Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking

Since the large occurrence of Brettanomyces yeasts in strict anaerobiosis environments (sparkling wines) has been found without an increase in acetic acid content, we evaluated the influence of the oxygen concentration on acetic acid production. Results showed that the oxygen concentration exerted a strong influence on both growth and acetic acid production by Brettanomyces yeasts in winemaking. Full aerobiosis lead to a large production of acetic acid causing a block of metabolic activity. Semi-aerobiosis resulted in the best condition for alcoholic fermentation (Custers effect) combined with acetic acid production. In anaerobic condition Brettanomyces yeasts did not result in high acetic acid production and a pure, even if slow, alcoholic fermentation occurred. The absence of an increase in acetic acid in wines, does not exclude the active presence of Brettanomyces yeast since the characteristic ‘high acetic producer’ in Brettanomyces yeast is linked to the presence of oxygen. ©1997 SCI     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

Humulus lupulus – a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α‐ and β‐acids) and the latest findings on the absolute configuration of the cis and trans iso‐α‐acids are discussed. Special attention is given to the hard resins; the known δ‐resin is reviewed and the ε‐resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well‐known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)₅ was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

ESTERASE ACTIVITY IN THE GENUS BRETTANOMYCES

During the spontaneous fermentation of Iambic and the production of the typical Belgian beer Gueuze, yeasts of the genus Brettanomyces are always found. They are especially active in the 6 to 12 months fermentation period and during refermentation in the bottle. It was found that Brettanomyces yeasts show esterase activity towards a large number of esters but especially towards esters normally found in Gueuze: ethyl acetate, ethyl lactate, iso-amyl acetate and phenethyl acetate. The esterase activity is expressed by whole cells of all Brettanomyces strains in our collection, but not by Saccharomyces or Kloeckera. The enzyme is partially purified by ammonium sulphate precipitation. It shows optimal activity at pH 7·6 for the hydrolysis of ethyl acetete and relative good stability at around 2°C. Levels of whole cell esterase activity are dependent on growth medium, aeration rate and temperature. Most interesting is the ester-synthesising activity of this esterase. Its role and influence on the ester fraction of Iambic and Gueuze seem very likely, which may then be considered as the first confirmed function of Brettanomyces during this special fermentation process.     

Barley Grain Non‐specific Lipid‐Transfer Proteins (ns‐LTPs) in Beer Production and Quality

Plant non‐specific lipid‐transfer proteins (ns‐LTPs) are known for their ability to transfer various lipids between membranes in vitro. These ubiquitous basic proteins, that all share alpha structure stabilized by four disulphide bridges, are characterized by the presence of a hydrophobic cavity able to accommodate lipid molecules. According to molecular mass, this multigene family is subdivided into two subfamilies, ns‐LTP1 (9 kDa) and ns‐LTP2 (7 kDa); both located in the aleurone layer of the cereal grain endosperm. Ns‐LTP1 is a prominent protein in barley grain, malt and beer. Numerous studies performed on its structure and function confirm its important role in grain protection, as well as brewing technology. As the major beer protein crucial for many aspects of brewing, ns‐LTP1 can affect beer production and quality. Comparatively, there is less data available on the less abundant ns‐LTP2. In this review, the focus is on recent progress on the structure, biological and technological function of barley grain ns‐LTP1 and ns‐LTP2, with an emphasis on their importance in brewing.     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

Biotransformation of hop derived compounds by Brettanomyces yeast strains

Several hop derived compounds in wort are known to be converted by yeast during fermentation, influencing the overall perception of the beer. A deeper understanding of such metabolic processes during fermentation is needed to achieve better control of the outcome. Here, the interaction between hop derived compounds and the yeast genera Brettanomyces was studied. Several Brettanomyces strains with different genomic backgrounds were selected, focusing on two traits: beta-glucosidase activity and nitrate assimilation. The role of three beta-glucosidases present in Brettanomyces bruxellensis and Brettanomyces anomalus and their impact on the final monoterpene alcohol profile was analysed. The beta-glucosidase activity was highly strain dependent, with B. anomalus CRL-49 exhibiting the highest conversion. Such activity could not be related to the release of aglycones from hops during fermentation, as a substantial part of such activity was intracellular. Nevertheless, the reduction of geraniol to β-citronellol was remarkably efficient for all Brettanomyces strains during fermentation, and it is suggested that two oxidoreductases BbHye2 and BbHye3 may have an influence. Moreover, the transfer of nitrate from hops to wort and its further assimilation by Brettanomyces species was analysed. The amount of nitrate in wort proved to be linearly proportional to the contact time of the hops with the wort. The level of nitrate assimilation by yeast was shown to be dependent on the nitrate assimilation cluster (YNR, YNI, YNT). Hence, the desired yeast strains may be selected according to the genetic make-up. © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

IDENTIFICATION OF LAMBIC SUPERATTENUATING MICRO-ORGANISMS BY THE USE OF SELECTIVE ANTIBIOTICS

The microbial population present in Iambic beer after one year of spontaneous fermentation consists mainly of Brettanomyces yeasts, lactic acid bacteria and acetic acid bacteria. The density of the wort by that time has decreased to around 3.5° Plato. At that time a period of superattentuation is initiated, resulting in a Iambic with sometimes less than 1° Plato. Such old Iambics are used in the production of gueuze. In order to find out which organisms are really necessary for this process. Iambic attenuated to around 3.5° Plato was pasteurized and re-inoculated with a mixed microbial population obtained from fermenting Iambic. By the addition of the antibiotics actidione, pimaricin, gentamycin, oxytetracycline and nisin it was found that Brettanomyces was the main organism responsible for superattenuation, although this was less pronounced when Pediococcus was absent. Acetic acid bacteria were not involved. Bacteria alone were not really superattenuating. The process with Iambic wort which had reached the 3.5° Plato value by a natural spontaneous fermentation was slower than with a Iambic wort pre-fermented to 3.5° Plato with S. cerevisiae. It was found that Brettanomyces but not Saccharomyces survives well under the conditions normally found for a 1 year old Iambic.     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.     

Use of coffee by‐products for the cultivation of Pleurotus citrinopileatus and Pleurotus salmoneo‐stramineus and its impact on biological properties of extracts thereof

Incorporating spent coffee grounds (SCGs), a by‐product from coffee brewing, in growth substrate of beneficial edible mushrooms is an approach that has to be further studied due to its potential positive outcomes: environmental impact mitigation, production costs reduction and beneficial impact on consumer health. Hence, cultivation of Pleurotus citrinopileatus and Pleurotus salmoneo‐stramineus was tested using SCG which enabled maximum production yield of P. citrinopileatus which was of 25.1% (w/w). Variable antidiabetic potential was observed between aqueous and enzymatic extracts (3.8%–29% inhibition) regardless species and substrates, whereas aqueous extract of P. citrinopileatus grown in substrate without SCG stood out presenting the highest antioxidant activity and inhibition activity of angiotensin I‐converting enzyme (IC₅₀ = 123 μg mL^?1). Ethanolic and aqueous extracts of both Pleurotus species grown in the presence or absence of SGC proved to be an interesting prebiotic source for growth of Bifidobacterium animalis Bo in comparison with fructooligosaccharides (FOS).     

Note — Preliminary Applications of Response Surface Modelling to the Evaluation of Optimal Growth Conditions for Beer‐spoiling Pediococcus damnosus

Beer‐spoiling lactic acid bacteria (LAB) belong to the most harmful contaminants in the brewing industry and various rapid molecular detection methods have been introduced for the detection of these organisms. However, the enrichment cultivation steps needed prior to detection by molecular methods can extend the duration of the analytical procedure by up to several days. The use of brewery‐specific enrichment cultivation media has been recommended due to the large variety within the group of LAB, as well as differences in the microbes encountered at the breweries. In contrast to using a general medium that may support only the growth of some LAB, the combination of several media could allow detection of all relevant LAB. The aim of the present study was to show the effects of shifts in the cultivation conditions and media on the growth of beer‐spoiling LAB using one of the most frequently encountered species, Pediococcus damnosus, as an example. Based on the variable analysis, significant factors could be recognized and their effects on lag‐times and growth rates were compared by means of response surface modelling.     

Microbial community changes in Makgeolli during brewing

Makgeolli, a traditional Korean alcoholic beverage, is produced from the simultaneous saccharification and fermentation of glutinous rice by microbes introduced through nuruk (fermentation starter). The microbial community in Makgeolli is complex, and changes in the community are important for quality control and product development of Makgeolli. In this study, changes in the microbial community of Makgeolli during brewing were monitored by pyrosequencing. Pyrosequencing of microbes in Makgeolli illustrated dynamic changes in the populations and species of bacteria and fungi during brewing. Saccharomyces, a fungal genus, dominated the fungal community in Makgeolli throughout brewing, and produced alcohol after sufficient production of reducing sugars. As the bacterial genera Pediococcus, Weissella, Lactobacillus and Enterococcus, categorized as lactic acid bacteria, grew in Makgeolli, the pH decreased. The fungal community in Makgeolli was not significantly changed during brewing, but there were dynamic changes in the bacterial community, especially during the first 2 days of brewing. Copyright © 2015 The Institute of Brewing & Distilling     

Biovalorization of brewers’ spent grain for the production of laccase and polyphenols

Brewers’ spent grain (BSG) is the main solid by‐product of the brewing process and is typically disposed of as cattle feed. In this study, BSG was evaluated as a substrate for the production of polyphenols and the lignin‐degrading enzyme laccase using fungal solid‐state fermentation by Trametes versicolor. Laccases are finding increasing applications in the food industry and polyphenols have benefits for human health. After 14 days of fermentation with T. versicolor, there was a 3.4‐fold increase in the extraction of total polyphenols compared with untreated BSG. Using BSG as the sole source of carbon and nitrogen, maximum laccase activity was achieved after seven days of treatment with an activity of 560 U/L. Based on these results, BSG is suggested to be a good lignocellulose waste material to produce value‐added products such as the enzyme laccase and polyphenols. Copyright © 2018 The Institute of Brewing & Distilling     

Breeding of lipoxygenase‐1‐less malting barley variety ‘Satuiku 2 go’

The lipoxygenase‐1‐less (LOX‐less) trait has positive effects on beer quality, in particular, improvement of flavour stability related to the reduction of beer‐deteriorating substances such as trans‐2‐nonenal. ‘Ryohfu’ is the only spring‐sown malting barley variety grown in Hokkaido, located in the northern part of Japan, and has been used in the Japanese brewing industry for over 20 years. ‘Satuiku 2 go’ was developed as the first LOX‐less malting barley variety in Japan by successive back‐crossing with molecular marker‐assisted selection to introduce the LOX‐less trait into the recurrent parent ‘Ryohfu’. The agronomic performance and general malt quality of ‘Satuiku 2 go’ were almost equivalent to those of ‘Ryohfu’. Wort and beer analyses at the pilot‐scale brewing trial indicated that the LOX‐less trait had little effect on the general characteristics. In contrast, the beers made from ‘Satuiku 2 go’ malt exhibited reduced levels of trans‐2‐nonenal and trihydroxyoctadecenoic acid. The sensory evaluation demonstrated the superiority of ‘Satuiku 2 go’ beers stored under differing conditions in terms of staleness. It can be concluded that the LOX‐less trait was effective in different genetic backgrounds of the recurrent parents used for the development of LOX‐less malting barley varieties. Copyright © 2018 The Institute of Brewing & Distilling     

Saccharomyces pastorianus: genomic insights inspiring innovation for industry

A combination of biological and non‐biological factors has led to the interspecific hybrid yeast species Saccharomyces pastorianus becoming one of the world's most important industrial organisms. This yeast is used in the production of lager‐style beers, the fermentation of which requires very low temperatures compared to other industrial fermentation processes. This group of organisms has benefited from both the whole‐genome duplication in its ancestral lineage and the subsequent hybridization event between S. cerevisiae and S. eubayanus, resulting in strong fermentative ability. The hybrid has key traits, such as cold tolerance and good maltose‐ and maltotriose‐utilizing ability, inherited either from the parental species or originating from genetic interactions between the parent genomes. Instability in the nascent allopolyploid hybrid genome may have contributed to rapid evolution of the yeast to tolerate conditions prevalent in the brewing environment. The recent discovery of S. eubayanus has provided new insights into the evolutionary history of S. pastorianus and may offer new opportunities for generating novel industrially‐beneficial lager yeast strains. Copyright © 2014 John Wiley & Sons, Ltd.