DIAGNÓSTICO DE RESULTADOS

6.3.1. Medium of isolation

The are several media with different components and protocols developed by several authors to isolate enzyme-producing bacteria. For instance, media for isolating extracellular amylase, protease, cellulase, lipase or alginate lyase have been described in previous studies by several authors (Hoshino et al., 1997; Bairagi et al., 2002; Tang et al., 2009; Zhou et al.,

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2009; Sirisha et al., 2010; Alnahdi, 2012; Hadi et al., 2014). Different media and protocols might support the growth of different bacteria. However, this present study only used one source of medium and protocol. This might be the reason why less diverse of enzyme- producing bacteria was obtained in this present study. Therefore, combination of several protocols is highly recommended in future study, to obtain more number and diverse results. LAB targeted for antimicrobial compound production are generally known to be auxotrophic bacteria, having limited capacity to synthesize nutrients such as amino acids from inorganic sources (LeBlanc et al., 2011). Thus, the bacterial strains that can be recovered is highly dependent on the growth medium used. This present study used two commercial media: De Man Rogosa Sharp (MRS, Oxoid) and M17 (Oxoid). Previously,

MRS medium has been described to support the growth of only 4 genera: Lactobacillus, Streptococcus, Pediococcus and Leuconostoc (De Man et al., 1960), and M17 medium for

Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactococcus lactis (Terzaghi and Sandine, 1975). Therefore, using only these two-commercial media may lead to an underestimate of the number and diversity of LAB associated with the GITs of aquatic species. The use of wider range of media, such as BM medium (Yanagida et al., 2008),

glucose yeast extract peptone (GYP) medium (Maeda et al., 2014), Enterococcus medium (Litsky et al., 1953), and Raka Ray (Abassah-Oppong et al., 2007) may have increased the diversity and number of isolates.

In addition, the use of molecular approaches might detect the presence of more LAB than were detected using culture-dependent methods. A study by González-Arenzana et al. (2013) demonstrated that polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) detected 9 out of 11 LAB, while culture-dependent method using a modified MRS medium could detect only 5 out of 11 LAB. Based on these studies, it is therefore recommended to use a polyphasic approach which combines molecular (e.g., PCR) and

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culture-dependent methods with more varieties of culture media, to give a more comprehensive profile especially to LAB associated with the GIT of aquatic animals.

6.3.2. Screening methods

Screening of antagonistic bacteria was performed using either the microtiter plate assay or well diffusion assay. Both method used only extracellular products in terms of CFS or CFSn of tested bacteria. Meanwhile, there are many other antagonistic mechanisms such as immunomodulation and competition for adhesion site and nutrients which should be tested using live cells (Kesarcodi-Watson et al., 2008). However, all these mechanisms cannot be investigated in this present study. Furthermore, the non-antagonistic bacteria (result from this present study) does not necessary means that they do not have capacity to produce antimicrobials. The non-antagonistic bacteria might carry antimicrobial-encoding genes but the expression of extracellular antimicrobial compounds (e.g., bacteriocin) might be inhibited by the culture condition or nutrient which were available in the culture medium. As consequence, screening of antagonistic bacteria using well diffusion or microtiter plate assay was frequently reported to underestimate the number of antimicrobial-producing bacteria (Miller and McMullen, 2014; Chanos and Mygind, 2016; Perin et al., 2016). Therefore, more screening methods including immunomodulation, competition for adhesion and nutrient, and molecular technique are highly recommended for future studies to get more comprehensive result.

Furthermore, the screening of digestive enzyme-producing bacteria was based on their capacity to utilize substrates: casein and gelatin (protease), alginate lyase (sodium alginate) and carboxymethyl cellulose (cellulase). The casein, gelatin and CMC used in this screening assay might be different to the substrates used in the natural diet and the manufactured diet of aquaculture animals. For instance, it was described that a protein substrate from different sources may have a different structure (Ochoa-Solano and Olmos-

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Soto, 2006). However, this current study did not investigate the capacity of the 24 enzyme- producing bacteria to digest fish meal or soybean meal by protease activity nor for cellulase activity on fresh Gracillaria sp. Selected bacterial strains based on these substrates (Gracillaria sp., fish meal or soybean meal) might give more applicable results for further in vivo studies, especially for abalone.

6.3.3. Characterization of digestive enzymes and antimicrobial substances

This present study identified 24 isolates which could produce one of these enzymes; cellulase, protease and alginate lyase. The screening assay was performed all by in vitro assay. Verschuere et al. (2000a) reviewed that results of in vitro studies should be confirmed by in vivo assay to get more valid result. However, this study did not investigate the 24 enzyme-producing bacteria by in vivo. For instance, whether the bacterial strains could produce digestive enzyme in environmental condition of GITs or in what conditions do the bacterial strains produce the digestive enzymes optimally. In addition, the specific mechanisms of protease, cellulase or alginate lyase which were produced by the isolated strains were not studied. For instance, there two types of alginate lyase, poly(α-L- guluronate)lyase which act on PolyG, and poly(β-D-mannuronate)lyase which act specifically on PolyM (Iwamoto et al., 2001). It has been described that abalone need poly(α-L-guluronate)lyase, because alginate lyase produced endogenously could only digest polyM (Sawabe et al., 1995). Thus, further characterization of digestive enzymes should be investigated in future studies.

Furthermore, the antimicrobial substances produced by the 22 endogenous bacteria and digestive enzymes produced by the 24 endogenous bacteria are still poorly characterized. In terms of antimicrobial substances, a number of studies have documented that LAB produce several antimicrobial substances such as organic acids (Goncalves et al., 1997;

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Vazquez et al., 2005), bacteriocins (Verschuere et al., 2000a; Lin et al., 2013), and hydrogen peroxide (Verschuere et al., 2000a). This current study confirmed the presence of organic acids, indicated by a lower pH in the cell-free supernatant of LAB culture (De Vuyst and Vandamme, 1994; Presser et al., 1997). The antagonistic activity of hydrogen peroxide was excluded since the CFS or CFS was obtained from anaerobic culture condition. The other possible antimicrobial substances are bacteriocin-like inhibitory substances (BLIS), indicated by the presence of antimicrobial activity after the CFS of each LAB was neutralized (pH ~6.5-6.8). However, quantification of organic acids by these 22 LAB was not studied. In addition, Pediocin PA-1 encoding gene detected from two pediococci, P. acidilactici MA160and P. pentosaceus MA169 should be further confirmed by sequencing the amplified genes. Furthermore, the expression of the bacteriocin-encoding gene should be tested by treating with proteinase K.

6.3.4. Viability of the supplemented probionts

The viability of three supplemented bacteria (B. amyloliquefaciens subsp. plantarum, E. ludwigii and P. acidilactici MA160) in the GIT of juvenile abalone was not monitored. Even though these three probiotic candidates were previously confirmed to have a high tolerance to the low pH in the stomach as well as to the bile contents in intestinal tract, the in vitro assay was done in a sterile environment (chapter 3 and 4). On the other hand, a diverse group of endogenous bacteria inhabit the abalone GIT and may interfere in terms of nutrient and space competition. One of the supplemented bacteria (P. acidilactici MA160) has also been confirmed to release antimicrobial substances against a broad range of bacterial pathogens, such as Vibrio, Yersinia, and Listeria. This current study did not evaluate whether P. acidilactici MA160 was antagonistic to the other two supplemented bacteria. All these factors may determine the contribution of supplemented probionts. While it was suggested

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that interactions among 2 or more probiotic strains should be taken into account when they were used in mixture (Makridis et al., 2000).

This present study did not quantify the amount of digestive enzymes, especially protease and alginate lyase, in the intestinal tracts of juvenile abalone in both treatments and the control group. The comparison of these enzyme amounts could be used to evaluate the activity and the capacity of the supplemented probionts to aid feed digestion (Wang, 2007; Kar and Ghosh, 2008; Askarian et al., 2011).