1
In Vitro Characterization of probiotic properties and detection of novel compounds
2
secreted by lactic acid bacteria isolated from kodo millet (Paspalum scrobiculatum) flour-–a
3
traditional Himalayan food
4
Anupama Gupta* and Nivedita Sharma
5
6
Department of Basic Science, Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan,
Himachal Pradesh 173 230 (India)
7
*Corresponding author: npm.gpt@gmail.com
8
9
Abstract
10
In the present study, Pediococcus pentosaceus C-1 was investigated and
11
characterized for probiotic attributes and found to possess acid and bile tolerance,
12
tolerance to gastrointestinal tract conditions, autoaggregation, coaggregation ability,
13
antimicrobial activity (bacteriocin production) and safety traits including antibiotic
14
resistance, non-hemolytic activity, no DNase and gelatinase enzyme production. The
15
antimicrobial potential was obtained against Listeria monocytogenes MTCC 839, Bacillus
16
cereus CRI and Clostridium perfringenes MTCC 1739. Out of 38, 15 bioactive compounds
17
with antimicrobial and potential therapeutic properties have been found and
18
9Octadecenamide, (Z) with anti-sleep disorder properties have been reported for the first
19
time by probiotic P. pentosaceus C-1. On the basis of these results, P. pentosaceus C-1 has
20
been found safe, can survive under gastrointestinal conditions and can be used as a
21
potential probiotic isolate that could be useful in food preservation, nutraceutical
22
preparations and in clinical use.
23
Keywords: Probiotic, Pediococcus, Kodo millet, Himachal Pradesh, 9Octadecenamide, (Z)
1
24
Introduction
25
Traditional fermented foods are very rich sources of microorganisms with potential probiotic
26
characteristics and LAB isolated from them are widely used for the production of a variety of
27
nutraceutical and functional foods with rich nutritional and therapeutic values. LAB also play an
28
important role in various fermented foods to prevent the growth of harmful bacteria by producing
29
organic acids and antimicrobial substances (bacteriocins) (1). Probiotics are defined by the
30
World Health Organization as ‘‘live microorganisms, which when administered in adequate
31
amounts, confer a health benefit upon the host’’ (2) and can be used as different types of food
32
product formulations which are highly beneficial to human beings. Probiotics include group of
33
Lactic acid bacteria, industrially important organisms, which are used for the production of
34
fermented and functional food products. Mainly, group of gram positive bacteria including the
35
genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus are probiotic
36
bacteria with commercial uses especially in fermented food products. Health profits of probiotic
37
LAB viz. reducing lactose intolerance, cholesterol reduction, immunomodulation, resistance
38
against gastrointestinal disorders, etc., are well known to influence positively in the
39
gastrointestinal tract of humans (3).
40
Pediococcus are gram positive cocci bacteria being able to resist upper gastrointestinal transit
41
and to colonize the digestive tract (4;5). Isolation and screening of lactic acid bacteria from
42
naturally occurring products and processes have always been the most powerful resources of
43
obtaining useful cultures for scientific as well as commercial purposes. The appropriate selection
44
and maintenance of lactic acid bacteria for fermentation process is critical for the manufactures
45
of fermented food products to get the desirable texture, flavor and appropriate nutrients (6;7). To
46
date, most studies on probiotic lactic acid bacteria have been performed on strains from dairy
2
47
fermented products or from the human/animal gastrointestinal tract. However, many studies (8;
48
9) have revealed the probiotic potential of plant-originated lactic acid bacterial isolates and have
49
reported that the isolates of food origin are more resistant to acidic environments and are able to
50
adhere efficiently to intestinal cells than animal-originated LAB. A very few studies on the Kodo
51
millet have been conducted, mainly on the nutritional evaluation and food products (10;11).
52
However, there are no reports cited in the literature on the composition and functional probiotic
53
attributes of LAB from kodo millet flour. Therefore, the objective of this study was to explore
54
kodo millet flour for the very first time for isolation and identification of LAB and to evaluate
55
the probiotic potential of the isolates, including their safety, tolerance towards gastrointestinal
56
juices, their antimicrobial potential against serious food borne and spoilage causing organisms
57
and to study their ability to secrete novel compounds with nutritional as well as therapeutic
58
properties.
59
2. Methods
60
2.1 Isolation of Lactic acid bacteria
61
Kodo millet (Paspalum scrobiculatum) flour– a novel and weaning food, rich in fiber and
62
minerals, of Himachal Pradesh has been explored for the first time to isolate probiotic lactic acid
63
bacteria on MRS agar by serial dilutions method and incubated at 35°C for 24-48 h anaerobically
64
(12). In total 4 isolates were obtained and were further screened by Gram reaction, catalase test,
65
cell morphology and antimicrobial activity. Isolate C-1 was selected for further study on the
66
basis of its broad antagonistic spectrum against spoilage and food borne pathogens viz. Listeria
67
monocytogenes MTCC 839, Clostridium perfringens MTCC 1739, Escherichia coli IGMC,
68
Bacillus cereus CRI, Staphylococcus aureus IGMC, Leuconostoc mesenteroides MTCC 107,
3
69
Enterococcus faecalis MTCC 2729, Pectobacterium carotovorum MTCC 1428 and
70
Pseudomonas syringae IGMC by using Bit/disc method.
71
All the isolates were tentatively identified at genus level according to their
72
morphological, cultural, physiological and biochemical characteristics. Molecular identification
73
upto species level of the selected isolate after screening (on the basis of biochemical and
74
antagonistic potential) was evaluated on the basis of 16S rRNA gene sequence. Alignment of the
75
16S rRNA sequence obtained was conducted by using the BLASTN program from NCBI web
76
site (http://www.ncbi.nlm.nih.gov). Based on maximum identity score, the sequences were
77
selected and aligned using multiple alignment software program Mega 6.
78
2.2 Safety assessment of LAB
79
One of the most important aspects in selecting probiotic strains for use in food and
80
pharmaceutical products is their safety. To evaluate the safety status, in vitro assays were
81
conducted to examine different intrinsic properties of the strains such as antibiotic susceptibility,
82
hemolysis, DNase and gelatinase production.
83
2.3 Assessment of probiotic attributes
84
2.3.1 Effect of Low Acid conditions on survival
85
Effect of low pH on the survival of the culture was done by following the method of Liong and
86
Shah (13) with slight modifications. Buffers of different pH values viz. 1, 2, 3 and 6.5 were
87
prepared and used to evaluate the survival of P. pentosaceus C-1 at low pH for 3h. Acid
88
tolerance was determined by comparing the final plate count after 3h with the initial plate count
89
at 0h.
90
2.3.2 Effect of bile salts on the growth rate of isolates
4
91
Growth and survival of P. pentosaceus C-1 in the presence of bile salts was studied by the
92
method of Gilliland and Walker (14). Viability of cells in MRS broth supplemented with 0.3, 1
93
and 2% bile salts upto 8h was observed by plating 100µl of culture onto MRS agar plates and
94
incubated at 35oC for 24h. Growth of bacteria was expressed in colony forming units per
95
milliliter (log CFU/ml) and the percent survival of strain was then calculated.
96
2.3.3 Survival in simulated in vitro digestion
97
Survival in simulated gastric and intestinal juice was determined following the method given
98
by Charteris et al. (15). Simulated gastric (pepsin, HiMedia) and intestinal juices (pancreatin
99
from porcine pancrease, HiMedia) were prepared to a final concentration of 3g/L (pH 2 and 3)
100
and 1g/L (pH 8), respectively. An aliquot of 0.1 ml from gastric and intestinal transit assay was
101
removed after 0, 60 and 240 min. The tolerance was assayed by the viable count estimation in
102
simulated gastrointestinal juices after the incubation up to 4 h.
103
2.3.4 BSH activity
104
Bile salt hydrolase activity of P. pentosaceus C-1 was tested according to method given by
105
Dashkevicz and Feighner (16). Spots of overnight grown culture were placed on MRS agar
106
plates supplemented with 0.5% (w/v) sodium salt of taurodeoxycholic acid (TDCA) (Sigma,
107
India). Plates were incubated at 37∘C for 72h, after which the diameter of the precipitation zones
108
was measured.
109
2.3.5 Autoaggregation
110
Autoaggregation ability of selected isolate was assessed by the method of Del Re et al.
111
(17) with minor modifications. An aliquot of 0.1ml of the upper suspension was taken and 3.9ml
5
112
of PBS was added to it. Autoaggregation % was measured as 1- (At/A0) × 100, where At
113
represents the absorbance at time t=1, 2, 3, 4, 5 h and A0 the absorbance at t = 0 h (i.e. 0.5).
114
2.3.6 Co-aggregation
115
Coaggregation ability of P. pentosaceus C-1 with pathogenic bacteria was determined by
116
following the method given by Del Re et al. (17). Mixtures were made for C-1 with pathogenic
117
bacteria viz. L. monocytogenes MTCC 839, C. perfringenes MTCC 1739 and B. cereus CRI at
118
1:1 ratio. Probiotic bacterial cells and indicator bacteria were kept as control and were incubated
119
at 35oC for 4 h. Absorbance at λ = 600 nm was observed for mixture and each of individual
120
strain. Co-aggregation % was calculated according to Handley’s equation.
121
2.3.7 Bacteriocin production
122
Antimicrobial activity of cell free supernatant of P. pentosaceus C-1 was checked against
123
L. monocytogenes MTCC 839, L. mesenteroides MTCC 107, E. faecalis MTCC 2729, B.cereus
124
CRI, C. perfringenes MTCC 1739, P. caratovorum MTCC 1428, E. coli IGMC, P. syringae
125
IGMC and S. aureus IGMC. The wells with the holding volume of 150μL were made in the plate
126
using well cutter and 24h old culture of isolate was centrifuged at 12,000 x g for 10 min. An
127
aliquot of 150μL of the un-neutralized supernatant for overall antimicrobial potential and
128
neutralized with 0.1N NaOH to a final pH of 6.5 and with catalase to remove the effect of H2O2
129
for bacteriocin production was loaded in the wells and the plates were incubated at 35ºC for 24h.
130
The antibacterial activity in the form of bacteriocin production was determined and zone of
131
inhibition was measured in millimeter (mm) (18). Arbitrary Unit/ml (AU/ml) was calculated as
132
the inverse of the highest two-fold dilution which induced definite inhibition (19).
133
2.3.8 HPLC- determination of lactic acid
6
134
The major end product of the metabolism of lactic acid bacteria especially Pediococcus
135
(homofermentative) is lactic acid which mainly contributes to their antimicrobial potential.
136
Production of lactic acid by P. pentosaceus C-1 was detected by using HPLC (Novapak C-18)
137
column, 490E multi-wavelength UV detector, Millennium 2010 data processor and Rheodyne
138
injector with 20µl loop. Whatman stainless steel syringe assembly with 0.22µm Durapore
139
membrane filter was used to inject the sample. Mobile phase used was Methanol:Water (double
140
distilled) (95:5). Standard organic acid solution i.e. 5% of lactic acid (Sigma Aldrich) was
141
prepared in double distilled water. Twenty four hour old culture grown in MRS broth was
142
centrifuged to get the culture supernatant. HPLC analysis was firstly performed with standard
143
organic solution followed by the samples.
144
2.3.9 H2O2 production
145
Another factor responsible for the antimicrobial activity of the culture supernatant is
146
Hydrogen Peroxide. Quantitative estimation of Hydrogen Peroxide (H2O2) was done by
147
following the method described in AOAC (20). To the 24 h old culture broth, dil. H2SO4 (0.1 M,
148
20ml) was added gently. Suspension was titrated against 0.1 N KMnO4 until the suspension
149
become colorless. Each ml of 0.1 N KMnO4 is equivalent to 1.070 mg of H2O2.
150
2.3.10 Metabolic fingerprinting of P. pentosaceus C-1
151
Metabolic fingerprint of the isolate was elucidated to evaluate the presence of novel as
152
well as therapeutic compounds. Metabolites were extracted by following the method given by
153
Coucheney et al. (21) with minor modifications using GC-MS. Cell suspensions obtained after
154
sonication were centrifuged for 10 min at 10,000 rpm in order to separate the extra as well as
155
intracellular solution from the cells. The supernatant was used for extracting the compounds with
7
156
methanol:water:chloroform mixture (2:0.8:1):2.5 ml of cold chloroform. Five ml of cold
157
methanol (-20oC) was added to the mixture and the phases were allowed to separate. Metabolic
158
fingerprints were measured in the aqueous phase after freeze drying while in the organic phase;
159
the dried chemical extracts obtained after complete evaporation of the solvent was used for
160
metabolic profiling.
161
3. Results and discussion
162
Isolating and screening microorganisms from traditional fermented food products has
163
always been the most prevailing way of obtaining novel and genetically stable strains for
164
industrial uses. Lactic acid bacteria have always been important in the food industry because of
165
their important metabolic products i.e. lactic acid and bacteriocins which acts as natural
166
preservatives as well as flavor enhancers (25). Consumption of food supplemented with live
167
probiotic bacteria may impart many health benefits viz. intestinal microbial homeostasis,
168
regulation of immune response/modulation, improvement of gastrointestinal health, decrease of
169
cholesterol, cancer and lactose intolerance and improvement of immune and mucosal barrier
170
functions (26). A total of 4 isolates were obtained from Kodo millet flour on MRS agar. All
171
isolates were primarily observed on the basis of their colony morphology as well as some
172
biochemical characteristics. Microscopically, well defined gram positive bacilli and coccus
173
which were distributed either in groups or individually were observed. Biochemical
174
characteristics have shown the cultures to be non-motile, catalase negative and non-spore
175
forming. The bit/disc technique was used to assess the production of antimicrobial compounds
176
for initial screening of antagonistic potential against important serious food borne pathogens and
177
spoilage causing microorganisms. The inhibitory spectrum of antimicrobial compounds secreted
178
by LAB has been shown in (Fig. 1).
8
179
Three out of four LAB isolates were able to inhibit growth of all the indicator strains used
180
in the study. The zones of inhibitions varied from 15-24.6 mm. Strain C-1 showed a relatively
181
wide inhibition spectrum, inhibiting the growth of a number of pathogenic bacteria both Gram
182
positive and gram negative bacteria and was selected for the further study. The inhibitory activity
183
of isolates might be due to the secretion of antimicrobial compounds viz. organic acids,
184
bacteriocins, H2O2, etc. Of the four LAB isolates obtained from Kodo millet flour, C-1 was
185
selected for the further study on the basis of antagonistic potential against serious food borne
186
pathogens and spoilage causing microorganisms. The main revelation of the study was that the
187
strain has been active against both gram positive and gram negative bacteria. Antagonistic
188
activity was correlated with the diameter inhibition zone by bit/disc method which was used to
189
screen antimicrobial activity. The antimicrobial potential of isolate C-1 is mainly due to the
190
lactic acid, proteinceous compounds i.e. bacteriocin, hydrogen peroxide and other compounds.
191
Similar results regarding the antimicrobial activity of LAB from fermented food product
192
(vacuumed-packed meat) have been reported by Mandal et al. (27) where Pediococcus
193
acidilactici LAB5 was observed to exhibit antimicrobial potential against some of the highly
194
pathogenic species i.e. Listeria, Leuconostoc and Staphylococcus spp. to human beings. Thirteen
195
LAB isolates out of 307 isolated from Thai indigenous chicken were found to possess
196
antibacterial activity against pathogenic bacteria [28].
197
The selected isolate was characterized at species level by 16S rRNA sequencing as
198
Pediococcus pentosaceus and the sequences have been submitted to NCBI gene bank with
199
accession number KM251461. The phylogenetic analysis depicted the sequence to cluster with
200
Pediococcus pentosaceus ATCC 25745 with 98% sequence homology. This isolate is reported
9
201
for the very first time from a novel, nutritionally enriched but weaning food of Himachal Pradesh
202
with a very good probiotic potential.
203
Safety assessment of P. pentosaceus C-1 was done for its successful use in food and
204
fermentation industry as potential probiotic candidate. Susceptibility towards antibiotics is the
205
main criterion to be considered for the selection and safe use of the probiotic strains in food
206
products. In the present study, P. pentosaceus C-1 was found to be susceptible to all the
207
antibiotics used (Table 1) except for Co-trimoxazole. Selection of probiotics is based on some
208
desirable characteristics which provide a specific benefit to the host. However, certain properties
209
have become priority in order to select safe and effective probiotics for food industry. In this
210
sense probiotics must be safe for the host and humans (as last consumers), not possess antibiotic
211
resistance genes, show potential colonization and replication within the host, be able to reach the
212
location where the effect is required to take place and actually work in vivo conditions (29). P.
213
pentosaceus C-1 was assessed for the important safety criteria for its safe and efficient use in
214
foods and fermentation processes. Isolate has been found to be sensitive to most of the antibiotic
215
used in the study, thereby revealing its ability not to take up antibiotic resistant traits.
216
Other parameters to be considered for the safety assessment of probiotic isolates were
217
DNase, gelatinase and hemolytic ability. The hemolysis test showed that, P. pentosaceus C-1 did
218
not produce hemolysis on blood agar and showed a negative response in the production of
219
gelatinase and DNase enzymes as pathogenicity factors. Therefore, the selected LAB isolate with
220
probiotic properties used in this study may be used as starter culture in fermented food. Cells of
221
probiotic isolate have been assessed for their hemolytic ability and were observed to be non-
222
hemolytic. Non-hemolytic activity and antibiotic resistance traits are considered as a safety
223
prerequisite for the selection of a probiotic strain. Antibiotic resistance and hemolytic potential
10
224
of lactic acid bacteria isolated from curd was studied and it was found that all isolates did not
225
exhibit β-hemolysis and some of the isolates were sensitive while other exhibited resistance to
226
antibiotics used (6). Other factors for the consideration of safe status of probiotic bacteria are
227
production of DNase and gelatinase enzymes. Gelatinase is a metalloendopeptidase capable of
228
hydrolyzing insulin, casein, hemoglobin, fibrinogen, collagen and gelatin, thus affecting the
229
membrane integrity and DNase are the enzymes which affect the ribonucleotide pool of the host.
230
P. pentosaceus C-1 has been found negative for the production of these two factors, thereby
231
revealing its safe status. Similar results were obtained by Anas et al. (30) where the potentially
232
probiotic Lactobacillus plantarum (P6) was assayed for gelatinase activity and hemolysis and the
233
isolate showed no activity of gelatinase but slightly positive haemolysis.
234
The most important properties for a probiotic to provide health benefits is its ability to
235
overcome physical as well as chemical barriers viz. acid and bile salts in the gastrointestinal
236
tract. P. pentosaceus C-1 survived 3h exposure to pH 3.0 and 1.0 h to pH 2 (Table 2) and showed
237
less reduction of viable cells compared to control (pH 6.5) at pH 3 showing good survival under
238
acidic conditions. Selected isolate survived in MRS broth containing 2% of Ox-bile for 8h (Fig.
239
3). P. pentosaceus TMU457 showed a sharp decline in its growth rate immediately after
240
exposure to pH 2.0, L. fermentum TMU121 showed moderate resistance to pH 2.0 value and lost
241
about half of its viable counts within an hour of incubation and L. rhamnosus TMU094 was able
242
to resist low pH values of 2.0 and showed maximum growth at this pH within an hour of
243
incubation (31). Tolerance to low pH and bile salt concentration the general criteria for selecting
244
probiotic microorganisms and are successfully fulfilled by the selected isolate for its use as
245
effective probiotic in food as well as pharmaceutical preparations.
11
246
The survival of C-1 at pH 2.0, 3.0 containing pepsin (depicting stomach conditions) and
247
pH 8.0 containing pancreatin (depicting intestinal conditions) was observed for 4 h. Isolate E.
248
faecium Ch-1 exhibited good survival at pH 3 (48.4% survival) upto 4h and retained a moderate
249
rate of survival at pH 2.0 (45.6% survival) after 1 h of incubation (Table 3) while percent
250
survival in intestinal juice was observed to be 65.60%, revealing its good ability to survive the
251
gastric transit and survival in intestinal conditions.
252
BSH are the class of enzymes which catalyses the deconjugation of bile salts. Free
253
(deconjugated) bile salts possess lower solubility at low pH and precipitate due to the
254
fermentative metabolism of LAB. The ability of probiotic strains to hydrolyze bile salts has often
255
been included among the criteria for probiotic strain selection. However, microbial BSH activity
256
has also been mooted to be potentially detrimental to the human host and it is so far not entirely
257
clear whether BSH activity is in fact a desirable trait in a probiotic bacterium. P. pentosaceus C-
258
1 strain in this study was unable to deconjugate bile salts as it did not produce any precipitation
259
on MRS supplemented with TDCA.
260
Auto-aggregation ability is another criterion for probiotic properties which have been
261
associated with the adherence potential of these bacteria. Autoagrregation of probiotic strains is
262
referred to the clumping together of bacterial cells and could be functional in forming biofilms in
263
gastrointestinal (GI) tract to form a barrier against colonization by pathogens (22). As shown in
264
(Fig. 4), P. pentosaceus C-1 exhibited a strong autoaggregation after 5 h of incubation (99.0%).
265
Similar percent autoaggregation was observed where the autoaggregation ability of probiotic
266
lactic acid bacteria isolated from traditional fermented foods of Western Himalayas was in a
267
range of 51.15-87.69% (32). Coagrregation helps in eliminating/destruction of pathogens in GI
268
tract by optimizing the effect of antibacterial substances secreted against them by probiotic
12
269
bacteria during aggregation. P. pentosaceus C-1 showed coaggregation with L. monocytogenes
270
(18.36%), B. cereus (14.2%) and C. perfringens (14.0%) (Fig. 5). The results thereby reveal the
271
ability to of C-1 to compete with pathogenic bacteria for adhesion sites and inhibit their
272
colonization in GI tract. Coaggregation ability of lactic acid bacteria isolated from cooked meat
273
products was studied and it was found that all the isolates coaggregated efficiently with E. coli
274
O139:H26 and S. parera IV O11:Z4Z23 (0.48%-32.71%) (33). It has been suggested that
275
probiotic microorganisms that have the ability to coaggregate with pathogens may be better able
276
to kill pathogenic bacteria because they could produce antimicrobial substances in very close
277
proximity to them (23).
278
The inhibitory activity of cell free supernatant was studied by agar well diffusion method
279
against serious food borne pathogens (Fig. 6). P. pentosaceus C-1 displayed the highest
280
antimicrobial activity against E. coli with inhibition zone of 25 mm while lowest antimicrobial
281
activity was shown against P. caratovorum with inhibition zone of 16.6 mm. Lin et al. (24)
282
suggested that the antimicrobial activity of lactic acid bacteria relies on acidity, lactic acid or
283
other antimicrobial products. Other possible factors might be bacteriocins which play roles at
284
low pH values. Lactic acid bacteria are known for their ability to produce antibacterial peptides
285
and other small proteins called bacteriocins enabling them to inhibit the pathogenic bacteria in
286
the environment (34). The production of antibacterial substances viz. bacteriocin, lactic acid and
287
H2O2 advocate the ability of isolate to compete with the microorganisms in the surrounding
288
environment and its successful survival in harsh conditions.
289
Bacteriocin production of C-1 against L. monocytogenes was studied after neutralizing the cell
290
free supernatant with 0.1N NaOH and catalase by serial two fold dilution method. Isolate C-1
291
produced proteinceous compound that exhibited activity i.e. 666.6 AU/ml (less activity as
13
292
compared to un-neutralized supernatant) as the activity was lost after treatment with proteolytic
293
enzyme (trypsin). This suggests that the bacteriocin was also responsible for the antagonism
294
against the pathogenic bacteria along with other antimicrobial substances. P. pentosaceus C-1
295
inhibited important food borne pathogen viz. L. monocytogenes which is a commonly found in
296
fermented foods rendering them as unacceptable and the antagonism against pathogens suggests
297
that P. pentosaceus Ch-1 may have potential applications in food biopreservation.
298
Bacteriocin producing probiotic lactic acid bacteria isolated from Thia indigenous
299
chicken were screened and 14 strains displayed bacteriocin production and strong inhibitory
300
activity against indicator strains (35). The inhibition off pathogens suggests that P. pentosaceus
301
C-1 may have potential application in food industry especially in biopreservation and
302
enhancement of shelf stability of food products.
303
Pediococcus spp. being homofermentative probiotic bacteria produces more than 85%
304
lactic acid, the major product of this fermentation, from glucose. The cell free supernatant
305
obtained in this study has been found to have low pH (3.40) and 0.09 % titratable acids. Lactic
306
acid was quantified by HPLC spectra system (Novapak C-18). P. pentosaceus C-1 produced
307
8.304 mg/l of lactic acid after 24h of incubation. (Fig. 7) shows typical HPLC chromatograms of
308
standard solution and lactic acid extracted from culture supernatant of P. pentosaceus C-1
309
revealing the main role of lactic acid in its antimicrobial potential.
310
Metabolic fingerprinting of P. pentosaceus C-1 was done using GC-MS to explore novel
311
compounds with various beneficial and therapeutic characteristics. In the current study, intra-as
312
well as extracellular metabolites of P. pentosaceus C-1 were assessed by GC-MS analysis. P.
313
pentosaceus C-1 secreted total 38 compounds out of which 15 compounds were reported with
14
314
various antimicrobial and therapeutic properties (Table 4). L-Lactic acid (86.40%),
315
Benzaldehyde, 2,4dimethyl (1.29%), Tetradecane (1.77%), Phenol, 2,4bis (1,1dimethylethyl)
316
(7.84%) and nNonadecanol1 (1.83%) were reported to have antimicrobial properties. Dodecane
317
(1.88%), Tetradecane
318
pyrazine1,4dione, hexahydro3( 2methylpropyl) (4.03%) and Eicosane (1.54%) have been
319
reported for their antioxidant potential. Hexadecanoic acid, 2methylpropyl ester (2.33%),
320
Pyrrolo[1,2a] pyrazine1,4dione, hexahydro3(2methylpropyl) (4.03%), Isopropyl myristate
321
(2.73%) and Octadecane (2.23%) have been observed and were found to have anti-inflammatory,
322
anti-cancerous, medicinal activity in skin disorders and ability to Lower LDL cholesterol.
323
Hexadecane, 2,6,11,15 tetramethyl (0.59% ) and Benzaldehyde, 2,4dimethyl (1.29%) were
324
reported to be natural food additive and flavor agent, respectively. 9Octadecenamide, (Z)
325
(9.34%) with anti-sleep disorder (anti depression agent) properties have been reported for the
326
first time by P. pentosaceus C-1. GC-MS chromatogram of metabolites secreted by P.
327
pentosaceus C-1 has been shown in (Fig. 8).
328
Conclusions
(1.77%), Pyrrolo[1,2a]
pyrazine1,4dione (1.50%), Pyrrolo[1,2a]
329
P. pentosaceus C-1 isolated and reported for the first time from kodo millet flour was
330
evaluated for its safety as well as probiotic potential by using in vitro tests. The strain C-1 was
331
found to possess potential probiotic attributes and did not possessed any undesirable properties.
332
P. pentosaceus C-1 possessed the best probiotic properties viz. tolerance to acid and bile salts,
333
remarkable autoagrgregation and coaggregation abilities, broad antagonism against serious food
334
borne and spoilage causing organisms. P. pentosaceus C-1 has been reported among the
335
probiotic Pediococci bacteria for the very first time to produced 9Octadecenamide, (Z) which is
336
having anti-sleep disorder (anti depression agent) properties. Thus, the probiotic isolate can be
15
337
recommended for the nutraceutical/functional food preparation with anti-depression potential.
338
Though, in vitro studies advocate the probiotic potential of P. pentosaceus C-1, yet in vivo
339
studies and clinical trials are required for further confirmation of the results.
340
References
341
1. Oh YJ, Jung DS. Evaluation of probiotic properties of Lactobacillus and Pediococcus
342
strains isolated from Omegisool, a traditionally fermented Millet alcoholic beverage in
343
Korea. LWT - Food Science and Technology. 2015; doi: 10.1016/j.lwt.2015.03.005.
344
2. FAO/WHO. Guidelines for the evaluation of probiotics in food. London, Ontario, Food
345
and Agriculture Organization of the United Nations and World Health Organization
346
Working Group Report, 2002; pp. 1-11.
347
3. Soccol CR, de Souza Vandenberghe LP, Spier MR, Medeiros ABP, Yamaguishi CT, De
348
Dea Lindner J, Pan A, Thomaz-Soccol V. The potential of probiotics: A review. Food
349
Technology. 2010; 48(4): 413-434.
350
4. Ahmadi S, Soltani M, Shamsaie M, Islami HR, Peyghan R. Comparative effect of
351
Pediococcus acidilactici as probiotic and Vitamin C on survival, growth performance and
352
enzyme activities of white leg shrimp (Litopenaeus vannamei). Journal of Animal and
353
Veterinary Advances. 2014; 13(14): 877-885.
354
355
5. Klaenhammer TR. Genetics of bacteriocins produced by lactic acid bacteria. FEMS
Microbiol. Rev. 1983; 12: 39-85.
356
6. Hawaz E. Isolation and identification of probiotic lactic acid bacteria from curd and in
357
vitro evaluation of its growth inhibition activities against pathogenic bacteria. African
358
Journal of Microbiological Research. 2014; 8(13): 1419-1425.
16
359
360
7. Sanders ME. Consideration for use of probiotic bacteria to modulate human health.
Journal Nutr. 2000; 130: 384-390.
361
8. Argyri AA, Zoumpopoulou G, Karatzas KG, Tsakalidou E, Nychas GE, Panagou EZ,
362
Tassou CC. Selection of potential probiotic lactic acid bacteria from fermented olives by
363
in vitro tests. Food Microbiology. 2013; 33: 282-291.
364
9. Kimoto H, Nomura M, Kobayashi M, Okamoto T, Ohmomo S. Identification and
365
probiotic characteristics of Lactococcus strains from plant materials, Japan Agricultural
366
Research Quarterly. 2004; 38:111-118.
367
368
369
370
371
372
373
374
10. Amadou I, Gounga ME, Le GW. Millets: Nutritional composition, some health benefits
and processing- A review. Emir. J. Food Agric. 2013; 25(7): 501-508.
11. Verma V, Patel S. Value added products from nutri-cereals: Finger millet (Eleusine
coracana). Emir. J. Food Agric. 2013; 25(3): 169-176.
12. De Man J, Rogosa M, Sharpe M. A medium for the cultivation of lactobacilli. Journal of
Applied Bacteriology 1960; 3: 13-135.
13. Liong MT, Shah NP. Acid and bile tolerance and cholesterol removal ability of
Lactobacilli strains. Journal of Dairy Science. 2004; 88: 55-56.
375
14. Gilliland SE, Walker DK. Factors to consider when selecting a culture of L. acidophilus
376
as a dietary adjunct to produce a hypercholesterolemic effect in humans, Journal of Dairy
377
Science. 1990; 73: 905-909.
378
15. Charteris WP, Kelly PM, Morelli L, Collins JK. Development and application of an in
379
vitro methodology to determine the transit tolerance of potentially probiotics Lactobacilli
380
and Bifidobacterium species in the upper human gastrointestinal tract, Journal of Applied
381
Microbiology. 1998; 84: 759–768.
17
382
16. Dashkevicz MP, Feighner SD. Development of a differential medium for bile salt
383
hydrolase-active Lactobacillus spp. Applied and Environmental Microbiology. 1989; 55:
384
11-16.
385
17. Del Re B, Sgorbati B, Miglioli M, Palenzona D. Adhesion, autoaggregation and
386
hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied
387
Microbiology. 2000; 31: 438-442.
388
18. Kimura H, Sashihara T, Matsusaki H, Sonomoto K, Ishizaki A. Novel bacteriocin of
389
Pediococcus sp. ISK-1 isolated from well – aged bed of fermented rice bran. Annals of
390
New York Academy of Science. 1998; 864: 345-348.
391
19. Barefoot SF, Klaenhammer TR. Detection and activity of Lactacin B, a bacteriocin
392
produced by Lactobacillus acidophilu. Applied and Environmental Microbiology. 1983;
393
45: 1808-181.
394
20. AOAC. Official methods of analysis of association of official analytical chemists, 16 th
395
edn. Vol I and II. Association of Official Analytical Chemists. Arlington, Virginia, USA
396
(1995).
397
21. Coucheney E, Daniell TJ, Chenu C, Nunan N. Gas chromatographic metabolic profiling:
398
A sensitive tool for functional microbial ecology. Journal of Microbiological Methods.
399
2008; 75: 491–500.
400
22. Savedboworn W, Riansa-ngawong W, Sinlapacharoen W, Pajakang S, Patcharajarukit B.
401
Assessment of probiotic properties in lactic acid bacteria isolated from fermented
402
vegetables. KMUTNB International Journal of Applied Science and Technology. 2014;
403
7(4): 53-65.
18
404
23. Osmanagaoglu O, Kiran F, Ataoglu H. Evaluation of in vitro probiotic potential of
405
Pediococcus pentosaceus OZF isolated from human breast milk, Probiotics and
406
Antimicrobial Proteins. 2010; 2(3): 162-174.
407
408
24. Lin WJ, Savaiano DA, Harlander SK. A method for determining b-galactosidase activity
of yogurt cultures in skim milk. Journal of Dairy Science. 1989; 72: 351.
409
25. Adnan AFM, Tan IKP. Isolation of lactic acid bacteria from Malaysian foods and
410
assessment of the isolates for industrial potential. Bioresource Technology. 2007; 98:
411
1380-1385.
412
26. Powthong P, Suntornthiticharoen P. Isolation, identification and analysis of probiotic
413
properties of Lactic acid bacteria from selective various traditional Thai fermented foods.
414
Pakistan Journal of Nutrition. 2015; 14(2): 67-74.
415
27. Mandal V, Sen SK, Mandal NC. Optimized culture conditions for bacteriocin production
416
by Pediococcus acidilactici LAB 5 and its characterization, Indian Journal of
417
Biochemistry and Biophysics. 2008; 45: 106-110.
418
28. Musikasang H, Sohsomboon N, Tani A, Maneerat S. Bacteriocin producing lactic acid
419
bacteria as a probiotic potential from Thai indigenous chicken. Czech Journal of Animal
420
Science. 2012; 57: 137-149.
421
29. Sanchez-Ortiz AC, Luna-Gonzalez A, Campa-Cordova AI, Escamilla-Montes R, Flores-
422
Miranda MC, Mazón-Suástegui JM. Isolation and characterization of potential probiotic
423
bacteria from pustulose ark (Anadara tuberculosa) suitable for shrimp farming. Lat. Am.
424
J. Aquat. Res. 2015; 43(1): 123-136.
19
425
30. Anas M, Ahmed K, Mebrouk K. Study of the Antimicrobial and Probiotic Effect of
426
Lactobacillus plantarum (P6) Isolated from Raw Goat's Milk from the Region of Western
427
Algeria. World Applied Sciences Journal. 2014; 32 (7): 1304-1310.
428
31. Karimi Torshizi MA, Rahimi S, Mojgani N, Esmaeilkhanian S, Grimes JL. Screening of
429
Indigenous Strains of Lactic Acid Bacteria for Development of a Probiotic for Poultry.
430
Asian-Aust. J. Anim. Sci. 2008; 21(10): 1495 – 1500.
431
32. Sourabh A, Kanwar SS, Sharm PN. Diversity of bacterial probiotics in traditional
432
fermented foods of Western Himalayas. International Journal of Probiotics and
433
Prebiotics. 2010; 5(4): 193-202.
434
33. Ramirez-Chavarin ML, Wacher C, Eslava-Campos CA, Perez-Chabela ML. Probiotic
435
potential of thermotolerent lactic acid bacteria strains isolated from cooked meat
436
products. International Food Research Journal. 2013; 20(2): 991-1000.
437
34. Messaoudi S, Manai M, Kergourlay G, Prevost H, Connil N, Chobert JM, Dousset X.
438
Lactobacillus salivaris: Bacteriocin and probiotic activity. Food Microbiology. 2013; 36:
439
296-304.
440
35. Musikasang H, Sohsomboon N, Tani A, Maneerat S. Bacteriocin-producing lactic acid
441
bacteria as a potential from Thai indigenous chicken. Czech Journal of Animal Science.
442
2012; 57(3): 137-149.
443
Table 1 Antibiotic sensitivity of P. pentosaceus C-1
S. No
Antibiotics
Concentration (µg)
Sensitive/Resistance
1.
Ampicillin (AMP)
30
S
2.
Augmentin (AMC)
30
S
3.
Gentamicin (GEN)
10
S
20
4.
Cephalothin (CEP)
30
S
5.
Cloxacillin (COX)
1
S
6.
Cefotaxime (CTX)
30
S
7.
Cefoxitin (CX)
30
S
8.
Lincomycin (L)
2
S
9.
Tetracycline (TE)
30
S
10.
Amoxyclav (AMC)
30
S
11.
Co-trimoxazole (COT)
25
R
12.
Cefuroxime (CXM)
30
S
% Sensitivity
444
91.66%
Table 2 Acid tolerance of P. pentosaceus C-1
pH
1.0
2.0
3.0
Control
Mean
Incubation time (min)
Cell survival (log CFU/ml)*
**% Cell Survival
0
60
120
180
Mean
60
120
180
9.91
0.00
0.00
0.00
0.00
0.00
0.00
2.47
(0.00)#
(0.00)
(0.00)
9.95
4.00
0.00
0.00
39.23
0.00
0.00
3.48
(38.76)
(0.00)
(0.00)
10.03 10.0
9.70
9.70
99.23
95.19
94.63
9.85
(84.93)
(77.30)
(76.57)
10.13 10.18 10.19 10.25 10.18
100
100
100
(89.96)
(89.96)
(89.96)
10.00 6.04
4.97
4.98
59.61
48.79
48.65
(53.41)
(41.81)
(41.63)
445
446
Treatment (0.481)
Incubation Time (0.481)
TxI (0.962)
*log CFU/ml: Mean of results from three separate experiments
**% Survivability = (log cfu pH 1.2.3/ log cfu pH.65 ) × 100
447
# Transformed values (Arcsign transformation)
Mean
0.0
(0.0)
13.07
(12.92)
96.35
(79.60)
100
(89.96)
Treatment (0.026)
Incubation Time (0.023)
TxI (0.045)
CD0.05
448
449
Table 3 Tolerance of P. pentosaceus C-1 to gastrointestinal transit
Gastrointestinal
juices
pH 2
pH3
Cell survival (log
0
1
4
9.6
5.7
0.0
9.17
7.87
8.2
Incubation Time (h)
Cell survival (%)*
Mean 1
4
Mean
#
5.1 53.37 (46.91) 0.00 (0.00)
26.68 (23.45)
73.68
(59.11)
75.64
(60.40)
8.41
74.66 (59.75)
CFU/ml)*
21
pH8
Control
Mean
CD0.05
450
451
10.12
10.04
9.77
10.50
10.68
10.84
9.84
8.57
7.20
9.97 94.00 (75.79)
10.67 100 (89.96)
80.26 (67.94)
Treatment (0.179)
Incubation Time (0.155)
TxI (0.310)
*Log cfu/ml: Mean of results from three separate experiments
**% Cell Survival = (log cfu/ml pH2,3,8/ log cfu/ml pH 6.5) × 100
90.12 (71.65) 92.06 (73.72)
100 (89.96)
100 (89.96)
66.44 (55.50)
Treatment (0.187)
Incubation Time (0.132)
TxI (0.265)
452
#
453
Table 4 GC-MS analysis of extra- and intra-cellular metabolites of P. pentosaceus C-1
Transformed values (Arcsign transformation)
S. No.
Compound Name
RT
Molecular
Formula
3.04
C9H18O3
2.
Ethyl 2methylpentyl
carbonate
L-Lactic acid
5.42
3.
9-Octadecenamide, (Z)
4.
C-1
Peak Area
Area
%
Peak
height
Biological
activity
8180419.72
0.88
2757310.35
-
C3H6O3
80045968.18
86.40
Antibacterial
17.60
C18H35NO
86510467.38
9.34
45393302.7
7
6335307.22
Trichloromethane
3.09
CHCl3
2033530858.
24
14.40
348422839.
60
5.
Butane,
2ethoxy2methyl
3.56
C7H16O
991514725.8
6
7.02
78800446.3
2
-
6.
Diethyl carbonate
4.30
C5H10O3
115472676.2
4
8.15
134314332.
61
-
7.
Butane,
2,3dichloro2methyl
4.68
C5H10Cl2
97735870.77
0.69
11418482.7
1
-
8.
Benzene, chloro-
5.01
C6H5Cl
708365490.8
7
5.02
64945566.8
0
-
9.
Cyclopentane,
1,3dichloro, cis
6.64
C5H8Cl2
369385252.9
1
2.62
38117379.0
7
-
10.
Propanal,
2,3dichloro2methyl
7.31
C4H6Cl2O
306416150.4
6
2.17
34650637.7
7
-
11.
Undecane
7.98
C11H24
133098984.6
1
0.94
25469962.2
6
-
12.
Dodecane
9.02
C12H26
264865809.2
4
1.88
42422379.1
9
Antioxidant
13.
Benzaldehyde,
2,4dimethyl
9.31
C9H10O
182353894.2
0
1.29
34350995.2
0
Antibacterial,
Flavor agent
14.
Benzene,
1,3bis(1,1dimethylethyl
9.62
C14H22
573886351.2
2
4.06
149665476.
93
-
1.
22
Anti depression
agent)
-
)
15.
Dodecane,
2,6,11trimethyl
10.23
C15H32
128002725.6
2
0.91
11950202.5
6
-
16.
Tetradecane
10.90
C14H30
249375686.6
8
1.77
59554296.2
4
Antioxidant,
antibacterial
17.
Phenol, 2,4bis(
1,1dimethylethyl)
11.92
C14H22O 9
1107816603.
75
7.84
272063789.
73
Antimicrobial
18.
Hexadecane,
2,6,11,15tetramethyl
12.10
C20H42
83632247.3
0.59
15439374.3
7
Natural food
additive
19.
Dodecanoic acid
12.34
C12H24O2
92062583.10
0.65
14941984.4
4
-
20.
Pentadecane, 7methyl
12.56
C16H34
225692967.5
8
1.60
50030072.1
8
-
21.
Hexadecane, 1,1bis(
dodecyloxy
12.65
C40H82O2
89591108.98
0.63
18400772.5
9
-
22.
Ethanol, 2(
octadecyloxy)
13.75
C20H42O2
175888227.4
1
1.25
18140705.3
6
-
23.
Octadecane
14.06
C18H38
315004752.5
8
2.23
43031709.8
4
Lower LDL
cholesterol
24.
Isopropyl myristate
14.24
C17H34O2
386036937.9
5
2.73
97904440.9
2
Medicinal
activity in skin
25.
Pyrrolo[1,2a]
pyrazine1,4dione
14.38
C11H18N2
O2
211926755.3
7
1.50
46863717.8
8
Antioxidant
26.
Phthalic acid, butyl
tetradecyl ester
14.65
C26H42O4
185754847.2
0
1.32
21939968.4
3
-
27. 1Hexadecanol, 2methyl
15.03
C17H36O
140518707.8
3
1.00
25826700.4
9
-
28.
Pyrrolo[1,2a]
pyrazine1,4dione,
hexahydro3(
2methylpropyl)
15.25
C11H18N2
O2
569021007.2
9
4.03
57297572.0
5
Antioxidant,
anti-cancerous
29.
Eicosane
15.41
C20H42
217293414.1
6
1.54
34411109.0
5
Antioxidant
30.
Isopropyl palmitate
15.58
C19H38O2
80536906.97
0.57
19557742.4
4
-
31.
nNonadecanol1
15.98
C19H40O
258729362.7
2
1.83
29795237.7
5
Antimicrobial
32.
2Ethylhexyl
trans4methoxycinnamat
16.60
C18H26O3
545433382.8
3.86
49961564.0
-
23
e
3
6
33. Z5Methyl6heneicosen1
1one
17.00
C22H42O
89629621.17
0.63
14159635.4
3
-
34.
2Propenoic acid, 3(
4methoxyphenyl)2ethylhexyl ester
17.82
C18H26O3
229422327.6
5
1.62
45967532.8
5
-
35.
Tetracosane
18.38
C24H50
184288567.7
1
1.31
31912641.8
4
-
36.
Hexadecanoic acid,
2methylpropyl ester
19.60
C20H40O2
328809068.0
0
2.33
38893174.2
2
Antiinflammatory
37.
Phthalic acid,
di(2propylpentyl)ester
20.33
C24H38O4
310842310.8
5
2.20
51830380.3
3
-
38.
Trichloromethane
20.91
C34H70
199845509.6
8
1.42
31261996.8
8
454
455
456
457
458
459
24
460
461
C. perfringens
B. cereus
L.
monocytogenes
0
5
10
462
25
15
20
463
464
465
466
467
a
26
468
469
b
470
Figure 1 Antimicrobial activity of LAB isolated from Kodo millet flour by Bit/disc method
471
Figure 3 Bile salt tolerance of P. pentosaceus C-1
472
Figure 4 Autoaggregation of P. pentosaceus C-1
473
Figure 5 Coaggregation of P. pentosaceus C-1
474
Figure 6 Antimicrobial activity of P. pentosaceus C-1 against indicator strains at un-neutralized
475
pH
476
Figure 7 HPLC chromatogram of lactic acid produced by P. pentosaceus C-1
477
Figure 8 GC-MS chromatogram of C-1 a) aqueous phase metabolites b) organic phase
478
metabolites
27