Halotolerance and Survival Kinetics of Lactic Acid

Bacteria Isolated from Jalapeño Pepper

(Capsicum annuum L.) Fermentation
Génesis Karendash González-Quijano, Lidia Dorantes-Alvarez, Humberto Hernández-Sánchez, Marı́a Eugenia Jaramillo-Flores,
Marı́a de Jesús Perea-Flores, Arturo Vera-Ponce de León, and César Hernández-Rodrı́guez

Abstract: The microbiota associated with spontaneous fermentation of vegetables in a saline substrate may represent an
important group of bacteria in the food industry. In this work, the lactic acid bacteria (LAB) Weissella cibaria, Lactobacillus
plantarum, Lactobacillus paraplantarum, and Leuconostoc citreum were identified by partial 16S rRNA gene sequence analysis. In
addition, entophytic bacteria such as Pantoea eucalypti, Pantoea anthophila, Enterobacter cowanii, and Enterobacter asburiae were
detected, but they were irrelevant for the fermentation process and were inhibited after 12 h of fermentation when the
pH decreased from 6.5 to 4.9. Moreover, 2 species of yeast were isolated and identified as Hanseniaspora pseudoguilliermondii
and Kodamaea ohmeri by their partial 26S rRNA gene sequence. The growth of LAB was evaluated at different sodium
chloride contents. L. citreum was the most halotolerant species followed by L. plantarum and W. cibaria with a concentration
index to obtain a 50% population reduction (IC50 ) of 7.2%, 6.6%, and 5.2%, respectively. Furthermore, the growth of
LAB and Escherichia coli O157:H7 was evaluated in the presence of the main phenylpropanoids from chilli peppers such
as p-coumaric and ferulic acid. It was determined that LAB can grow in both acids at 4 mM, unlike E. coli O157:H7,
whose growth is inhibited in the presence of these acids.

M: Food Microbiology
Keywords: capsicum, fermentation, jalapeño pepper, lactobacillus, leuconostoc, weissella

& Safety
Practical Application: Fermentation of the jalapeño pepper occurs spontaneously in traditional Mexican cuisine practices
and adds new flavors while reducing the initial pungency of the pepper. The primary bacteria isolated from chilli
fermentation are lactic acid bacteria. These bacterial species exhibit a high resistance to phenolic acids and sodium
chloride concentrations and can therefore be used for the fermentation of many other food products. In addition, chilli
pepper fermentation has antibacterial and antifungal properties that enhance shelf life and help to avoid subsequent
contamination with pathogenic or saprophytic microorganisms.

Introduction to the oral burn sensation. Norcapsaicin, nordihydrocapsaicin, ho-

The chilli pepper is an important component of the Mexican mocapsaicin, homodihydrocapsaicin, nornordihydrocapsaicin, and
diet. It is consumed fresh, as an ingredient in traditional dishes or nornorcapsaicin are only minor flavor components (Liu and others
in a wide variety of meals and processed products such as sauces 2013).
and pickles. The jalapeño pepper (Capsicum annuum L.) is one In addition, chilli peppers have phenolic compounds such as
of the most widely commercialized species in various products p-coumaric and ferulic acids. These compounds are phenyl-
(Jaramillo-Flores and others 2010). propanoids derived from hydroxycinnamic acids that have an-
The principal characteristic of the chilli pepper is its pun- tioxidant and antimicrobial properties (Dorantes and others 2000;
gency, which is due to capsaicinoids. These compounds consist Acero-Ortega and others 2005, Conforti and others 2007). Phe-
of an aromatic moiety, vanillylamine, linked to an acyl residue nolic acids are often bound, forming complex molecules such as
with C8–C13 fatty acids (Whiting and others 2012). Capsaici- lignins and tannins, and thus it is difficult to find them in free form.
noids, capsaicin, and dihydrocapsaicin are the main contributors Processes such as freezing, sterilization, and fermentation could
promote the formation of free phenolics (Fernandez-Orozco and
others 2009). The addition of external starter cultures and the in-
duction of naturally associated microbiota are widely used in lactic
MS 20140118 Submitted 1/22/2014, Accepted 4/14/2014. Authors González-
Quijano, Dorantes-Alvarez, Hernández-Sánchez, and Jaramillo-Flores are with Depto.
acid fermentation of foods (Di Cagno and others 2009). Lactic
de Graduados en Alimentos, Escuela Nacional de Ciencias Biológicas, Inst. Politécnico fermentation requires at least a fermentable sugar, anoxic condi-
Nacional. Prolongación de Carpio y Plan de Ayala. Col. Sto. Tomás, México, Distrito tions, and the presence of anaerobic lactic acid bacteria (LAB;
Federal, C.P. 11340. México. Author Perea-Flores is with Centro de Nanociencias y Karovičová and Kohajdová 2005). In addition, several foods can
Micro y Nanotecnologı́as del Inst. Politécnico Nacional, México, Distrito Federal. Au- undergo lactic fermentation at high sodium chloride concentra-
thors Vera-Ponce de León and Hernández-Rodrı́guez are with Depto. de Microbiologı́a,
Escuela Nacional de Ciencias Biológicas, Inst. Politécnico Nacional. Prolongación de tions (Di Cagno and others 2008).
Carpio y Plan de Ayala. Col. Sto. Tomás, México, Distrito Federal, C.P. 11340. LAB decrease pH by producing organic acids such as lactic,
México. Direct inquiries to authors Dorantes-Alvarez and Hernández-Rodrı́guez (E- formic, phenyl acetic, and caproic acids, and these bacteria can
mails: idoran@ipn.mx, chdez38@hotmail.com). produce ethanol and carbon dioxide through different metabolic

C 2014 Institute of Food Technologists

 R

doi: 10.1111/1750-3841.12498 Vol. 79, Nr. 8, 2014 r Journal of Food Science M1545
Further reproduction without permission is prohibited
 Halotolerance and survival kinetics of LAB . . .

Table 1–Phylogenetic approach and nucleotide similitude for the species identified from spontaneous fermentation of (Capsicum
annuum L.).

GenBank sequence with Similitude Microbial group

Phylogenetic group Representative clonea highest homologyb (%)c membershipd
W. cibaria 2M, 3M, 11M, 12M, 13M, 15M W. cibaria (AJ295989) 99.86 W. cibaria
L. citreum 6M, 7M, 16M, 17M, 18M, 19M, 20M L. citreum (AB022923) 100 L. citreum
L. paraplantarum P19, 5M, 14M, 9M, P18, P20 L. paraplantarum (AB191250) 99.19 L. paraplantarum
L. plantarum 1M, P22, 4M, 10M L. plantarum (M58827) 99.93 L. plantarum
P. anthophila P7, P12, P8 P. anthophila (EF688010) 99.42 P. anthophila
P. eucalypti P2, P3 P. eucalypti (KC139445) 99.14 P. eucalypti
E. cowanii P10 E. cowanii (HQ407245) 99.57 E. cowanii
E. asburiae P11, P13 E. asburiae (JQ582973) 99.50 E. asburiae
H. pseudoguilliermondii P1, P25 H. pseudoguilliermondii (AB525689) 99.65 H. pseudoguilliermondii
K. ohmeri 8M, P5, P15, P16, P23 K. ohmeri (AB281301) 100 K. ohmeri
a
Selected by morphotypes.
b
Sequence with highest homology in phylogenetic analysis.
c
Percentage similitude.
d
Determination of genus and specie according with the similitude percentage.

pathways. LABs are acidophilic and can grow in low pH environ- Rogosa-Sharpe (MRS) and Yeast Peptone and Dextrose (YPD)
ments whereas other competitor bacteria are inhibited (Granito media. Plates were incubated at 32 °C for 48 h; each colony type
and Alvarez 2006; Gálvez and others 2007). was categorized and counted. Axenic cultures of 5 isolates of each
The objectives of this work were to determine the microbial colonial morphotype observed were obtained and stored in 30%
succession during the spontaneous fermentation of the jalapeño glycerol at −70 °C for further analysis.
pepper and to examine the isolation and molecular identification
DNA extraction
M: Food Microbiology

of the microorganisms involved in the process. Moreover, due to

biotechnological interest in phenolic acids, the tolerance to the A total of 1.5 mL of bacterial and yeast cultures incubated for
& Safety

main phenylpropanoids present in the jalapeño and the halotol- 24 h at 37 ˚C were collected by centrifugation. DNA extraction
erance for the predominant LAB identified in this work were was performed as described by Morales-Jiménez and others (2009).
studied.
Polymerase chain reaction
Materials and Methods Specific gene fragments of the 16S rRNA-coding region of
the bacterial isolates were amplified by polymerase chain reac-
Conditioning and optimization tion (PCR) using the primers 8 forward (5 GCG GAT CCG
Fresh, ripe jalapeño peppers were bought in a supermarket CGG CCG CTG CAG AGT TTG ATC CTG GCT CAG 3 )
in Mexico City. They were washed with sterilized water, de- and 1492 reverse (5 GGC TCG AGC GGC CGC CCG GGT
stemmed, and then cut into uniform slices. The optimization of TAC CTT GTT ACG ACT T 3 ; Morales-Jiménez and others
jalapeño pepper fermentation was carried out using a central com- 2009). In addition, the D1/D2 26S rRNA regions of yeast were
posite design with 5 replications of the central point varying salt amplified using the primers 26S-A1 forward (5 CAT ATC AAT
concentration and temperature; the central point values for salt AAG CGG AGC AAA AG 3 ) and 26S-A2 reverse (5 CAG TTC
concentration and temperature were 5.75% and 35 °C, respec- TGC TTA CCA AAA ATGG 3 ), which were designed for this
tively. Response variables were pH and titratable acidity reported study. In both PCR procedures, a total of 25 μL per reaction was
as lactic acid. The optimal conditions obtained with the software mixed, which contained 10 ng of DNA, 0.8 pM of each primer,
Design-Expert R
Version 7.0.3 software (Stat-Ease, Minneapolis, 0.2 mM dNTPs, 2.5 mM MgCl2 , 1U of Taq polymerase, and 1×
Minn., U.S.A.) were 8.42% sodium chloride at an incubation Taq polymerase buffer (Invitrogen Life Technologies, Sao Paulo,
temperature of 31 ± 1 °C, to obtain a final pH of 4.27 (González- Brazil). The reaction conditions were 94 °C for 7 min; 35 cycles
Quijano and others 2012). of 60 s at 94 °C, 60 s at 55 °C, and 60 s at 72 °C; and a final
extension at 72 °C for 7 min. PCR products were purified using
Fermentation kinetics the DNA Clean and Concentrator Kit (Zymo Research, Irvine,
Fermentation was carried out at the optimal conditions listed Calif., U.S.A.) and sequenced in an ABI PRISM 310 genetic an-
above. Uniform slices were placed into flasks and covered with alyzer (Applied Biosystems, Foster City, Calif., U.S.A.) using the
sterile saline solution (8.42%). The pH drop was measured with a same specific primers described above.
pH meter (Smart monitor Milwaukee SMS110) every 2 h in the
brine until the pH was 4.2 ± 1 after 116 h of fermentation. Microbial identification by phylogenetic analysis and
nucleotide similitude of bacterial 16S rRNA and yeast 26S
Isolation of microorganisms rRNA genes and phenotypic tests
The isolation of microorganisms from both chilli pepper and The identification of the isolated bacteria and yeast was per-
brine was carried out by sampling every 2 h in the first stage formed using a phylogenetic approach and nucleotide similitude.
of fermentation when the pH decreased rapidly (0 to 12 h) and Sequences from isolated bacteria and yeast were compared in Gen-
then every 8 h when the pH was less variable (20 to 116 h). Ten Bank using the Basic Local Alignment Search Tool (BLAST) avail-
grams of each sample were homogenized in flasks with 90 mL able at http://blast.ncbi.nlm.nih.gov/Blast.cgi.
of sterilized water. Ten-fold serial dilutions were performed and A collection of taxonomically related sequences was obtained
0.1 mL of 10−6 , 10−7 , and 10−8 dilutions were spread on Man from the Natl. Centre for Biotechnology Information Taxonomy

M1546 Journal of Food Science r Vol. 79, Nr. 8, 2014

Halotolerance and survival kinetics of LAB . . .

homepage (http://www.ncbi.nlm.nih.gov/Taxonomy/-taxono- was performed in tubes containing 10 mL of MRS broth at one of

myhome.html/). Multiple alignments of query and related se- the following concentrations of sodium chloride: 0%, 2%, 4%, 6%,
quences were performed by BioEdit Sequence Editor v.7.0.9.0 8%, 10%, 12%, 14%, and 16% (Patil and others 2010). The initial
(Hall 1999) and edited using Sea View v.4.3.3 (Gouy and others inoculum was at 1 × 105 CFU/mL, and growth was measured by
2010). Neighbor-joining and nucleotide similitude analyses were optical density (OD560 ) for 48 h. The maximum growth (24 h) of
performed using MEGA 5.0.5 software (Tamura and others 2011). all the concentrations was modeled by My Stat v12 (Systat Soft-
The confidence at each node was assessed by 1000 bootstrap repli- ware, Washington, Chicago, Ill., U.S.A.) to obtain the parameters
cates. Due to the high similarity of 16S rRNA sequences within for the logistic equation Y = C + ([D − C]/[1 + (x/IC50 )b ]),
the Lactobacillus genus, a biochemical confirmation test using the where Y is the absorbance, C is the absorbance in maximum salt
API 50CHL Kit was necessary (API-bioMerieux, Marcy l’Etoile, concentration, D is the absorbance of the control, x is the sodium
France). chloride concentration in percentage (%), IC50 is the concentra-
tion necessary to obtain a half of total population (D/2), and b is
Halotolerance assay of LAB the Hill slope or slope factor constant and refers to the steepness
According to the identification, one isolate of each specie was of the curve. As the absolute value of the Hill slope increases, so
selected to use in the halotolerance tests in triplicate. LAB growth does the steepness of the curve.

Figure 1–Neighbor-joining phylogenetic tree of

16S rRNA from microorganisms isolated from the
fermentation of the jalapeño chilli pepper
(Capsicum annuum L.). The sequence of
Actinomyces israelii (NR_026227) was used as an
external group. The bar indicates the number of
substitution changes per site according to Kimura
2-parameter distances. GenBank accession

M: Food Microbiology
numbers are in brackets. Bootstrap values are
indicated on each node of the tree (percentage of
1000 pseudoreplicates).

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Vol. 79, Nr. 8, 2014 r Journal of Food Science M1547

 Halotolerance and survival kinetics of LAB . . .

Phenylpropanoids effect on growth of LAB ple comparisons by Duncan and Tukey’s tests were conducted to
The effect of phenolics on LAB growth was performed in tripli- determine the differences between samples and controls.
cate. LAB growth was performed in tubes containing MRS broth
supplemented with 4 mM phenolic salts for one isolate of each Results
species. Escherichia coli O157:H7 was used as a pathogen control
and cultured in soy trypticase broth containing 4 mM phenolic Isolation and identification of bacteria and yeast from
salts. The inoculum was at 1 × 105 CFU/mL and the growth ki- fermented jalapeño peppers and brine
netics were monitored using optical density (OD560 ) for 36 h and A total of 38 microorganisms were isolated from fermented
by measuring total viable counts in the mentioned solid media. peppers and brine, all of which were identified by PCR. LAB
only grew in MRS, but gram-negative bacteria and yeast were
isolated from YPD medium.
Fluorescence microscopic analysis of bacteria viability in Table 1 shows the identification of isolated strains using a phy-
phenylpropanoids logenetic approach and nucleotide similitude. The clustered phy-
The effect of phenolics on bacterial viability was determined logenetic analysis of the LAB isolates for the sequences of the
by confocal laser scanning microscopy (CLSM) using a selective strains showed the following bacteria: Weissella cibaria, Leuconostoc
fluorescent dye. Low concentrations of acridine orange (AO) and citreum, Lactobacillus plantarum, and Lactobacillus paraplantarum. Both
propidium iodide (PI; 3 mM; Sigma Aldrich, St. Louis, Mo., Lactobacillus species are closely related, and their identification was
U.S.A.) were used to visualize living and dead cells simultaneously. confirmed by API 50CHL tests. In addition, the Gram-negative
AO is a weak base that readily enters living cells and functions bacteria were identified as Pantoea eucalypti, Pantoea anthophila, En-
as an inclusion dye. At a low concentration, AO causes live cells terobacter cowanii, and Enterobacter asburiae (Figure 1). Finally, the
to fluoresce green. In this assay, PI functions as an exclusion dye yeasts Hanseniaspora pseudoguilliermondii and Kodamaea ohmeri were
that cannot penetrate living cells but readily enters dead or dying identified (Figure 2).
LAB cells with damaged membranes and causes cells to fluoresce
red (Bank 1988; Yániz and others 2013). In the CLSM assay, Microbial diversity changes during jalapeño pepper
cell suspensions were incubated with phenolics and collected by
M: Food Microbiology

fermentation
centrifugation at different times of incubation, 100 μL of the cells
The total LAB count of the morphotypes identified in jalapeños
were mixed with 1 μL of each dye for 15 min on ice in the
& Safety

reached a maximum of 8.06 ± 0.25 log CFU/mL after 28 h

dark, and then cells were trapped between a slide and an 18-mm
of fermentation. Lactobacillus spp. were the predominant species
square coverslip. The samples were analyzed by CLSM (LSM 710;
in the fermentation, achieving 7.83 ± 0.01 log CFU/mL at
Carl Zeiss, Oberkochen, Germany) using Plan Apochromat 63×
20 h, whereas L. citreum reached 7.51 ± 0.06 log CFU/mL
(numerical aperture, 1.30). The wavelengths of laser tracks 1 and
at 20 h and W. cibaria reached 7.48 ± 0.05 log CFU/mL after
2 were set at 488 and 405 nm, respectively. The images obtained
36 h of fermentation. L. citreum decreased after 44 h of fermen-
were grouped, projected, and analyzed with Zen 2010 software.
tation whereas Lactobacillus spp. showed growth and W. cibaria de-
creased after 92 h of fermentation (Figure 3A). In the jalapeño
Statistical analysis pepper after 20 h of fermentation, relative counts of LAB showed
All data are expressed as the mean and standard deviations. One- that Lactobacillus spp. were dominant and comprised 56.65% of
way analysis of variance (ANOVA) was conducted in GraphPad the total population, whereas L. citreum and W. cibaria accounted
Prism 5.0 using a significance level of 0.05. In addition, multi- for 25.75% and 17.60%, respectively. Apparently, LABs were

Figure 2–Neighbor-joining phylogenetic tree

of 26S rRNA from yeasts associated with the
fermentation of chilli pepper (Capsicum
annuum L.). The sequence of Rhodotorula
mucilaginosa (DQ832198) was used as an
external group. The bar indicates the number
of substitution changes per site according to
Kimura 2-parameter distances. GenBank
accession numbers are in brackets. Bootstrap
values are indicated on each node of the tree
(percentage of 1000 pseudoreplicates).

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Halotolerance and survival kinetics of LAB . . .

mobilized by the brine during fermentation to reach 7.87 ± 0.03 Table 2–Parameters for the behavior of LAB in the presence of
log CFU/mL at 60 h because the isolation of these microorganisms sodium chloride.
at higher concentrations was possible. However, the population of D−C
LAB in the brine was lower than in the pepper matrix itself (Fig- Microorganism C (OD560 ) (OD560 ) IC50 (%) b R2
ure 3B). L. plantarum 0.16 1.14 6.60 9.88 0.98
W. cibaria 0.31 1.52 5.20 3.14 0.98
Halotolerance assay L. citreum 0.43 0.97 7.20 9.79 0.98
L. plantarum, W. cibaria, and L. citreum had a notably reduced
growth and an extended lag phase in the presence of sodium chloride. However, all isolates exhibited a significant growth in 6%
NaCl, confirming their halotolerant character. L. citreum displayed
a larger lag phase and the highest tolerance to NaCl by growing
even in 8% and 16% NaCl. The maximum growth of the strains at
different concentrations of NaCl was modeled and their behavior
was described as a decreasing logistic curve. Lactobacillus spp. and L.
citreum had similar growths and their range of tolerance was from
6% to 8% sodium chloride whereas W. cibaria was less tolerant with
its limit at 5.2% (Figure 4). The values of the modeling are shown
in Table 2.

Phenylpropanoids tolerance assay

L. plantarum reached its maximum growth peak at 9.07 ± 0.02
log CFU/mL after 24 h of incubation in MRS broth. When fer-
ulate was added, the strains were able to reach their maximum

M: Food Microbiology
growth but only after a longer incubation time (36 h). When
p-coumarate was added to the culture medium, the maximum
growth for L. plantarum was 7.09 ± 0.01 log CFU/mL after

& Safety
36 h (Figure 5A). L. paraplantarum was inhibited after 36 hours
of incubation with p-coumarate reaching a growth of 5.11 ± 0.03
log CFU/mL, and both strains demonstrated similar behavior.
Both bacteria reached maximum growth with a longer incubation
time (data not shown).
Likewise, L. citreum exhibited a maximum growth peak at
8.93 ± 0.05 log CFU/mL after 24 h, but when ferulate was
present its maximum growth was 6.7 ± 0.05 log CFU/mL after
24 h of incubation, reaching the stationary phase. The compound
p-coumarate displayed a bacteriostatic effect against L. citreum by
inhibiting its growth after 36 h and reducing the population by
5 orders of magnitude to 2.62 ± 0.33 (Figure 5B), which was
significantly different from control (P < 0.05).
Although W. cibaria exhibited the highest growth among LAB
(10 log ± 0.02 CFU/mL), this maximum growth was not reached
Figure 3–Microbial growth of LAB during fermentation: (A) in jalapeño when phenolics were present in the culture medium. In the pres-
chilli pepper matrix and (B) in pepper brine. ence of ferulate, its maximum growth was 8.90 log CFU/mL, and
with p-coumarate it was 6.78 ± 0.03 log CFU/mL (Figure 5C).
However, when the data of the control curve was compared to
the sample curves when ferulate and p-coumarate were added,
there was not a significant difference between the control and the
samples (P < 0.05).
E. coli O157:H7 had a maximum growth peak at approxi-
mately 9.96 ± 0.23 log CFU/mL in the control medium. With
this bacterium, ferulate and p-coumarate exerted a severe bac-
teriostatic effect by 24 h of incubation. After 36 h of incu-
bation in the presence of phenolics, its growth decreased to
2.87 ± 0.11 and 2.48 ± 0.36 log CFU/mL for ferulic and p-
coumaric acid, respectively (Figure 5D). These effects were signif-
icantly different from control according to Duncan’s test (P < 0.05)
and among E. coli and LAB isolates according to Tukey’s test
(P < 0.05).
In the fluorescence assay, we observed the same trends in LAB
and E. coli O157:H7 kinetics. L. plantarum had 77% viable cells
Figure 4–Modeling of halotolerance of LAB isolated from fermented after 3 h of incubation in the presence of ferulate, but E. coli
jalapeño chilli pepper (Capsicum annuum L.).
O157:H7 had only 37% viable cells. Similarly, the inhibitory effect

Vol. 79, Nr. 8, 2014 r Journal of Food Science M1549

 Halotolerance and survival kinetics of LAB . . .

of p-coumaric acid on L. plantarum was weaker than on E. coli have been broadly recognized as rice, citrus, sugarcane, and potato
O157:H7, which retained 58% and 8% viable cells, respectively plant endophytes (Asis and Adachi 2004; Andreote and others
(Fig. 6A and B). 2008; Brady and others 2009; Quecine and others 2012). To our
knowledge, none of these endophytic species have been previously
Discussion isolated from chilli peppers, but related species of both genera
The fermentation of the jalapeño pepper was performed at high have been isolated from chilli pepper species (Kang and others
sodium chloride concentrations (8.42%), which is an environmen- 2007).
tal condition that, combined with anoxic conditions, may induce It is possible that the endophytes isolated from the jalapeño
the growth of anaerobic, aerotolerant, and halotolerant LAB. Stud- pepper fermentation play an important role in plant growth but
ies have reported that some LAB have the ability to survive under no relevant participation in chilli pepper fermentation, as demon-
stress conditions in different environments (Bautista-Gallego and strated by the growth kinetics in saline conditions. Endophytic
others 2008). isolates of Pantoea spp. are potentially capable of fixing nitrogen as
In this study, fermentation is a dynamic system that results in endophytes (Feng and others 2006), but apparently no relevant ni-
changes in the microbial populations due to the diffusion of nutri- trogen fixation occurs under fermentation conditions. Pantoea spp.
ents and the accumulation of primary metabolites. Once the chilli were detected a low populations at the beginning of fermentation,
matrix loses stiffness, sodium chloride is able to move into the tis- but eventually were not detected further along in the fermenta-
sues and the nutrients begin to spread to the medium, becoming tion process. Possibly, these bacteria were unable to compete as a
more bioavailable. This allows mobilization of LAB from the chilli result of the synergic effect of the free phenolic acids, high sodium
pepper to the brine. chloride concentration, acidic pH, and competition with LAB for
In this work, we isolated and identified some enterobacteria nutrients. In addition, some LAB are able to produce antimicro-
such as P. eucalypti, P. anthophila, E. cowanii, and E. asburiae at bial metabolites as bacteriocins (Rodrı́guez-Pazo and others 2013).
the beginning of the jalapeño fermentation, suggesting that all When fermentation conditions are supplied, the proliferation of
those bacteria are pepper bacterial endophytes. All those species LAB quickly overcomes the growth of endophytic enterobacteria,
M: Food Microbiology
& Safety

Figure 5–Survival kinetics of bacteria isolated from jalapeño pepper fermentation in the presence of ferulate and p-coumarate (4 mM). (A) L. plantarum
1M; (B) L. citreum 6M; (C) W. cibaria 3M; (D) E. coli O157:H7.

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Halotolerance and survival kinetics of LAB . . .

a cluster of nonhalotolerant microorganisms poorly adapted to typically differentiated by the L. plantarum fermenting capacity
high NaCl conditions (Betts and others 2000; Di Cagno and oth- of α-methyl-d-mannoside and melezitose (Pot and Tsakalidou
ers 2008). 2009).
During lactic acid fermentation of the jalapeño pepper, In addition, obligate heterofermentative bacteria such as Weis-
some enterobacteria are not desirable because of their potential sella and Leuconostoc species were isolated from chilli fermentation.
pathogenic impact on the human population (Brady and others In other foods, both bacteria produce desirable exopolysaccharides
2009). However, although several Enterobacter species are recog- that improve the rheological and physical properties of the dough
nized as important pathogens, E. cowanii and E. asburiae isolated (moisture, hardness, volume, and crumb color), sensory properties
in the initial fermentation have not been recognized as relevant and shelf life of bread. In addition, these genera are commonly
human pathogens. used as food additives for bread production (Di Cagno and others
In addition, low populations of H. pseudoguilliermondii and K. 2006; Bounaix and others 2010).
ohmeri yeasts were identified in the fermentation of the jalapeño In particular, the presence of W. cibaria has been reported in
pepper. Hanseniaspora and Kodamaea species have been reported baked products and other vegetable fermentations such as green
in spontaneous alcoholic fermentations of wine, cacao seeds, and smoothies (Russo and others 2010; Di Cagno and others 2011a).
apples (Granchi and others 2002; Daniel and others 2009). The However, L. citreum is not commonly reported in vegetable fer-
interaction between yeasts and LABs present in jalapeño fermen- mentations, where the main strain present is L. mesenteroides as
tation was not studied. However, the isolation indicated that fer- shown in kefir fermentation (fermented milk product), shalgam (tra-
mentation conditions were not adequate for their growth despite ditional Turkish fermented beverage), and sweet cherry (Di Cagno
the fact that they are often reported as fermentable yeasts. and others 2011b; Gulitz and others 2011; Tanguler and Erten
Among the LAB isolated, facultative heterofermentative bacte- 2012).
ria L. plantarum and L. paraplantarum were detected during fer- In the case of kimchi (fermented cabbage product), L. citreum was
mentation. Both phylogenetically related species can be pheno- the predominant bacteria during the first stage of fermentation
(In-Kwon and others 2003), whereas in jalapeño pepper fermen-
tation, the main microorganisms were Lactobacillus spp.

M: Food Microbiology
In this work, L. citreum was the most halotolerant bacterium,
so this strain harbors a potential application for salty food prod-

& Safety
ucts such as fermented meat and cheeses. These products have
high concentrations of NaCl, the biochemical changes are related
to the proteolytic activity and they also have a ripening process
where flavor and aroma are developed. One potential use for L.
citreum could be fermented sausage (comminuted meat and fat,
mixed with salt, nitrate and/or nitrite, sugar, and spices), which
is stuffed into casings and subjected to fermentation and dry-
ing. Another potential application may be in ripe cheeses such
as cotija, where halotolerant bacteria such as Lactobacillus acidipis-
cis, Tetragenococcus halophilus, Weissella thailandensis, and Lactobacillus
pentosus are present (Hugas and Monfort 1997; Morales and others
2011).
LABs isolated in this work followed behaviors similar to those
of different species of Lactobacillus and yeasts when upon exposure
to high salt concentrations. The tolerance to chloride salts is im-
portant for future applications in foods as mentioned by Bautista-
Gallego and others (2008). They found that L. pentosus was more
sensitive to NaCl and CaCl2 than MgCl2 and KCl, which are salts
commonly used in fermented products.
LABs tolerate hydroxycinnamic acid derivatives such as p-
coumaric and ferulic acid release into the system. It has been
reported that some species of Lactobacillus display the capabil-
ity to produce specific enzymes such as tannase, phenolic acid
decarboxylase (PAD), and benzyl alcohol dehydrogenase (Cavin
and others 1997; Landete and others 2008; Rodrı́guez and others
2008). In particular, PAD of L. plantarum transformed p-coumaric
and ferulic acid substrates into 4-vinylphenol and 4-vinylguaiacol
derivatives, which are approved by the Joint Expert Committee on
Food Additives (JECFA) as flavoring agents (JECFA 2000; Lorca
and De Valdez 2009; Rodrı́guez and others 2009).
LAB tolerance to phenolic compounds is a selective advan-
tage over sensitive enterobacteria like E. coli O157:H7. In Gram-
negative bacteria, inhibition due to phenolic acids is associated
with the competitive inhibition between phenolic acids and pro-
Figure 6–Confocal laser scanning microscopy images of bacteria viability line because phenolics act as proline analogs for the proline-
assay in the presence of p-coumarate after 3-h incubation. (A) L. plantarum dehydrogenase enzyme and are responsible for growth-inhibiting
1M and (B) E. coli O157:H7.
effects (Lin and others 2005).

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 Halotolerance and survival kinetics of LAB . . .

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