Hindawi Publishing Corporation
Gastroenterology Research and Practice
Volume 2012, Article ID 457150, 6pages
Development of Probiotic Candidate in Combination with
Essential Oils from Medicinal Plant and Their Effect on Enteric
Pathogens: A Review
Shipradeep,1Sourish Karmakar,1Rashmi Sahay Khare,2Sumedha Ojha,1
Kanika Kundu,2and Subir Kundu1
1School of Biochemical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India
2Chemistry Section, MMV, Banaras Hindu University, Varanasi 221005, India
Correspondence should be addressed to Subir Kundu, email@example.com
Received 17 March 2012; Revised 4 May 2012; Accepted 12 May 2012
Academic Editor: Antonio Gasbarrini
Copyright © 2012 Shipradeep et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Medicinal plants and probiotics both have very high potential in terms of their antimicrobial activity against antibiotic-resistant
enteric pathogens. The probiotics being enteric microorganism do not have any parasitic eﬀect on human beings. They have been
an integral part of daily food for centuries. They have been shown to have health beneﬁciary properties. The probiotics retard the
growth of the microorganisms, while essential oil kills them. Combining the eﬀect of medicinal plant extract and probiotics may
be a new approach due to their complementary antimicrobial eﬀects and practically no side eﬀects. The synergistic eﬀect of the
essential oil and probiotics will be necessarily higher than using them alone as health product.
The plants have been used in Ayurvedic medicines from
ancient times. The extracts from these plants have shown
potent antimicrobial eﬀect. Recently, much work has been
done on extraction of chemicals responsible for the antimi-
crobial eﬀect from these plant species. It has been reported
that the essential oils extracted from these plants have potent
activity against microorganisms . However, the studies
have shown that these essential oils have very high MIC
(minimal inhibitory concentration) against beneﬁcial enteric
bacteria known as probiotics [2,3].
Probiotic is the term as per WHO deﬁnition denotes
“live microbial feed supplement which beneﬁcially aﬀects the
host animal by improving its intestinal microbial balance.”
As the deﬁnition clearly indicates, most of the intestinal
bacteria have an important role to play in the digestive
system. Earlier, probiotics were given to animals to improve
their health, but later much research has been put in
the development of the probiotics for human health. The
major probiotics that are taken in the diets belongs to the
genera of Lactobacilli and Biﬁdobacteria . Apart from
that, the gut ﬂora predominately has obligate anaerobes that
include Biﬁdobacteria, Clostridia, Eubacteria, Fusobacteria,
of these bacteria are facultative anaerobes belonging to the
genera of Lactobacilli, Escherichia coli, Klebsiella, Streptococ-
cus, Staphylococcus,andBacilli. In the case of newborns,
food habits play a major role in the development of enteric
ﬂora. The breastfed babies normally have abundance of
Biﬁdobacteria, while the others have complex microﬂora
in their enteric system. Biﬁdobacterium sp. can be isolated
mostly from the feces of infant milk feed baby. However,
in the case of infants fed on normal formula based food
products the gut ﬂora is found to be rich in Enterobacteria,
Lactobacilli, Bacteroides, Clostridia,andStreptococci. These
gut ﬂora help to digest the milk-based food and oﬀer the
primary line of defense against the pathogenic bacteria.
The infants have weak but developing immune system .
These enteric bacteria help the infantile immune system to
ﬁght against pathogenic enteric bacteria by lowering the pH
of the gut, rendering it unsuitable for pathogenic bacteria
to survive . Even the medical practitioners recommend
probiotics-based supplement to both patients suﬀering from
2Gastroenterology Research and Practice
enteric diseases. The most popular probiotics supplements
belong to the genera of Lactobacilli and Biﬁdobacteria. The
recommended dosage of 109–1010 CFU is considered a
minimum for healthy enteric system .
Present review emphasizes on the synergistic antimicro-
bial eﬀect of essential oil of the Lamiaceae family and probi-
otics administered together as ﬂavored fermented milk prod-
ucts. The advantage of using such a combination is its beneﬁ-
cial eﬀect with its antimicrobial property. The probiotics can
help in improving the gut epithelial conditions while essen-
tial oil acts on killing the pathogens present in the human
2. Health Beneﬁts of Probiotics
Probiotics, though recently popular, have been an integral
part of the human diet for centuries. All the civilizations from
ancient times have documented the beneﬁts of curd in the
human diet. The lactose-tolerant people are always advised
to take curd with their diet. The curd is rich in Lactobacillus
sp. and Streptococcus sp. These microorganisms utilize the
lactose present in milk-based food and convert it to lactic
intolerant individuals is also observed with fatty acid. The
carbohydrates that are not fully digested due to lack of
certain enzymes in human being can also be digested with
probiotics. These carbohydrates are fermented into short-
chain acids such as butyric acid, lactic acid, or acetic acid
. These acids are readily utilized in by human cells for
ATP metabolism providing energy to the individuals. The
lactic acid also helps in protein metabolism by coagulating
the protein chunks from meat inside the intestine .
Formation of hydrogen peroxide is also prevented by cata-
lases produced from probiotics preventing protein-caused
rancidity [11–13]. Hydrolysis of sarcoplasmic protein was
also observed with many species of Lactobacillus genus [14–
16]. Coprecipitation of cholesterol with bile salts at lactic
acid-induced lower pH is also observed in in vitro conditions
Probiotic microorganisms are also found to be involved
in synthesis of vitamins. The probiotics microorganisms are
known to synthesize biotin and vitamin K . Apart from
that, they are also involved in the ions absorption such as
The probiotic microorganisms are also involved in the
enhancement of expression of certain pattern recognition
receptors. Pattern recognition receptors such as TLRs have
active role in wound healing process. The intestinal cells have
high need for these receptors for supporting their process of
proliferation and diﬀerentiation, healing the wounds made
due to irregular bowel movement . The short-chain
fatty acid produced from carbohydrate metabolism also
enhances the process of proliferation and diﬀerentiation of
gut epithelial cells.
The probiotics have also a major role to play in preven-
tion of allergies in children . However, the connection
of probiotics and immune system regulation is still under
investigation. It has been observed that with allergy-prone
adults and children, the count of Lactobacilli and Biﬁdobac-
teria is lower. It has been also observed that administration
of probiotic strains during prenatal stage can decrease the
chance of atopic eczema. In addition, the production of pat-
tern recognition receptors, interleukin, and growth factors
from the probiotic microorganisms in gut epithelia also play
an important role in prevention of allergies. Therefore, it
can be inferred that these microorganisms have direct role
in immune system regulation . Apart from that, these
microorganisms also play a role in immune response mod-
ulation. The probiotic microorganisms interact with the gut-
associated lymphoid tissue (GALT) . The probiotics are
involved in cytokine synthesis, that plays an important role in
immune system regulation. However, due to insuﬃcient clin-
ical trial, administration of probiotics in immunosuppressed
individuals is still prohibited.
It has been also observed from both in vitro and in vivo
studies that probiotics may prevent cancer . It has
been found that daily intake of fermented milk products
substantially decrease the concentration of nitroreductases,
azoreductases, and β-glucuronidase in the gut. These micro-
bial enzymes are associated with carcinogen production
in the gut . Lactobacillus casei have also shown an
antigenotoxic eﬀect. It prevents inducible DNA damage in
the tumor target tissues of gastrointestinal tract of rats.
3. Antimicrobial Effect and
Mechanism of Action
Probiotics have a known antimicrobial eﬀect. They are
very potent against pathogens. There are several proposed
mechanisms for the antimicrobial action of the probiotics.
Bacteriocins, organic acids, hydrogen peroxide, diacetyl, and
other inhibitory chemicals are released by the probiotics .
All of these chemicals are known for their potent antimi-
crobial eﬀects. Bacteriocins are toxic chemicals released by
the probiotics, that are highly potent against most of the
bacteria. However, the most feasible mode of action seems
to be lowering of pH with release of organic acids such as
lactic acid [24,25]. In the limiting condition of available
substrates inside the intestine, lowering the pH ensures the
survival of acidophilic micro-organisms only. The growth
of the pathogens gets inhibited at acidic conditions, slowing
the metabolic process in them. Lactobacillus strain GG has
been reported to produce inhibitory chemicals, possibly a
microcin, that have high activity against pathogenic microor-
ganisms. It has been found eﬀective against Clostridium spp.,
Bacteriodes spp., Enterobacteriaceae spp., Staphylococcus spp.,
and Pseudomonas spp. in microbiological assays. Lactocidin
released by strains of lactobacillus acidophilus is found active
against Staphylococcus aureus and Pseudomonas aeruginosa
. There has been a study that Lactobacillus acidophilus
LB release chemicals that are eﬀective against both gram
positive and gram negative microorganisms. These chemicals
released in the broth were eﬀective against Staphylococ-
cus aureus,Listeria spp.,Salmonella typhimurium,Shigella
ﬂexneri,E. coli,Klebsiella pneumoniae,Bacillus cereus,Pseu-
domonas aeruginosa,andEnterobacter spp. . However,
Gastroenterology Research and Practice 3
Tab l e 1: MICs of essential oil against known pathogenic microorganisms.
Bacterial strains Coleus aromaticus Shyama tulasi Rama tulasi
Providencia rettgeri ∼4μl/mL ∼3μl/mL ∼2μl/mL
Shigella ﬂexneri ∼1μl/mL ∼5μl/mL ∼5μl/mL
Shigella dysentery ∼3μl/mL — ∼5μl/mL
Vibrio parahaemolyticus ∼2μl/mL ∼5μl/mL ∼5μl/mL
Salmonella enteritis ∼4μl/mL ∼2μl/mL ∼3μl/mL
Salmonella typhi ∼0.5 μl/mL ∼2μl/mL ∼2μl/mL
Vibrio cholerae ∼2μl/mL ∼2μl/mL ∼1μl/mL
the chemical did not have any inhibitory eﬀect on probiotics
strains such as Lactobacillus and Biﬁdobacterium spp. This
can be explained by the similarity of survival conditions
of both these microorganisms. Some of the strains of
Biﬁdobacterium spp. have potent activity against Salmonella
typhimurium. However, not all the strains of Biﬁdobacterium
spp. have the activity against S. typhimurium. All of the pro-
biotics have higher survivability in low pH conditions. These
microorganisms produce acids by breaking the carbohydrate
present in the diet. The properties of acid production and
acid survivability increase their survivability in the toughest
of conditions [28,29]. The adherence property of the
probiotic microorganisms also ensures their longevity in the
human guts . However, the probiotic strains have shown
an eﬀective potential in inhibiting the adhesion of pathogen
such as E. coli and Salmonella enterica in in vitro conditions
. The potential of adhesion inhibition by the probiotics
is credited to the mucin production and competitive binding
to gut epithelial receptor sites. Lactobacillus acidophilus
LA1 has high calcium independent adhesive property that
inhibits the invasion of enteropathogenic bacteria. Mucins
are complex glycoprotein that inhibits the enterobacterial
adhesion by protection of intestinal epithelial cell receptors.
Both MUC2 and MUC3 produced by Lactobacillus spp. are
potent examples of Mucins that have adhesion inhibitory
activity against enteropathogens.
4. Antimicrobial Effects of Essential Oils from
There has been lot of studies in recent year that have
established the antimicrobial eﬀect of essential oils of
medicinal plants such as plants of the Lamiaceae family
[2,3]. The essential oils predominately present in the
leaves of the plant species have a pleasant aroma. They are
commonly used in ﬂavor enhancement in food industries,
as they are safe for human consumption. These essential
oils have been shown to have a bactericidal eﬀect. The
plant species of Lamiaceae family have been proven eﬀective
against Uropathogen . Tab l e 1 shows the MICs of Coleus
aromaticus and Ocimum sanctum (Rama Tulasi and Shayama
Tul a s i) against few known enteric pathogens . The
essential oil from the plants of Carum carvi, Coleus aro-
maticus, Rama Tulasi, Shyama Tulasi, Citrus aurantium var.
amara,foeniculumvulgare dulce, Illicium verum, Lavandula
angustifolia, Mentha arvensis, Mentha x piperita,andTra-
chyspermum copticum have been shown to be eﬀective against
variety of microorganisms. These plants extracts have been
found eﬀective against Bacteroides fragilis, Candida albicans,
Clostridium diﬃcile, Clostridium perfringens, Enterococcus
faecalis, Escherichia coli, Eubacterium limos, Staphylococ-
cus aureus, Klebsiella oxytoca, Proteus vulgaris, Escherichia
coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus
mirabilis,andPeptostreptococcus anaerobius . The MICs
against these microorganisms varies from 0.1 to 3%v/v.
The MICs of the same plant extracts against probiotic
microorganisms such as Biﬁdobacterium biﬁdum, Biﬁdobac-
terium longum, Lactobacillus acidophilus,andLactobacillus
plantarum are much higher in magnitude than the pathogens
. Therefore, if the dosage of essential oil is low, then
it eﬀectively wipes out the pathogens without harming the
5. Proposal on Synergistic Effect of Probiotics
and Essential Oil from Plants
The essential oils have high MIC values for probiotics,
while it is eﬀective in much lesser concentration against
the pathogens. The above phenomenon makes it possible
that both probiotics and essential oil can be administered
together to cure pathogenic infection in human gut. They
both can be combined to form essential oil-ﬂavored fer-
mented milk products such as ﬂavored curd beverages or
ﬂavored yogurt. Antibiotics coupled with probiotics are
already present in the market, but these medicines mostly
face stiﬀchallenge from antibiotic-resistant bacteria. Further
frequent use of the antibiotics may lead to the development
of antibiotic resistance in the pathogenic microorganisms
too. Hence, the strategic use of probiotics may be beneﬁcial
to curb the growing phenomenon of antibiotic resistance.
Probiotics have antimicrobial properties associated with the
production of bacteriocin-like chemicals. However, it mostly
arrests the proliferation of the pathogens by lowering the pH
in the gut environment. The pathogens do not normally have
any mechanism against the action of essential oils. Essential
oils are resistant against enzymatic activity of β-lactamase
produced as a countermeasure against β-lactam antibiotics.
The use of probiotics lowers the survivability chances of
pathogen, while the essential oil in lower dosage ensures
their complete killing inside the human digestive tract. The
probiotics may also impart its good beneﬁts discussed earlier.
4Gastroenterology Research and Practice
Tab l e 2: Test of the beverages made with mixing diﬀerent concentration of essential oil of Coleus aromaticus, Rama tulasi, and Shyama tulasi against common pathogens for 24hrs at 37◦C
(−sign denotes no pathogen; + sign denotes the presence of pathogen).
S. Bacteria Gram Beverage Beverage Beverage Beverage Beverage Beverage Beverage Beverage Beverage
(1) Citrobacter freundii −ve −−−−−−−−−
(2) Proteus mirabilis −ve + + + −−−+−−
(3) Klebsiellal pneumoniae −ve++++++++−
(4) E. coli ATCC 25922 −ve + −−++++−−
(5) Entero. faecalis ATCC 29912 +ve −−−−−−−−−
(6) Salmo. typhi MTCC 3216 −ve −−−−−−++−
(7) Staph. aureus ATCC 25923 +ve + + −++−+−−
(8) Salmonella typhimurium −ve + −−−−−−−−
(9) Salmonella paratyphi −ve + + −+−−++−
(10) Vibrio cholerae −ve −−−−−−−−−
(11) Pseudo. aeru. ATCC 27853 −ve −−−−−−−−−
(12) Proteus vulgaris −ve + + −−−−+−−
(13) Listeria monocytogenes +ve + + −++−+−−
(14) Shigella ﬂexneri −ve −−−+−− ++−
(15) Helicobacter pylori −ve −−−−−−−−−
(16) Strptococcus heamophila +ve + −−−−−−−−
Gastroenterology Research and Practice 5
Apart from that, the fermented milk product will surely
impart beneﬁts in terms of supplying nutrients such as
sugar, water, salt, and acid to the human body. Adding
essential oil will not only give an aromatic ﬂavor to these
fermented milk beverages or products, but also increase their
shelf like considerably by preventing the microbial spoilage.
The product will act as both probiotic health product and
preventive antimicrobial product against enteric pathogens.
In an independent study, beverages A, B, and C were
prepared with probiotic curd (109CFU/ml) withvary-
ing concentration of essential oil of Coleus aromaticus, Rama
Tul a s i and Shyama Tulasi,respectively. The beverages
A1, A2, and A3 were prepared with essential oil of Coleus
aromaticus; beverage B1, B2, and B3 with essential oil of
Shyama Tulasi and beverages C1, C2, and C3 with essential
of Rama Tulasi in varying concentrations of 1, 2, and 3 μl/ml
respectively. These beverages were then grown with common
enteric pathogens in equal concentration, measured by count
of CFU, in nutrient broth for 24 hours in airtight culture
vials at 37◦C to simulate the anaerobic condition prevailing
in the intestine . The individual vial was tested for the
traces of pathogen as seen in Tab l e 2 with −sign indicating
the cidal eﬀect of the beverage against the pathogen (no
growth of the pathogens), while + sign indicated the growth
of the pathogen. The sample beverages were found to be
highly eﬀective in inhibiting the growth of the pathogen. The
shelf life of the beverages was also found to be signiﬁcantly
higher than normal probiotics . The test results can be
interpreted as the beverage’s capacity for prevention against
enteric pathogens. The use of beverage does not need the
stringent FDA regulations, yet it will impart the beneﬁt of
Probiotics and essential oils both have a great potential
in terms of their beneﬁcial eﬀect against microbial gut
infection. They also show a synergistic eﬀect that is normally
higher than any known drug due to their complementary
actions. Since most of these medicinal plants are edible, their
extracts as food product do not have any side eﬀects with
low dosage. Therefore, these products may be very beneﬁcial
for human beings. However, much research is needed to be
put into these studies, as drug regulatory authorities still
have strong regulations against usage of plant extracts as
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