SYNTHESIS OF LIPIDS BY CERTAIN YEAST STRAINS GROWN ON WHEY PERMEATE

Four yeast strains, Apiotrichum curvatum ATCC 10567, Cryptococcus albidus ATCC 56297, Lipomyces starkeyi ATCC 12659, and Rhodosporidium toruloides ATCC 10788, were grown on whey permeate in order to synthesize lipid. Lipomyces starkeyii ATCC 12659 was identified as a high producer of lipid. It synthesized 36.9% lipids based on dry cell mass at a carbon:nitrogen ratio of 30:1 in shake-flask experiments. The significant (p < 0.05) effect of both carbon:nitrogen ratio and yeast strain was observed on dry cell mass yield and lipid biosynthesis. All yeast strains except Rhodosporidium toruloides ATCC 10788 produced more triacylglycerols than phospholipids.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY RESTRICTION ENDONUCLEASE ANALYSIS OF THEIR MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) was isolated from different strains of brewery yeast and digested with various restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. Of the twenty restriction endonucleases used, only two—Aval and Haelli—produced different restriction fragment patterns when applied to the mtDNA from two strains of Saccharomyces uvarum. The restriction fragment patterns produced by the other eighteen enzymes were identical. Analysis of mtDNA from a strain of Saccharomyces cerevisiae with the same twenty restriction endonucleases revealed several differences with respect to Saccharomyces uvarum. Taken together, these results indicate that restriction endonuclease fragmentation patterns of mtDNA are useful as diagnostic tools for distinguishing strains of ale and larger yeast.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY DNA FINGERPRINTING

A reliable and reproducible non-radioactive DNA fingerprinting technique has been used to differentiate between twelve production brewery yeast strains. The method comprises total DNA preparation from the yeast strains, enzymatic digestion of the DNA and analysis of the DNA fragments by “probing” with known DNA labelled with the plant steroid digoxigenin. A range of probes and several enzymes have been investigated. A successful combination is the restriction endonuclease Hind III and a probe derived from Ty1 (a yeast transposable element). The main advantages of this method are its unambiguity, reproducibility and suitability for routine brewery QC use.     

EFFECTS OF LINOLEIC ACID SUPPLEMENTS ON THE SYNTHESIS BY YEAST OF LIPIDS AND ACETATE ESTERS

The concentrations of acetate esters in beer were reduced by up to 85% by addition of linoleic acid to the fermentation or by pitching with yeast previously enriched with this unsaturated fatty acid. Linoleic acid was rapidly incorporated into yeast lipids and was effective in reducing the rate of ethyl acetate formation within 2 h. Addition of linoleic acid altered the pattern of synthesis of fatty acids by yeast, causing a shift from medium toward long chain acids. Secondly, the amount of squalene in yeast was reduced by up to 70% whereas that of lanosterol was increased threefold. Since total yeast lipid synthesis was reduced by up to 40%, we conclude that less acetylCoA is synthesized in the presence of linoleic acid. Further, high concentrations of linoleic acid decreased the proportion of acetylCoA consumed by the synthesis of acetate esters. Therefore linoleic acid may directly decrease acetate ester synthesis in addition to its effect via reduction of acetylCoA availability.     

PRODUCTION OF ESTERS BY DIFFERENT YEAST STRAINS IN SUGAR FERMENTATIONS

The production of the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₀ fatty acids was investigated in semiaerobic sugar fermentations by 56 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum. The S. cerevisiae yeasts generally produced more esters than the S. uvarum yeasts. Isoamyl acetate was the main component in the ester fractions examined and others in decreasing order, were ethyl caprylate, ethyl caproate, ethyl caprate and phenethyl acetate.     

ASSESSMENT OF POTENTIAL PROBIOTIC PROPERTIES OF LACTIC ACID BACTERIA AND YEAST STRAINS

Four lactic acid bacteria (LAB) and three yeast strains isolated from a traditional Bulgarian cereal-based fermented beverage were assessed for potential probiotic properties. Acid and bile resistance, antipathogenic activity and antibiotic resistance of the strains were evaluated. Tolerance to low pH values (2.0-3.0) and high bile concentrations (0.2-2.0%) of the LAB and yeast strains varied, but all strains kept viable throughout the experiments. Antagonistic activity towards most of the eight test-pathogens was observed for one LAB (Lactobacillus plantarum B28) and two yeast strains (Candida rugosa Y28 and Candida lambica Y30). Antibiotic resistance (39 antibiotics) of the LAB strains was variable, but showed their potential for therapeutic application.     

SPHEROPLAST FUSION OF BREWER'S YEAST STRAINS

Spheroplasts of brewing polyploid yeast strains have been successfully fused with spheroplasts of haploid yeast strains. After regeneration of the cell wall, stable fusion recombinants were isolated. Genetic analysis of these recombinants revealed that they contained the genotype of both parents, sporulated well with each ascus containing four spores and were indeed diploid. Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.     

ASSESSMENT OF POTENTIAL PROBIOTIC PROPERTIES OF LACTIC ACID BACTERIA AND YEAST STRAINS

ABSTRACT

Four lactic acid bacteria (LAB) and three yeast strains isolated from a traditional Bulgarian cereal-based fermented beverage were assessed for potential probiotic properties. Acid and bile resistance, antipathogenic activity and antibiotic resistance of the strains were evaluated. Tolerance to low pH values (2.0–3.0) and high bile concentrations (0.2–2.0%) of the LAB and yeast strains varied, but all strains kept viable throughout the experiments. Antagonistic activity towards most of the eight test-pathogens was observed for one LAB (Lactobacillus plantarum B28) and two yeast strains (Candida rugosa Y28 and Candida lambica Y30). Antibiotic resistance (39 antibiotics) of the LAB strains was variable, but showed their potential for therapeutic application.     

EFFECT OF LIPIDS AND OXYGEN ON YEAST GROWTH AND THE BIOSYNTHESIS OF ACETOIN DURING FERMENTATION

Brewery yeast needs traces of oxygen for the biosynthesis of unsaturated fatty acids and ergosterol. Owing to the increase in cell mass during primary fermentation the concentrations of these essential lipids decrease and thereby affect the physiological condition of the yeast. When the sterol concentration of whole cells has decreased to 0.2 to 0.3 mg per 100 mg dry yeast, the yeast changes its metabolism. This metabolic change is revealed by a decrease in acetoin concentration. The absorption of wort nutrients and consequently the efficiency of growth is at this point also greatly reduced. The ratio between yeast growth and mole ethanol formed (i.e. the molar growth yield) decreases greatly during wort fermentation. A close correlation between molar growth yield and the change in acetoin metabolism can be observed. This metabolic change occurs when the ratio between yeast growth and ethanol formed is in the range of 8.3 to 9.1, averaging 8.7 g/mole.     

VARIATION OF CELL WALL CONTENT IN FLOCCULENT AND NON-FLOCCULENT YEAST STRAINS

Estimation of the wall content of a number of flocculent and non-flocculent yeast strains taken in both the growth and stationary phases reveals that flocculent yeasts almost invariably increase their wall content to a greater extent as the cells pass to the stationary phase than do non-flocculent strains. This variation of increase is observed whether the yeasts are grown in hopped wort or in a synthetic malt-yeast-peptone-glucose medium. There are considerable variations in wall content from strain to strain in both types of yeast. Cells of flocculent strains are more difficult to disrupt than those of powdery strains. The nitrogen, phosphate and carbohydrate contents of the walls of all strains varied between the growth and stationary phases but no significant trends were noted in any of the components which could be related to flocculent or non-flocculent behaviour.     

ATTACHMENT OF CERTAIN BACTERIAL STRAINS TO CHICKEN AND BEEF MEAT

The attachment of bacteria to chicken and beef meat with and without fascia was studied. It was found that bacteria attach readily to the meat surfaces. The kinetics of attachment depend on the bacterial strain, as well as on the meat surface. Of the bacteria tested, Pseudomonas EBT/2/143 attached most readily to all meat surfaces examined. Chicken breast with fascia was the best surface for attachment.
A study was also made of the effects of storage on the multiplication of the attached bacteria and the feasibility of removing them. A high level of Salmonella infantis was found in comparison to the other bacteria after 24 h of storage at 20°C.
More investigations are needed to find out whether other serotypes of salmonellae will multiply to similar levels on these surfaces.
After micro-colonies began to form, the newly generated bacteria were easy to remove. The hygienic consequence of this phenomenon is discussed.     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

CHEMICAL CHARACTERIZATION OF DIFFERENCES BETWEEN ALES AND LAGERS

The chemical identification of the substances responsible for differences between ales and lagers has been investigated, using new as well as established techniques. The two types of beer are broadly similar in composition, but lagers are characterized particularly by higher levels of certain sulphur compounds, and ales by higher levels of certain heterocyclic compounds. Several compounds have been identified for the first time as beer components.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

RESTRICTION ANALYSIS OF 2 μm DNA FROM DIVERSE STRAINS OF YEAST

2 μm plasmids from an ale yeast, a lager yeast and a strain of Saccharomyces diastaticus have been characterised by restriction analysis. Plasmids were similar to each other and conformed to a pattern which has been reported for well-characterised genetic stocks. The structure of 2 μm DNA, therefore, has been conserved in diverse strains of Saccharomyces. Preparative procedures used here are likely to be applicable to the detection and characterisation of plasmid DNA from a range of yeast genera. The apparent yield of 2 μm was increased when strains were grown at elevated temperature.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

CHEMICAL AND SENSORY CHARACTERIZATION OF COMMERCIAL SAUERKRAUT1

Canned sauerkraut from eight U.S. companies was analyzed for salt, titra-table acidity (TA), fermentation substrates and end products, volatile sulfur compounds and sensory characteristics. The TA ranged from 0.9–1.5%, while salt content ranged from 1.4–2.0%, which was lower than in previous surveys. High performance liquid chromatography (HPLC) was used to monitor lactic, acetic, malic, succinic, propionic and butyric acids; mannitol, ethanol, propanol, glycerol, glucose, fructose and sucrose. Low concentrations of propionic acid, propanol and glycerol were found. These three compounds are not characteristic of lactic acid fermentations. No butyric acid was detected. GC analysis revealed seven main sulfur compounds (hydrogen sulfide, methanethiol, dimethyl sulfide, carbon disulfide, dimethyl disulfide, allyl isothiocyanate (AITC) and dimethyl trisulfide) and six other organic compounds (methanol, ethanol, n-propanol, 2propanol, acetaldehyde and ethyl acetate) in the headspace of sauerkraut juice. A profile panel characterized aroma, flavor and after-taste of sauerkraut with ten distinct notes. The sour, sulfur and salt notes had the greatest impact on sauerkraut flavor.     

DIFFERENTIATION OF ALE AND LAGER FLAVOURS BY PRINCIPAL COMPONENTS ANALYSIS OF FLAVOUR CHARACTERIZATION DATA

The application of principal components analysis to flavour characterization data has been examined by comparing and contrasting 32 beer samples. These comprised two samples of each of 16 brands of beer selected to represent 4 different types, contrasting ales and lager beers of two different strengths. Two-dimensional plots of results using the first two principal components as axes showed resolution of the four groups of beers and the close proximity of the majority of the duplicate samples. Differences between samples thus revealed are in accordance with known differences between the beer flavours.