PHILIPP AGRIC SCIENTIST ISSN 0031-7454

Vol. 95 No. 1, 22-32

March 2012

Resistance Profile of Probiotic Lactic Acid Bacteria Against

Inhibitory Effects of Selected Plant Extracts
Jennifer D. Saguibo* and Francisco B. Elegado
National Institutes of Molecular Biology and Biotechnology, University of the Philippines Los Baños, College, Laguna
4031, Philippines
*
Author for correspondence; e-mail: jen_saguibo@yahoo.com.ph

Local isolates of bacteriocin-producing lactic acid bacteria (LAB), namely, Pediococcus acidilactici
4E5, P. acidilactici AA5a, P. acidilactici 3G3, P. acidilactici K3A2-2, P. pentosaceous K2A2-3 and
Lactobacillus plantarum BS were subjected to spot-on-lawn antimicrobial assay against aqueous,
ethanol and methanol extracts of various plant parts of herbs and vegetables. For comparative
purposes, pathogenic Staphylococcus aureus and Salmonella enterica subsp. enterica serovar
Typhimurium (formerly known as S. typhimurium) were also used as test microorganisms and
three antibiotics (amoxycillin, ampicillin and erythromycin) as antimicrobial agents. Generally, the
tested LAB had selective resistance against inhibitory substances of plant part extracts. Methanol
extracts were more inhibitory than aqueous and ethanol extracts. The tested pediococci isolates
were more affected than the probiotic Lb. plantarum BS, which showed resistance against all plant
part extracts including the most potent methanol extract of avocado (Persea americana Mill.) and
malunggay (Moringa oleifera Lam.) leaves. All LAB and S. aureus were inhibited by all antibiotics
tested, while S. typhimurium was found resistant to erythromycin. Likewise, S. aureus was more
sensitive to various plant extracts than S. typhimurium. The antibiotic susceptibility and ability of
some LAB to grow on several natural plant extracts suggest the possibility of incorporating these
LAB in plant-based nutraceuticals.

Key Words: antibiotic resistance, antimicrobial assay, bacteriocin, lactic acid bacteria, nutraceuticals, plant extracts,
probiotics

Abbreviations: BIOTECH – National Institutes of Molecular Biology and Biotechnology; EFSA – European Food
Safety Authority; ELISA – Enzyme-Linked Immunosorbent Assay; GRAS – generally regarded as safe; LAB – lactic
acid bacteria; MRS – de Man, Rogosa, Sharpe; NA – nutrient agar

INTRODUCTION ingredients from these herbs as medicine or food

Among health-conscious consumers, there has been an
increasing preference for natural and minimally
processed foods. Nutritionists recommend that these
should consist of more than half of an individual’s total
food intake. Natural foods are undoubtedly packed with
countless healthful benefits, depending greatly on the
right amount used and the proper method of preparation.
Raw fruits, vegetables and herbs are rich in enzymes that
help body organs in food digestion. They are also full of
dietary fibers that help maintain a healthy gastrointestinal
tract, and are good sources of vitamins, minerals and
plant chemicals such as bioflavonoids that lower cancer
risk. Some of them have been used as a more organic
way of preventing or treating some bacterial infections
since early times. In the beginning of the 20th century,
however, scientists began to extract and purify essential
supplements. This trend led to the decline of alternative
medicine including herbalism. Interestingly, in the late
20th century, many people started to realize the negative
effects of purified pharmaceuticals, such as allergic
reactions and hepatotoxicity. Hence, whole plant parts or
their liquid extracts have been used again as
nutraceuticals (Brater and Daly 2000).
The Philippines is a tropical country blessed with
abundant natural resources, including various vegetables
and herbs that are cheap, nutritious and almost always
available all year-round. The Bureau of Plant Industry
promotes consumption of indigenous vegetables such as
malunggay (moringa), alugbati (Malabar spinach),
saluyot (jute mallow), kulitis (amaranth), ampalaya
(bitter gourd), labong (bamboo shoot), katuray
(sesbania), pakô (fern) and himbabao (alokon). Some of
the most common and easily available natural/herbal

22 The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)

Resistance of Lactic Acid Bacteria to Plant Extracts
medicines such as cayenne pepper, garlic, soursop or
guyabano, okra, papaya, malunggay leaves, tamarind and
watermelon are recommended by Kurian (2010). Herbs,
spices and condiments do not only impart flavor, but are
also excellent sources of antioxidants and bioflavonoids
which are of medicinal importance in the fight against
diabetes, cancer, diarrhea and respiratory diseases
including asthma. To date, 10 herbal medicines have
been approved by the Department of Health (DOH) since
1992. These include akapulko (Cassia alata Linn.),
ampalaya (Momordica charantia Linn.), garlic (Allium
sativum Linn.), guava (Psidium guajava Linn.), lagundi
(Vitex negundo Linn.), niyog-niyogan (Quisqualis indica
Linn.), sambong (Blumea balsamifera Linn.), tsaang
gubat (Ehretia micophylla Lam.), ulasimang bato or
pansit-pansitan (Peperomia pellucida Linn.) and yerba
buena [Clinopodium douglasii (Benth.) Kuntze].
Turmeric or luyang dilaw (Curcuma longa Linn.)
and banaba (Lagerstroemia speciosa Linn.) have also
been reported to exhibit medicinal value. Turmeric
contains curcumin which is responsible for inhibiting
inflammation and is a more potent antioxidant than
vitamin E (Natarajan and Bright 2002; Buhrmann et al.
2011). It has anti-coagulant and anti-carcinogenic
properties (Moghaddam et al. 2009). On the other hand,
banaba has penta-O-galloyl-glucopyranose, the most
potent gallotannin that lowers blood sugar (anti-diabetic).
According to a review by Klein et al. (2007), penta-O-
galloyl-glucopyranose has significantly higher glucose
transport stimulatory activity than Lagerstroemin, and
exhibits anti-adipogenic properties.
Natural foods, however, are not 100% safe for
consumption. Plants contain some chemicals that are
known to be toxic to both animals and humans. Some of
these chemicals evolve in plants to protect them from
insects, plant pathogens and other organisms (Pimentel
1988). Natural toxins include hydrazines, lectin proteins
(phytohemagglutinins), lathyrogens (NAS 1973),
protease inhibitors (NAS 1973; Bender 1987; Oser 1978),
cyanogenic glycosides (Strong 1974), and goitrogens
(glucosinolates) (Coon 1975; Heaney and Fenwick
1987). The most potent natural toxins responsible for
human health risks are the mycotoxins. These are not
strictly plant compounds but toxic metabolites produced
by fungi-infested foodstuffs, especially cereals and nuts
which have been stored under conditions of elevated
temperature and high humidity (NAS 1989). If not
properly composted, animal manure commonly used by
organic farmers might pose hazard to consumers through
infestations of the vegetable crops by various pathogens
such as E. coli and Salmonella (Gerba and Smith 2004).
Moreover, pesticide residues in plant crops pose hazards
to human and animal life (Reigart and Roberts 1999).
Infectious diseases are still one of the leading causes
of morbidity, especially in the developing countries.
The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)
Jennifer D. Saguibo and Francisco B. Elegado
Antibiotics are still the immediate resort of veterinarians
and physicians in treating bacterial infections in animals
and humans. Over the years, the amount of manufactured
antibiotics has rapidly increased (Col and O’Connor
1987). Their overuse and misuse has resulted in the
increase in antibiotic resistance (Dy 1997). In fact, there
has been a growing awareness of the emergence of
antibiotic-resistant microorganisms and the accumulation
of antibiotic residues that could be passed on to and
ingested by meat consumers. Several mechanisms
through which the bacteria become resistant to antibiotics
have been discovered (Alekshun and Levy 2007), but the
resistance frequently results from modification to or
disruptions of an individual gene (Liu and Pop 2008).
Other proven health-promoters are the probiotics.
Probiotic strains should be accurately identified, non-
pathogenic or “generally regarded as safe (GRAS)”,
stable under acid and bile conditions, viable throughout
manufacturing and storage, and able to produce
antimicrobial substances such as organic acids, hydrogen
peroxide, bacteriocins, adhesion inhibitors and other
small antimicrobial substances (Ishibashi and Yamazaki
2001). Such probiotic properties are strain-specific and
must be supported by strong scientific evidence. Lactic
acid bacteria (LAB) are Gram-(+), anaerobic bacteria
usually present in fermenting food materials (Rodriguez
et al. 2000), and produce lactic acid and other organic
acids through fermentation, thereby lowering the pH of
the fermented products. This pH-lowering effect and
antimicrobial compounds called bacteriocins are
inhibitory against bacterial pathogens, and thus, can be
used as substitute preservative in foods, enhancing their
shelf-life. LAB are also involved in food fermentations
that have a lot of economic value, as well as in helping
improve human health (Savadogo et al. 2006).
Consumption of lactic acid-fermented vegetable juice
is now becoming more popular in many countries.
Several fermented vegetable or herb drinks (such as
LactovitalleTM, NoniTM and VitaplusTM), either lactic
acid-fermented or not, are already being marketed in the
Philippines. For fermentation of juices, the most
important lactic acid bacteria belong to the genera
Lactobacillus, Pediococcus and Bifidobacteria and the
yeast Saccharomyces cerevisiae. Generally, fermentation
is carried out to enhance taste, aroma, shelf-life, texture,
nutritional value and other properties of food. It improves
the nutritional value of a particular food by affecting its
digestibility and nutrient content, particularly through
enhancing its nutrient density, and increasing the amount
and the bioavailability of nutrients (Svanberg and Lorri
1997). Lactic acid fermentation leads to a decrease in the
level of carbohydrates as well as some nondigestible
polysaccharides and oligosaccharides (Friend and
Shahani 1984). The latter reduces side effects such as
abdominal distension and flatulence (Nout and Motarjemi
23
Resistance of Lactic Acid Bacteria to Plant Extracts
1997). It also increases utilization of iron from food by
breaking away inorganic iron from complex substances
under the influence of vitamin C (Siegenberg et al. 1991;
Venkatesh Mannar and Diosady 1998). Acid-fermented
vegetables are important sources of vitamins and
minerals (Lee 1997). Vitamin C is better preserved in
lactic acid-fermented vegetable products, compared with
those processed through alternative methods (Nout and
Motarjemi 1997). However, despite these advantages,
improved safety, especially in the microbiological aspect,
is still an issue in food fermentation.
Due to several reported health claims regarding
functional foods and probiotic supplements, including
reduction of cancer risk, improvement of cardiac health
and stimulation of the immune system (Walker et al.
2006; Dimer and Gibson 1998), preference for fresh
organically grown or fermented foods over industrial
foods has increased. Interestingly, phytochemicals,
vitamins and other nutritive components are better
available in fermented foods.
In our previous studies, we isolated bacteriocin-
producing lactic acid bacteria from indigenous sources
and characterized those that have probiotic potentials
(Banaay et al. 2004; Elegado et al. 2003; Elegado et al.
2004; Gervasio and Lim 2007). The present study was
conducted to assess the susceptibility of local LAB
isolates to commercial antibiotics, and their possible
resistance to extracts of plant parts of common herbs and
vegetables. LAB isolates found to be resistant could then
be used as adjuncts to herbs, spices and nutritious green
leafy vegetables in developing nutraceuticals. Common
food pathogens such as Salmonella and Staphylococcus
were also tested to investigate their possible eradication
by the plant extract compared with the commercial
antibiotics.
METHODOLOGY
Microorganisms and Their Maintenance
The lactic acid bacteria used in this study included local
bacteriocin-producing isolates deposited at the Philippine
National Collection of Microorganisms (PNCM),
BIOTECH, UPLB, namely: Pediococcus acidilactici
4E5, P. acidilactici AA5a, P. pentosaceous K2A2-3, P.
acidilactici K3A2-2, Lactobacillus plantarum BS and P.
acidilactici 3G3. These were maintained in de Man,
Rogosa, Sharpe (MRS) agar stab culture and stored at
refrigerated temperature. For comparative purposes,
pathogenic microorganisms such as Salmonella enterica
subsp. enterica serovar Typhimurium (formerly S.
typhymurium BIOTECH 1826 and referred to as S.
typhimurium throughout this paper) and Staphylococcus
aureus Sa16 maintained on Nutrient Agar (NA) were also
subjected to antimicrobial assay. LAB isolates were
24
Jennifer D. Saguibo and Francisco B. Elegado
subcultured in MRS broth, incubated overnight and
plated onto MRS agar to determine colony count prior to
assay. S. typhimurium and S. aureus were subcultured in
Nutrient Broth, incubated overnight and were also plated
onto NA for colony count.
Formulation of Antibiotics
Three capsulated 500-mg commercial antibiotics,
namely, amoxicillin and ampicillin and erythromycin in
trihydrate form, were purchased from a local drug store.
These antibiotics were selected because they are
commonly used. Initially, different concentrations (0, 2,
4, 6, 8, 10 and 12 µg mL-1) were prepared from a 50 µg
mL-1 stock solution. The solutions were filter-sterilized.
Sterile distilled water was used in all preparations or
dilutions. Depending on the initial result of antimicrobial
assay, concentrations were increased (10, 20, 30, 40, 50,
60, 80, 120 and 140 µg mL-1). The antimicrobial assay
was repeated for each antibiotic concentration.
Extraction from Plant Samples
Table 1 shows the list of 27 herbal and vegetable leaves,
11 vegetable and fruit seeds, seven root crops and stems,
and four fleshy fruits that were collected and oven-dried.
Most of these plants were documented to exhibit
nutritional and pharmacological importance and are
commonly available in the backyard of every Filipino.
These plants include (in alphabetical order): aloe vera
(Aloe barbadensis Mill.), alugbati (Basella rubra Linn.),
avocado (Persea americana Mill.), banaba
(Lagerstroemia speciosa Linn.), bamboo (Bambusa
vulgaris Linn.), bitter gourd (Momordica charantia
Linn.), cacao (Theobroma cacao Linn.), calamansi
(Citrus microcarpa Bunge.), carrot (Daucus carota
Linn.), celery (Apium graveolens Linn. var. dulce (Mill)),
chili (Capsicum frutescens Linn.), cucumber (Cucumis
sativus Linn.), duhat (Syzygium jambolanum Lam. DC),
gabi/taro (Colocasia esculenta Linn. (Schott), garlic
(Allium sativum Linn.), ginger (Zingiber officinale
Roscoe), guava (Psidium guajava Linn.), guyabano
(Anona muricata Linn.), kangkong (Ipomoea aquatica
Torsk), kinchay or wansoy (Coriandrum sativum Linn.),
lagundi (Vitex negundo Linn.), laurel (Laurus nobilis
Linn.), leek (Allium porrum Linn.), lemon grass
(Andropogon citratus DC Stapf), luyang dilaw (Curcuma
longa Linn.), malunggay (Moringa oleifera Lam.),
mango (Mangifera indica Linn.), onion (Allium cepa
Linn.), okra (Abelmoschus esculentus Linn.), oregano
(Coleus aromaticus Linn.), pandan (Pandanus
odoratissimus Linn.), papaya (Carica papaya Linn.),
rosemary (Rosmarinus officinalis Linn.), saluyot
(Corchorus olitorius Linn.), sambong (Blumea
balsamifera Linn.), sayote (Sechium edule (Jacq.)
Swartz), serpentina (Rauvolfia serpentina (Linn.) Benth.
R. Baksha), spinach Baguio (Tetragonia expansa Murr.),
The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)
Resistance of Lactic Acid Bacteria to Plant Extracts Jennifer D. Saguibo and Francisco B. Elegado

Table 1. Susceptibility of bacteriocinogenic lactic acid bacteria (LAB), Salmonella typhimurium and Staphylococcus
aureus to various plant extracts.

Plant Extract 4E5 AA5a K2A2-3 BS 3G3 K3A2-2 S.t. S.a.

AQUEOUS EXTRACT
Seed:
Avocado - - - - - - - -
Bitter gourd + + + + - + - -
Cacao - - - - - - - -
Calamansi - - - - - - - -
Guyabano - - + - - - - -
Malunggay - - - - - - - -
Mango - - - - - + - +++
Papaya + + + + - + - -
Duhat - - - - - - - +++
Sayote - - - - - - - -
Star apple - - - - - - - -

Leaf:
Aloe vera - - - - - - - -
Avocado - - - - - - - -
Alugbati + + + - - + - -
Banaba - - - - - - - -
Bitter gourd - - - - + - - -
Celery + - + - + + + +
Chili - - - - - - - -
Guava - - - - - - - -
Kangkong - - - - - - - -
Kinchay - - - - - - - -
Lagundi - - - - - - - -
Laurel - - - - - - - +
Leek + + + - + - - -
Lemon grass - - - - - - - -
Malunggay - - - - - - - -
Onion + + + - + - - -
Oregano - - + - + - - +
Pandan - - - - - - - -
Rosemary - - - - + - - -
Saluyot - - - - - - - -
Sambong - - - - - - - -
Serpentina - - - - - - - -
Spinach Baguio - - - - - - - -
Squash - - - - - - - -
Sweet potato - - - - - - - -
Talilong - - - - - - - -
Wansoy - - - - - - - -

Root/Stem:
Bamboo shoot - - - - - - - -
Carrots - - - - - - - +
Gabi/Taro - - - - + + - -
Ginger - - - - - - - -
Luyang dilaw + - + - + + - +
Garlic - - - - + - + +
Sweet potato - - - - - + - -

Fleshy Fruit:
Cucumber - - - - - - - -
Okra - - - - - - - -
Tomato - - - - - - - -
Squash + - + - - - - -

The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012) 25

Resistance of Lactic Acid Bacteria to Plant Extracts Jennifer D. Saguibo and Francisco B. Elegado

Table 1. Continued...
Plant Extract 4E5 AA5a K2A2-3 BS 3G3 K3A2-2 S.t. S.a.
ETHANOL EXTRACT
Seed:
Avocado - - - - - - - -
Bitter gourd - - - - - - - -
Cacao - - - - - - - -
Calamansi - - - - - - - -
Guyabano - - - - - - - -
Malunggay - - - - - - - -
Mango - - - - - - ++ ++
Papaya - - - - - - - -
Duhat - - - - - - +++ +++
Sayote - - - - - - - -
Star apple - - - - - - - -

Leaf:
Aloe Vera - + - - - - - -
Avocado - - - - - - - -
Alugbati - - - - - - - +
Banaba - + + + - - - -
Bitter gourd - - - - - - - -
Celery - - - - - - - -
Chili - - - - - - - -
Guava - - + - - - - +++
Kangkong - - - - - - - -
Kinchay - - - - - - - -
Lagundi - - - - - - - -
Laurel - - - - - - - -
Leek - - - - - - - -
Lemon grass - - - - - - - -
Malunggay - - - - - - - ++
Onion - - - - - - - -
Oregano + + + + + - - ++
Pandan - - - - - - - -
Rosemary + + + - - - - +
Saluyot - - - - - - - -
Sambong + - + - - - - -
Serpentina - + - - - - - -
Spinach Baguio - - - - - - - +
Squash - - - - - - - -
Sweet potato - - - - - - - -
Talilong - - - - - - - -
Wansoy - - - - - - - -

Root/Stem:
Bamboo Shoot - - - - - - - -
Carrot - - - - + - - -
Gabi/Taro + + + - + - - +
Garlic - - - - + - - -
Ginger - - - + - - - -
Luyang dilaw - - - - - - - +
Sweet potato - - - - + - - -

Fleshy Fruit:
Cucumber - - - - + - - -
Okra - - - - - - - -
Tomato - - - - - - - -
Squash - - - - - - - -

26 The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)

Resistance of Lactic Acid Bacteria to Plant Extracts Jennifer D. Saguibo and Francisco B. Elegado

Table 1. Continued...
Plant Extract 4E5 AA5a K2A2-3 BS 3G3 K3A2-2 S.t. S.a.
METHANOL EXTRACT
Seed:
Avocado +++ - - +++ - - - -
Bitter gourd - - - - - - - -
Cacao - + + + - - - +++
Calamansi - - - - - - - -
Guyabano - - - - - - - -
Malunggay - - - - - - - -
Mango - - - - - - ++ ++
Papaya - - - - - - - -
Duhat - + - - - - - ++
Sayote - - - - - - - -
Star apple - + + + - - - +++
Leaf:
Aloe Vera - - - - - - - -
Avocado - ++ ++++ - - +++ - +++
Alugbati - - - - - - - -
Banaba - - - - - - - -
Bitter gourd + - - - - - - +++
Celery - - - - - - - -
Chili - - - - - - - -
Guava +++ - +++ +++ +++ +++ - +++
Kangkong - + - - - - - -
Kinchay - - - - - - - -
Lagundi - - - - - - - -
Laurel - - - - - - - -
Leek - - - - - - - -
Lemon grass - - - - - - - -
Malunggay ++++ +++ - - - - - +++
Onion - - - - - - - -
Oregano - + - - - - - +
Pandan - - - - - - - -
Rosemary + + - - - - - +
Saluyot - - - - - - - -
Sambong - - - - - - - -
Serpentina - - - - - - - -
Spinach Baguio - - - - - - - -
Squash - - - - - - - -
Sweet potato - - - - - - - -
Talilong - - - - - - - -
Wansoy - - - - - - - -

Root/Stem:
Bamboo Shoot - - - - - - - -
Carrot + - - - - - - -
Gabi/Taro + - + - - - - -
Garlic - - - - - - - -
Ginger - - - - - - - -
Luyang dilaw + + + - - - - +
Sweet potato - - + - - - - +
Fleshy Fruit:
Cucumber - - - - - - - -
Okra - - - - - - - -
Tomato - - - - - - - -
Squash - + - - - - - -
Legend: -, resistant without zone of inhibition; +, slightly susceptible with 3–5 mm diameter zone of inhibition; ++, susceptible with greater than 5–10 mm
diameter zone of inhibition; +++, very susceptible with greater than 10–15 mm diameter zone of inhibition; ++++, extremely susceptible with greater than
15 mm diameter zone of inhibition
Code of bacterial strains: 4E5 – Pediococcus acidilactici; AA5a – P. acidilactici; K2A2-3 – Pediococcus pentosaceous; BS – Lactobacillus plantarum;
3G3 – P. acidilactici; K3A2-2 – P. acidilactici; S.t. – Salmonella typhimurium; S.a. – Staphylococcus aureus

The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012) 27

Resistance of Lactic Acid Bacteria to Plant Extracts
squash (Cucurbita maxima Linn.), starapple
(Chrysophyllum cainito Linn.), sweet potato (Ipomoea
batatas (L.) Lam.), talilong (Liza macrolepis Smith) and
tomato (Lycopersicon esculentum Mill.).
To 1 g of each sample was added 5 mL of either
distilled water, ethanol or methanol. Then, the mixture
was ground using a mortar and pestle. The extracts were
initially kept in the freezer and then thawed prior to
antimicrobial assay.
Antimicrobial Assay
The spot-on-lawn antimicrobial assay (USP 1990) was
employed in this study. Initially, the number of the lawn
microorganisms in colony-forming units (cfu) mL-1 was
determined following the method of Miles and Misra
(1938) on MRS agar medium or NA medium. Briefly,
100 µL overnight culture of the microorganism (1 x 108
to 1 x 109 cfu mL-1) was suspended in 5 mL molten soft
agar (MRS broth or Nutrient Broth plus 8 g mL-1 agar),
mixed, and then overlaid on solidified MRSA or NA on
plate. Ten microliter (10-µL) samples were equidistantly
dropped or spotted into duplicates, on the solidified soft
agar with the test microorganism. Sterile distilled water
was also spotted to serve as the negative control. For
plant samples, water extracts were directly spotted onto
plate with microbial lawn, while ethanol and methanol
extracts were placed on ELISA plate, air-dried in the
fumehood and resuspended with an equal volume of
water prior to assay. The zones of inhibition were
measured after 24–48 h of incubation at 40 °C for LAB
isolates, and at 37 °C for the pathogenic microorganisms.
RESULTS AND DISCUSSION
Propensities of Plant Extracts to Inhibit Growth of
Lactic Acid Bacteria
Table 1 summarizes the propensities of all plant extracts
to inhibit the growth of the lactic acid bacteria and
pathogenic microorganisms tested. Resistance or
susceptibility was measured by the inhibition zone
diameter. Diameter variation of the spotted extract, the
surface tension and hydropathy did not affect the zone of
inhibition. The well-dried media prevented the spotted
extract from scattering and fusing with other spots. Thus,
the observed inhibition was due mainly to the diffused
active component of the extract. The wider the inhibition
zone diameter, the more susceptible the microorganism
was to the plant extract as shown in Figure 1. Resistance
of an organism to a particular plant extract is not the
same in all the extracting agents tested.
In general, one or more LAB showed resistance to a
particular plant part, whether extracted with aqueous,
ethanol or methanol. Interestingly, various herbal and
28
Jennifer D. Saguibo and Francisco B. Elegado
Fig. 1. Spot-on-lawn assay showing zones of inhibition
of different methanol plant extracts against
Pediococcus acidilactici 4E5: (a) sterile distilled
water as negative control; (b) avocado leaf and
(c) oregano leaf without inhibition; and (d)
avocado seed, (e) moringa leaf and (f) guava
leaf showing ++++ or greater than 15-mm
diameter zones of inhibition.
nutritious plant extracts were compatible or did not affect
the growth of the LAB isolates tested, which means that
such LAB isolates are resistant to antimicrobial and other
phytochemicals present in a particular herb or vegetable
extract. Among all plant extracts, methanol extracts of
avocado, guava and malunggay leaves were the most
potent. Zones of inhibition of representatives of the least
and most potent plant extracts tested against P.
acidilactici 4E5 are shown in Figure 1.
With aqueous and ethanol extracts, all the tested
LAB were either resistant (able to grow as depicted in the
absence of zone of inhibition) or slightly inhibited (Table
1). On the other hand, methanol extracts were most
inhibitory to some LAB compared with the other two
extracting agents tested. Strong inhibition was observed
in methanol extracts of avocado seeds against P.
acidilactici 4E5 and Lb. plantarum BS; avocado leaf
extract against P. pentosaceous K2A2-3 and P.
acidilactici K3A2-2; guava leaf extract against all the
LAB tested except P. acidilactici; and malunggay leaf
extract against P. acidilactici 4E5 and P. acidilactici
AA5A.
Lb. plantarum BS showed good resistance to several
herbal fruit and vegetable extracts. According to
literature, lactobacilli are commonly associated with
plant herbage (Siezen et al. 2010), which explains their
viability in most of the plant extracts. Based on previous
studies, these two bacilli possess good probiotic
properties including bacteriocin activity and viability,
The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)
Resistance of Lactic Acid Bacteria to Plant Extracts
and survival on artificial gastric juice and simulated
intestinal fluid (Banaay et al. 2004; Gervasio and Lim
2007).
In this study, most of the bacteriocin-producing LAB
were found to be resistant to several natural plant
extracts, suggesting the possibility of incorporating them
in functional foods or nutraceuticals such as herb-
probiotic products. Preliminary studies conducted on the
formulation and animal testing of anti-diarrheal, anti-
diabetic (Saguibo and Elegado 2011a) herbals and
vegetable juice (Saguibo and Elegado 2011b) fermented
with local lactic acid bacteria showed promising results.
Resistance/Susceptibility of Salmonella and
Staphylococcus to Plant Extracts
For comparative purposes, plant extracts were also tested
against common food-borne pathogens particularly the
Gram (-) S. typhimurium BIOTECH 1826 which causes
salmonellosis by food infection, and the Gram (+) S.
aureus Sa16 which causes enterotoxicosis by food
intoxication. Listeria monocytogenes was not included as
this has already been tested by other researchers (Elegado
et al. 2007; Marilao et al. 2007). The susceptibility or
resistance is mainly based on the growth inhibition test
conducted regardless of the standard set by the European
Food Safety Authority (EFSA 2008, 2011). The results of
the resistance or susceptibility of S. typhimurium and S.
aureus are also shown in Table 1.
S. typhimurium was susceptible (10–15 mm diameter
zone of inhibition) to ethanol and methanol extracts of
mango seeds. S. aureus was susceptible to ethanol extract
of mango seeds, and methanol extracts of duhat and
mango seeds. Garlic and celery, which are commonly
used as condiments in cooking, also exhibited slight
inhibition against the growth of both pathogens.
In general, the Gram (+) S. aureus was susceptible to
numerous plant extracts regardless of the extracting agent
used, while the Gram (-) S. typhimurium was susceptible
to fewer plant extracts. The difference in susceptibility
could be due to the differences in the cell wall of Gram
(-) and Gram (+) bacteria. The cell wall of Gram (+)
bacteria is thick and exposed while the cell wall of Gram
(-) bacteria is thin and enclosed by an outer membrane of
lipopolysaccharides. This difference in the chemical
composition of the bacterial cell wall accounts for the
selective activity of some drugs including antibiotics
(Keeton and Gould 1993), and possibly, of inhibitory
substances of plant extracts.
Effect of Common Antibiotics
Antibiotics are utilized by the health care industry to
fight bacterial infections. They are also used as growth
promoter in feeds, and for plant protection and
prophylaxis. However, pathogenic and commensal
bacteria, including lactic acid bacteria, are highly
The Philippine Agricultural Scientist Vol. 95 No. 1 (March 2012)
Jennifer D. Saguibo and Francisco B. Elegado
adaptable organisms and are capable of developing
resistance to antibiotics. Recently, many investigators
have speculated that LAB may act as reservoirs of
antibiotic resistance genes that could be transferred to
intestinal microorganisms and pathogenic bacteria. Genes
conferring resistance to tetracycline, erythromycin and
vancomycin have been reported in Lactococcus lactis,
Enterococci and Lactobacillus species isolated from
fermented meat and milk products (Mathur and Singh
2005).
According to Gallagher and MacDougall (2011),
amoxicillin, ampicillin and erythromycin are among the
most commonly used antibiotics. Amoxicillin is a
moderate-spectrum, bacteriolytic, β-lactam antibiotic that
acts by inhibiting the synthesis of bacterial cell walls,
particularly by inhibiting cross-linkages between the
linear peptidoglycan chains that make up a major
component of the cell walls of both Gram (+) and Gram
(-) bacteria. Ampicillin is also a β-lactam antibiotic and
has an advantage over other β-lactam antibiotics due to
the presence of amino group which helps the drug
penetrate the outer membrane of Gram (-) bacteria. It acts
as a competitive inhibitor of the enzyme transpeptidase
needed for cell wall synthesis, inhibiting the third and
final stage of bacterial cell wall synthesis in binary
fission, which ultimately leads to cell lysis. Lastly,
erythromycin or ilosone is a macrolide antibiotic which is
often used for people who are allergic to penicillins.
Although its mechanism of action is not fully understood,
it is postulated that when the antibiotic binds with the 50s
subunit of the bacterial 70s rRNA complex, protein
synthesis and subsequent structure and function processes
critical for life or replication are inhibited.
The resistance or susceptibility of LAB and
pathogenic control microorganisms to common
antibiotics such as amoxicillin, ampicillin and
erythromycin was also tested at 100 µg mL-1
concentration. Results showed that all tested LAB were
inhibited by the antibiotics under study (Fig. 2). The most
pronounced inhibition was observed with the use of
erythromycin against all the LAB isolates. Similar to the
Gram (+) LAB isolates, S. aureus was susceptible to all
the tested antibiotics, but was greatly inhibited by
ampicillin and amoxicillin, and least inhibited by
erythromycin. The Gram (-) S. typhimurium, on the other
hand, was largely inhibited by ampicillin, followed by
amoxicillin. However, it was resistant to erythromycin
because it was able to grow on media after spotting of the
antibiotic and overnight incubation at 37 °C. The
resistance of S. typhimurium to erythromycin concurs
with previous reports (Cardoso et al. 2006; Malik et al.
2010).
29
Resistance of Lactic Acid Bacteria to Plant Extracts
Legend:
A- Pediococcus acidilactici 4E5
B- Pediococcus acidilactici AA5a
C- Pediococcus pentosaceous K2A2-3
D- Lactobacillus plantarum BS
E- Pediococcus acidilactici 3G3
F- Pediococcus acidilactici K3A2-2
G- Staphylococcus aureus Sa16
H- Salmonella typhimurium BIOTECH 1826
Fig. 2. Zones of inhibition of local bacteriocinogenic
LAB isolates and pathogens against common
commercial antibiotics (100 µg mL-1). Values
are average of two replications ± SD.
CONCLUSION
Based on spot-on-lawn antimicrobial assay, local LAB
from BIOTECH, namely, P. acidilactici 4E5, P.
acidilactici AA5a, P. pentosaceous K2A2-3, Lb.
plantarum BS, P. acidilactici 3G3 and P. acidilactici
K3A2-2 were susceptible to common antibiotics such as
amoxicillin, ampicillin and erythromycin because they
were unable to grow on media upon exposure to
antibiotic solution. More importantly, their resistance to
several natural plant extracts suggests the possibility of
incorporating these LAB to develop different functional
foods. Health claims on the therapeutic properties of the
tested extracts can be enhanced by the inclusion of one or
more lactic acid bacteria that provide protection against
several diseases including diabetes, cancers, intestinal
disorders and respiratory diseases. Results of the study
showed that our local LAB are able to survive even in
methanol extracts of the most potent guava and avocado
leaves. Future studies are suggested on the formulation of
various nutraceutical products such as anti-diabetic, anti-
diarrheal and anti-cancer probiotic drinks/beverages that
can provide a safer, cheaper and more available remedy
to various lethal diseases for animal, and eventually,
human consumption, if backed with clinical and non-
clinical tests. Nutraceutical drinks can also be formulated
by combining the probiotic LAB and plant extracts with
promising beneficial effects.
30
Jennifer D. Saguibo and Francisco B. Elegado
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