Gram Staining
Beneficial microorganisms were isolated from fermented soy cake and shrimp paste. Gram staining was conducted immediately after cultivating the isolated microorganisms from the first culture media. Gram staining is one of the most important microbiological staining procedures. Staining the slide with crystal violet dye was the first step for this analysis. To prevent the color from being easily removed, the subsequent process, often known as fixing the dye, requires the use of iodine to generate a crystal violet-iodine complex. After that, the dye was removed with a decolorizer such as ethanol.
Nineteen samples were labeled as follows: Sample of bacteria = SB1, SB2, SB3, SB4, SB5, SB6, SB7, SB8, SB9, SB10; Sample of yeast = SY1, SY2, SY3, SY4, SY5, SY6, SY7, SY8, SY9. Tables 1 summarizes the Gram staining results obtained from this study for bacteria, while Fig. 1depicts the morphology images of bacteria. In general, Gram positive organisms are organisms that preserve their primary color and appear purple-brown under the microscope. Meanwhile, Gram negative organisms are those that do not take up primary stain and appear red under the microscope.
Table 1
Gram staining results of bacteria samples
Sample
|
Color
|
Shape
|
Gram Strain
|
Culture Characteristic
|
SB1
|
Brownish
|
Coccus
|
Positive
|
Smooth, round, raised, matte, crateriform
|
SB2
|
Creamy white
|
Bacillus
|
Positive
|
Wavy, flat, smooth, moist, irregular
|
SB3
|
Off white/ Milky white
|
Oval shaped/ cocci
|
Positive
|
Matte, filamentous
|
SB4
|
White
|
Oval shaped/ cocci
|
Positive
|
Round, convex, dry, not smooth
|
SB5
|
Milky white
|
Coccus
|
Positive
|
Round, punctiform, smooth, shiny
|
SB6
|
Creamy white
|
Bacillus
|
Positive
|
Wavy, convex, dry, filamentous
|
SB7
|
Reddish
|
Coccus
|
Positive
|
Pulvinate, circular, smooth, shiny
|
SB8
|
Yellowish white
|
Streptobacilli
|
Positive
|
Round, matte, lobate
|
SB9
|
Creamy white
|
Coccus
|
Positive
|
Smooth, slimy, moist, irregular
|
SB10
|
Matte milky white
|
Coccus
|
Positive
|
Irregular, curled, matte
|
From the results obtained, all the bacteria samples showed Gram positivity in Gram staining analysis and appeared as bacillus, cocci and streptobacillus. Conversely, Table 2 shows the results of Gram staining for yeast samples, and Fig. 2 depicts the images of yeast samples. Intact yeast cells are Gram positive, while ruptured or disrupted cells are Gram negative [15]. It is difficult to determine Gram positive or Gram negative; for yeast cell, the color depends on its cell wall. Yeast cells appeared oval and round in shape, with and without hyphae.
Table 2
Gram staining results of yeast samples
Sample
|
Color
|
Type
|
Colour After Gram Strain
|
Culture Characteristic
|
SY1
|
White
|
Oval shaped cell with hyphae
|
Purple
|
Irregular, filamentous, flat, wrinkled
|
SY2
|
White
|
Round/ Oval yeast cells
|
Purple
|
Circular, pulvinate, smooth, glistening
|
SY3
|
Creamy White
|
Round yeast cell with spore
|
Pinkish/ Reddish
|
Circular, moist, smooth, raised
|
SY4
|
Off-white
|
Oval yeast cell with hyphae
|
Red
|
Round, curled, raised, shiny
|
SY5
|
Yellowish white
|
Oval shaped yeast cell
|
Purple
|
Dry, flat, lobate, rhizoid
|
SY6
|
Creamy white
|
Separated hyphae cell
|
Pinkish purple
|
Moist, flat, shiny, irregular
|
SY7
|
Creamy white
|
Round/oval with hyphae
|
Dark Purple
|
Moist, flat, curled
|
SY8
|
White
|
Oval with Hyphae
|
Light Purple
|
Irregular, flat, matte
|
SY9
|
Yellow
|
Oval
|
Red
|
Irregular, moist, flat
|
In Vitro Test (Growth Kinetics, Cell Concentration and Survivability)
Among 19 microorganisms, several of them were unable to grow in chickens which had different GIT pH balances. The optimum pH range required for microbial growth could be from pH 6 to pH 8 [16]. At different pH levels, yeast and bacteria have different growth curves. Although bacteria could grow in a neutral pH, yeast requires a slightly acidic condition to grow. The best pH range for bacteria growth is between 6.5 to 7, and pH 4.0–6.0 is ideal for yeast growth [17]. The growth curves were generated by combining the OD and time for each microbe as exhibited in Fig. 3. On the other hand, Fig. 4 presents bacteria and yeast maximum cell concentrations.
Tables 3 and 4 summarize the results of maximum biomass, specific growth rate, doubling time, and survivability percentage of bacteria and yeast samples. Between SB3 and SB5, higher survivability was demonstrated by SB5 ranging from 12–98% within 24 hours of anaerobic fermentation. This indicated that SB5 has higher chance of surviving in acidic conditions of chicken GIT compared to SB3. Moreover, SB5 has faster doubling time in all pH values compared to SB3. Although the doubling time for SB10 was faster than that of SB5, SB10 had lower survivability of not more than 68% and low maximum cell concentration produced. As observed in Fig. 1, SB8 and SB7 showed a decreasing growth rate as the pH increased.
Table 3
Specific growth rate (µ), maximum biomass, doubling time (Td) and survivability of bacteria strain fermented at different pH values
Sample Code
|
pH
|
µ
(hr− 1)
|
Maximum Biomass
(g/ml)
|
Td
(hr)
|
Survivability (%)
|
SB1
|
2.5
|
0.0191
|
0.0002
|
36.29
|
3
|
3.5
|
0.0307
|
0.0002
|
22.58
|
5
|
5.0
|
0.0595
|
0.0007
|
11.65
|
16
|
6.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.5
|
0.0782
|
0.0029
|
8.864
|
41
|
8.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
SB2
|
2.5
|
0.1099
|
0.0005
|
6.307
|
1
|
3.5
|
0.0813
|
0.0023
|
8.526
|
12
|
5.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
6.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.0
|
0.4331
|
0.1342
|
1.600
|
88
|
7.5
|
0.3716
|
0.1739
|
1.865
|
89
|
8.0
|
0.3256
|
0.1830
|
2.129
|
87
|
SB3
|
2.5
|
0.0213
|
0.0034
|
32.54
|
10
|
3.5
|
0.0282
|
0.0033
|
24.58
|
12
|
5.0
|
0.0904
|
0.0429
|
7.668
|
75
|
6.5
|
0.1243
|
0.0627
|
5.576
|
79
|
7.0
|
0.1876
|
0.1416
|
3.695
|
88
|
7.5
|
0.2350
|
0.1201
|
2.950
|
87
|
8.0
|
0.3408
|
0.1233
|
2.034
|
84
|
SB4
|
2.5
|
0.0589
|
0.0003
|
11.77
|
1
|
3.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
5.0
|
0.0601
|
0.0020
|
11.53
|
8
|
6.5
|
0.0642
|
0.0037
|
10.80
|
12
|
7.0
|
0.0649
|
0.0029
|
10.68
|
9
|
7.5
|
0.0306
|
0.0040
|
22.65
|
8
|
8.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
SB5
|
2.5
|
0.0584
|
0.0006
|
11.87
|
12
|
3.5
|
0.0249
|
0.0016
|
27.84
|
14
|
5.0
|
0.0434
|
0.0008
|
15.97
|
19
|
6.5
|
0.2024
|
0.2239
|
3.425
|
98
|
7.0
|
0.2637
|
0.2714
|
2.629
|
97
|
7.5
|
0.2565
|
0.2787
|
2.702
|
98
|
8.0
|
0.2162
|
0.3107
|
3.206
|
95
|
SB6
|
2.5
|
0.0403
|
0.0007
|
17.20
|
4
|
3.5
|
0.0711
|
0.0018
|
9.749
|
3
|
5.0
|
0.0796
|
0.0052
|
8.708
|
25
|
6.5
|
0.1224
|
0.0152
|
5.663
|
48
|
7.0
|
0.0829
|
0.0077
|
8.361
|
32
|
7.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
8.0
|
0.0749
|
0.0094
|
9.254
|
35
|
SB7
|
2.5
|
0.0779
|
0.0011
|
8.898
|
9
|
3.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
5.0
|
0.0431
|
0.0046
|
16.08
|
23
|
6.5
|
0.0577
|
0.0272
|
12.01
|
50
|
7.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.5
|
0.0671
|
0.0329
|
10.33
|
55
|
8.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
SB8
|
2.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
3.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
5.0
|
0.0522
|
0.0100
|
13.28
|
18
|
6.5
|
0.0527
|
0.0050
|
13.15
|
13
|
7.0
|
0.0468
|
0.0173
|
14.81
|
23
|
7.5
|
0.0312
|
0.0131
|
22.22
|
12
|
8.0
|
0.0121
|
0.0123
|
57.28
|
13
|
SB9
|
2.5
|
0.0225
|
0.0018
|
30.81
|
3
|
3.5
|
0.0458
|
0.0007
|
15.13
|
2
|
5.0
|
0.0606
|
0.0043
|
11.44
|
2
|
6.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.0
|
0.0277
|
0.0015
|
25.02
|
1
|
7.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
8.0
|
0.0166
|
0.0069
|
41.76
|
13
|
SB10
|
2.5
|
0.0108
|
0.0003
|
64.18
|
2
|
3.5
|
0.0910
|
0.0019
|
7.617
|
18
|
5.0
|
0.0422
|
0.0022
|
16.43
|
20
|
6.5
|
0.6267
|
0.0181
|
1.106
|
66
|
7.0
|
0.7638
|
0.0200
|
0.907
|
68
|
7.5
|
0.6941
|
0.0199
|
0.999
|
66
|
8.0
|
0.7040
|
0.0216
|
0.985
|
66
|
*NG = No Growth |
Based on the results obtained, SB8 and SB7 were still able to grow at high pH but not as fast as other strains, and they also preferred low pH over high pH. Furthermore, they also showed low survivability percentage of less than 55% and long doubling time of at least 7 hr. Subsequently, SB2 performed well at pH more than 7 with survivability of at least 88% (Table 3) and high growth rate between 0.5000 to 0.3000 hr− 1 (Fig. 3), which were higher than those of SB3 and SB5. Its maximum cell concentration produced was also high at the same pH. However, since it has no survivability in pH 5 and 6.5, thus it has no potential to be used as probiotics as it would disappear before reaching the end of the chicken GIT. Their maximum cell concentrations are demonstrated in Figs. 5 and 6.
Table 4
Specific growth rate (µ), maximum biomass, doubling time (Td) and survivability of yeast strains fermented at different pH
Sample Code
|
pH
|
µ
(hr− 1)
|
Maximum Biomass
(g/ml)
|
Td
(hr)
|
Survivability (%)
|
SY1
|
2.5
|
0.0228
|
0.0012
|
30.40
|
12
|
3.5
|
0.1091
|
0.0091
|
6.353
|
57
|
5.0
|
0.0428
|
0.0016
|
16.20
|
17
|
6.5
|
0.1798
|
0.0079
|
3.855
|
55
|
7.0
|
0.0633
|
0.0006
|
10.95
|
8
|
7.5
|
0.1474
|
0.0079
|
4.702
|
53
|
8.0
|
0.1259
|
0.0056
|
5.506
|
43
|
SY2
|
2.5
|
0.0321
|
0.0021
|
21.59
|
21
|
3.5
|
0.0400
|
0.0012
|
17.33
|
16
|
5.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
6.5
|
0.0389
|
0.0044
|
17.82
|
42
|
7.0
|
0.0423
|
0.0056
|
16.39
|
50
|
7.5
|
0.0727
|
0.0068
|
9.534
|
53
|
8.0
|
0.0471
|
0.0038
|
14.72
|
38
|
SY3
|
2.5
|
0.0381
|
0.0024
|
18.19
|
21
|
3.5
|
0.0176
|
0.0044
|
39.38
|
37
|
5.0
|
0.0307
|
0.0061
|
22.58
|
48
|
6.5
|
0.0477
|
0.0067
|
14.53
|
49
|
7.0
|
0.0374
|
0.0063
|
18.53
|
50
|
7.5
|
0.0337
|
0.0072
|
20.57
|
50
|
8.0
|
0.0253
|
0.0054
|
27.40
|
44
|
SY4
|
2.5
|
0.0377
|
0.0003
|
18.39
|
2
|
3.5
|
0.0257
|
0.0008
|
26.97
|
11
|
5.0
|
0.1234
|
0.0075
|
5.617
|
57
|
6.5
|
0.1096
|
0.0067
|
6.324
|
55
|
7.0
|
0.1044
|
0.0010
|
6.639
|
14
|
7.5
|
0.1144
|
0.0062
|
6.059
|
52
|
8.0
|
0.0946
|
0.0073
|
7.327
|
55
|
SY5
|
2.5
|
0.0719
|
0.0010
|
9.640
|
11
|
3.5
|
0.0611
|
0.0017
|
11.34
|
18
|
5.0
|
0.0505
|
0.0038
|
13.73
|
34
|
6.5
|
0.0368
|
0.0014
|
18.84
|
15
|
7.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.5
|
0.0452
|
0.0038
|
15.34
|
34
|
8.0
|
0.1051
|
0.0029
|
6.595
|
27
|
SY6
|
2.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
3.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
5.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
6.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.0
|
0.5304
|
0.0116
|
1.307
|
58
|
7.5
|
0.1438
|
0.0006
|
4.820
|
2
|
8.0
|
0.4377
|
0.0036
|
1.584
|
23
|
SY7
|
2.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
3.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
5.0
|
0.0289
|
0.0021
|
23.98
|
16
|
6.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.5
|
0.0466
|
0.0034
|
14.87
|
26
|
8.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
SY8
|
2.5
|
0.1020
|
0.3665
|
6.796
|
89
|
3.5
|
0.1295
|
0.4813
|
5.352
|
93
|
5.0
|
0.1446
|
0.4702
|
4.794
|
91
|
6.5
|
0.1201
|
0.4575
|
5.771
|
86
|
7.0
|
0.2770
|
0.3554
|
2.502
|
93
|
7.5
|
0.1046
|
0.3648
|
6.627
|
80
|
8.0
|
0.1131
|
0.4124
|
6.129
|
86
|
SY9
|
2.5
|
0.0000
|
0.0000
|
0.000
|
NG
|
3.5
|
0.4694
|
0.0458
|
1.477
|
61
|
5.0
|
0.1289
|
0.0021
|
5.377
|
7
|
6.5
|
0.1614
|
0.0030
|
4.295
|
10
|
7.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
7.5
|
0.1438
|
0.0039
|
4.820
|
9
|
8.0
|
0.0000
|
0.0000
|
0.000
|
NG
|
*NG = No Growth |
Initial yeast cell concentration was not standardized as this study evaluated the growth rate of the isolated yeasts to investigate isolated yeasts which could survive acidic conditions mimicking chicken GIT. Most isolated yeast strains showed the ability to grow well in acidic condition during anaerobic fermentation for 24 hours at 37℃, except at pH 7 which had low specific growth rate range of about 0.1044 hr− 1 to 0.0159 hr− 1 (Fig. 5). In contrast to SY6 and SY8, they grew well with high specific growth rates of 0.5304 hr− 1 and 0.2770 hr− 1, respectively, at pH 7. Thus, it can be inferred that pH 7 was the optimum pH for SY6 fermentation as it has the highest survivability (58%), lowest doubling time (1.31 hr) and maximum cell concentration produced (0.0116 g/ml) in less than 24 hr among other pH values. Nevertheless, SY6 was not a promising candidate to be used as probiotics as it could not survive in other pH values without survivability percentage (Table 4). The probiotic candidates should be able to survive in acidic condition as it will pass through the acidity of the chicken GIT from the mouth to the cloaca.
Meanwhile, SY1, SY3, SY4 and SY8 showed high survivability in all pH values of anaerobic fermentation for 24 hours at 37℃ (Table 4). Therefore, these strains have the potential to be used as probiotics as they only require approximately less than 3.5 hours to pass through the GIT [18]. Among these four strains, SY8 showed the most promising probiotic potential with the highest specific growth rate (0.1020 hr− 1), maximum cell concentration (0.3554 g/ml), doubling time (2.50 hr) and survivability (80%) (Table 4). Although SY8 did not exhibit the highest specific growth rate as shown in Fig. 2, it was able to survive in all pH values. Some of the strains showed low survivability at certain pH, making them unsuitable to be used as probiotics in broilers, even though high specific growth rates were demonstrated at other pH values.
On another note, Hsiung also reported that yeast strains that are able to tolerate high pH conditions have the potential to be used as probiotics in the chicken GIT [19]. They also claimed that yeast had beneficial effect on the chicken GIT which could not be achieved by bacteria. Apart from the crucial probiotic survivability in the chicken GIT, high growth rate of probiotics also plays an important role in ensuring that broilers would gain maximum benefit from the probiotic in limited time. This is due to the fact that probiotics are only available in the GIT in less than 3.5 hours depending on the chicken metabolism [18].
Identification of Toxic Compounds Produced by Yeast and Bacteria During In Vitro Analysis
Pre-selection of isolated yeast and bacteria strains based on their physiological properties was then further analysed to identify whether the strains produced any toxic compounds during anaerobic fermentation that may harm broilers. Based on the results, all isolated strains produced at least one toxic compound during anaerobic fermentation under conditions mimicking the chicken GIT. The toxic compounds analysed by GC-MS are shown in Tables 5 and 6. From the results obtained, SB10 produced only one toxic compound, while the highest toxic compounds were produced by SB3 (five toxic compounds) as shown in Table 5. On the other hand, SB5 produced three toxic compounds during anaerobic fermentation at 37℃ (Table 5). It also survived well at different pH values but it did not exhibit probiotic potential for chicken GIT as it produced several toxic compounds. In contrast, SY8 and SY4 produced eight and nine toxic compounds, respectively, whereas SY2 and SY3 each produced five toxic compounds and SY1 produced seven toxic compounds (Table 6). As discussed earlier, SY8 showed more promising probiotic potential compared to the other three strains. However, the toxic compounds produced by SY8 may reduce its potential for use as probiotic for the chicken GIT as it would affect the health of broilers.
Table 5 Identification of toxic compounds produced by bacteria in a condition mimicking the gastrointestinal tract (GIT) of chicken
Table 6 Identification of toxic compounds produced by yeast in a condition mimicking the gastrointestinal tract (GIT) of chicken
From the toxicity result, dioxin was one of the toxic compounds produced by SY4. Dioxin is a food contaminant commonly found in organic pesticides. This compound tends to accumulate in chicken meat which could affect human health. In a previous study conducted by Gerber et al [20], it was reported that toxic compound, such as dioxin, may has adverse effects on chicken health, namely high blood pressure, glucose intolerance, disturbance in mental and motor development, cancer, diabetes and endocrine disruption [20].
Identification of Microbes (rRNA analysis):
According to rRNA identification data from CCB, the bacteria sample has 100% similarity to Staphylococcus hominis and the yeast sample has 99.52% similarity to Cutaneotrichosporon arboriforme.
The process of rRNA identification analysis is depicted in Fig. 7. Figures 8 and 9 illustrate the phylogenetic tree resulting from distance analysis of bacteria and yeast rRNA gene sequences, respectively.
Animal growth rate:
From batch B chicken and Y chicken, B chicken and Y chicken grew to almost the same size and weight, the control batch, grew less and also gained less weight. In 36 days of monitoring, all chickens reached 2 kilograms of weight. 2 kilograms of weight is considered to be their ideal weight. Y chicken showed the highest growth, and B chicken showed the 2nd highest growth (Fig. 10)