Beta-glucosidase activity of wine yeasts and its impacts on wine volatiles and phenolics: A mini-review

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Food Microbiology 100 (2021) 103859

Available online 10 June 2021

Beta-glucosidase activity of wine yeasts and its impacts on wine volatiles and phenolics: A mini-review

Pangzhen Zhang

^a^,^*

, Ruige Zhang

^a

, Sameera Sirisena

^a

, Renyou Gan

^b^,^c

, Zhongxiang Fang

^a

aSchool of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, 3030, Australia

bResearch Center for Plants and Human Health, Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu, 610213, China

cKey Laboratory of Coarse Cereal Processing (Ministry of Agriculture and Rural Affairs), Sichuan Engineering & Technology Research Center of Coarse Cereal Industrialization, Chengdu University, Chengdu, 610106, China

A R T I C L E I N F O Keywords:

β-glucosidase activity Grape

Non-Saccharomyces yeasts Phenolics

Terpene Yeast

A B S T R A C T

Beta-glucosidase is an important enzyme for the hydrolysis of grape glycosides in the course of winemaking.

Yeasts are the main producers of β-glucosidase in winemaking, therefore play an important role in determining wine aroma and flavour. This article discusses common methods for β-glucosidase evaluation, the β-glucosidase activity of different Saccharomyces and non- Saccharomyces yeasts and the influences of winemaking conditions, such as glucose and ethanol concentration, low pH environment, fermentation temperature and SO2 level, on their activity. This review further highlights the roles of β-glucosidase in promoting the release of free volatile compounds especially terpenes and the modification of wine phenolic composition during the winemaking process. Furthermore, this review proposes future research direction in this area and guides wine professionals in yeast selection to improve wine quality.

1. Introduction

Beta-glucosidase is an important enzyme in the winemaking process.

It is involved in the hydrolysis of glycosidic bonds connected to the terminal non-reducing glucosides and oligosaccharides and the release of glucose glycone (Liang et al., 2020). β-Glucosidase is naturally present in plant materials such as grapes and can be produced by some microorganisms (Maicas and Mateo, 2005). However, the former sources are of less importance to the winemaking process (Ar´evalo Villena et al., 2005), and yeast and lactic acid bacteria have been suggested as the main source of this enzyme (Blasco et al., 2006; Matthews et al., 2004).

Modern winemaking techniques often use exogenous enzymes to compensate the insufficient enzyme activity in grapes (Cordero Otero et al., 2003), mostly by the addition of specific strains of Saccharomyces or non-Saccharomyces yeasts with known β-glucosidase activity (Palmeri and Spagna, 2007).

Both Saccharomyces and non-Saccharomyces yeast strains can produce β-glucosidase during fermentation to hydrolyse glycosides (Villena et al., 2007). Their β-glucosidase activities are commonly tested using p-nitrophenyl-β-^D-glucopyranoside or 4-methylumbelliferyl-β-^D-glucose assays (Hall et al., 2017; Porter et al., 2019a). It is important to note that the activity of β-glucosidase varies significantly depending on the yeast

species, and some strains may not produce this enzyme at all (Carrau et al., 2005). Furthermore, yeasts may produce β-glucosidase at different locations such as cytosol, cell membrane and cell wall (Ar´evalo Villena et al., 2005) and the intracellular β-glucosidase activity of yeasts is of little value for industrial application. Therefore, researches on yeast β-glucosidase activity mainly focus on its activity of whole cell and yeast supernatant, in other words, extracellular β-glucosidase activity. In addition, wine processing conditions such as glucose concentration, ethanol concentration, pH level, fermentation temperature and SO2

level could also affect its activity (Madrigal et al., 2013).

An important role of β-glucosidase in the winemaking process is to promote the production of aromatic compounds of wine, thereby affecting the aroma and flavour of the product. β-Glucosidase contrib- utes to the hydrolysis of different types of glycosidic bonds such as 1,4-β and 1,6-α linkages, and promotes the formation of free terpenes, phe- nylpropenes and specific aliphatic ester during wine fermentation (Liang et al., 2020). Among these aromatic volatiles, terpenes, especially monoterpenes such as geraniol, nerol, citronellol, linalool and α-terpineol are the most important chemicals due to the abundance of their precursors in grape (Mateo and Jim´enez, 2000). In addition, β-glucosidase also modifies the composition of phenolic compounds in wine through hydrolysis of their parent compounds. For example,

* Corresponding author.

E-mail address: [email protected] (P. Zhang).

Contents lists available at ScienceDirect

Food Microbiology

journal homepage: www.elsevier.com/locate/fm

https://doi.org/10.1016/j.fm.2021.103859

Received 16 December 2020; Received in revised form 4 June 2021; Accepted 6 June 2021

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different Saccharomyces and non-Saccharomyces yeast strains with different β-glucosidase activity could influence the concentration of resveratrol and anthocyanins in the resultant wine (Todaro et al., 2008).

Some specific S. cerevisiae strains such as AS11 could reduce the content of anthocyanins by hydrolysing them into anthocyanidin aglycones, eventually resulting in loss of wine colour (Rodríguez et al., 2004).

To date, there is lack of literature comparing the extracellular β-glucosidase activity of different wine yeasts. This article summarises the previous studies on the extracellular β-glucosidase activity of wine- associated yeasts by comparing and analysing the characteristics of each yeast strain, which allows the identification of yeasts with strong application potential for winemaking. This review further highlights the role of β-glucosidase in altering the volatile and phenolic composition of wine and thus the product quality. Importantly, this review proposes future research aspects on yeast-originated β-glucosidase, and guides wine industry in yeast selection.

2. Comparison of β-glucosidase activity from different yeasts Both Saccharomyces and non-Saccharomyces yeasts have the ability to produce β-glucosidase (Strauss et al., 2001). Specific strains of common yeasts such as S. cerevisiae, Hanseniapora uvarum (Ar´evalo Villena et al., 2005), Wickerhamomyces anomalus (Manzanares et al., 2000), Tricho- sporon asahii (Wang et al., 2015) have been reported to have high β-glucosidase activity during alcohol fermentation. It is suggested that S. cerevisiae is not the major source of extracellular β-glucosidase enzymes, while non-Saccharomyces yeasts are more likely to produce extracellular glycosidase (Charoenchai et al., 1997).

2.1. Common methods for β-glucosidase activity evaluation

Beta-glucosidase activity of yeasts is generally measured in vitro using an artificial substrate, which can be converted into a coloured product and then detected using spectrophotometer. The most commonly used method for determining β-glucosidase activity is the p- nitrophenyl-β-D-glucopyranoside (p-NPG) assay (Hall et al., 2017), which is to measure the concentration of p-nitrophenol (p-NP) released from p-NPG catalysed by the β-glucosidase of microorganisms (Hu et al., 2016). This assay is widely used to evaluate the intensity of β-glucosidase activity and hydrolytic capacity during the fermentation process (Hu et al., 2016; Mateo and Jim´enez, 2000; Swangkeaw et al., 2009).

Strains are often cultivated in YPD medium (1% yeast extract, 2%

peptone and 2% glucose) with pH adjusted to 3.5 before subjected to p-NPG analysis (Wang et al., 2015). The release of p-nitrophenol is measured by spectrophotometry at 400 nm and the β-glucosidase activity can be calculated using p-nitrophenol standard curve (Hall et al., 2017). Based on the p-NPG assay, researchers often determine the extracellular β-glucosidase activity of yeasts by studying the supernatant and/or pellet of yeast after it is cultured in specific broth media, and the result is usually expressed as supernatant β-glucosidase and whole cell β-glucosidase, respectively (Hern´andez et al., 2003). Alternatively, Ar´evalo Villena et al. (2005) proposed a method which used cellobiose as the hydrolysis substrate and measured the release of glucose to reflect the β-glucosidase activity, and this method is able to measure the β-glucosidase activity of different cell fractions. Furthermore, other researchers screened the release of 4-methylumbelliferone from 4-methylumbelliferyl-β-D-glucose (4-MUG), a fluorescent compound, by irradiating long-wave ultraviolet light, which reflect β-glucosidase activity (Charoenchai et al., 1997; Porter et al., 2019a). This method (MUG assay) is often used for the identification rather than the quantification of β-glucosidase activity of yeasts. Plate screen of β-glucosidase can be carried out using 4-MUG assay, which is especially important for the fast and large-scale screening of yeast strains when quantification of β-glucosidase activity is not required (Manzanares et al., 2000; Lopez ´ et al., 2015). In addition to MUG, arbutin (hydroquinone β-D-glucopyranoside) and esculin (esculetin 6-β-D-glucoside) are the mostly used

chromogenic substrates for plate screening of yeasts with β-glucosidase activity, which can produce brown-blackish pigments in the presence of β-glucosidase (Kwon et al., 1994; Rosi et al., 1994). Later, P´erez et al.

(2011) demonstrated the suitability of esculin-glycerol agar (EGA) solid medium for the screening of wild yeasts with β-glucosidase activity, where the halo diameter in EGA medium could indicate the strength β-glucosidase activity. EGA medium can be utilised by wine industry professionals and researchers as a fast-screening method of indigenous yeasts, where yeast strains showing positive β-glucosidase activity can be further quantified using the p-NPG assay. Furthermore, Zhou et al.

(2009) developed a high-speed screening system using calcium alginate micro-beads and flow sorting for the screening of β-glucosidase activity on yeast cell surface. This method allows the high-throughput selection and isolation of specific yeasts cells with high enzymatic activity on the cell surface, thus a more efficient way to obtain high performance strains. Nevertheless, this method requires more technical skills than using the EGA medium, and thus less suitable for industry application.

More recently, Chen et al. (2021) developed a simple and portable β-glucosidase activity assay based on the β-glucosidase-mediated cascade reaction in personal glucose meter (PGM) device. D-(− )-salicin was used as the reacting substrate and can be hydrolysed by β-glucosidase into saligenin, which could trigger the ferricyanide-ferrocyanide conversion in the glucose strips and display reading in the PGM device. Future commercialisation of this newly developed method may offer an alternative option for wine industry professionals to perform fast β-glucosidase activity screening of wild yeasts.

2.2. β-Glucosidase activity of Saccharomyces yeasts

Although yeasts are generally considered as a source of glucosidases, there are contradictory reports on the activity of β-glucosidase produced by different Saccharomyces yeasts. The structural genes for the biosyn- thesis of β-glucosidase present in the genome of Saccharomyces spp., however the yield and activity of β-glucosidase reported in previous studies is often low or even absent. Significant variations in β-glucosidase activity were observed among different S. cerevisiae strains, where AL41 showed the strongest exogenous β-glucosidase activity compared to other 5 strains including CBS1171, DIPROVL220, 6527, 6527-1D and 7070 (Spagna et al., 2002b). Vernocchi et al. (2011) compared the extracellular and whole cell β-glucosidase activity between commercial and wild S. cerevisiae strains. Results showed that wild S. cerevisiae strains AS11, AS15 and BV14 had significantly higher extracellular and whole cell β-glucosidase activity than that of the commercial strain CY.

Among these wild strains, AS11 showed the highest β-glucosidase activity, followed by AS15 and BV14, while strain BV12 had the lowest activity similar to CY. This result suggested good commercial values for AS11 and AS15 strains. In addition to S. cerevisiae, S. uvarum are often used in winemaking, where S. uvarum B2EN2, VA42, CRY11, CRY14, CRY24, GRAS13 and GRAS14 have been found to have stronger whole cell β-glucosidase activity than S. cerevisiae AL41 (Bonciani et al., 2018).

The same study also found S. cerevisiae IperR had comparable whole cell β-glucosidase activity as that of S. uvarum strains, while no activity was detected in S. cerevisiae UMCC2617. In addition to natural yeasts, engineered S. cerevisiae have also been developed to achieve higher β-glucosidase producing ability (Bae et al., 2014). Nevertheless, the present review focuses on natural yeast rather than genetically modified yeast.

Overall, high β-glucosidase activity is rare in indigenous S. cerevisiae strains, which generally had lower activity than that of non-Saccharo- myces yeasts strains (Maicas and Mateo, 2005). Therefore, research ef- forts have been made to screen non-Saccharomyces yeasts with high β-glucosidase activity or study the influence of mixed-culture fermentation of Saccharomyces and non-Saccharomyces yeasts on the β-glucosidase activity during winemaking.

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2.3. β-Glucosidase activity of Non-Saccharomyces yeasts

Non-Saccharomyces yeasts have been used in the wine fermentation process as a source of exogenous glycosidases (Palmeri and Spagna, 2007). Up to date, the reported non-Saccharomyces yeasts with extracellular and/or whole cell β-glucosidase activity include Candida (Charoenchai et al., 1997), Brettanomyces (Fia et al., 2005), Debar- yomyces (Rosi et al., 1994), Hanseniaspora (Rosi et al., 1994), Lachancea (Porter et al., 2019a), Metschnikowia (Rosi et al., 1994), Pichia (Char- oenchai et al., 1997), Rhodotorula (Ar´evalo Villena et al., 2005), Tri- chosporon (Wang et al., 2011), Torulaspora (Charoenchai et al., 1997), Wickerhamomyces (Madrigal et al., 2013) and Zygosaccharomyces (Manzanares et al., 2000), although the levels of β-glucosidase activity are different (Table 1).

Yeasts in Candida genus are generally considered as good producers of extracellular and/or whole cell β-glucosidase. With the recent changes in nomenclature, some of the member species are now classified into different genera (Schoch et al., 2020). Despite of this, we try to discuss the current and former Candida species together. Charoenchai et al. (1997) observed the presence of high extracellular β-glucosidase activity in three strains of Starmerella stellata (homotypic synonym:

Candida stellata) 504, 800 and 8008. There are variations in the β-glucosidase activity of different species and strains, where C. cantarelli, C. domerquiae, Trigonopsis vinaria (previously C. vinaria) and Kre- gervanrija fluxuum (heterotypic synonym: C. vini) were found with extracellular β-glucosidase activity, but no activity was detected in the two strains of S. stellata (C. stellata) 11108 and 11109 (Manzanares et al., 2000). Furthermore, three strains of C. dattila including 10559, 1962 and 10387 were observed with low extracellular β-glucosidase activity, but no activity was observed in C. dattila 10652 (Manzanares et al., 2000), C. parapsilosis F25 and Starmerella bacillaris (heterotypic synonym: Candida zemplinina) F38 (Wang et al., 2011). Wang et al. (2011) also observed low extracellular β-glucosidase activity in C. railenensis F72. Among these different species, Kuraishia molischiana (gomotypic genbank synonym: Candida molischiana) seems to have the strongest extracellular and/or whole cell β-glucosidase activity and therefore is often used as a positive control in yeast β-glucosidase studies (Ar´evalo Villena et al., 2005; Fernandez-Gonz´ ´alez et al., 2003).

High whole cell β-glucosidase activity was also found in Brettano- myces spp., where all of the eight tested strains exhibited moderate to strong β-glucosidase activity (Fia et al., 2005). Low whole cell β-glucosidase activity was detected in Brettanomyces bruxellensis (heterotypic synonym: Dekkera intermedia; homotypic synonym: Dekkera brux- ellensis), but no activity was observed in the supernatant of the same microorganism (Rosi et al., 1994). Similarly, Manzanares et al. (2000) did not detect extracellular activity in B. bruxellensis 11045 strain.

Debaryomyces hansenii (also known as Candida famata) and Schwannio- myces polymorphus (homotypic synonym: Debaryomyces polymorphus) were reported with both extracellular and whole cell β-glucosidase activities, where D. hansenii was more frequently studied (Rosi et al., 1994). Clear variations were observed between different strains of D. hansenii, where D. hansenii 4025 was reported to have strong β-glucosidase activities, while D. hansenii 533, 6010, 618, 1 and 1M seemed to have lower activity (Arévalo Villena et al., 2005; Charoenchai et al., 1997; Fernández-González et al., 2003; Rosi et al., 1994). Another widely studied genus is Hanseniaspora, where H. valbyensis, H. uvarum, H. guilliermondii, H. osmophila, and H. vineae all have been found with β-glucosidase activity (Arévalo Villena et al., 2005; Hall et al., 2017; Hu et al., 2018; Manzanares et al., 2000; Maturano et al., 2012). Among them, H. uvarum is most studied, where the activity is highly dependent on the strain and some H. uvarum strains such as 24 Mt and 52 Mt were reported with positive β-glucosidase activity, while others without the activity (Arévalo Villena et al., 2005). A recent study screened 196 strains of Hanseniaspora and observed high β-glucosidase activity in H. guilliermondii than H. uvarum, indicating the potential of H. guilliermondii as a contributor of β-glucosidase for wine fermentation

(Testa et al., 2020).

Lachancea spp. is attracting more research attention due to its oenological significance (Porter et al., 2019b). The two most common Lachancea species are L. thermotolerans (homotypic synonym: Kluyver- omyces thermotolerans) and L. fermentati, both having been found to produce β-glucosidase when fermenting grape (Porter et al., 2019a). The same study also reported slightly lower β-glucosidase activity in L. lanzarotensis in comparison to the forementioned two species. While specific strains of L. thermotolerans such as Y940 and Concerto hold strong β-glucosidase activity, others such as 85, 32C and 30 Mt have much lower β-glucosidase activity, showing significant variations among strains (Arévalo Villena et al., 2005; Fernández-González et al., 2003; Porter et al., 2019a). Many studies reported the β-glucosidase activity in Metschnikowia genus, mostly M. pulcherrima, and its activity was validated in both MUG and p-NPG assays (Morata et al., 2019).

Nevertheless, the extracellular β-glucosidase activity of M. pulcherrima also depends on the strain, and some strains such as 11202 may not have the activity at all (Manzanares et al., 2000). Pichia genus has also been widely studied as a common non-Saccharomyces yeast involved in wine fermentation, and many species in this genus such as P. fermentans and P.

membranifaciens (heterotypic synonym: P. membranaefaciens) have been characterised for their moderate to high β-glucosidase producing ability (Ar´evalo Villena et al., 2005; Wang et al., 2011), while others such as P. kudriavzevii (heterotypic synonym: I. orientalis) has low whole cell β-glucosidase activity (Charoenchai et al., 1997). Another yeast species Meyerozyma guilliermondii that formerly classified as Pichia guilliermondii has also been found to possess low to high β-glucosidase activity, where M. guilliermondii G1.2 strain is a good producer of high β-glucosidase (da Silva et al., 2019; Wang et al., 2011). Recent research found that another species of Pichia genus, i.e. Pichia terricola (formerly Issatchenkia terri- cola), can effectively produce β-glucosidase and increase the volatile content in Cabernet Sauvignon wine (de Ovalle et al., 2018).

Only a few strains of Rhodotorula mucilaginosa were found with extracellular β-glucosidase activity, while most studies reported nega- tive result for Rhodotorula yeasts (Ar´evalo Villena et al., 2005; Wang et al., 2011). Amongst different strains of R. mucilaginosa, BEI29 strain was reported with high extracellular β-glucosidase activity (Hu et al., 2016), as well as other types of glycosidase activity such as β -D-galac- tosidase, α-L-rhamnosidase and α-L-arabinosidase activities (Wang et al., 2017). These researchers also identified a specific strain of Trichosporon ashii F6 with strong extracellular β-glucosidase activity (Hu et al., 2016).

Torulaspora delbrueckii has been reported by many studies, and specific strains such as BTd259 has strong β-glucosidase producing capacity, while some strains such as 2585 does not produce extracellular β-glucosidase (Fia et al., 2005; Maturano et al., 2012). Wickerhamomyces anomalus, also known as Pichia anomala and Hansenula anomala, has been well recognised as a good contributor of β-glucosidase. Researchers observed moderate to high extracellular and whole cell β-glucosidase activities in W. anomalus depending on the specific strain, where strong activity was observed in 97M strain (Ar´evalo Villena et al., 2005;

Madrigal et al., 2013; Rosi et al., 1994). Zygosaccharomyces spp. is less studied and generally show low or no extracellular β-glucosidase activity (Manzanares et al., 2000).

Beta-glucosidase activity of Saccharomyces and non-Saccharomyces yeasts were compared in many studies. Hall et al. (2017) compared the extracellular β-glucosidase activity of two S. cerevisiae isolates with that of M. pulcherrima GU080051, H. uvarum EU386753, L. thermotolerans U69581, and observed the lowest activity in these two S. cerevisiae isolates, while H. uvarum EU386753 showed the strongest activity. How- ever, Saccharomyces strains with the strongest β-glucosidase activity, such as S. cerevisiae IperR and S. uvarum GRAS14, showed lower activity compared with that of W. anomalus BS81 strain (Bonciani et al., 2018).

Charoenchai et al. (1997) reported extracellular β-glucosidase activity in two S. cerevisiae strains, HB350 and V1118, which were slightly weaker than that of D. hansenii 6010, T. delbrueckii 9003, M. pulcherrima 1000, S.

stellata 504 and 8008, W. anomalus 703300 and 209, but similar to that

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Table 1

Comparison of β-glucosidase activity from different yeast.

Yeast Species Strains Code β-Glucosidase

activity^a Experimental condition Result Reference

Saccharomyces cerevisiae CBS 1171 + Yeast whole cell and supernatant

solution was analysed separately using p-NPG assay (citrate-phosphate buffer, pH 5.0, incubated at 30 ^◦C).

•AL 41 strain had the highest extracellular and whole cell β-glucosidase activity;

•Other strains had similar β-glucosidase activity.

Spagna et al. (2002b) DIPROVAL 220 +

6527 +

6527-1D +

7070 +

AL41 ++

S. cerevisiae commercial

strains CY + Yeast whole cell and supernatant

solution was analysed separately using p-NPG assay (Supernatant sample mixed with acetic acid buffer, p-NPG, pH 4.0 incubated at 50 ^◦C for 25 min, then absorbance measured at 400 nm;

whole cell sample incubated in citrate–phosphate buffer, p-NPG, pH 4.0 incubated at 40 ^◦C for 25 min, then absorbance measured at 400 nm).

•Strains AS11 had the highest extracellular and whole cell β-glucosidase activity, followed by AS15, BV14, BV12, while CY had the lowest.

Vernocchi et al.

(2011) Saccharomyces cerevisiae

wild strains AS11 +++

AS15 ++

BV12 +

BV14 ++

Saccharomyces cerevisiae UMCC 2617 – Yeast whole cell, supernatant and cell lysates analysed separately using p- NPG assay (citrate-phosphate buffer, pH 5.0, incubated at 30 ^◦C).

•W. anomalus strain BS81 showed the higher whole cell β-glucosidase activity than S. uvarum strains;

•Selected S. uvarum strains had similar whole cell β-glucosidase activity as that of S. cerevisiae IperR.

(Bonciani et al., 2018)

AL41 +

IperR ++

Saccharomyces uvarum B2EN2 ++

VA42 ++

CRY11 ++

CRY14 ++

CRY24 ++

GRAS13 ++

GRAS14 ++

Wickerhamomyces anomalus(Hansenula anomala and Pichia anomala)

BS81 +++

Debaryomyces hansenii

(Candida famata) 4025 +++ Yeast culture supernatant, whole and

permeabilized cell were measured separately using p-NPG assay (citrate- phosphate buffer, pH 5.0, incubated at 30 ^◦C for 1 hr).

•The highest supernatant and whole cell β-glucosidase activity was found in D. hansenii, followed by S. polymorphus (D. polymorphus);

•No β-glucosidase activity was found in the supernatant of wine spoilage yeasts B. bruxellensis, W. anomalus and S. ludwigii, and low activity was found in their whole cell samples. In contrast, these yeasts have higher intracellular β-glucosidase activity in the permeabilised samples.

Rosi et al. (1994) Schwanniomyces

polymorphus (Debaryomyces polymorphus)

3631 ++

Brettanomyces bruxellensis (Dekkera intermedia, Dekkera bruxellensis)

16 +

Hanseniaspora valbyensis 3138 ++

Wickerhamomyces anomalus (Hansenula anomala and Pichia anomala)

4045 ++

Hanseniaspora uvarum

(Kloeckera apiculata) 4045 +

Metschnikowia pulcherrima 3563 +

Saccharomycodes ludwigii 3991 +

Starmerella stellata

(Candida stellata) 504, 800, 8008 ++ Yeast whole cell was analysed using using p-NPG assay (pH 3.5. Glucose content in medium 0 g/L or 5 g/L.

Incubated at 25 ^◦C for 48 hrs).

•At 5 g/L glucose condition, all strains exhibited extracellular β-glucosidase activity;

•Clear variation in extracellular β-glucosidase activity were observed among different strains of T. delbrueckii, M. pulcherrima and H. uvarum.

Charoenchai et al.

(1997) Debaryomyces hansenii

(Candida famata) 533, 6010, 618 +

Hanseniaspora uvarum 401 ++

610, FRR2164,

FRR2168 +

Pichia kudriavzevii

(Issatchenkia orientalis) 304, 8001 +

Metschnikowia pulcherrima 1000 ++

2000 +

703300, 209 ++

Torulaspora delbrueckii 502, 3005 +

9003, 6005, 9512

++

Saccharomyces cerevisiae HB350, V1118 +

Candida cantarelli 11150, 11170 + Yeast suspension inoculated in sodium

phosphate buffer (3.5) and p-NPG, incubated at 30 ^◦C, then absorbance measured at 405 nm.

•Strongest total extracellular and cell wall β-glucosidase activity was observed in W. anomalus and Hanseniaspora;

Manzanares et al.

(2000)

Candida dattila 10559, 1962,

10387

+

10652 –

Candida domerquiae 10650 +

11108, 11109 –

(continued on next page)

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Table 1 (continued)

•No β-glucosidase activity was observed in Brettanomyces, Rhodotorula and Schizosaccharomyces genera;

•W. anomalus 10590 had the strongest total extracellular and cell wall β-glucosidase activity;

•The total extracellular and cell wall β-glucosidase activity of yeast highly depended on the species and strains.

Starmerella stellata (Candida stellata) Trigonopsis vinaria

(Candida vinaria) 11177 +

Kregervanrija fluxuum

(Candida vini) 10053 +

Brettanomyces bruxellensis (Dekkera intermedia, Dekkera bruxellensis)

11045 –

Hanseniaspora

guilliermondii 11104 ++

11027 –

Hanseniaspora osmophila 11206, 11207 ++

Hanseniaspora uvarum 11106 ++

1444, 10389, 11105, 11107, 11156, 10388, 10408

–

11202 –

10320, 10410, 10591

++

10590 +++

Pichia fermentans 10064 –

Pichia membranifaciens (Pichia

membranaefaciens)

10037, 10570 + 100113, 10568 – Sterigmatomyces elviae

(Rhodotorula acuta) 11175 –

Rhodotorula glutinis 10145 –

Schizosaccharomyces

pombe 1375, 1376,

1377 –

Zygosaccharomyces bailii 11040 +

11041, 11042 –

Zygosaccharomyces mellis 11149 +

Zygosaccharomyces rouxii 11040, 11149, 10137, 11136, 11189, 10381, 10445

+

1231, 10425 – Kuraishia molischiana

(Candida molischiana) CBS136 +++ Yeast whole cell was measured separately using p-NPG assay (citrate- phosphate buffer, pH 5.0, incubated at 30 ^◦C for 1 hr).

•Strongest whole cell β-glucosidase activity was observed in K. molischiana CBS136;

•Similar whole cell β-glucosidase activity was observed in other strains.

Fern´andez-Gonz´alez et al. (2003) Debaryomyces hansenii

(Candida famata) 1 +

Hanseniaspora uvarum 31 +

Lachancea thermotolerans (Kluyveromyces thermotolerans)

85 +

Pichia kluyveri 84 +

Saccharomyces cerevisiae 39 +

301 +

UCLM325 +

Kuraishia molischiana (Candida molischiana)

+++ Yeast whole cell, supernatant, cell wall, cytosol and membrane were analysed separately. Cellobiose was mixed with cell extract and citrate phosphate buffer (pH 3.5) then glucose produced was measured using glucose kit at 505 nm to reflect β-glucosidase activity.

•Wickerhamomyces, Pichia and Debaryomyces strains exhibited the strongest whole cell β-glucosidase activity, while strains from H. uvarum, H. osmophila, L. thermotolerans and M. pulcherrima had low or no whole cell β-glucosidase activity;

•H. uvarum, H. osmophila, L. thermotolerans and M. pulcherrima strains had significantly higher intracellular β-glucosidase activity than in the cytosol;

•All strains had significantly higher intracellular β-glucosidase activity than whole cell β-glucosidase activity, except for P. membranifaciens 98M and 6 Mt, T. delbrueckii 34C and K. molischiana, where whole cell β-glucosidase activity was stronger.

Ar´evalo Villena et al.

(2005) Debaryomyces hansenii

(Candida famata) 1M +

32C, 30 Mt +

Hanseniaspora uvarum 24 Mt, 52 Mt ++

31 Mt –

Hanseniaspora osmophila 7C, 9C, 45C –

44C +

Mestchnikowia pulcherrima 132M, 9 Mt + Wickerhamomyces

anomalus (Hansenula anomala and Pichia anomala)

97M +++

membranaefaciens)

98M +++

6 MT +++

Rhodotorula mucilaginosa 341C ++

2 Mt –

Torulaspora delbrueckii 4 Mt +

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Table 1 (continued)

33C, 34C +++

Saccharomyces cerevisiae UCLMS325 ++

41C ++

Brettanomyces spp 233, 539, 543 +++ Whole cell β-glucosidase activity

measured using 4-methylumbelliferyl- β-D-glucose (4-MUG) assay at 37 ^◦C, pH 5.0.

•High whole cell β-glucosidase activity was observed in specific strains of Brettanomyces, Debaryomyces, Hanseniaspora, Metschnikowia and Saccharomyces.

Fia et al. (2005)

345 ++

470 ++

464, 571, 572 ++++

Debaryomyces hansenii

(Candida famata) 4025 ++++

Hanseniaspora spp 300, 474 +++

301, 316, 319,

324, 327 ++++

653 –

Metschnikowia spp 103, 645 ++++

145, 146, 471, 803

+

553 –

562, 563, 756 +++

Torulaspora dulbrueckii 2585 –

Saccharomyces cerevisiae 91 +

416, 56, 60, 66, 45

++++

83 +++

89 –

744 +

Aureobasidium pullulans F82 – Yeast culture supernatant measured

using p-NPG assay in citrate-phosphate buffer at pH 5.0

•The strongest extracellular

β-glucosidase activity was identified in T. asahii, while moderate activities were detected in H. uvarum, P.

fermentans and S. cerevisiae F27.

C. railenensis and M. guilliermondii had the lowest activity.

Wang et al. (2011)

Candida parapsilosis F25 –

Starmerella bacillaris

(Candida zemplinina) F38 –

Candida railenensis F72 +

Hanseniaspora uvarum F35 ++

Pichia fermentans F42 ++

Meyerozyma guilliermondii

(Pichia guilliermondii) F47 +

Rhodotorula mucilaginosa F32 –

Trichosporon asahii F6 +++

Saccharomyces cerevisiae F27 ++

Saccharomyces cerevisiae BSc562 +++ Pure and mixed yeast culture were

used to ferment Pedro Gim´enez grape and β-glucosidase in the ferment was measured using p-NPG assay. The total fermentation time was 36 days. The Area Under the Enzyme Curve (AUEC) was calculated as the area under the plotted graph of measured enzyme level against time, from day 0 to day 36.

•Pure culture fermentation of S. cerevisiae BSc562 and T. delbrueckii BTd259 showed higher AUEC value than H. vineae BHv438;

•Despite of similar AUEC between S. cerevisiae BSc562 and T. delbrueckii BTd259, the peak β-glucosidase value was higher in BSc562.

•All mixed fermentation resulted in lower AUEC compared to pure culture fermentation using S. cerevisiae BSc562.

Maturano et al. (2012)

Hanseniaspora vineae BHv438 ++

Torulaspora delbrueckii BTd259 +++

S.cerevisiae and H.vineae 1% BSc562 + 99% BHv438

++

S.cerevisiae and H.vineae 10% BSc56 vs 90% BHv438

++

S.cerevisiae and

T. delbrueckii 1% BSc562 vs

99% BTd259 ++

S.cerevisiae and

T. delbrueckii 10% BSc562 vs

90% BTd259 ++

Pichia fermentans Isolate 1 – Whole cell β-glucosidase activity

measured using 4-methylumbelliferyl- β-D-glucose (4-MUG) assay at 28 ^◦C and incubated for 3 days.

•W. anomalus were detected with high β-glucosidase activity;

•No β-glucosidase activity was detected in P.fermentans isolates;

•All strains of P. membranifaciens have β-glucosidase activity, among them P. membranifaciens isolate 7 had the strongest activity.

Madrigal et al. (2013)

Isolate 2 –

membranaefaciens)

Isolate 1 +

Isolate 2 +

Isolate 7 +++

Isolate 1 ++

Isolate 2 ++

Hanseniaspora uvarum F35 ++ Yeast culture supernatant measured

using p-NPG assay in citrate-phosphate buffer at pH 5.0

•High extracellular β-glucosidase activity was observed in T. asahii at low glucose (2%) condition in comparison to other strains.

Wang et al. (2015)

Pichia fermentans F16, F42 +

Trichosporon asahii F6 +++

Saccharomyces cerevisiae F30 ++

Hanseniaspora uvarum EU386753 + Yeast whole cell was analysed using

using p-NPG assay (pH 3.5. Sugar content in medium 5 g/L glucose or 100 g/L glucose and fructose.

Incubated at 8 or 25 ^◦C for 48 hrs).

•H. uvarum showed significantly higher extracellular β-glucosidase activity under sugar restriction culture condition (5 g/L glucose, pH3.5 and 25 ^◦C) compared to other strains;

•In the condition without sugar restriction (100 g/L glucose, 100 g/L fructose, pH3.5 and 25 ^◦C), M. pulcherrima and two S. cerevisiae strains had similar extracellular β-glucosidase activity higher than that of H. uvarum and L. thermotolerans.

Hall et al. (2017) Lachancea thermotolerans

(Kluyveromyces thermotolerans)

U69581 +

Metschnikowia pulcherrima GU080051 ++

Saccharomyces cerevisiae isolate 1 ++

Saccharomyces cerevisiae isolate 2 ++

Y940 ++++ Whole cell associated β-glucosidase

activity measured during fermentation

•Lachancea strains exhibited stronger

β-glucosidase activity when fermenting Porter et al. (2019a)

Concerto +++

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of D. hansenii 533, M. pulcherrima 2000, H. uvarum 401 and FRR2164 (Charoenchai et al., 1997). Fern´andez-Gonz´alez et al. (2003) also identified three S. cerevisiae strains UCLM325, 39 and 301 with whole cell β-glucosidase activity similar to that of D. hansenii 1, H. uvarum 31, M. pulcherrima 9, L. thermotolerans 85 and P. kluyveri 84, however their activities are much weaker compared to that of K. molischiana CBS136.

Ar´evalo Villena et al. (2005) further examined the location of β-glucosidase activity in two S. cerevisiae strains 41C and UCLMS325 and compared that with non-Saccharomyces. Moderate to high whole cell β-glucosidase activity was observed in 41C and UCLMS325 respectively, while low β-glucosidase activity was detected in the supernatant of UCLMS325, but absent in the supernatant of 41C. Interestingly, dramatically higher β-glucosidase activity was detected in the cytosol and membrane of these two strains in comparison to that of whole cell, suggesting strong intracellular β-glucosidase activity. A similar phe- nomenon was also observed in some non-Saccharomyces such as H. uvarum, H. osmophila, L. thermotolerans and M. pulcherrima strains where higher β-glucosidase activity was detected in intracellular com- ponents than extracellular samples. On the other hand, some specific non-Saccharomyces yeasts including Pichia and Candida genera were exceptionally high in extracellular activity in comparison to their intracellular activity, which indicated the practical value for their application in wine industry (Ar´evalo Villena et al., 2005).

Overall, S. cerevisiae strains including AL41, AS11, AS15, BV14 and BSc562 and non-Saccharomyces yeasts such as K. molischiana CBS136, D. hansenii 4025, L. thermotolerans Y940, W. anomalus 10590 and 97M, P. membranifaciens 98M and 6 MT, R. mucilaginosa BEI29 and T. delbrueckii 33C, 34C BTd259 are good sources of β-glucosidase and may be used for winemaking. When selecting yeasts, winemakers should consider wine must environmental factors that influence the activity of β-glucosidase such as glucose concentration, ethanol concentration, pH level, temperature and SO2 level (Session 3) and the contribution of yeasts to the aroma and phenolic profile of the wine products (Session 4). With a good understanding of the extracellular β-glucosidase producing capacity of specific Saccharomyces and non-Saccharomyces yeasts strains, most studies in the past decade have directly evaluated the influence of mixed culture fermentation on wine quality traits, while less focus was placed on screening β-glucosidase activity of yeasts (Binati et al., 2020). Understanding the fermentation outcome is certainly important for the industry from a practical point of view. It is also important to understand the mechanism, i.e. how yeasts influence wine quality traits through their β-glucosidase activity. Monitoring enzyme production by specific yeasts using separate fermentation trials is the

most effective way in assessing their individual enzyme production ca- pacities, which could be included in future oenology studies.

3. Factors influencing the β-glucosidase activity of yeasts Many studies have indicated that changes in the winemaking conditions such as concentrations of glucose and ethanol, and pH level in the grape must are the key factors that may influence the activity of β-glucosidase produced by yeasts (Table 2).

3.1. Glucose concentration

Beta-glucosidase is a rate-limiting enzyme and its activity can be regulated by the glucose concentration it releases as a result of hydrolysis (Madrigal et al., 2013). Generally, when high amount of glucose is present in the must, the enzyme activity is reduced, and the hydrolysis of substrates slows down. In addition, yeast grown at a higher glucose concentration condition may produce too much alcohol that inhibits β-glucosidase activity (da Silva et al., 2019). The effects of alcohol concentration is discussed in section 3.2.

In general, the high concentrations of glucose inhibits the activity of β-glucosidase produced by both Saccharomyces and non-Saccharomyces yeasts. However, it was demonstrated in several studies that S. cerevisiae strains maintain a good level of β-glucosidase activity under high glucose concentrations. The level of glucose tolerance is considered an important criterion in yeast selection for wine production, as grape must inherently contains significant levels of glucose. Spagna et al. (2002b) isolated a S. cerevisiae strain AL41 and found that the β-glucosidase produced by this strain was not influenced by glucose and fructose at the concentration range of 0%–20% w/v, and higher fructose concentration at 15% w/v may in fact increase β-glucosidase activity by 30% compared to that at 0% sugar condition. Hall et al. (2017) compared the sugar tolerance of β-glucosidases secreted by Saccharomyces and non-- Saccharomyces strains, and reported higher β-glucosidase activity retained at high sugar level in the two S. cerevisiae isolates compared to M. pulcherrima, H. uvarum, L. thermotolerans. These two S. cerevisiae isolates were able to retain 50–88% of β-glucosidase activity in high sugar condition (100 g/L glucose and 100 g/L fructose) than low sugar condition (5 g/L glucose), while M. pulcherrima, H. uvarum, L. thermo- tolerans only retain 20%, 2% and 30% activity respectively.

High concentration of glucose generally has more impacts on the β-glucosidase activity produced by non-Saccharomyce yeasts compared to that secreted by Saccharomyce. Nevertheless, many non- Table 1 (continued)

of Muscate grape juice using 4- methylumbelliferyl-β-D-glucose (4- MUG) assay.

Muscate grape juice compared to S.

cerevisiae VIN13 and the highest activity was observed in the eighth day of fermentation.

Lachancea lanzarotensis CBS 12615 ++

Y992-5 ++

Lachancea fermentati Y515 +++

Saccharomyces cerevisiae VIN13 +

Hanseniaspora uvarum CBS279 + Whole cell associated β-glucosidase

activity was qualitatively screened using arbutin; Yeast whole cell, supernatant, cell wall and

permeabilised cell were quantitatively analysed separately using p-NPG assay.

•A total of 196 strains of Hanseniaspora were qualitatively screen for β-glucosidase activity, most H.uvarum and H. guilliermondii exhibited weak β-glucosidase activity;

•Within the 123 H. guilliermondii strains screened, 1 strain had strong activity and 39 strains had moderate activity;

•None of the H.uvarum strains had moderate nor high β-glucosidase activity.

Testa et al. (2020)

CBS5914 +

70 new strains - or + Hanseniaspora

guilliermondii CBS 2574 ++

123 new strains - or +or ++or +++

Hanseniaspora osmophila CBS 313 +

aRelative β-glucosidase activity: very weak activity (+); weak activity (++); strong activity (++ +); very strong activity (++ + +); The relatively β-glucosidase activity of different yeasts can only be compared with strains of the same study as the test conditions were different among studies.

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Table 2

Influence of glucose concentration, ethanol concentration and pH on β-glucosidase activity produced by yeasts.

Yeast strains β-glucosidase activity at different experimental conditions Reference

Glucose concentration (% w/v)

0% 5% 10% 15% 20% (Spagna et al., 2002b))

S. cerevisiase AL41 +++++ +++++ +++++ +++++ +++++

0% 5% 10% 15% 20% Spagna et al. (2002a)

W. anomalus (P. anomala) AL112 +++++ ++++ +++ ++++ +++

0% 5% 10% 15% 20% Swangkeaw et al. (2009)

W. anomalus (P. anomala) MDD24 +++++ ++ + + +

0% 5% 10% 15% 20% Madrigal et al. (2013)

P. membranifaciens 7 +++++ ++++ +++ +++ +++

W. anomalus (P. anomala) 6 +++++ +++ ++ ++ +

0% 5% 10% 15% 20% Hu et al. (2016)

H. uvarum YUN268 +++++ +++++ +++++ +++++ ++++

P. membranifaciens NAN13 +++++ +++++ +++++ ++++ ++++

R. mucilaginosa BEI29 +++++ ++++ ++++ ++++ +++

AR200 (commercial enzyme) +++++ + + + +

0% 1.8% 3.6% 9% L´opez et al. (2015)

H. uvarum Hu8 +++++ +++++ +++++ +++++

H. vineae Hv3 +++++ +++ ++ ++

P. membranifaciens Pm7 +++++ +++ ++ ++

W.anomalus (P. anomala) Wa1 +++++ ++ ++ ++

0% 5% 10% 18% da Silva et al. (2019)

M. guilliermondii (P. guilliermondii) G1.2 +++++ ++++ +++ +++

0% 10% 20% 30% 40% Wang et al. (2012)

T. asahii F6 +++++ +++++ ++++ ++++ +++

0% 1.8% 3.6% 7.2% 10.8% Baffi et al. (2013)

S. pararoseus SP8A +++++ +++ ++ ++ +

0% 4% 8% 15% Cordero Otero et al. (2003)

Brettanomyces spp + – – –

C. oleophila + + ++ ++++

S. polymorphus (D. polymorphus) + ++ ++ ++

D. pseudopolymorphus + ++ ++++ +++++

Ethanol concentration (% v/v)

0% 5% 10% 15% 20% (Spagna et al., 2002b))

S. cerevisiase AL41 +++ ++++ +++++ +++++ +++++

0% 5% 10% 15% 20% Spagna et al. (2002a)

W. anomalus (P. anomala) AL112 + +++++ +++++ +++++ ++++

0% 4% 8% 16% 20% Swangkeaw et al. (2009)

W. anomalus (P. anomala) MDD24 ++++ ++++ +++++ +++++ +++++

0% 5% 10% 15% 20% Madrigal et al. (2013)

P. membranifaciens 7 +++++ +++++ +++++ +++++ +++++

W. anomalus (P. anomala) 6 +++++ ++++ +++++ ++++ ++++

0% 10% 20% 30% da Silva et al. (2019)

M. guilliermondii (P. guilliermondii) G1.2 +++++ +++++ +++++ +++++

0% 5% 10% 20% L´opez et al. (2015)

H. uvarum Hu8 +++++ +++++ +++++ ++++

H. vineae Hv3 +++++ +++++ +++++ ++++

P. membranifaciens Pm7 +++++ +++++ ++++ ++++

W.anomalus (P. anomala) Wa1 +++++ +++++ ++++ ++++

0% 5% 10% 15% Hu et al. (2016)

H. uvarum YUN268 +++++ +++++ ++++ ++++

P. membranifaciens NAN13 +++++ ++++ ++++ ++++

R. mucilaginosa BEI29 +++++ ++++ ++++ +++

AR200 (commercial enzyme) +++++ ++++ ++++ +++

0% 5% 10% 15% 20% Wang et al. (2012)

T. asahii F6 +++++ +++++ +++++ +++++ +++++

0% 10% 12% 14% Cordero Otero et al. (2003)

Brettanomyces spp +++++ +++++ ++++ +++

C. oleophila +++++ ++ + +

S. polymorphus (D. polymorphus) +++++ ++ ++ ++

D. pseudopolymorphus +++++ +++++ +++++ +++

0% 5% 10% 15% 20% Baffi et al. (2013)

S. pararoseus SP8A ++++ +++++ +++++ ++ +

pH

3 4 5 6 7 Spagna et al. (2002b)

S. cerevisiae AL41 +++ +++++ ++++ ++ +

3 4 5 6 7 Hern´andez et al. (2003)

S. cerevisiae 39 ++++ +++++ +++++ ++++ ++

3 4 5 6 8 L´opez et al. (2015)

H. uvarum Hu8 +++++ +++++ +++++ +++++ +++++

H. vineae Hv3 +++ ++++ +++++ +++++ +++

P. membranifaciens Pm7 + ++++ +++++ +++++ ++++

W.anomalus (P. anomala) Wa1 ++ ++ ++++ +++++ +++

3 3.5 4 4.5 5 Hu et al. (2016)

H. uvarum YUN268 +++++ +++++ +++++ +++++ +++++