Results
Chapter - 4 Results Round, yellow seeded soy beans were used for the preparation soy milk and soy mash (Plate 1 a - c). 4.1 ISOLATION AND IDENTIFICATION From twenty commercially available fermented food (curd 8, cheese 5 and yoghurt 7) and thirty faecal samples were collected from infants (2-4 months aged). Among 20 samples, forty one isolates were identified as LAB based on the cultural characteristics (MRS agar - white rounded, raised/ flat, colonies about 0.1 0.5 mm in diameter), the isolates were purified (Plate 1d). They are catalase negative, oxidase negative, nitrate reduction negative, cocci (7 isolates) and rods (34 isolates) were placed under carbohydrate fermentation profile for further identification. The isolates belong to 4 species in the same genus such as Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus bulgaricus, Lactobacillus helviticus and Streptococcus (Table 1 and 2; Fig. 1; Plate 1 e-i, Plate 2). Creamy white punctiform colonies (0.1 0.5) were selected from MRSc agar. From 30 faecal samples, under anaerobic incubation, 63 isolates were separated, and presumed as LAB (Plate 3a, b). F-6-PPK, test was used to confirm the Bifidobacterium strains. Twenty six strains showed positive result (appearance of red colour) and they belong to the genus Bifidobacterium (Table 1 and 3; Fig. 2; Plate 3a-d and Plate 4). Species level identification was performed based on the carbohydrate fermentation pattern. Bifidobacterium Species such P. Bhuvaneswari and Dr. S. Ahmed John 36
Table 1. Characteristic features of the isolated strains Characteristic Features Gram staining S1 S2 S3 S4 S5 S6 S7 Positive rod Positive rod Positive rod Positive rod Positive bifid form Positive bifid/rod form Positive cocci in chains Motility - ve - ve - ve - ve - ve - ve - ve Catalase - ve - ve - ve - ve - ve - ve - ve Oxidase - ve - ve - ve - ve - ve - ve - ve Nitrate reduction - ve - ve - ve - ve - ve - ve - ve Carbohydrate fermentation pattern Arabinose +ve +ve +ve - ve - ve +ve - ve Cellobiose +ve +ve +ve - ve - ve - ve - ve Esculin +ve +ve +ve +ve - ve - ve +ve Fructose +ve +ve +ve +ve +ve +ve +ve Galactose +ve +ve - ve +ve +ve - ve - ve Glucose +ve +ve +ve +ve +ve +ve +ve Inulin +ve +ve - ve - ve - ve - ve - ve Lactose +ve +ve +ve +ve +ve +ve +ve Maltose +ve +ve +ve - ve - ve +ve - ve Mannose +ve - ve +ve - ve - ve +ve - ve Melibiose - ve +ve +ve +ve +ve +ve - ve Raffinose - ve +ve +ve - ve - ve +ve - ve Rhamnose - ve +ve +ve - ve - ve - ve - ve Ribose - ve +ve +ve - ve - ve +ve - ve Salicin +ve +ve +ve - ve - ve - ve - ve Sorbitol +ve +ve +ve - ve - ve - ve - ve Sucrose +ve - ve +ve +ve +ve +ve - ve Trehalose +ve +ve - ve - ve - ve - ve - ve Xylose +ve +ve +ve - ve - ve +ve - ve S1 Lactobacillus acidophilus S2 Lactobacillus lactis S3 Lactobacillus bulgaricus S4 Lactobacillus helviticus S5 - ve Bifidobacterium bifidum S6 Bifidobacterium longum S7 Streptococcus
Table 2. Number of isolates from commercially available fermented products Samples Isolated organisms No. Of isolates Curd Cheese Yoghurt Lactobacillus acidophilus Lactobacillus casei Lactobacillus lactis Lactobacillus acidophilus Lactobacillus bulgaricus Lactobacillus helviticus Streptococcus 11 6 8 3 9 3 7 Table 3. Isolation of Bifidobacterium from Infant faecal samples F- 6- PPK POSITIVE (Bifidobacterium) TOTAL NO. OF ISOLATES No. Of Acid/ bile resistant strain B. bifidum B. longum No. of Acid/ bile sensitive strais F- 6- PPK NEGATIVE 63 5 2 19 37
Streptococcus 15% Lactobacillus acidophilus- Curd 24% Lactobacillus helviticus 6% Lactobacillus bulgaricus 19% Lactobacillus acidophilus - Cheese 6% Lactobacillus lactis 17% Lactobacillus casei 13% Figure 1. Number of isolates from commercially available fermented products
Plate 1 Soy substrate and isolated strains from commercially available products a b c d e f g h i a. Dried and soaked soy bean, b. Soy milk, c. Soy mash, d. Spread plate in MRS agar, e. Lactobacillus acidophilus in MRS agar, f. L. lactus in MRS agar, g. L. bulgaricus in MRS agar, h. Streptococcus in M-17 agar, i. Gram staining of Lactobacillus acidophilus
Plate 2 Carbohydrate Fermentation Test a b c d a. Carbohydrate fermentation profile for Lactobacillus acidophilus b. Carbohydrate fermentation profile for Lactobacillus lactus c. Carbohydrate fermentation profile for Lactobacillus bulgaricus d. Carbohydrate fermentation profile for Streptococcus
B. bifidum 8% B. longum 3% F- 6- PPK NEGATIVE 59% No. of Acid/ bile sensitive strais 30% Figure 2. Isolation of Bifidobacterium from Infant faecal samples
Plate 3 Strains isolated from infant faecal material and Rizhobus oligosporous a b c ve +ve d e a. Spread plate MRSc agar, b. Bifidobacterium bifidum in MRSc agar, c. F-6- PPT Test, d. Gram staining of Bifidobacterium bifidum, e. Rhizopus oligosporous in Malt Extract Agar
Plate 4 Carbohydrate Fermentation Test a b c a. Carbohydrate fermentation profile for Bifidobacterium bifidum b. Carbohydrate fermentation profile for Bifidobacterium longum
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. as bifidum and longum were identified. Rhizopus oligosporous (MTCC 556) culture was maintained in Malt Extract agar (Plate 3e). 4.2 ACID AND BILE TOLERANCE TEST Viability and activity of the bacteria are important considerations because the bacteria must survive in the food during short life and during transit through the acidic conditions of the stomach, and resist degradation by hydrolytic enzymes and bile salts in the small intestine. According to the survival rate under acid and bile, the efficient strains identified and tabulated (Table 4 and 5; Fig. 3 and 4). There was from 41 isolates, only one strains was selected based on maximum % of survivability in acid and bile, where as twenty six strains of Bifidobacterium, only 7 strains (Bifidobacterium bifidum 5 and Bifidobacterium longum 2) showed maximum survival rate. 4.3 FERMENTATION One 1% log phase bacterial strains were preferred for soy milk fermentation whereas 2% sporangiospore suspension was used for tempeh preparation. After inoculation of the respective cultures to the soymilk and soy mash, incubated for 24 hrs. Plate 5 showed fermented soy milk and tempeh. 4.4 GROWTH KINETICS Viable count, in the first 6 hrs, all the strains which showed minimum CFU due to adaptation (lag phase). 12 hrs, and 18 hrs exhibit the range of growth will surprisingly faster especially Streptococcus showed enormous growth. Compared to Lactobacillus acidphilus, Bifidobacterium showed faster growth pattern. Consequently, mixed fermentation having P. Bhuvaneswari and Dr. S. Ahmed John 37
Table 4. Effect of acid on the isolated strains S. No Fermented with % of survivability at ph 2.5 with different interval 1 Hr 2 Hrs 3 Hrs 4 Hrs 1. Lactobacillus acidophilus 89.18±2.06 80.16±3.05 76.94±2.11 68.01±2.43 2 Lactobacillus lactis 86.77±1.54 78.46±2.97 69.14±2.04 56.43±1.06 3. Lactobacillus bulgaricus 87.84±3.67 74.73±2.63 61.56±3.05 52.14±2.86 4. Bifidobacterium bifidum 94.44±2.79 77.77±2.55 66.67±2.66 55.55±4.93 5. Streptococcus 88.65±1.04 80.15±1.95 70.99±2.87 58.75±2.60 Mean ± Standard deviation of three independent replicates
120 1 Hr 2 Hrs 3 Hrs 4 Hrs 100 80 60 40 20 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Streptococcus Figure 3. Effect of acid on the isolated strains
Table 5. Effect of different concentration of bile on the isolates S. No Fermented with % of survivability at different concentration of oxgall with different interval 0.5% 1.0 % 1 Hr 2 Hrs 3 Hrs 4 Hrs 1 Hr 2 Hrs 3 Hrs 4 Hrs 1. Lactobacillus acidophilus 94.13 ± 1.21 86.03± 2.54 71.99± 3.21 59.82± 2.43 86.83± 2.45 74.95± 4.32 66.03± 3.02 48.73± 1.04 2 Lactobacillus lactis 91.02 ± 2.01 84.33± 3.44 69.84± 1.89 54.92± 5.01 82.98± 3.02 69.74± 2.03 59.87± 3.74 33.86± 2.04 3. Lactobacillus bulgaricus 89.92 ± 3.43 78.01± 3.11 68.47± 2.78 53.65± 3.05 79.03± 2.54 63.82± 2.78 53.38± 2.03 39.55± 2.07 4. Bifidobacterium bifidum 96.33 ± 1.43 90.31± 4.01 81.07± 2.57 69.83± 3.67 87.73± 2.85 76.67± 3.02 68.74± 2.74 52.84± 3.10 5. Streptococcus 89.18 ± 2.88 79.03± 2.67 64.83± 2.04 58.04± 5.43 84.23± 2.76 70.74± 2.54 58.93± 2.82 46.26± 2.48 Mean ± Standard deviation of three independent replicates
120 100 Lactobacillus acidophilus Lactobacillus bulgaricus Streptococcus Lactobacillus lactis Bifidobacterium bifidum 80 60 40 20 0 1 Hr 2 Hrs 3 Hrs 4 Hrs 1 Hr 2 Hrs 3 Hrs 4 Hrs 0.5% 1.0% Figure 4. Effect of different concentration of bile on the isolates
Plate 5 Fermentation (Soy milk & Soy mash) a. Before fermentation b. after fermentation c. Tempeh 1. Lactobacillus acidophilus, 2. Lactobacillus lacus 3. Lactobacillus bulgaricus, 4. Lipidobacterium lipidum 5. Streptococcus
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. organisms showed more number of colonies that indicated that symbiotic relationship may be taken place between the organisms (Table 6). 4.5 ph AND TITRATABLE ACIDITY ph and titratable acidity were measured at different interval. Decrease of ph when fermentation time increases whereas acidity (% of lactic acid) increased in mixed culture fermentation than mono culture fermentation process (Table 7 and 8; Fig. 5 and 6). More ph reduction, more acid production was noted in the soy milk fermented with Lactobacillus bulgaricus + Streptococcus, where as in contrast tempeh (Soy mash fermented with Rhizopus oligosporous) showed slight alkaline ph at the end of the fermentation due to the breakdown process of protein by protease enzyme. 4.6 ESTIMATION OF CRUDE PROTEIN Quantity of crude protein was estimated after fermentation and was compared with unfermented soy milk and soy base. (Table 9; Fig. 7). Fermented products exhibit more quantity of crude protein than control. Fermented soymilk (Lactobacillus bulgaricus + Streptococcus ) showed 5.60 % of crude protein i.e. compared to unfermented soy milk 14.07% increase the quantity of crude protein. 4.7 QUANTIFICATION OF SUGAR Sugars such as glucose+ galactose, fructose and sucrose were estimated. (Table 10, 11 and 12; Fig. 8, 9 and 10). After fermentation glucose + galactose and fructose quantity gradually increased subsequently whereas sucrose level increased. This was due to utilization of fructo oligosaccharide such as P. Bhuvaneswari and Dr. S. Ahmed John 38
Table 6. Quantification of viable cells at different time interval in the fermented food S. No Fermented with Viable count of the fermented food with different interval (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 1.2 10 5 3.4 10 5 1.1 10 6 3.2 10 6 2 Lactobacillus lactis 1.3 10 5 2.1 10 5 1.6 10 6 8.3 10 5 3. Lactobacillus bulgaricus 1.1 10 5 1.9 10 5 1.4 10 6 2.3 10 6 4. Bifidobacterium bifidum 1.2 10 5 2.1 10 5 1.6 10 6 2.8 10 6 5. 6. 7. 8. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus 1.7 10 5 2.1 10 5 1.7 10 6 2.1 10 6 1.6 10 5 2.4 10 5 1.8 10 6 2.6 10 7 1.3 10 5 1.9 10 5 1.4 10 6 1.7 10 6 1.4 10 5 2.4 10 5 1.8 10 6 2.6 10 6 1.2 10 5 1.9 10 5 1.6 10 6 1.2 10 6 1.4 10 5 2.4 10 5 1.8 10 6 2.3 10 7 1.6 10 5 2.2 10 5 17 10 6 1.6 10 6 1.3 10 5 2.8 10 6 1.9 10 7 3.4 10 8
Table 7. ph of the fermented soy milk and tempeh at different time interval S. No Fermented with ph of the fermented food at different interval (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 6.17±0.03 5.32±0.02 4.99±0.02 4.73±0.02 2 Lactobacillus lactis 6.42±0.04 5.90±0.01 5.16±0.04 4.47±0.01 3. Lactobacillus bulgaricus 6.16±0.02 5.87±0.04 5.28±0.02 4.90±0.02 4. Bifidobacterium bifidum 6.44±0.01 5.61±0.05 5.43±0.02 5.01±0.04 5. 6. 7. 8. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus 6.32±0.11 4.84±0.03 4.67±0.01 4.55±0.02 6.16±0.03 5.11±0.02 4.96±0.03 4.76±0.01 5.92±0.05 4.98±0.03 4.52±0.02 4.13±0.04 6.10±0.02 5.76±0.01 4.80±0.03 4.27±0.05 9. Rhizopus oligosporous 5.6±0.08 6.1±0.03 6.5±0.06 7.1±0.03 Mean ± Standard deviation of three independent replicates
8 7 6 hrs 12 hrs 18 hrs 24 hrs 6 5 4 3 2 1 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus Rhizopus oligosporous Figure 5. ph of the fermented soy milk and tempeh at different time interval
Table 8. Titratable acidity of the fermented soy milk and tempeh (at different time interval) S. No Fermented with Titratable acidity of the fermented food with different interval (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 0.16±0.02 0.24±0.03 0.32±0.02 0.45±0.03 2 Lactobacillus lactis 0.14±0.01 0.21±0.04 0.38±0.04 0.42±0.05 3. Lactobacillus bulgaricus 0.11±0.03 0.19±0.02 0.26±0.01 0.34±0.01 4. Bifidobacterium bifidum 0.09±0.02 0.19±0.01 0.24±0.03 0.33±0.03 5. 6. 7. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus 0.23±0.04 0.32±0.02 0.47±0.01 0.51±0.04 0.19±0.06 0.28±0.04 0.39±0.04 0.46±0.04 0.24±0.03 0.35±0.11 0.56±0.03 0.64±0.02 8. Bifidobacterium bifidum + Streptococcus 0.21±0.02 0.31±0.04 0.49±0.02 0.61±0.01 9. Rhizopus oligosporous 0.08±0.03 0.04±0.01 0.02±0.01 - Mean ± Standard deviation of three independent replicates
0.7 6 hrs 12 hrs 18 hrs 24 hrs 0.6 0.5 0.4 0.3 0.2 0.1 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus Rhizopus oligosporous Figure 6. Titratable acidity of the fermented soy milk and tempeh (at different time interval)
Table 9. Concentration of Crude protein of fermented soy milk and tempeh S.No Fermented with Protein (%) 1. Lactobacillus acidophilus 4.37 2 Lactobacillus lactis 4.11 3. Lactobacillus bulgaricus 4.26 4. Bifidobacterium bifidum 4.01 5. Lactobacillus acidophilus+ Streptococcus 6. Lactobacillus lactis +Streptococcus 7. Lactobacillus bulgaricus + Streptococcus 8. Bifidobacterium bifidum + Streptococcus 4.28 5.16 5.60 4.96 9. Rhizopus oligosporous (Tempeh) 6.63 Control (Protein %) Soy milk - 3.98 Soy mash 5.53
7 6 5 4 3 2 1 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis +Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus Rhizopus oligosporous (Tempeh) Figure 7. Concentration of Crude protein of fermented soy milk and tempeh
Table 10. Changes of glucose and galactose content in soy milk fermented with mono and mixed culture and tempeh S. No Fermented with Glucose + galactose content (mmol/l) after fermentation (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 1.25±0.06 1.46±0.11 2.02±0.04 2.58±0.04 2 Lactobacillus lactis 1.19±0.01 1.87±0.03 1.98±0.08 2.43±0.03 3. Lactobacillus bulgaricus 1.14±0.04 1.74±0.08 1.84±0.04 2.31±0.03 4. Bifidobacterium bifidum 1.16±0.03 1.37±0.06 1.92±0.05 2.53±0.11 5. 6. 7. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis +Streptococcus Lactobacillus bulgaricus + Streptococcus 1.37±0.03 1.46±.014 2.02±0.02 2.99±0.01 1.14±0.06 1.26±0.01 2.57±0.11 2.88±0.01 1.37±0.03 1.46±0.14 2.03±0.04 3.53±0.01 8. Bifidobacterium bifidum + Streptococcus 1.19±0.03 1.87±0.03 2.99±0.12 3.51±0.02 9. Rhizopus oligosporous 1.48±0.01 1.51±0.03 1.53±0.02 1.61±0.05 Mean ± Standard deviation of three independent replicates Control : Soy milk - 1.03 mmol/lit Soy mash 1.45 mmol/lit
4 6 hrs 12 hrs 18 hrs 24 hrs 3.5 3 2.5 2 1.5 1 0.5 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis +Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus Rhizopus oligosporous Figure 8. Changes of glucose and galactose content in soy milk fermented with mono and mixed culture and tempeh
Table 11. Changes of fructose content in soy milk fermented with mono and mixed culture and tempeh S.No Fermented with Fructose content (mmol/l) after fermentation (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 1.37±0.04 1.81±0.01 2.07±0.13 2.26±0.03 2 Lactobacillus lactis 1.16±0.07 1.30±0.11 1.6±0.01 1.81±0.04 3. Lactobacillus bulgaricus 1.18±0.14 1.45±0.03 1.74±0.05 1.97±0.02 4. Bifidobacterium bifidum 1.29±0.03 1.76±0.04 1.98±0.06 2.11±0.04 5. 6. 7. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus 1.67±0.07 1.98±0.05 2.04±0.02 2.96±0.03 1.25±0.11 1.84±0.04 2.11±0.03 2.85±0.01 1.67±0.10 1.98±0.06 2.75±0.01 3.26±0.02 8. Bifidobacterium bifidum + Streptococcus 1.48±0.09 1.88±0.08 2.95±0.02 3.12±0.05 9. Rhizopus oligosporous 1.46±0.06 1.51±0.01 1.53±0.02 1.60±0.04 Mean ± Standard deviation of three independent replicates Control : Soy milk - 1.02 mmol/lit Soy mash 1.42 mmol/lit
3.5 6 hrs 12 hrs 18 hrs 24 hrs 3 2.5 2 1.5 1 0.5 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidm + Streptococcus Rhizopus oligosporous Figure 9. Changes of fructose content in soy milk fermented with mono and mixed culture and tempeh
Table 12. Changes of sucrose content in soy milk fermented with mono and mixed strains and tempeh S.No Fermented with Sucrose content (mmol/l) after fermentation (hrs) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 14.21±0.02 13.28±0.04 12.36±0.01 11.57±0.11 2 Lactobacillus lactis 14.29±0.02 13.96±0.01 12.54±0.08 12.87±0.09 3. Lactobacillus bulgaricus 14.87±0.04 13.15±0.08 13.01±0.06 12.87±0.12 4. Bifidobacterium bifidum 14.57±0.11 13.32±0.08 12.48±0.05 11.98±0.07 5. 6. 7. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus 14.41±0.05 13.25±0.11 12.44±0.04 11.69±0.06 14.30±0.04 13.46±0.13 12.42±0.06 11.98±0.04 13.85±0.02 12.34±0.01 11.53±0.05 10.20±0.03 8. Bifidobacterium bifidum + Streptococcus 13.83±0.03 12.53±0.06 11.31±0.03 10.67±0.05 9. Rhizopus oligosporous 16.98±0.03 16.12±0.04 15.14±0.01 14.21±0.02 Mean ± Standard deviation of three independent replicates Control : Soy milk 15.02 mmol/lit Soy mash 17.02 mmol/lit
18 6 hrs 12 hrs 18 hrs 24 hrs 16 14 12 10 8 6 4 2 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidm + Streptococcus Rhizopus oligosporous Figure 10. Changes of sucrose content in soy milk fermented with mono and mixed strains and tempeh
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. stachyose and raffinose that release mono and disaccharide that leads to the release of monosaccharide and disaccharide. Among the eight fermented soy milk and tempeh, Lactobacillus bulgaricus + Streptococcus fermented soymilk showed high quantity of glucose+ galactose and fructose was noted. Simultaneously the quantity of sucrose also reduced. 4.8 TOTAL PHENOLIC CONTENT When fermentation time increases, total phenolic content was increased (Table 13; Fig. 11). After 24 hrs fermentation, Soy milk fermented with Bifidobacterium bifidum + Streptococcus themophilus showed maximum quantity of total phenolic content (0.46 mg/lit). Compared with 6 hrs of fermentation all the fermented products had drastic level increment of phenolic content at 24 hrs. 4.9 ANTIMICROBIAL ACTIVITY Antimicrobial activity of nine fermented products were tested against nine pathogens such as Escherichia coli, Bacillus cereus, Salmonella typhimurium, Shigella dysenteriae, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Vibrio cholera and Candida albicans. Fermented Soy milk (Bifidobacterium bifidum + Streptococcus ) showed more activity against E.coli, Bacillus cereus, Salmonella typhimurium and Vibrio cholera where as Candida albicans effectively inhibited by Soymilk fermented with L.bulgaricus and St.. Fermented soy milk (L.acidophilus + St.) produce more zone of inhibition against Klebsiella pneumoniae, Tempeh which showed maximum zone of inhibition P. Bhuvaneswari and Dr. S. Ahmed John 39
Table 13. Total phenolic content of soy milk fermented with mono and mixed strains and tempeh S.No Fermented with Total Phenolic Content (mg/ lit) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 0.17 0.21 0.26 0.31 2 Lactobacillus lactis 0.11 0.18 0.20 0.23 3. Lactobacillus bulgaricus 0.14 0.19 0.23 0.28 4. Bifidobacterium bifidum 0.16 0.21 0.28 0.32 5. 6. 7. 8. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum + Streptococcus 0.13 0.20 0.27 0.34 0.12 0.18 0.21 0.26 0.16 0.23 0.39 0.37 0.19 0.25 0.36 0.46 9. Tempeh 0.18 0.23 0.38 0.41 Control : Soy milk 0.09 mmol/lit Soy mash 0.16 mmol/lit
0.5 6 hrs 12 hrs 18 hrs 24 hrs 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidm + Streptococcus Tempeh Figure 11. Total phenolic content of soy milk fermented with mono and mixed strains and tempeh
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. against Shigella dysentriae and Pseudomonas aeruginosa (Table 14; Plate 6 and 7). 4.10 IDENTIFICATION OF BIO ACTIVE COMPONENTS Among nine fermented products mixed fermentation of soy milk and tempeh showed more total phenolic content, antimicrobial activities were noted. Bio active components such as muramic acid,cyclopentene, 1- (2-propenyl)-, 2, 3- butane diol,2-pyrrolindinone, hexadeconoic acid methyl ester (palmitic acid), cyclo hexane, methoxy acetyl chloride etc., were observed from the five fermented products through GC-MS analysis (Plate 8). The components were showed in the Table. 15-19. 4.11 SENSORY EVALUATION Sensory evaluation was done by fifty panellists. The report was noted as mean value. Most of the persons like the appearance, aroma, mouth feel and taste of the soy milk fermented with Bifidobacterium bifidum + Streptococcus (Table 20). 4.12 ANTIOXIDANT ACTIVITY 4.12.1 In vitro Radical scavenging activity of fermented products were analysed at different intervals of incubation by DPPH method. Soy milk fermented with Bifidobacterium bifidum + Streptococcus showed maximum percentage of radical scavenging activity. This sample can be used for in vivo analysis of antioxidant activity (Table 21; Fig. 12). Correlation between total P. Bhuvaneswari and Dr. S. Ahmed John 40
Table 14. Antimicrobial activity of fermented soy milk and tempeh Zone of Inhibition (mm in dm) Pathogens Soy milk fermented with S1 S2 S3 S4 S(1+5) S(2+5) S(3+5) S(4+5) Tempeh Escherichia coli 12 13 15 14 17 15 14 28 24 Bacillus cereus 12 16 13 15 13 18 17 35 26 Salmonella typhimurium Shigella dysentriae Staphylococcus aureus Klepsiella pneumonia Pseudomonas aeruginosa 14 14 18 21 22 24 21 28 19 13 13 20 18 21 18 24 28 29 18 20 22 24 20 23 22 27 26 14 18 17 17 25 20 17 17 18 22 23 13 15 14 17 18 16 25 Vibrio cholera 16 16 24 18 22 20 14 24 17 Candida albicans 17 18 16 15 18 17 21 16 18
Plate 6 Antimicrobial activity of fermented soy milk against pathogens a b c d a & b. Escherichia coli a. 1. Lactobacillus acidophilus, 2. Lactobacillus lacus 3. Lactobacillus bulgaricus, 4. Lipidobacterium lipidum 5. 1 + 5 (Streptococcus ) b. 1. 2 + 5, 2. 3 + 5, 4 + 5 c & d. Candida albicans c. 1. Lactobacillus acidophilus, 2. Lactobacillus lacus 3. Lactobacillus bulgaricus, 4. Lipidobacterium lipidum 5. 1 + 5 (Streptococcus ) d. 1. 2 + 5, 2. 3 + 5, 4 + 5
Plate 7 Antimicrobial of tempeh milk against pathogens a b c d e f g h i a. Escherichia coli b. Bacillus cereus c. Salmonella typhimiurium d. Staphylococcus aureus e. Klebsiella pneumonia f. Vibrio cholera g. Candida albicans h. Pseudomonas aeruginosa i. Shigella dysenriae
Table 15. Bioactive components in soy milk fermented with Lactobacillus acidophilus and Streptococcus S. No RT Area% 1. 20.537 0.92 Identified component 2- Ethoxyethyl. Diethylamine Biological activity Anesthetic, anticholesteremic, antagonistic 2. 2.222 0.43 2,3, Butanol Antimalarial, antibacterial, oxytoxic Table 16. Bioactive components in soy milk fermented with Lactobacillus lactis and Streptococcus S. No RT Area% Identified component Biological activity 1. 2.860 6.85 Muramic acid Antibacterial, immunostimulatory 2. 2.899 0.56 Methoxy acetyl chloride Antibacterial, anticandidal 3. 3.966 5.48 4. 4.427 6.57 Benzene acetaldehyde 2- Pyrrolindinone 2- methoxy- 4- vinyl phenol Antioxidant, antifungal, anti tumour Anti inflammatory, anticonvulsant 5. 7.127 4.52 Cyclohexane, pentyl Antifungal, antimicrobial 6. 11.388 3.57 7. 18.098 4.86 8. 20.409 2.11 9. 21.279 1.82 Hexadecanoic acid, Methyl ester N- hexa decanoic acid 5,10-Dioxatricyclo decane Cyclopentene, 1- (2-propenyl)- Anti proliferative, antimicrobial (antibacterial, antiviral, antimalarial) Antimicrobial Anti tuberculosis, antiviral, analgesic, anti inflammatory, antimalarial Antagonistic, antioxidant, anticancer, analgesic, anti inflammatory, antipyretic.
Table 17. Bioactive components in soy milk fermented with Lactobacillus bulgaricus and Streptococcus S. No RT Area% 1. 2.135 1.63 Identified component Acetic acid, methoxy- Biological activity Analgesic, anti inflammatory 2 2.309 2.82 2,3-Butanol Antimalarial, oxytoxic 3 2.967 70.66 Methyl tartonic acid Mild anesthetic, antibacterial 4 3.170 3.43 2, Pentanol Antifungal, anti candidal 5 4.639 1.82 Cyclopropyl carbinol Antidiabetic, hypolipidemic 6 4.765 2.13 Maltol Antioxidant, anticonvulsant Table 18. Bioactive components in soy milk fermented with Bifidobacterium bifidum and Streptococcus S. No RT Area% 1. 2.203 1.83 Identified component Hydrazine, 1,2 dimethyl - 2. 2.348 36.72 2,3 Butane diol 3. 2.725 4.31 4 2.938 1.62 5 3.934 4.38 Ethanol 2, (2- ethylenyloxy ethoxy)- Butane 1,4, methoxy- Benzene acetaldehyde 6 4.427 3.80 2, Pyrolindinone 7 4.649 2.55 Urea trimethyl- 8 7.115 9.03 9 11.381 1.80 10. 17.228 3.75 2, methoxy 4- vinyl phenol Azabicyclo (3,1.0) hexane Hexadecanoic acid, methyl ester- Biological activity Antibacterial, anticarcinogenic Anti tumour, mild analgesic Antidiabetic, antibacterial Antimicrobial, sedative activity Antitumour, antioxidant, antifungal Anti inflammatory, anticonvulsant Antibacterial, antitubercular, antifungal Antioxidant, antitumour Antibacterial, anti malarial Antimalarial, antibacterial, antiviral, anti helminthic,
Table 19. Bioactive components in Tempeh S. No RT Area% Identified component Biological activity 1. 2.155 30.73 2,3-Butan diol Antimalarial, Oxytoxic 3. 5.377 0.56 2,3- Butanol Antimalarial, Oxytoxic 4. 12.780 1.80 3, cyclohexene- 1- ethanol beta, 4- dimethyl Antagonistic activity against E.coli 5. 17.586 0.82 Ethoxy acetylene Antagonistic activity 6. 20.893 0.72 Aminoguanidine Anti proliferative Table 20. Sensory evaluations of fermented soy milk and tempeh Characteristics Soy milk fermented with S1 S2 S3 S4 S(1+5) S(2+5) S(3+5) S(4+5) Tempeh Appearance 3.1 2.8 4.1 4.0 3.3 3.1 4.2 4.3 3.9 Aroma 3.5 1.9 3.2 3.6 3.7 2.8 3.9 4.2 2.9 Colour 3.9 3.1 3.5 3.6 3.9 3.4 3.8 3.8 3.3 Mouth feel 2.8 3.2 3.4 3.7 3.1 3.3 3.9 3.9 3.1 Taste 3.2 3.0 3.1 3.8 3.4 3.2 4.1 4.3 3.1 0 1 Very much dislike, 1 2 Dislike, 2 3 Dislike or like, 3 4 Like, 4-5 Very much Like (mean of 50 individual values) S1- Lactobacillus acidophilus S2 - Lactobacillus lactis S3 - Lactobacillus bulgaricus S4 - Bifidobacterium bifidum S5 Streptococcus
Plate 8 Mass spectrum a. 20Ethoxyethyl diethylamine 2,3-butanediol c. Muramic acid
d. Methoxyacetyl chloride e. Benzeneacetaldehyde f. 2-Pyrrolidinone g. 2-Methoxy-4-vinylphenol
h. Cyclohexane phenyl i. Hexadecanoic acid, methyl ester j. n-hexadecanoic acid k. 5,10-Dioxatricyclo (7.1.0.0(4,6))decane
l. Cyclopentene,1(2-propenyl) m. Hydrazine, 1,2-dimethyl
n. Ethanol, 2-[2-(ethenyloxy)ethoxy]- o. Butane,1-4-dimethoxy- p. 2-pyrrolidinone
q. Urea, trimethyl- r. 2-Methoxy-4-vinylphenol s. 1-Azabicyclo(3.1.0) hexane t. Acetic acid, methoxy-
u. Methyltartronic acid v. 2-Pentanol w. Cyclopropyl carbinol x. Maltol
y. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl z. 3-cyclohexene-1-ethanol, beta,4-dimethyl-
Table 21. Anti oxidant activity (%) activity of the fermented soy milk and tempeh S. No Fermented with Radical scavenging activity (%) 6 hrs 12 hrs 18 hrs 24 hrs 1. Lactobacillus acidophilus 69.74 71.21 73.01 76.22 2. Lactobacillus lactis 58.13 59.06 61.16 71.88 3. Lactobacillus bulgaricus 61.81 62.52 63.24 68.97 4. Bifidobacterium bifidum 60.85 69.42 71.36 73.74 5. 6. 7. 8. 9. Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum +Streptococcus Rhizopus oligosporous (Tempeh) 71.21 70.45 76.84 83.07 68.78 70.21 70.45 71.69 67.11 67.35 78.78 82.60 74.92 80.02 86.21 89.31 76.23 79.62 82.26 88.04 Control : Soy milk 56.06 % Soy mash 64.67 Table 21a. Correlation between Total phenolic content and antioxidant potentiality TPC (mg/lit) Antioxidant (%) Correlations TPC (mg/lit) Antioxidant (%) Pearson Correlation 1.928 ** Sig. (2-tailed).000 N 9 9 Pearson Correlation.928 ** 1 Sig. (2-tailed).000 N 9 9 **. Correlation is significant at the 0.01 level (2-tailed).
90 6 hrs 12 hrs 18 hrs 24 hrs 80 70 60 50 40 30 20 10 0 Lactobacillus acidophilus Lactobacillus lactis Lactobacillus bulgaricus Bifidobacterium bifidum Lactobacillus acidophilus+ Streptococcus Lactobacillus lactis + Streptococcus Lactobacillus bulgaricus + Streptococcus Bifidobacterium bifidum +Streptococcus Tempeh Figure 12. Anti oxidant activity (%) activity of the fermented soy milk and tempeh
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. phenolic content and radicals scavenging activity showed (p < 0.01) significance (Table 21a). 4.12.2 In vivo Antioxidant activities of fermented soy milk on wister albino rats (in vivo model) The enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GSH-Px) activities and the values of TBARS in liver and kidney (Table 22 and 23; Fig. 13; Plate 9). Compared with the normal rats (negative control), the SOD activity and the CAT activity of the experimental rats treated with 0.0025% Vitamin E (positive control) increased significantly in liver (p < 0.05), and experimental rats treated with Fermented soy milk also produced significant increase (p < 0.05). The SOD activity of Fermented soy milk treated rats in liver was significantly higher than the positive control (p < 0.05). But, the GSH-Px activities (in positive, fermented soy milk treated rats) of liver increased compared with negative control group, but there was no significant difference (p > 0.05). The significant increase of the SOD activities and the GSH-Px activities in kidney was observed in the positive, Fermented soy milk treated rats (p < 0.05), compared with the negative control. The effects in the SOD activities and the GSH-Px activities of Fermented soy milk group were almost equivalent to the positive control group in kidney. The CAT activities (in positive and Fermented soy milk treated rats) of kidney increased compared with the negative control group, but there was no significant difference (p > 0.05). The values of TBARS in the positive control group, Fermented soy milk treated group all P. Bhuvaneswari and Dr. S. Ahmed John 41
Table 22. The SOD, CAT, GSH-Px activities and the values of TBARS in liver of cholesterol Fed rats supplemented with Vit-E or fermented soy milk (Bifidobacterium bifidum + Streptococcus ) Groups SOD (nm/mg protein) CAT (U/g protein) GSH-Px (U/g protein) TBARS (nm/mg protein) Liver Control rats (negative control) Experimental rats treated with fermented soy milk Experimental rats treated with 0.0025% Vitamin E (positive control) 80. 14 ± 3.56 a 320.03 ± 18.46 b 168.18 ± 14.12 a 6.93 ± 0.41 a 137.87 ± 6.40 a 348.23 ± 9.32 a 198.45 ± 7.85 a 4.29 ± 0.33 b 110.33 ± 15.86 b 323.30 ± 6.53 a 197.71 ± 12.79 a 4.63 ± 0.31 b a Value represents the mean standard deviation of duplicate assays in six animals in each group b Value in column with different superscript were significantly different (p< 0.05)
Table 23. The SOD, CAT, GSH-Px activities and the values of TBARS in liver of cholesterol Fed rats supplemented with Vit-E or fermented soy milk (Bifidobacterium bifidum + Streptococcus ) Groups SOD (nm/mg protein) CAT (U/g protein) GSH-Px (U/g protein) TBARS (nm/mg protein) Kidney Control rats (negative control) Experimental rats treated with fermented soy milk Experimental rats treated with 0.0025% Vitamin E (positive control) 66.68 ± 5.73 b 280.23 ± 32.18 a 141.23 ± 37.03 b 5.31 ± 1.38 a 98.72 ± 20.02 a 281.04 ± 51.58 a 196.21 ± 24.71 a 4.25± 1.09 b 105.28 ± 21.28 a 316.32 ± 23.60 a 181.42 ± 29.94 a 4.85 ± 0.85 b a Value represents the mean standard deviation of duplicate assays in six animals in each group b Value in column with different superscript were significantly different (p< 0.05)
350 Control rats (negative control) 300 Experimental rats treated with fermented soy milk Experimental rats treated with 0.0025% Vitamin E (positive control) 250 200 150 100 50 0 SOD (nm/mg protein) CAT (U/g protein) GSH-Px (U/g protein) TBARS (nm/mg protein) SOD (nm/mg protein) CAT (U/g protein) GSH-Px (U/g protein) TBARS (nm/mg protein) Liver Kidney Figure 13. The SOD, CAT, GSH-Px activities and the values of TBARS in liver and kidney of cholesterol Fed rats supplemented with Vit-E or fermented soy milk
Plate 9 In vivo analysis of anti-oxidant activity a b a. animal model; b. Weighing animal model Section of liver negative control Section of liver soy milk treated Section of liver positive control Vitamin E
Chapter 4 Investigation of the Antimicrobial and Antioxidant activities. significantly decreased in liver and kidney compared with the negative control (p < 0.05). 4.12.3 Histopahtological study Histopathological effect on the liver cells was examined in each group of rats treated with negative control, positive control and treated with fermented soy milk. The section from the liver of negative control group illustrated the fatty degeneration of hepatocytes was remarkable. Incontrary in positive control group (f) fatty degeneration of hepatocytes was less obvious. But in the case of fermented soy milk (Bifidobacterium bifidum + Streptococcus ) treated group showed, the degree of fatty degeneration of hepatocytes are milder. Hence microscopic images suggested that the soy milk might play a role in preventing the progression of the lipidosis of hepatocyts. But in the case of Kidney, SOD and GSH Px increased significantly. CAT activities exhibited no change in the kidney tissue. These results showed that fermented soy milk might have different activities on different antioxidant enzymes in the liver and kidney. P. Bhuvaneswari and Dr. S. Ahmed John 42