活性干酵母在杨梅酒酿制中的应用

研究了活性干酵母应用于发酵型杨梅酒的特性。试验结果表明 ,SO2 浓度和活性干酵母的添加量显著影响杨梅酒的发酵速率和口感。文中采用了单因素发酵性能试验和比较试验。结果表明 ,当发酵用杨梅原汁SO2 质量浓度在 5 0～ 10 0mg/L ,活性干酵母的添加量在10 6 ～ 10 7/mL条件下 ,杨梅汁发酵迅速 ,得到的酒液品质与使用As 14 5 0鲜酵母发酵得到的杨梅酒相近 ,口感较佳。活性干酵母适用于杨梅果酒的酿造     

Dominance and influence of selected Saccharomyces cerevisiae strains on the analytical profile of craft beer refermentation

This study investigated the influence of Saccharomyces cerevisiae strains inoculated at the refermentation phase on the analytical profile of craft beer. After preliminary screening of 33 S. cerevisiae strains, four were selected and used in bottle refermentation trials. To attribute the flavour profile of the refermented beers to the inoculated strains, molecular characterization was carried out using a mini‐satellite protocol of amplification with inter‐δ primers. Fingerprinting analysis of 500 isolates showed that all of the inoculated strains used in the bottle refermentation dominated the process. The main analytical characters across the bottle refermentation trials were similar, while there were distinctive and significant variations in the volatile compounds. In particular, when compared with the starter strain used as control, the bottle refermentation trials using the DBVPG 2170, DBVPG 2187 and L951 S. cerevisiae strains showed significantly higher levels of the volatile compounds that are responsible for fruity and flowery aromas (i.e. isoamyl acetate, ethyl octanoate, ethyl dodecanoate phenyl ethyl acetate, β‐phenyl ethanol). This study indicates that the proper refermentation process results in the dominance of the selected starter strain, which produces fermentation compounds that show a specific analytical profile and give the craft beer its distinctive bioflavour. Copyright © 2014 The Institute of Brewing & Distilling     

CHILL PROOFING BY A STRAIN OF SACCHAROMYCES CEREVISIAE SECRETING PROTEOLYTIC ENZYMES

The attempted exploitation of a strain of yeast with extracellular protease activity in the fermentation of brewers' wort is described. S. cerevisiae strain YS01 was previously shown to secrete proteases with activity against a variety of substrates. It is shown here that these included some of the tannin precipitable proteins involved in the formation of chill haze in beer and consequently, more stable beers resulted from incubation with added YS01 cell-free culture supernatants. The proteases were also produced in an active form at low levels during fermentation of brewers' wort using established commercial conditions. Concomitantly, a marginally more stable beer resulted. Respiratory deficient, auxotrophic rare-mating was used to transfer the extracellular proteolytic characteristic (epr1.1) to a commercial ale yeast. Segregants of the recombinants obtained showed great instability in protease secretion and were unsuitable for brewing purposes. The potential advantages and drawbacks to this approach are discussed.     

Construction of recombinant industrial <Emphasis Type="Italic">S. cerevisiae</Emphasis> strain with barley lipid-transfer protein 1 secretion capability and lower PrA activity

Beer barley LTP1 in beer is an important component of beer foam, and it participates in the formation of beer foam. The digestion of beer barley LTP1 by proteinase A from brewing yeast leads to the decline of beer foam stability, especially for the unpasteurized beer. The objective of this study was to construct an industrial brewing yeast strain to secrete recombinant barley LTP1 into fermenting wort during beer fermentation for the foam stability improvement. We constructed barley LTP1 expression cassette and transformed into the host industrial yeast cells to replace partial PEP4 alleles using homologous recombination method. The expression of b-LTP1 was under control of the constitutive yeast ADH1 promoter, and the concentration of recombinant barley LTP1 secreted by recombinants reached 26.23 mg/L after incubation in YEPD medium for 120 h. The PrA activity of the recombinant strain declined compared with the host strain. The head retention of beer brewed with the recombinant industrial strain (326 ± 12 s) was improved when the host strain WZ65 (238 ± 7 s) and the constructed strain S.c-P-1 (273 ± 10 s) with partial PEP4 gene deficiency were used as control. The present study may provide reference for brewing industries and researches on beer foam stability.     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

Optimisation of High Gravity and Diet Beer Production in a German Brewery by Flow Cytometry

Increasing the quantity of beer production without diminishing the quality of the product is a key concern of the beer producing industry. Modifications to the brewery's equipment and settings are the most commonly used methods to improve the brewing process, while the supreme importance of the physiological state of the beer producing organisms, the yeast cells, for the productivity of the brewing process is often poorly recognised. The work described here was designed to optimise two processes: the inoculation regime used to produce high gravity bottom-fermenting beer, and the production of high quality diet beer. To achieve these aims, flow cytometry was used to follow changes in the distribution of DNA, neutral lipid and 3β-hydroxsterol contents in Saccharomyces carlsbergensis strains during inoculation, fermentation and storage. This allowed potential time-saving alterations in the process to be identified. Double staining techniques proved that vigorous fermentative activity and long-term survival capacity during main and secondary fermentation requires intense multiplication of the yeast cells during inoculation. The production of high gravity beer was then enhanced by altering the schedule of the wort additions, and thus increasing the yeast's activities related to multiplication. To produce diet beer, oligosaccharides that remain after the standard brewing process are degraded by adding small amounts of wort, usually during secondary fermentation. However, during this period of fermentation the physiological activity of the yeast cells is hampered by low carbon and high ethanol concentrations. Adding small batches of wort at carefully defined time points and in optimised amounts, even during the main fermentation, improves the physiological state of the yeast cells and rapidly decreases the carbon concentration within the fermentation tank. Both of these factors help to promote quick fermentation to a high quality diet beer. Thus, the flow cytometric investigations provided a reliable basis for identifying effective means of improving the process regime for brewing both of these products.     

ON THE DIFFERING RATES OF FRUCTOSE AND GLUCOSE UTILISATION IN SACCHAROMYCES CEREVISIAE

Fructose is utilised slower than glucose when the two sugars are fermented separately. This phenomenon occurs in a growth promoting medium as well as in brewers wort when using the brewing yeast Saccharomyces cerevisiae. The use of a fructose adjunct in wort at concentrations of 2% w/v and above, may result in residual concentrations of fructose to remain at the end of fermentation and consequently taint the beer with a sweet off-flavour. Glucose and fructose have no effect on maltose utilisation. Thus they do not exert catabolite repression on the maltose membrane transport system in the particular brewing strain of S. cerevisiae under investigation, when fermented in brewers wort.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

NISIN: A BACTERIOCIN WITH A POTENTIAL USE IN BREWING

Depending on the amount used and the strain of bacteria involved, nisin either kills lactic acid bacteria or inhibits their growth. In medium inoculated with approximately 10⁵ cells ml^?1 of a sensitive strain of Lactobacillus (BSO 375) nisin, added at levels recommended for commercial use (100 International Units ml^?1, killed all the cells in less than 6 h. In the absence of nisin this inoculum grew to a concentration of 10¹⁰ cells ml^?1 in about 50 h. Lower nisin concentrations killed fewer cells but inhibited the growth of those still viable. For the more resistant strain Lactobacillus (BSO 343) growth was only inhibited at the higher nisin concentrations. Nisin maintained its activity against lactic acid bacteria in brewing fermentations. It had no effect on the growth and fermentative performance of the 9 brewing yeast strains tested, and, in a pilot brewery fermentation, had no deleterious affect on the taste of the beer produced. Nisin could be used either as a preventative measure by regular addition to fermentations, or as a remedial measure once contamination by lactic acid bacteria had been detected.     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Beer metabolomics: molecular details of the brewing process and the differential effects of late and dry hopping on yeast purine metabolism

The flavour of beer is complex, based upon changes at the molecular level in the key raw materials, notably grain, hops and yeast, as well as during the process stages that comprise malting and brewing. As analytical techniques evolve in their sophistication and sensitivity, there are opportunities to delve ever more deeply into the fate of small molecules in brewing. To this end, ¹H nuclear magnetic resonance (NMR) metabolomics was used to follow the progression of 76 metabolites in four different late or dry hopped beers (brewed in triplicate) at five time points throughout the brewing process. The majority of the metabolites identified, including sugars, amino acids and nucleotides, significantly decreased in concentration from the start of the boil to post‐secondary fermentation, whereas energy‐related and fatty acid associated metabolites significantly increased in concentration as wort nutrients were consumed by the yeast. Adenine was significantly higher in the dry hopped brews than in the late hopped brews after both primary (p = 2.1 × 10^?6) and secondary (p = 2.7 × 10^?9) fermentation, while 2′‐deoxyadenosine (after primary, p = 1.1 × 10^?2, after secondary, p = 3.2 × 10^?5) and adenosine (after primary, p = 2.6 × 10^?8; after secondary, p = 3.1 × 10^?7)were significantly lower in the dry hopped beers at these time points. These results give molecular insight into the brewing process and the differential effects of hopping methods on yeast purine metabolism. Copyright © 2016 The Institute of Brewing & Distilling     

Inhibition of A. carbonarius growth and reduction of ochratoxin A by bacteria and yeast composites of technological importance in culture media and beverages

Five composites of yeast and six of bacterial isolates from fermented products were studied, in order to assess their ability to inhibit Aspergillus carbonarius growth and reduce OTA concentration in culture media and beverages. The antagonistic effect of the above composites against A. carbonarius growth was studied in synthetic grape medium of pH 3.5 and a_w 0.98, 0.95, 0.92 after incubation at 25 °C. Different combinations of initial inocula of bacteria or yeast composites and fungi were used (10² cfu/mL vs 10⁵ spores/mL; 10⁵ cfu/mL vs 10² spores/mL; and 10⁵ cfu/mL vs 10⁵ spores/mL). Regarding the OTA reduction experiment, 10³ and 10⁷ cfu/mL of the bacteria and yeast composites were inoculated in liquid media of different pH (3.0, 4.0, 5.0, and 6.1 or 6.5) and initial OTA concentration (50 and 100 μg/L) and incubated at 30 °C. Moreover, grape juice, red wine, and beer were supplemented with 100 μg/L of OTA and inoculated with composites of 16 yeasts (16YM) and 29 bacterial (29BM) strains (10⁷ cfu/mL) to estimate the kinetics of OTA reduction at 25 °C for 5 days. Fungal inhibition and OTA reduction were calculated in comparison to control samples. None of the bacterial composites inhibited A. carbonarius growth. The high inoculum of yeast composites (10⁵ cfu/mL) showed more efficient fungal inhibition compared to cell density of 10² cfu/mL. All yeast composites showed higher OTA reduction (up to 65%) compared to bacteria (2-25%), at all studied assays. The maximum OTA reduction was obtained at pH 3.0 by almost all yeast composites. For all studied beverages the decrease in OTA concentration was higher by yeasts (16YM) compared to bacteria (29BM). The highest OTA reduction was observed in grape juice (ca 32%) followed by wine (ca 22%), and beer (ca 12%). The present findings may assist in the control of A. carbonarius growth and OTA production in fermented foodstuffs by the use of proper strains of technological importance.     

Populations and potential association of Saccharomyces cerevisiae with lactic acid bacteria in naturally fermented Korean rice wine

The microbial populations focused on predominant yeast species and lactic acid bacteria (LAB) in 15 commercial makgeolli brands, where a fungal starter nuruk was used were examined. Viable yeast counts were obtained on yeast potato dextrose (YPD) and MRS agar containing sodium azide. MRS-C (0.1% cycloheximide supplemented) was used for selective counts of LAB. Saccharomyces cerevisiae was found to be predominant in the 15 samples tested, with an average count of 4.6×10⁷ CFU/mL. Contrary to the earlier studies, Lactobacillus plantarum and Weissella cibaria were shown as predominant LAB species with an average count of 1.7×10⁷ CFU/mL. Surprisingly, as many as 7 log viable cells/mL were present at the ethyl alcohol concentration of 6–7%. The data from real-time PCR also indicated that the yeast populations remains almost constant during the refrigerated storage of 12 days, while that of LAB decrease slightly first 9 days and increase after then, despite the overall increase in acidity. Data from the differential microbial counts suggest that yeast S. cerevisiae might be associated with 2 LAB species, L. plantarum and W. cibaria, under ethyl alcohol stress during the turbid rice wine fermentation.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Influence of Zinc on Distiller's Yeast: Cellular Accumulation of Zinc and Impact on Spirit Congeners

Accumulation of zinc by a whisky distilling yeast strain of Saccharomyces cerevisiae was studied during fermentation of malt wort and synthetic defined medium. Zinc uptake by yeast cells was very rapid in malt wort, as zinc (0.32 μg/mL) was completely removed from the fermentation medium within one hour. The type of fermentable carbohydrate had an impact on the kinetics of zinc accumulation, with maltose most effective at enhancing metal uptake at zinc concentrations above 3.2 μg/mL. Enriching yeast cells with zinc by “preconditioning” impacted on the production of flavour congeners in the distillates produced from fermented cultures. Such distillates were characterised by an altered flavour and aroma profile. In particular, the production of some higher alcohols increased when yeast cells were preconditioned with zinc. This phenomenon is yeast strain related. Industrial fermentation processes, including brewing and distilling, may benefit from optimization of zinc bioavailability in yeast cultures resulting in more efficient fermentations and improved product quality.     

类胡萝卜素降解对枸杞酒特征香气的影响

为探究类胡萝卜素降解后产生的降异戊二烯类化合物对枸杞酒特征香气的影响,采用一株可降解类胡萝卜素的库特氏菌进行实验。分别在枸杞渣中接种库特氏菌,枸杞清汁中接入工业酿酒酵母,发酵结束后混合;在枸杞汁中加入库特氏菌酶制剂,辅助工业酿酒酵母发酵;将高压灭菌的枸杞渣加入枸杞清汁后接种工业酿酒酵母发酵,并以正常发酵工艺酿制的枸杞酒作为对照。采用顶空固相微萃取及气相色谱质谱联用（GC-MS）法,对枸杞酒香气成分进行检测,并通过感官评分法评价3种降解类胡萝卜素方式对枸杞酒香气的影响。结果表明,库特氏菌酶制剂辅助发酵组枸杞酒香气和口感均为最佳,高压灭菌后发酵组澄清度最好。因此,在枸杞酒酿造过程中,可以采用库特氏菌酶制剂对枸杞汁进行降解处理,在此之前对枸杞渣可适度配合使用高压灭菌,从而改进枸杞酒的酿造工艺,提升枸杞酒香气。     

Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity

The characteristic buttery taste of diacetyl has long been a major problem in the brewing industry, and the foam stability of unpasteurized beer is often influenced by proteinase A (PrA), which is encoded by PEP4 and released from yeast cells into beer during brewing. A recombinant industrial brewer’s yeast strain that reduces the diacetyl content of beer and improves foam stability was constructed. We constructed a PGK1p-ILV5-PGK1t expression cassette, which was introduced into one of the PEP4 alleles via PCR-mediated homologous recombination. Then, the second PEP4 allele was disrupted using the Cre-loxP recombination system, and the recombinant strain was designated as S-CSIK12. The results show that the diacetyl production of S-CSIK12 is always lower than that of the host strain at all stages of beer fermentation. In addition, brewing with S-CSIK12 reduced the PrA activity of the final beer by 44 % compared with that using the wild-type strain. The head retention of the beer brewed with S-CSIK12 (260 ± 2 s) was better than that of the host strain S-6 (212 ± 3 s). Considering that more PrA is released from yeast cells during the final stage of main fermentation and that the timing of yeast cropping is determined by diacetyl reduction, brewing with strains that have low diacetyl production also reduced the PrA activity of the beer and improved its head retention. The present study provides reference for the brewing industry as well as research on the diacetyl reduction and foam stability of beer.     

双歧杆菌酸奶生产工艺的优化

通过研究婴儿双歧杆菌和长双歧杆菌在脱脂乳中的生长特性、影响其发酵的因素以及发酵方式 ,优化出双歧杆菌酸奶的最佳生产工艺。研究结果显示 ,选用长双歧杆菌制作酸奶时 ,在乳中添加 0 3 %酵母粉 ,低聚糖取代 10 %的蔗糖 ,接菌量为 10 % ;选用婴儿双歧杆菌制作酸奶时 ,在乳中添加 0 6%的酵母粉 ,以低聚糖取代 2 0 %的蔗糖 ,接菌量为 15 %。 3 9℃条件下发酵 ,然后再按 1∶1的比例与普通酸奶混合 ,所制得的酸奶在贮存期间 ,活菌数可达到10 8cfu/mL以上。     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.^15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.