The application of astaxanthin producing bacteria in poultry feed

Conradie, Tersia Andrea (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: In the food industry, the colour of the product is important to the consumer as it gives an indication of the freshness and quality of the product. Hens are not able to produce pigments and absorb pigments through their diet. This has led to a rapidly emerging trend in poultry farming to enhance egg yolk colour as the yolk colour is influenced by the diet of the hen. Over the years, natural or synthetic carotenoids have been added to poultry feed. Several studies have focused on using astaxanthin producing microorganisms, such as the microalga, Haematococcus pluvialis, and yeast, Xanthophyllomyces dendrorhous. However, the production costs are expensive and the thick cell walls of the microalga and yeast limits its whole cell application as a feed additive. Some bacterial species are also able to produce astaxanthin, including the bacterium Paracoccus marcusii, and have previously not been used as a feed additive to enhance yolk colour. The purpose of this study was, therefore, to determine the whole cell application of P. marcusii as a feed additive to enhance egg yolk colour, without the need to homogenise the cells or extract the pigment. In the first experimental chapter (Chapter 2), the growth conditions and astaxanthin production of P. marcusii was optimised. Furthermore, the stability of the astaxanthin molecule under different storage conditions, namely lyophilisation and microencapsulation, was determined. The optimum growth conditions for P. marcusii and for astaxanthin production was at 26 °C in a specialised medium containing yeast extract (5 g/L), bacteriological peptone (10 g/L) and NaCl (3%) at a pH between 6 – 7. Astaxanthin is a valuable compound with several health promoting benefits for humans and animals. However, the molecule is unstable when exposed to oxygen, light and temperature. After lyophilisation in sucrose (10% m/v), there was an 85% loss in astaxanthin concentration after 3 weeks. However, the loss in cell viability was low. When P. marcusii was microencapsulated in calcium alginate beads, cell viability significantly decreased when stored at 20 °C compared to 4 °C. However, only 30% of the total astaxanthin concentration was lost after 3 weeks at both storage temperatures. The microencapsulation significantly improved the stability of astaxanthin under storage. The highest concentration of astaxanthin obtained was 24.25 μg/g dry cell weight. Chapter 3 examined the pigmentation effect of P. marcusii when fed to laying hens to enhance egg yolk colour. Paracoccus marcusii was fed to hens daily either in a sucrose solution (10% m/v) or microencapsulated in calcium alginate beads. After the pilot study, it was clear that a diet free of all pigments was needed to effectively determine the pigmentation effect of P. marcusii. In all the experimental trials there was a significant increase in yolk colour and no negative effect on egg quality, laying rate or hen weight was observed. There was also an increase in whole egg and yolk weight when compared to the control groups. Furthermore, the microbial communities of the duodenum and caeca were investigated after a prolonged feeding of P. marcusii to detect any changes that might have occurred (Chapter 4). The microbial community of the hen’s gastro-intestinal tract (GIT) starts out as a simple community in the small intestines which becomes increasingly diverse and complex further down the intestinal tract. The findings in this study revealed a similar pattern when considering the results obtained from the Shannon diversity index and total number of operational taxonomic units (OTUs). Starting in the duodenum, the index ranged between 2.14 – 2.59 and increased in the caeca to between 2.45 – 3.03. OTUs increased from 21.44 – 28.60 in the duodenum to 28.30 – 38.00 in the caeca. A significant difference was only observed for the OTUs of the experimental group compared to the control groups in both the duodenum and caeca. There was no significant difference observed in the microbial community structure of the duodenum. However, distinct patterns and clusters formed in the caeca between the experimental diet group compared to the control diet groups. Since no mortalities were recorded during the trial and all hens appeared in excellent health, it is safe to assume that the change in microbial community structure of the caeca was not negative. Therefore, P. marcusii is safe to use as a feed additive for laying hens. Finally, Chapter 5 evaluated the costs associated with the small-scale production of P. marcusii microencapsulated in calcium alginate beads and its feasibility in the poultry industry. Based on the economic assessment, the total cost for one month’s production of 210 g calcium alginate beads is estimated at R2912.88. This is too expensive and not practical to be used by poultry farmers. Possible solutions can include the use of inexpensive peptones, production on a larger scale and also increasing the concentration of bacterium encapsulated in the bead.

AFRIKAANSE OPSOMMING: In die voedselbedryf is die kleur van ʼn produk baie belangrik vir die verbruiker, aangesien dit ‘n indruk skep van ‘n vars produk van goeie gehalte. Hoenders is nie daartoe instaat om self pigmente te produseer nie en is afhanklik van hul dieet om dit in te neem. Dit het gelei tot ‘n toenemende neiging onder pluimveeboere om die kleur van die eiergeel te manipuleer deur natuurlike of sintetiese kleurmiddels, byvoorbeeld karotenoïede, by die voer te meng. Karotenoïede word oor ‘n lang tydperk reeds by pluimveevoer gevoeg. Meeste van vandag se navorsing fokus op die gebruik van ‘n astazantien produserende mikroalge, Haematococcus pluvialis, en gis, Xanthophyllomyces dendrorhous. Die gebruik van hierdie mikroörganismes as ‘n bymiddel word egter beperk aangesien die produksie kostes baie hoog is. Hierdie mikroörganismes het ook ‘n baie dik selwand wat die vrystelling van astazantien bemoeilik. Sommige bakterieë, byvoorbeeld die bakterium Paracoccus marcusii, is ook daartoe instaat om astazantien te produseer, maar is voorheen nog nie gebruik as a bymiddel nie. Die doel van hierdie studie was dus om te bepaal of P. marcusii gebruik kan word as ‘n bymiddel om die kleur van eiergeel te manipuleer sonder om die selle te homogeniseer of die pigmente uit te haal. In die eerste eksperimentele hoofstuk (Hoofstuk 2), is die optimale groeitoestande van P. marcusii vir ‘n hoë produksie astazantien bepaal. Verder is die stabiliteit van die astazantien molekule onder verskillende berging metodes ook bepaal, naamlik vriesdroging en mikroënkapsulering. Die optimale groeitoestande van P. marcusii was by 26 °C in ‘n gespesialiseerde medium met ‘n pH van tussen 6 – 7, wat gis ekstrak (5 g/L), bakteriologiese peptone (10 g/L) en NaCl (3%) bevat. Astazantien is ‘n baie waardevolle pigment met verskeie gesondheids voordele vir beide mense en diere. Hierdie molekuul is egter onstabiel wanneer dit blootgestel word aan suurstof, lig en hoë temperature. Drie weke na vriesdroging in sukrose (10% m/v) was daar ‘n 85% verlies in astazantien konsentrasie, maar die lewensvatbaarheid van die selle was nog hoog. Die mikroënkapsulering van P. marcusii in kalsiumalginaat-balletjies het ‘n laer sellewensvatbaarheid gehad by 20 °C in vergelyking met 4 °C en slegs 30% van die astazantien konsentrasie het verlore gegaan na drie weke by albei temperature. Die mikroënkapsulering het dus die stabiliteit van die astazantien molekule aansienlik verbeter. Die hoogste konsentrasie astazantien wat verkry is, was 24.25 μg/g droë sel gewig. Hoofstuk 3 het gekyk na die effek wat P. marcusii op die kleur van die eiergeel uitoefen wanneer dit vir lê-henne gevoer word. Die henne het daagliks ‘n dosis van die bakterium gekry in óf ‘n sukrose oplossing (10% m/v) óf gemikroënkapsuleer in kalsiumalginaat-balletjies. Na die loodsstudie was dit duidelik dat ‘n voer sonder enige pigmente nodig is om die kleuringseffek van die bakterium te kan bepaal. Daar was ‘n beduidende toename in die kleur van die eiergeel by al die eksperimentele proewe en geen newe-effekte is waargeneem in terme van eierkwaliteit, hoeveelheid eiers wat gelê is of die gewig van die hoender nie. In vergelyking met die kontrolegroepe was daar ‘n effense toename in die gewig van die eier en die eiergeel. Verder is daar ook gekyk na die effek van die bakterium op die mikrobiese gemeenskap van die duodenum en seka (Hoofstuk 4). Die mikrobiese gemeenskap van die spysverteringskanaal (SVK) van die hoender begin as ‘n eenvoudinge samestelling van bakterieë in die dunderm wat meer kompleks en divers raak verder af in die SVK tot by die seka. Die bevindinge in hierdie studie het gedui op soortgelyke patrone deur die resultate van die Shannon indeks en totale aantal operasionele taksonomiese eenhede (OTE) te bestudeer. Die indeks van die duodenum was tussen 2.14 – 2.59 en het toegeneem tot 2.45 – 3.03 in die seka. Die OTE het ook toegeneem van 21.44 – 28.60 tot 28.30 – 38.00. ‘n Beduidende verskil was slegs waargeneem tussen die OTE waar die eksperimentele groep ‘n laer OTE gehad het as die kontrole groepe. Daar was geen verskil tussen die mikrobiese samestellings van die duodenum nie, maar duidelike patrone en groepe was waargeneem tussen die eksperimentele groep en kontroles van die seka. Aangesien daar geen mortaliteite was nie en alle hoenders gesondheid was, kan afgelei word dat die verandering in die mikrobiese samestelling van die seka nie negatief was nie. Daarom is dit veilig om P. marcusii te gebruik as ‘n bymiddel vir lê-henne. In die finale hoofstuk (Hoofstuk 5) is die kostes verbonde aan die produksie van mikrogeënkapsuleerde P. marcusii in kalsiumalginaat-balletjies, en die ekonomiese haalbaarheid daarvan in die pluimveebedryf, bespreek. Die maandelike koste van 210 g kalsiumalginaat-balletjies beloop tans R2912.88. Die kostes is egter baie duur en nie haalbaar vir ‘n pluimveeboer nie. Moontlike oplossings kan die gebruik van ‘n goedkoper bron van peptone insluit, asook grootskaalse produksie en ‘n verhoogde konsentrasie van die bakterium in die kalsiumalginaat-balletjies.

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