3.1 Molecular identification and strain characterization of E. faecium
Once sequencing of the 16S-rRNA PCR-amplified fragment (1500 bp) was completed and its conformity with the sequences deposited in GenBank has been assessed, the strain isolated from the ewe colostrum was identified with 99 to 100% homology as E. faecium (Accession number: MK367697). In accordance with FAO/WHO guidelines, molecular identification of probiotic strains by 16S-rRNA sequencing (FAO, WHO, 2002) the threshold value for bacterial taxonomic studies was approx. 97% and based on the results in this study where the 16S-rRNA sequencing with 99-100% similarity, which was performed as an accurate and validated method according to Deng et al. (2008) 23.
Probiotic bacteria require specific characteristics to be efficient in improving host health and according to scientific agreements, the evaluation and assessment of the probiotic properties of bacteria should be completed according to standard in vitro experiments, which include the susceptibility to antibiotics, anti-pathogenic activity and resistance to acid and bile in the digestive tract 24. In this study, the E. faecium showed appropriate antibiotic susceptibility and acceptable anti-pathogenic activity (Table 1), and was sensitive to all of the seven antibiotics assessed and inhibited growth of all five pathogens assessed. However, the tolerance to high bile salt (53%) concentrations and low pH (44%) was poor. Therefore, to compensate for this weakness, the viability of microencapsulation, using differing biopolymer-prebiotic formulations, was evaluated using in-vitro assessment in this study.
Table 1: Antibiotic susceptibility of isolated E. faecium against the high consumption antibiotics performed by disk diffusion assay and antimicrobial activity of isolate against the pathogenic bacteria
Strain
|
Antibiotic susceptibility (Clear zone (mm))
|
Oxytetracycline
|
Tetracycline
|
Amoxicillin
|
Ampicillin
|
Erythromycin
|
Sulphonamides
|
Oxolinic acid
|
E. faecium
|
20
|
21
|
23
|
18
|
25
|
16
|
17
|
Strain
|
Antimicrobial activity (Clear zone (mm))
|
Streptococcus agalactiae
|
Salmonella enterica
|
Streptococcus iniae
|
Yersinia ruckeri
|
Clostridium botulinum
|
E. faecium
|
19
|
13
|
18
|
13
|
14
|
3.2 Water activity, moisture content, and encapsulation efficiency (EE) of beads
In this study the ALG-PG (F1) and control (ALG) formulations showed the greatest water activity value (P<0.05) compared with the other formulated blends. On the other hand, the water activity values for the ALG-PG + Fk (F5 to F7) formulations were lower than other formulations (Table 2). The moisture content of all the beads prepared in this study was below 3.29% (w/w) and there were no differences in the moisture content of the seven gels and control (ALG) formulations in this study, which was in keeping with other researchers who found similarly low water activity and moisture content in the beads that were created during microencapsulation. This low water activity and residual water content has been shown to improve the stability and storage capacity of probiotic-containing beads 18.
Table 2: Compositions, size, water activity, moisture content (%), and encapsulation efficiency (%) of microencapsulated E. faecium with various gel and prebiotic concentrations. Alginate-encapsulated cells (2% (w/v)) were used as control. F1-F7: Various gel formulations. Values shown are means ± standard deviations (n = 3)
Formulation
|
Alginate
(% w/v)
|
PG
(% w/v)
|
Prebiotic
(FOS)
(% w/v)
|
Prebiotic
(Fenugreek)
(% w/v)
|
Diameter (µm)
(n = 50)
|
Water Activity
|
Moisture
Content
(%)
|
Encapsulation Efficiency
(%)
|
ALG
|
2
|
_
|
_
|
_
|
790 - 980
|
0.55 ± 0.002a*
|
3.22 ± 0.04a*
|
99.1a*
|
F1
|
1.5
|
0.5
|
_
|
_
|
320 - 350
|
0.48 ± 0.003b
|
3.25 ± 0.06a
|
98.8a
|
F2
|
1.5
|
0.5
|
1
|
_
|
360 - 370
|
0.37 ± 0.001c
|
3.12 ± 0.03a
|
99.4a
|
F3
|
1.5
|
0.5
|
1.5
|
_
|
360 - 380
|
0.36 ± 0.004c
|
3.29 ± 0.06a
|
99.6a
|
F4
|
1.5
|
0.5
|
2
|
_
|
390 - 410
|
0.34 ± 0.005c
|
2.98 ± 0.02a
|
99.0a
|
F5
|
1.5
|
0.5
|
_
|
1
|
540 - 580
|
0.27 ± 0.006d
|
3.18 ± 0.07a
|
98.6a
|
F6
|
1.5
|
0.5
|
_
|
1.5
|
570 - 600
|
0.25 ± 0.007d
|
2.92 ± 0.05a
|
99.3a
|
F7
|
1.5
|
0.5
|
_
|
2
|
640 - 670
|
0.24 ± 0.002d
|
3.08 ± 0.08a
|
99.5a
|
*Values followed by the same letters are not significantly different (P<0.05). Statistical analysis of each formulation was done separately.
ALG: alginate-encapsulated cells. PG: Persian gum. FOS: fructooligosaccharides.
In terms of the encapsulation efficiency in this study, there were no difference between the seven gel and ALG as control (Table 2) formulations, all of which showed a high encapsulation efficiency (>98.6%) that indicated the successful entrapment of viable probiotic cells within the beads that were prepared, which facilitates the efficient release of viable probiotic cells (1-2 × 108 CFU/g) at the required site of impact. Similarly, high rates of encapsulation efficiency, close to 100%, have been reported by other researchers through different encapsulation methods 25. The results from this study showed that the encapsulation efficiency was independent of formulation, while some sources have reported that the polymer concentration and composition affected encapsulation efficiency 26,27.
3.3 Survival rate of E. faecium under fish simulated digestive conditions
The results from this study show that the un-encapsulated E. faecium were highly sensitive to simulated fish digestive conditions and the survival rate and viability were low, changing from an initial cell count of 9.87 ± 0.02 Log CFU/g to 3.85 ± 0.05 log CFU/g following incubation harsh conditions, resulting in a survival rate of approximately 39% (Table 3). This survival rate was similar to results from other studies demonstrating a substantial loss of free probiotics cells in simulated digestion conditions 28,29.
Table 3: Survival rates of microencapsulated E. faecium with various gel and prebiotic concentrations after incubation in simulated fish gastric juices (0.08M HCl containing 0.2% NaCl, pH 1.4) for 0, 30, 60, 90 and 120 min and sequentially in stimulated fish intestinal juice containing (0.5% w/v oxgall, pH 8 at 37 °C for 120 min). Alginate-encapsulated cells (2% (w/v)) were used as control. F1-F7: various gel formulations. Values shown are means ± standard deviations (n = 3)
Formulation
|
Prebiotics
|
Con. (%)
|
Mean count of cells after incubation (log CFU/g)
|
SR (%)
|
0 min
|
30 min
|
60 min
|
90 min
|
120 min
|
Un-microencapsulated Cells
|
_
|
_
|
9.87±0.02
|
5.12±0.04
|
4.73±0.05
|
4.18±0.01
|
3.85±0.05
|
39 a*
|
ALG
|
_
|
_
|
9.76±0.04
|
5.04±0.01
|
4.77±0.06
|
4.59±0.07
|
4.39±0.06
|
45b
|
F1
|
_
|
_
|
9.69±0.02
|
6.23±0.04
|
5.94±0.03
|
5.46±0.08
|
5.14±0.01
|
53c
|
F2
|
FOS
|
1
|
9.92±0.07
|
7.18±0.03
|
6.74±0.02
|
6.51±0.04
|
6.05±0.04
|
61d
|
F3
|
FOS
|
1.5
|
9.79±0.03
|
7.42±0.01
|
7.09±0.09
|
6.58±0.04
|
6.17±0.03
|
63d
|
F4
|
FOS
|
2
|
9.94±0.04
|
7.58±0.03
|
7.26±0.02
|
6.89±0.07
|
6.36±0.02
|
64d
|
F5
|
fenugreek
|
1
|
9.57±0.05
|
7.97±0.04
|
7.56±0.03
|
7.36±0.04
|
6.99±0.07
|
73e
|
F6
|
fenugreek
|
1.5
|
9.91 ±0.06
|
8.12±0.01
|
7.91±0.04
|
7.74±0.07
|
7.43±0.05
|
75e
|
F7
|
fenugreek
|
2
|
9.83±0.01
|
8.59±0.02
|
8.33±0.06
|
8.12±0.05
|
7.96±0.04
|
81f
|
*Values followed by the same letters are not significantly different (P<0.05). Statistical analysis of each formulation was done separately.
ALG: alginate-encapsulated cells. FOS: fructooligosaccharides. Con: Concentration, SR: Survival Rate.
In this study all these seven gel formulations had a greater survival rates than ALG-encapsulated beads (control) following the exposure to simulated fish digestive conditions, however, the highest microencapsulated cell survival rates were observed in the F5, F6, and F7 (Table 3). The E. faecium survival rates in microencapsulated in ALG-PG blend with 1%, 1.5%, and 2% Fk were 73%, 75%, and 81% respectively and the survival rate for ALG-PG blend with 2% Fk (F7) was the greatest in this study, showing a 1.87 log decrease in the cell CFU counts within the first 2 h of incubation, while other blends showed a continuous decrease of between 2.48 and 5.37 log in the cell CFU (Table 3). Similarly well protected formulations, with the ability to survive harsh digestive condition, have been reported when psyllium 9, whey protein 30 and milk 30 were incorporation with ALG.
In this study, combining FOS and Fk with ALG-PG mixture resulted in a 22 to 42% increase in probiotic cell viability, and increasing the prebiotic content from 1 to 2% increased cell viability even more, possibly due to prebiotics' protective and nutritive functions. Fk, on the other hand, had a stronger beneficial impact in a simulated digestive situation (34-42% ) than FOS (22-25%). Fk's great protective ability can be attributed to the higher density of beads produced by its robust structure. The binding of glucuronic acid to divalent cations is primarily responsible for the crosslinking of ALG molecules. The stability and permeability of ALG-biopolymer membranes are influenced by their molecular weight and chemical content. As a result, combining ALG with flexible biopolymers like Fk enhances the strength of synthetic blends. The stability of the ALG-biopolymer is determined by the precursor structure, biopolymer molar ratio, and addition sequence 31.
3.4 Storage stability of microencapsulated E. faecium in food pellet
Feed products that can be used as probiotic-carriers, such as pelleted fish feed , are usually stored for six to eight weeks at room temperature (25 ºC), which has informed storage stability experiments 32. The free E. faecium cell viability in un-microencapsulated E. faecium decreased from 9.73 to 2.87 log CFU/g during the whole seven week storage period, which was greatest during the first week followed by a gentle consistent decline, which was likely to be due to a temperature shock (25 ºC) for the cells during the first week, followed by an adaptation process (Fig 1). A similar decrease trend was observed by Haghshenas et al. (2015) 11, in which the survival rate of free E. durans 39 °C cells and following one month of storage, the cell count in yogurt lowered from 9.52 to 2.83 log CFU/g.
The results from this study showed that E. faecium microencapsulated in ALG (control) and all seven gel formulations had good storage stability, which was similar to other researchers who found that the low temperature storage stability (25 ℃) of encapsulated probiotics in ALG-gum Arabic 33, ALG-chitosan (Chavari et al., 2010) and ALG-psyllium 9. In this study, the ALG-PG (F1) and ALG-PG blended with FOS (F2-F4) showed moderate protection, with a 0.10 to 1.09 log increase in CFU/g, while an excellent cell survival rate during storage (>115%) was found when ALG-PG + Fk formulations (F5 to F7) were evaluated. Moreover, the gel formulation formulated with greater Fk concentrations (F7) in this study, showed a greater protective capacity compared to lower Fk concentrations (F5 and F6) (Fig. 1), due to greater Fk (2%) concentration forming a dense, strong membrane and growth-stimulating activity of Fk, however the extrusion of highly concentrated blends are difficult to press through the nozzle gage, which lowers the encapsulation efficiency.
3.5 Release assay of microencapsulated E. faecium
The effective microencapsulated of probiotics into feed is required to have animal health-promoting effects and to achieve the probiotic cells must be released in sufficient quantity and within an appropriate time frame. Thus, the time-dependent release of probiotic cells from the microencapsulation beads within the simulated intestine solution is a critical 21 and, in this study, an initial number (1 × 107 CFU/g) of E. faecium was selected for release assay. However, previous researches have indicated that the concentration and composition of polymers used in the microencapsulation process influence the release of cells from the beads 34. In this study the Log CFU/g, for released E. faecium from ALG blend (control), was stable (7 to 7.2) and there were no significant changes in the rate bacterial growth (Fig 2), which was similar to results previously reported by Mandal et al. (2006) 21 and Nami et al. (2017) 33 in terms of the sustained and continuous release of cells from ALG.
Once the cell release was complete, in this study, there were significant additive release rates observed from the prepared formulations (F1 to F7), resulting in a greater rate of release due to the growth-stimulating effects of PG, FOS or Fk, and as potential prebiotics, these can release and deliver a greater population of probiotic cells to the active sites in the intestine. In this study, the microencapsulation of E. faecium with ALG-PG or Fk (F5 to F7) was able to release between 33 and 44% of the beads after one hour of incubation and was fully release after two hours. Log CFU / g for F5, F6, and F7 formulations increased from 7 to 9.9, which was greater than the release rates from the other formulations (7 to 8.1), while greater concentration of Fk (2.0%) (F7) lowered the rate of probiotic cell release (33%) within the first hour, however the full release was complete after 2 h (Fig 2), showing how the addition of Fk to the ALG-PG blend lowered the rate of probiotic cell release from the beads. Similar results have been observed previously 7,35, which were probably due to a more dense membrane on the beads that were covered by the rigid structure of Fk.
In this study the ALG-PG (F1) and ALG-PG + FOS blends (F2-F4) released between 54 and 61% of the probiotic cells within one hour, while the full release was completed after two hours. The greater early rate of release compared with Fk in this study was probably due to the erosion of loose networks in formulations.
3.6 Anti-pathogenicity against Streptococcus agalactiae and histopathological assay
The results from this part of the study highlighted how the different analytical assay of microencapsulated probiotic beads indicated high encapsulation efficiency and acceptable viability of probiotic cells in simulated fish digestive as well as the greater stability of viable cells in all the experimental gel formulations. It also demonstrated that the findings in the in vivo challenge reported in this paper demonstrated that the G05 to G011 microencapsulated E. faecium probiotic formulations could be used to improve the health and viability of tilapia fish infected Streptococcus agalactiae. This research, in this paper, provides a novel investigation of the microencapsulated E. faecium probiotic-supplemented diet and its application in controlling the mortality rate of tilapia fish infected streptococcus agalactiae.
In this study, tilapia fish infected showed hemorrhage and red skin, particularly around the anus and eyes, exophthalmia, cloudy eyes and erratic swimming movement, in addition some hemorrhage and ulceration near the mouth were observed (Fig. 3A and B) and similar clinical signs were observed by other researchers 36,37. The tissue samples collected from the eye, kidney, liver, brain, spleen, and skin of fish infected and showing clinical signs of Streptococcosis and histopathological changes, which included hemorrhage and congestion of blood vessels in the brain, eye, kidney, liver, and spleen. Melanomacrophage centers (MMCs) in the kidney, liver, and spleen was observed. In the kidney, dissolution, and degeneration of some tubules, necrosis of tubular cells, glomeruli degeneration, attachment to bowman capsule, and congestion of renal vessels were observed. Liver changes included swelling, degeneration, and necrosis of hepatocytes and nuclear pyknosis (Fig. 3C and E). Also, the organs of the fish in probiotic treated and control group which showed no infection were chosen for histopathology test as normal organs (Fig 3D and F). The observed clinical signs were similar to those in other studies, confirming that the tilapia fish were infected by causative agents of streptococosis (S. agalactiae) 38. The infected organs also were sampled and after biochemical and molecular identification, it was obtained that the cause of infection was S. agalactiae bacteria.
Previous researches have shown that probiotics improved the survival rates of fish to pathogenic Aeromonas hydrophila, Flavobacterium psychrophilum, Streptococcus iniae, Vibrio harveyi, and Pseudomonas fluorescens 39-41 are some of the strains that have been identified. The survival rates of the treated red hybrid tilapia with free or varied concentrations of probiotic cells (17-63%) were higher than those of the infected fish that were not treated with probiotics (CON+) (4%). Furthermore, the ALG-PG + 2% Fk formulation (F7) had a decent survival rate (63%) while the control group injected with PBS (CON) had no mortality during the trial period (Table 4). Other researchers found that complex dietary probiotics containing Bacillus and Pediococcus spp. had low anti-pathogenic activity against S. agalactiae in red hybrid tilapia 42; however, according to the findings of this study, probiotic E. faecium isolated from ewe colostrum demonstrated excellent resistance to S. agalactiae in red hybrid tilapia for the first time. Other studies, on the other hand, demonstrated that B. subtilis-containing diets had no anti-pathogenic activity against streptococcal agents 43.
Table 4: Mortality and survival rates of fish infected with S. agalactiae (1.6 × 108 CFU/mL) after 90 days of feeding with food pellets containing E. faecium microencapsulated with various gel and prebiotic concentrations. The control group (CON) injected with 0.1 mL of phosphate-buffered saline (PBS, pH 7.4). CON+ group injected with 1.6 × 108 CFU/mL of S. agalactiae without probiotic treatment. F1-F7: various gel formulations. Values shown are means ± standard deviations (n = 3)
Formulation
|
Alginate
(% w/v)
|
PG
(% w/v)
|
Prebiotic
(FOS)
(% w/v)
|
Prebiotic
(Fenugreek)
(% w/v)
|
Probiotic strain
|
Pathogen strain
|
Fish mortality
|
survival
rates
(%)
|
CON
|
_
|
_
|
_
|
_
|
_
|
_
|
0
|
100 a*
|
CON+
|
_
|
_
|
_
|
_
|
_
|
S. agalactiae
|
29/30
|
4 ±1.15 b
|
Free Cell
|
_
|
_
|
_
|
_
|
E. faecium
|
S. agalactiae
|
25/30
|
17 ± 1.25c
|
ALG
|
2
|
_
|
_
|
_
|
E. faecium
|
S. agalactiae
|
23/30
|
24 ± 2.0d
|
F1
|
1.5
|
0.5
|
_
|
_
|
E. faecium
|
S. agalactiae
|
20/30
|
34 ±2.20e
|
F2
|
1.5
|
0.5
|
1
|
_
|
E. faecium
|
S. agalactiae
|
18/30
|
40 ± 1.75f
|
F3
|
1.5
|
0.5
|
1.5
|
_
|
E. faecium
|
S. agalactiae
|
18/30
|
40 ±1.55f
|
F4
|
1.5
|
0.5
|
2
|
_
|
E. faecium
|
S. agalactiae
|
17/30
|
43 ±1.85f
|
F5
|
1.5
|
0.5
|
_
|
1
|
E. faecium
|
S. agalactiae
|
15/30
|
50 ± 2.15g
|
F6
|
1.5
|
0.5
|
_
|
1.5
|
E. faecium
|
S. agalactiae
|
15/30
|
50 ± 2.23g
|
F7
|
1.5
|
0.5
|
_
|
2
|
E. faecium
|
S. agalactiae
|
11/30
|
63 ± 1.75h
|
*Values followed by the same letters are not significantly different (P<0.05). Statistical analysis of each formulation was done separately.
Free Cell: Un-encapsulated probiotic cells. ALG: alginate-encapsulated probiotic cells. PG: Persian gum. FOS: fructooligosaccharides.
Because probiotics can help fish cope with diseases by increasing their immune systems, these disparities in tilapia fish findings could be related to differences in probiotic strains, culture systems, and treatment methods 44. The most effective tested formulation in this study for protecting tilapia against the highly pathogenic S. agalactiae was dietary E. faecium encapsulated with ALG-PG + 2% Fk. Because survival rates did not improve to 100%, more research into E. faecium in conjunction with other probiotic strains or in various encapsulation matrixes may be worthwhile. The use of dietary probiotics and continued study in this area are likely to help lower the incidence of harmful bacteria in tilapia aquaculture, allowing for a more ecologically friendly expansion of the tilapia breeding business. The observed results also can highlight in two part. First), different analytical assay of microencapsulated probiotic beads indicated high encapsulation efficiency and acceptable viability of probiotic cells in simulated fish digestive as well as high stability of viable cells in all experimental gel formulations. Second) the finding of in vivo challenge test the present report demonstrates that the G05 to G011 microencapsulated E. faecium probiotic treatment could be used for treating infected streptococcus agalactiae tilapia fish. This research represents a novel investigation to use microencapsulated E. faecium probiotic-supplemented diet to control mortality rate of infected streptococcus agalactiae tilapia fish. In conclusion, the probiotic E. faecium cells were successfully microencapsulated in appropriate sizes and shapes utilizing ALG-PG blends with varied concentrations of FOS and Fk. According to the findings, the ALG-PG + 2 percent Fk (F7) formulation had the highest encapsulation efficiency, viability in gastrointestinal conditions and during storage time, increased cell release, and excellent anti-pathogenicity against S. agalactiae. Local herbal gums such as PG and Fk, in combination with ALG, are recommended as a suitable scaffold and an ideal matrix for probiotic encapsulation. As a prebiotic, these herbal gums promote the growth of probiotic cells in the food environment and digestive tract.