CONVERSION OF α-ACETOLACTATE AND REMOVAL OF DIACETYL A KINETIC STUDY

Brewers' yeast does not form diacetyl during fermentation, but α-acetolactate which spontaneously is converted to diacetyl. This non-enzymic reaction is mainly dependent upon pH, temperature and substrate concentration. The present kinetic studies demonstrate that temperature and pH have a dominating effect on the spontaneous conversion of α-acetolactate during primary fermentation. From the reported kinetic studies it is possible to predict the conversion rate of α-acetolactate present in the fermenting liquid, and to calculate the time necessary to convert a given amount of α-acetolactate to diacetyl. As long as yeast is present in the fermenting liquid, diacetyl can not be detected analytically due to the high diacetyl-reducing activity of the yeast cells. The specific activity is strain-dependent and decreases gradually during fermentation. The diacetyl-reducing activity of the yeast will, however, normally be high enough to compete with the diacetyl formed from α-acetolactate.     

PRODUCTION OF BEER USING IMMOBILIZED YEAST ENCODING α-ACETOLACTATE DECARBOXYLASE

Genetically modified brewer's yeast encoding α-acetolactate decarboxylase (α-ALDC) was tested in immobilized yeast bioreactors for main fermentation of beer. The α-ALDC enzyme produced by the transformant catalyzes the direct conversion of α-acetolactate to acetoin without formation of diacetyl. The long lagering period required for beer maturation in conventional brewing can thus be shortened or even omitted. Three different packed bed bioreactors were employed, with volumes of 1.6 dm³, 5 dm³ and 25 dm³. The 5 dm³ column had a slightly conical geometry in contrast to the others which had cylindrical shapes. Sintered glass beads were chosen as the carrier material on the basis of experiments with the parent strain. The brewing performance of the transformant compared well with that of the parent strain in the immobilized system. Fermentation, utilization of amino acids (including isoleucine, valine and leucine) and flavour formation were practically identical with both strains, the only difference being a marked decrease in the formation of diacetyl by the transformant. Small differences were, however, observed in the long-term biochemical stability. By using yeast encoding α-ALDC in the immobilized yeast system the total (primary and secondary) fermentation time could be reduced to approximately 2–6 days, compared with 3–6 weeks in a conventional batch process.     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

PITCHING RATE IN HIGH GRAVITY BREWING

The optimal pitching rate in high gravity worts (12–16°P) was about 0.3 g/l wet weight (2.3 × 10⁶ counted cells/ml) and per one percent of original wort gravity. In very high gravity worts (20–23°P) the corresponding figure was 0.4 g/l (2.9 × 10⁶ cells/ml). Higher amounts of yeast did not improve the fermentation rate. With increased original wort gravity, flocculation of the yeast weakened and the amount of cropped yeast decreased. The viability of the crop yeast was good. In the conditions used, excessive production of acetate esters occurred only with pitching rates lower than the recommended rate. As the original wort gravity increased, more fermentable extract was metabolized to ethanol rather than utilized for yeast growth. The highest ethanol yield obtained was 10.9% (v/v).     

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     

黄酒固定化发酵过程及香气物质形成的研究

将黄酒酵母用海藻酸钙包埋制成固定化酵母,研究游离酵母与固定化酵母的黄酒发酵过程理化指标和香气物质生成量的差异,结果表明:经过主发酵5 d(28℃),后发酵15 d(15℃),固定化酵母发酵过程的酒精度、总糖、总酸变化曲线与游离发酵过程曲线差异不大,固定化酵母发酵性能稳定,杂菌污染可控。用气相色谱(GC)分析乙酸乙酯等9种黄酒特征性香气物质,发现游离发酵黄酒正丙醇、异丁醇、异戊醇、β-苯乙醇的总高级醇含量为680.91 mg/L,固定化发酵第1,2,3,4,5批黄酒的总高级醇含量分别为633.63,649.79,658.17,637.63,614.68 mg/L,说明固定化发酵有利于控制黄酒中的高级醇含量。固定化发酵酯类总量高于游离酵母,且随着固定化酵母发酵批次的增加而递增,即:固定化第5批(115.90 mg/L)>第4批(90.81 mg/L)>第1~3批(80.10~81.81 mg/L)>游离酵母(55.66 mg/L),表明固定化发酵使黄酒的酯类香气愈加浓郁。根据香气阈值计算的固定化发酵酿得的黄酒的香气不比游离酵母酿得的黄酒差,固定化酵母发酵生产黄酒有商业化应用的前景。     

利用固定化酵母进行黄酒主发酵的初步研究

该文以海藻酸钙凝胶为固定化载体,从酵母浓度、海藻酸钠浓度、氯化钙浓度3方面来探究黄酒主发酵阶段的最佳固定化条件,确定了酵母浓度为109个/mL,海藻酸钠浓度为2.0%,氯化钙浓度为4%。同时将其与传统游离酵母的发酵曲线进行对比,并且在实验室条件下连续进行了12次固定化酵母发酵,结果显示主发酵结束时酒样的酒精度在7.4%vol左右(与游离发酵结果类似),稳定性较为理想。     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

Production and Characterization of Wine with Sugarcane Piece Immobilized Yeast Biocatalyst

In the present study, an economically cheap and sustainable food-grade yeast biocatalyst for wine production was successfully prepared. It was prepared through absorption technique by using fresh sugarcane (Saccharum officinarum L.) pieces as immobilizing support for yeast, Saccharomyces cerevisiae CFTRI 101. Immobilization was confirmed by scanning electron microscopy. Suitability of biocatalyst was investigated at low and room temperatures by using it in repeated batch. The fermentation rate and other parameters were compared with free yeast cells at different temperatures. In all cases, fermentation time was short (24 h at 30 °C and 98 h at 10 °C) and ethanol productivities were high as 4.2 g/l/h (3.33-fold at 30 °C and 2.31-fold at 10 °C). The volatile compounds methanol, ethyl acetate, propanol-1, isobutanol (2-methyl-1-propanol), and amyl alcohols (2-methyl-1-butanol and 3-methyl-1-butanol) that formed during fermentation were analyzed with the help of a gas chromatograph–flame ionization detector. The concentrations of ethyl acetate and methanol were not more than 100 mg/l in all cases, indicating an improvement in the product quality. Cell metabolism of immobilized yeast was not negatively affected by immobilization process. It was found that the immobilization of yeast cells on sugar pieces increased the fermentation rate and viability of yeast cells. Preliminary sensory tests recognized the fruity aroma, fine taste, and the overall improved quality of the produced wines.     

Fermentation of banana media by using kappa-carrageenan immobilized Lactobacillus acidophilus

Fermentation media were prepared by using banana as the raw material, and cell immobilization of Lactobacillus acidophilus by kappa-carrageenan entrapment was applied to enhance the fermentation efficiency. Gel beads of diameters around 3.0 mm were prepared for the immobilized cells, ripe bananas were used for preparation of media, and both free and immobilized cells were employed to carry out the fermentation for 80 h. Cells leaked out from the gel beads and proliferated in the medium solution during the fermentation of immobilized cells. The final viable cell number reached 10(5) CFU/ml in the medium suspension, over 10(8) CFU/(ml gel) in gel beads for the immobilized cell fermentation and around 10(6) CFU/ml for the free cell fermentation. Immobilized cells withstand the adverse conditions in banana media resulting in better fermentation efficiency compared to free cells. Variation of fructooligosaccharides (FOS) in banana media was not significant in immobilized cell fermentation compared to free cell. Immobilized L. acidophilus fermented banana medium was found to possess synbiotic properties.     

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

Abstract: The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11^th day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC‐MS analysis.     

固定化酵母-膜生物反应器连续发酵生产乙醇的研究

对固定化酿酒活性干酵母-硅橡胶膜生物反应器连续发酵生产乙醇进行了实验研究。对固定化酵母和游离酵母的发酵能力进行了比较,实验研究了系统在长期运行过程中的发酵反应动力学参数和膜传质动力学参数等基本性能。结果表明,酵母细胞固定化后,其发酵速度有明显提高;膜渗透汽化分离产物乙醇使发酵反应能够长时间连续稳定地进行,葡萄糖消耗率和乙醇体积产率都较间歇发酵有明显的提高;在连续运行中,发酵液中葡萄糖浓度控制在20～50g/L的范围内,乙醇体积产率为4.0g/（L·h）,罐内乙醇浓度基本保持在45g/L左右,固定化酵母浓度维持在1.05×10^10～2.15×10^10个/克胶,发酵液中游离酵母浓度维持在0.02×10^10～0.098×10^10个/mL,膜的渗透通量和分离因子分别为300～500g/（m^2·h）和3.6～7.2;实验系统连续运行了294h,收得渗透冷凝液4841.7g,平均质量分数为21.3%。     

Continuous Fermentation of Worcestershire Sauce by Trickle Bed Bioreactor Using Comb-Shaped Ceramic Carriers

A two-step fermentation process using Saccharomyces cerevisiae OC-2 (wine yeast) was studied. The first step, to multiply and immobilize yeast cells, was carried out by batch method (7 days) in a trickle bed bioreac-tor. In a second step, the substrate was continuously fermented by immobilized and free yeast cells in the same bioreactor. Continuous fermentation resulted in retention of 3% (W/V) of ethanol, 50 mg/L of isoamyl alcohol and 6 mg/L of β-phenethyl alcohol (the major aromatic components of fermented Worcestershire sauce). Maximum ethanol productivity was retained at 4.1–4.2 g/L/hr under continuous operation with immobilized yeasts, 3.2-fold higher than with the batch system.     

SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

The efficacy of sand-immobilized organoclays as filtration bed materials for bacteria

Previous research has shown that montmorillonite clays exchanged with cationic surfactants including cetylpyridinium (CP), hexadecyltrimethylammonium (HDTMA), and cetyldimethylethylammonium (CDEA) were effective in reducing Salmonella enteritidis colony counts in bulk antibacterial assays. In order to increase the porosity (and hydraulic conductivity) of these materials for use in filtration beds, organoclays were tightly adhered and immobilized onto the surface of sand. Bulk antibacterial assays performed with 10 mg of the sand-immobilized organoclay (S/IOC) showed that the composite materials retained their antibacterial activity after processing. Reductions in plate colony counts ranged from 16.5% for sand-immobilized CP-exchanged sodium montmorillonite to 83.9% for immobilized CDEA-exchanged low pH montmorillonite. The sand/adhesive matrix and sand-immobilized activated charcoal failed to produce any significant decreases. Pilot studies with columns containing 1.0 g of S/IOC showed that maximal filtration efficiency was obtained at a filtration rate of 1.0 ml/min. Initial column studies with Salmonella suspensions at a concentration of 3.0×10⁸ cfu/ml, produced reductions in colony plate counts varying from 28.5% for immobilized CP-exchanged calcium montmorillonite to 59.5% for immobilized CDEA-exchanged low pH montmorillonite. The sand/adhesive matrix and immobilized activated charcoal yielded much lower reductions (7.6% and 10.4%, respectively). Studies with Escherichia coli suspensions (3.0×10⁷ cfu/ml) indicated reductions varying from 59.3% for immobilized CP-exchanged calcium montmorillonite to 99.9% for immobilized CDEA-exchanged low pH montmorillonite. These initial results suggest that S/IOC can be effective in reducing microbial numbers in wastewater following some modifications.     

Production of acetic and propionic acids from labneh whey by fermentation with propionibacteria

Whey produced during the manufacture of labneh was supplemented with yeast extract (10 g/1), and then fortified with lactose, treated with β-galactosidase or fermented with Lactobacillus helveticus, prior to inoculation with free living cells of Propionibacterium freudenreichii ssp shermanii or Propionibacterium acidipropionici or cells immobilized in aliginate beads. Under anaerobic batch conditions, fermentation of the whey with Lb helveticus followed by P acidipropionici (free cell system) for 2.5 days at 32°C gave a broth with 5.9 g/l of propionic acid and 2.4 gll of acetic acid, while immobilized cells of the same organisms gave a broth with 11.0 gll propionic acid and 3.2 g/l acetic acid over 4 days. These latter values were the maximum levels recorded with any of the treatments, and it is suggested that such yields might make recovery economically feasible in certain countries.     

无机盐对新型固定化酵母蔗汁酒精发酵的影响

以甘蔗作酒精酵母的固定化载体,以蔗汁为发酵培养基进行酒精发酵,通过电镜观察,酒精酵母不仅能吸附在甘蔗外表面,同时也能较好地吸附在甘蔗内部,比较固定化酵母与游离酵母数,发现固定化酵母数达到1011个/g,而游离细胞数只有107个/mL.通过考察不同浓度的CaCl2、MgSO4和KH2PO4对酵母酒精发酵性能的影响,发现1.0 g/L CaCl2和1.5 g/L KH2PO4可在最短时间内使二氧化碳生成速率达到最大值;而KH2PO4可明显地提高酒精发酵效率.     

固定化酵母薯干原料酒精发酵中试研究

本文论述了固定化酵母薯干原料带渣酒精发酵的中试结果。中试生物反应器容积为１０００Ｌ，采用改性海藻酸钙凝胶为固定化酵母细胞的载体。中试研究中设计了较合理的中试工艺流程，优选出较佳的发酵条件，并考察了生物反应器的性能。试验获得了如下结果：中试装置已运转１９２天，固定化酵母仍保持高活力，并可继续运转；发酵成熟醪酒精含量达６．５～８．０％（ｖ），它比同期传统式酒精发酵高出０．３～０．４％；淀粉利用率达９１～９２％；固定化酵母生物反应器的乙醇生产能力为９．５ＫｇＥｔＯＨ／Ｍ￣３·ｈ，它可比传统式发酵罐的生产能力提高１０～１２倍。