Fractionation and characerisation of a commercial yeast extract to facilitate acceleration of yogurt fermentation

Abstract

English: In order to study the decrease of yogurt fermentation time, the effects of a wide range of supplements on yogurt fermentation time were evaluated. YE was identified as the only supplement which showed potential. Unfortunately it resulted in a product with an unacceptable flavour. It was therefore important to identify and isolate the specific component responsible for the decrease in yogurt fermentation time. YE was fractionated with size exclusion chromatography and it was subsequently determined that the accelerating fraction had a low molecular weight. It was important to establish whether the accelerating component was of mineral, vitamin or amino acid origin. Three cocktails containing the most common minerals, vitamins and amino acids were prepared and their respective effects on yogurt fermentation were determined. Results indicated that when compared to the respective controls, no decrease in fermentation time was observed with the mineral and vitamin cocktails. A decrease in fermentation time was observed with the amino acid cocktail, indicating that the accelerating component present in YE was of amino acid origin. It was however not clear whether it was a single amino acid or a peptide. The accelerating fraction was further analysed by SDS-PAGE and due to no visible bands in the respective region (<1kDa), it could not be analysed with mass spectrometry. The fraction obtained directly after size exclusion chromatography was however analysed by using mass spectrometry in order to determine the total amino acid content of the accelerating fraction after which it was evident that the fraction containing the accelerant contained an abundance of peptides. The individual effects of the 17 identified amino acids were determined in respective yogurt fermentations. Results indicated that no single amino acid was responsible for the decrease in yogurt fermentation time. Although a combination of the 17 amino acids in one fermentation run resulted in a decrease in fermentation time in comparison to unsupplemented yogurt, the decrease was not as considerable as that of the accelerating fraction obtained after size exclusion chromatography. Due to the fact that it was not practical to evaluate the effect of all possible combinations of the 17 amino acids, the combinations that were evaluated were based on literature reports on stimulation of Streptococcus thermophilus growth. Focus was placed on the growth of Streptococcus thermophilus due to this organism being the growth limiting organism between the two starter organisms used for yogurt fermentation. None of the evaluated amino acid combinations decreased yogurt fermentation time, and it was therefore concluded that the accelerating component was a peptide and not a free amino acid. In order to establish the mechanism of acceleration of the isolated YE fraction, it was important to determine whether the addition of YE to milk increased the rate of starter bacteria growth or whether it completed the growth requirements of starter bacteria. The latter could either result in earlier initiation of lactic acid production or in increased levels of lactic acid production. Results indicated that YE had no effect on the total growth rate of the starter bacteria. However, when examining the effect on the respective bacteria individually, it became clear that YE increased Streptococcus thermophilus cell numbers in comparison to the unsupplemented control. Using resazurin, it seems YE increased the metabolic rate of the starter bacteria. Although supplementation of yogurt with YE did not influence combined starter bacteria growth, it influenced lactic acid production. The addition of YE to yogurt resulted in increased lactic acid levels as well as an increase in lactic acid production rate. Lactic acid was also initiated earlier in the fermentation process, resulting in higher lactic acid levels in comparison to unsupplemented yogurt. It was therefore evident from this study that YE did not provide nutrients that are not already present in milk, but rather provided nutrients in a readily available form at the beginning of the yogurt fermentation process resulting in the reduction of the lag phase of lactic acid production.

Afrikaans: Ten einde die afname van jogurt fermentasie tyd aan te spreek, is die effek van 'n wye verskeidenheid van aanvullings op jogurt fermentasie tyd geëvalueer. Gisekstrak is geïdentifiseer as die enigste aanvulling wat potensiaal getoon het alhoewel dit ongelukkig gelei tot 'n produk met onaanvaarbare smaak. Dit was dus noodsaaklik om die spesifieke komponent wat verantwoordelik is vir die afname in jogurt fermentasie tyd, te identifiseer en te isoleer. Gisekstrak is gefraksioneer met grootteuitsluitingschromatografie en dit is daarna vasgestel dat die versnellende fraksie 'n lae molekulêre massa het. Die versnellende fraksie is verder ontleed deur SDS-PAGE en as gevolg van geen sigbare bande in die onderskeie area (< 1kDa), kon dit nie met massaspektrometrie geanaliseer word nie. Die fraksie wat direk na die grootteuitsluitingschromatografie opgevang is, is egter geanaliseer deur die gebruik van massaspektrometrie waarna dit was duidelik was dat die versnellende fraksie 'n oorvloed van peptiede bevat het. Dit was belangrik om vas te stel of die versnellende komponent van mineral-, vitamien- of aminosuur oorsprong is. Drie mengsels met die mees algemene minerale, vitamiene en aminosure is voorberei en hul onderskeie effekte op jogurt fermentasie is bepaal. Resultate het aangedui dat in vergelyking met die onderskeie kontroles, die versnellende komponent teenwoordig in gisekstrak van aminosuur oorsprong is. Wat egter nie duidelik was nie, is of dit 'n enkele aminosuur of 'n peptied is. Ten einde dit te bevestig is die versnellende fraksie verkry na grootteuitsluitingschromatografie gehidroliseer en die aminosuurinhoud is bepaal. Die individuele effekte van die 17 aminosure geïdentifiseer is bepaal in onderskeie jogurt fermentasies. Resultate het aangedui dat geen enkele aminosuur verantwoordelik was vir die afname in jogurt fermentasie tyd nie. Hoewel 'n kombinasie van die 17 aminosure in een fermentasie lopie gelei het tot 'n afname in die fermentasie tyd in vergelyking met gewone jogurt, was die afname in tyd nie so groot soos dié van die versnelling fraksie verkry na grootteuitsluitingschromatografie nie. As gevolg van die feit dat dit prakties nie uitvoerbaar was om die effek van alle moontlike kombinasies van die 17 aminosure te evalueer nie, is die kombinasies wat geëvalueer is gebaseer op literatuurverslae oor die stimulasie van Streptococcus thermophilus groei. Die fokus is op die groei van Streptococcus thermophilus geplaas as gevolg van die feit dat hierdie organisme die groei beperkende organisme tussen die twee suurselorganismes is. Nie een van die geëvalueerde kombinasies het jogurt fermentasie verkort nie, en daar is dus tot die gevolgtrekking gekom dat die versnellende komponent 'n peptied en nie 'n aminosuur is nie. Ten einde die meganisme van gisekstrakversnelling te vestig, was dit belangrik om te bepaal of die toevoeging van gisekstrak tot melk die tempo van groei van bakterieë verhoog en of dit die groeivereistes van suurselbakterieë wat lei tot verhoogde vlakke van melksuur produksie aanvul. Resultate het aangedui dat gisekstrak geen effek op die totale groeitempo van die suurselbakterieë het nie. Maar, tydens die ondersoek van die uitwerking op die onderskeie individuele bakterieë was dit duidelik dat gisekstrak die Streptococcus thermophilus selgetalle verhoog het in vergelyking met die gewone kontrole. Dit was ook duidelik dat gisekstrak die metaboliese tempo van die suurselbakterieë verhoog het. Hoewel die aanvulling van jogurt met gisekstrak nie die groei van gekombineerde suursel bakterie beïnvloed het nie, het dit die melksuurproduksie beïnvloed. Die byvoeging van gisekstrak tot jogurt het gelei tot verhoogde melksuur vlakke, sowel as 'n toename in melksuur produksietempo. Melksuur is ook vroeër gedurende die fermentasie proses geïnisieer wat gelei het tot hoër vlakke melksuur as in die gewone jogurt. Dit was dus duidelik uit hierdie studie dat gisekstrak nie voedingstowwe verskaf wat nie in melk teenwoordig is nie, maar eerder voedingstowwe in 'n geredelik beskikbare vorm verskaf aan die begin van die jogurt fermentasie proses, wat dan die sloerfase van melksuurproduksie verkort.