Saccharomyces unisporus: Biotechnological Potential and Present Status

Authors


Direct inquiries to author Tyagi (E-mail: Rd.tyagi@ete.inrs.ca).

Abstract

 The yeast species of the Saccharomyces genus have a long history of traditional applications and beneficial effects. Among these presence of the Saccharomyces unisporus has been documented in various dairy products and has become a subject of interest and great importance. S. unisporus has shown a significant role in the ripening of cheese and production of fermented milk products such as kefir and koumiss. The absence of pseudohyphae during the life cycle of S. unisporus is an indication of nonpathogenicity. Significance has been laid on the presence of S. unisporus in food-grade products and a close proximity of S. unisporus to S. florentinus and both of these species are accepted by the International Dairy Federation and the European Food and Feed Cultures Association for food and feed applications. Since over the years, S. unisporus has already become a part of various dairy products, S. unisporus can be considered as a potential candidate for generally regarded as safe status. S. unisporus has the capacity to convert ketoisophorone to levodione, which is an important pharmaceutical precursor. S. unisporus are considered as the potential producers of farnesol which eventually controls filamentation of pathogenic microorganisms. Apart from that, S. unisporus produces certain omega unsaturated fatty acids which combat diseases. Henceforth, the areas which S. unisporus can be possibly exploited for its useful intermediates are the enzymes and fatty acids it produces. In this context, this review attempts to describe and discuss the ubiquity of S. unisporus in food products, cellular composition, regulatory pathways, and its synthesis of fatty acids and enzymes.

Introduction

Yeasts are known for their biotechnological applications and therefore interest in searching and characterizing new yeast species has increased over the years. Yeasts are mainly used for the production of beverages, cereal-based food, enzymes, fine chemicals, single-cell protein, and flavoring compounds (Eijk and Johannes 1995; Gatto and Torriani 2004; Wang 2008). Yeasts are also used for research in genetics, molecular biology, and cell biology, and most of the focuses are on the 2 species Saccharomyces cerevisiae and Schizosaccharomyces pombe. However, some nonconventional yeast species such as Saccharomyces unisporus draw interest because of their biotechnological potentials due to ubiquitous presence, nonpathogenicity, and probiotic nature (Sinnott 2010). For the possible benefits of human and animal health, organisms, which are termed as probiotic can be incorporated into dietary adjuncts for the maintenance of a healthy gastrointestional balance (Lourens-Hattingh and Viljoen 2001). Mostly incorporated organisms are Lactobacillus along with Saccharomyces. S. unisporus yeast was discovered by Holm (Jorgensen 1920). Under the genus Saccharomyces, 10 species are accepted of which S. unisporus, which is also called as Kazachstania unispora (Lu and others 2004) is one of them. S. unisporus is an auxotrophic organism (Vogl and others 2008) and it is found to be the dominant yeast in traditional dairy products (Montanari and Grazia 1997; Coppola and others 2008; Rahman and others 2009; Yildiz 2010; Bourdichon and others 2006). A unique synchronization of Kluyveromyces marxianus and S. unisporus (Kazachstania) species have been found in milk-fermented products, such as dahi, suusac, gariss, kefir, shubat, and koumiss (Narvhus and Gadaga 2003; Lore and others 2005; Abdelgadir and others 2008; Rizk and others 2008).

S. unisporus is the slowest producer of ethanol and performs a clean fermentation in milk and whey (Montanari and others 1996). Oxygen is required in very minute amounts for the yeasts Saccharomyces to synthesize sterols and unsaturated fatty acids. A specialty about Saccharomyces cells is that they cannot ferment sugars, especially under anaerobic conditions over a long period because they require preformed lipid precursors for growth, which are synthesized under aerobic condition. In laboratory practices, ergosterol and Tween 80 (defined source of oleic acid which is an unsaturated fatty acid) are added to the medium to study Saccharomyces species anaerobically (Ingledew 1999). Yeast organisms extracted from Spanish blue-veined cabrales cheese have been found to contribute toward the maturation and ripening of the cheese (Callon and others 2006).

Most of the studies have been done on kefir grains and by-products, where it is mentioned that metabolites from yeast, such as S. unisporus give taste to the products and also provide a milieu for the growth of bacteria (Maalouf and others 2011). However, the significance of consumption of these metabolites is not yet known. S. unisporus grows efficiently in cheese; however the enzymes and extracellular enzymes produced by this yeast still remain a subject of study. Furthermore, there is a debate whether this organism is generally regarded as safe (GRAS) in food products and animal feed. Therefore, this review intends to summarize the research available on the existence of S. unisporus in natural food and feed, as well as other pertinent information regarding its safe use with other potential applications.

Taxonomy and Biology

Taxonomy

S. unisporus can be classified as belonging to the fungi, which are the largest eukaryotic group consisting of yeasts and molds, of which one of the phylum is the sac fungi known as Ascomycota. A subphylum of Ascomycota is Saccharomycotina and 1 class there is Saccharomycetes; Saccharomycetales is an order, which falls under the class Saccharomycetes, since they reproduce by budding. Family is Saccharomycetaceae, and genus is Kazachstania. However, S. unisporus has also been placed under species Zygosaccharomyces (Alvarez-Martin and others 2007). Scientific classification has been presented in Figure 1.

Figure 1.

Scientific classification of S. unisporus.

Species of the genus Saccharomyces are divided into a group of petite-positive and petite-negative. A typical specialty of a Saccharomyces species is that, when it is allowed to grow in the absence of oxygen, it generates respiratory-deficient mitochondrial mutants, which are called petites. Similar observations have been recorded for S. unisporus. This is a unique trait of Saccharomyces clade. A petite-positive organism can efficiently grow in the absence of oxygen and its survival is not dependent on the loss of mtDNA. A petite-negative organism cannot grow on fermented carbon sources and it cannot grow after the loss of mtDNA (Wang 2008). Petite-positive species are further divided into sensu stricto (species with narrow circumscription) and sensu lato species (species with wider circumscription). S. unisporus falls into the category of Saccharomyces sensu lato species (Broker and Harthus 1997; Piskur and others 1998; Loureiro and Querol 1999; Marinoni and others 1999; Groth and others 2000; Cliften and others 2005) along with other sensu lato species which are Saccharomyces turicensis, Saccharomyces bulderi, Saccharomyces zonatus, and Saccharomyces spencerorum (Mikata and others 2001). Although Saccharomyces bayanus and Saccharomyces eubayanus falls in the sensu stricto group (Libkind and others 2011). Other Saccharomyces spp., which is Saccharomyces chevalieri and Saccharomyces ellipsoideus have close associations with S. cerevisiae (Soltesova and others 2000; Hoff 2012). Hence a phylogenetic tree (Figure 2) has been drawn to identify the S. sensu stricto and sensu lato spp. in clusters and Table 1 has been constructed to reflect the distance matrix and evolutionary distance of 1 Saccharomyces spp. from another Saccharomyces spp.

Table 1. Observed evolutionary (percentage) distances of one Saccharomyces spp. to the other in matrix–technological relationships using standard distance matrix
 12345678910111213141516
  1. 1, S. bayanus MCYC 623; 2, S. bulderi 30–1; 3, S. cariocanus NCYC 2890T; 4, S. cerevisiae AG4; 5, S. chevalieri MUCL 27815; 6, S. dairenensis NRRL Y-12639; 7, S. ellipsoideus MUCL 38888; 8, S. eubayanus CBS 1538; 9, S. exiguus 2–3; 10, S. florentinus NRRL Y-1560; 11, S. martiniae NRRL Y-409; 12, S. spencerorum NRRL Y-17920; 13, S. turicensis NRRL Y-27345; 14, S. unisporus SA21S02; 15, S. zonatus NBRC 100504; 16, S. castellii NRRL Y-12630.

1 5654978845881009459579558959495
256 9554534353565466655665565095
35495 57534253545363655767545295
4975457 904590979458599462949595
588535390 4499888961618562859086
64543424544 44464153564552404786
7885353909944 888961628662859086
8100565497884688 9459579558959495
99454539489418994 64609362949392
10596663586153615964 916494606493
1157656559615662576091 5894575894
129556579485458695936458 60949695
13586567626252625862949460 625996
1495565494854085959460579462 9596
159450529590479094936458965995 90
16959595958686869592939495969690 
Figure 2.

A phylogenetic tree based on maximum parsimony method clustering Saccharomyces sensu stricto and sensu lato spp. Scale bar indicates 0.4 nucleotide substitution per site.

Biology

Morphology and reproduction

S. unisporus ascospores are refractive and round with an average diameter of 4.5 μm and length of 3 μm. Vegetative cells are transformed directly into asci containing 1 and occasionally 2 globose to subglobose ascospores (Yarrow 1984). The yeast actively grows in presence of yeast malt agar and sporulation is observed in the absence of acetate. The spores appear at the end of 40 h at 25 °C and after 72 h at 15 °C (Yarrow 1984). This yeast does not form a scum but shows simply a ring in old cultures. The external morphology consists of a cream, flat layer. It is usually glossy and smooth, sometimes with light striations (Yarrow 1984). Morphology of S. unisporus is shown in Figure 3 obtained through scanning electron microscopy (SEM).

Figure 3.

S. unisporus cells through scanning electron microscopy (SEM): (A) Colony formation of S. unisporus. The S. unisporus cells, oval-shaped, are joined through small clusters. (SEM original magnification 2000), (B) S. unisporus cells conjoint with each other and budding simultaneously (SEM original magnification 30000).

The cell components are coenzyme Q6 (Kurtzman 2003) and%Mol G+C (guanine + cytosine) range is 32.4 (Vaughan-Martini and Kurtzman 1988). However, in the genus Saccharomyces there is less G+C content. The size of the mitochondrial DNA is detected as 29 kilobase (kb) pairs, with an identification of sites for MspI gene/type-2 restriction enzyme after 11 kb pairs and after subsequent 8 kb pairs HaeIII gene/type-2 restriction enzyme. The other important gene site (Ori/rep gene) has not been detected. Composition of S. unisporus can be identified based on the most popular organism S. cerevisiae with which S. unisporus has a 59.6% identity match. In all genetic comparisons, S. cerevisiae is taken as the base organism. Out of 59.6%, the percentage of noncoded region is 69.5 and the percentage of coded region is 40.4 (when compared with S. cerevisiae). The predicted protein types are hypothetical protein, polyprotein, and homolog of cofactor B. S. unisporus has chromosomes [Chr2:363049(start)-362771(end), Chr3:100839 (start)-101343(end), and Chr5:551117(start)-550863(end)] and they match with those of Caenorhabditis elegans and Schizosaccharomyces pombe (Piskur and others 1998). The total genomic size up to the year 1999 was 11560 kb (Piskur and others 1998; Cliften and others 2001).

The external morphology of S. unisporus is entire or undulating; pseudohyphae are not formed. Pseudohyphae are morphologically different from hyphae. The pseudohyphae or hyphae are virulent factors, and hence an organism having pseudohyphae or hyphae is considered as pathogenic (Sudbery and others 2004). However, hyphae are far more pathogenic than pseudohyphae. During yeast growth, 1st stage is the formation of pseudohyphae, which may or may not convert into hyphae. Since S. unisporus does not even form pseudohyphae, therefore it cannot be considered pathogenic. Also, pseudohyphae are formed when yeast cells do not fully separate after every cell cycle completion (Sudbery and others 2004). On the other hand, S. cerevisiae can form pseudohyphae but hyphae are not formed (Bastidas and Heitman 2009). However, all strains of S. cerevisiae do not form pseudohyphae (Llanos and others 2006; Perez-Torrado and others 2012); only diploid strains of S. cerevisiae develop pseudohyphae under nitrogen starvation condition (Strudwick and others 2010). Single copies of genes responsible for the development of pseudohyphae in haploid cells of yeasts are a1/α2 repressor. These repressors, which are transcriptional repressors, stimulate the pseudophyphal pathways in yeasts when environmental condition is devoid of nitrogen. In case of diploid cells, when MAT locus as MATa/MATα, MATα/MATα, and MATa/MATa are present in the yeast cells, pseudohyphae are likely to form (Lo and others 1997; Wolfe 2006). Thus, based on pseudohyphae/hyphae formation characteristics, a phylogenetic tree was drawn (Figure 4) to correlate the pathogenicity, where maximum parsimony method has been used for aligning species of Saccharomyces complex and Candida albicans ATCC 18804.

Figure 4.

Maximum parsimony method for Saccharomyces complex and K. marxianus 26S rDNA partial sequences; where S. unisporus NRRL Y-1556, S. martiniae NRRL Y-409, S. florentinus NRRL Y-1560, and S. dairenesis NRRL Y-12639 fall into the same subcluster. S. unisporus NRRL Y-1556 is relatively closer to S. cerevisiae YJM789 and distant from C. albicans ATCC 18804 and K. marxianus PUMY014; where C. albicans is a known pathogen and S. cerevisiae and K. marxianus fall under the generally regarded as safe (GRAS) yeasts. Scale bar indicates 0.002 nucleotide substitution per site.

Studies have been carried out to know whether S. unisporus could sustain extreme conditions and whether the organism needs any extra chemicals for sustaining the drastic conditions. Merico and others (2006) tried to grow S. unisporus in synthetic medium with fortification of antimycin A (concentration range of 0.5 to 25 μM). S. unisporus grew efficiently at all dosages of antimycin A up to the 7th d. However, other strains of Saccharomyces species could grow only when the medium was supplemented with lysine (Mikata and others 2001), glutamic acid, and acetoin (Loureiro and Querol 1999; Merico and others 2006). Antimycin A (secondary metabolite) is produced by Streptomyces, a genus of Actinobacteria. This chemical compound binds to the cytochrome c reductase and thus inhibits the oxidation of ubiquinol (Neft and Farley 1972).

Growth conditions and substrate (carbon, nitrogen, and vitamin requirements)

S. unisporus is a nonlactose fermenting yeast (Montanari and others 2000; Merenstein and others 2009). The compounds which S. unisporus can efficiently consume are listed in Table 2. S. unisporus ISA 1097 where ISA stands for Instituto Superior de Agronomia grew efficiently when the carbon sources were glucose, sucrose, maltose, cycloheximide, and sorbic acid (Rodriques and others 2001). It does not grow on sucrose, maltose, or lactose (Wang and others 2008) and produces succinic acid (Sahasrabudhe and Sankpal 2001). Considering this organism's sugars metabolism, it could be related to Saccharomyces mali duclauxi. Further, S. unisporus is capable of utilizing nitrogenous compounds that are found in the environment (except nitrates and nitrites) as the sole nitrogen source. S. unisporus assimilates ethylamine (Middelhoven 2002), cadaverine, and lysine (Andorra and others 2010), which are not usually consumed by all Saccharomyces (James and others 1997).

Table 2. Consumption of different carbon, nitrogen, and others substrates by S. unisporus
CompoundAssimilationFermentationReference
  1. (+) indicates positive growth and (–) indicates no growth.

Glucose(+)(+)Rodriques and others 2001
Lactose(+) By certain S. unisporus(–)Cheirsilp and others 2003
Galactose(+)(+)Fröhlich-Wyder 2001
Trehalose(+)(–)Mikata and others 2001
Sorbic acid(+)(+)Rodriques and others 2001
Sucrose(+)(+)Rodriques and others 2001; Qureshi and others 2007
Maltose(+)(+)Rodriques and others 2001; Qureshi and others 2007
Lactic acid(–)(–)Fröhlich-Wyder 2001
Succinate(+)(–)Mikata and others 2001
Lysine(+)(–)Mikata and others 2001
Cadaverine(+)(–)Mikata and others 2001; Lu and others 2004
Cycloheximide(+)(+)Rodriques and others 2001; Middelhoven 2002
Ethanol(+)(+)Rodriques and others 2001
p-Coumaric acid(+)(+)Rodriques and others 2001
Glycerol(+)(+)Rodriques and others 2001
Ethylamide(+)(+)Mikata and others 2001; Middelhoven 2002
Vitamins(+)(+)Middelhoven 2002
Max. growth T (°C)  Middelhoven 2002; Qureshi and others 2007

Thiamine is the only vitamin required as exogenous source for growth of S. unisporus. S. unisporus grows faster in the presence of thiamine in culture media rather than in the absence of thiamine. It is classified auxotrophic because it fails to synthesize thiamine as an essential compound (Wightman and Meacock 2003). It is corelated with S. servazzi and S. castellii because of the lack of gene THI5/enzyme involved in the synthesis of thiamine precursor. It follows HMP (4-amino-5-hydroxylmethyl-2-methylpyrimidine) and HET (5-(2-hydroxy-ethyl)-4-methylthiazole) pathways. These pathways undergo phosphorylation and are condensed to thiamine monophosphate (Vogl and others 2008). However, S. unisporus cannot use thiamine monophosphate for its growth.

THI9 gene is strongly upregulated (activated) by a supply of thiamine and that implies thiamine transport. This also specifies the absence of another significant gene, BSU1, which is upregulated when pyridoxine and thiamine are present endogenously. The minimum concentration of thiamine required for growth of S. unisporus is 1.2 μM (Vogl and others 2008), however THI9 starts expressing even when a lower concentration of thiamine is present in the medium. Lower glucose concentration or absence of glucose in the medium enables a strong reduction in thiamine uptake. Thiamine-dependent S. unisporus also requires efficient plasma membrane transport proteins for the acquisition of thiamine. However, in most of the organisms, thiamine is not produced in higher quantities than are being required for thiamine diphosphate (TDP) biosynthesis.

Thiamine plays a major role in carbohydrate metabolism in living cells. In S. unisporus, thiamine represses 60 or more genes and most of them are involved in thiamine metabolism. Vogl and others (2008) demonstrated a current model of thiamine-sensing, where TDP works as the intracellular thiamine signal. It is an important cofactor of transketolase enzyme complexes in the oxidative decarboxylation of oxo-acids (Vogl and others 2008).

Potential applications

Presence and role in milk-derived products

Graziella and Grazia (1997) studied the yeast microflora of 94 samples of Central Asian koumiss and found S. unisporus as a dominant species (in 68% of the samples examined). The species was tested for sugar fermentation and it was found that S. unisporus is comparatively less capable of producing alcohol from grape must. S. unisporus has been found on the surface of different types of cheese and has been shown to be important for cheese quality as it performs cheese ripening and maturation (Corsetti and others 2001; Callon and others 2006; Jacques and Casaregola 2008). It was also stated that S. unisporus was the only isolated species of the Saccharomyces genus and constituted 5% of all the identified yeast from cheese (Tornadijo and others 1998). Furthermore, S. unisporus was identified by Nunez and others (1981) from Cabrales cheese, and its population was 3% of total isolates obtained from this cheese.

Kefir production is started using kefir grains, which consists of a rubbery matrix embedded with different types of microorganisms. Kefir grains contain lactic-acid bacteria, acetic acid bacteria, lactose-fermenting yeasts of Kluyveromyces spp. which are K. marxianus and Kluyveromyces lactis, and nonlactose fermenting yeasts are S. unisporus, S. cerevisiae, and Saccharomyces exiguus (Angulo and others 1993; Pintado and others 1996; Wyder and Puhan 1997; Wyder and others 1997; Marquina and others 2002; Heras-Vazquez and others 2003; Farnworth and Mainville 2003; Farnworth 2005; Mainville and others 2006; Latorre-Garcıa and others 2007; Păucean and Socaciu 2008; CodexAlimentarius 2011). A broader spectrum of yeast species was found among samples of homemade kefir samples, of which Issatchenkia orientalis, S. unisporus, S. exiguous, and Saccharomyces humaticus were the most representative species (Latorre-Garcıa and others 2007). S. unisporus is also involved in a unique process patented in the United States for the production of kefir-like fermented milk (Saita and others 1991).

Viili is a category of fermented milk, which originated in Scandinavia, it is claimed to possess functional benefits and health-improving potential. K. marxianus, S. unisporus, and Pichia fermentans were identified in viili starters corresponding to 58%, 11%, and 31% of total cell counts, respectively (Wang and others 2008). In a study by Canibe and others (2010) fermented liquid feed samples for feeding pigs from 40 Danish farms were prepared and further microbial and biochemical variations were determined. The feed group was divided into 2 groups, a high-intake group and low-intake group. In both groups, compositions of yeasts constituted 85% to 91% of the total isolates of microorganisms. Variable isolated yeasts were S. unisporus along with other Saccharomyces species.

Among the fermented milk products, koumiss (2% alcohol at pH 4.0) is prepared from mare's milk; its microflora is dominated by thermophilic lactic acid bacteria (LAB) along with K. marxianus and Saccharomyces species, specifically S. unisporus (Hansen and Jakobsen 2004). In kefir, the microorganisms often found are Saccharomyces florentinus, Saccharomyces globosus, along with S. unisporus (Coppola and others 2008). S. unisporus has shown close relation with S. florentinus, and both of these organisms come under the category of species which are accepted by the International Dairy Federation (IDF) and European Food and Feed Cultures Association (EFFCA) as suitable organisms for consumption by human beings or animals (Bourdichon and others 2012).

Even for edible biomass production, K. marxianus and S. unisporus are the common yeast species exploited (Lewandowski 2011). Apart from that, S. unisporus has shown presence in dahi (a milk fermented product from India), gariss (which is a fermented camel milk product), shubat (which is similar to gariss), and suusac along with other beneficial yeasts and bacteria (Rizk and others 2008).

Interaction with LAB

S. unisporus is present in dairy products, but usually at a lower concentration than other lactose-fermenting yeast species, such as Kluyveromyces species (Wouters and others 2002). S. unisporus has a marked presence in koumiss, a product produced in Central Asia and the former Soviet Union derived from the lactic and alcoholic fermentation of mare's milk (Curadi and others 2001). The microflora of koumiss consists mainly of thermophilic LAB and species of Saccharomyces. The yeasts consist of lactose-fermenting and lactose-nonfermenting species. The yeast producing mare milk fermentation (alcoholic) is a lactose-fermenting species from the Kluyveromyces genus and, more often, nonlactose-fermenting strains belonging to Saccharomyces genus (Montanari and Grazia 1997; Fröhlich-Wyder 2003; Cagno and others 2004).

In Saccharomyces, a frequently found species is S. unisporus, which vigorously ferments galactose (Montanari and Grazia 1997; Wouters and others 2002). Currently, koumiss is manufactured at an industrial level in many countries (Tamime and others 1999; Litopoulou-Tzanetaki and Tzanetakis 2000). The interaction between yeast and LAB may be stimulation or inhibition of each other in a coculture. They compete for growth nutrients or they will produce certain metabolic products that stimulate growth of the other organism (Neviani and others 2001; Lopitz-Otsoa and others 2006). S. unisporus is one of the isolates from different batches of kefir grains. Apparently, other Lactobacillus spp. are also isolated from the same. The unique interaction between yeasts and LAB, for example, Lactobacillus kefiranofaciens demonstrates stability in a mixed culture of kefir or any other milk fermentation product (Lopitz-Otsoa and others 2006).

Certain yeasts in kefir are lactose-positive and most of them consume galactose, including S. unisporus (Yarrow 1984). On the contrary, yeasts may produce vitamins, which may enhance the growth of LAB (Lopitz-Otsoa and others 2006). Commensalism between yeast and LAB is taken into consideration for the production of single-cell protein (SCP) in cheese whey (a by-product of the cheese industry). LAB produces dl-pyro-glutamic acid and lactic acid; however, none of the yeasts produces glutamic acid (Gadaga and others 2001). In kefir grains, when complex microfloras were considered, often they were classified as homofermentative LAB, heterrofermentative LAB, lactose-assimilating yeast, and nonlactose-assimilating yeast. During the production of kefirs, homofermentative LAB were simulated by the intermediates produced from the other 3 groups. For the kefir production and enhancement, presence of yeasts, for example, S. unisporus is a must as excessive accumulation of lactic acid can inhibit L. kefiranofaciens, while gradual removal of lactic acid enhances the production of kefir. Yeasts otherwise provide vitamins, amino acids, and other growth factors which influence the bacterial growth (Cheirsilp and others 2003).

Presence in other fermented food

S. unisporus was found in fermented orange fruit and juice and was identified and classified by different molecular techniques (Heras-Vazquez and others 2003; Canibe and others 2010). The presence of S. unisporus has also been observed in sugarcane juice (Qureshi and others 2007). Apart from those, vegetable juice favors growth of S. unisporus (Savard and others 2002b, 2002b). The role of S. unisporus was found important in whey vinegar production (Rainieri and Zambonelli 2009). However, the exact role of S. unisporus for its existence in these items is not identified and further research is required to exactly define its function.

Eijk and Johannes (1995) invented the process of preparing substrate-limited dough (a mixture of flour and water), where S. unisporus was one of the proposed yeast for the preparation of yeast-leavened bread dough (Gatto and Torriani 2004). In such dough preparations, the proposed yeasts or S. unisporus consumes the fermentable sugars, whereas the nonfermentable sugars are added additionally to the bread dough for adjusting the sweetness of the bread dough. An example of such additive, which acts as artificial sugar supplement, is whey permeate; it is added in the range of 0.1% to 10% (w/w) to the bread dough.

Production of volatile compounds

S. unisporus IFO (Inst. for Fermentation, Japan) 0298 strain has the capacity to convert ketoisophorone into levodione (a volatile organic compound). Levodione is involved in oxidoreductase activity and also plays a significant role in the commercial synthesis of carotenoids. In the presence of other yeasts, S. unisporus converts 2,6,6-trimethyl-2-cyclohexane-1,4-dione into (6R)-2,2,6-trimethylcyclohexane-1,4-dione (an isomer of levodione) (Fukuoka and others 2002) as shown in Figure 5. The average time needed for levodione production by S. unisporus was 16 h, with a final production rate of 16.7 g levodione/kg yeast cells/h; whereas the production rate was only 8.8 g levodione/kg yeast cells/h when S. cerevisiae was used (Fukuoka and others 2002).

Figure 5.

Biotransformation of ketoisophorone to levodione by S. unisporus.

Prenyl alcohol is the major component of odorants in roasted coffee and essential oils. S. unisporus cells produce a significant amount of prenyl alcohol in a nutrient medium. It also has the potential to produce geranylgeraniol (which is an intermediate in the biosynthesis of vitamins E and K) and farnesol, which are typical members of the prenyl alcohol family (Obata and others 2005). Farnesol (one of the major natural alcohols) is produced by S. unisporus. Farnesol has been considered as an antitumor agent (Obata and others 2005). If a wild-type strain of yeast which produces geranylgeraniol or farnesol naturally, yeast growth can be stimulated and geranylgeraniol or farnesol can be produced at a much higher rate in a medium which will be inexpensive and economic, leading to mass production of the intermediates. Along with S. unisporus, S. cerevisiae, and S. dairensis are certain species of Saccharomyces, which are the natural producers of farnesol (Muramatsu and others 2002). Extensive production of farnesol controls filamentation in fungi, which are polymorphic and pathogenic, for example, C. albicans, henceforth destroying the harshest of opportunistic organisms (Albuquerque and Casadevall 2012).

The entire Saccharomyces complex shows a well-balanced fermentative metabolism. S. unisporus produces mutants when allowed to grow under drastic conditions with rearranged mtDNA molecules. This condition is called petite phenotype and S. unisporus is petite-positive (Merico and others 2006; Fekete and others 2007). Petite-positives can survive well anaerobically. Under anaerobic conditions, respiratory biochemical pathways are shut down and the only way the yeast cells generate energy is through substrate-level phosphorylation. Under such conditions, cells maintain their redox balance by the production of glycerol through glycerol 3-phosphate dehydrogenase and production of succinate by fumarate reductase. During such conditions, the mitochondrion usually does not play a significant role in energy metabolism (Merico and others 2006).

Savard and others (2007) stated that food products containing a minimum concentration of ethanol at pH 3 to 11 were more prone to microbial spoilage. S. unisporus can efficiently grow in this range of pH (3 to 9) (Loureiro and Querol 1999) and produce low ethanol concentration and, therefore, is mainly considered as a food spoiler (Savard and others 2007). The strains of S. unisporus do not perform clean alcohol fermentation as they produce some other minor byproducts, such as glycerol, succinic acid, and acetic acid (Montanari and Grazia 1997). Two strains of S. unisporus, BR174 and BR180 isolated from mangrove bromeliads and tested for alcohol fermentation were found to be less tolerant to high ethanol concentration (5 g/L) (Morais and others 1996).

S. unisporus does not produce any phenolic odor during the entire incubation period as presence of phenolic aroma in a fermented food product can be considered as a food spoiler. However, it generates a phenolic aroma whenever it efficiently consumes p-coumaric acid (derivative of 4-ethylphenol) and produces 4-ethylphenol as an intermediate (Loureiro and Querol 1999; Rodriques and others 2001). The amount of 4-ethylphenol produced is proportional to the concentration of p-coumaric acid in the medium. The 4-ethylphenol is a volatile phenol responsible for wine spoilage (Rodriques and others 2001). S. unisporus along with other yeasts produces 0.7% to 2.5% (w/v) of ethanol (Zajsek and Gorsek 2010). S. unisporus also demonstrates the “crabtree” effect, which means it produces ethanol, pyruvate, acetate, succinate, and glycerols following the tricarboxylic acid (TCA) cycle (Merico and others 2006). Production of volatile and nonvolatile flavoring compounds by S. unisporus in fermented food products has a positive impact on any food and feed.

Fatty acids profile of S. unisporus

Over the years, SCPs have become well known for their high nutritive value as they have higher protein, vitamin, and fatty acid contents; and they are used as a supplement in food and feed (Lewandowski 2011). Fatty acids constitute 5% of the overall cell weight of S. unisporus (Kock and others 1986). S. unisporus produces palmitic acid and oleic acid. Middle chain fatty acids (saturated or unsaturated) are present in S. unisporus ranging from carbon 14:0 to 18:1. When compared to other Saccharomyces spp., S. unisporus cells contain a high percentage of palmitoleate, followed by palmitic acid, and oleic acid. Other fatty acids like myristic (Kock and others 1986) and linolenic acids are absent in this species of Saccharomyces (Grillitsch and others 2011). Palmitoleic acid is an omega-7 monounsaturated fatty acid, which is also a major constituent of human adipose tissues. Current-day research shows that palmitoleic acid (which S. unisporus is known to produce) acts as the signaling molecule to help fight weight gain (Yarrow 1984). Palmitate is the 1st product of lipogenesis (fatty acid synthesis) and is considered as an antioxidant and a source of vitamin A (Beare-Rogers and others 2001).

Furthermore, no unusual fatty acid chain has been reported in S. unisporus as yet. S. unisporus produces high concentration of fatty acids in the presence of glucose, sucrose, and low concentrations of lower-chain fatty acids have been recorded in the presence of maltose and galactose, whereas it does not produce any fatty acid in the presence of lactose at temperatures of 25 and 30 °C (Obata and others 2005).

S. unisporus did not produce linoleic acid (C18:2) and linolenic acid (C18:3) (Kock and others 1986; Loureiro and Querol 1999). However, it is not known if S. unisporus produces other C18 fatty acids. The absence of C18 polyunsaturated fatty acids is considered as a chemical marker of a strain being a pathogen or a food spoiler in the food industry. Therefore, production of C18 fatty acids by S. unisporus must be investigated.

Biochemical sensitivity of S. unisporus

Unlike other Saccharomyces species, S. unisporus is resistant to organic acids. The strain S. unisporus Y-42 was tested against a set of organic acids including acetic acid, lactic acid, and propionic acid, individually and in mixture. A mixture of lactic acid 0.7% (w/v), acetic acid 0.3% (w/v), and propionic acid at 0.2% (w/v), when added to a vegetable juice medium, completely inhibited growth of S. unisporus (Savard and others 2002a, 2002b). Another effective inhibitory effect of S. unisporus has been noticed by an easily hydrolyzable chitosan-acid complex comprised of a chitosan oligomer complexed with an acid radical or an acid with a molecular weight of 0.5 to 1.2 kDa. S.unisporus Y-42 also exhibited sensitivity to chitosan hydroxylates at pH 3.8. S. unisporus is resistant when the pH is low. A microscopic view of the inhibited S. unisporus by chitosan-acid complex showed an irregular coating around its surface, which generalizes the phenomenon of cell suffocation. The mechanism proposed is the reactive amino groups in chitosan, which interacts with various anionic groups on the yeast cell surface and thus creating a layer (Savard and others 2007).

S. unisporus has been found to partially inhibit a food pathogen Listeria monocytogenes. Goerges and others (2006) showed in an agar-membrane assay that S. unisporus produces a toxin responsible for eradicating this common food pathogen, which has not been biochemically characterized. Haruji and others (2008) claimed the use of yeast (Saccharomyces and non-Saccharomyces) to produce an inhibitor for the production of secondary bile acid. S. unisporus was also found to be a significant producer of an inhibitor which inhibits secondary bile acid production. Deoxycholic acid is a bile acid and S. unisporus absorbs up to 65.5% of this acid. Another bile acid, chenodeoxycholic acid, is also absorbed by S. unisporus up to 58.8% (Haruji and others 2008).

Biochemistry and Molecular Biology

Intracellular proteins and intergenic sequences

Mitochondrial DNA (mtDNA ) of S. sensu lato species, for example, S. unisporus, contains fewer guanine–cytosine (GC) clusters and length is usually smaller than 50 kb. Some researchers have also reported GC clusters of less than 100 bps (Groth and others 2000; Spirek and others 2003). The size of mtDNA for S. unisporus is 29 kb and GC cluster content is 23% in mtDNA (Piskur and others 1998). Groth and others (2000) studied 2 mitochondrial genes (SSU and ATP9) from various Saccharomyces species and phylogenetic trees were drawn based on mtDNA molecules, of which S. unisporus was found to have close relationship with S. exiguus species in SSU gene; whereas S. unisporus exhibited close relatedness with S. servazzii in ATP9 gene (Groth and others 2000).

The gene order in Saccharomyces spp. differs among each other (Keogh and others 1998). Gene order of the same species is karyotype but is more heterogeneous. The chromosome numbers in Saccharomyces spp. vary from 8 to 16, whereas S. sensu is a strict group, which is interfertile and S. unisporus chromosome numbers are usually 11 to 13 (Petersen and others 1999). In the S. sensu lato species, small chromosomes (less than 0.5 Mb) are present; very different from the Saccharomyces sensu stricto species, which has larger mtDNA molecules and highly extensive intergenic regions (Marinoni and others 1999; Spirek and others 2003).

Ho protein is an intracellular protein, highly conserved in all Saccharomyces species including S. unisporus. The latter has 2 domains of nuclear localization sequences (NLS1 and NLS2), where NLS1 is inside the endonuclease domain, and NLS2 is inside the zinc finger domain. Ho proteins are induced by domestication of VMA1 interin (Bakhrat and others 2006). S. unisporus produces VMA1-derived endonucleases known as VDE (Okuda and others 2003; Posey and others 2004). Okuda and others (2003) studied endonuclease activity of S. unisporus IFO0316 and showed that VDE proteins were expressed; however, no endonuclease activity was recorded. S. unisporus contains 45.6% hydrophobic, 25.9% neutral, and 28.0% hydrophilic amino acids in VMA1 inteins (Okuda and others 2003).

Bakhrat and others (2006) studied common sequential regions of Ho and VMA1 strains. Ho and VMA1 are intracellular proteins and fall into 2 different phylogenetic groups. Ho is marked as the proteosome substrate within the nucleus. In a bootstrap alignment, VMA1 interim sequence present in S. unisporus is closely related to S. cariocanus, S. castellii, and S. dairenensis species (Bakhrat and others 2006). Posey and others (2004) speculated duplication of endonuclease gene followed by their fusion which furnished monomeric proteins (like PI-SceI) for S. cerevisiae. In the case of S. unisporus this process gave an enzyme known as PI-SunIP. Percentage identity for this monomeric protein (PI-SceI) is 33% and the region conserved at position 301st is lysine, 341st position is threonine, and 403rd position it is lysine again. PI-SunIP is an active homing enzyme (Posey and others 2004).

Presence or absence of ori-rep-tra sequences has divided the Saccharomyces species into 2 different categories of sensu stricto and sensu lato (Piskur and others 1995). In mtDNA of S. cerevisiae, a unique class of intergenic sequences is formed by the ori-rep-tra sequences, which are available in multiple copies. These sequences are supposed to be conserved in the same genus or species. However, these set of sequences are absent in S. unisporus and other yeasts of the same species. The sequence of ori-rep-tra is specific only for a limited number of closely related species.

In a process, where levodione is converted to actinol with the help of ketoisophorone reductase gene, which are derived from the group of organisms, one of them is S. unisporus IFO0298. Further ahead, actinol is extracted from the reaction mixture, where a recombinant microorganism is derived by direct transformation of the host microorganisms, for example, Saccharomyces and Candida (Hoshino and others 2005). Actinol is a useful compound as it prevents osteoporosis.

A phylogenetic tree (Figure 6) has been drawn for S. sensu lato species based on the D1/D2 domain sequences of 26S rDNA S. unisporus NRRL Y-1556 was found closely related to S. servazzii and S. martiniae, while S. cariocanus, S. dairenesis, and S. castelli are distantly related. Most of the species from S. sensu lato are nonsporulating, for that reason interfertility tests were not performed on the organisms falling into this group (Spirek and others 2003). Such closeness with other important organisms of the Saccharomyces spp. makes S. unisporus a potential candidate for recombinant DNA technology. Apart from that, the presence of S. unisporus in various fermented milk products and fermented fruit products, along with other species, reveals that S. unisporus was the most conspicuous species of yeast found in kefir (Latorre-Garcia and others 2007) of which rRNA genes has been sequenced.

Figure 6.

UPGMA method for the Saccharomyces complex, where S. unisporus are aligned against S. cariocanus, S. castelli, S. servazzii, S. cerevisiae, and S. dairenesis. S. unisporus NRRL Y-1556 was closely related to S. servazzii ATCC 58439 though S. dairenesis NRRL Y-12639 fall into the same cluster as S. unisporus NRRL Y-1556 and S. servazzii ATCC 58439. S. unisporus is distantly related to S. castellii and S. cariocanus, when 26S rDNA is compared based on D1/D2 domains. Scale bar indicates 0.05 nucleotide substitution per site.

Regulatory pathways

When 18S rDNA of S. unisporus is aligned along with 18S rDNA of other yeast, the nearest 2 organisms, which were highlighted were K. sinensis and S. transvaalensis which have a 97% sequence match (Blast N) (James and others 1997). Another nearest organism is S. servazzii with which S. unisporus exhibits 99% sequence similarity (Blast N) (James and others 1997). Some strains of Saccharomyces genus have 97% coverage with S. cerevisiae, and the metabolic pathways that S. cerevisiae follows are phenylalanine, tyrosine, and tryptophan biosynthesis; histidine and lysine biosynthesis; fatty acid biosynthesis; fatty acid elongation; triglyceride biosynthesis; sphingolipid biosynthesis; and phospholipid biosynthesis. These are a few of the major pathways which S. unisporus might be following, but they are not exactly known and, therefore, further research is required on this topic.

Potential of recombinant DNA technology

Advancement in recombinant DNA (rDNA) technology has become a potential molecular biology tool, which is used to exploit industrially potential microorganism to increase the yield or productivity of a desired product. The application of rDNA technology sometimes becomes essential for an effective industrial process. S. unisporus possesses much higher active biotransformation of ketoisophorone to levodione, with the help of old yellow enzyme (OYE)/ketoisophorone reductase, as compared to S. cerevisiae. The gene responsible for the enzyme, OYE (EC 1.6.99.1), has been cloned from S. cerevisiae and expressed in Escherichia coli (Wada and others 2003). However, S. unisporus OYE has not been cloned yet and expressed until now. The other possibility is the insertion of levodione reductase gene to make recombinant S. unisporus. The recombinant S. unisporus, in that case, could convert ketoisophorone directly to actinol and complete the entire process in a single step (Hoshino and others 2005).

Similar applications can be achieved when other potential genes of S. unisporus can be cloned for the purpose of producing useful and beneficial compounds, which otherwise S. unisporus is producing only in minimal quantities. The scope of rDNA technology has a wide perspective when certain genes are considered for cloning, which otherwise might be producing significant compounds or proteins, and in the long run must satisfy the concept of genetically modified organism for long-time-frame usage.

Conclusions

The ubiquitous presence of S. unisporus in fermented milk (viili), cheese, and certain fruits (grapes and oranges) has been long observed. S. unisporus is also one of the important yeasts found in food products like koumiss and kefir-based milk products. S. unisporus grows efficiently in a medium where carbon sources are glucose, galactose, sucrose, maltose, sorbic acid, and cycloheximide, but the yeast does not consume lactose. Growth of S. unisporus takes a boost in the presence of thiamine. It also produces thiamine monophosphate but cannot consume it.

A unique interaction between LAB and S. unisporus has been observed in certain milk products like kefir and koumiss. S. unisporus produces metabolites which LAB consumes and, on the other hand, LAB produces glutamic acid and lactic acid which S. unisporus consumes. S. unisporus efficiently produces middle-chain fatty acids upto C18:1. Other important fatty acids produced by the organism are palmitoleic, palmitic, and oleic acids. It is not known if it produces polyunsaturated C18 fatty acid to class it as safe or unsafe. Absence of C18 polyunsaturated fatty acid chains are considered as chemical markers of an organism being a pathogen.

It is a significant producer of levodione and actinol, which shows oxidoreductase activity and synthesis of carotenoids. Other essential volatile compounds are prenyl alcohol and farnesol. Certain sets of organic acids, which are lactic acid, acetic acid, and propionic acid, can inhibit S. unisporus. An importance of S. unisporus is that it inhibits certain food pathogens like L. monocytogenes and degrade secondary bile acids effectively. Regulatory metabolic pathways of S. unisporus have not been well defined until now and further research is required. Whole ranges of intergenic and intragenic sequences have been studied in S. unisporus to get it placed at the right subspecies of Saccharomyces and classified as a sensu lato species. Closeness of S. unisporus with S. florentinus has been observed, and both the organisms are accepted under IDF and EFFCA as food cultures to be present in food and feed; these aspects predict that S. unisporus can be considered as a GRAS organism.

Future perspective

Aspects of using S. unisporus commercially in food products should be in concordance with the benefits and its natural occurrence in food items. Certain patented compounds, which include levodione, have human health benefits, but many other compounds are still unknown which S. unisporus might be producing under different culture media, for example, in cheese whey. The study of proteins also remains a significant researchable aspect because so far only limited numbers of extracellular proteins have been reported. Certain toxins are also produced by S. unisporus and they kill pathogens; more and more could be produced at a commercial scale and thus would require further research. By listing all the properties of S. unisporus it can be categorized as a useful organism. Production of middle-chain fatty acids and significant amounts of intermediates by S. unisporus, when grown along with LAB, good-quality SCP can be produced which can be used for animal feed or food. It is an abundant occurring organism in certain milk-based products and fruits that are being produced efficiently now by the food industry.

Acknowledgment

The authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (Grant A4984, RDCPJ 379601, Canada Research Chair) for financial support. The views and opinions expressed in this article are strictly those of the authors.

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