Selection of Probiotic Lactic Acid Bacteria Isolated from Fermented Plant Beverages
Screening for probiotic bacteria from non-human sources
were performed in this study. Three hundred and twenty-seven strains of
Lactic Acid Bacteria (LAB) were isolated from 90 samples of Fermented
Plant Beverages (FPBs) and pickles collected from around Thailand. Potentially
useful probiotic properties were investigated in vitro in parallel
with a commercial probiotic strain of Lactobacillus casei, R obtained
from a dairy probiotic product in Thailand. An isolate SS2, selected from
a fermented star fruit beverage, survived in the human biological barriers
(0.15 and 0.30% bile salt, pH values between 3-8, presence or absence
of oxygen), resistance to some antibiotics in general use and showed other
benefits to the host (antibacterial activity, utilizations of protein
and starch). The isolate SS2 had a higher specific growth rate and better
inhibitory properties against food borne pathogenic bacteria and spoilage
organisms than the commercial probiotic R strain. It also grew well in
MRS and the SPY2 medium that is free from animal-derived ingredients,
(r>0.8). The isolate SS2 was therefore considered to be a potentially
useful probiotic LAB for a non dairy product such as FPBs and was provisionally
identified as a strain of Lactobacillus plantarum.
Probiotics are known as live
microbial feed supplements that provide many benefits to the host by improving
its microbial intestinal balance (Guarner and Schaafsma, 1998). In general,
the best probiotic strains to give to humans are those that originated
from the human intestine as they may be assumed to be the true probiotics
(Surawicz 1998; Marteau and Rambaud, 1993). Most probiotic microbes are
LAB such as Lactobacillus casei, L. plantarum and Streptococcus
lactis (Sindhu and Khetarpaul, 2001). Probiotic LAB are normally used
as starter cultures for fermented dairy products like yogurt. In recent
years, consumers who are lactose intolerant and worry about the cholesterol
content in milk as well as vegetarians increasingly demand for non-dairy-based
probiotics. In Thailand, fermented plant beverages are considered to be
non-alcoholic and people who consume these FPBs believe that plant beverages
are healthy due to their high nutritional value and presence of bioactive
compounds derived from the plant substrates used and also during the fermentation
processes (Kantachote and Charernjiratrakul, 2007). However, scaling up
the beverage production for commercial use can have a problem with a high
contamination by yeasts. To restrict this, starter cultures have been
advocated (Prachyakij et al., 2007). Consequently, the aims of
this study were to isolate and select an appropriate probiotic LAB from
fermented plant products to use as a starter culture and restrict yeast
growth for the production of FPBs.
MATERIALS AND METHODS
Selection and isolation of probiotic LAB: LAB strains were isolated
from 90 samples of fermented plant beverages and pickles collected from
different areas in Thailand and they were used to investigate their probiotics
during year 2005-2006. The selection was conducted to investigate probiotic
properties of LAB in order steps as following provided while any strains
which showed a good performance for each probiotic property were again
selected for a next step. Possible biological barriers were investigated
as described by Conway et al. (1987), such as survival in 0.15
and 0.30% (w/v) bile salt and also tolerance to pH values of 2, 3, 4,
5, 8 and 9. Briefly, LAB strains were grown in MRS (de Man Rogosa and
Sharp) broth at 37°C for 24 h and were inoculated by streak on bile salt
agar plates, whereas acid-base tolerance was tested in MRS broth with
various pH. All plates and tubes were incubated at the same condition
as above to observe their growth. Utilizations of protein, starch and
lipid were examined using agar plates of milk, starch and tributyrin,
respectively. Utilization ability was observed as a clear zone around
bacterial colonies in each agar medium. Growth as facultative anaerobe
was investigated in MRS broth and then the culture tubes were separated
to incubate in an anaerobic jar while another set was separated to incubate
in an aerobic incubator at 37°C for 24 h. Bacterial growth was measured
using spectrophotometer (OD660 nm). Requirement for vitamin
B12 was tested in vitamin B12 assay medium and the growth was compared
with Lactobacillus delbrueckii subsp. lactis growth. Antibiotic
susceptibility tests to 15 antibiotics in regular use (Table 1) and antagonistic
activity against 13 strains of food borne potentially pathogenic bacteria
and spoilage organisms (Table 2) were carried out according to standard
methods as described by Seeley et al. (1991) and Spelhaug and Halander
(1989). Bacterial indicators used in this study were provided by Department
of Microbiology, Faculty of Science, Prince of Songkla University, Thailand.
In addition, an experiment examined the possibility that the isolates
could grow as well in a medium without animal derived ingredients (SPY2)
as they did in MRS. A commercial probiotic dairy product in Thailand,
Lactobacillus casei namely R was used as a reference strain (Lee et
RESULTS AND DISCUSSION
One probiotic lactic acid bacterium, isolate SS2, was selected from 327
strains of LAB isolated from samples of FPBs and pickles. The isolate
SS2 was provisionally identified as Lactobacillus plantarum (data
not shown) and it was isolated from a fermented star fruit (Averrhoa
carambola) beverage from Si-Sa-Ket province in the Northeast part
of Thailand. As shown in Table 1, the selected strain
SS2 and a commercial strain R were able to grow with both 0.15 and 0.30%
bile salt in a modified MRS medium. Both strains had survival rates of
more than 90% at pH 3 and 4 after a 3 h incubation time. It was surprising
that more than 50% of the isolate SS2 survived at pH 8, but the R strain
died. However, neither grew at pH 2 or 9. These tests for screening probiotic
bacteria are appropriate because normally they must transit through the
acidic condition of the stomach and bile in the intestine (Lee and Salminen,
1995). In addition, oxygen, one of the human physiological barriers, has
a strong negative effect on the survival of possible probiotics bacteria
in commercial products (Shah et al., 1995). Fortunately, isolate
SS2 and the commercial strain showed no significant differences in their
growth in the presence
Comparisons of properties
of the isolate SS2 and the probiotic commercial strain, R
a: Protein and
starch utilizations were determined in 3% milk agar plates and
0.2% starch agar plates, respectively, b: No significant
difference (p>0.01) between growth with or without oxygen present,
c: VC: Vancomycin, BC: Bacitracin, GT: Gentamycin,
KC: Kanamycin, ST: Streptomycin, NF: Norfloxacin, PB: Polymyxin
B; other tested antibiotics were ampicillin (10 mcg), cephalothin
(30 mcg), ceftazidime (30 mcg), chloramphenical (30 mcg), erythromycin
(15 mcg), pencillin G (10 U), ceferperazone (75 mcg), tetracycline
The antagonistic activity
of the isolate SS2 and the commercial probiotic strain R after
18 h of incubation time
1: Productions of organic
acids and H2
, 2: No control of organic
acid production but a limitation of H2
3: Control of organic acid
s and H2
+++: >=10 mm inhibition zone, ++ : 7-9 mm inhibition zone, +:
<7 mm inhibition zone and -: No inhibition zone
or absence of oxygen. In addition, both strains achieved
an OD660 nm>2.00 in a cobalamin deficient medium so neither
had a growth requirement for vitamin B12. Hence, the isolate
SS2 should not need to compete with the human body for cobalamin. This
is especially important for vegetarians whose vitamin B12 levels
are normally low because of its absence in their foods (Herbert, 1973).
Utilization of protein and starch was observed in the milk and starch
agar plates by the isolate SS2 but the R strain utilized only protein.
This meant that the selected strain may provide more benefit to human
digestion than the commercial strain.
The isolate SS2 had a higher antibacterial activity against
13 strains of food borne pathogenic bacteria and spoilage organisms than
that found with the R strain in all conditions tested (Table
2). The antagonist activity of probiotics has long been used to preserve
foods. The antimicrobial substances produced by probiotics bacteria act
to improve the composition and the activity of the normal microbiota in
the intestine (Ouwehand and Vesterlund, 2004). In addition, the antibiotic
susceptibility tests showed that the SS2 and R strains were resistant
to 7 of the antibiotics used, vancomycin (30 mcg), bacitracin (10 mcg),
gentamicin (10 mcg), kanamycin (30 mcg), streptomycin (10 mcg), norfloxacin
(10 mcg) and polymyxin (300 mcg). Results of a clinical trial indicated
the advantages of administering both antibiotics and a probiotic strain
to patients with recurrent Clostridium difficile infections (McFarland
et al., 1994). Besides, co-administration may benefit patients
whose normal intestinal microbiota has become unbalanced or greatly reduced
in numbers due to the administration of various antimicrobial agents (Salminen
et al., 1998). Both SS2 and R strains grew well in the MRS and
SPY2 broths with a Pearson`s correlation level r >0.8. That means both
strains could grow well in media containing either animal or plant ingredients.
The isolate SS2 had a generation time of 51 min in SPY2 medium, whereas
the R strain took 71 min for one generation. This could indicate that
SS2 could respond more quickly than R to any potential antagonist activity.
We conclude that the selected strain, L. plantarum
SS2, could be developed as an effective starter culture for use as a probiotic
in a fermented plant beverage produced especially for vegetarians or others
preferring to use a fermented plant product free from animal products.
This study was fully supported by National Science
and Technology Development Agency (NSTDA), Thailand and the project number
is CO-B-22-2C-10-483. We would like to thank Dr. Brian Hodgson for critical
reading the manuscript.
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