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THE EFFECTS OF PROBIOTICS AND PREBIOTICS ON RAINBOW TROUT
(Oncorhynchus mykiss) INTESTINAL FLORA
Gonca Alaka1, Sükriye Aras Hisarb
aAgricultural
Biotechnology Department, Agricultural Faculty, Atatürk
University, 25240-Erzurum, Turkey
b
Fishing and Processing Technology Department, Faculty of Fisheries,
Çanakkale 18 Mart University, 17000-Çanakkale, Turkey
ABSTRACT
In this study, the effects of probiotic and/or prebiotic use in raising rainbow trout on intestinal
flora. Feeding with probiotic and/or prebiotic added bait was effective on aerobic mesophilic
bacteria, psychotrophic bacteria, lactic acid bacteria, Enterobacteriaceae at the p<0,01 or
p<0,05 level. Feed mixtures including the probiotic caused a 1 logarithmic unit increase in
the number of lactic acid bacteria. The number of enterobacteriaceae was found to be at a
lower level in the fish fed with the probiotic, including feed compared to control and prebiotic
groups.
Keywords: Rainbow trout, probiotic, prebiotic, lactic acid bacteria
1
Agricultural Biotechnology Department, Agricultural Faculty, Atatürk University, 25240-Erzurum, Turkey
[email protected] Phone:
+90 4422311426; Fax: +90 4422361128
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INTRODUCTION
Some microorganisms present in the intestinal flora play an important role in the rotting of
fish. The intestinal micro flora of aquatic animals is likely to change fast with the ingestion of
microorganisms in the water. Some animal and plant derived inhibitor elements (probiotic,
prebiotic, organic acids and various enzymes) that do not have any negative effects on health
are widely used to decrease the efficiency of some microorganisms present in the fish’s
intestinal flora (Barug et al., 2006).
Probiotic and prebiotic usage in aquaculture is common; however, use in Turkey is very new.
In aquaculture, research on the usage of probiotics in decreasing the pathological bacteria load
and improving the water quality by balancing the bacteria population in the water have been
increasing and the use of probiotics has become more and more common (Irianto and Austin
2002; Korkut et al., 2003; Vine et al., 2006). Especially during recent years, biological
principles-oriented control systems (microbial cells such as enzymes, lectins, receptors, and
probiotics) are used in the application of food safety. These studies are generally performed
in vitro in the food sector.
Many research studies have been performed on the effects of probiotic and prebiotic use in
aquaculture on increased live weight, feed utilization, and the immune system. ; however, the
effects of probiotics and prebiotics on the duration of the cold preservation of the fish has not
been studied in vivo yet. This present study is built and implemented to observe the changes
in the intestinal flora of fish during periods of feeding with probiotics and prebiotics that will
have a very important place in aquaculture in the future.
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MATERIAL AND METHOD
Rainbow trout (Oncorhynchus mykiss), with an average weight of 100±10g, supplied by
Ataturk University Faculty of Aquaculture, Research and Production Facility were used as
fish material in this research. A probiotic bacteria level of 1x109 kob/g was used to prepare
feed with the probiotic (Lactobacillus rhamnosus DSM 7133), and the 2% prebiotic rate was
used to prepare feed with the prebiotic (Mannan-Oligosaccharides). The same rates were used
in preparing feed with probiotic + prebiotic. Assays consisted of four groups, including one
control and three treatments. Four hundred fish were randomly distributed in 16 tanks, each
consisting of 25 fish. The assay, based on five different days (0, 15, 30, 45 and 60th day) and
four different treatment (control, probiotic, prebiotic, and probiotic + prebiotic) in 5 x 4
factorial array, was implemented with double repetition according to the assay plan related to
full chance. The feeding program was calculated according to water temperature (10 ±10C)
and fish weight for each diet, and feeding was performed twice daily, during morning and
afternoon. Samples were taken on certain days of feeding by sampling in this assay and
intestinal flora was microbiologically counted.
Microbial analysis
10 g fish intestinal flora was removed aseptically and homogenized for 1 min in a Stomacher
400 (Lab Stomacher Blander 400-BA7021, Sewardmedical) bag containing 0.85% NaCl
solution. Further decimal dilutions were made and then 0.1 ml of each dilution was pipetted
onto the surface of plate count agar (PCA, Merck). PCA plates were then incubated for 7 days
at 100C for psychrotorophic bacteria count and for 2 days at 370C for mesophilic bacteria
count. Enterobacteriaceae was determined in Violet-Red-Bile-Glucose agar (VRBG-agar,
Merck) plates incubated anaerobically at 300C for 2 days. Lactic acid bacteria were
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determined in MRS Agar (Oxoid) plates incubated anaerobically at 300C for 72 h. All counts
were expressed as log10 CFU/g.
Statistical analysis
Data obtained were analyzed using variance analysis in SPSS 18.0 package program, and
means of significant variation origins were compared with the Duncan multi comparison test.
However, for the statistical analysis of Enterobacteriaceae counts, <2 log kob/g were taken as
2 log kob/g.
RESULT AND DISCUSSION
In the intestinal samples of rainbow trout (O. mykiss) that were fed by different feeds, the
total number of aerobic bacteria at the beginning of the feeding was at the 105-106 kob/g level.
The number was again at the 105-106 kob/g level after a 60-day period. According to these
results, the bacteria load in mesophilic characteristics in the intestinal flora of rainbow trout
were around 105-106 kob/g. Diler and Diler (1998) found similar results in a study performed
on carp. However, the number of psychotrophic bacteria that were in a lower level at the
beginning of feeding compared to mesophilic bacteria increased parallel to the time elapsed.
These results are thought to be due to the temperature of the water which was around 100C
during feeding. At the end of the feeding period, there were differences among the groups;
however, the number of psychotrophic bacteria had increased up to levels of 108 kob/g and
109 kob/g. Bacteria load and dominant species in the intestinal flora change according to the
feeding regimen. In a study in which sea bass larvae were fed with different feeds
(commercial diet, including artemia and iron) for 20 days, bacteria levels were found to be 109
kob/g, 107 kob/g and 108 kob/g, respectively, and dominant species differed in every group
(Reuter, 2001).
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At the beginning of the feeding, the number of lactic acid bacteria was at a level of 10 4 kob/g,
there was an increase in the groups fed by feeds containing probiotic, and probiotic +
prebiotic during the feeding period. The number of lactic acid bacteria stayed the same at the
end of the 60-day feeding period in the control group. In the groups containing probiotic, the
number of lactic acid bacteria increased during the feeding period and reached a level of
approximately 106 kob/g. When K1 species of Carnobacterium sp. was added to the feed by
5x1010 kob/g as probiotic, it was reported that the number of probiotic bacteria in the
intestinal flora at the beginning was lower than the identifiable level, and the number reached
a maximum (1,3x108 kob/g) on day 7, and decreased to a level of 2,9 x 105 on the last day of
feeding (Jöborn et al., 1997). The number of lactic acid bacteria of the intestinal samples of
trout during feeding increased until the 30th day of feeding, decreased on the 45th day, and
increased again on the 60th day and reached a level of 5,06 log kob/g. The lowest and the
highest mean values were identified on day 0 and day 60, respectively. The values that belong
to the 15th and 30th days of the feeding were found to be statistically insignificant (p>0,05)
(Figure1).
Similar to our results, Dulluç (2010), reported that the number of probiotic bacteria of baby
tilapias that were fed by feeds containing probiotic bacteria at different levels (105, 106 and
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107kob/g) were 4,37 log kob/g, 3,70 log kob/g, and 5,08 log kob/g, respectively. The results
of a study investigating the effects of Carnobacterium spp. on Atlantic salmon and rainbow
trout intestinal load were in parallel with our study (Robertson et al., 2000).
The level of Enterobacteriaceae was found to be at a level of 103 kob/g on the first day of
feeding in all treatment groups. The numbers remained at the same level in the groups fed by
feeds containing prebiotic and controls during the feeding period. On the other hand, in
groups containing probiotic, the number of Enterobacteriaceae decreased to a lower than
measurable level on the 15th day. The same results were obtained on the other days of feeding
in these groups (p<0,01). These results demonstrate that probiotics have an important
inhibiting effect on Gram negative bacteria. According to this, the differences of the means of
the groups of probiotic + prebiotic and the control group are higher than 1 logarithmic unit.
According to this, probiotics cause a remarkable reduction in the number of
Enterobacteriaceae (Figure2).
Similar results were identified by Burr and Gatlin (2005), and these researchers reported that
probiotic bacteria in fish intestinal flora have an inhibiting effect on Enterobectericeae and
coliform bacteria, and by adherence to the intestinal lumen of some microorganisms, they
inhibit their growth. Similarly, Öztürk (2008), reported that feeding carp with feed containing
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probiotic (Enterococcus faecium) had an inhibiting effect on Enterobacteriaceae and that the
number of Enterobacteriaceae decreased to a lower than measurable level due to probiotic
use. Bagheri et al. (2008), suggested that lactic acid bacteria compete with the pathogeneous
microorganisms in the intestinal flora and inhibit their growth.
CONCLUSION
The period of feeding was effective on the total number of aerobic mesophilic and
psychotrophic bacteria in the samples of the intestines of trout fed with different feeds at a
very significant level (p<0,01). Treatment had a significant effect (p<0,05)
on these
microorganism groups. Although the lowest number of mesophilic bacteria in the intestines
was identified in the probiotic group, the average value of this group was similar to the
average value of the prebiotic group. The lowest value of psychotrophic bacteria was
identified again in the probiotic group; however, this mean value and the mean value of the
probiotic + prebiotic group was not significantly different. The number of psychotrophic
bacteria in the treatment groups was found to be approximately 2 logarithmic units higher
than the number of mesophilic bacteria in the treatment groups. The number of lactic acid
bacteria in the intestines increased until the 30th day of feeding, decreased on the 45th day
and demonstrated an increase on the 60th day. Adding probiotic to the rainbow trout was
found to increase the number of lactic acid bacteria by 1 logarithmic unit. Probiotic
(Lactobacillus
rhamnosus)
caused
a
remarkable
reduction
in
the
number
of
Enterobacteriaceae.
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