Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

Screening Test of Microbes

Potent bacteria for production of chillproofing enzyme were isolated during screening tests on 1670 strains of microorganisms.

All but one of these bacteria were classified as Serratia marcescens and the exceptional strain was tentatively designated as B–103. These bacteria produced an extracellular proteolytic enzyme which prevented chill haze of beer.     

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12° all-malt wort supplemented with sorbitol to reach osmolarity equal to 16° and 20°. Three pitchings were performed in 12°/12°/12°, 16°/16°/12°, and 20°/20°/12° worts. Fermentations in 16° and 20° worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd “back to normal” pitching into 12° wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16° and 20° worts was delayed but increased about two times at fermentation end relative to that in 12° wort. Third “back-to-normal” pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.     

Accumulation and cellular distribution of zinc by brewing yeast

This study focused on the interactions between yeast and zinc in relation to beer fermentations. Yeast accumulation of zinc from growth media, including malt wort, was found to be rapid following inoculation with a brewing strain of Saccharomyces carlsbergensis. In contrast, at the onset of the fermentation, the uptake of other divalent cations such as magnesium and calcium was not as pronounced compared with zinc. At the end of fermentation, both growth media and yeast cells became zinc-depleted, the latter due to dilution of zinc to daughter cells following growth and cell division. In addition, in brewing fermenters, the levels of intracellular zinc were much higher in suspended yeast cells compared with cells that sedimented in the yeast cone at the end of fermentation. This may result in impaired yeast performance in subsequent fermentations if yeast is recycled into low zinc media and if the sub-population is composed by zinc-depleted daughter cells. Cellular uptake of zinc was mediated by a metabolism-dependent mechanism as evidenced by impaired uptake following heat shock. Zinc was thereafter localised in the yeast cell vacuole. As industrial fermentation processes may occasionally be suppressed due to zinc deficiencies, the findings of this study are pertinent for several yeast-based industries, especially beer production.     

Identification of Potent Bacterium for Production of Chillproofing Enzyme

A bacterium was isolated which produced an extracellular proteolytic enzyme preventing chill haze of beer. This strain was named B–103.

Taxonomical studies on strain B–103 indicated that it was Gram-negative, aerobic, motile, and catalase-positive. Cells were single rods (0.2~0.6×1.0~1.8 μ) with a peritrichous flagella. The bacterium produced nitrite from nitrate, extracellular DNase and 5′-nucleotide from nucleoside. It was capable of nitrate respiration and of oxidative or fermentative cleavage of carbohydrates.

From these characteristics, it was identified as a strain of Serratia marcescens.     

Filtration, haze and foam characteristics of fermented wort mediated by yeast strain

AIMS: To investigate the influence of the choice of yeast strain on the haze, shelf life, filterability and foam quality characteristics of fermented products. METHODS AND RESULTS: Twelve strains were used to ferment a chemically defined wort and hopped ale or stout wort. Fermented products were assessed for foam using the Rudin apparatus, and filterability and haze characteristics using the European Brewing Convention methods, to reveal differences in these parameters as a consequence of the choice of yeast strain and growth medium. CONCLUSIONS: Under the conditions used, the choice of strain of Saccharomyces cerevisiae effecting the primary fermentation has an impact on all of the parameters investigated, most notably when the fermentation medium is devoid of macromolecular material. SIGNIFICANCE AND IMPACT OF THE STUDY: The filtration of fermented products has a large cost implication for many brewers and wine makers, and the haze of the resulting filtrate is a key quality criterion. Also of importance to the quality of beer and some wines is the foaming and head retention of these beverages. The foam characteristics, filterability and potential for haze formation in a fermented product have long been known to be dependant on the raw materials used, as well as other production parameters. The choice of Saccharomyces cerevisiae strain used to ferment has itself been shown here to influence these parameters.     

Identification of two key genes controlling chill haze stability of beer in barley (Hordeum vulgare L)

Background

In bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer.

Results

In this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding.

Conclusions

We identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1683-1) contains supplementary material, which is available to authorized users.     

啤酒酵母ECM25/YJL201W基因敲除对啤酒风味稳定性的影响

以pUG6为模板, 设计含有与ECM25基因两侧序列同源的长引物, 构建了带有卡那抗性基因(kanMX)破坏盒, 转化啤酒酵母G-03, 获得一株G-03/a转化菌, 遗传稳定性良好, 测序结果证实ECM25基因敲除是成功的。有氧条件下11oC和28oC培养时转化菌G-03/a的胞外谷胱甘肽(GSH)分泌量在对数生长期分别比原菌高21.4%和14.7%。在锥形瓶中连续发酵4代后, 与原菌株相比, 转化菌G-03/a发酵液、成品酒中GSH含量分别提高32.1%和13.8%, 发酵液和成品啤酒SI系数分别提高7.7%和5.3%, 成品啤酒RSV值提高45.0%。EBC管发酵6 d后, 与原菌株相比, 转化菌G-03/a发酵液中GSH含量提高34.0%。转化菌G-03/a与G-03所酿制成品啤酒的常规指标没有显著差别。表明G-03/a是一株具有抗老化能力的优良啤酒酵母, 能够提高啤酒的风味稳定性。     

几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。     

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.     

Nutrient depletion modifies cell wall adsorption activity of wine yeast

Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions—early, mid, ‘adjusted’ for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in ‘adjusted’ for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters.     

高温高浓发酵工业啤酒酵母菌种的构建

高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

Continuous beer production was investigated in a high cell-density culture system which consisted of two stages for the fermentation and sedimentation of yeast cells. The continuous culture was carried out for a fermentation time of 5,500 h without contamination, at varying dilution rates and fermentation temperatures in the ranges of 0.017-0.033 h⁻¹ and 6.5–8.5°C, respectively. This process was found to be suitable for continuous and stable beer brewing. Under these conditions, the cell concentration in the first stage was about 80 times as high as that in the exit of the second stage. Concentrations of viable cells, sugar and ethanol were maintained at 1.3 × 10⁹ cells/ml, 25 and 36 g/l, respectively, and were hardly affected by fermentation temperature. Concentrations of ethyl acetate, isoamyl alcohol and isoamyl acetate were similar in the fermentation temperature ranges of 6.5–8.5°C, and the amounts at a fermentation temperature of 7°C were comparable to those of lager-type beer. Diacetyl flavor, which is known to be an effluent component that causes deterioration in the second stag e (young beer), was maintained at 1.2 ppm at a dilution rate and fermentation temperature of 0.022 h⁻¹ and 7°C, respectively. The diacetyl flavor was due to the accumulation of vicinal diketone, the precursor of which is acetohydroxy acid. The acetohydroxy acid was converted to vicinal diketone by pretreatment at 60°C for 30 min. The vicinal diketone was then consumed by the yeast during after-fermentation at a fermentation temperature of 3°C. Using this method, total vicinal diketone decreased below 0.3 ppm for an after-fermentation time of 6.8 h, which was 225 times as fast as that of after-fermentation without the pretreatment. This process may make it possible to achieve continuous beer fermentation from the fermentation stage to after-fermentation for diacetyl removal.     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

The Structure of Calreticulin C-terminal Domain Is Modulated by Physiological Variations of Calcium Concentration

Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.     

Influence of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains on wine total antioxidant capacity evaluated by photochemiluminescence

A preliminary investigation on 20 Aglianico del Vulture commercial wines from the Basilicata region proved the existence of a significant variability in total antioxidant capacity which can exert a potential impact on wine quality. Nineteen Saccharomyces cerevisiae strains were tested in Aglianico del Vulture on pilot scale fermentation and the experimental wines obtained were evaluated for the antioxidant capacity, ethanol and total polyphenols. At the ninth day of fermentation the experimental wines had an antioxidant capacity, measured by photochemiluminescence, between 2.88 and 6.25 mM of ascorbic acid equivalent, ethanol concentration, measured by GC, between 5.49% and 10.99% and total polyphenols, determined by Folin Ciocalteau reagent, from 1153 to 1867 mg catechins/l. After 12 days the total antioxidant capacity was increased in most wines but decreased in some wines. These results, statistically analysed by principal component analysis, revealed a significant influence of S. cerevisiae strain on total antioxidant capacity and total polyphenols content of wine.     

Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast

Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc-ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc-ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc-ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.     

Enhanced direct ethanol production by cofactor optimization of cell surface‐displayed xylose isomerase in yeast

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d ‐xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC‐displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co²⁺) especially enhanced the activity by 46‐fold. Co²⁺ also contributed to d ‐xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0‐ and 2.7‐fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC‐displaying yeast showed the faster d ‐xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co‐culture system. A xylan‐degrading yeast strain was established by displaying two kinds of xylanases; endo‐1,4‐β‐xylanase (Xyn11B) from Saccharophagus degradans, and β‐xylosidase (XlnD) from Aspergillus niger. The yeast co‐culture system enabled fine‐tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)‐displaying yeast and XylC‐displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10¹³: 1.39 × 10¹³: 2.78 × 10¹³ molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co‐culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068–1076, 2017     

Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain

The sulfuric acid hydrolysate of lignocellulosic biomass, such as wood chips, from the forest industry is an important material for fuel bioethanol production. In this study, we constructed a recombinant yeast strain that can ferment xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from Pichia stipitis, and xylulokinase from Saccharomyces cerevisiae and a gene for displaying β-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of the sulfuric acid hydrolysate of wood chips, xylose and cellooligosaccharides were completely fermented after 36 h by the recombinant strain, and then about 30 g/l ethanol was produced from 73 g/l total sugar added at the beginning. In this case, the ethanol yield of this recombinant yeast was much higher than that of the control yeast. These results demonstrate that the fermentation of the lignocellulose hydrolysate is performed efficiently by the recombinant Saccharomyces strain with abilities for xylose assimilation and cellooligosaccharide degradation.