Isolation of potential probiotic and carotenoid producing bacteria and their application in aquaculture

De Bruyn, Anneke (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: The ocean’s fish resources are declining mainly because of irresponsible exploitation. Fish is a vital source of protein for humans and growing world populations are threatening the sustainability of commercial fisheries. This has led to the rapid growth of aquaculture worldwide. In South Africa, aquaculture of both fresh and marine species is expanding and is now practised in all nine provinces of the country. One of the major problems in aquaculture is the economic losses as a result of diseases. Viruses, bacteria, fungi and parasites are well known to infect fish, with bacteria causing the majority of diseases. Antibiotics were commonly used to control diseases, however, due to their negative impact on the environment, the use of these agents is questioned. This has led to the search for probiotics as an alternative way to control bacterial diseases in aquaculture. Probiotics used in aquatic environments can be defined as live microbial supplements which have beneficial effects on the host by altering the microbial communities associated with the host and the immediate environment. Probiotics have a variety of different mechanisms of action, including competition with pathogens, production of beneficial compounds, enhancement of host immune response and antiviral effects. This study aimed to isolate potential probiotic bacteria from the gastrointestinal tract (GIT) of the South African abalone (Haliotis midae). Nine different bacterial species were isolated and identified as Corynebacterium variabilei, Staphylococcus carnosus, Staphylococcus equorum, Staphylococcus cohniii, Vibrio aestuarianus, Vibrio nigripulchritudo, Vibrio cyclitrophicus, Photobacterium leiognathi, and Paracoccus marcusii (Chapter 2). One of these isolates, P. marcusii (isolate 6.15), showed promising probiotic properties together with the potential to be used as a pigmentation source due to its production of the carotenoid astaxanthin. Aquatic animals are not able to synthesize astaxanthin and under aquaculture conditions do not come into contact with natural pigment sources. This results in dark grey meat which is unappealing for consumers. Therefore, astaxanthin is included in the feed of a variety of aquaculture species such as salmon, trout, red see bream and shrimp to give the meat a pink/orange colour. Astaxanthin also plays an important role in other essential biological functions of fish such as increasing the defence potential against oxidative stress and enhancing sexual maturity, embryo development, and egg survival. Mozambique tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), two important aquaculture species in South Africa, were used to evaluate the probiotic and pigmentation effect of P. marcusii (isolate 6.15). Fish feed was coated with freeze dried bacterial cells (107 CFU/kg feed) and administrated to tilapia and trout. Because tilapia cannot incorporate astaxanthin into their meat, no pigmentation effect of P. marcusii (isolate 6.15) was evaluated for this species. However, tilapia showed significant improvement in growth and immune parameters. Fish supplemented with P. marcusii (isolate 6.15) had a higher percentage increase in body weight and a better feed conversion ratio for the duration of the trial. Enhanced lysozyme activity in the blood serum of the fish was also seen (Chapter 3). In contrast, P. marcusii (isolate 6.15) did not have any probiotic or pigmentation effect on rainbow trout. A possible reason for this may be that the concentration of P. marcusii (isolate 6.15) added to the feed was too low. More probably, it is suspected that no pigmentation was observed due to the destruction of the astaxanthin before being ingested by the trout, because astaxanthin is a very unstable molecule. Furthermore, the GIT microbial communities of trout were investigated over the duration of the trial for the different treatments. No similarities in community structures were observed betwee the different treatments, however, bacterial communities in the GIT of fish sampled at the same time were very similar (Chapter 4).

AFRIKAANSE OPSOMMING: Die oseaan se vis hulpbronne is besig om af te neem as gevolg van die onverantwoordelike gebruik daarvan. Vis is ‘n belangrike bron van proteïene vir mense en die toenemende wêreld populasie bedreig die volhoubaarheid van kommersiële visserye. As gevolg hiervan is daar ‘n drastiese toename in die akwakultuur industrie wêreldwyd. Ook in Suid Afrika brei die akwakultuur van beide vars water en mariene vis spesies uit. Een van die grootste probleme in akwakultuur is ekonomiese verliese as gevolg van siektes wat veroorsaak word deur virusse, bakterieë, fungi en parasiete. Bakterieë veroorsaak die meerderheid van die siektes en antibiotika word algemeen gebruik vir die beheer van bakteriële siektes. Die gebruik van antibiotika word egter bevraagteken omdat dit verskeie negatiewe implikasies vir die omgewing inhou Daarom word probiotika oorweeg as ‘n alternatief tot antibiotika om bakteriële siektes te voorkom en te behandel. Probiotika wat in akwatiese omgewings toegedien word kan gedefinieer word as a lewende mikrobiese aanvulling wat ‘n positiewe effek op die gasheer het, deur die mikrobiese gemeenskappe geassosieer met die gasheer en die ommidellike omgewing te verander. Hierdie mikrobiese aanvulling verbeter die gesondheid van die visse deur verskeie meganismes wat insluit kompetisie met patogene, produksie van voordelige chemiese verbindings, verhoging van die gasheer se immuniteit en antivirale effekte. Die doel van hierdie studie was om potensiële probiotika te isoleer uit die spysverterings kanaal (SVK) van die Suid Afrikaanse perlemoen spesie, Haliotis midae. Tydens die studie is daar nege verskillende bakteriële spesies geïsoleer en geidentifiseer as stamme verteenwoordegend van Corynebacterium variabilei, Staphylococcus carnosus, Staphylococcus equorum, Staphylococcus cohniii, Vibrio aestuarianus, Vibrio nigripulchritudo, Vibrio cyclitrophicus, Photobacterium leiognathi en Paracoccus marcusii (Hoofstuk 2). Een van die isolate, P. marcusii, het belowende probiotika en potensiële pigmentering eienskappe getoon a.g.v. die produksie van die karotenoïed astazantien. Akwatiese diere is nie daartoe instaat om hierdie pigment te produseer nie en onder akwakultuur toestande kom die visse ook nie in kontak met natuurlike bronne van hierdie pigment nie. Dit lei daartoe dat die vleis van visspesies soos forel en salm grys word wat dit onaantreklik vir verbruikers maak. Daarom word astazantien bygevoeg by visvoer om sodoende ‘n pienk/oranje kleur te verseker.Daar benewens speel astazantien ook ‘n rol in belangrike biologiese funksies van visse. Dit sluit in die verhoging in beskerming teen oksidatiewe stres, bevordering van seksuele volwassenheid, embrio ontwikkeling en eier oorlewing. Twee belangrike akwakultuur spesies in Suid Afrika, Mosambiek tilapia (Oreochromis mossambicus) en reënboog forel (Oncorhynchus mykiss), was in hierdie studie gebruik. Die probiotiese en pigmentasie effek van P. marcusii op reënboog forel was gëevalueer terwyl slegs die probiotiese effek op tilapia geëvalueer weens die onvermoeë van tilapia om die pigment in hul vleis te inkorpereer. Visvoer korrels was omhul met gevriesdroogde bakteriële selle (107 CFU/kg kos) en vir die visse gevoer. Daar was ‘n duidelike verbetering in groei en immuun parameters van tilapia. Visse toegedien met P. marcusii het ‘n hoër persentasie vermeerdering in liggaamsgewig en ‘n beter voedsel omsettings verhouding gehad tydens die verloop van die proef in vergelyking met die kontroles (Hoofstuk 3). In kontras hiermee kon daar geen probiotiese of pigmenterings effekte waargeneem word by die reënboog forel nie. ‘n Moontlike rede hiervoor kon wees dat die konsentrasie van P. marcusii wat by die kos gevoeg is te laag was. Dit is egter ook moontlik dat die astazantien vernietig was voordat dit deur die forel opgeneem is aangesien astazantien ‘n baie onstabiele molekuul is. Verder het ons die impak van verskillende visvoer behandelings op die mikrobiese gemeenskappe in die spysverteringskanaal (SVK) van forel tydens die verloop van die proef bestudeer. Geen ooreenkomste in mikrobiese gemeenskap strukture in die forel SVK is waargeneem tussen die verskillende voer behandelings nie, maar daar is wel ooreenkomste gevind tussen die mikrobiese gemeenskappe van visse by spesifieke tyd intervalle (Hoofstuk 4).

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