Production of Bioingredients from Kluyveromyces marxianus Grown on Whey: An Alternative

Whey waste is a major problem for the dairy industry. Finding alternative means to reduce its pollution potential and produce high value-added bioingredients has been attempted by many researchers. Kluyveromyces marxianus var. marxianus is a dairy yeast that produces β-galactosidase, allowing for whey fermentation. Also, K. marxianus has been proposed as a source of: (1) oligonucleotides, used as flavor enhancers in food products; (2) oligosaccharides, used as prebiotics to stimulate the growth of Bifidobacterium sp. in the animal and human intestines; and (3) oligopeptides, immunostimulators added to dairy products that are released in the wort after whey protein proteolysis. Fed-batch fermentation can be used as an alternative process to avoid increases in lactose concentration and prevent the catabolite repression of the respiratory enzyme synthesis during aerobic fermentation, thus allowing for high biomass yields. The relevance of these factors on yeast fermentation of whey is summarized in this critical review.     

Apple pieces as immobilization support of various microorganisms

The biocatalyst prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on apple pieces was used for fermentation of grape must both in batch and continuous system. The immobilized yeast showed an important operational stability without any decrease of its activity. In continuous process, the bioreactor was operated for 95 days without any diminution of the ethanol productivity. Concentrations of higher and amyl alcohols were low indicating an improved quality product.Apple pieces were also used as support for immobilizing Kluyveromyces marxianus IMB3 for high-temperature (45 °C) wine-making. The fermented grape must contained 3-4% alcohol and semi-sweet wines were produced by the addition of potable alcohol.Preliminary sensory evaluation of the produced wines ascertained the fruity aroma and the overall improved quality compared to other commercially available products.Lactobaccillus casei cells immobilized on apple pieces were used for probiotic, fermented milk and lactic acid production. Apple-supported L. casei used for successive fermentation batches of whey proved to be very effective and suitable for food-grade lactic acid production. The immobilized biocatalyst was also used for milk fermentation and was able to ferment after storage for 15, 98 and 129 days at 4 °C, while no infection was reported during storage periods.     

SUPPRESSION OF α-ACETOLACTATE FORMATION IN BREWING WITH IMMOBILIZED YEAST

Immobilized yeast cells extensively produced the diacetyl precursor, α-acetolactate, during alcohol fermentation. The activity of acetohydroxy acid synthetase, which is responsible for the formation of α-acetolactate from pyruvic acid, was high in cell-free extracts of immobilized yeast cells compared with that of free yeast cells. It was suggested that the expression of AHA synthase of immobilized yeast cells was increased during growth in the carrier as compared with free yeast cells. When the initial immobilizing yeast cell concentration was changed from 1.0 × 10⁶ cells/ml to 1.0 × 10⁹ cells/ml, production of α-acetolactate was reduced from 0.94 mg/l to 0.30 mg/l. Furthermore, during continuous fermentation for 10 d, the concentration of α-acetolactate in beer was 0.30 mg/l.     

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

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。     

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

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.     

Diversity and Stability of Yeast Species During the Fermentation of Tarhana

Tarhana is a traditional cereal-based fermented food produced with a mixture of yoghurt and flour. The main microbiota in the fermentation of tarhana is yeast, together with lactic acid bacteria. In this study, the yeast microbiota of home-made tarhana (HMT) and plant-type tarhana (PTT) dough samples was evaluated and compared during fermentation. Culture-dependent LSU and ITS-5.8S rDNA sequence analysis of yeast isolates collected during the tarhana dough fermentation clarified 45 selected isolates representing different clusters. These yeast isolates displayed high homologywith species Pichia kudriavzevii (11), Candida glabrata (11), Candida humilis (10), Saccharomyces cerevisiae (7), Kluyveromyces marxianus (4), Kazachstania servazzi (1), and Kazachstania unispora (1). Additionally, both culture-dependent and PCR-Denaturated Gradient Gel Electrophoresis (PCR-DGGE) analyses showed that S. cerevisiae, P. kudriavzevii and K. marxianus were abundant in the fermentation of HMT dough samples whereas P. kudriavzevii, C. humilis, and C. glabrata dominated the PTT dough samples. It was concluded that tarhana fermentation was accomplished with the presence of a wide variety of yeast species that mainly included P. kudriavzevii in both HMT and PTT dough samples.     

Effects of magnesium ions on both VHG batch and continuous fruit wine fermentations

In Poland, fruit wines or aromatized fruit wines are semi‐sweet or sweet and contain approximately 15–16% (v/v) ethanol. Their production can be classified as a very high‐gravity (VHG) fermentation. Magnesium has a beneficial effect on VHG fermentations as it protects the yeast cells against ethanol, osmotic and temperature stress. The effect of the magnesium concentration in an apple must, containing 32% sugars, on the fermentative parameters of batch and continuous fermentations was assessed. In the batch process, a magnesium concentration of ~8.5 mg/L resulted in decreased the ethanol production in comparison to a magnesium concentration of ~250, 490 and 970 mg Mg²⁺/L. The highest amount of Mg also caused a metallic taste. A continuous fermentation was carried out for 2.5 months in a four‐column packed‐bed fermentor. The medium contained ~50, 250 and 490 mg Mg²⁺/L and the yeast was immobilized on foam glass. During the continuous fermentation, no differences at p ≤ 0.05 in terms of fermentative parameters were seen with magnesium additions. The same beginning amount of magnesium ions in the medium led to a similar use of this element, both in batch and in continuous fermentation. The more Mg²⁺ that was present in the medium, the more Mg²⁺ was used by the yeast. The results suggest that the minimal dose of magnesium, under the described conditions, is 50 mg/L, corresponding to the amount of Mg in the medium prepared using concentrated apple juice and tap water. This finding has industrial significance, as Polish wine companies prepare their fruit musts using tap water. Copyright © 2014 The Institute of Brewing & Distilling     

十二烷醇对酵母细胞影响的研究

以残糖和细胞生长OD值为分析指标,探讨了十二烷醇对酵母游离细胞和固定化细胞的影响。结果表明:萃取剂对游离细胞毒害性比较大,发酵残糖含量高;固定化细胞采用外部随程萃取发酵则可以忽略其对细胞的毒害作用,发酵残糖几乎没有。      

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。     

Potential of yeast isolated from apple juice to adhere to stainless steel surfaces in the apple juice processing industry

The aim of this study was to assess the potential of four yeast strains to initiate a biofilm on stainless steel. The yeasts were isolated from apple juice and were identified as Kluyveromyces marxianus, Candida krusei, Zygosaccharomyces sp. and Rhodotorula rubra. The physiochemical properties of cell surface were determined under different conditions. The adhesion capacity of the yeast strains to stainless steel was assayed in presence of apple juice. Cell surfaces were always negatively charged except for Zygosaccharomyces sp., whose isoelectric point was around 3.0. Neither electrophoretic mobility nor flocculation coefficient correlated with the capacity of attachment to stainless steel. The hydrophobicity expressed by the yeast surfaces at pH 3.0, correlated positively with the rate of adhesion (number of cells/min) of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied contaminant yeast strains to stainless steel surfaces common to the apple juice processing plant.     

Characterization of Osmotolerant Yeasts and Yeast‐Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast‐like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S‐ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD‐PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast‐like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.     

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.     

Production of white wine by Saccharomyces cerevisiae immobilized on grape pomace

White wine was produced with Saccharomyces cerevisiae cells immobilized on grape pomace, by natural adsorption. The support, the main solid waste from the wine industry, consisted of the skins, seeds and stems. Immobilization was tested using different media, namely complex culture medium, raw grape must and diluted grape must. Grape pomace was revealed to be an appropriate support for yeast cell immobilization. Moreover, grape must was shown to be the most suitable medium as immobilized cells became adapted to the conditions in the subsequent alcoholic fermentation in the wine‐making process. The wines produced, either with immobilized cells or with free cells, were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds); additionally, colour (CIELab) and sensory analysis were performed. The use of immobilized systems to conduct alcoholic fermentation in white wine production proved to be a more rapid and a more efficient process, especially when large amounts of SO₂ were present in the must. Furthermore, the final wines obtained with immobilized cells demonstrated improved sensory properties related to the larger amounts of ethanol and volatile compounds produced. The more intense colour of these wines could be a drawback, which could be hindered by the reutilization of the biocatalyst in successive fermentations. Copyright © 2012 The Institute of Brewing & Distilling     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules

Kluyveromyces marxianus is emerging as a new platform organism for the production of flavour and fragrance (F&F) compounds. This food‐grade yeast has advantageous traits, such as thermotolerance and rapid growth, that make it attractive for cell factory applications. The major impediment to its development has been limited fundamental knowledge of its genetics and physiology, but this is rapidly changing. K. marxianus produces a wide array of volatile molecules and contributes to the flavour of a range of different fermented beverages. Advantage is now being taken of this to develop strains for the production of metabolites such as 2‐phenylethanol and ethyl acetate. Strains that were selected from initial screens were used to optimize processes for production of these F&F molecules. Most developments have focused on optimizing growth conditions and the fermentation process, including product removal, with future advancement likely to involve development of new strains through the application of evolutionary or rational engineering strategies. This is being facilitated by new genomic and molecular tools. Furthermore, synthetic biology offers a route to introduce new biosynthetic pathways into this yeast for F&F production. Consumer demand for biologically‐synthesized molecules for use in foods and other products creates an opportunity to exploit the unique potential of K. marxianus for this cell factory application. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of Fermentation Temperature on Key Aroma Compounds and Sensory Properties of Apple Wine

Fermentation temperature strongly affects yeast metabolism during apple wine making and thus aromatic and quality profiles. In this study, the temperature effect during apple wine making on both the key aroma compounds and sensory properties of apple wine were investigated. The concentration of nine key aroma compounds (ethyl acetate, isobutyl acetate, isopentylacetate, ethyl caprylate, ethyl 4‐hydroxybutanoate, isobutylalcohol, isopentylalcohol, 3‐methylthio‐1‐propanol, and benzeneethanol) in apple wine significantly increased with the increase of fermentation temperature from 17 to 20 °C, and then eight out of the nine key aroma compounds with an exception of ethyl 4‐hydroxybutanoate, decreased when the temperature goes up 20 to 26 °C. Sensory analysis showed that the apple wine fermented at 20 °C had the highest acceptance for consumers. Fermentation at the temperature of 20 °C was therefore considered to be the most suitable condition using the selected yeast strain (Saccharomyces cerevisiae AP05) for apple wine making. Changes in the fermentation temperature can considerably affect the production of key aroma compounds and sensory profiles of apple wine. These results could help apple wine producers make better quality production for consumers at the optimal fermentation temperature.