Food Chemistry 397 (2022) 133774

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Heat, cold, acid, and bile salt induced differential proteomic responses of a
novel potential probiotic Lactococcus garvieae C47 isolated from camel milk
Mohd Affan Baig a, Mark S. Turner b, Shao-Quan Liu c, Nagendra N. Shah d, Mutamed
M. Ayyash a, *
a
Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates University (UAEU), Al Ain, United Arab Emirates
b
School of Agriculture and Food Sciences, The University of Queensland (UQ), Brisbane, Australia
c
Department of Food Science and Technology, Faculty of Science, National University of Singapore, Science Drive 2, Singapore 117542, Singapore
d
Food and Nutritional Science, School of Biological Sciences, the University of Hong Kong, Pokfulam Road, Hong Kong

A R T I C L E I N F O A B S T R A C T

Keywords: Probiotics encounter various stresses during food processing and digestion. This study evaluated the differential
Environmental stress proteomic responses of a newly identified potential probiotic lactic acid bacteria, Lactococcus garvieae, isolated
Proteomics from camel milk. Lc. garvieae C47 was exposed to heat, cold, acid, and bile conditions, and stress-responsive
Lactic acid bacteria
proteins were identified. The proteomic analysis was done using 2D-IEF SDS PAGE and nano-LC-MS/MS. Out
Nano-LC-MS/MS
of 91 differentially expressed proteins, 20 upregulated and 27 downregulated proteins were shared among the
stresses. The multivariate data analysis revealed abundance of elongation factor Ts (spot C42), uridine phos­
phorylase, fructose-bisphosphate aldolase, peptidase T, cobalt ECF transporter T component CbiQ, UDP-N-ace­
tylmuramate-L-alanine ligase, uncharacterized protein, aspartokinase, chaperone protein DnaK, IGP synthase
cyclase subunit, probable nicotinate-nucleotide adenylyltransferase, NADH-quinone oxidoreductase, holo-[acyl-
carrier-protein] synthase, L-lactate dehydrogenase, and uncharacterized protein. The maximum number of
differentially expressed proteins belonged to carbohydrate and protein metabolism, which indicates Lc. garvieae
shifts towards growth and energy metabolism for resistance against stress conditions.

1. Introduction microorganisms need to confer main health benefits on the host to be

The functional food sector has gained tremendous growth in recent
years due to its health-promoting benefits. Foods containing probiotics
are considered functional foods. Consumption of probiotics can
contribute to overall well-being and protection against several diseases
or disorders like diarrhea, constipation, inflammatory bowel diseases,
allergies, cancer, etc. (Ayyash et al., 2020; Ayyash et al., 2021). The use
of probiotic bacteria as food starter cultures or supplements poses many
challenges regarding their function, growth, viability, and survival due
to food processing, storage, and consumption. It has been observed that
the probiotics obtained from different origins have different tolerances
to stress conditions. The probiotics isolated from foods have a higher
tolerance to changing pH and temperature during food processing but
less tolerance to the gastrointestinal environment compared to pro­
biotics isolated from the gut (Min, Bunt, Mason, & Hussain, 2019).
According to The Food and Agriculture Organization (FAO), the
termed a probiotic (Menconi et al., 2011). The capability of surviving
the passage through the digestive tract and proliferation inside the gut
are the two prerequisites of these microorganisms. This means they must
be able to replenish in the presence of bile and gastric juices under the
existing environmental conditions or be consumed along with the food
matrix to permit survival and passage through the gut (Ayyash et al.,
2021).
Lactococcus spp. are generally gram-positive LAB, non-colonizing
facultative anaerobe, and well known as fermenting agents used in
yogurt, ripened cheeses, and kefir (Qi et al., 2020). Moreover, the spe­
cies of the Lactococcus genus (e.g., L. lactis, L. cremoris, and
L. diacetylactis) are regarded for its economic value and probiotic
properties. Most the Lactococcus members are considered as Generally
Recognized As Safe (GRAS) and possess capacity of surviving during the
gut passage (Qi et al., 2020).
Lactococcus garvieae is an indigenous lactic acid bacterium (LAB) of

* Corresponding author at: Department of Food Science, College of Agriculture and Veterinary Medicine (CVAM), United Arab Emirates University (UAEU), United
Arab Emirates.
E-mail address: mutamed.ayyash@uaeu.ac.ae (M.M. Ayyash).

https://doi.org/10.1016/j.foodchem.2022.133774
Received 19 February 2022; Received in revised form 20 July 2022; Accepted 21 July 2022
Available online 23 July 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
M.A. Baig et al.
the microbiota of raw milk and its dairy products (Gibello et al., 2016).
Lc. garvieae strains isolated from dairy sources have not been reported to
be involved in foodborne disease outbreaks (Delpech et al., 2017). Lc.
garvieae strains having important biological and technological proper­
ties have been isolated from various dairy products such as raw milk and
cheeses and are shown to be non-pathogenic (Morandi, Silvetti, Miranda
Lopez, & Brasca, 2015). The avirulent strains of Lc. garvieae are non-
hazardous to human health (Flórez et al., 2012). Some strains of Lc.
garvieae may be used as probiotics due to their ability to inhibit the
activity of various pathogenic bacteria (Zhang, Xie, Zhang, Fu, & Zhang,
2016). Probiotics are known to improve the digestion of nutrients and
growth performance (Ayyash et al., 2021).
Various approaches such as genomics, transcriptomics, and prote­
omics can be applied to study the tolerance and responses of probiotics
under stress conditions. Proteomics has been widely used to compare
differentially expressed proteins among different strains of the same
bacterial species or between the same strains under various experi­
mental conditions. The 2-DE maps of identified proteins can provide
information on the mechanism(s) involved in bacterial pathogenicity,
resistance to antibiotics, responses to environmental stress, and adap­
tation to the host immune system (Baig et al., 2021). Probiotics
including Lactococcus spp. are susceptible to different stresses due to
changes in their environmental conditions or industrial processing,
affecting their growth and survival (Budin-Verneuil, Pichereau, Auffray,
Ehrlich, & Maguin, 2005).
Lc. garvieae C47 was a newly identified potential probiotic LAB from
camel milk (Abushelaibi, Al-Mahadin, El-Tarabily, Shah, & Ayyash,
2017), and its exopolysaccharides exhibited remarkable antioxidant,
antidiabetic, antimicrobial, and anticancer activities (Ayyash et al.,
2020). To the best of our knowledge, there is no information available
about the proteomic responses of Lc. garvieae C47 under different stress
conditions. In this study, the Lc. garvieae C47 proteins expressed under
heat, cold, acid, and bile stresses were identified using 2D IEF SDS PAGE
and nano-LC MS/MS technique.
2. Materials and methods
2.1. Bacterial propagations
Lc. garvieae C47 cultures stored at − 80 ◦ C in 50 % glycerol solution
were revived in MRS broth (de Mann Rogosa Sharpe broth, Lancashire,
UK) incubated at 37 ◦ C for 20 h under anaerobic conditions. Two
consecutive sub-culturing in MRS broth were performed prior to stress
treatment.
2.2. Stresses treatment
Preliminary investigations were performed to determine the stress
point of each stress factor according to the procedure detailed in (Chen,
Tang, & Chiang, 2017). The pelleted culture was heated for 2 h at 40 ◦ C
to 65 ◦ C at 5 ◦ C/min intervals. For cold stress, the pelleted culture was
stored at 4 ◦ C for 1 h to 5 h with 1 h intervals. The acid stress was
determined by incubating the pelleted culture under acid conditions pH
2.0 to 4.0 with 0.5 h intervals for 2 h. For the bile stress, the pelleted
culture was subjected to different concentrations of a bile salts mixture
(6.0 mmol/L sodium glycocholate, 6.0 mmol/L sodium taurocholate, or
conjugated bile salt mixture containing 6.0 mmol/L glycocholic acid,
glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic
acid, and taurodeoxycholic acid) (0.01 % to 0.1 % w/v) (Sigma, St.
Louis, Missouri, USA) with 0.01 % intervals for 2 h. The point that
achieved a 1.0 log reduction or less was considered a stress point (data
not shown).
The Lc. garvieae C47 cultures in MRS broth were subjected to heat
stress at 50 ◦ C for 2 h. The cold stress treatment was performed by
incubating the freshly activated cultures in MRS broth at 4 ◦ C for 2 h. For
acid and bile stress treatments, the cultures were centrifuged at 5000 × g
2
Food Chemistry 397 (2022) 133774
at 4 ◦ C for 10 min, and the pellets were resuscitated in 0.1 mol/L
phosphate buffer of pH 3.0 for acid stress or supplemented with 0.05 %
(w/v) bile salts mixture for bile stress. The cultures were incubated at
37 ◦ C for 2 h. One set of culture tubes without stress was considered as
the control. After stress treatment, all the tubes were centrifuged at
5000 × g at 4 ◦ C for 10 min, and the pellets were washed with the same
phosphate buffer and stored at − 20 ◦ C until protein extraction.
2.3. Protein extraction
ReadyPrep™ total protein extraction kit (Bio-Rad, Hercules, CA,
USA) was employed to extract proteins from Lc. garvieae C47 after the
exposure to the stress treatment. The pellets were suspended in 0.5 mL of
2-D sample buffer (7 mol/L urea, 2 mol/L thiourea, 40 mmol/L tris base,
1 % w/v amidosulfobetaine-14 detergent, and 0.001 % w/v bromo­
phenol blue). The pellet suspension tubes were placed in an ice-bath,
and cells were ruptured by sonication using an ultrasonic probe at 20
kHz (Sonifier SFX550 Model, Branson, CT, USA). The sonication was
performed 4 times using 30 s bursts. Chilling the pellets between each
ultrasonication treatment was crucial to avoid protein denaturation
caused by generated heat. The tubes were centrifuged at 18 ◦ C and
15000 × g for 20 min. The supernatant containing bacterial proteins was
then transferred to a clean microcentrifuge tube. Bradford assay was
carried out to assess the protein concentration after the sonication
treatment (Bradford, 1976).
2.4. Two-dimensional electrophoresis (2D-IEF SDS-PAGE)
For isoelectric focusing (IEF), 400 μg of bacterial proteins in a total
volume of 300 μL in sample buffer was loaded onto 17 cm, pH 4–7, non-
linear, IPG strips (Ready Strip™, Bio-Rad, USA) in a rehydration tray.
Two mL of mineral oil was applied to each strip and kept overnight at
20 ◦ C for passive rehydration of proteins. The strips were then trans­
ferred to an IEF tray (Protean II IEF™ cell, Bio-Rad, USA), and protein
focusing was performed at 20 ◦ C with a total 65,000 Vh current supply.
Following IEF, the strips were applied with reduction and alkylation
buffers for 20 min each and then rinsed with ultrapure water. For the
second dimension SDS-PAGE, the strips were placed on 12 % poly­
acrylamide gels, and electrophoresis was performed in Protean II XL
Cell™ (Bio-Rad, USA) with a total current flow of 200 V at 10 ◦ C. The
gels were stained with Coomassie blue silver stain for protein visuali­
zation. The gels were scanned with a gel documentation system (Gel
Doc™, Bio-Rad). The spots were analyzed using MelanieTM Version
9.2.3 (Swiss Institute of Bioinformatics, Lausanne, Switzerland). The
protein spot intensities having more than 1.5-fold and lower than 0.5-
fold changes were considered upregulated and downregulated, respec­
tively, compared to the control. The selected spots were picked in 0.2-
mL PCR tubes, and 100 μL of ultrapure water was added and stored at
4 ◦ C.
2.5. Enzymatic digestion and protein identification
The dye from the spots was removed by washing with 1 mL of a
decoloring solution (50 % acetonitrile + 25 mmol/L ammonium bicar­
bonate solution) several times until the gel spot was decolorized. After
cleaning, 500 μL of acetonitrile was added to the gel spots for dehy­
dration. The supernatant was removed, and the gel spots were dried in
the air. To reduce the disulfide bonds, the gel spots were added with 10
mmol/L DTT (dithiothreitol) at 56℃ for 60 min. The supernatant was
removed, and 55 mmol/L IAM (iodoacetamide) was added in a dark
room for 45 min to perform alkylation of cysteine. The supernatant was
removed, and the gel spots were dried in the air. A 0.1 μg/μL trypsin
solution was added, and the tubes were kept on ice for 30 min. An
aliquot of 25 mmol/L ammonium bicarbonate solution was added to
cover the gel spots and digested overnight at 37℃. The remaining liquid
outside the gel spots was transferred to a new centrifuge tube. The
M.A. Baig et al.
peptide was extracted once from the gel spots with 50 % (v/v) aceto­
nitrile–water and again with 100 % acetonitrile. The two extracted so­
lutions were pooled with the trypsin digested solution and lyophilized.
2.6. Phase nano-LC-MS/MS analysis
The dried peptide samples were reconstituted with mobile phase A
(2 % ACN, 0.1 % FA), shaken, and centrifuged, and the supernatant was
taken. The samples were separated on a Shimadzu LC-20AD Nano
nanoliter liquid chromatography. After being enriched and desalted in
the trap column, the sample was directed into a tandem self-packed C18
column (75 µm internal diameter, 3 µm column size, 15 cm column
length). The following effective gradient was used to separate the
samples at a flow rate of 300 nL/min: 0–6 min, 6 % mobile phase B (98 %
ACN, 0.1 % FA); between 6 and 40 min, mobile phase B increased lin­
early from 6 % to 25 %. Mobile phase B increased from 25 % to 40 %
between 40 and 48 min; between 48 and 51 min, mobile phase B
climbed from 40 % to 90 %; mobile phase B: 90 % between 51 and 55
min; mobile phase B: 6 % between 55.5 and 60 min. The nanoliter liquid
chromatograph end was directly connected to a mass spectrometer LTQ
Orbitrap Velos (Thermo Fisher, Santa Clara, USA), and the ion source
was nano-ESI. In the data acquisition, the positive ion scanning mode
was adopted, the resolution of the MS1 was 30,000, and the scanning
mass range was 350 to 1,500 m/z. Based on the MS1 scanning infor­
mation and according to the ion intensity in the order MS1 from high to
low, the top six were selected for fragmentation, and the MS2 infor­
mation was scanned in an Orbitrap analyzer. The resolution of the MS2
was 7,500, and the fragmentation mode was HCD.
Fig. 1. 2DE gel images for protein expression profiles of Lc. garvieae C47 under heat, cold, acid and bile stresses. A, control; B, heat; C, cold; D, acid; E, bile. The arrow
indicates proteins quantitatively analyzed by MelanieTM Version 9.2.3 (Swiss Institute of Bioinformatics, Lausanne, Switzerland). PhaseNano-LC-MS/MS analysis
identified the protein spots and assigned identities/functions are listed in Table 1. (may place figures horizontally/in landscape format, to make them larger/
more visible).
3
Food Chemistry 397 (2022) 133774
2.7. Spot analysis and data processing
The spot intensities were quantitatively determined using MelanieTM
Version 9.2.3 (Swiss Institute of Bioinformatics, Lausanne, Switzerland).
Protein gel spot identification was based on the mass spectrometry data
generated by the experiment and matched with the simulated theoret­
ical mass spectrometry data obtained from database to acquire the
protein identification result. The selection of database is an important
step in MS based protein identification, and the final identified protein
sequences were from the selected database. The tandem mass spectra
were searched against the MASCOT v2.3.02 server database (www.
matrixscience.com/) and UniProt protein database (https://www.
uniprot.org/). The following parameters were employed for the Uni­
Prot search: enzyme digestion—trypsin, peptide mass tolerance—20
ppm, fragment mass tolerance—0.1 Da, fixed mod­
ifications—carbamidomethyl (C), variable modifications—oxidation
(M) —Gln->pyro-Glu (N-term Q)—deamidated (NQ), max missed
cleavages—1, instrument type—LTQ-Orbitrap velos, and taxonomy
search—bacteria. The search threshold for accepting each individual
spectrum was set to the default value, with a false discovery rate (FDR)
< 1 % and peptide minimum score greater than 40 were considered
significant. The spot intensities for all the differentially expressed pro­
teins in 2D gels were analyzed statistically using Metaboanalyst 5.0
software (Chong et al., 2018). For correlation analysis, the spot in­
tensities were Log2 transformed for performing the principal component
analysis (PCA), partial least squares-discriminant analysis (PLS-DA),
variable importance in projection (VIP), and heatmap.
M.A. Baig et al.
3. Result and discussion
3.1. Identification of differentially expressed proteins in Lc. Garvieae C47
and their functional categorization
Fig. 1A–1E exhibits the total 91 protein spots identified in the con­
trol, heat, cold, acid, and bile stress treatments. Out of which, 36 pro­
teins under acid stress (23 upregulated and 13 downregulated), 47
proteins under bile stress (22 upregulated and 25 downregulated), 45
proteins under heat stress (29 upregulated and 16 downregulated), and
39 proteins under cold stress (29 upregulated and 10 downregulated)
were differentially expressed compared to the control. Eleven upregu­
lated proteins were common among all the stress treatments, which
were Elongation factor G (spot C1), HTH-type transcriptional regulator
FruR (spot C14), Citrate (pro-3S)-lyase subunit beta (spot C32), Prob­
able dihydroxyacetone kinase regulator (spot C38), PTS mannose/
fructose/sorbose transporter subunit IIB (spot C56), Uroporphyrinogen
decarboxylase (spot C60), Ribosome maturation factor RimP (spot C67),
NADH-quinone oxidoreductase (spot C69), Cobalt ECF transporter T
component CbiQ (spot C73), Aspartate–ammonia ligase (spot C74), and
SAP domain-containing protein (spot C79). Two downregulated proteins
Malate dehydrogenase (spot C64) and Molybdopterin/thiamine
biosynthesis adenylyltransferase (spot C94) were common among all the
stress treatments (Table 1).
For functional categorization, the identified protein ID’s were
searched against the UniProt database, and the proteins were assigned to
different cellular functions (Fig. 2). Maximum 22 % of identified pro­
teins were found to be associated with carbohydrate metabolism, fol­
lowed by 20 % protein metabolism, 13 % amino acid metabolism, 16 %
transport, 11 % nucleotide metabolism, 9 % oxidation–reduction, 4 %
vitamin metabolism, 3 % stress response, and 2 % cell wall synthesis.
3.2. Multivariate data analysis
For analyzing correlations between protein profiles and changes
between the control, heat, cold, acid, and bile treatments, multivariate
data analysis was performed via MetaboAnalyst software. Before anal­
ysis, the differences in spot intensities for all the proteins were
normalized by log transformation (Fig. 3, A–F). An unsupervised PCA
biplot was created from the loadings plot, representing different
stresses’ influence on principal components (PCs) (Fig. 3, A–D). The PCA
score represents the variation in protein profile due to the influence of
different stress treatments. The PCA plots showed clear separation of
proteins among different PCs, representing the differential expression of
proteins. The first principal component (PC1) showed 40.6 % variation
among all the protein intensities, while the second component (PC2)
showed 30.6 % variation (Fig. 3B and 3C). The differentially expressed
proteins with maximum abundance were identified with variable
importance in a projection (VIP) plot with VIP scores ranging from 1.45
to 1.65 (Fig. 3F). The relative changes between differentially expressed
proteins under different stress treatments were observed by hierarchical
clustering and a correlation heatmap (Fig. 3E).
3.3. Differential proteomic responses of Lc. Garvieae C47 under heat,
cold, acid, and bile stresses
The fermented milk-based foods and foods with a short shelf life
require cold storage, which exposes the probiotic LAB to cold stress. The
adaptation and survival of LAB to a cold environment is a prerequisite
for their commercial chilled transportation and storage. LAB strains are
commonly freeze-dried into powder for their commercial supply (Zhang,
Liu, Chen, Du, & Chen, 2016). In our study L-lactate dehydrogenase (spot
C46) was expressed with maximum abundance among all the stress
treatments, which shows the protective role of this enzyme under
different stress conditions. The heat-shock treatment significantly
affected the survival rate of Lactobacillus acidophilus after freeze-drying,
4
Food Chemistry 397 (2022) 133774
which altered the activity of lactate dehydrogenase (Zhen et al., 2020).
In another study by Liu et al. (2021), the proteomic response of freeze-
dried L. acidophilus ATCC4356 following heat-shock treatment was
assessed. The authors suggested that proteins of amino acid metabolism,
glycolysis, ABC transportation, transcription, and translation provide
tolerance to the freeze-dried in L. acidophilus ATCC4356. For adaptation
to stressful environments, bacteria can synthesize or increase the pro­
duction of stress-responsive proteins. Lactobacillus kefiranofaciens M1
developed an adaptation to stressful environments by increased pro­
duction of heat shock proteins (HSPs), chaperones, phosphotransferases,
kinases, and cofactors, which play essential roles in proper folding, post-
translational modification, and translocation of newly synthesized pro­
teins (Chen et al., 2017).
The low pH environment of the stomach challenges the survival of
LAB. However, the viability of LAB at acidic pH has been reported in
several strains (Feng et al., 2019). These LAB strains have developed
mechanisms for survival at the low pH of their environment, such as the
stomach (pH 1.5–3) and fermented dairy foods (pH 4.6, pH 4–5.3)
(Maillot, Honoré, Byrne, Méjean, & Genest, 2019). Nonetheless, the
acidic pH exposes the LAB to severe stress. During LAB fermentation, a
proton translocating ATPase is secreted, which acts as a protective
molecule by maintaining the internal cellular pH in response to the low
pH of the external environment (Li, Tao, Teixeira, Shu-Wei Su, & Gänzle,
2020).
The majority of bacterial cell membrane protein structures consist of
ATP-binding cassette (ABC) transporters which regulate the inward and
outward transport of different biomolecules such as peptides, amino
acids, antibiotics, sugars, and micronutrients (Durmort & Brown, 2015).
Some ABC transporters promote biofilm formation in response to acid
and salt stresses. In our study, a putative ABC transporter ATP-binding
protein YheS (spot C2 & C85) was upregulated under acid and bile
stresses while downregulated under cold stress. The ribose ABC trans­
porter (spot C28) was downregulated under all the stress treatments.
The sugar ABC transporter (spot C44) was upregulated under bile stress
while being downregulated under acid and cold stresses.
Aminopeptidases hydrolyze the peptide bonds in proteins and
polypeptides at the N-terminus. In lactic acid bacteria, hydrolysis of
proline-containing peptides is mediated by specialized aminopeptidases,
which also regulate the utilization of caseins (Kunji, Mierau, Hagting,
Poolman, & Konings, 1996). The cytosol aminopeptidase (spot C57)
upregulation occurred probably under all the stress treatments. The
casein-derived peptides of Lactobacillus helveticus R211 and R389
generated by aminopeptidases cause inhibition of the angiotensin-
converting enzyme (Seppo, Jauhiainen, Poussa, & Korpela, 2003).
Bacteria can resist osmotic and temperature stresses by producing
compatible solutes like ectoine and hydroxyectoine, which are synthe­
sized by L-aspartate-beta-semialdehyde. Aspartokinase catalyzes the
formation of L-aspartate-beta-semialdehyde from L-aspartate (Stöveken
et al., 2011). In our study, aspartokinase (spot C17) was upregulated
under all the stress treatments, which could help Lc. garvieae C47 in
protection against these stresses.
In many bacteria, the sigma factors of RNA polymerase regulate the
responses towards environmental stress conditions. Kormanec and
Sevcikova (2002) identified a promoter recognized by stress-responsive
sigma factor σH in Streptomyces coelicolor. This promoter controls the
expression of the gltB gene, which encodes a protein that is homologous
to a large subunit of glutamate synthase; this suggests the control of
stress-response σH over the Streptomyces coelicolor gltB gene. The small
subunit GltB (spot C7) of glutamate synthase [NADPH] was upregulated
under cold stress and downregulated under heat stress. The adverse
environmental stresses such as extreme temperatures can affect the
bacterial metabolic pathways and cellular components such as decreases
in membrane fluidity, reduction in transcription and translation due to
the formation of RNA secondary structures, and diminished protein
export and folding. Bacterial cells have developed several mechanisms
to counteract these adverse effects, such as alterations in their
M.A. Baig et al. Food Chemistry 397 (2022) 133774

Table 1
Identified proteins in Lc. garvieae C47 with their molecular weight, pI values, protein spot intensities under heat, cold, acid and bile stresses. The proteins were
categorized according to their biological functions.
Function Homologous protein Molecular weight (kDa) pI Spot No. Induction factor (intensity)

Heat Cold Acid Bile

Stress response Chaperone protein DnaK 64515.28 4.30 C05 27.6 34.0 39.8 27.0
60 kDa chaperonin 57395.94 4.43 C08 52.4 48.0 23.4 53.0
Carbohydrate metabolism Enolase 47718.05 4.46 C26 49.7 48.0 40.7 53.0
Phosphoglycerate kinase 41867.02 4.59 C30 40.5 39.0 30.3 31.0
Citrate (pro-3S)-lyase subunit beta 33337.82 4.73 C32 60.7 95.0 73.6 89.0
Polysaccharide deacetylase family protein 24076.34 5.57 C35 24.8 29.0 23.4 22.0
Glyceraldehyde-3-phosphate dehydrogenase 35940.26 5.09 C39 67.2 108.0 74.4 94.0
UTP–glucose-1-phosphate uridylyltransferase 34235.20 6.13 C45 17.5 24.0 23.4 16.0
L-lactate dehydrogenase 34127.80 4.78 C46 97.5 85.0 56.3 40.0
ATP-dependent 6-phosphofructokinase 35752.50 5.64 C50 20.2 53.0 32.9 66.0
Fructose-bisphosphate aldolase 31302.00 4.56 C54 91.1 93.0 128.0 110.0
2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 28426.00 4.79 C62 41.4 34.0 19.9 26.0
Malate dehydrogenase 32378.00 4.56 C64 29.4 27.0 10.4 30.0
Maltokinase 49449.00 6.01 C71 138.0 124.0 145.0 102.0
Amino acid metabolism IGP synthase cyclase subunit 25129.60 4.19 C06 27.6 34.0 39.8 27.0
Glutamate synthase [NADPH] small subunit GltB 53052.85 5.19 C07 25.8 33.0 26.8 29.0
Aspartokinase 43729.10 8.09 C17 80.0 61.0 58.0 50.0
Ornithine carbamoyltransferase 38401.78 6.43 C33 51.5 58.0 54.5 62.0
Indole-3-glycerol phosphate synthase 29990.00 6.01 C55 99.4 120.0 13.0 15.0
Aspartate–ammonia ligase 36814.00 4.56 C74 95.7 75.0 58.9 90.0
Glutamine amidotransferase 18343.41 4.66 C86 41.4 51.0 14.7 49.0
Protein metabolism 30S ribosomal protein S1 44530.40 4.71 C12 20.2 34.0 29.4 42.0
HTH-type transcriptional regulator FruR 27470.82 9.73 C14 57.0 47.0 60.6 51.0
Elongation factor Tu 43232.01 4.60 C19 62.6 82.0 64.1 58.0
Probable cytosol aminopeptidase 52124.00 6.01 C20 77.3 66.0 37.2 33.0
Elongation factor Ts 32045.45 4.67 C42 45.1 39.0 19.9 29.0
Transcriptional regulator 34464.98 9.89 C58 13.8 29.0 28.6 6.8.0
Ribosome maturation factor RimP 17776.00 6.01 C67 86.5 56.0 100.0 31.0
Ribosomal RNA small subunit methyltransferase G 27117.26 5.92 C76 30.4 24.0 27.7 22.0
Ribosomal RNA large subunit methyltransferase H 17750.00 6.01 C80 38.6 30.0 13.9 70.0
50S ribosomal protein 17803.00 4.98 C82 27.6 30.0 26.0 19.0
Elongation factor G 76235.22 4.57 C01 18.4 22.0 48.5 18.0
Nucleotide metabolism Primosomal protein DnaI 33401.10 6.01 C09 58.9 76.0 42.4 73.0
Bifunctional protein PyrR 19712.58 4.78 C61 22.1 28.0 40.7 38.0
Adenylate kinase 23661.13 4.75 C65 42.3 39.0 39.8 34.0
Probable nicotinate-nucleotide adenylyltransferase 24253.00 6.01 C78 36.8 33.0 26.0 21.0
Uridine phosphorylase 27313.00 4.56 C84 52.4 68.0 106.0 63.0
Cytidylate kinase 23946.71 4.68 C88 33.1 40.0 49.3 19.0
Transport Putative ABC transporter ATP-binding protein YheS 71530.75 4.69 C02 11.0 7.0 48.5 15.0
Phosphoenolpyruvate-protein phosphotransferase 62604.67 4.41 C10 22.1 38.0 32.0 8.0
ATP synthase subunit beta 50932.19 4.48 C15 20.2 36.0 34.6 25.0
Rgg/GadR/MutR family transcriptional regulator 34324.77 7.71 C16 16.6 49.0 84.0 63.0
PTS transporter subunit EIIC 70656.40 7.70 C24 61.6 61.0 52.8 75.0
Ribose ABC transporter (ATP-binding protein) 54006.28 5.11 C28 128.0 108.0 93.5 86.0
Cobalt ECF transporter T component CbiQ, cbiQ 29796.50 10.86 C73 69.9 97.0 22.5 87.0
Sugar ABC transporter ATP-binding protein 57040.85 5.78 C44 144.0 127.0 100.0 145.0
ESX-5 secretion system protein EccC5 152816.00 4.19 C53 43.2 38.0 72.7 56.0
PTS mannose/fructose/sorbose transporter subunit IIB 16754.04 9.38 C56 69.0 49.0 58.0 24.0
Vitamin metabolism 3-methyl-2-oxobutanoate hydroxymethyltransferase 31123.00 9.73 C21 80.0 70.0 50.2 48.0
Biotin synthase 35935.00 4.56 C49 39.6 37.0 29.4 37.0
Cell wall synthesis UDP-N-acetylmuramate–L-alanine ligase 59868.00 4.60 C34 29.4 17.0 12.1 14.0
Oxidoreductase 4-hydroxymandelate oxidase 39738.36 4.79 C11 21.2 17.0 37.2 39.0
Thioredoxin reductase 33861.71 4.31 C52 25.8 20.0 34.6 16.0
4-methylaminobutanoate oxidase 89818.00. 5.19 C68 40.5 19.0 8.66 34.0
NADH-quinone oxidoreductase 19498.00 6.03 C69 103.0 80.0 68.4 80.0
Aldo/keto reductase 32618.57 5.22 C91 34.0 31.0 19.9 35.0
Miscellaneous proteins Molybdopterin/thiamine biosynthesis adenylyltransferase 37696.98 4.56 C94 27.6 34.0 19.9 24.0
DEAD/DEAH box helicase family protein 74299.91 4.80 C23 52.4 63.0 25.1 53.0
Peptidase T 45899.03 4.44 C25 38.6 27.0 21.6 27.0
C-di-GMP-specific phosphodiesterase 32304.73 8.36 C27 74.5 64.0 42.4 84.0
UPF0210 protein FVP42_08325 46539.45 4.96 C31 70.8 80.0 63.2 75.0
Isoleucine–tRNA ligase 106283.00 5.65 C36 32.2 27.0 31.2 56.0
Probable dihydroxyacetone kinase regulator 23756.08 6.03 C38 86.5 123.0 73.6 128.0
C alpha-dehydrogenase 32380.00 8.86 C40 62.6 89.0 110.0 54.0
Probable cytosol aminopeptidase 52124.00 4.56 C57 31.3 22.0 35.5 6.8.0
Uroporphyrinogen decarboxylase 40187.00 6.01 C60 18.4 13.0 22.5 10.0
CRISPR-associated endonuclease Cas9 172850.00 8.85 C66 12.9 17.0 33.8 5.7.0
DUF669 domain-containing protein 19033.50 4.40 C70 152.0 136.0 142.0 136.0
Uncharacterized hydrolase YsaA 29509.21 4.39 C75 121.0 113.0 138.0 94.0
UPF0260 protein YcgN 17894.00 4.79 C77 38.6 32.0 47.6 6.8.0
SAP domain-containing protein 44729.02 10.3 C79 18.4 17.0 37.2 17.0
Deoxyribose-phosphate aldolase 27944.00 4.98 C83 100.0 88.0 90.0 87.0
(continued on next page)

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M.A. Baig et al. Food Chemistry 397 (2022) 133774

Table 1 (continued )
Function Homologous protein Molecular weight (kDa) pI Spot No. Induction factor (intensity)

Heat Cold Acid Bile

Aldo/keto reductase 32618.57 5.22 C91 34.0 31.0 19.9 35.0

Fumarate reductase 13155.00 6.55 C92 39.6 55.0 44.1 34.0
Holo-[acyl-carrier-protein] synthase 14343.00 6.03 C93 32.2 33.0 26.0 22.0
Uncharacterized proteins Uncharacterized protein 39294.2 4.62 C4 68.1 83.0 60.6 96.0
Uncharacterized protein 24566.92 6.55 C13 33.1 29.0 48.5 35.0
Uncharacterized protein 6129.38 11.80 C41 36.8 38.0 39.0 31.0
Uncharacterized protein 43487.51 11.26 C48 6.44 14.0 19.9 17.0
Uncharacterized protein 16718.72 8.86 C81 67.2 67.0 38.1 100.0
Uncharacterized protein 13637.92 6.26 C90 50.6 54.0 6.93 45.0

Fig. 2. Functional categorization of Lc. garvieae C47 proteins identified under heat, cold, acid and bile stresses.

membrane composition to maintain viscosity.
Under normal and stressed conditions, chaperones play an important
role in stabilizing membrane structure and integrity (Chen et al., 2017).
Chaperone proteins protect the cells under cold stress and regulate the
folding of substrates. Maillot et al. (2019) studied the role of a major
ATP-dependent chaperone DnaK (Hsp 70), and its associated co-
chaperones in E. coli. These chaperones regulate proteostasis in the
majority of organisms. The absence of either DnaK or its associated co-
chaperones caused cold-sensitive phenotypes in E. coli. This indicates
the role of these chaperones in maintaining cell homeostasis at low
temperatures. Chaperone protein DnaK (spot C5) was downregulated
under all the stress treatments. DnaK negatively regulates heat shock
response which might induce heat shock proteins in response to stress
(Da Silva et al., 2003).
The biosynthesis of histidine is widely characterized due to its
pivotal role in cellular metabolism and its function as an intermediate
between nitrogen metabolism and purine biosynthesis. This intercon­
nection is catalyzed by imidazole glycerol phosphate synthase (IGP
synthase). Glutamine amidotransferase and a cyclase are the products of
two His genes, hisH, and hisF, which form the heterodimeric enzyme IGP
synthase (Chioccioli et al., 2020). The IGP synthase cyclase subunit (spot
C6) was downregulated under all the stress treatments in our study.
Phosphorylation is a necessary process that regulates protein func­
tioning and mediates the transport of molecules across the membrane,
and the transport of sugars across the membrane is essential for cellular
functioning. A multi-component phosphoenolpyruvate phospho­
transferase system (PEP-PTS) assists in the phosphorylation and trans­
port of specific sugars such as N-acetylglucosamine, sucrose, mannose,
and glucose (Houot, Chang, Pickering, Absalon, & Watnick, 2010). PEP-
PTS (spot C10) was upregulated under cold stress while being
downregulated under heat and bile stress.
Several transcription factors mediate the regulation of transcription.
Some transcription factors are specific to bacteria and archaea, which
help survive under various stress conditions. FruR is a bacterial HTH-
type transcriptional regulator belonging to the MerR family of tran­
scription regulators with two distinct DNA binding domains, N-terminal
and C-terminal effector domains (CTD). Most CTDs are responsive to
environmental stresses and activate the transcription of downstream
genes for the survival of bacteria under stresses induced by oxidative
radicals, metals, antibiotics, and drugs (Singh, Sevalkar, Sarkar, &
Karthikeyan, 2018). In our study, the upregulation of the HTH-type
transcriptional regulator (spot C14) under all stress treatments in­
dicates the possible activation of stress-responsive genes by Lc. garvieae
C47.
The attachment of probiotic bacteria to the intestinal mucosal sur­
face is vital for survival and functioning. Mucins are high molecular
weight proteins expressed on the mucosal surface and help in the
attachment of bacteria, including lactobacillus strains on the mucosal
surface. Some intestinal bacteria express adhesins which are specialized
surface proteins. Limosilactobacillus fermentum 104R has a mucus
adhesion-promoting protein (MAPP), and L. johnsonii NCC533 has
elongation factor Tu (EF-Tu) on their surface. In our study, EF-Tu (spot
C19) was upregulated under all the stress treatments.
Enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
are glycolytic enzymes secreted by several Gram-positive bacteria on
their surface, mediating pathogen-host interactions. The extracellular
proteome of several Lactobacillus species of the acidophilus group, such
as Lactobacillus crispatus, has enolase and GAPDH as their major con­
stituents at neutral pH. Enolase and GAPDH are plasminogen-binding
proteins localized on the cell surface of L. crispatus at pH 5. An

6
M.A. Baig et al. Food Chemistry 397 (2022) 133774

Fig. 3. Multivariate data analysis using MetaboAnalyst software. A, 3D-loading plot for PCA (colored plots from white to red indicate the intensity differentially
expressed proteins); B, 3D-score plot PCA exhibiting differences between stresses. C, PCA biplot for variation among differentially expressed proteins; D, score plot
PCA showing variation between stresses. E, hierarchical clustering and correlation heatmap (each colored cell in heatmap represents an average peak intensity value
in Table 1, the column represents stress treatments and rows represents proteins) and D, VIP score plot for proteins with maximum abundance. The color red in­
dicates increased, and blue indicates decreased Z scores, representing standard deviations from the mean compared to the control. (For interpretation of the ref­
erences to color in this figure legend, the reader is referred to the web version of this article.)

increase in the pH causes the release of these cell surface proteins into 2017). Enolase (spot C29) was upregulated under acid stress while being
the environment. At low isoelectric pH, these surface proteins bind to downregulated under bile and cold stresses. GAPDH (spot C39) was
lipoteichoic acids. It has been observed that the surface properties of upregulated under cold stress and downregulated under acid and heat
lactobacilli modify rapidly in response to pH fluctuations (Chen et al., stresses.

7
M.A. Baig et al.
In bacterial cells, the motility and adhesion are controlled by the
global second messenger cyclic diguanylic acid (c-di-GMP) (spot C27).
Diguanylate cyclases (DGC) and c-di-GMP-specific phosphodiesterases
(PDE) catalyze the synthesis and degradation of c-di-GMP, respectively
(Christen, Christen, Folcher, Schauerte, & Jenal, 2005). c-di-GMP was
downregulated under all the stress treatments, which might affect the
motility and adhesion of Lc. garvieae C47 under stress conditions. Argi­
nine is an important amino acid for bacteria. In addition to its role in
protein synthesis, it is a source of nitrogen, carbon, and energy for many
bacteria. The bacteria synthesize polyamines by utilizing arginine as a
substrate which provides acid resistance in E. coli (Charlier & Bervoets,
2019).
The de novo pathway for arginine biosynthesis involves the conver­
sion of ornithine into citrulline, which is catalyzed by ornithine carba­
moyltransferase (OTCase). This pathway is also utilized by detoxifying
the urea cycle. The enzyme OTCase was purified from a strictly psy­
chrophilic and piezophilic bacterium Moritella abyssi which exhibited
maximum activity at low temperatures compared to other cold-active
enzymes. OTCase from Moritella abyssi is less thermoresistant than its
counterparts from other thermophilic bacteria (Xu, Feller, Gerday, &
Glansdorff, 2003). In our study, OTCase (spot C33) was downregulated
under heat, cold and acid stresses. UDP-N-acetylmuramate-L-alanine
ligase (spot C34) catalyzes the synthesis of peptidoglycan for biogenesis
of bacterial cell wall and outer membrane. Thermo-acidophilic Alicy­
clobacillus acidoterrestris can grow at pH 3 and 20 ◦ C. UDP-N-ace­
tylmuramate-L-alanine ligase was differentially expressed in
A. acidoterrestris DSM 3922 when exposed to heat stress (Feng et al.,
2019). In our study, this enzyme was upregulated under heat and cold
stresses.
Lactiplantibacillus pentosus MP-10 is a potential probiotic bacterium
adapted to the human GIT. The molecular mechanism behind this
adaptation was studied using genome sequence (Abriouel et al., 2017).
The genes encoding proteins related to carbohydrate metabolism and
bacteria–host interactions were studied. In our study, a polysaccharide
deacetylase family protein (spot C35) was downregulated under all the
stress treatments. Carbohydrate-modifying enzymes such as poly­
saccharide deacetylase and other enzymes which metabolize carbohy­
drates such as raffinose, starch, and levan were identified. These
enzymes regulate bacterial adaptation to GIT by assimilating carbohy­
drates that are not digested by humans (Abriouel et al., 2017).
Biotin is an essential vitamin and micronutrient which works as a
cofactor for enzymes that require biotin. Bacteria can either synthesize
or import biotin from external sources, and Biotin is essential for the
survival of bacteria. The conversion of dethiobiotin to biotin is catalyzed
by biotin synthase. Transcriptomic analysis of L. helveticus CICC 22,171
showed the vital role of biotin in growth and energy metabolism. Biotin
regulates the fatty acid synthesis, energy supply, and intracellular
transport in bacterial cells (Yao, Chou, Wang, Zhao, & Zhang, 2018).
Biotin synthase (spot C49) was upregulated under heat, cold, and bile
stresses while being downregulated under acid stress.
Oxidoreductases are enzymes that catalyze several types of cellular
redox reactions. Oxidative damage due to environmental stresses can
affect bacterial cell structures and impairments protein functions (Wang,
Cui, & Qu, 2018). Thioredoxin (TRX) is a disulfide oxidoreductase that
can protect bacteria from oxidative stress. Thioredoxin gene trxB1 in
Lactiplantibacillus plantarum WCFS1 was overexpressed under oxidative
stress and protected the bacteria from oxidative damage. A 3-fold higher
thioredoxin reductase (TR) activity was observed in L. plantarum WCFS1
compared to a wild strain (Serrano et al., 2007). In our study thioredoxin
reductase (spot C52) was upregulated under acid stress while being
downregulated under heat, cold, and bile stresses.
Environmental stresses can affect cellular metabolism due to changes
in gene expression, which cause alterations in the activity of different
enzymes (Baig et al., 2021). Arginine catabolism causes ammonia pro­
duction via the transformation of aspartate into arginine, which is
catalyzed by aspartate-ammonia ligase. This process causes an elevation
8
Food Chemistry 397 (2022) 133774
in intracellular pH and the production of ATP. In L. plantarum ZDY2013,
the aspartate-ammonia ligase gene (asnA) was downregulated under
acid stress (Huang et al., 2016). In our study, aspartate-ammonia ligase
(spot C74) was upregulated under all the stress treatments.
In bacteria, bacterial glutamine-amidohydrolase (glutaminase) cat­
alyzes the conversion of glutamine to glutamate. The deamination of
glutamine assists in synthesizing bioactive metabolites such as γ-glu­
tamyl peptides and γ-aminobutyrate (GABA), provides acid resistance.
In Lactobacillus taiwanensis and Limosilactobacillus reuteri, glutamine can
cause increased acid resistance. In lactobacilli, the glutamine-
amidotransferase contributes less to glutamine metabolism and con­
tributes more towards acid resistance (Li et al., 2020). In our study,
glutamine-amidohydrolases (spot C86) were downregulated under acid,
bile, and heat stresses.
4. Conclusions
The intracellular proteome of Lc. garvieae C47 under different envi­
ronmental stresses was established for the first time. The differentially
expressed proteins were mainly classified as the main component and
stress response proteins of intermediary and central metabolism. This
allows withstanding extreme and sudden environmental fluctuations
and a clear understanding of the development of cross-protection
against multiple stresses. The 2DE referral maps can infer further
studies and provide a detailed insight into the metabolic activity and
processes of the cell under variable circumstances for industrial pur­
poses. In order to deal with the upcoming technological advancement
and enhance the survival of probiotic bacteria in unfavorable condi­
tions, the proteomics of Lc. gravieae could be useful.
Lc. garvieae C47 responded to heat, cold, acid, and bile stresses by
overexpression of proteins involved in carbohydrate and protein meta­
bolism, which indicates Lc. garvieae C47 shifts towards growth and en­
ergy metabolism for resistance against these stresses. Proteins such as
aminopeptidases, HTH-type transcriptional regulator, elongation factor
Tu, biotin synthase, and aspartate-ammonia ligase were overexpressed
in at least three out of four stress treatments. The overexpression of these
proteins could provide resistance to Lc. garvieae C47 against different
environmental stresses. The downregulated proteins such as cyclic
diguanylic acid can affect the motility and adhesion of bacteria, the IGP
synthase cyclase subunit can affect the biosynthesis of histidine, and
chaperone DnaK negatively regulates HSPs under stress conditions.
These proteins could be studied for their mechanism of action for
enhancement of viability of Lc. garvieae C47 under different stress con­
ditions and their potential use as probiotic markers. To the best of our
knowledge, this is the first study that demonstrated the proteomic re­
sponses of Lc. garvieae C47 under different environmental stresses.
Funding
Authors thank United Arab Emirates University (UAEU) for funding
this project via fund code 31F101.
CRediT authorship contribution statement
Mohd Affan Baig: Writing – original draft, Investigation, Data
curation, Formal analysis. Mark S. Turner: Conceptualization, Writing
– review & editing. Shao-Quan Liu: Writing – review & editing.
Nagendra N. Shah: Conceptualization, Writing – review & editing.
Mutamed M. Ayyash: Conceptualization, Writing – review & editing,
Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
M.A. Baig et al.
Data availability
Data will be made available on request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.133774.
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