Among the various food preservation methods, fermentation is commonly used
by the food industries for the production of infection free products. Indigenous
raw and fermented foods are strongly linked to culture and tradition. Among
the most commonly used microorganisms, Lactic Acid Bacteria (LAB) play an important
role in preserving milk and milk products. Their common occurrence in foods
coupled with their long historical use contribute to their acceptance as Generally
Recognized As Safe (GRAS) for human consumption and also are approved for Qualified
Presumption of Safety (QPS) (Liu et al., 2011;
Jans et al., 2012). Lactococcus, Lactobacillus,
Pedicoccus, Leuconostoc, Streptococcus, Enterococcus and Bifidobacterium
are important LAB genera (Liu et al., 2011).
Some researchers reported that LAB exist in many food products and are part
of natural population of Gastrointestinal (GI) tract (De
Vuyst and Leroy, 2007).
Presently, it is considered that LAB act against the pathogenetic organisms
to improve the quality of preserved foods for human consumption (De
Vuyst and Leroy, 2007; Settanni and Corsetti, 2008).
Those antimicrobials produced by Gram-positive bacteria, particularly LAB, have
been largely studied with the perspective of food protection against pathogenic
and spoilage microorganisms (Malheiros et al., 2012).
Many factors such as varying pH, growth of bacteriocins, nutrients competition
and displacing pathogens affect the protective mechanism of LAB for the control
of various pathogens of food in the GI. Bacteriocins are antimicrobial peptides
produced among bacteria that may show varied antimicrobial spectra. Also, bacteriocin
producers have the immunity to their own bacteriocins (Chen
and Hoover, 2003; De Vuyst and Leroy, 2007; Settanni
and Corsetti, 2008). They are also lethal to some food-borne pathogens and
spoilage bacteria (Chen and Hoover, 2003; Cheikhyoussef
et al., 2008). Food-borne pathogens and spoilage microorganisms such
as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus
and Clostridium tyrobutyricum were inhibited by bacteriocins of LAB
(Alegria et al., 2010).
Among the various LAB present in preserved foods, some LAB such as probiotics
Lactobacillus rhamnosus tended to reduce the adhesion and viability of
adherent Staphylococcus aureus (Vesterlund et
al., 2006). The health benefits of the consumption of fermented milk
containing viable or non-viable LAB have been extensively documented. Particularly,
it has been reported that the administration of fermented dairy products can
confer enhanced resistance against infections by enteric pathogens (Millette
et al., 2008).
The raw milk microbiota are an essential components of many traditional fermented
milk products and play important roles during manufacturing and ripening of
fermented products such as cheeses. The quantitative evolution of lactococci,
lactobacilli, leuconostocs and enterococci from milk to finished products like
cheese followed similar trends. In traditional fermentation, microflora dominated
by LAB probably originating from the vessel surface are used in fermentation
(Jans et al., 2012). In Europe, Africa and other
regions of the world, traditional food such camels milk were explored
as natural sources for LAB. However, studies on isolating active LAB from camels
milk are scarce or lacking. Therefore, the present study was aimed to isolate
active LAB from raw camels milk.
MATERIALS AND METHODS
Milk samples: A total of thirty five samples of raw camel's milk were
obtained by manual milking of camels. The milk samples were analyzed immediately
or, when necessary, stored refrigerated overnight prior to experiments.
Isolation of lab from traditional food: A 10 mL portion from each experimental
milk sample was stomached with 90 mL sterile peptone water (Oxoid, UK). Serial
dilutions were further prepared using the same diluent. MRS agar plates (Oxoid,
UK) were spread with 0.1 mL diluted samples with a sterile glass rod. Plates
were incubated anaerobically (Gas Pak) at 37°C for 48-72 h. Representative
colonies were aseptically picked from plates containing 20-100 colonies. Gram-positive
coccids/rods and catalase negative bacteria were considered as presumptive LAB.
Isolates and indicators were maintained in the respective broth with 15% glycerol
Antibacterial activity of isolates: Well diffusion assay Cell-Free Supernatants
(CFS) of isolates were obtained by centrifugation (9000 g, 10 min at 5°C)
of cultures grown in broths (MRS) for 48 h followed by 0.45 μm membrane
filtration (Nalgene, USA). MRS agar is often abbreviated to MRS, this type of
bacterial growth medium is so-named by its inventors: de Man, Rogosa and Sharpe
(MRS). Eight milliliters of soft agar media (MRS or BHI plus 0.75% agar) seeded
with 105 CFU mL-1 of indicator bacteria was overlaid onto
MRS agar plates (Nikolic et al., 2008). Wells
(5 mm) were made in the agar media and filled with 100 μL of CFS. Plates
were incubated at 37°C for 18-48 h. Clear inhibition zones around wells
in the lawn of indicators were measured in mm.
Effect of proteases: The CFS producing inhibition zones were tested
for susceptibility to α-Chymotrypsin (Sigma), Trypsin (Sigma) and Lipase
(Sigma) using the well assay mentioned earlier. L. monocytogenes ATCC
7644 was the indicator. Each enzyme (5 mg mL-1) was separately spotted
(20 μL) adjacent to the edge of well prior to incubation at 37°C for
20-48 h. Untreated CFS were used as control. The activity of isolates Enterococcus
(LG), Lactococcus (C8) and Lactobacillus (F3) was determined (well
assay) to evaluate effects of proteolytic enzymes on the antagonistic activity
of their CFS.
Activity of isolates in packaging materials: The method of Ercolini
et al. (2010) with some modifications was adopted for evaluating
the inhibition activity of CFS of isolates (LG, F3 and C8) against the pathogen
L. monocytogenes ATCC 7644. Plastic discs of 1 cm diameter were aseptically
dipped in CFS of isolates and left to dry under ambient temperatures. Discs
were aseptically put onto agar media (Tryptic soy agar +0.6% yeast extract)
and plates were incubated at 37°C for up to 24 h. The activity of CFS against
L. monocytogenes was also determined as described by Ercolini
et al. (2010).
Phenotypic characterization of active isolates: The isolates (presumptive
LAB) which produced CFS sensitive to proteolytic enzyme (bacteriocins) were
characterized using the phenotypical and biochemical tests (Holt,
1977; Morandi and Brasca, 2012).
Carbohydrate assimilation: Carbohydrate fermentation tests of the above
isolates were carried out by using API 50CH strips and API 50 CHL medium according
to manufacturer's instructions (BioMerieux, Marcy-l'Etoile, France).
Probiotic activity of isolates
Bile tolerance: The isolates were tested for their ability as probiotics
by tolerating bile salts following the procedures of some previous publications
(Ricci et al., 2011; Ripamonti
et al., 2011). Tolerance to bile salts was tested at 37°C by
inoculation of fresh cultures in MRS broth enriched with 0.3% Oxgall (Oxoid,
UK). Resistance to bile salts (Oxygall) was assessed in terms of viable count,
enumerated (in MRS agar, Gas Pak at 37°C) after incubation with bile salts
for 0 and 2 h.
Antibiotic resistance: Antimicrobial susceptibility was determined by
the standardized agar diffusion test on MRS agar (Morandi
and Brasca, 2012). Sterilized and tempered (50°C) agar media were seeded
with test isolates at 105 CFU mL-1. Antibiotic discs were
placed on agar surfaces. Plates were incubated for 12-48 h at 37°C. LAB
isolates were screened for their susceptibility to amikacin (30 μg/disc),
ampicillin (10 μg/disc), amoxicillin (30 μg/disc), bacitracin (10
IU disc-1), cefuroxime (30 μg/disc), chloramphenicol (30 μg/disc),
colistin (10 μg/disc) and gentamicin (10 μg/disc). After incubation,
inhibition zones were measured in mm.
Animal experiments: The impact of ingested probiotics (in vivo trails)
on fecal population was evaluated by the oral administration (bottle feeding)
of probiotics to animal models (albino mice). The experimental trails using
mice and other necessary items for feeding were conducted in the animal laboratory
of the College of Veterinary Medicine, King Faisal University (KFU). The modulation
of intestinal microbiota was assessed according to the procedures of Medici
et al. (2005) and Millette et al. (2008).
Animal experiments (feeding trials for 3-16 days) were conducted to evaluate
the health benefits (fecal microbiota modulation) of probiotic LAB (108
CFU mL-1 milk) isolated from samples. Prior to feeding experiments,
mice were kept for a 14-day acclimatization period (day zero) as recommended
by Shu et al. (1999).
Albino mice (25 g) were obtained from the random bred colony kept by the College
of Veterinary Medicine (KFU). The treated and control mice were fed ad libitum
(barley, corn, alfalfa, wheat bran, soybean meal, date syrup, vitamins and minerals)
from ARASCO, Saudi Arabia. The diet contained 13% crude protein, 9% crude fiber,
1% fat (crude) and 0.7% minerals. Mice were housed in groups of five per plastic
cage and kept under ambient conditions (20-23°C) in the animal laboratory.
Experimental mice were divided into six groups: (1): Control group were fed
on normal diet and water (2): three groups were fed on normal diet plus diluted
experimental probiotic yoghurt (108 CFU mL-1 of F3, LG
or C8). Meanwhile, two additional groups of mice were fed with UHT milk (blank
without probiotics) or a commercial probiotic dairy product (as reference).
From preliminary trails, it was observed that one isolate from camel's milk
(coded LG) caused aggressive behaviors in mice and thus was excluded from further
animal experiments. LG was later identified as Enterococcus.
Experimental mice were examined at regular intervals for any signs of behavioral
perturbation during the experiments. After defecation, the stool (controls,
treatments and reference) was aseptically collected and tested for selected
microorganisms; LAB (MRS agar), E. coli (EMB agar), Staphylococcus
aureus (Baird-Parker agar with egg yolk-tellurite supplement) (Medici
et al., 2005; Millette et al., 2008).
Statistical analysis: Data from triplicate experiments were evaluated
for statistical significance at 5% level of significance using ANOVA according
to SAS Institute (2001).
RESULTS AND DISCUSSION
The isolation and screening of microorganisms from natural sources have always
been the most powerful means for obtaining useful and genetically-stable strains
for industrially-important products. LAB are important in the food and dairy
industries because their metabolites (bacteriocins) act as natural preservatives
as well as flavor enhancers. Meanwhile, LAB find increasing acceptance as probiotics
which aid in stimulating immune responses, preventing infection by enteropathogenic
bacteria and treating and preventing diarrhea (Adnan and
Tan, 2007; Giraffa, 2011; Jiang
et al., 2012).
Presumptive LAB isolated from camel's milk samples from local farms were screened
for antagonistic activity against pathogenic microorganisms (e.g., L. monocytogenes
ATCC 7644, Staphylococcus aureus ATCC 29213).
Well assay: The three LAB isolates (Table 1) gave
clear inhibition zones (14-25 mm) against indicators (gram positive and negative
bacteria) in the well assay using the CFS. It is known that some LAB produce
bacteriocins which are inhibitory for closely related gram positive bacteria
(i.e., LAB) and well-known pathogens such as L. monocytogens. Up to date,
nisin is a bacteriocin accepted as a safe food preservative by over 45 countries.
It is the most widely used commercial bacteriocin and remains the only bacteriocin
that may be added to U.S. foods. Microbes produce an extraordinary array of
microbial defense systems. Metabolic by-products such as bacteriocins are loosely
defined as biologically active protein moieties with a bactericidal mode of
|| Inhibition zones in mm (well assay) of presumptive LAB isolates
against pathogens and reference LAB
|+: 14<zone<20 mm, ++: >20-25 mm
|| Effect of proteolytic enzymes and lipase on inhibitive activity
(well assay) of food isolates against L. monocytogenes ATCC 7644
|+: Inhibition zones>14-25 mm, -: No inhibition zones
The bacteriocin-producing strains have a natural immunity to their own bacteriocins.
LAB can inhibit the growth of other bacteria by synthesizing a wide array of
antimicrobials and bacteriocins. Their bio-preservation value is reflected in
their inhibition of food spoilage and pathogenic microorganisms (Liu
et al., 2011).
Effect of proteases: CFS of active isolates were subjected to various
proteases and a lipase. The results in Table 2 show that CFS
were sensitive to proteolytic enzymes indicating the proteinaceous nature (i.e.,
bacteriocins) of inhibitors. As expected, lipase did not affect the inhibition
activity of CFS against L. monocytogenes ATCC 7644. It is stated that
LAB bacteriocins are proteins and thus were degraded by proteases such as α-Chymotrypsin
(Alegria et al., 2010; Chen
and Hoover, 2003).
Activity of CFS in food packaging materials: Many studies revealed the
antimicrobial activities of packaging films containing bacteriocins (e.g., nisin)
or plant materials (e.g., cinnamaldehyde) against E. coli O157:H7, S.
enterica and L. monocytogenes in poultry and meat (Ercolini
et al., 2010; Ravishankar et al., 2009).
Therefore, CFS of some active isolates were tested for activity in food-contacting
surfaces (packaging) against L. monocytogenes ATCC 7644. As shown in
Table 3, inhibitory substances for L. monocytogenes
ATCC 7644 of CFS from isolates (F3, C8 and LG) gave clear zones under the plastic
discs (plastic wrapping of food). Meanwhile, no inhibition halos were detected
against the pathogen around the plastic discs. Evidently, this finding indicated
that inhibitory substances present in CFS did not migrate from the discs (plastic
packages) and thus the adsorbed components imposing only their inhibitory activity
against L. monocytogenes ATCC 7644 in the area underneath the plastic
films. This finding was in a complete agreement with results reported by Ercolini
et al. (2010).
Phenotypic characteristics of active isolates: Some of the phenotypic
characteristics of the LAB isolates are presented in Table 4.
As per assumption, the LAB isolates were gram-positive, catalase negative, coccids
or rods bacteria. They were homo-fermentative LAB producing only lactic acid
from glucose. Lactobacillus plantarum and Lactococcus lactis were
isolated from camel's milk in Kuwait (Yateem et al.,
|| Inhibitory activity against L. monocytogenes ATCC
7644 of CFS (LAB isolates) adsorbed to food packaging plastic films
|+: Inhibition, -: No inhibition, LG: Enterococcus,
F3: Lactobacillus, C8: Lactococcus
|| Some characteristics of presumptive LAB isolated from camel's
|-: Negative, +: Positive, c: Cocci, r: Rods, W: Weak. LG:
Enterococcus, C8: Lactococcus, F3: Lactobacillus
Isolates lowered the pH of MRS broths and skim milk to below 5 when incubated
at 22°C as a result of producing lactic acid. The three isolates (LG, F3
and C8) had no β-hemolytic activity (Table 4).
Utilization of carbohydrates: The carbohydrate assimilation (API 50)
pattern of test isolates is presented in Table 5. Some isolates
were identified (89-95%) as Lactococcus (C8) and Lactobacillus
(F3) using the API software (BioMerieux Model Mini API).
Probiotic properties of LAB isolates: Probiotic traits (e.g., bile salt
tolerance) of selected isolates showing better inhibition against the pathogen
L. monocytogenes (Table 1) were evaluated as described
Bile tolerance of isolates: As seen in Fig. 1 and
2, counts (log10 CFU mL-1) of test isolates
were not significantly (p>0.05) affected by bile slats in MRS broth. Similarly,
other LAB (i.e., L. helveticus, L. paracasei and L. rhamnosus)
isolated from artisanal Italian cheeses were resistant to 0.3% bile salts (Ricci
et al., 2011). These results highlight the potential of the strains
of dairy origin to survive under gastrointestinal conditions. Bile salts did
not significantly affect the viability of Bifidobacterium longum and
Lactobacillus paracasei subsp. paracasei (Ripamonti
et al., 2011). Bile tolerance, being an important character, enables
the probiotic strains to survive, grow and exert their beneficial effects in
the host (Ricci et al., 2011; Ripamonti
et al., 2011).
Antibiotic sensitivity of isolates: Table 6 lists
the inhibition zones produced by the antibiotic discs. Lactococcus lactis
subsp. Lactis ATCC was used as a reference.
|| Carbohydrate assimilation of Isolates using the API 50 CH
|+: Positive, -: Negative, w: weak reaction, Isolates. LG:
Enterococcus, F3: Lactobacillus, C8: Lactococcus
||Growth (log10 CFU mL-1) of LAB isolates
in MRS broth (without bile salt) during incubation at 37°C for 0 to
All isolates were sensitive to ampicillin, amoxicillin, bacitracin and chloramphenicol.
The range of some important isolates was 40, 30 and 26 of LG, C8 and F3, respectively
for Ampicillin; 45, 34 and 28 of LG, C8 and F3, respectively for Amoxicillin;
25, 13 and 12 of LG, C8 and F3, respectively for Bacterian whereas as it was
30, 25 and 24 of LG, C8 and F3, respectively for Chloemphenicol.
|| Diameter of inhibition zones (mm) for presumptive LAB isolates
using disc diffusion test of 8 antibiotics
|Lactococcus lactis ssp. lactis ATCC 11955,
a-: No inhibition. Isolates: LG: Enterococcus, F3: Lactobacillus,
||Survival (log10 CFU mL-1) of LAB isolates
in MRS broth with 0.3% bile salts during incubation at 37°C for 0 to
On the other hand, colistin had no activity against isolates. The LAB displays
a wide range of natural antibiotic resistances but in most cases antibiotic
resistance is not of the transmissible type. Lactobacillus strains with
non-transmissible antibiotic resistances are not usually of a safety concern.
It has to be considered that antibiotic resistance observed in LAB strains is
often intrinsic and non-transmissible because it is chromosomally encoded (Ripamonti
et al., 2011). Due to the indiscriminate use of antibiotics in human
and veterinary medicine as well as in animal growth promoters, antibiotic resistance
has become an increasingly common characteristic in microorganisms. However,
checking the ability of a proposed probiotic strain to act as a donor of antibiotic
resistance genes may be a further prudent precaution (Ricci
et al., 2011).
Animal feeding experiments: Now a days, there is a growing interest
in using some LAB as probiotics in food products to enhance immunity and prevent
gastrointestinal infections. According to the WHO, probiotics are live microorganisms
and when administered in adequate amounts confer a health benefit on the host
(Giraffa, 2011; Millette et al.,
2008; Vesterlund et al., 2006). Since, January
2006, the European ban of Growth Promoter Antibiotic (GPA) has resulted in an
increased interest in the use of probiotics as feed additives to optimize gut
health and animal performance (Ripamonti et al.,
|| Counts (log10 CFU g-1 feces) of microorganisms
in mice groups fed daily with LAB isolates used as probiotics for sixteen
|Zero day counts (log10 CFU g-1), LAB
8.2, E. coli 6.6, S. aureus 6.9. n.d.: Not done. *p<0.05.
Isolates: F3: Lactobacillus, C8: Lactococcus
In the present investigation, there were no abnormal signs in both the control
and treated mice during the 16 day period of experiments. Although F3-treated
mice consumed more milk (100 mL daily), yet there were no significant differences
(p>0.05) in consumption of liquids between untreated (water or blank milk)
and treated (F3 or C8 probiotic milks) mice. This finding completely agreed
with data reported by Shu et al. (1999).
As depicted in Table 7, F3 and C8 possessed antagonistic
activities against some of GI microflora of mice. In fact, there are several
reports showing that specific probiotic strains protect against gastrointestinal
infections (Millette et al., 2008; Vesterlund
et al., 2006). The safety of different probiotic LAB, such as Lactobacillus
acidophilus and Bifidobacterium was studied in mice fed with doses
of test strains.
Although the safety status of traditional LAB strains used in food products
has been defined by extensive studies, it is nevertheless important to confirm
the safety of any newly identified probiotic strains (Shu
et al., 1999). Based on the above reports, the safety and efficacy
of some isolated LAB from test food (camels milk) were carried out using
mice feeding trails.
In the present study, there was no significant difference (p>0.05) in weight
gain among control and treated mice. The average gain was 1.2 g during the trail
periods of 16 days. These findings were in complete agreement with a previous
investigation by Vesterlund et al. (2006) regarding
detachment of pathogens to GI, F3 and C8 prevented adhesion of staphylococci
in the intestine of mice. The counts of staphylococci in feces of mice fed test
isolates were significantly (p<0.05) higher comparing to the control or blank
milk (Table 7). Additionally, probiotic isolates antagonized
growth of E. coli (Table 7) since the counts of those
enterics were less in feces of mice fed test isolates (F3 or C8). The commercial
probiotic yoghurt used in experiments contained Bifidobacterium acitiregularis
(as indicated in label) had lesser activity, compared to control or blank, against
E. coli (Table 7).
Probiotic-containing foods can be categorized as functional foods and are often
associated with prebiotics which are non-digestible carbohydrates that act as
food for probiotics. Functional foods contain beneficial properties over and
above their normal nutritional value. In this framework, probiotics are actually
being functional products. Probiotics are obtained by the action of microorganisms,
usually LAB and yeasts. Those microorganisms are useful in assisting the gastrointestinal
tract by breaking down sugars and carbohydrates to promote good digestion, boost
the immune system and maintain proper intestinal pH. When probiotics and prebiotics
are combined, they form a synbiotic. Yoghurt is considered a synbiotic food
because it contains live bacteria (Giraffa, 2011). When
choosing a probiotic strain, it must meet different requisites, including product
safety for human and animal consumption (GRAS) and survival in the Gastrointestinal
(GI) tract. Probiotics are chosen from the European list of accepted additives;
the reference law for the permission to introduce and employ additives in feedstuff
or pre-mixtures for feedstuff is the EU Regulation 1831/2003. For this reason,
the development of new probiotic products that could be licensed for animal
use is receiving considerable interest (Ripamonti et
The present study showed varying levels of probiotics in the camels
milk. The CFS were sensitive to proteolytic enzymes indicating the proteinaceous
nature (i.e., bacteriocins) of inhibitors. There was no significant difference
(p>0.05) in weight gain among control and treated mice. The counts (log10
CFU mL-1) of test isolates were not significantly (p>0.05) affected
by bile slats in MRS broth. The LAB isolates exhibited probiotic activities
in albino mice by preventing the adhesion of Staphylococcus aureus and
inhibiting E. coli in GI.
The authors thank the King Abdul-Aziz City for Science and Technology (KACST)
for the partial support of this research project no. ARP-29-141.