Food Microbiology 63 (2017) 28e34

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

The effect of bacteriophages on the acidication of a vegetable juice

medium by microencapsulated Lactobacillus plantarum
Claude P. Champagne a, b, *, Sylvain Moineau c, d, Sonia Laeur a, Tony Savard a
a
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada, 3600, Casavant Blvd W., Saint-Hyacinthe, Quebec, J2S 8E3, Canada
b
Institute of Nutrition and Functional Foods (INAF), Universit e Laval, 2440, Hochelaga Blvd., Quebec City, Quebec, G1V 0A6, Canada
c 
Departement de Biochimie, Microbiologie et bio-informatique, Facult e des Sciences et g
enie, Universit
e Laval, 1045, Avenue de la M
edecine, Quebec City,
Quebec, G1V 0A6, Canada
d 
Felix dH
erelle Reference Center for Bacterial Viruses, Facult
e de Medecine Dentaire, Universit e Laval, 2420, Rue de la Terrasse, Quebec City, Quebec, G1V
0A6, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Starter cultures are increasingly being used for the production of sauerkraut, kimchi and other fermented
Received 8 August 2016 vegetables. The goal of this study was to determine whether the microencapsulation of a bacterial culture
Received in revised form can prevent phage infection during vegetable fermentation. Lactobacillus plantarum HER1325 was
18 October 2016
microencapsulated in alginate beads. Some beads were used without further processing, while others
Accepted 28 October 2016
Available online 28 October 2016
were freeze-dried prior to testing. Fresh beads (diameter of 2 mm) and dried cultures of the lactobacilli
(particle size of 53e1000 mm) were added to a vegetable juice medium (VJM) at 1
107 CFU/mL. The
virulent phage HER325 was added at an initial titer of 1
104 PFU/mL. In the absence of phages, the pH
Keywords:
Fermented vegetables
of the vegetable juice dropped to 4.2 after 40 h of fermentation at 19  C. In the presence of phage
Lactic acid bacteria HER325, acidication by both the non-microencapsulated and microencapsulated starter cultures
Microencapsulation stopped after 24 h. In all assays, the alginate particles dissolved during the 40 h of VJM fermentation.
Alginate When 15 g/L of calcium chloride was added to the VJM, the alginate beads did not dissolve and signicant
Bacteriophage phage protection was observed. The results suggest that phage-protected microencapsulated starter
cultures can be used for vegetable fermentation if means are taken to prevent them from dissolving
during acidication.
2016 Published by Elsevier Ltd.

1. Introduction (Jung et al., 2013; Yoon et al., 2001).

Lactic fermentation of vegetables can be carried out by a variety
of bacteria, but the main genera are Lactobacillus and Leuconostoc
(Jung et al., 2013). It is well documented that bacterial food
fermentation can be impaired by virulent bacteriophages (phages)
(Moineau and Le vesque, 2005). For example, several phages active
against Lactobacillus (for a review, see Villion and Moineau, 2009)
and Leuconostoc (for a review, see Kot et al., 2014), have been iso-
lated. It is therefore not surprising that various studies have re-
ported the presence of phages in sauerkraut (Barrangou et al., 2002;
Lu et al., 2003a; Yoon et al., 2002; Zhang et al., 2015), fermented
cucumbers (Lu et al., 2003b, 2010; Yoon et al., 2007), and kimchi
* Corresponding author. Saint-Hyacinthe Research and Development Centre,
Agriculture and Agri-Food Canada, 3600, Casavant Blvd W., Saint-Hyacinthe,
Quebec, J2S 8E3, Canada.
E-mail address: claude.champagne@agr.gc.ca (C.P. Champagne).
As a result, phages are likely to contribute to the microbial
ecology of sauerkraut fermentation. When a starter-free fermen-
tation based on the wild microbiota of the vegetable is carried out,
the microbial diversity allows fermentation to proceed in the
presence of phages. Microbial selection also occurs, including the
emergence of phage-resistant strains. However, such microbial
adaptation takes time and could be responsible for some of the
variability observed in this type of fermentation (Barrangou et al.,
2002).
There is growing interest in avoiding the variability linked to
spontaneous fermentation caused by indigenous vegetable micro-
biota. To standardize fermentation processes, starter cultures con-
taining lactic acid bacteria have been developed for sauerkraut
(Mudgal et al., 2006; Gardner et al., 2001) and kimchi (Moon et al.,
2013). Furthermore, with the development of low-sodium sauer-
kraut, the selective effect of salt favoring the lactic acid bacterial
population is reduced, and inoculation with a starter culture

http://dx.doi.org/10.1016/j.fm.2016.10.036
0740-0020/ 2016 Published by Elsevier Ltd.
 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34
becomes desirable (Yoon et al., 2002; Mudgal et al., 2006). How-
ever, if the same lactic starter culture is repeatedly inoculated, it is
expected that specic virulent phages could emerge and adversely
affect the vegetable fermentation process. This would result in
killing the added starter cultures (Samson and Moineau, 2013), as
has been observed in the dairy industry (Pujato et al., 2015; Johnson
and Lucey, 2006), although the stability of phages in plant-based
matrices and environments is not well documented. Moreover,
phages of Leuconostoc and Lactobacillus have mostly specic host
ranges (Barrangou et al., 2002; Kleppen et al., 2011; Johnson and
Lucey, 2006; Marco  et al., 2012), suggesting that industrial acidi-
cation problems linked to phage infection could be addressed by
inoculating with carefully selected strains, by using a mixture of
strains and/or by rotating the cultures (Moineau and Le vesque,
2005). As with fermented dairy products, these approaches will
likely generate variability in the acidication and avour proles.
It has been shown that encapsulating lactic acid bacteria in
alginate gels protects them against phages (Steenson et al., 1987)
and that milk can successfully be fermented by alginate-
microencapsulated (ME) lactococci in the presence of phages
(Champagne et al., 1992). What is not known, however, is whether
this strategy can also be used to ferment vegetables. Alginate gel
particles do not affect the sensory properties of dairy foods if the
particle size is below 70 mm (Hansen et al., 2002; Sheu et al., 1993),
but they are more acceptable in foods with a heterogeneous
structure, such as dry sausages (Muthukumarasamy and Holley,
2006). This could be the case with sauerkraut or kimchi, but it is
unknown to what extent the particle size of ME starters will in-
uence the acidication rate and, potentially, consumer acceptance
of fermented vegetables.
The goal of this study was to determine whether a Lactobacillus
plantarum culture microencapsulated in alginate particles of vary-
ing sizes can successfully complete lactic acid fermentation in a
vegetable-based medium in the presence or absence of lytic
phages.
2. Materials and methods
2.1. Production of Lactobacillus plantarum reference cultures
Lactobacillus plantarum HER 1325 was obtained from the Fe lix
dHerelle Reference Center for Bacterial Viruses (www.phage.
ulaval.ca). The strain was plated on MRS (deMan-Rogosa-Sharpe)
culture medium (Difco, Becton Dickinson, Franklin Lakes, NJ, USA)
at an inoculation rate of 1% from a milk-glycerol stock culture
stored in vials and frozen at 80  C. The stock culture was prepared
by mixing the following at a ratio of 1:2.5:2.5: Lactobacillus culture,
20% (w/w) reconstituted skim milk, and 20% (w/v) glycerol. The
skim milk and glycerol were heated beforehand at 115  C for
10 min. For autoclaving, a temperature probe was placed in a
control bottle that was same size and held the same volume of
liquid as the microbiological medium. The heating times reported
are therefore the actual holding times for the liquids at the tem-
perature indicated. One thawed vial was used to inoculate 100 mL
of MRS medium, and the resulting suspension was incubated at
30  C for 14 h. At the end of the incubation period, the pH was
4.4 0.2, and the viable count was 1.9 0.5
109 CFU/mL.
2.2. Immobilization of a fresh culture of Lb. plantarum in alginate
(fresh alginate beads)
A sterile alginate solution (20 g/L; Sobalg FD 126, Grindsted) was
mixed with an equal volume a fresh MRS-grown Lb. plantarum
culture prepared as described above. The resulting suspension was
added dropwise, using a syringe and a 20-gauge needle, to a stirred
29
solution (with a magnetic stirring bar) of 11 g/L calcium chloride
dihydrate (VWR, Montreal, QC, Canada) to which 1 g/L peptone (BD,
Franklin Lakes, NJ, USA) was added, and the pH was adjusted to 7.0.
The ratio of bacterial suspension to alginate:CaCl2 solution was 1:5.
The beads were left without agitation in the CaCl2epeptone solu-
tion at 4  C for 30 min and then collected in a sterile sieve.
2.3. Production of Lb. plantarum biomass (starters)
The MRS for the fermentations was prepared as recommended
by the manufacturer. After sterilization and just prior to inocula-
tion, 10 mL of a 500 g/L CaCl2 solution was added. This 2.5 g/L
supplementation was used to maintain rmness of the beads dur-
ing fermentation. Two biomass production processes were used: 1)
traditional free-cell fermentation followed by centrifugation to
concentrate the Lb. plantarum culture; and 2) addition of cell-
containing alginate particles to the growth medium followed by
an incubation period. For the alginate-based process, the fermen-
tation medium (MRS) was inoculated with the ME cells at a rate of
9% (200 g of cell-containing beads added to 2 L of medium). For
free-cell fermentation, to ensure inoculation with the same volume
and quantity of cells, 100 mL of fresh MRS culture was mixed with
100 mL of 1 g/L peptone, and this 200 mL cell suspension was
placed in the fermentor. This resulted in similar total cells inoc-
ulated in the fermentor (Table 1), but the CFUs per mL of the free
cell system is lower than in the alginate system, because the free
cells distribute themselves throughout the growth medium, while
the cells in the beads remain concentrated in the alginate gel.
The fermentations were carried out in BioFlo 3000 systems
(New Brunswick Scientic, Eneld, CT, USA) at 30  C for 15 h, and
the pH was maintained at 6.0 by the addition of 5 M KOH and 5 M
NH4OH in a 5:1 proportion. The medium was agitated at 60 rpm,
but agitation was increased to 100 rpm during neutralization by the
addition of alkali. At the end of fermentation, the concentrated
suspension of ME cells was obtained by collecting 200 g of alginate
beads using a sterile sieve. For the concentrated suspension of free
cells, 2.2 L of the fermentation medium was centrifuged (Avanti J-
20 XPI, Beckman Coulter, Mississauga, ON, Canada) at 5000g for
15 min. The cell pellet was then mixed with 200 mL of sterile 1 g/L
peptone.
2.4. Freeze-drying
The freeze-drying medium was prepared (w/w) with the
following nal concentrations: 200 g/L skim milk powder (Crino
low-heat skim milk powder, Agropur, Granby, QC, Canada), 50 g/L
sucrose, and 10 g/L casitone hydrolysate (Difco). The medium was
heat-treated at 60  C for 30 min and stored at 4  C for a maximum
of 24 h. Next, the concentrated free cells or beads were mixed with
the freeze-drying medium (1:1) supplemented with 1.75 g/L
ascorbic acid (Sigma, St. Louis, MI, USA). Each mixture was agitated
at 25  C for 30 min and then stored at 40  C before being freeze-
dried (Caltech FTS Systems, Stone Ridge, NY, USA). The freeze-
drying conditions were as follows: 4 h at 40  C under atmo-
spheric pressure, 16 h at 0  C under 100 mTorr vacuum, 16 h at 20  C
under 100 mTorr vacuum, and nally 24 h at 20  C under 10 mTorr
vacuum. After freeze-drying, the free-cell culture contained
7.5
1010 CFU/g, and the immobilized-cell culture contained
6.7
1010 CFU/g. The two dried cakes thus obtained were then
ground and sieved (Canadian Standard Sieve Series, W.S. Tyler, St.
Catharines, ON, Canada) to retain powders with particle sizes be-
tween 53 and 90 mm (small particle size) and between 500 and
1000 mm (large particle size).
Taken together, the following Lb. plantarum culture powders
were obtained: free-cell powder with a small particle size (FPSm),
30 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34

Table 1
Effect of the fermentation technology (free-cell or microencapsulated/alginate bead states) on biomass production of Lactobacillus plantarum HER1325 in MRS medium
supplemented with 2.5 g/L of CaCl2.

Parameter Alginate bead system Free-cell system

CFU/g of CFU/mL of Total CFUs in fermentor: 200 g beads 2 L CFU/mL of Total CFUs in fermentor: 2.2 L of
beads medium medium medium medium

Initial population 1.6
109 0 3.2
1011 1.5
108 3.3
1011
Population at the end of the 3.2
1010 6.3
108 7.8
1012 6.1
109 1.3
1013
fermentation
% of free cells 17% 100%

free-cell powder with a large particle size (FPLa), alginate-bead

powder with a small particle size (APSm), and alginate-bead
powder with a large particle size (APLa). The powders were
placed in glass bottles under nitrogen ush and stored at 4  C until
use.

2.5. Preparation of the phage lysates

Lb. plantarum phage HER325 was also obtained from the Fe lix
dHerelle Reference Center for Bacterial Viruses (www.phage.
ulaval.ca). Phage lysates were obtained as follows. MRS medium
supplemented with 1.1 g/L CaCl2 and 0.95 g/L MnCl2 was inoculated
at 1% with a fresh culture of Lb. plantarum HER1325 and 0.5% (v/v)
of the phage suspension. The culture was incubated at 30  C for 16 h
or until lysis. After centrifugation, the supernatant was ltered
(0.45 mm) and stored at 4  C until use.

2.6. Preparation of vegetable juice medium

Fig. 1. Effect of the addition of 5 g/L yeast extracts (YE), 0.2 g/L minerals (M) and 1 g/L
Organic onions, carrots, and cabbages (certied organic by the Tween 80 (T) to the vegetable juice (VJ) base medium on the acidication of Lacto-
Organic Crop Improvement Association) were rst processed bacillus plantarum HER1325. Error bars represent the standard error of the mean
individually into juice using a juice extractor (model 40768, Santos, (SEM).
Vaulx-en-Velin, France), and the juices were coarsely ltered to
remove as much pulp as possible. The juices were then stored
was not detrimental to cell viability (Gardner et al., 2001). Certain
at 20  C and thawed in the refrigerator (4  C) just prior to use. A
assays were also carried out using fresh cultures that had not been
vegetable juice blend was prepared from 612 g of carrot juice, 118 g
centrifuged or freeze-dried. These fresh cultures of free cells were
of cabbage juice, 35 g of onion juice, 20 g of salt Windsor; Pointe
inoculated directly into the VJM in what was designated as the
Claire, QC, Canada, and 235 g of deionized water. A blend of vege-
liquid free-cell (LiF) treatment. Cells freshly microencapsulated in
table juices was prepared instead of a single juice. This blend of
alginate were also prepared, with beads containing lactobacilli at
vegetables was prepared in line with commercial products (Cald-
1.6
109 CFU/g; this constituted the alginate bead (AB) inoculum.
well BioFermentation, Canada Ste-Edwidge, QC, Canada). Using a
The diameter of the fresh AB cultures was approximately 2 mm,
blend of vegetable substrates is also in line with many Kimchi
which was between two and four times larger than the diameter of
products are also made of multiple vegetables. In some assays, to
the APLa (alginate-encapsulated cells in powder with large parti-
improve bacterial growth and acidication, the blend was supple-
cles) cultures. The bottles containing 250 mL of heat-treated VJM
mented with 5 g/L yeast extract (Difco), 1 g/L Tween 80 (Fisher,
were inoculated with the rehydrated culture powders (FPSm,
Ottawa, ON, Canada), 0.2 g/L MgSO4 (Mallinckrodt, Chestereld,
APSm, FPLa, APLa), as well as the LiF or AB cultures, to obtain an
Derbyshire, UK), and 0.05 g/L MnSO4 (Mallinckrodt) (Fig. 1). This
initial population of 1
107 CFU/mL of Lb. plantarum. Half of these
blend was referred to as the vegetable juice medium (VJM). The
inoculated VJM bottles were supplemented with the phage lysate to
VJM was adjusted to pH 6.7, bottled in 250-mL portions, and heat-
obtain an initial titer of 1
104 PFU/mL (multiplicity of infection of
treated at 60  C for 30 min before being stored at 4  C for a
0.001).
maximum of 24 h. Unless otherwise stated, the VJM was not further
enriched. However, for certain assays designed to prevent bead
dissolution, the VJM was supplemented with CaCl2 at 15 g/L. In 2.8. Measurement of acidication
commercial sauerkraut or kimchi productions, there is no heat
treatment of vegetables. However we carried out the above mild The acidication tests were carried out using the BioGe nie
thermization process of VJM to better control the wild microbial automated pH measurement system (ID 3.00; Quebec City, QC,
population during the fermentation. Canada). The system consists of six electrodes (pHC2401-8; London
Scientic, St. Thomas, ON, Canada) that had been disinfected with
2.7. Inoculation of vegetable juice medium 300-ppm chlorine and six small stainless steel containers (steril-
ized at 121  C for 10 min) placed in an incubator (ThermoFisher
The dried cultures (1 g) (FPSm, APSm, FPLa, or APLa) were Scientic, Waltham, MA, USA) maintained at 19  C. The containers
rehydrated for 10 min in 10 mL of sterile NaCl (20 g/L) at 25  C being were placed on a stir plate (Labline Instruments, Tripunithura,
agitated with a magnetic stirring bar. This rehydration procedure Kochi, India). To each container was added 250 mL of heat-treated
 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34
VJM, along with a magnetic stirring bar to ensure the gentlest
agitation possible. The pH electrodes were inserted into the con-
tainers, and everything was kept at the same height by the
container lids. The pH measurements were taken at 15-min in-
tervals until 64 h of incubation had passed. Before the pH elec-
trodes were inserted, they were calibrated individually using two
buffer solutions (pH 4.0 and 7.0). To prevent phage contamination,
the electrodes and containers were immersed in a 300-ppm chlo-
rine solution for 30 min after each use.
2.9. Bacteriophage counts
Phage titers were determined using the classical double layer
method. The bottom layer consisted of MRS supplemented with
1.5% agar (Difco), 1.1 g/L of CaCl2, and 0.95 g/L MnCl2 (MRS-Ca-Mn).
The top layer consisted of MRS supplemented with 0.75% agar, 1.1 g/
L CaCl2, 0.95 g/L MnCl2, and 2.5 g/L glycine (Mallinckrodt). The
glycine was added to increase the size of the phage plaques.
An aliquot (5 mL) of VJM to which the phages had been added
was ltered (0.45-mm) (Millex, Millipore, Etobicoke, ON, Canada) to
remove bacteria. The ltrate was serially diluted in sterile 1 g/L
peptone, and 0.1 mL of each dilution was added to warm (50  C)
MRS-Ca-Mn top agar containing 50 mL of a fresh culture of Lb.
plantarum HER1325. The medium was then mixed and poured
uniformly onto the bottom agar layer. Plates were incubated at
30  C for 48 h, after which the phage plaques were counted.
2.10. Bacterial counts
The bacterial counts were performed at the end of incubation,
i.e. after 40 h for the VJM and after 64 h for the VJM supplemented
with CaCl2 per the recommendations of Champagne et al. (2011).
Two analyses were required to estimate populations in the freeze-
dried alginate powders: analysis of the total bacterial population
and analysis of the free-cell population. The difference between
those two counts provided an estimate of the population of
immobilized cells. To estimate the free-cell fraction, samples were
recovered by ltering the juice through a sterile sieve with a pore
size of 50 mm. Because the two series of alginate particles were
larger than 50 mm, the sieve retained the immobilized cells, and
only the free cells (or the cells released by the depolymerization of
alginate particles) were present in the ltrate.
For the analyses, 1 g of beads, or 1 mL of the sample (ltered or
unltered), was suspended in 100 mL of 10 g/L sodium citrate (A&C,
Montreal, QC, Canada) at pH 6.0 for 15 min. This incubation was
followed by a 30 s of high-shear homogenization at 27,000 rpm
(Omni Kennesaw, GA, USA). The resulting cell suspensions then
underwent serial 1:9 dilutions in 1 g/L peptone water (Difco).
Enumeration of the diluted cell suspensions was carried out in MRS
Agar (Difco) using the pour plate method. Plates were incubated at
30  C for 48 h in an aerobic atmosphere. The subsequent dilutions
of these samples were performed in 0.1% peptone.
2.11. Determination of lactic acid by HPLC
Acids were determined at time 0 as well as at the end of the
incubation period. The VJM samples were centrifuged (GS-6R
centrifuge; Beckman Coulter) at 3750 rpm (20 min, 4  C), and the
supernatant was ltered (0.45 mm). Acids were determined by
high-performance liquid chromatography (HPLC) using a Waters
717plus autosampler (Mississauga, ON, Canada), an Aminex HPX-
87H column (Bio-Rad, Mississauga, ON, Canada) maintained at
45  C and coupled with a Micro-Guard Cation H precolumn (Bio-
Rad) and a Waters 996 photodiode array detector (analysis at
214 nm). The eluent was 0.008 N sulfuric acid with a ow rate of
31
0.4 mL/min through a Waters 600 pump. Lactic acids was deter-
mined using external standards, and samples were integrated by
means of the Waters Empower data acquisition system.
2.12. Statistical analyses
All experiments were independently replicated three times.
Statistical analyses were carried out GraphPad InStat (San Diego,
CA, USA) software, version 3.0 for Windows. The data in scientic
format were converted to logarithmic format before undergoing
One-way ANOVAS (Student-Neuman-Keuls test). Signicant dif-
ferences were reported at an alpha of 5%.
3. Results and discussion
3.1. Biomass production
In the traditional process with free cells, the Lb. plantarum
population increased 40-fold (Table 1). This yield is on the high side
for typical MRS-grown cultures (Kreft et al., 2001) and is likely due
to the use of pH control during fermentation. Overall, similar CFUs
in the two bioreactors were obtained (Table 1). In the alginate bead
system, most of the biomass was concentrated in beads repre-
senting 9% of the volume of the fermentor (on a w/w basis), which
resulted in high CFU/g counts in the beads themselves and low
counts of free cells (Table 1). Even higher viable cell counts could be
obtained in the beads if the spent medium was replaced by a fresh
medium and another fermentation was carried out (Maitrot et al.,
1997). However, optimal bead biomass was not the goal of this
study, and the process was limited to a single fermentation.
Overall, in the 2.2 L of the fermentor, there was no signicant
difference in total viable Lb. plantarum counts between the free-cell
system and the alginate-based system. However, there was a 17%
content of free cells in the alginate-based system, mainly owing to
cell release from the beads (Champagne et al., 1993). The cell-
release level was lower than previously recorded with a yogurt
starter (Champagne et al., 1993), which suggested that yields could
be strain-dependent. It should also be mentioned that, in addition
to the phage protection, this ME technology offers other benets
over free cells which are 1) biomass production without centrifu-
gation, 2) the possibility to air-dry the cultures instead of the
traditional freeze-drying process, 3) ME cells can be more stable
during storage and 4) increased survival to gastro-intestinal con-
ditions (Champagne, 2006).
Here, the beads were recovered and the free cells from the
alginate-based system were discarded. Since only the beads were
dried, free-cell and ME CFUs of the dried products showed that 97%
of the cells in the APLa and APSm cultures were microencapsulated.
3.2. Control assays on bacteriophages
Because the VJM was heat-treated at only 60  C and for only
30 min (slightly below pasteurization conditions) and phages of
lactic acid bacteria can survive that kind of heat treatment (Chopin,
1980), there was concern that Lb. plantarum phages might naturally
be found in the VJM. However, no phage plaques on Lb. plantarum
HER1325 were found in the VJM used for our assays (data not
shown). Thus, there were no wild phages against Lb. plantarum in
our VJM which could interfere with our experiments.
Sample treatment and phage dilutions are typically carried out
by agitating bottles or test tubes, which are relatively gentle ho-
mogenization procedures. Analysis of lactic cultures requires strong
homogenization to break down the food matrix and clumps or
chains of cells (Champagne et al., 2011). We therefore examined
whether the high-shear homogenization carried out in our sample
32 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34

analysis technique (generator probes operated at 27,000 rpm)

affected the phage titers. Only a 10% drop in plaque-forming units
was observed after the 2-min high-shear homogenization treat-
ment, indicating that phage HER325 was stable in the analytical
methodology used.
Lastly, we examined whether the VJM itself contained com-
pounds that would naturally inactivate phage HER325, thereby
reducing its infectivity. The VJM was inoculated with phage
HER325 and incubated at 19  C for 48 h. The initial phage count was
2.8
103 PFU/mL. The count remained identical without agitation
of the VJM during the incubation period but dropped to
1.2
103 PFU/mL when constant agitation with a magnetic stirring
bar was carried out. These data suggest that phage HER325 was
stable in VJM under our experimental conditions.

3.3. Vegetable juice medium formulation

Acidication of the initial vegetable juice blend was not rapid

(Fig. 1) with the strain Lb. plantarum HER1325, even though the
inoculation level of the FPLa (free cells in powder with large par-
ticles) cultures was 1
107 CFU/mL (Fig. 1). This inoculation level is
relatively high for typical processes (Gardner et al., 2001), and we
decided not to increase it. Nevertheless, we kept this bacterial
strain because we had a virulent phage that was capable of
infecting it and that could persist in our experimental conditions
(see above).
Yet there was a concern that an extended incubation period
would allow microbial contaminants to develop. Hence, attempts
were made to improve the acidication rate. Supplementation with
yeast extract, Tween and minerals signicantly improved the
acidication rate. These ingredients were selected because of their
recognized benets to the growth of lactobacilli in commercial Fig. 2. Effect of the addition of phage HER325 on vegetable juice medium (VJM)
media such as MRS. Therefore, the nal VJM used contained the acidication by Lactobacillus plantarum HER1325. Note: A Alginate-micro-
various vegetable juices as well as the supplements. Because of encapsulated cells; B Beads; F Free cells; Li Liquid; P Powder; Sm Small
particles. Error bars represent the standard error of the mean (SEM).
costs, adding these ingredients would not be recommended in a
commercial production formulation, but it was useful in this proof
of concept study to assess the effect of phages on growth and Table 2
acidication of Lb. plantarum. Phage HER325 titers in the fermented media.

Medium Culture type Phage count (PFU/mL)

3.4. Acidication in vegetable juice medium
VJM None 1.2
103 d
FPSm 2.7
109 a
Adding alginate particles to a food matrix will inuence its VJM FPLa 5.1
109 ab
appearance and texture. Although reducing particle size can miti- APSm 2.9
109 a
gate sensory problems, smaller particles could be less efcient at APLa 3.0
109 a
exploiting the benets of microencapsulation (Sheu et al., 1993; LiF 1.7
1010 b
AB 5.8
109 ab
Sheu and Marshall, 1993). Different particle sizes and formats
VJM CaCl2 AB 2.3
108 c
were therefore tested to assess their efciency in protecting against
A Alginate-microencapsulated cells; B Beads; F Free cells; La Large particles;
phages.
Li Liquid; P Powder; Sm Small particles.
In the absence of phages, the cultures acidied the VJM to pH 4.4 a-d: Phage count values followed by the same letter are not statistically different
within 40 h. To mimic commercial conditions where vegetables are (P > 0.05; one-way ANOVA).
not sterilized prior to fermentation, VJM was only heated at 60  C
for 30 min. When no starter culture was added, the residual
microbiota remained below 102 CFU/mL and did not contribute to the acidication was stopped. Lactic acid concentrations were in
acidication (Table 3). The acidication rate of the fresh Lb. plan- line with the pH observations (Table 3; Fig. 2). A visual examination
tarum cultures was slightly faster than the rate of the dried ones of the products fermented by the APSm, APLa and AB cultures did
(Fig. 2). There was no major difference between the dried cultures not reveal bead particles, strongly suggesting that the alginate gel
with small APSm and FPSm (Fig. 2) and large (APLa and FPLa) particles dissolved during fermentation.
particles (data not shown). When phages were added, acidication The cell counts of the fermented products in the absence of
stopped at pH values above 5.4 in all cases (Fig. 2). Phage titers phages conrmed the visual observations. Although the level of
reached well above 109 PFU/mL in all VJM, clearly indicating that free cells was about 3% in the inoculated APLa and APSm cultures, it
signicant phage amplication had occurred (Table 2). was close to 100% in the fermented products (Table 3). These data
Previous reports have shown that lactic cultures in alginate gels conrmed that the alginate particles dissolve during the VJM
can be protected from phage infections (Steenson et al., 1987; fermentation. Presumably, by releasing the cells, the alginate ma-
Champagne et al., 1992). Therefore, it was a surprise that the trix lost its protective effect against phages.
APSm, AB (Fig. 2), and APLa cultures were inhibited by phages when
 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34 33

Table 3
Effect of the addition of CaCl2 to the vegetable juice medium (VJM) on the total (free and microencapsulated) and free populations of Lactobacillus plantarum HER1325 after
fermentation (40 h in VJM or 64 h in VJM CaCl2) at 19  C in the absence of phages.

Medium Culture type Free cells (CFU/mL) Total population (CFU/mL) Lactic acid (g/L)

VJM Nonea < 102 b < 102 b 0.1 a

VJM FPSm 1.4
109 a 1.3
109 a 0.6 b
FPLa 1.8
109 a 1.7
109 a 0.9 b
APSm 1.8
109 a 1.8
109 a 0.9 b
APLa 1.6
109 a 1.6
109 a 1.0 b
LiF 2.0
109 a 2.1
109 a 2.5 b
AB 1.9
109 a 1.8
109 a 2.3 b
VJM CaCl2 AB 1.1
109 a 1.7
109 a 4.5 c

Inoculation level was of 1
107 CFU/mL in treatments where starter inoculation was carried out.
A Alginate-microencapsulated cells; B Beads; F Free cells; La Large particles; Li Liquid; P Powder; Sm Small particles.
a-c: In a given column, values followed by the same letter are not statistically different (P > 0.05 in one-way ANOVAs).
a
No inoculation with a starter culture was carried out in this treatment; CFUs represent the residual microbiota of the heat-treated matrix.

3.5. Effect of addition of CaCl2 to vegetable juice medium
The lack of phage protection by alginate beads during VJM
fermentation is in disagreement with previous studies involving
microencapsulated Lactococcus lactis in milk fermentation
(Champagne et al., 1992). Because calcium stabilizes the alginate
matrix (Chandramouli et al., 2004) and milk contains high levels of
calcium, we hypothesized that the absence of high calcium content
in the VJM was linked to bead dissolution during fermentation.
Therefore, we carried out fermentation assays in CaCl2-supple-
mented VJM. A level of 15 g/L was arbitrarily selected because it is
known that at least 11 g/L ensures good alginate bead formation
(Section 2.2).
The addition of CaCl2 at 15 g/L reduced the pH of the VJM to 6.1;
thus, calcium chloride affects the buffering properties of the me-
dium. The acidication of the CaCl2-supplemented VJM medium by
Lb. plantarum was slower than the acidication of the VJM alone
(Figs. 2 and 3). However, the presence of phage HER 325 did not
affect the fermentation of the AB culture (Fig. 3) when CaCl2 was
added. The development of phages still occurred in the VJM CaCl2
medium (Table 2) but was 1 log lower than in the VJM. Presumably,
the phage multiplied at the surface of the beads or on released cells.
The large (2 mm) alginate beads could still be observed in the
fermented VJM CaCl2 medium.
Taken together, our data suggest that microencapsulated cul-
tures are protected from phages if means are taken to prevent the
particles from dissolving during fermentation of the vegetables.
Even in the presence of the AB culture, high level of phages
remained (Table 2), which raised the question of their effect during
storage. The free cells would presumably continue to lyse and only
those found in the alginate gel would remain protected and viable.
It remains to be seen, however, if phage endolysins released by free
cells outside the gel particle could diffuse into the gel and affect
lactobacilli viability. Further data are needed to ascertain the effect
of phages on the ME microbiota during its subsequent storage.
3.6. Practical considerations
Consumer acceptance of visible particles of cultures can vary
according to the function of the food. Alginate particles are
acceptable in foods with a heterogeneous structure, such as dry
sausages (Muthukumarasamy and Holley, 2006). Further studies
are needed to assess whether this observation extends to sauer-
kraut or kimchi products. Sensory and consumer acceptance
studies are also needed to examine the effect of adding calcium
salts to these fermented foods.
Clearly, alginate-based microencapsulation requires CaCl2 in the
medium to maintain efcacy, and pectin gels would presumably
have the same requirement. However, other matrices that do not
dissolve during lactic fermentation could be considered. Thermo-
formed gels such as agar or gelatin may remain intact during the
process. Further research is needed to ascertain the efciency of
other microencapsulation matrices that would provide protection
against phages but do not require the addition of CaCl2 to the
medium.
Previous studies revealed that lactococci in alginate gels are
protected against phages in dairy applications (Steenson et al.,
1987; Champagne et al., 1992). This study extends the previous
observations to a different bacterial species as well as food appli-
cation. Therefore, it is expected that our observations would apply
to other cultures used in vegetable fermentations.

4. Conclusion

This was a proof-of-concept study intended to demonstrate that

Fig. 3. Effect of the addition of 15 g/L calcium chloride to vegetable juice medium
(VJM) on the acidication pattern of fresh alginate bead (AB) cultures of Lactobacillus
plantarum HER1325 in the presence or absence of phage HER 325. Error bars represent
the standard error of the mean (SEM).
microencapsulated cultures are protected from phage infection
during vegetable fermentation only if means are taken to prevent
the particles from dissolving during the process. The addition of
CaCl2 to the vegetable juice prevented the 2-mm alginate beads
from dissolving and partially protected the culture from phage
infection. We demonstrated that phages can multiply (>109 PFU/m)
and remain stable in vegetable juice media, in addition to blocking
34 C.P. Champagne et al. / Food Microbiology 63 (2017) 28e34
the lactic fermentation process if no anti-phage control strategies
are employed. We also showed that phages can be treated by high-
shear homogenization for up to 2 min without being signicantly
inactivated and that the growth of lactobacilli in vegetable juice
media can be enhanced by supplementation with yeast extract,
Tween and minerals.
There is concern that the reuse of salt brines or fermentation
vessels, in combination with unvarying starter inoculation prac-
tices, could result in acidication problems for fermented vegetable
products. Microencapsulation of the lactic culture can prevent
phage infection if CaCl2 is added to the medium. This strategy has
the disadvantage of increasing costs, but it could be viewed
favourably by consumers because of the health benets of calcium.
It remains to be determined what minimal CaCl2 addition level
sufces and whether this supplementation will affect the texture
and avour of the fermented vegetables. Other matrices besides
alginate that would not dissolve during lactic fermentation should
also be considered.
Acknowledgments
This study was funded by Caldwell Bio Fermentation Canada,
Inc. and AAFCs Matching Investment Initiative Research Program
(SIGPI 1810). Gratitude is expressed Caldwell BioFermentation
Canada for providing raw materials and market support, as well as
to Nancy Gardner and Yves Raymond for technological assistance.
S.M. holds a Tier 1 Canada Research Chair in Bacteriophages.
Editorial assistance in English was provided by Julia Yeung-Yam-
Wah.
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