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International Journal of Food Microbiology 217 (2016) 170176

Contents lists available at ScienceDirect

International Journal of Food Microbiology


journal homepage: www.elsevier.com/locate/ijfoodmicro

Effect of milk fermentation by ker grains and selected single strains of


lactic acid bacteria on the survival of Mycobacterium bovis BCG
C.L.S. Macuamule a, I.J. Wiid b, P.D. van Helden b, M. Tanner c, R.C. Witthuhn d,
a

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.

In developed countries, where milk is pasteurised, the digestive


route of infection by M. bovis has become less important (Thoen et al.,
2006). In contrast, M. bovis is still considered a serious threat to public
health in sub-Saharan Africa (Mller et al., 2013). The high risk of infection in Africa is the result of the high prevalence and endemic status of
the disease in cattle, where control measures are either less stringent or
not applied (Cosivi et al., 1995; Marcotty et al., 2009; Michel et al., 2010;
OIE, 2012) in relation to the consumption of raw milk and derived
products (Ayele et al., 2004; Fetene et al., 2011).
Although surveys on the occurrence of human TB of animal origin in
African countries are scarce, zoonotic TB has been documented in
Ethiopia, Ghana, Mali, Nigeria, Tanzania and Uganda (Mller et al.,
2013). The proportions of M. bovis cases ranged from 0.8 to 20.5%
of the human TB cases (Addo et al., 2007; Cadmus et al., 2006;
Cleaveland et al., 2007; Fetene et al., 2011; Garba et al., 2004; Gumi
et al., 2012; Kazwala et al., 2001; Kidane et al., 2004; Mnanga et al.,
2004; Regassa et al., 2008; OIE, 2012; Oloya et al., 2008). However,
these gures for the prevalence of M. bovis infections in humans are believed to be an underestimated in most countries because of the inadequate isolation and identication procedures being used (Berg et al.,
2009; de la Rua-Domenech, 2006; Rowe and Donaghy, 2008; Thoen
et al., 2006; Wedlock et al., 2002).

C.L.S. Macuamule et al. / International Journal of Food Microbiology 217 (2016) 170176

In Africa, households and small-scale dairies traditionally ferment a


considerable proportion of raw milk using lactic acid fermentation.
The products prepared in this way are consumed in the community or
sold in peri-urban areas (Akabanda et al., 2010; Ashena, 1996; Bille
et al., 2007; FAO, 1990; Gonfa et al., 2001; Isono et al., 1994;
Mugampoza et al., 2011; Parry-Hanson et al., 2009; Savadogo et al.,
2004). Low hygienic standards during milking and the uncontrolled
disease status of cattle make consumption of milk and derivatives
risky for consumers. However, this risk can be reduced by the presence
of lactic acid bacteria (LAB) and their metabolites during the fermentation process (Adams and Nicolaides, 1997; Sun et al., 2011).
LAB exert antagonistic activity against many microorganisms,
including food spoilage micro-organisms and pathogens. The antimicrobial action of LAB in food is attributed to the combined action of a range
of antimicrobial factors produced during the fermentation process,
namely low acidity, organic acids, rapid acid production, bacteriocins,
carbon dioxide, hydrogen peroxide, ethanol, diacetyl, low redox potential and nutrient depletion (Adams and Nicolaides, 1997; Cintas et al.,
2001; Reis et al., 2012; Sun et al., 2011). Acidity is perhaps the most
important inhibitory agent. The major metabolite of LAB is lactic acid
which is responsible for signicant pH changes in their growth environment and is sufcient to antagonise a wide range of microorganisms.
However, other antimicrobial factors may be critically important in
the course of fermentation (Adams and Nicolaides, 1997; Reis et al.,
2012; Sun et al., 2011;). Food antimicrobials are used increasingly as a
primary intervention for inhibition or inactivation of pathogenic microorganisms in foods. The use of naturally occurring anti-microbials in the
food industry can help to reduce the addition of chemical preservatives,
as well as the intensity of heat treatment resulting in food that is more
naturally preserved and better in sensorial and nutritional properties.
LAB have shown to be natural effective anti-microbials in foods, including some traditional fermented African products. For example, it was
shown that during the production of ergo, an Ethiopian fermented
milk, Salmonella enteritedis was reduced by 4 log after 48 h, whereas
Escherichia coli decreased by 3.2 log CFU/ml after 72 h of fermentation
(Tesfaye et al., 2011; Tsegaye and Ashena, 2005). Reductions of E. coli
by 2 to 3 logs were also observed after 48 h of milk fermentation
1using single and paired starter strain combinations obtained from
Zimbabwean traditional fermented milk (Mufandaedza et al., 2006).
Five days of fermentation were necessary to reduce E. coli by 2.7 log
during the production of madila, a traditional milk in Botswana
(Parry-Hanson et al., 2009). Listeria monocytogenes, inoculated into
raw milk, was eliminated after 60 h of fermentation (Ashena, 1994).
The sensitivity of pathogenic and spoilage bacteria to milk fermentation
seems to vary according to the bacterial characteristics, LAB starter
present (Mufandaedza et al., 2006; Tesfaye et al., 2011) as well as
temperature of fermentation and the concentration of unwanted microorganism (Michel et al., 2015).
Limited research has been focused on the behaviour of M. bovis during
the fermentation process. A previous study done on the interaction of
M. bovis with single strains of mesophylic LAB from Ethiopian fermented
milk (Mariam, 2009), showed M. bovis reductions of 5 log after 7 to
14 days of fermentation. It was suggested that the elimination of
M. bovis could be accelerated in the presence of several LAB strains. The
aim of this study was to determine the survival of M. bovis BCG when
exposed to products of ker fermentation collected throughout milk fermentation using either ker grains or selected single-strain LAB cultures
as fermentative starters, in order to evaluate whether milk fermentation
could be an effective measure for controlling the spread of zoonotic TB.
Ker is a fermented milk originating from the Caucasian mountains that
is produced worldwide. In this study, ker was selected for being an
extensively investigated fermented milk, being easy to reproduce (by
using ker grains as starters) without considerable variations between
batches, and having a claimed antimicrobial activity against various food
pathogens (Anselmo et al., 2010; Garrote et al., 2000; Van Wyk, 2001).
As observed in most of African fermented milks, the wide diversity

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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C.L.S. Macuamule et al. / International Journal of Food Microbiology 217 (2016) 170176

MillexRGP 0.22 m syringe lters (Millipore, Ireland). In parallel, 50 ml


of fermented ker were taken at time intervals (6, 12, 18, 24, 36 and
48 h), autoclaved (121 C, 15 min) and stored under refrigeration for a
maximum of 48 h. Samples were evaluated in triplicate. 30 ml of supernatants and sterile ker samples were inoculated with M. bovis BCG (5
6 log CFU/ml nal concentration) and incubated at 37 C for 7 days. Sterile milk inoculated in the same way was used as control culture. The survival of M. bovis BCG was monitored for 12, 24, 46, 60 and 72 h.
To assess the survival of M. bovis BCG and to evaluate the inuence of
the initial level of milk contamination during milk fermentation by a
consortium of mesophilic fermentative strains, dilution series of active
cultures of M bovis BCG were prepared in phosphate buffer (BBLTM
Mycroprep) and 500 l added to the milk at different nal concentrations, namely: (A) 6.1 log CFU/ml, (B) 4 log CFU/ml; and (C) 2.2 log CFU/
ml, and gently mixed at the beginning of ker manufacture. Triplicate
batches of each concentration were prepared and evaluated.
To assess the interaction of M. bovis BCG with individual strains of
LAB during milk fermentation, 500 l of active M. bovis BCG culture
was added to 500 ml of the inoculated milk samples to a nal concentration of approximately 6 log CFU/ml. After a gentle agitation, the cultured
milks were left to ferment at 30 C for a maximum of 72 h. The experiments were repeated in triplicate.
The survival of M. bovis BCG was monitored by enumerating viable
bacteria in each fermenting milk sample over time (at 18 h, 24 h, 36 h
48 h, 60 h and 72 h) after gentle agitation and sampling by pouring
10 ml of the milks in a sterile sampling ask.
2.4. Bacterial enumerations
Samples were taken from dairy products (ker supernatants, sterile
ker, ker and milk fermented by LAB starters) throughout incubation
time and M. bovis BCG enumerated. For this purpose, serial dilutions in
sterile saline solution (0.8% w/v) were spread plated on Middlebrook
7H10 agar supplemented with BACTEC PANTA PLUS (ker and
fermented milk) or not (ker supernatants, sterile ker). Incubation
was performed at 37 C for 21 days in absence and for 28 days in the
presence of the antibiotic supplement, respectively. The detection
limit of the enumeration assays was 10 CFU/ml. All the assays were
performed in triplicate. For lactic acid bacteria counting, appropriate
dilutions were pour plated on MRS agar plates and incubated at 30 C
for 48 h in anaerobiosis (AnaerocultR A Merck, Germany). pH evolution
of the fermenting milks were monitored using a HI 221 Microprocessorbased pH metre at 25 C.
3. Results and discussion
3.1. Effect of ker metabolites on the growth of M. bovis BCG
The survival of M. bovis BCG when exposed to ker supernatant and
ker sterilised by autoclaving is shown in Fig. 1 (A and B). It is evident
that ker supernatant and sterilised ker reduced M. bovis BCG viable
cell counts.
The reduction of M. bovis BCG viable counts increased with the incubation time after which the ker supernatants were collected. In fact,
18 h-old supernatants reduced BCG counts by 0.5 log CFU/ml after
24 h of incubation and 3.3 log CFU/ml remained still viable after 72 h.
24 h-old supernatants reduced BCG by 0.9 log CFU/ml after 12 h of incubation and no viable counts were detected after 24 h. Finally, 48 h-old
supernatants completely inactivated the microorganism as no viable
counts were detected after 12 h (Fig. 1A). It was not possible to collect
ker supernatant at 6 and 12 h of fermentation, as the protein precipitation in the milk occurs at a pH of 4.6, and this pH was reached after
14-16 h of fermentation.
In a similar way, the reduction of M. bovis BCG increased as longer
the incubation time after which the sterile ker samples were collected.
In 6 and 12 h-old sterile ker, BCG counts decreased by 0.2 and 0.7 log

CFU/ml after 12 and 24 h of incubation, respectively, viable counts


being kept over 3 log CFU/ml after 72 h (Fig. 1-B). With 24 h-old sterile
ker, counts were reduced by 3 log CFU/ml after 12 h.
When exposed to the sterilised ker fermented for 48 or 36 h,
M. bovis BCG was eliminated after 12 h of incubation. When exposed
to the sterilised ker for a shorter fermentation period of 24 h, viable
counts were reduced by 3 log (50%) after 12 h and were not detected
after 24 h of incubation. Ker fermented for 18 h reduced M. bovis
BCG counts by 3.7 log (37%) after 24 h of incubation, while after 48 h
of incubation the bacilli were reduced by 4.6 log. The bacteria could
not be detected after 60 h. Sterile ker fermented for 6 and 12 h had a
minor effect in reducing M. bovis BCG counts. In this group, the bacteria
was reduced by less than 0.2 log after 12 h of incubation and by less than
0.7 log after 24 h of incubation. The bacilli remained at concentrations
greater than 3 log CFU/ml after 72 h (Fig. 1-B). The results showed clearly that the degree of inhibition of M. bovis BCG increased with prolonged
fermentation by the ker, while the bacteria persisted in the control
sample of sterile milk for more than 7 days.
The ker extracts collected after 24 h dramatically reduced the viability of M. bovis BCG. The inhibiting compounds remained active after
heat-inactivation of ker by autoclaving, indicating thermo-stability of
the antimicrobial compounds.
In this study, it was shown that direct interaction of LAB and M. bovis is
not a necessary condition to induce antimicrobial effect on the pathogen,
as the lter-sterilised ker was active against M. bovis BCG. Bacteriocin
production and accumulation of different organic acids resulting from
heterolactic fermentation of ker may have contributed to the lethal
effect. A bacteriocin produced by Lactobacillus plantarum isolated from
ker has been identied previously (Powell et al., 2006). This bacteriocin,
which was found to be stable to autoclaving conditions, as well as to low
pH values, could have contributed to the lethal effect on M. bovis BCG.

3.2. Inhibition of M. bovis BCG by fermenting ker


Fig. 2 indicates the results of the inhibitory effect of natural fermentation of ker on M. bovis BCG. When the mycobacterium growth medium
was inoculated at the beginning of fermentation, at high bacterial concentration (106 CFU/ml), medium concentration (104 CFU/ml) and low
concentration (102 CFU/ml), a slight reduction of 0.4 log of M. bovis
BCG was observed after 24 h of fermentation, irrespective of the initial
load. After 24 h, the response differed between the three groups. A reduction of approximately 1.5 log CFU/ml was observed at 48 h in the high
and medium inoculums, while in the low inoculum size group, M. bovis
BCG was not detected. Greater reduction of M. bovis BCG was observed
when the fermentation times increased, and the fermentation time
required for total elimination of the M. bovis BCG was 60 h. The fermentation time, and consequently, the accumulated metabolites exerted a
higher effect on M. bovis BCG survival than the initial contamination
level. The fermentation time required for anti-mycobacterial effect may
be related to the continued replication of LAB population during the
course of fermentation. According to Adams and Nicolaides (1997) to
produce sufcient acid to achieve a notorious inhibition of bacterial
pathogens requires relatively large numbers of LAB to be present and
the resulting decrease of pH. Analysing the pH changes during ker fermentation, we observed that with the reduction of pH values from 6.7 to
4.4 (at 24 h) only a slight growth reduction of 0.4 log CFU/ml of M. bovis
was registered. At 48 h of fermentation and a pH of 3.9, reductions of 1.5
to 2 log CFU/ml occurred, while no M. bovis BCG survived when the pH
decreased to 3.7 at 60 h of fermentation (Fig. 2). This is an indication of
the inuence of pH on the fate of M. bovis BCG. However, it is still not
clear whether the lethality is due to the fast decrease of pH or to the
long exposition of the mycobacteria to certain acid environments.
The growth of the LAB population during ker fermentation was
studied by Liu and Lin (2000). They observed that from an initial
concentration of 6 log CFU/ml, the LAB population increased in only 1

C.L.S. Macuamule et al. / International Journal of Food Microbiology 217 (2016) 170176

173

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.

3.2.1. Inhibition of M. bovis BCG during milk fermentation by single-strain


LAB starters
Fig. 3 shows the anti-microbial activity against M. bovis BCG during
milk fermentation using pure cultures of Lb. paracasei subsp. paracasei
CHB 2121; Lactobacillus casei NWL63 (B) and Lc. lactis subsp. lactis
NM161-4 (C). When the single-strain cultures were used as starters
for fermentation of contaminated milk, a slight reduction on M. bovis
BCG viability was observed after 24 h of fermentation, as M. bovis BCG
reduced by 0.26 log, 0.2 log and 0.36 log cycles in the groups A, B and
C, respectively. Similar reduction of M. bovis BCG viability (over 1 log
CFU/ml) was observed after 36 h in the groups A and B, and a slight decrease of viable counts occurred in group C. After 48 h of fermentation,
milk fermented with Lb. paracasei subsp. paracasei reduced M. bovis by

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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.

C.L.S. Macuamule et al. / International Journal of Food Microbiology 217 (2016) 170176

At 60 h of fermentation, the pH of the milks was around 3.7. M. bovis is a


robust microorganism and its tolerance to acidic environments is well
documented. Rao et al. (2001), investigating the biochemical and physiological response of some species of mycobacteria to acidic pH, found
that M. bovis BCG adopts an intracellular pH homeostasis able to regulate the internal pH near neutral, even when exposed to decreasing external pH. However, the maximum variation tolerated was by
approximately 2.3 units. In the present study, the variation of the initial
pH in more than 3.0 units occurred after 48 h, which proved to be critical
for the survival of M. bovis BCG. Although an acidic environment leads to
inhibition of bacterial cell growth, other cell wall damaging factors
present in a fermenting environment such as bacteriocins and organic
acids may also have an effect on cell viability.
The results found in the current study are not in complete agreement
with the study performed by Mariam (2009). In that study, M. bovis was
eliminated by fermenting milk for a minimum of 7 days at room
temperature. The inoculum size of the LAB starter cultures used was
0.01%, which is a twofold lower than the concentration used in this
study, and could have retarded the fermentation process. Strainrelated differences may also occur, since the reference strain M. bovis
ATCC 19210 was used in that study.
3.4. Effect of fermentation time on the survival of M. bovis BCG
Similar to the observations during ker fermentation, by using the
two lactobacilli strains as starters, the average reductions of M. bovis
BCG CFU's achieved at 18 h and 24 h of fermentation were 0.4
and 1.2 logs, respectively, while the minimum time required to achieve
undetectable levels of M. bovis BCG was 60 h. This highlights the importance of long-term fermentation in the control of this slow growing
mycobacterium. The maximum inhibition of M. bovis BCG observed at
24 h of fermentation, namely 0.4 log, indicated that when raw milk
contains viable mycobacteria, short-term fermentation for a period of
24 h or less may still represent a health risk. Results from this study
show that long term fermentation under certain conditions has the potential to inactivate slow growing mycobacteria. Published information
about African traditional fermented milk indicates that 68% of these
products are fermented for 3 days or more. Additionally, some traditional
practises such as adding fresh raw milk to the curd in order to reduce the
bitterness, might re-introduce the eliminated pathogen even during
extended fermentation.
There is a lack of published information that estimates the amount of
M. bovis excreted into milk from infected cows. Data on the amount
necessary to cause human disease by ingestion is not available.
O'Reilly and Daborn (1995) summarised research ndings based on
animal models, which suggested that 3 to 300 cells of M. bovis were
able to cause pulmonary infection by inhalation, whereas one thousand
to 18 million times larger doses would be required to cause TB by ingestion. These required doses are expected to be much lower in immune
decient populations that characterise most of sub-Saharan African
countries. Such information would be necessary to accurately evaluate
the risk of TB by ingestion of indigenous fermented milk. Furthermore,
fermented milks represent a considerable part of the diet of most
African communities, with more extensive consumption patterns
throughout Eastern African countries where this product is a staple
food and a traditional weaning food for children. In some of the pastoralist communities, the consumption of fermented milk can increase
to 3 to 5 l daily (Isono et al., 1994, Mathara et al., 2004), meaning that
when viable M. bovis is present, even in low concentrations, the high
daily intake of contaminated products could increase the risk of zoonotic
TB from M. bovis by ingestion.
4. Conclusion
It is evident that the fermentation process of raw milk by lactic
acid bacteria contributes to reducing the viability of M. bovis BCG

175

present in milk. The inhibitory effect is achieved by increasing the


fermentation time. Fermentation for 24 h or shorter did not ensure
the inactivation of M. bovis BCG, even when the bacterium was present
in low concentrations.
The inhibition of M. bovis BCG may not be attributed to a single
factor. Non-bacterial, heat-stable components produced during milk
fermentation, as well as the LAB populations predominant in the starter
cultures, are factors that play a key role in the fate of M. bovis BCG. The
inhibition of M. bovis BCG by mixed fermentative strains (present in
ker grains), as well as by Lactobacillus fermented milks, was greater
than that manifested by Lactococcus fermented milks.
The fermentation of milk contaminated with M. bovis may be a measure to reduce the spread of zoonotic TB to humans. However, to ensure
safety of naturally fermented milk in Africa, fermentation should be
combined with other hurdle technologies such as milk pasteurisation
or boiling.
Acknowledgements
The authors thank the German Research Foundation (DFG) Grant
number TA 668/1-1, for the nancial support of this study. The technical
support from Dr. Michelle Cameron and Bertus Viljoen from the Department of Food Science and the Division of Molecular Biology and Human
Genetics, Faculty of Health Sciences, all at the Stellenbosch University is
greatly acknowledged.
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