MICROBIOLOGICAL SHELF‐LIFE STUDIES ON COMMERCIALLY MANUFACTURED YEAST

ABSTRACT

During three replicate studies, cream, compressed and dry yeast samples were stored for 21 days at 4, 10, 25 and 37C. At 3‐day intervals, bacteria were quantified using standard plate counting procedures, and 1,044 predominant colonies isolated and characterized. The highest counts of aerobic bacteria and Enterococcus spp., up to 7 and 8 log colony‐forming units (cfu) per mL or g, were found when cream and compressed yeast samples were exposed to elevated temperatures (25 and 37C), while lower counts (4 and 6 log cfu/mL or g) were obtained from refrigerated samples (4C). At 10C, counts of aerobic bacteria and Enterococcus spp. increased from 4 to 7 log cfu/mL or g, highlighting the importance of temperature control during the storage and distribution of perishable cream and compressed yeast. Vacuum‐packaging of dry yeast reduced the growth of aerobic populations. Throughout storage, Lactobacilli dominated the bacterial populations in both cream and compressed yeast (45 to 78%), while Enterococcaceae predominated in vacuum‐packaged dry yeast (54 to 68%), suggesting they were primarily responsible for the spoilage of commercial yeast products.

PRACTICAL APPLICATIONS

In addition to producing high quality yeast for baking and brewing, it is an important objective for yeast manufacturers to increase product shelf life. Temperatures chosen for this shelf‐life study simulated various elevated temperatures of practical use for yeast processors and downstream consumers. For example, 4C (recommended refrigeration temperature), 10C (potential abuse temperature during transportation), 25C (wine‐making temperature) and 37C (temperatures often encountered in bakeries). Results from our study showed that storage temperature and time considerably influenced bacterial growth patterns associated with commercially manufactured fresh yeast. Bacterial spoilage and sensory deterioration were observed during lengthened storage periods, and especially at higher storage temperatures. Even when stored at 10C, cream and compressed yeast samples became bacteriologically and visually spoiled. It was therefore apparent that any break in the cold chain during processing, storage or distribution could affect the development of spoilage bacteria resulting in shortened product shelf life of commercially manufactured fresh yeast. Conversely, this study found vacuum‐packaged dry yeast to be the most bacteriologically stable manufactured product.     

Rapid Removal of Lactic Acid from Wastewater by a Flocculent Yeast

A process has been developed for rapid removal of the lactic acid in sauerkraut processing wastewater by a flocculent strain of Kluyveromyces fragilis. Under optimum conditions, the yeast reduced the lactic acid by 95–97% in 4 hr. and the net increase in yeast dry weight was slightly higher than 1,000 mg/liter. The yield of yeast based on the amount of lactic acid consumed was approximately 34%. The yeast cells settled rapidly and compacted well and greater than 99% of them settled in 1 hr. The results of this study indicate that using K. fragilis to reduce the lactic acid in the wastewater may have economic value in waste disposal and in the production of feed yeast.     

Yeast derived from lignocellulosic biomass as a sustainable feed resource for use in aquaculture

The global expansion in aquaculture production implies an emerging need of suitable and sustainable protein sources. Currently, the fish feed industry is dependent on high‐quality protein sources of marine and plant origin. Yeast derived from processing of low‐value and non‐food lignocellulosic biomass is a potential sustainable source of protein in fish diets. Following enzymatic hydrolysis, the hexose and pentose sugars of lignocellulosic substrates and supplementary nutrients can be converted into protein‐rich yeast biomass by fermentation. Studies have shown that yeasts such as Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus have favourable amino acid composition and excellent properties as protein sources in diets for fish, including carnivorous species such as Atlantic salmon and rainbow trout. Suitable downstream processing of the biomass to disrupt cell walls is required to secure high nutrient digestibility. A number of studies have shown various immunological and health benefits from feeding fish low levels of yeast and yeast‐derived cell wall fractions. This review summarises current literature on the potential of yeast from lignocellulosic biomass as an alternative protein source for the aquaculture industry. It is concluded that further research and development within yeast production can be important to secure the future sustainability and economic viability of intensive aquaculture. © 2016 Society of Chemical Industry     

Disc Stack Centrifuge Operating Parameters and Their Impact on Yeast Physiology

Hydrodynamic stresses imposed on brewing cells of Saccharomyces cerevisiae during beer processing can have a detrimental impact on beer quality. The use of centrifuges has become an efficient way to increase brewery throughput as they decrease clarification times and improve fermenter and tank conditioning efficiency. The effect of a disc stack centrifuge on yeast and beer physical stability has been investigated. In this study, a commercial ale yeast strain has been subjected to different operating conditions during centrifugation. Cell viability and intracellular pH decreased due to processing conditions encountered during yeast cropping with a centrifuge. A relationship has been established that yeast cell wall mannan, an unfilterable haze constituent, as a function of G‐force and centrifugation cycles, is released from the cell wall while concurrently, particle sizes between 0.5‐2.8 μm and beer haze increased. Furthermore, yeast intracellular glycogen and trehalose levels were depleted as a result of centrifugation.     

Effect of various types of fermentation on in vitro protein and starch digestibility of differently processed pearl millet

Pearl millet (Pennisetum typhoideum) grains were fermented with Lactobacilli and yeast alone, in combination and with natural flora at 30 °C for 48 h after giving various processing treatments viz, fine and coarse grinding, soaking, debranning, dry heat treatment, germination and autoclaving after adding of water. Fermentation was carried out with Lactobacillus acidophilus and Rhodotorula isolated from naturally fermented pearl millet and Lactobacillus acidophilus, Candida utilis and natural fermentation using freshly ground pearl millet flour as inoculum. All the processing treatments except coarse grinding improved the protein and starch digestibility. Autoclaving enhanced the digestibilities of processed samples which was further improved by different types of fermentation, the maximum being in case of germinated and naturally fermented pearl millet. A combination of Lactobacilli and yeast was more effective in increasing the protein as well as starch digestibility as compared to pure culture fermentation.     

The Biotechnology of Wine Yeast

Abstract

The production of wine from grapes is one of the world's oldest biotechnological processes. The alcoholic fermentation, the conversion of grape sugars to ethanol, is conducted by yeast of the genus Saccharomyces. In addition to wide-ranging roles in beverage and food processing, Saccharomyces is also a premier research model system because of its genetic tractability. Its genome has been sequenced, and an extensive array of molecular technologies has been developed for the genetic manipulation of this organism. In spite of the availability of these molecular tools, genetically engineered yeast strains are not yet in use in the commercial wine industry. Anxiety over consumer acceptance is one reason genetically modified strains are not employed, but there are other concerns as well. Wine production is unique in many respects. The wine production process is not conducted under sterile conditions, and any modified organism has the potential to become an enduring resident of the winery flora. Further, wine production is both science and art. There is concern that many of the proposed genetic modifications of yeast are designed to correct processing errors or incompetence of the winemaker rather than being useful tools to enhance the artistry of wine making. In spite of these concerns, the efficacy of genetic modification of wine yeast has been demonstrated.     

Aneuploidy,copy number variation and unique chromosomal structures in bottom‐fermenting yeast revealed by array‐CGH

DNA microarray for comparative genome hybridization (CGH) of bottom‐fermenting yeast was performed based on our in‐house DNA sequence data. Aneuploidy, copy number variation and unique chromosomal structures were observed among bottom‐fermenting yeast strains. Our array experiments revealed a correlation between copy number variation and mRNA expression levels. Chromosomal structures in a Saccharomyces carlsbergensis‐type strain and in a S. monacensis‐type strain that both belong to S. pastorianus phylogenetically differed greatly from those in contemporary industrial bottom‐fermenting yeast strains. The knowledge gained in this study contributes to a more precise genomic characterization of bottom‐fermenting yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae

The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducible GAL7 promoter. The enzyme α-galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP. In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned. In most cases the genes are efficiently expressed and the products secreted. The expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non-yeast source. The latter requires a separate processing step after yeast cultivation to obtain mature AFP. In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP-oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers.     

ZYMOMONAS AND ACETALDEHYDE LEVELS IN BEER

High acetaldehyde levels in beers during processing were found to be due to infection with Zymomonas anaerobia. Acetaldehyde only appeared in the absence of yeast and the level was correlated with the concentration of Z. anaerobia as determined by the time taken to produce hydrogen sulphide in a primed beer forcing test.     

The Symba Process

In processing potatoes, wheat, tapioca, maize or other raw materials containing starch, considerable amounts of valuable substances are unavoidably lost into the waste-streams. The Symba process permits utilisation of these wastes. It converts starch and other carbohydrates into Candida and Torula yeast by the symbiotic growth of two yeasts, Endomycopsis fibuliger and Candida utilis, on these materials. Endomycopsis produces the amylases which break down the starch into sugars. Candida utilis being unable to feed on starch, quickly “steals” the sugar, thereby growing and producing a yeast product which consists mainly of Candida utilis. By this symbiotic action most of the carbohydrates in the starchy waste streams are utilised. The process could be applied for waste waters from the manufacture of many different products. The first commercial plant was built in Sweden at a factory processing potatoes — mostly in the form of potato granules and “French Fries”. Waste streams as well as solid wastes from the factory are treated, streams are purified to 90%.     

Identification and characterization of a drug‐sensitive strain enables puromycin‐based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6?, pdr1? and pdr3? (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright © 2014 John Wiley & Sons, Ltd.     

History of genome editing in yeast

For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30–40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non‐conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high‐throughput PCR‐based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this ‘Budding topic’ we discuss the significant developments of genome editing in yeast, mainly focusing on Cre‐loxP mediated recombination, delitto perfetto and CRISPR/Cas.     

Characterization and quantification of folates produced by yeast strains isolated from kefir granules

For the first time to our knowledge, distribution and content of individual folate forms in kefir yeast strains were investigated. This was done using a validated method based on reversed-phase high performance liquid chromatography (HPLC) with fluorescence and diode array (DAD) detection. Eight kefir yeast strains, belonging to different Candida and Saccharomyces species, were isolated from Russian kefir granules. They were grown in synthetic media at standardized conditions before analysis. The average folate content for these yeast strains was 10,780±550 μg/100 g dry matter. In all yeast strains tested, the most abundant folate forms as percentages were 5-methyltetrahydrofolate (43–59%), and 5-formyltetrahydrofolate (23–38%), whereas tetrahydrafolate occurred in a lesser proportion (19–23%).     

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end‐products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast‐based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1·0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA‐1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA‐1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA‐1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1_P‐MFα1_S‐pedA‐ADH1_T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1_P) and terminator (ADH1_T). Secretion of the PED1‐encoded pediocin PA‐1 was directed by the yeast mating pheromone α‐factor's secretion signal (MFα1_S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4·6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria. Copyright © 1999 John Wiley & Sons, Ltd.     

The Sequence of 32 kb on the Left Arm of Yeast Chromosome XII Reveals Six Known Genes,a New Member of the Seripauperins Family and a New ABC Transporter Homologous to the Human Multidrug Resistance Protein

The analysis of a 32 kb DNA fragment from cosmid 2G12 on the left arm of chromosome XII identifies 14 open reading frames (ORFs) numbered L0948 to L1325, a new tRNA for proline, a delta remnant and two putative ARS. Six ORFs have been previously identified: HSP104, SSA2, SPA2, KNS1, DPS1/APS and SDC25. Three putative ORFs have significant homology with known proteins: L0968 is a new member of the very large ‘seripauperins’ family, comprising at least 20 yeast members; L1313 is a new ABC transporter highly homologous to the yeast cadmium resistance protein Ycf1p and to the human multidrug resistance protein hMRP1; the C-terminal part of L1325 present in our sequence is very homologous to the fruit fly abdominal segment formation protein Pumilio. Finally, two ORFs, L1201 and L1205, have weak homology with two yeast hypothetical proteins of unknown function identified by the yeast systematic sequencing genome. Since our nucleotide sequence overlaps by 11·6 kb the cosmid 2B18 sequenced by Miosga and Zimmerman (1996) on the right end, we have not reported here the analysis of the ORFs L1313, L1321 and L1325. The complete nucleotide sequence of 32,088 bp and the deduced ORFs were submitted to the EMBL database under Accession Number X97560.© 1997 John Wiley & Sons, Ltd.     

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-α-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-α-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.     

Microbiological,chemical and sensory aspects of bread supplemented with different percentages of the culinary mushroom Pleurotus eryngii in powder form

Pleurotus eryngii (DC.) Quél. powder was used in bread production. Three dough trials (0, 5 and 10% of mushroom) were obtained with commercial baker's yeast. P. eryngii powder was first tested against several yeast species; 10% P. eryngii trial was characterised by the highest pH and total titratable acidity. P. eryngii did not influence negatively the fermentation process, since all trials reached yeast levels of 10⁸ CFU g⁻¹. Mushroom powder decreased bread height and softness, increased crust redness and crumb void fraction and cell density and, although the breads were scored diverse, the overall assessment was comparable. The final breads provided higher concentrations of thiamin, riboflavin and pantothenic acid than control breads and, mostly importantly, supplied biotin, cobalamin and cholecalciferol generally absent in wheat bread. P. eryngii can be cultivated on food residues. Thus, its inclusion in functional bread production represents an optimal strategy for the valorisation of food processing by-products.     

CONTAMINATION OF IMMOBILIZED YEAST BIOREACTORS

Microbiological contamination of immobilized yeast bioreactors for lagering of beer was studied. Four common brewery contaminants and two contaminants isolated from the continuous processing system were tested for their ability to survive and grow in the packed bed DEAE-cellulose bioreactors. Bacterial contaminants were washed out within less than six weeks without causing any alterations in the flavour composition of the beer. The initial contamination levels were 10⁴–10⁶ cells ml^?1. Gram positive lactic acid bacteria, Lactobacillus brevis and Pediococcus damnosus, were more persistent than a Gram negative wort bacterium, Enterobacter agglomerans. The wild yeasts Saccharomyces cerevisiae (ex. diastaticus) and Candida krusei were the most persistent contaminants and the only ones able to grow in the reactors. Another wild yeast, Rhodotorula rubra, did not survive in competition with brewer's yeast and was washed out within a few weeks. C. krusei and R. rubra were previously isolated from a spontaneously contaminated system. None of the contaminants caused formation of phenolic or other off-flavour compounds in detectable levels.