactic acid, was observed after 32 h.
The growth of LAB and acid production were also monitored during
the processing of traditional madila, fermented milk from Botswana.
The LAB grew from an initial concentration of 7.3 log CFU/ml to a peak
of 9.3 log CFU/ml and a titratable acidity of 0.74 after 24 h of fermentation (Parry-Hanson et al., 2009). After that point, maximum amounts of
antimicrobial compounds accumulated in the media and consequently a
greater antimicrobial effect is expected.
Fig. 2. Survival of M. bovis BCG during fermentation of ker inoculated with M. bovis at
concentrations of 106 CFU/ml (), 104 CFU/ml () and 102 CFU/ml (). pH
changes in ker fermentation is represented by the dotted line.
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Fig. 3. Mycobacterium bovis BCG viable counts (.) and the changes in the pH (-) during milk fermentation in the presence of different LAB starters, namely Lactobacillus paracasei
subsp. paracasei (A), Lactobacillus casei (B), Lc. lactis subsp. lactis (C), and the control in the absence of starter culture (D).
2.2 log, and the pathogen was not detected after 60 h. Similar trends
were observed in the milk inoculated with Lb. casei. M. bovis BCG counts
decreased by 1.8 logs after 48 h of fermentation and was eliminated
after 60 h. In contrast, when Lc. lactis subsp. lactis was used, a 1.1 log
reduction was observed after 48 h of fermentation and 3 log CFU/ml
of BCG was still detected after 72 h of incubation. In milk fermented
without the addition of LAB M. bovis BCG was reduced less than 1 log
after 72 h of incubation.
Milk fermented with Lactobacillus starters has a pronounced inhibitory effect on the M. bovis BCG. According to the carbohydrates fermentation pathway, Lc. lactis is an obligate homofermentative bacterium,
which produces almost exclusively lactic acid from the metabolism of
hexoses, while Lb. paracasei subsp. paracasei and L. casei are hetero fermentative (Ward and Timmins, 1999). Heterofermentative lactobacilli
are characterised by the production of a wide range of compounds as
end products of fermentation, including acetate and ethanol in addition
to the lactic acid (Cintas et al., 2001; Earnshaw, 1992). Therefore, the
differences in respect of the effect of various starters on M. bovis BCG
may be explained by the type of acid present in the medium. Lactic
acid, which has a lower pKa than acetic and propionic acid, will exert
weaker anti-microbial effect than that exerted by a mixture of two or
more organic acids at the same pH environment (Earnshaw, 1992).
Lb. paracasei subsp. paracasei and L. casei are also used as probiotics
and can produce bacteriocins with antimicrobial activity against several
pathogens (Atanassova et al., 2003; Mojgani et al., 2010). The role of
fermentation in the prevention of milk-borne TB not only depends on
the fermentation time but also on the LAB strains predominant in the
starter culture.
The genera Lactobacillus is of great signicance in African dairy
products and was found to be the largest microbial population in most
of the African indigenous fermented milks, including kule naoto, a
Maasai traditional fermented milk, kwerionie and chekapmkaica from
Uganda, nunu (from Ghana and Nigeria) and masse from Mozambique
(Akabanda et al., 2010; Schutte, 2013).
Lc. lactis is well known as a fast acid producer and this was observed
by the shortest coagulation time of 12 h and a fast decrease in the pH
(Fig. 3). Since Lc. lactis is a fast acid producer, it has been suggested
that the use of this strain as a starter would hasten the fermentation
process and, due to the initial acid shock on pathogen cells, would
shorten the survival of the pathogenic microorganisms (Mufandaedza
et al., 2006). However, our results indicated that fast acid production
is the least relevant factor in terms of the viability of M. bovis BCG. Lc.
lactis subsp. lactis is also known as a nisin producer, and this bacteriocin
has been shown to inhibit Mycobacterium sp. (Carroll et al., 2010).
However, the presence of bacteriocins in the fermented products has
not been tested in this study.
3.3. Effect of pH changes during fermentation on M. bovis BCG
The pH changes during fermentation by Lb. paracasei subsp.
paracasei and Lb. casei was very similar and reduced from 6.5 to 5.3
after 24 h. After 60 h, when no M. bovis BCG was detected, the pH of
these milks was 3.6 (Fig. 3). A different pH prole was observed for Lc.
lactis. The pH in this fermented milk decreased suddenly (from 6.7 to
4.43) in the rst 18 h, causing a thick coagulum. The control also showed
a drop in pH although not inoculated with a culture and is possibly the
result of the presence of an inherently present LAB. However, the results
indicate that although the reduction in the pH (due to the presence of
organic acids) generates an unfavourable environment to the M. bovis
BCG, the pH is not the only inhibitory factor on the Mycobacterium.
A steady decrease (of about 1 log every 24 h) in the rst 48 h of
fermentation, followed by a sudden decrease in viable M. bovis BCG
cells (of about 3.8 log) in the interval between 48 h and 60 h, was the
typical behaviour of M. bovis BCG during the fermentation process
when using either ker grains or Lactobacillus strains as starters. In
both cases, undetectable levels of the pathogen were observed after
60 h. The interval between 48 h and 60 h of fermentation was found
to be an important stage for ensuring the safety of the milk product.
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