A questionnaire based study was conducted among villagers of Nam Phake village (fig: 1) to document type of traditional food, mode of preparation, variations in mode of preparations, their role in socio-cultural gatherings and therapeutic value.
The daily food intake of Tai-Phake includes a range of prepared food such as freshly prepared chutneys, herb juices, steamed cereals wrapped in leaves, boiled vegetables, preserved fruits and vegetables, fermented fish and meat, using raw materials which are locally available and economically encouraging (Fig.2) . This study gave an insight on the traditional knowledge of the Tai-Phake community in accessing nature’s biodiversity for preparation of TFF and maintains a sustainable association with the surrounding ecosystem. Nine different fermented foods, based on vegetables, fruits, and fish, were selected and listed in Table 1.
Table 1: Characteristics of some of the traditional fermented food of Tai-Phake community
|
Sl. No
|
Local name of the sample
|
Main Ingredients
|
Colour
|
Consistency
|
Smell
|
1
|
No-cha
|
Bamboo shoot
|
White
|
Watery-chunks
|
Very Acidic
|
2
|
No-Chom
|
Bamboo shoot
|
White
|
Watery-chopped
|
Very Acidic
|
3
|
Nohi-U
|
Bamboo shoot
|
Brown
|
Dry-shredded
|
Very Acidic
|
4
|
Pakkat-su
|
Lai Patta-Rice starch
|
Dark-Greenish (Cloudy)
|
Watery-semi solid
|
Foul
|
5
|
Tenga
|
Lemon and salt
|
Dark brown to Black
|
Solid with little water
|
Slightly Acidic
|
6
|
Manang
|
Garcinia fruit
|
Black
|
Dry
|
Slightly Acidic
|
7
|
Ne-tek
|
Meat
|
Dark brown
|
Semi solid
|
Pungent
|
8
|
Pa-su
|
Fish
|
Brown
|
Sticky and semi-solid
|
Pungent
|
9
|
Pa-nao
|
Fish
|
Light brown
|
Dry
|
Pungent
|
The traditional fermented food/preserved food from Tai-Phake Community which comprised of Bamboo shoot based ( No-cha, No-chom, Nohi-U), leafy vegetable based (Pakkat Su) , Fruit based (Tenga, Manang ), Meat based (Ne-tek) and Fish based (Pa-su, Pa-nao)
|
Vegetable based fermented food- ‘Pakkat Su’
Pakkat su’, Brassica juncea based fermented food was selected from the listed 9 TFF, to further isolate and characterize potential probiotic microorganisms. ‘Pakkat Su’ is prepared from tender leaves of Brassica juncea (Lai Xaak in Assamese language) and rice starch. Leaves are washed, dried, chopped and knead to get the water out. Then it is kept with rice starch for 3 to 4 days for souring (Fig.3). It can be kept for a year and eaten with rice or other food items as side dish. It is cloudy light greenish in colour, has sour taste semi-solid and has a very strong, ‘not so pleasant’ smell.
Screening of microorganisms and Preparation of pure culture
A total of 29 isolates were isolated from ‘Pakkat su’, leafy vegetable ‘Brassica juncea’ based fermented food and purified using serial dilution method and plating on MRS medium. Isolates were screened on the basis of colony morphology (colour, size, margin and shape of the colony), gram staining, endospore staining and catalase test. Only Gram positive, non spore former and catalase negative 13 isolates were selected. Isolates obtained from ‘Pakkat Su’ are TPI-1, TPI-2, TPI-4, TPI-6, TPI-7, TPI-9 TPI-12, TPI-15, TPI-16, TPI-17, TPI-20, TPI-21and TPI-23. Purified isolates were studied according to Bergey’s manual and observed that TPI-1, TPI-9, TPI-12, TPI-15, TPI-17, TPI-22 showed smaller, white to creamish, opaque colonies with smooth margins. TPI-7 and TPI-20 showed larger yellowish colonies.
In vitro phenotypic test to test viability of potent isolates in Acidic and Alkaline environment
Only three TPI-4, TPI-7, and TPI- 9 showed broad range of pH tolerance and able to grow in MRS media with pH 8.0, 7.0, 5.0, 3.0 and 2.0. TPI-2 TPI-6 TPI-12 TPI- 15 TPI-16, TPI-17, TPI-20, TPI-21 and TPI-23 could tolerate and able to grow in pH 5.0 and pH 6 but were unable to grow below pH 5 except forTPI-12.
Salt Tolerance assay
Salt tolerance assay of all the tested cultures showed significant tolerance (Table 7). One of the isolates, IL-9 showed the highest salt tolerance.
Bile Tolerance assay
We tested 13 isolates for bile tolerance test. Only 3 isolates were able to survive in presence of bile salts (0.125%, 0.25%, 0.5%) for hrs, which was confirmed by plating. 0.25% Bile salt was tolerated by TPI-1, TPI-4, TPI-6, TPI-7, TPI-9, TPI-15, TPI-16, TPI-17 and TPI-20 but there was a sharp decline in viability of Isolates at 0.5% conc. of Bile salt except for TPI-4, TPI-7 and TPI-9 which showed maximum tolerance for bile (Fig.4).
Antagonistic effect
We tested the most potent Isolates TPI-4, TPI-7 and TPI-9 for antimicrobial compounds. Zone of inhibition showed that crude extracts from each isolate had antimicrobial effect against each tested pathogen (Table-2). TPI-9 displayed highest antagonistic activity against E.coli ATCC-10536 and Staphylococcus aureus ATCC-BAA-1026. However, TPI-4 showed a minimum inhibition zone of diameter ranging from 0.7 to 0.8 cm against the indicator microorganisms.
Table 2: Antagonistic activity by Isolates against test pathogens
|
Sl. no
|
Isolate
|
Zone of Inhibition (cm) [Mean±SD]
|
E. coli
|
S. aureus
|
1
|
TPI-4
|
0.8±0.029
|
0.7±0.056
|
2
|
TPI-7
|
1.3±0.09
|
1.4±0.087
|
3
|
TPI-9
|
1.2±0.049
|
1.6±0.039
|
All three Isolates showed strong antagonistic activity against Staphylococuus aureus and E.coli. ZOI of TPI-4 was less than 1 cm against E.coli. TPI-7 and TPI-9 showed ZOI more than 1 cm against both pathogens. These results are from three independent observations.
|
Table 2: Antagonistic activity by Isolates aginst test pathogen
Hemolytic test
The tested strains displayed no hemolysis (γ-hemolysis) when inoculated on Blood Agar Plates. Pathogenic bacteria Staphylococcus aurues and E.coli showed β Hemolysis while LAB Isolates TPI- 4, TPI- 7 and TPI-9 showed no hemolysis which indicates their non-pathogenic character (Fig.5).
Antibiotic susceptibility
Antibiotic susceptibility test was conducted using nine broad spectrum antibiotics. Overnight grown cultures of three isolates showed susceptibility of variable degrees (Table.3) to all the antibiotics except Isolate TPI- 7 showed resistance to Ampicillin (Fig.6). TPI-9 isolates showed resistance against Vancomycin.
Table 3: Zone of Inhibition by nine antibiotic (disc) against three isolates
|
Isolates
|
Zone of Inhibition (cm) [Mean±SD]
|
E
|
VA
|
TE
|
K
|
CIP
|
AMP
|
C
|
GEN
|
CD
|
TPI-4
|
1.5±0.057
|
1.5±0.02
|
2.1±0.1
|
2.5±0.15
|
2.2±0.09
|
1.4±0.04
|
2.1±0.09
|
1.5±0.09
|
1±0.058
|
TPI-7
|
2.3±0.029
|
2.2±0.01
|
2.2±0.08
|
2.1±0.05
|
2.4±0.03
|
0
|
2.5±0.12
|
2±0.12
|
2.8±0.068
|
TPI- 9
|
1.2±0.015
|
0
|
1.3±0.02
|
1.0±0.2
|
0.8±0.06
|
1.2±0.03
|
1.5±0.08
|
1.3±0.09
|
1.3±0.071
|
TPI-4, TPI-7, TPI-9 were susceptible to all the broad spectrum antibiotics except isolate TPI-7 which showed resistance against Ampicillin (2 µg) and TPI-9 which showed resistance against Vancomycin (10 µg).
E: Erythromycin (5 µg),VA: Vancomycin (10 µg), TE: Tetracyclin (10 µg), K: Kanamycin (30 µg), CIP: Ciprofloxacin (30 µg), AMP: Ampicillin (2 µg), C: Chloramphenicol (25-µg), GEN: Gentamycin (50 µg), and CD: Clindamycin (10 µg)
|
4.2.1 Hydrophobicity assay and Autoaggregation test
Bioassay on cell surface hydrophobicity of all three isolates to their adhesion ability to tolune displayed good degree of hydrophobicity and autoaggrgation. TPI-9 was with the highest hydrophobicity of 76.00%, while other two isolates 4 and 7 showed 56% and 64% after 3 hrs of incubation. Autoaggregation was highest in TPI-7 and TPI-9, followed by TPI-4 after 24 hours of incubation at 37 °C in static condition (Figure.6).
4.2.3 Response to simulated stomach duodenum passage (SSDP)
Three isolates which exhibited Strong Stress tolerance viz NaCl, pH and Bile tolerance, were screened for “Response to simulated stomach duodenum passage (SSDP)”. The SSDP assay on these three isolates, showed positive results even after 3 hrs (Viable cell counts are calculated to check the survival rate as CFU/ml) which can predict, survival of LAB in stomach and duodenum (of human). TPI-9 showed maximum viability while TPI-4 and TPI-7 showed lower viability.
4.2.4: Mucin adhesion test
Relative adhesion values of LAB were calculated from CFU/mL after performing Mucin adhesion test. The analysis shows that all three strains were able to adhere to Mucin and survive. Bacterial adhesion was measured by CFU/mL and plotted against serial dilutions performed (Table-4)
Table 4 : CFU/mL after adhesion to Mucin showed the adherence of TPI-4, TPI-7 and TPI-9
Isolates
|
10-2
|
10-4
|
10-6
|
TPI-4
|
534±12
|
467±23
|
202±10
|
TPI-7
|
490±26
|
479±05
|
154±03
|
TPI-9
|
450±11
|
708±06
|
354±04
|
CFU/mL after adhesion to Mucin showed the adherence of TPI-4, TPI-7 and TPI-9
4.2.5: Molecular identification
The partial 16S-rRNA sequences of three isolates, i.e., isolate TPI- 1, isolate TPI2, and isolate 3, after BLASTn analyses at NCBI, were observed in separate clade in respective phylogenetic analyses (Fig.8). The respective accession numbers were assigned as given below after submission to GenBank Database.