Food Chemistry 152 (2014) 1–10

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Review

Food-related applications of Yarrowia lipolytica Smita S. Zinjarde ⇑ Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India

a r t i c l e

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Article history: Received 3 September 2013 Received in revised form 16 October 2013 Accepted 20 November 2013 Available online 27 November 2013 Keywords: Yarrowia lipolytica Dairy products Food-additives Single cell oil Waste degradation

a b s t r a c t Yarrowia lipolytica is a non-pathogenic generally regarded as safe yeast. It displays unique physiological as well as biochemical properties that are relevant in food-related applications. Strains naturally associated with meat and dairy products contribute towards specific textures and flavours. On some occasions they cause food spoilage. They produce food-additives such as aroma compounds, organic acids, polyalcohols, emulsifiers and surfactants. The yeast biomass has been projected as single cell oil and single cell protein. Y. lipolytica degrades or upgrades different types of food wastes and in some cases, value-added products have also been obtained. The yeast is thus involved in the manufacture of food stuffs, making of food ingredients, generation of biomass that can be used as food or feed and in the effective treatment of food wastes. On account of all these features, this versatile yeast is of considerable significance in foodrelated applications. Ó 2013 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Y. lipolytica strains in meat and dairy products . Production of c-decalactone by Y. lipolytica . . . . . . . . . . . . . . . . Y. lipolytica strains as spoilage yeasts and their control . . . . . . Synthesis of organic acids by Y. lipolytica . . . . . . . . . . . . . . . . . . Role of Y. lipolytica strains in the production of polyalcohols . . Y. lipolytica mediated synthesis of surfactants/emulsifiers . . . . Application of Y. lipolytica as single cell oil (SCO) . . . . . . . . . . . Y. lipolytica biomass as single cell protein (SCP) . . . . . . . . . . . . Food industry waste degradation by Y. lipolytica . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Yarrowia lipolytica is an ascomycetous yeast species that has been intensively studied. The yeast usually inhabits hydrophobic substrate-containing environments. This is possible because the yeast inherently harbours several multi-gene families that play a role in the efficient degradation of these substrates (Fukuda, 2013). The yeast also utilises a limited range of sugars, alcohols, sugar alcohols and organic acids. It tolerates physical parameters such as the presence of salt, low temperatures, acidic and alkaline ⇑ Tel.: +91 20 25601385; fax: +91 20 25690087. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.117

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pH. Moreover, it inherently produces extracellular enzymes such as proteases, lipases, esterases, phosphatases and RNases that aid its growth under different conditions. The yeast displays distinct physiological, metabolic and genomic features that differentiate it from other systems such as Saccharomyces cerevisiae. On account of these characteristics, several research groups all over the world have been using this system for basic research and in different biotechnological applications. With respect to fundamental biological aspects, the yeast has been proposed as a model system to study dimorphism, the mitochondrial complex, peroxisomes, lipid accumulation, lipase production and as a generic tool for understanding the molecular evolution of different enzymes. With regard to applications, the

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yeast has been used in the biotransformation of hydroxy fatty acids, heterologous expression of proteins and their secretion, synthesis of organic acids, production of bio-oil, for tackling several industrial and environmental issues and for developing cell surface display techniques with respect to bio-refinery applications. When microorganisms are to be used in food applications, they should be non-pathogenic and with a GRAS (generally regarded as safe) status. Recently, the safety issues of this yeast have been thoroughly evaluated and it has been labelled as a ‘‘safe-to-use’’ organism (Groenewald et al., 2013). The aspects regarding the safety of the yeast are evident because (i) it is inherently associated with dairy, poultry and meat products (ii) yeast biomass is a safe nutritional supplement (iii) it is consumed as food and feed (iv) foodgrade additives have been obtained from this yeast. Although there are published articles on several basic and applied aspects of this yeast, an exclusive summary on the extensive food-related applications is lacking. The purpose of this review is to bring together a selection of data from different laboratories and provide a recent update on such applications of Y. lipolytica. Included here are the following points (i) significance in meat, poultry and dairy products (ii) role in the production of aroma compounds (iii) importance as spoilage microorganism (iv) production of organic acids and polyalcohols (v) synthesis of surfactants and emulsifiers (vi) use as single cell oil and single cell protein and (vii) for waste degradation or upgradation.

2. Importance of Y. lipolytica strains in meat and dairy products Yarrowia lipolytica strains are present in different types of foods due to their nutritional versatility and physiological aspects. These are usually encountered in meat, poultry and dairy products where they play a variety of roles. The subsequent sections will summarise the occurrence and significance of Y. lipolytica in different food products. The flavours associated with dry sausages are due to the acids produced during fermentation, salt and molecules derived from the catabolism of sugars, proteins and lipids. A variety of yeasts play a significant role in the ripening of dried fermented sausages. Their presence in such foods has been confirmed by classical culture-based methods and by molecular techniques (Andrade, Rodr´ıguez, Sánchez, Aranda, & Córdoba, 2006). In general, the lipolytic and proteolytic activities associated with these yeasts mediate the breakdown of fatty tissues and proteins and thereby contribute towards taste and flavour. The selection of new, functional starter cultures with desirable industrial or nutritional functionalities is critical in this regard. The choice of microorganisms capable of generating aroma compounds, health-promoting molecules, bacteriocins and antimicrobial compounds is particularly important. Moreover, microorganisms contributing to cured meat colour, probiotic qualities and lacking undesirable properties (production of biogenic amines and toxic compounds) are advantageous. The effect of surface inoculation of Debaryomyces hansenii and Y. lipolytica strains (along with Lactobacillus plantarum) on sausage characteristics has been evaluated. Patrignani et al. (2007) observed that yeasts mediated a rapid decrease in available water (aw) during ripening. It was postulated that water loss, microstructural and compositional modifications during ripening accounted for this decrease. The lytic activities of the enzymes and the consequent formation of molecules with low molecular weight were mainly responsible for reducing aw. The lipolytic and proteolytic activities affected the final sensory properties. In addition, the study also revealed that Y. lipolytica strains resulted in the formation of free fatty acids such as pentadecanoic, palmitic, margaric, stearic, oleic, linoleic and behenic acids that contributed towards

the final flavour. The effect of these yeast species on the sensory quality and biogenic amine content has also been evaluated (Iucci et al., 2007). Y. lipolytica strains produced a variety of flavourcontributing compounds. The major ones included acids (acetic acid, hexanoic acid); alcohols (2-methylbutan-1-ol, 2-nonen-1-ol, 1-octen-3-ol and 2-ethylhexan-1-ol); aldehydes [2(E)-decenal, 2,4(EE)-decadienal]; ketone [3-hydroxybutan-2-one]; terpenes (delta-3-carene, L-limonene, cimene); alkanes (pentane and octane) and cyclic hexane. Putrescine, cadaverine, histamine, tyramine, spermidine and spermine were the biogenic amines that were detected. These substances however, resulted in the generation of typical flavours that were less appreciated by panellists during subsequent sensory evaluation tests. When different lipolytic strains were inoculated in pork fat, a variety of fatty acid profiles were obtained (Patrignani, Vannini, Gardini, Guerzoni, & Lanciotti, 2011). Palmitic, stearic, oleic and linoelic acids were some of the predominant free fatty acids that were produced. These were thought to be important in flavour development. This study thus suggested the possible use of some of these strains in enhancing flavour. A large number of yeasts are widely associated with dairy environments (milk, air, surfaces, implements, brine and water). Such strains tolerate low temperatures and high salt contents. They can assimilate lactose, galactose, lactate and citric acid. Y. lipolytica displays strong proteolytic and lipolytic activities that contribute towards texture and aroma development during the ripening process. The physiological and metabolic attributes associated with Y. lipolytica are summarised in Fig. 1. These features are responsible for the following changes that occur during the process of cheesemaking. Hydrolysis of casein requires the action of proteases that are often provided by cheese-associated strains of Y. lipolytica. Proteolytic cleavage of as1-casein and b-casein by Y. lipolytica proteases generates peptides and free amino acids (De Wit, Osthoff, Viljoen, & Hugo, 2005). These end products are of particular significance in the making of blue cheeses (Curioni & Bosset, 2002). Penicillium species associated with these varieties metabolize amino acids and produce NH3. This in turn de-acidifies the curd and favours curing. Some strains also display good aminobiogenic potential and decarboxylate ornithine, phenylalanine, tyrosine and lysine (Gardini et al., 2006). Y. lipolytica produces several types of lipases and esterases. This yeast is thus considered to be a major contributor towards lipolytic activities associated with cheese-ripening processes. Some strains also display strong lipolytic activities at low temperatures. As a result, large quantities of free fatty acids (propionic, butyric, myristic, palmitic, palmitoleic, stearic and oleic) have been observed (De Wit et al., 2005). Some of these free fatty acids are said to be responsible for sensory characteristics. The breakdown of tributyrin to butanoic acid is also responsible for flavours associated with varieties such as Cheddar and Camembert cheese (Curioni & Bosset, 2002). Cheese aroma is generated due to a variety of volatile compounds, which individually do not reflect the overall odour. These compounds are produced by the action of microbial enzymes on lactose, lipids and proteins in the curd. In natural biological systems such as cheese maturing in brine, the microflora can give rise to a range of volatile metabolites that can contribute to the aroma and flavour of the finished cheese. In general, alcohols, aldehydes, ketones and esters contribute towards flavour. Y. lipolytica strains also produce volatile sulphur compounds (VSCs) that add to cheese flavours. Sulphur metabolism in Y. lipolytica based on a comparison between high- and low-sulphur supplies (sulphate, methionine or cystine) by combined approaches (transcriptomics, metabolite profiling, and VSC analysis) has been recently reviewed (Hébert et al., 2013). The yeast has the inherent ability to synthesise several structurally diverse sulphur containing

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Fig. 1. Summary of the attributes of Yarrowia lipolytica that are important in its association with cheese varieties.

Table 1 Yarrowia lipolytica associated with cheese varieties: occurrence and role. Cheese variety

Comment

References

Blue Serbian and Croatian Fresh soft artisanal Danish farmhouse Slovakian bryndza

In synergy with Penicillium roqueforti imparts cheese odour Lipolytic activity

Gkatzionis et al. (2013) Golic´ et al. (2013)

Identified by molecular techniques Detected by culture-independent approach

Livarot

Dominant yeast along with Candida catenulate, Geotrichum spp. and Candida intermedia Lipolytic activity Dominant with Debaryomyces hansenii during later stages of maturation

Gori, Ryssel, Arneborg, and Jespersen (2013) Chebenˇová-Turcovská, Zˇenišová, Kuchta, Pangallo, and Brezˇná (2011) Mounier, Monnet, Jacques, Antoinette, and Irlinger (2009) De Freitas, Pinon, Maubois, Lortal, and Thierry (2009) Gardini et al. (2006)

Cantalet Pecorino Crotonese

molecules that can contribute towards aromatic notes. In a study with Y. lipolytica CBS 2075, Sørensen, Gori, Petersen, Jespersen, and Arneborg (2011) mainly observed the presence of sulphides, furans and short-chain ketones as cheese-associated aroma compounds. Dimethylsulfide (DMS), dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) were some specific VSCs that have also been reported. Literature survey shows that Y. lipolytica isolates have been associated with Gouda, Picante, Apulian, Camembert, blue-veined, Cheddar, Brie and Feta cheese varieties to mention a few. Some of the recent reports on its occurrence and significance in cheese varieties are summarised in Table 1.

3. Production of c-decalactone by Y. lipolytica Lactones are constituents of essential oils and plant volatiles that contribute towards taste and aroma of foods. c-Decalactone (lactone of 4-hydroxydecanoic acid) has a peachy flavour. This has been approved by FDA as a food-additive. There are several reports on Y. lipolytica producing c-decalactone wherein the conversion biochemistry and process parameter optimisation have been described. Some earlier reports on the role of b-oxidation genes, size of substrate droplets, their microscopic observations and the role of other parameters involved in lactone production by Y. lipolytica have been summarised in a review (Waché, Aguedo, Nicaud, & Belin, 2003). The more recent work on this topic is described below.

Fig. 2. Growth of Yarrowia lipolytica on edible oil stained with (a) STYO 9: specific for nucleic acids (b) Nile Red: specific for lipids (c) Superimposed images (oil droplets: orange; cells green). Single arrow points to cells and double arrows to oil droplets. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Peroxisomal b-oxidation is responsible for lactone bioconversion in Y. lipolytica. One of the strategies to enhance lactone production has been the disruption of some of the POX genes coding for acyl coenzyme A (acyl-CoA) oxidase isozymes. Some of the observations regarding c-decalactone production have been reconfirmed recently. For example, the role of the POX3 gene product in decreasing production of c-decalactone has been earlier established (Waché et al., 2003). The observation that the enzyme Aox3 (product of POX3 gene) degrades the lactone has also been recently confirmed in another strain CGMCC 2.1405 (Guo, Song, Wang, & Ding, 2012). Moreover, in a recent study, Gomes, Waché, Teixeira, and Belo (2011) have concluded that larger droplets of the substrate favoured aroma production. Y. lipolytica cells are known to adhere to large droplets of hydrophobic substrates such as oils and alkanes as depicted in Fig. 2. Process parameter optimisation plays an important role in enhancing the yields of products such as c-decalactone (Gomes, Teixeira, & Belo, 2010). Constant medium feeding rate and intermittent fed-batch fermentation strategies were compared with the traditional batch modes by these authors. Although the productivity of c-decalactone was considerably higher in the batch mode (168 mg l 1 h 1), substrate conversion to lactone (73 mg g 1) was greater in the intermittent fed-batch giving 6.8 g L 1. Moradi, Asadollahi, and Nahvi (2013) have used Y. lipolytica (DSM 3286) for the production of this flavour compound from castor oil. The initial yields of 65 and 70 mg L 1 could be enhanced to 220 mg L 1 when fed-batch fermentations were used. Cell immobilization has also been effective in increasing yields of c-lactone (Braga & Belo, 2013). Immobilization of cells onto a polymer (DupUMÒ) yielded high aroma contents (1597 ± 34 mg L 1 after 264 h corresponding to a molar conversion of 29%. Free cells on the other hand yielded 954 ± 7 mg L 1 in 120 h. Immobilization prevented re-consumption of the aroma lactone by the cells. In microbial processes involving high-cell density cultures, productivity is often limited by the transport of oxygen. Oxygen transfer rates (OTR) in a system mainly depend on the volumetric mass transfer coefficients, kLa, and the oxygen solubility in the medium. OTR can be increased by increasing agitation and aeration rates. In a study conducted by Gomes, Aguedo, Teixeira, and Belo (2007), the effect of kLa values on the production of c-decalactone were determined. Highest content of c-decalactone (141 ± 21 mg L 1) was achieved when a kLa value of 70 h 1. This was obtained by maintaining agitation and aeration rates at 400 rpm and 0.6 vvm, respectively. In an earlier study, it was shown that by growing the cultures under high-pressure, oxygen solubility could be increased (Aguedo et al., 2005). Under these conditions, although growth was accelerated, c-decalactone production was less. To obtain enhanced yields, a decrease in oxygen supply when cdecalactone concentrations were high was suggested. In the absence of oxygen, the conversion of the lactone to other products was inhibited. Recently, Y. lipolytica NCYC 3825 has been reported to produce another flavour compound 2-phenylethanol with a rose-like odour (Celin´ska, Kubiak, Białas, Dziadas, & Grajek, 2013). The yields obtained under non-optimised culture conditions were as high as 2 g L 1.

4. Y. lipolytica strains as spoilage yeasts and their control Y. lipolytica isolates are associated with discolourations and spoilage of food. Surface discolouration is an undesirable flaw observed in cheese varieties. In this regard, some strains are known to catabolise tyrosine and produce the brown pigment ‘melanin’. This in turn is responsible for the aforementioned surface

discolouration (Carreira, Paloma, & Loureiro, 1998). In another report, samples of raw, marinated, smoked, or roasted chicken and turkey products were analysed for the presence of different spoilage yeasts. Y. lipolytica was isolated on several occasions and it played a prominent role in the spoilage of the tested poultry products that were stored at 5 °C. It must be noted that some Y. lipolytica strains are psychrotrophic and can bring about spoilage of food products at low temperatures. There are some reports on the efforts suggested to control spoilage-associated Y. lipolytica strains. Immersion of poultry (wings) in 2% lactic acid (with or without 0.2% potassium sorbate or sodium benzoate) or 4% trisodium phosphate caused a significant reduction in Y. lipolytica numbers (Ismail, Deak, Abd El-Rahman, Yassien, & Beuchat, 2001). These authors have also shown that dipping in decoctions of basil, marjoram, sage and thyme controlled the yeast. In another report, Karanika, Komaitis, and Aggelis (2001) have observed that extracts of Origanum dictamnus and Rosmarinus officinallis extended the yeast lag phase considerably and decreased specific growth rates. It is well-known that Y. lipolytica exhibits the biofilm mode of growth under certain conditions (Dusane, Nancharaiah, Venugopalan, Kumar, & Zinjarde, 2008a). Biofilms of this organism have been effectively disrupted by lauroyl glucose, a food-additive (Dusane et al., 2008b). Some essential oils and their constituents have been effective in controlling the growth of this yeast. For example, limonene, myrcene, b-pinene, a-pinene and a-terpineol were identified as the major essential oil components of Citrus sinensis cv. New Hall – Citrus aurantium (Papanikolaou et al., 2008a). This oil inhibited Y. lipolytica by decreasing the final biomass content and increasing the lag phase. Y. lipolytica and other yeast species associated with food-spoilage have been controlled by other essential oils (Kunicka-Styczyn´ska, 2011). Thyme oil followed by marjoram, peppermint and basil oil were found to be useful. Origanum vulgare L. essential oil (with carvacrol and thymol as the major components) is another oil that has been effective in controlling the growth of Y. lipolytica (Chatzifragkou, Petrou, Gardeli, Komaitis & Papanikolaou, 2011). Natamycin is a natural antimycotic polyene produced by Streptomyces natalensis. This has also been employed in dairy-based food products to prevent yeast and mold contamination. Natamycin at a concentration 50 ppm effectively controlled the growth of Y. lipolytica (Ollé Resa, Jagus, & Gerschenson, 2013). Thus it is evident that strains of Y. lipolytica are associated with different foods and they can be controlled by simple means.

5. Synthesis of organic acids by Y. lipolytica Y. lipolytica secretes large quantities of organic acids under conditions of growth-limitation and carbon-excess. On account of these properties there has been an increased interest in the yeast for this food-related application. In the following sections, production and optimisation of parameters for the synthesis of citric, isocitric, a-ketoglutaric (a-KGA), pyruvic, succinic acid are described. Citric acid is commercially used as an acidity regulator and flavour enhancer in beverages and in the manufacture of pharmaceutical products. Citric acid production by Y. lipolytica has been documented for a very long time. Choice of substrates, use of additives, optimisation of process parameters, use of mutants (for enhancing production or improving citrate:isocitrate ratios) and generation of recombinant strains (for increasing yields or extending substrate range) are some of the parameters that are significant in citric acid synthesis by Y. lipolytica (Fig. 3). Although some earlier reports have applied hydrocarbons and ethanol for citric acid production, the current trend has been the use of alternative inexpensive substrates. Raw glycerol (the major low-price waste product generated during biodiesel production) has become a popular substrate. In a recent review, Rywin´ska et al.

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Fig. 3. Production of citric acid by Yarrowia lipolytica: a summary of the strategies involved.

(2013) have in detail, summarised the reports on Y. lipolytica mediated conversion of crude glycerol into value-added products including organic acids. For more details on the use of this substrate for organic acid production, the reader is directed to refer to this earlier review and thus the references cited there have been excluded here. Some agricultural residues and wastes have been employed as alternative substrates for the production of citric acid. For example, Wang, Wang, Liu, and Chi (2013a) have proposed the use of Jerusalem artichoke tuber extract and a genetically engineered strain. During typical fermentations using the extract (containing 84.3 g L 1 total sugars), 68.3 g L 1 citric acid was produced. The yield was 0.91 g g 1 within 336 h. Up to 67.2% of the citric acid with 96 % purity could be recovered. Olive-mill wastewater (O.M.W.) based media have been also been used (Papanikolaou, Galiotou-Panayotou, Fakas, Komaitis, & Aggelis, 2008b). Y. lipolytica (ACA-DC 50109) was cultivated with an enriched media supplemented with commercial industrial glucose. In diluted O.M.Ws enriched with glucose (initial sugar concentration, 65 g L 1), total citric acid obtained was 28.9 g L 1. O.M.W. based media thus stimulated the production of citric acid. The final citric acid concentration and conversion yield per unit of sugar consumed were higher in the presence of O.M.W. when compared to the results obtained in its absence. Y. lipolytica strains effectively use plant oils and synthesise citric acid. For example, sunflower oil was evaluated for the production of this acid by Y. lipolytica strain UOFS Y-1701 (Venter et al., 2004). When acetate was added to media containing this oil, a remarkable increase in the production of citric acid was observed. In the absence and presence of acetate, 0.5 and 18.7 g L 1 of citric acid were produced, respectively. The ratio of citric acid:isocitric acid also increased from 1.7:1 in the absence of acetate, to 3.7:1 in its presence after 240 h of incubation. Y. lipolytica DSM 3286 was cultivated on different plant oils (Darvishi, Nahvi, Zarkesh-Esfahani, & Momenbeik, 2009). The production of valuable metabolites such as lipase, citric acid and single-cell protein were evaluated. Olive oil proved to be the best carbon supplement for lipase and citric acid production. Some recombinant strains have been generated for the production of citric acid. Two main strategies have been employed (i) metabolic pathway involved in the synthesis has been manipulated in some cases and (ii) the substrate range has been extended. When the ATP-citrate lyase gene (ACL1) was deleted and the copy number of isocitrate lyase gene (ICL1) was increased in a marine strain (SWJ-1b) displaying recombinant inulinase, the lipid and isocitric acid content were greatly reduced (Liu, Chi, Liu, Madzak, & Chi, 2013). Citric acid production on the other hand was greatly enhanced. One transformant (30) was subjected to batch fermentations in media containing 10.0% inulin. This produced 84.0 g L 1 of citric acid and 1.8 g L 1 of isocitric acid within 214 h. At the end of the process only 0.36% of the residual reducing sugar and 1.0% of

the residual total sugar were left in the fermented medium. This suggested that 89.6% of the total sugar was used for citric acid production and cell growth. A recombinant strain Y. lipolytica [H222-S4(p67ICL1) T5], harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15) was constructed (Förster, Aurich, Mauersberger, & Barth, 2007). Citric acid production by this recombinant in a fedbatch manner on sucrose medium was evaluated. At pH 6.0–6.8, citric acid produced was 127–140 g L 1 corresponding to a yield of 0.75–0.82 g g 1. Another recombinant (SUC+) strain of Y. lipolytica (A-101-B56–5) was also evaluated for citric acid production on different carbon sources (Lazar, Walczak, & Robak, 2011). Among the tested substrates, the highest concentration of citric acid and high yields were obtained with glycerol (57.15 g L 1 and 0.6 g g 1, respectively). With sucrose, the citric acid secreted was 45 g L 1 and the yield was slightly higher (0.643 g g 1). The isocitrate content was below 2% of total citrates. Another value-added product obtained was the invertase enzyme. Chemical surfactants such as Triton X-100 affect the permeability of yeast cells and citric acid production. In Y. lipolytica strains DSM 3286 and M7 permeabilized with the surfactant, citric acid formation increased 1.4 to 1.8-fold (Mirbagheri, Nahvi, Emtiazi, & Darvishi, 2011). The final concentrations ranged between 75 and 85 g l 1 (corresponding to 0.80–0.84 g g 1 in terms of conversion yields). It was concluded that Triton X-100 could be used to increase the efficiency of citric acid production by Y. lipolytica strains. The ability of Y. lipolytica to synthesise a-KGA was discovered in late 1960s. The a-KGA-producing strains of Y. lipolytica are unable to synthesise the pyrimidine structure in the thiamine molecule. Under conditions of thiamine deficiency, although the yeast is unable to grow, it oxidises the substrate and produces a-KGA. In a recent report, under optimal conditions, 49 g L 1 of a-KGA was produced by strain VKM Y-2412 (Kamzolova, Chiglintseva, Lunina, & Morgunov, 2012). Production of a-KGA from ethanol was higher in the presence of zinc and iron ions when aeration rates were high and pH was 3.5. With further optimisation of conditions, 72 g L 1 of a-KGA was obtained. The possibility of using rapeseed oil as a carbon source for the production of a-KGA was studied (Kamzolova & Morgunov, 2013). Among the twenty-six strains of Y. lipolytica that were screened, strain Y. lipolytica VKM Y-2412 was selected. When the thiamine concentration, medium pH, temperature, aeration and oil concentration were optimised [thiamine concentration: 0.063 lg g cells 1; pH: 3.5; temperature: 30 °C; pO2: 50%; oil concentration: 20–60 g L 1], 102.5 g L 1 of a-KGA with the mass yield coefficient of 0.95 g g 1 and volumetric productivity of 0.8 g L 1 h 1 were obtained. Some recombinant strains have been constructed and evaluated for altered a-KGA production. The alpha-ketoglutarate dehydrogenase

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(KGDH) complex in S. cerevisiae consists of three subunits: alphaketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase and lipoamide dehydrogenase. The subunit encoding genes were over-expressed in Y. lipolytica to containing multiple copies (Holz et al., 2011). This recombinant was inefficient in the production of a-KGA although the authors have reported an elevated production of pyruvic acid. A thiamine-auxotrophic strain of Y. lipolytica (WSH-Z06) over-produced a-KGA (Yin, Madzak, Du, Zhou, & Chen, 2012). However, by-products such as pyruvate in large concentrations limited its application on an industrial scale. In order to increase a-KGA production and decrease pyruvate accumulation, pyruvate carboxylase genes (ScPYC1) from S. cerevisiae and RoPYC2 from Rhizopus oryzae were over-expressed in Y. lipolytica WSH-Z06. The yields of a-KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5% and 35.3%, and the yields of pyruvate decreased by 51.9% and 69.8% in shake flasks, respectively. On further controlling the pH in fermentors, the maximum concentration of a-KGA that Y. lipolytica-RoPYC2 could produce was 62.5 g L 1. The pyruvate yield decreased from 35.2 to 13.5 gL 1. In an attempt to improve the a-KGA production by Y. lipolytica H355 and reduce the by-products (fumarate and pyruvate), recombinants harbouring multiple copies of the fumarase (FUM1), pyruvate carboxylase (PYC1) or both FUM1 and PYC1 genes were constructed (Otto, Yovkova, Aurich, Mauersberger, & Barth, 2012). These were evaluated for a-KGA production on raw glycerol as a carbon source in bioreactors. The production of a-KGA with the multi-copy strains H355A (FUM1) and H355A (FUM1-PYC1) was comparable with the wild-type strain and slightly lower with H355 (PYC1). Although a-KGA productivity was not altered significantly, selectivity of the main product, a-KGA was increased in H355A (FUM1). There are reports on the application of Y. lipolytica in the production of succinic acid (by chemical oxidation of a-KGA) as well. A two-step method for the production of this acid has been developed (Kamzolova et al., 2009). In the first step, the yeast mediated the synthesis of a-KGA from ethanol. This was subsequently oxidised by hydrogen peroxide to succinic acid. The maximum concentration of the acid was 63.4 g L 1 and the yield on the basis of the ethanol consumed was 58%. The quality of the acid produced by the method met the biochemical grade definitions. The production of this organic acid by a recombinant strain of Y. lipolytica (with a deletion in the gene coding for one of the succinate dehydrogenase subunits) has been reported (Yuzbashev et al., 2010). These transformants did not grow on glucose but grew on glycerol and produced succinate in the presence of a buffering agent. Subsequent studies allowed the selection of a strain capable of accumulating 45 and 17 g L 1 succinate under shake flask conditions with and without buffering, respectively. The authors concluded that this recombinant could be evaluated on different substrates to develop an industrially feasible process.

6. Role of Y. lipolytica strains in the production of polyalcohols Erythritol is an FDA-approved food-additive that is used throughout the world. It is widely employed to replace sucrose in different applications. It offers several advantages (i) is 60–70% as sweet as sucrose (ii) is almost non-caloric with an energy value (0.2 kcal g 1) much lower than sucrose (4 kcal g 1) and other sugar alcohols (2.4 kcal g 1) (iii) is non-cariogenic (iv) generally free of gastric side-effects and (v) does not affect blood sugar. Among other microorganisms, Y. lipolytica has been employed for the synthesis of this artificial sweetener. The reports on Y. lipolytica strains producing this polyol are not included here as they have been reviewed recently (Rywin´ska et al., 2013). A thorough examination of the reports reviewed in this section suggests that Y. lipolytica has tremendous potential in the production of different food-additives. 7. Y. lipolytica mediated synthesis of surfactants/emulsifiers Several microorganisms produce emulsifiers and surfactants. These amphiphilic biomolecules exhibit emulsifying properties and surface activity. They find several applications in the food and beverage industries. Some of the older reports on strains of Y. lipolytica producing emulsifiers have been reviewed earlier (Bankar, Kumar, & Zinjarde, 2009). The reports on in the synthesis and characterisation of surfactants/emulsifiers during the past five years are summarised in Table 2. 8. Application of Y. lipolytica as single cell oil (SCO) Single-cell oils are oils derived from microorganisms. Their composition is similar to oils and fats obtained from plants or animals. They are being regarded as products capable of supplying major polyunsaturated fatty acids (PUFA) that are essential human nutrients. Y. lipolytica has been used for the production of singlecell oils. This yeast belongs to the group of oleaginous microorganisms that can accumulate large quantities of lipid in their biomass (Kosa & Ragauskas, 2011). Moreover, Y. lipolytica has been projected as a model for bio-oil production (Beopoulos, Chardot, & Nicaud, 2009b; Beopoulos et al., 2009a). In the current review, the recent work on this topic has been categorised on the basis of substrates used, application of recombinant strains and their use as dietary supplements. A large number of substrates including low-cost ones have been used for SCO production (Huang et al., 2013). For example, volatile fatty acids have been volarized into microbial lipids by Y. lipolytica (Fontanille, Kumar, Christophe, Nouaille, & Larroche, 2012). The yeast was initially cultivated on glucose or glycerol. After exhaustion of the carbon source, acetic acid under nitrogen limiting

Table 2 Summary of the biosurfactants/ emulsifiers produced by Y. lipolytica. Y. lipolytica

Composition/properties

Application

References

UCP 0988

Surfactant ‘‘Rufisan’’ obtained on ground nut refinery residue medium Surfactant ‘‘Yansan’’, factorial design approach employed to increase productivity Surfactant ‘‘BS-I’’ produced on whey wastewaters Surfactant ‘‘Rufisan’’grown on soybean oil refinery residue Surfactant ‘‘Yansan’’ based perfluoro-n-hexane in acetate buffer emulsions studied

Reduced surface tension to 25.3 mN m 1, CMC: 0.03% displayed antimicrobial and anti-adhesive properties

Rufino et al. (2011)

Emulsification index (81.3%) and surface tension variation (Delta ST) 19.5 mN m 1

Fontes, Amaral, Nele, and Coelho (2010)

Value added product

Yilmaz, Ergene, Yalçin, and Tan (2009)

IMUFRJ 50682

MFW5 UCP 0988 IMUFRJ 50682

Surface tension reduced to 25.29 mN m

1

pH dependent stability of emulsions demonstrated

Rufino, Sarubbo, Neto, and Campos-Takaki (2008) Trindade et al. (2008)

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conditions was sequentially added. Typically with 40 g L 1 of glucose, 31 g L 1 biomass, lipid concentrations of 12.4 g L 1, (corresponding to a lipid content of the biomass close to 40%) were obtained. Other volatile fatty acids (butyric and propionic acids) or their mixtures were also effective. The yeast lipid composition was similar to that of vegetable oils. In a recent report, five strains of Y. lipolytica were evaluated as potential SCO candidates (Katre, Joshi, Khot, Zinjarde, & Kumar, 2012). All the strains accumulated more than 20% (w/w) of lipid in their dry cell mass. Glucose and wastes such as waste cooking oil or waste motor oil were used as carbon sources. Amongst all the strains tested, Y. lipolytica NCIM 3589, a tropical marine strain exhibited maximal lipid/biomass coefficient with 30 g L 1 of glucose (0.29 g g 1) and 100 g L 1 of waste cooking oil (0.43 g g 1). The content of saturated and monounsaturated fatty acids were high and similar to conventional vegetable oils. Inexpensive renewable raw materials (palm oil mill effluent, serum latex and crude glycerol) have also been evaluated for cultivating different strains of Y. lipolytica (Louhasakul & Cheirsilp, 2013). When crude glycerol and the effluent were used as cosubstrates, the biomass produced was 3.21 g L 1 and lipid content was found to be 2.21 g L 1 (68% of the dry weight). With scale-up and process improvement, the biomass and lipid could be increased to 5.53 and 2.81 g L 1, respectively. A semi-continuous mode of operation was effective for biomass and a fed-batch one for lipid enhancement. Food waste and municipal wastewater were used for the production of microbial lipid (Chi, Zheng, Jiang, & Chen, 2011). Different oleaginous yeasts including Y. lipolytica displayed comparable growth on hydrolysed food waste and glucose (as control). These strains were also further evaluated to grow in municipal primary wastewater. Some agricultural residues have been employed for SCO production. In this regard, detoxified sugarcane bagasse hydrolysate (DSCBH) has been evaluated to culture Y. lipolytica Po1g for microbial oil production (Tsigie, Wang, Truong, & Ju, 2011). Hydrolysed sugarcane bagasse contained 21.38 g L 1 of sugar. This was detoxified with calcium hydroxide and used for growing Y. lipolytica. The use of peptone as the nitrogen source yielded biomass contents of 11.42 g L 1. The maximum lipid content, lipid yield and lipid productivity of the culture grown in DSCBH and peptone were 58.5%, 6.68 g L 1 and 1.76 g L 1 day 1, respectively. Y. lipolytica based oil has also been obtained from defatted rice bran hydrolysates (Tsigie et al., 2012). The hydrolysis conditions were optimised (3% sulphuric acid at 90 °C for 6 h). Glucose was the predominant sugar in the hydrolysates (43.20 ± 0.28 g L 1) followed by xylose (4.93 ± 0.03 g L 1) and arabinose (2.09 ± 0.01 g L 1). The hydrolysates were neutralised and used for culturing Y. lipolytica. Dry cell mass and lipid content of the yeast grown in these hydrolysates under optimum conditions were 10.75 g L 1 and 48.02%, respectively. The hydrolysate of wheat straw has also been evaluated for the microbial production of oil (Yu, Zheng, Dorgan, & Chen, 2011). The resulting hydrolysate was composed of pentoses (24.3 g L 1) and hexoses (4.9 g L 1), along with some other degradation products, such as acetic acid, furfural and hydroxymethylfurfural (HMF). Y. lipolytica along with four other yeast strains used this hydrolysate as substrates for the lipid production. During the production of biofuels from renewable resources, high conversion yields are desirable. To achieve this, Y. lipolytica has been subjected to metabolic engineering (Tai & Stephanopoulos, 2013). An efficient expression platform was used for the over-expression of the enzyme involved in the final step of the triglyceride synthesis pathway (diacyl glycerol acyltransferase DGA1). This caused a fourfold increase (33.8% of dry cell weight) in lipid production over control cells. The over-expression of acetyl-CoA carboxylase (ACC1), the enzyme catalysing the first com-

7

mitted step of fatty acid synthesis, increased the lipid content twofold over the control (17.9% lipid content). Simultaneous co-expression of DGA1 and ACC1, further increased lipid content to 41.4%. In bioreactors, this recombinant gave 61.7% lipid content after 120 h. In another study, the MIG1 gene (encoding for a key component of the glucose repression pathway) in Y. lipolytica ACA-DC 50109 was disrupted (Wang, Xu, Wang, Chi, & Chi, 2013b). The recombinant (M25) when grow in yeast nitrogen base-N5000 medium without uracil or the medium with 2-deoxyd-glucose produced more lipid content [48.7% (w/w) of oil based on its cell weight] than the parent strain [36.0% (w/w)]. Transcript levels of several genes related to lipid biosynthesis were higher in the in the knockout compared to those in the parent yeast. A recombinant strain of Y. lipolytica ACA-DC 50109 [Z31 with the exo-inulinase (INU1) gene sequence from Kluyveromyces marxianus CBS 6556] has been projected as a SCO producer (Zhao, Cui, Liu, Chi, & Madzak, 2010). The inulinase activity was found to be 41.7 units ml 1 after 78 h. After optimisation of culture conditions for SCO production, this transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L 1 within 78 h at the flask level. In fermentors, 48.3% (w/ w) cell oil and 13.3 g L 1 cell dry weight was obtained within 78 h. An oil content of 50.6% (w/w) was observed when extract of Jerusalem artichoke tubers were used and cell dry weight was 14.6 g L 1 within 78 h. Over 91.5% of the fatty acids in the transformant grown on these extracts were composed of palmitic, oleic and linoleic acid. SCO can be used in the form of dietary supplements enriched in docosahexaenoic acid, arachidonic acid and c-linolenic acid (Ratledge, 2005). There are a few studies wherein the enzymatic capabilities of Y. lipolytica have been manipulated to display desirable fatty acid profiles. For example, the bifunctional D12/x3 desaturase from Fusarium moniliformis was expressed in Y. lipolytica (Damude et al., 2006). When the fatty acid profiles were evaluated, it was observed that the recombinant strain produced a-linolenic acid corresponding to 28% of the cellular dry mass. The D6 and D12 desaturases from Mortierella alpina were expressed in Y. lipolytica under the control of a strong constitutive promoter to enhance c-linolenic acid production (Chang et al., 2009). Picataggio et al. (pending US patent application Ser. No. 10/840,579) have engineered Y. lipolytica for the production of different x-3 and x-6 fatty acids. Heterologous genes encoding the proteins of the x-3/x-6 pathways were introduced and expressed in this oleaginous host to achieve the enhanced production of the unsaturated fatty acids. Dupont de Nemours have identified genes coding for acytransferases that aid the transfer of these PUFAs into triacyl glycerides. These modified strains have been patented (US Patents 7267976, 7238482, 7256033) and promoted as PUFA-enriched dietary supplements. Conjugated linoleic acid (CLA) has several beneficial medicinal and nutritional effects. During a study, Zhang et al. (2012) have expressed the linoleic acid isomerase gene from Propionibacterium acnes in Y. lipolytica Polh. On codon usage optimisation the percentage of trans-10, cis-12 conjugated linoleic acid was found to be six times higher than in the native yeast. With both codon usage optimisation and multi-copy integration, the production yield was enhanced 30-fold. The content of trans-10, cis-12 CLA reached 5.9% of total fatty acid yield in the transformed Y. lipolytica. Y. lipolytica has been effectively employed as a model system for the production of tailor-made lipids (Papanikolaou & Aggelis, 2010). Particularly, the yeast is known to spontaneously accumulate high levels of stearic (18:0) acid inside the cells. Other yeasts employed for making cocoa butter substitutes lack this property and the feature was regarded unusual and exclusive to Y. lipolytica. It must be noted that although most of the other oleaginous yeasts accumulate large contents of oleic and linoleic acids, stearic acid

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levels are much lower. On account of this unique feature, strains of Y. lipolytica have been used to produce cocoa butter substitutes. In particular, the lipid composition and growth of Y. lipolytica strain ACA-DC 50109 on an industrial derivative of animal fat called stearin, raw glycerol and glucose was monitored (Papanikolaou, Muniglia, Chevalot, Aggelis, & Marc, 2003). When glycerol and stearin were employed as co-substrates, the production of lipid and citric acid were enhanced. The lipids contained (on a % w/w basis) mainly stearic acid (50–70%). The contents of palmitic (15–20%), oleic (7–20%), and linoleic (2–7%) acids were lower. This composition mimicked cocoa-butter. Eventually, up to 3.4 g L 1 lipid and 14 g L 1 of citric acid (as by-product) were produced.

were 1.37 and 10.89 g L 1, respectively. The hydrolysates thus generated were further used as feed-stocks for ethanol fermentation by S. cerevisiae (Ethanol RedÒ). After 48 h, the ethanol concentration was found to be 13.39 g L 1, 98.05% of glucose was consumed and the yield was thus 0.38 g g 1. The saccharification yield for glucose was 0.224 g g 1 dry weight and the ethanol yield was 0.084 g g 1 dry weight. From the foregoing discussion it is evident that Y. lipolytica is emerging as a promising candidate for the production of SCO and SCP. The fact that it can use a variety of inexpensive substrates and can be genetically modified has made it a popular system.

9. Y. lipolytica biomass as single cell protein (SCP)

10. Food industry waste degradation by Y. lipolytica

Y. lipolytica has been employed as single cell protein for a long time. Recently, the potential of some newly isolated strains of Y. lipolytica for biomass production using glycerol wastes has been evaluated (Juszczyk, Tomaszewska, Kita, & Rymowicz, 2013). Strain S6 yielded 11.7 and 12.3 g L 1 of biomass with 1.30 and 1.37 g L 1 h 1 productivity, respectively, when pure and raw glycerol was used. The contents of lysine, threonine and phenylalanine/tyrosine in the biomass were higher than those observed in standard egg proteins. In another recent study, Y. lipolytica was used for the effective expression of an antimicrobial peptide (Zhao, Chi, Chi, & Madzak, 2013). This transformant also had high protein contents (45– 49 g% w/w) and dry cell weights obtained were between 9.9 and 12 g L 1. To extend substrate range, the exo-inulinase gene from K. marxianus was expressed in Y. lipolytica strain with high protein contents (Cui et al., 2011). These engineered cells grew in inulincontaining media and the crude protein in cells and cell mass obtained were 47.5% and 20.1 g L 1, respectively. The biomass of Y. lipolytica has been used as an alternative source of eicosapentaenoic acid (EPA) during salmon aquaculture (Hatlen, Berge, Odom, Mundheim, & Ruyter, 2012). When Atlantic salmon were fed three dietary levels (10%, 20% and 30%) of heatkilled dried yeast biomass containing 6% EPA and 20% oil by weight for 95 days (replacing fish meal, rapeseed oil and wheat meal in the diets) fish weight increased from 180 to 400 g. This was comparable to control groups fed diets with either rapeseed oil or a mix of rapeseed and fish oil. Inclusion rates of up to 20% yeast biomass in the feed resulted in growth, feed conversion ratio and protein and energy retention comparable to the control diets for fish up to about 400 g. However, it was suggested that complete disruption of cells may be required to obtain full benefit of the biomass. Apart from its use as SCP, in a recent study the biomass of Y. lipolytica strain Po1g was acid hydrolysed and applied as a feedstock for ethanol production (Tsigie, Wu, Huynh, Ismadji, & Ju, 2013). Glucose was found to be the most abundant sugar in the defatted cell hydrolysate. The highest concentration (35.89 g L 1) of this sugar was obtained when hydrolysis was carried out at 120 °C. At lower temperatures (90 and 100 °C) the glucose yields

Y. lipolytica has been used to tackle food industry wastes. In general, oily wastes (olive mill, palm oil mill) and solid wastes (pineapple, fish) have been effectively treated (Bankar et al., 2009). In some cases, chemical oxygen demand (COD) has been reduced and in others, value-added products have been obtained. A summary of the outcome and strategies adapted for treating food-related wastes is depicted in Fig. 4. Some of the recent developments are listed below. Y. lipolytica was genetically manipulated to displaying lipRS (Rhizopus stolonifer) on the cell surface by using the flocculation functional domain of S. cerevisiae (Flo1p, encoded by FLO) as the protein anchor. This recombinant was effective in treating oily waste water (Song et al., 2011). In open activated sludge bioreactors, the recombinant resulted in the removal of 96.9% of oil and 97.6% of COD. A solid waste (crude coconut fat) was subjected to solid-state fermentation with Y. lipolytica (RO13). The effect of water activity and time on hydrolysis was determined (Parfene, Horincar, Tyagi, Malik, & Bahrim, 2013). As a result of fermentation, the hydrolysates contained high contents (70%) of lauric acid. The fatty acid containing hydrolysates displayed antimicrobial activity against some food-borne pathogens. Y. lipolytica could thus mediate the synthesis of coconut fat hydrolysates with bio-preservative effects. Agro-industrial wastes have been used to isolate microorganisms capable of producing lipases and citric acid (Mafakher et al., 2010). Two isolates of Y. lipolytica (M1 and M2) produced lipase levels of 11 and 8.3 units ml 1, respectively, on olive oil. The levels of citric acid obtained were 27 and 8 g L 1, respectively, in the fermentation medium used. The authors have suggested the use of agro-industrial and hydrocarbon wastes for the production of such value-added products. This would facilitate a significant reduction in substrate and waste treatment costs for industry. Although most of these processes are effective and bring about a considerable reduction in COD or result in the synthesis of a value-added product, they have been restricted to lab-scale levels. Further optimisation of parameters for scale-up is still required. An integrated approach in this regard could result in the development of viable processes for waste treatment in the future.

Fig. 4. Application of Yarrowia lipolytica in degradation of wastes.

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11. Conclusion This yeast with distinct physiological features and enzymatic systems has contributed to the food industry in a very diverse manner. A large number of laboratories around the globe have been applying this for several food-related applications. Its contribution to the development of flavours and aromas in meat and dairy products is phenomenal. Apart from its role in the ‘‘in situ’’ production of such compounds in food-stuff, this yeast has also been used for making of aroma compounds such as c-decalactones. Its association with foods at times is also detrimental and means of controlling growth have also been established. Organic acids and polyalcohols that have huge markets in the food industry have been manufactured by using this GRAS organism. In addition, the biomass of this relatively safe yeast has special properties (high unsaturated fatty acids and protein) that allow its use as food and feed in the form of SCO and SCP. While growing on different types of hydrophobic substrates, this yeast produces a variety of surfactants and emulsifiers which in turn can find applications in the food industry. Waste generated from food industries have been effectively degraded by different strains. The yeast has thus carved a niche for itself and has evolved as an efficient microbial factory playing diverse roles in the ever-expanding food industry. Acknowledgments SZ thanks University Grants Commission for funding under UPE Phase II. References Aguedo, M., Gomes, N., Garcia, E. E., Waché, Y., Mota, M., Teixeira, J. A., et al. (2005). Decalactone production by Yarrowia lipolytica under increased O2 transfer rates. Biotechnology Letters, 27, 1617–1621. Andrade, M. J., Rodr´ıguez, M., Sánchez, B., Aranda, E., & Córdoba, J. J. (2006). DNA typing methods for differentiation of yeasts related to dry-cured meat products. International Journal of Food Microbiology, 107, 48–58. Bankar, A. V., Kumar, A. R., & Zinjarde, S. S. (2009). Environmental and industrial applications of Yarrowia lipolytica. Applied Microbiology and Biotechnology, 84, 847–865. Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J. L., Molina-Jouve, C., & Nicaud, J. M. (2009a). Yarrowia lipolytica as a model for bio-oil production. Progress in Lipid Research, 48, 375–387. Beopoulos, A., Chardot, T., & Nicaud, J. M. (2009b). Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie, 91, 692–696. Braga, A., & Belo, I. (2013). Immobilization of Yarrowia lipolytica for aroma production from castor oil. Applied Biochemistry and Biotechnology, 169, 2202–2211. Carreira, A., Paloma, L., & Loureiro, V. (1998). Pigment producing yeasts involved in the brown surface discoloration of ewes’ cheese. International Journal of Food Microbiology, 41, 223–230. Celin´ska, E., Kubiak, P., Białas, W., Dziadas, M., & Grajek, W. (2013). Yarrowia lipolytica: the novel and promising 2-phenylethanol producer. Journal of Industrial Microbiology and Biotechnology, 40, 389–392. Chang, L., Chen, D., Chen, Y., Nicaud, J., Madzak, C., & Huang, Y. (2009). Production of functional c-linonlenic acid (GLA) by expression of fungal D12- and D6desaturase genes in the oleaginous yeast Yarrowia lipolytica. In J. F. Ching (Ed.), Biocatalysis and agricultural biotechnology (pp. 164–179). Boca Raton: CRC Press, Taylor & Francis Group. Chatzifragkou, A., Petrou, I., Gardeli, C., Komaitis, M., & Papanikolaou, S. (2011). Effect of Origanum vulgare L. essential oil on growth and lipid profile of Yarrowia lipolytica cultivated on glycerol-based media. Journal of the American Oil Chemists’ Society, 88, 1955–1964. Chebenˇová-Turcovská, V., Zˇenišová, K., Kuchta, T., Pangallo, D., & Brezˇná, B. (2011). Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese. International Journal of Food Microbiology, 150, 73–78. Chi, Z., Zheng, Y., Jiang, A., & Chen, S. (2011). Lipid production by culturing oleaginous yeast and algae with food waste and municipal wastewater in an integrated process. Applied Biochemistry and Biotechnology, 165, 442–453. Cui, W., Wang, Q., Zhang, F., Zhang, S. C., Chi, Z. M., & Madzak, C. (2011). Direct conversion of inulin into single cell protein by the engineered Yarrowia lipolytica carrying inulinase gene. Process Biochemistry, 46, 1442–1448. Curioni, P. M. G., & Bosset, J. O. (2002). Key odorants in various cheese types as determined by gas chromatography-olfactometry. International Dairy Journal, 12, 959–984.

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Food-related applications of Yarrowia lipolytica.

Yarrowia lipolytica is a non-pathogenic generally regarded as safe yeast. It displays unique physiological as well as biochemical properties that are ...
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