Evaluation of Beer Fermentation with a Novel Yeast Williopsis saturnus

Beer is traditionally brewed from malted barley, water and hops using Saccharomyces cerevisiae, which produces fl avour-active substances during wort fermentation, giving beer its fl avour profi le. These substances include esters, alcohols, aldehydes and ketones, volatile sulphur compounds and fatt y acids, etc. Among the fl avour volatiles, esters are one of the most important aroma compounds found in beer, which provide fruity aroma (1,2).


Introduction
Beer is traditionally brewed from malted barley, water and hops using Saccharomyces cerevisiae, which produces fl avour-active substances during wort fermentation, giving beer its fl avour profi le.These substances include esters, alcohols, aldehydes and ketones, volatile sulphur compounds and fatt y acids, etc.Among the fl avour volatiles, esters are one of the most important aroma compounds found in beer, which provide fruity aroma (1,2).
Esters may be broadly categorised into two main groups, namely acetate esters and ethyl esters.Acetate esters are produced via alcoholysis from alcohols and acetyl coenzyme A (CoA) catalysed by alcohol acetyl transferases.Important acetate esters in beer include ethyl acetate, isoamyl acetate and 2-phenylethyl acetate (3).On the other hand, ethyl esters are secondary metabolites produced by both S. cerevisiae and non-Saccharomyces yeasts.These ethyl esters can be formed by esterifi cation or alcoholysis.Esterifi cation forms esters via condensation reactions between ethanol and carboxylic acids catalysed by fatt y acid ethyl ester (FAEE) synthases.In contrast, alcoholysis is a transesterifi cation reaction between fatt y acyl-CoAs and ethanol catalysed by fatt y acyl transferases (4).Important ethyl esters in beer include ethyl octanoate, ethyl hexanoate and ethyl decanoate (5).
At an industrial level, S. cerevisiae is the preferred yeast to perform beer fermentation because of its effi cient conversion of sugars to ethanol, high ethanol tolerance and ability to carry out fermentation under acidic conditions as in wort (6).However, consistence in fermentation practices results in litt le fl avour diff erentiation.To drive product diff erentiation, various microbreweries are dedi-cated to the production of specialty beer.Examples include fruit, honey and herb or spiced beer.To boost the fruity aroma in fruit beer, brewers have come up with different methods and styles of brewing by adding fruit (7).Fruits can also provide fl avours, aromas and colours that cannot be obtained from grains.
The resultant fruit beer oft en lacks traditional beer fl avours (7) so research has been directed at adding fruitiness to beer by enhancing the formation of fl avour-active esters with the use of S. cerevisiae (1) or genetically modifi ed yeasts (4,5).However, given the controversial nature of genetic modifi cation in food application, it would be of interest to impart fruitiness to beer using natural yeasts including non-Saccharomyces yeasts.
Non-Saccharomyces yeasts such as Brett anomyces sp.produce volatile phenols and play important positive roles in the spicy and smoky fl avour profi les of specialty beer such as Lambic and Gueuze (8).Other non-Saccharomyces yeasts such as Candida and Pichia are well-known in sherry production for their role in oxidising ethanol to acetaldehyde (giving oxidised off -fl avour) and have also been reported in mixed culture fermentation of acidic ales (8).Nevertheless, non-Saccharomyces yeasts can cause damage to beer and wine fl avour, depending on the yeast and style of an alcoholic beverage.
One of the non-Saccharomyces yeasts that have received much research att ention is from the genus Williopsis, which includes the saturn-shaped Williopsis saturnus (9).W. saturnus is known to possess high activities for syn thesising fruity acetate esters, besides antifungal properties (10).Yilmaztekin et al. (11) studied the growth of W. saturnus var.saturnus on beet molasses and found that the yeast produced signifi cant amounts of isoamyl acetate.Fermentation studies in sake using W. saturnus var.mrakii showed that it was a potent producer of acetate esters (12,13).
Williopsis yeasts are not usually associated with beer fermentation, but have found potential applications in wine and sake fermentations (12)(13)(14).Furthermore, the ability of Williopsis yeasts to produce esters, especially acetate esters, while producing small amounts of ethanol in wine suggests a feasible way for production of fruity, low-alcohol beer (14).Therefore, the aim of this study is to evaluate the potential of W. saturnus var.mrakii NCYC 500 to produce fruity beer.Fermentation performance of W. mrakii NCYC 500 was compared against that of S. cerevisiae Safale US-05.Findings of this study could off er a novel biotechnological direction in brewing extra fruity, low-alcohol beer.

Wort preparation
Wort was prepared by dissolving 1.1 L of Light Malt Extract (Coopers Brewery, Regency Park, Australia) in 6 L of water.A mass of 450 g of milled specialty malt (150 g of Carapils, 150 g of Crystal 10L and 150 g of Aromatic; Briess Malt & Ingredients Co., Chilton, WI, USA) was steeped in 1.5 L of water at 70 °C for 30 min for extraction of fl avour compounds from the grains.The extract was then added to the dissolved malt extract to make up 8 L of solution, which was boiled for 60 min.Once hot break had occurred, 113 g of glucose (brewing sugar) were mixed into the wort.Then, kett le hopping was carried out.At 60 min before the end of boiling, 14 g of Cascade hop pellets (iBrew, Schaumburg, IL, USA) were added to the wort.At 5 min before the end of boiling, 7 g of Perle hop pellets (iBrew) were added.Aft er boiling, cold distilled water (Ice Mountain, Singapore) was added to the remaining wort to make up to 5 L. The wort was then rapidly cooled down.Cooled wort was then bott led into 240--mL bott les and stored at 4 °C.Before inoculation, the wort was centrifuged (29 703×g, 3-18K centrifuge; Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) for 15 min and the supernatant was collected.Sterile fi ltration was carried out via peristaltic pumping of wort through a sterile 0.65-μm fi lter and then a sterile 0.45-μm fi lter.

Fermentation conditions
Triplicate fermentations were carried out in 500-mL sterile conical fl asks (plugged with cott on wool, then wrapped in aluminium foil and parafi lm) containing 400 mL of sterile wort at 21 °C.Inoculation conditions were as follows.For Safale US-05 strain, yeast cream was prepared following manufacturer's instructions: 0.92 g of dry yeast was reconstituted in 10 times its mass in wort (8 mL).Yeast cream was then inoculated at 0.5 %, by volume (equivalent to approx.10 7 CFU/mL).A preculture of NCYC 500 strain grown for 48 h in nutrient broth was inoculated at 1 %, by volume (equivalent to approx.10 5 CFU/mL).Fermentation was carried out statically for 14 days.

Measurement of pH and °Brix
The total soluble solids (°Brix) and pH were measured using a refractometer (ATAGO Co., Ltd, Tokyo, Japan) and a pH meter (Metrohm, Zofingen, Switzerland), respectively.

Analysis of sugars and organic acids
Sugars were separated and quantifi ed using a Shimadzu UFLC (Shimadzu, Kyoto, Japan) equipped with a Pinnacle II amino column (150 mm×4.6 mm; Restek, Bellefonte, PA, USA).The mobile phase consisted of aceto ni-trile and water (80:20) that was sonicated for 30 min before use.Elution was carried out at 40 °C with flow rate of 1 mL/min (isocratic mode).An evaporative light-scatt ering detector (ELSD; Shimadzu, Kyoto, Japan) was used as the detector.Organic acids were determined by a Shimadzu UFLC (same as above), using a Supelcogel C-610H column (Supelco, Bellefonte, PA, USA).A photodiode array detector at UV spectrum (210 nm) was used.The mobile phase contained 0.1 % (by volume) sulphuric acid that was sonicated for 30 min before use.Elution was carried out at 40 °C with flow rate of 0.4 mL/min (isocratic mode).

Volatile analysis by HS-SPME-GC-MS⁄FID
Volatiles were analysed using headspace (HS) solid--phase microextraction (SPME) and a Carboxen/PDMS fibre (85 μm; Supelco, Sigma-Aldrich, Barcelona, Spain).This was coupled with gas chromatograph-mass spectrometer (GC-MS) and fl ame ionisation detector (FID) (Agilent Technologies, Santa Clara, CA, USA).Sample size was 5 mL (with pH adjusted to 2.5 using 1 M HCl).Each sample was extracted by HS-SPME at 60 °C for 50 min using 250-rpm agitation.Thermal desorption of the fi bre was carried out at the injection port of Agilent 7890A gas chromatograph (Agilent Technologies) coupled to Agilent 5975C triple-axis MS at 250 °C for 3 min.Elution and separation of compounds were carried out with helium as the carrier gas in a capillary column (Agilent DB--FFAP) with dimensions of 60 m×0.25 mm i.d.The column had a 0.25-μm coating of polyethylene glycol modifi ed with nitroterephthalic acid.Oven temperature was held at 50 °C for 5 min before a gradual increase of 5 °C per min until 230 °C, which was held for 30 min.Volatiles were identifi ed by comparing their mass spectra with NIST08 Mass Spectral Library (15) and Wiley 275 Library (16).Identifi cation of volatiles was verifi ed using standard compound and/or linear retention indices (LRI).

Statistical analysis
Data collected from triplicate fermentations were subjected to statistical analysis.To compare values, analysis of variance (ANOVA) using Microsoft Office Excel (Microsoft , Redmond, WA, USA) was used.If the p-value was below 0.05, diff erences were deemed statistically signifi cant.

Yeast growth, total soluble solids and pH changes
The two yeasts showed signifi cant diff erences in terms of growth, total soluble solid content (°Brix) and pH changes (Fig. 1).S. cerevisiae Safale US-05 reached the maximum cell count on day 2 at 4.44•10 7 CFU/mL.The maximum W. saturnus var.saturnus NCYC 500 cell count of 2.26•10 7 CFU/mL was reached on day 6 (Fig. 1a).The total soluble solid content reduced only slightly in the beer fermented with NCYC 500 strain relative to that of the beer fermented with Safale US-05 strain; the latt er total soluble solid content decreased from 13.8 to 5.9 % by day 6 (Fig. 1b).The pH decrease in the beer fermented with NCYC 500 strain was more gradual than that of the beer fermented with Safale US-05 strain; the latt er pH decreased markedly from day 0 to day (Fig. 1c).
Other researchers have also observed the comparatively slower growth rates of Williopsis yeasts (17,18).It has been shown that the intensity of fermentation (defi ned as the average number of days elapsing between inoculation of the glucose fermentation tube and the reduction of gas volume in the insert) with W. saturnus var.mrakii takes about 5 days (19).Thus, growth of NCYC 500 strain observed here corresponded to that reported for this yeast.

Sugar utilisation
Safale US-05 strain utilised glucose, fructose and sucrose rapidly by day 2 (Figs.2a-c).This corresponded to  the large decreases in °Brix, pH and maximal growth of Safale US-05 strain from day 0 to day 2, indicating the effi cient fermentation of these sugars by the yeast.On the other hand, the utilisation of these sugars by NCYC 500 strain was much slower, with glucose being preferred but with few disaccharides being fermented.Maltose utilisation by Safale US-05 strain was the slowest amongst the four sugars (Fig. 2d), with only about half of the sugar being utilised by day 2 as opposed to the virtually complete utilisation of the other three sugars by the yeast during that time.
Utilisation of sugars by S. cerevisiae is well-studied with easily fermentable monosaccharides such as d-fructose and d-glucose, which are utilised fi rst (20).Williopsis yeasts have also been reported to be glucophillic, consuming glucose faster than fructose (11).Sucrose was depleted by Safale US-05 strain by day 2 (Fig. 2c).It is not transported into the yeast cell but it is hydrolysed by invertase into fructose and glucose in the periplasmic space (21).Also, sucrose may be broken down via acid hydrolysis into the monosaccharides followed by intracellular utilisation.Unlike Safale US-05, the wort fermented by NCYC 500 strain had a high content of residual sucrose, likely due to its weak fermentative ability.
Utilisation of maltose by S. cerevisiae was slower than of fructose or glucose.This could be due to catabolite repression by glucose, which represses maltase gene expression (22).Fermentation of maltose with NCYC 500 strain was not signifi cant (Fig. 2d).Non-utilisation of maltose is apparently characteristic of Williopsis saturnus yeasts (23).Maltose is a quantitatively dominant sugar in wort and its utilisation (or lack of) has a direct infl uence on the alcohol content in the fi nal beer, as shown in this study.

Organic acid metabolism
Among the organic acids in beer fermented by Safale US-05 strain, those that showed signifi cant increases in concentration were pyruvic, α-ketoglutaric, lactic and acetic acids (Fig. 3).When using NCYC 500 strain, the pyruvic and acetic acid concentrations increased signifi cantly.The concentration of the remaining acids stayed relatively constant (Fig. 3).
Pyruvic acid concentration increased signifi cantly in the beer fermented with Safale US-05 strain (Fig. 3a).The beer fermented with NCYC 500 strain also showed an increase in pyruvic acid concentration, but it was not as signifi cant.In both yeasts, the increase in pyruvic acid concentration was due to the Embden-Meyerhof pathway, which converts available sugars into pyruvic acid, yielding adenosine triphosphate (ATP).Fermentation rate by Safale US-05 was much faster than by NCYC 500 strain, which might explain why pyruvic acid concentration in the Safale US-05-fermented beer increased signifi cantly by day 2, while there was a much more gradual increase in pyruvic acid concentration in the beer fermented with NCYC 500.In addition, the higher concentration of pyruvic acid in the beer fermented with Safale US-05 could also be because S. cerevisiae carries out anaerobic fermentation The initial concentration of α-ketoglutaric acid was low (Fig. 3b).In the beer fermented with Safale US-05 strain, α-ketoglutaric acid concentration increased.In the beer fermented with NCYC 500 strain, α-ketoglutaric acid concentration showed minor fl uctuations.α-Ketoglutaric acid is synthesised from the Krebs cycle or from amino acid catabolism and is a minor organic acid found in beer (24).Thus, it was expected that in the beer fermented with Safale US-05 the increase of the concentration of this acid was greater because amino acid catabolism was more extensive in S. cerevisiae than in NCYC 500 strain (data not shown).
Lactic acid concentration increased slightly throughout fermentation in the beer fermented with Safale US-05 (Fig. 3c).In the beer fermented with NCYC 500 strain, there were minor fl uctuations in lactic acid concentration.Strain NCYC 500, being more oxidative, would likely carry out litt le, or none at all, anaerobic conversion of pyruvic to lactic acid, which might explain the insignifi cant concentration of lactic acid in the beer fermented with NCYC 500.Acetic acid concentration in the beer fermented with both types of yeast increased, with the production rate being higher by NCYC-500 than by Safale US-05 (Fig. 3d).However, the fi nal concentration of acetic acid was a litt le higher in the beer fermented with Safale US-05 (870 mg/L) than in the beer fermented with NCYC 500 (800 mg/L).Acetic acid can be formed in S. cerevisiae by the oxidation of acetaldehyde, an intermediate in the alcoholic fermentation pathway, and co-reduction of NAD + catalysed by aldehyde dehydrogenase.The oxidation of acetaldehyde to acetic acid acts as a redox sink for anabolic and physiological stress reactions.Pyruvic acid can also be reabsorbed by the yeast and metabolised to acetate (25).Excess acetic acid is secreted into the fermentation medium (26).Acetic acid is not desired in alcoholic beverages as it can impart a sour vinegary off -odour if its concentration is too high.Since the fl avour threshold of acetic acid in beer is 130 mg/L, the detected concentrations of acetic acid in the two beers could impart a vinegary odour, although this was not discernible organoleptically in informal sensory evaluation by sniffi ng.

Changes in volatile composition
A total of 8 alcohols, 11 acids, 41 esters, 9 aldehydes, 8 ketones, 21 terpenes and terpenoids, 5 Maillard reaction products (furans, pyrans and pyrroles) and 2 volatile phenolic compounds were detected in the beer samples.Selected major volatiles in wort and beer are presented in Table 1.
The biggest group of volatiles detected in the beer samples were esters (Table 1).Fermentation altered the volatile composition of wort drastically, with a large variety of esters reduced or produced via the yeast metabolism.Ethyl and acetate esters were the two most significant types of esters detected.In the wort, there were only a few ethyl esters detected.However, Safale US-05 strain produced a wider variety of ethyl esters in higher concentrations relative to NCYC 500 strain aft er fermentation.Major ethyl esters were ethyl octanoate, decanoate, 9-decenoate and laurate.Fatt y acid ethyl esters are secondary metabolites produced by S. cerevisiae (4).Esterifi cation of the medium-chain fatt y acids that are thought to be toxic to yeast can be considered as a means to allow the diff usion of these toxic compounds out of the cell (4).In terms of fl avour, ethyl octanoate with a very low detection threshold is expected to contribute signifi cantly to the desired fruity aspect of beer (3).
Acetate esters were the next major type of esters formed by strains Safale US-05 and NCYC 500.The major acetate esters were ethyl, isoamyl and 2-phenylethyl acetate.The concentrations of ethyl and isoamyl acetate de-  (11,12).The concentration of isoamyl acetate in the beer fermented with Safale US-05 was approx.20 times lower than in that fermented with NCYC 500.2-Phenylethyl acetate production by Safale US-05 was 5 times lower than that by NCYC 500 strain.Thus, usage of Williopsis to ferment wort would result in a beer with a more distinct fruity, banana-like and fl oral aroma imparted by higher concentrations of acetate esters (11).However, the higher production of ethyl acetate by NCYC 500 strain could lead to a glue-like off --fl avour in beer (27).
Methyl esters were produced at low concentrations in both beer samples fermented with Safale US-05 and NCYC 500.These methyl esters were likely products of the esterifi cation of methanol and organic acids.The presence of methanol in fruit and grain-based beverages has been att ributed to the demethylation of methylated pectins, possibly due to acid hydrolysis (28) or enzyme action.
The major alcohol produced by both yeasts was ethanol, with Safale US-05 strain producing about 5 times more ethanol than NCYC 500 strain (Table 1).Based on o Brix approximation, the ethanol volume fraction in the beer fermented with Safale US-05 was 6.9 %, while that of the beer fermented with NCYC 500 strain was 1.7 %.This result was expected, as NCYC 500 strain has been reported to carry out fermentation with low ethanol production (11,29).Williopsis yeasts seem to predominantly utilize sugars via oxidative respiration, producing carbon dioxide and water, but only litt le ethanol (29).Other major alcohols detected in both beer samples included isobutanol, active amyl, isoamyl and 2-phenethyl alcohol, which are mainly produced via branched-chain and aromatic amino acid metabolism (30).Short-to medium-chain fatt y acids such as hexanoic, octanoic and decanoic acids were detected in the highest concentrations in both beer samples (Table 1).The fatt y acids may be products of yeast metabolism such as β-oxidation of higher fatt y acids (31).These fatt y acids are important for beer flavour because they themselves are odour-active and can lead to rancid off -flavour at high concentrations.Moreover, they are also one of the starting materials for the synthesis of volatile esters (32).In general, fatt y acid production by Safale US-05 was higher than by NCYC 500 strain.
Terpenes and terpenoids in wort are mainly derived from added hops.Terpene and terpenoid concentrations generally decreased from day 0 to day 14 in the beer fermented with either Safale US-05 or NCYC 500, with some terpenes becoming undetectable (Table 1).The exception was p-cymene, which was not detected in the wort, but was detected in both beer samples fermented with US-05 or NCYC 500 strain.
The most signifi cant terpenoid detected in the wort and the fermented beer samples was linalool (Table 1).Linalool is one of the key odourants that impart a fl oral, fruity and citrus note to the Cascade and Perle hops (33,34) used in this study.More importantly, linalool is also one of the hop-derived odourants in hopped beer, indicating that the presence of this terpenoid in beer comes at least partly from wort (35,36).However, the concentration of linalool found in the beer can be aff ected by yeast metabolism (37).S. cerevisiae is able to convert the odourless linalyl glycoside to linalool via the action of β-glucosidases (38).Yeast has been shown to be able to convert geraniol to linalool (37,39), although geraniol was not detected in the wort.
Linalool content could also decrease due to yeast metabolism, as indicated by our results (Table 1).It has been shown that linalool can be converted into other terpenoids such as α-terpineol (37,39), which was detected in the fi nal beer samples albeit at levels lower than those in the wort.Linalool could also have been transformed into linalool oxide detected in the beer fermented with NCYC 500 strain.Hence, it must be pointed out that the fi nal concentration of linalool is a net balance of production and utilisation.
Two terpenoid esters were detected in the fermented beer, but not in the wort.Specifi cally, citronellyl acetate was detected in both beer samples fermented with Safale US-05 and NCYC 500, but geranyl acetate was detected only in the beer fermented with NCYC 500 strain (Table 1).Citronellol was not detected in the wort, but it is possible that citronellyl acetate could have been formed via reduction of geranyl acetate (37).In addition, although geraniol was also not detected in all the samples, geranyl acetate was still formed.It has been shown that the geraniol to linalool biotransformation reaction is reversible; thus, some linalool present in the wort could have been converted into geraniol via yeast metabolism (37).Strain NCYC 500, with its higher acetate ester-synthesising activity than that of Safale US-05, could then form geranyl acetate, whereas the latt er could not.
Acetaldehyde was the major aldehyde detected in the beer fermented with Safale US-05 strain, but it was not detected in the beer fermented with NCYC 500 strain (Table 1).In S. cerevisiae, anaerobic catabolism of sugars to produce ethanol involves production of acetaldehyde as an intermediate.Although most of the acetaldehyde is metabolised, some molecules would still diff use out of the cell (40).Strain NCYC 500 has a low anaerobic fermentative ability; hence, the alcoholic fermentative pathway would not be carried out as extensively as by Safale US-05, resulting in only trace amounts of acetaldehyde formed.
Benzaldehyde was also another major aldehyde detect ed in the beer fermented by either Safale US-05 or NCYC 500 (Table 1).Benzaldehyde may originate from the oxidative degra dation of phenylpyruvic acid during phenylalanine meta bo lism of yeast (41).3-Methylbutanal, hexa nal, 2-phenylacetaldehyde and m-tolualdehyde (3-methylbenzaldehyde) were detected in the wort but not in the beer, probably due to yeast-mediated reduction of aldehydes to alcohols.Nonanal, (E)-2-decenal and o-tolualdehyde (2-methylbenzaldehyde) identifi ed in both beer samples could be the result of autoxidation of unsaturated fatt y acids.

Conclusions
The fermentation of sweet wort by a brewer's yeast and a Williopsis saturnus yeast was compared.W. saturnus var.mrakii NCYC 500 was able to produce higher concentrations of acetate esters and retain bett er the original terpenoid content.Fermentation with this yeast could be a novel biotechnological way of increasing the desirable fruity and fl oral fl avour of beer while retaining the characteristic hop aroma of the wort.Given that its fermentative ability of sugars is low, this could be a way of brewing extra-fruity low-alcohol beer.

Table 1 .
Selected major volatile compounds (GC-FID peak area•10 6 ) and their relative peak areas (RPA) identified in wort and beer fermented with S. cerevisiae Safale US-05 or W. saturnus var.mrakii NCYC 500 aft er 14 days of fermentation

Table 1 .
-continued tected in the beer fermented with NCYC 500 strain were signifi cantly higher than those detected in the beer fermented with Safale US-05.Williopsis yeasts are able to produce high concentrations of acetate esters, in particular isoamyl, ethyl and 2-phenylethyl acetate