Wpływ ph podłoża hodowlanego oraz glicerolu jako źródła węgla na biosyntezę polimerów ściany komórkowej drożdży Candida utilis i Kluyveromyces fragilis

Celem badań było określenie możliwości wykorzystania glicerolu w hodowli drożdży Candida utilis oraz Kluyveromyces fragilis ukierunkowanej na biosyntezę β-glukanu i/lub mannoprotein tworzących strukturę ściany komórkowej tych grzybów. Preparaty ścian uzyskiwano na drodze autolizy komórek drożdży. Poddawano je następnie frakcjonowaniu w warunkach alkalicznych na poszczególne polisacharydy. Stwierdzono zróżnicowanie zawartości β(1,3)-/β(1,6)-glukanów i mannoprotein w ścianach badanych drożdży, zależne od szczepu, stężenia glicerolu oraz pH podłoży hodowlanych. Istotne zwiększenie zawartości cukrów ogółem i β(1,3)-/β(1,6)-glukanów w ścianie drożdży Candida utilis ATTC 9950 odnotowano po hodowli w podłożu o pH 4,0 zawierającym 2% glicerolu. W preparatach ścian omawianego szczepu stwierdzono wówczas około 75% cukrów ogółem i około 53% β(1,3)-/(1,6)-glukanu. Drożdże Kluyveromyces fragilis R11 okazały się lepszym źródłem mannoprotein. W preparatach komórek z podłoży o pH 7, zawierających 3 lub 5% glicerolu, odnotowano około 30,5% tych polimerów.

The structure of yeast cell wall is mainly composed of polymers such as β-glucans and α-mannan (present as mannoprotein). It is well known that yeast β-glucans and mannoprotein exhibit a range of bioactive properties in humans and animals, like anti-toxic, anti-mutagenic, anticancerous and anti-oxidative activity, stimulation of immunological response as well as antibacterial properties. The content of β-glucans and mannoprotein in cell wall of unicellular fungi is connected to growth conditions. This work reports on the infl uence of glycerol as a carbon source and pH of the medium on structural cell wall polymers (β-glucan and mannoprotein) biosynthesis of Candida utilis ATTC 9950 and Kluyveromyces fragilis R11. The experimental cultivations of investigated yeast strains were performed in control YPD medium and in a model mediums where glucose was replaced with glycerol in the amount of 2, 3 and 5% (w/v). All mediums were prepared in three pH variants i.e. 4.0, 5.0 and 7.0. The preparations of cell walls of yeasts from the experimental cultivations were achieved via 24-h cell autolysis. The obtained cell wall preparations were subjected to fractionation on particular structural polysaccharides, i.e. β(1,3)-glucan, β(1,6)-glucan and mannoproteiny, by extraction in alkaline conditions. The content of reducing carbohydrates (as glucose) was analyzed using colorimetric method (λ = 540 nm) with 3,5-dinitrosalicylic acid after hydrolysis of particular fractions in acidic conditions. Results of the investigation demonstrated that cultivation of Candida utilis ATTC 9950 and Kluyveromyces fragilis R11 on glicerol, low-cost substrates from biodiesel production, may intensify the biosynthesis of cell wall polymers. The tendency depended on cultivation medium and strain. For yeast of genus Candida the portion of polysaccharides in total, as well as β(1,3)/(1,6)-glucan content were highest after cultivation on medium containing 2% of glycerol and pH 4.0. It was 75% and 53% respectively. Depending on growth conditions, the Candida utilis ATTC 9950 strain contained from 2 to 4 times more β(1,3)/(1,6)-glucan in cell wall structure comparing with Kluyveromyces fragilis yeasts. At the same time, Kluyveromyces fragilis R11 strain was more effi cient source of mannoprotein comparing with Candida yeast. Preparations of cell walls of Kluyveromyces fragilis after yeast cultivation in mediums with 3 and 5% of glycerol pH 7.0 contained app. 30.5% of mannoproteins. The results confi rmed that cultivation in medium with glycerol as a carbon source contributed to obtaining the biomass of Candida utilis and Kluyveromyces fragilis with increased portion of functional cell wall polymers. Further studies should be oriented towards an optimization of cultivation conditions for effi cient biosynthesis of β-glucans or mannoprotein and to establish their functional properties.