Biocontrol and Probiotic Function of Non-saccharomyces Yeasts: New Insights in Agri-Food Industry

1. Introduction

Traditional industrial attributes to Saccharomyces cerevisiae the most important role as biotechnological organism more involved in worldwide fermentation products such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. However, in the last 20 years, the world of research and in parallel the industrial one, started to re-evaluate the potential positive contribution of non-Saccharomyces yeasts (NSYs). They have found a pro-technological use in traditional fermentations, where can impart peculiar and distinctive characteristics to the product, but also in other applications as: biomedical or fundamental biological research, environmental biotechnology, heterologous protein production, biocontrol and food and feed sectors [1]. The utility of NSYs in food field is accelerating due to a wide number of metabolic abilities that confer aroma and structure to the final products but also due to their plasticity in assimilating different substrates. They generally show low fermentation yields, and higher sensitivity to ethanol stress if compared with S. cerevisiae. On the other hand. NSYs may display a great range of fermentation metabolites and end products in providing distinctive features [2].

In any case, the positive or negative contribution that each non-Saccharomyces species gives to the product strictly depends on the matrix and fermentation context (Figure1).

Figure 1. Identification of key strengths (S), weaknesses (W), opportunities (O) and threats (T) related to the use of non-Saccharomyces yeasts (NSYs) in fermented foods and beverages.

Figure 1. Identification of key strengths (S), weaknesses (W), opportunities (O) and threats (T) related to the use of non-Saccharomyces yeasts (NSYs) in fermented foods and beverages.

Until a few decades ago, in the traditional fermented beverages, NSYs were considered synonymous of spoilage yeasts. Differently, today many researchers clearly highlighted their potential for specie-specific or strain-specific traits, and it is unequivocally accepted that NSYs represent an important resource with economic repercussions in the market of fermented products. In the worldwide market, there is a growing interest in “non-conventional” yeast strains, another way to define NSYs, that can help generate the diversity and the complexity desired in diversified and aware consumers. Effectively, selected strains of NSYs contribute to set up innovative fermentations in food and beverages field, starting to winemaking, continuing in brewing where impart specific “bioflavouring” or functionalizing the beverages and reducing the final ethanol content. In addition, NSYs may be used as alternative starters to promote biodiversity and quality of other different fermented foods such as bakery products. In a recent paper, some selected non-Saccharomyces wine strains were assayed in the production of leavened doughs [3]. Several NSYs species as Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia kudriavzevii, Torulaspora delbruekii, and Zygotorulaspora florentina could be advantageous in the bakery industry, providing greater diversity than S. cerevisiae-based products, and may be useful in reducing or avoiding yeast intolerance.

Some NSYs including Kluyveromyces lactis, have been able to survive in the conditions that mimic the gastrointestinal environment and can form biofilms on an abiotic medium such as polystyrene. These yeast strains also exhibited highly hydrophilic cell wall surface properties and adhesion ability to intestinal Caco-2 cells, thus increasing their potential as probiotic strains. Effectively, in the pharmacological industry, the rational design of probiotics includes, in addition to their health claims, several other criteria: stability during manufacturing processes, viability during gastrointestinal transit, and functionality at the desired target site. Host safety and the management of their formulation and viability are also determining characteristics in the selection of probiotics. In relation to these multiple traits, the last decade has seen much evidence regarding NSYs as probiotic candidates. For example, a report by Li and colleagues [4] clarified the GRAS status of several Kluyveromyces species. In a general picture, yeast may offer more benefits than bacteria, as they are insensitive to antibiotics and can be easily used for the treatment of antibiotic-associated diarrhea.

The probiotic and biocontrol features as well as the general interest that revolves around the characteristics and the use of NSYs in the agri-food industry are the concerns of the present review. In this regard, an updated survey of the main recent publications focusing on this topic with scientific and market implications is prepared. The antimicrobial and probiotic attitudes is analyzed with particular attention to pointing out the recent findings, indicating the perspectives and emphasizing the fields of interest that are still poor investigated.

2. Antimicrobial activity of non-Saccharomyces yeasts (NSYs)

Already more than twenty years ago some authors [5,6] anticipated the potential use of NSYs during the first step of fermentation applied to improve the final flavor of wines. Today, knowledge has exponentially increased and the aromatic enhancement of wines due to the use of NSYs is only one of the many other features identified for their potential application. In view of the possible use in agri-food industry, it is a common prerogative that NSYs must possess specific traits to be selected. Indeed, after the general revaluation of the role of NSYs, several studies have focused the attention on multiple advantages particularly in winemaking. Among these, the antagonistic activity against undesired microorganisms is of paramount importance.

It has been widely demonstrated that some NSYs strains in winemaking can control spoilage yeasts or filamentous fungi both in vineyard and during the early stages of fermentation [7-9]. In this regard, spoilage species producing off-flavours or films in bulk wine such as Zygosaccharomyces rouxii, Kloeckera apiculata, Pichia spp., Candida spp. may be prevented by the use NSYs through active antimicrobial extracellular molecules released [10]. Effectively, the growing interest in reducing and rationalizing the use of SO₂ in winemaking has urged scientific investigations on the antimicrobial activity of NSYs as alternative to chemical conventional additives [11, 12]. About this, natural control strategies may involve the use of killer toxins (mycocins), antimicrobial peptides such as Lactoferricin B or volatile compounds produced by NSYs as biological strategy to counteract contamination [13].

2.1. Biological control

The term “biocontrol” related to the use of microorganisms as natural biological agents was defined as the reduction of pathogen or disease activities through organism or their molecules. In agri-food this concept is related to an alternative strategy to the use of chemical products, and the use of microorganisms with antagonist action against other microorganisms lowering the use of pesticides and boosting food quality and safety [14-16].

In recent years, to avoid the huge losses of fruit and vegetables due to pathogens, biological strategies have been studied as alternatives to products of a chemical nature. The addition of microorganisms as bio-protective agents or their antimicrobial products have already been identified as “bio-protection”. This practice is firstly used in agriculture and then in food industries for the control of fruits decay from post-harvest spoilage microorganisms, and the relative extension of their shelf life. This strategy consists in the inoculation of viable antagonistic microorganisms [bacteria, yeasts, or a mixture of them) or the addition of their antimicrobial products in complete or partial purified form, during, at the end or after the chain production [17-19]. Biological control based on the use of beneficial microorganisms is receiving increasing recognition, although the number of registered and approved marketed bioproducts containing bioactive yeasts or bacteria remains scarce. Czajkowski and collaborators [20] argues that this situation arises from objective difficulties encountered during registration and marketing, but also from problems of understanding the specific roles of each member of a consortium and their biological activity. Regarding to the application of NSYs in bio-protection strategies against grapevine trunk diseases in the vineyard [the fungal pathogen Botrytis cinerea causing bunch sour rot] has increased significantly in recent years [21,22]. In addition, there is a growing interest in the application of this approach on the protection against undesirable microorganisms during all wine production chain as well as during the conservation and maturation phases [23,24]. The renewed interest in biological control is due to the growing attention to the use of sulphites in food and particularly in wine. In this regard NSYs has been proposed as a possible effective natural alternative. Several recent studies have been carried out by using selected strains of M. pulcherrima and T. delbrueckii at the prefermentative stage in the red winemaking process [23,25-26]. Other studies focused the attention on the use of other selected strains of M. pulcherrima during cold clarification stage of Italian Verdicchio white variety underlining the double role of this yeast as biocontrol agent and wine aroma enhancer [27].

The antimicrobial activity of NSYs has been investigated also in other sectors since the re-evaluation of the use of these yeasts with biocontrol purposes has been extended across the food industry. Some applicative studies showed their effectiveness also in bread-making or cheesemaking. Valsaraj and colleagues [28] highlighted the biological load of some NSYs and how their toxic killer effects could be used in the food and beverage industry, maintaining food quality, and improving the safety in beer, cheese, or bread. They suggested that the biocontrol strategy of NSYs isolated from food and beverage naturally fermented, may be effective in suppressing wild yeast strains during another fermentation.

No less important is the involvement of the antimicrobial role of yeast strains in the medical field. For over thirty years, Polonelli and Morace [29] reported the killer phenomenon against bacteria, seeing the possibility of using these toxins to counteract the growing phenomenon of antibiotic resistance. In a more recent study, the yeasts D. hansenii, Pichia fermentans, Candida tropicalis and Wickerhamomyces anomalus have been shown to induce bacterial lysis [30]. Chen et al. [31] isolated mycocin-producing strains of Kluyveromyces marxianus and demonstrated that crude extracts were effective in preventing Escherichia coli disease in mice. In addition, the yeasts Kluyveromyces lactis and K. marxianus, isolated from cheese, were able to inhibit the growth of pathogenic microorganisms, such as Listeria monocytogenes and Candida albicans [32]. Finally, C. albicans strains isolated from children had inhibitory effects on strains of E. coli ATCC 25 922 and Staphylococcus aureus ATCC 25 923 [33].

2.2. Modalities of antimicrobial action of NSYs

The regulation and the control of the growth of undesired microorganisms could be exploited through different mechanisms as space and nutrient competition, cell to cell contact or antagonistic action mediated by antimicrobial compounds production as mycocins, small peptides, VOCs or extracellular vesicles.

2.2.1. Antagonistic action: competition of nutrients and space

The main mode of action of yeasts in biocontrol is the competition for space and nutrients [34]. Most organisms can starve pathogens or parasites through iron withdrawal [35]. Aureobasidium pullulans, possesses a siderophore identified as fusarinin C which has been shown to exhibit antibacterial activity [36,37]. Pulcherriminic acid is a cyclic dipeptide that complexes iron in M. pulcherrima. Studies have shown that pigment-deficient mutants of M. pulcherrima exhibited reduced or null antifungal activity, and iron deprivation of the fungal pathogen. This suggested that the production of this pigment is one of several mechanisms by which this yeast antagonizes plant pathogenic fungi [38]. However, mutants lacking the ability to synthesize pulcherriminic acid still strongly inhibited filamentous fungi, confirming that the antifungal activity was only due to iron deprivation. Therefore, the exact contribution of iron chelators to the yeast biocontrol activity remains to be clarified in detail. Another competition for nutrients was found in Saccharomycopsis schoenii that it is unable to assimilate sulfur, a specific feature of the genus Saccharomycopsis. In addition, phytopathogenic fungi and Trichoderma species exhibited a similar phenomenon, which may indicate that methionine is an important target for such organisms and has been hotly contested [39]. Pioneering experiments aimed at evaluating the suitability of an easily transformable Pichia (formerly Ogataea) angusta haploid strain to identify biocontrol-minus mutant clones: while the wild-type strain proved effective in reducing brown rot lesion caused by Monilinia fructicola on apple fruit, its derivate leucine-auxotrophic mutant L1 had no significant effect in controlling the pathogen. The addition of exogenous leucine fully restored the biocontrol capability of mutant L1, whereas a leucine stand-alone treatment showed no significant biocontrol effect [40].

Another strategy to compete for the space, is the formation of biofilm. Biofilms are microbial communities consisting of a single or more species and are considered virulence factors for pathogenic microbes [41,42]. The development of a yeast biofilm begins with the adhesion of single cells to a surface and usually involves cell wall modifications, secretion of an extracellular matrix, and often the formation of hyphae or pseudo-hyphae [43]. In yeasts to be used in biocontrol activity, biofilm formation in wounds is now considered an important mode of action even though the molecular basis of the process and the composition of different biofilms have only been studied in detail for P. fermentans. Biofilm formation of P. fermentans in apple wounds protects against post-harvest disease, while on peaches it changes from yeast-like to hyphal growth form and causes rapid decay of inoculated fruit in the absence of a plant pathogen [44]. Biofilm formation has been demonstrated besides that S. cerevisiae also in P. kudriavzevii, W. anomalus and M. pulcherrima [45-48].

The saprophytic yeast-like fungus A. pullulans has been well documented for over 60 years in microbiological literature for its ubiquitarian presence in soil, water, rock surfaces, in both cold and warm areas. A lot of A. pullulans strains are known to produce a wide range of natural antimicrobials useful for biocontrol applications against plant pathogens mediated by nutrient and space competition and/or VOC production (see paragraph 2.1.6). A. pullulans may can be used at both vineyards, for the management of grey mold disease caused by B. cinerea, and winery levels, through the synthesis of antifungal compounds, providing a versatile tool for the viticulturist/farmer as well as for the oenologist to combat problems in the field and create a high-quality wine.

2.2.2. Mycocins

Mycocins (killer toxins) are the most investigated yeast antimicrobial compounds. Since Bevan and Makower [49] discovered the killer phenomenon in S. cerevisiae, several other yeast species have been found to produce a toxic proteinaceous factor that kills sensitive strains [50,51]. Several potential applications for the killer phenomenon have been suggested in food industry to control spoilage yeasts in the preservation of food and beverages [52,53].

The mycocin Kpkt produced by Tetrapisispora phaffii was first described as anti-spoilage yeasts [52]. Kpkt acts through a specific β-glucanase activity causing irreversible modifications on the cell wall structure and it is codified by TpBGL2 chromosomal gene [17, 54,55]. The recombinant toxin (rKpkt) was recently obtained by transferring the Kpkt coding gene in Komagataella phaffii (formerly Pichia pastoris) [56]. The recombinant rKpkt, when expressed in K. phaffii, displayed a wider spectrum of action than its native yeast [57], reinforcing the idea of the possible application of mycocins in the food and beverages industries. T. phaffii was used in mixed fermentation at pre-fermentative stage to control wild yeasts such as Hanseniaspora, Zygosaccharomyces and Saccharomycodes in the place of sulfur dioxide [11].

Several works have been focused on the study of NSYs able to counteract the development of Brettanomyces spp., a relevant dangerous yeast in winemaking [58]. In this context, Pikt and Kwkt mycocins, produced by W. anomalus (formerly Pichia anomala) and Kluyveromyces wickerhamii, respectively [54], able to counteract Dekkera/Brettanomyces. Pikt is an ubiquitin-like protein of about 8 kDa able to interact with β-1,6-glucan of cell wall of sensitive yeasts [59] while Kwkt, a protein of about 72 kDa of molecular mass, without any glycosyl residue [11] and β-1,6-glucosidase activity that seems to be in involved in to act blocking the cell cycle function of sensitive yeasts [12]. Cytofluorimetric evaluation showed that both Pikt and Kwkt caused irreversible death of this yeast and in a different way from sulphur dioxide that induced a viable but non-cultivable (VBNC) state of Brettanomyces with consequence recovery of yeasts when fresh medium was replaced [12]. Another P. membranifaciens strain showed a killer action producing two mycocins denominated PMKT and PMKT2. PMKT binds linear (1→6)-β-d-glucans in the cell wall and Cwp2p plasma membrane receptor of sensitive yeasts leading alterations in ionic exchange via plasma membrane [60]. PMKT2, a protein with an apparent molecular mass of 30 kDa, binds mannoproteins and induces cell cycle blockage an early S-phase of sensitive yeasts and stimulates markers of cellular apoptosis such as the cytochrome c release, DNA strand breaks, metacaspase activation and production of reactive oxygen species at low dose [61]. The killer activity of P. membranifaciens was exploited in wine making in mixed fermentation with S. cerevisiae and B. bruxellensis [inoculum ratio of 1:1]. P. membranifaciens inhibited B. bruxellensis growth without any effects on the fermentation activity of S. cerevisiae.

Other mycocins (CpKT1 and CpKT2) active against B. bruxellensis are produced by Candida pyralidae. Both mycocins were active and stable at pH 3.5–4.5 and with the general conditions of winemaking environment [62]. Both mycocin CpKT1 and C. pyralidae viable yeasts were used in mixed fermentation in red grape juice containing B. bruxellensis determining a decrease in spoilage yeast concentration [63].

Also, a strain of W. anomalus was proposed as a biocontrol agent against Brettanomyces/Dekkera spp. [18]. The killer activity of W. anomalus is expressed through the release of KTCf20, a mycocin. This mycocin was able to counteract the growth of different spoilage yeasts such as Brettanomyces/Dekkera, Pichia guilliermondii and P. membranifaciens. Moreover, they showed that W. anomalus in mixed fermentation did not negatively affect S. cerevisiae strains. Another mycocin named WA18 and active against B. bruxellensis is produced by an autochthonous W. anomalus strain isolated from soil pit, exhibited 99 % identity with UDP-glycosyltransferase protein [64]. In accordance with de Ullivarri and co-workers [18] the compatibility of this W. anomalus strain in mixed fermentation with S. cerevisiae yeast was confirmed. Another killer strain belonging to W. anomalus has been investigated for its wide potential of antibacterial activity against numerous human pathogenic agents [65]. Interest in these antibacterial mycocins was revealed by Muccilli and Restuccia, [66] that highlighted the potential use against pathogens, resistant to conventional antibiotics, such as Staphylococcus aureus.

In a recent work, a comparative evaluation of formulates (semi-purified and lyophilized broth) of three well characterized mycocins were assessed against B. bruxellensis. The absence of dangerous effects toward human epithelial cells, opening the way for their possible commercial application [67]. Another NSYs that shows antimicrobial activity is T. delbrueckii where a mycocin exhibited glucanase and chitinase activities, it is stable in wine environmental condition and it is active against B. bruxellensis, and other potential wine spoilage yeasts. Moreover, Ramírez et al. [68] isolated and selected T. delbrueckii strain that produces a mycocin [Kbarr-1]. This toxin is encoded by a dsRNA, TdV-Mbarr-1, structurally like M dsRNAs of S. cerevisiae, both seem to be evolutionarily related [69].

2.2.3. Antimicrobial peptides (AMPs)

Some peptides produced by yeasts have shown antimicrobial effects against several grape-must/wine contaminating yeasts. In general, these peptides show lengths of up to 100 amino acids, sorted into variable sequences and the mode of action involves the disruption of the cell wall in sensitive strains. For example, small peptides with molecular mass below 5 kDa produced by Candida intermedia have shown greater antimicrobial specific effects against B. bruxellensis [70]. In addition, it was demonstrated the antibacterial activity of the same strain against Escherichia coli, L. monocytogenes and S. typhimurium [71]. Similar observations have been reported by Younis et al. [72], where three isolates of C. intermedia from raw milk and fruit yoghurt showed antimicrobial activity against E. coli, S. aureus and Pseudomonas aeruginosa.

Another mechanism of interaction in mixed fermentation is the possible involvement of extracellular vesicles (EVs). In this regard, a recent work on exo- proteome of EV-enriched fractions in pure and mixed fermentation with six different species of NSYs and S. cerevisiae showed a wide diversity of proteins secreted indicating the presence of interactions and the possible involvement of EVs [73]. The EV-enriched fractions from different species such as S. cerevisiae, T. delbrueckii and Lachancea thermotolerans showed enrichment in glycolytic enzymes and cell-wall-related proteins, particularly the enzyme exo-1,3-β-glucanase. However, this protein was not involved in the here-observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions.

2.2.4. Secreted enzymes

Some enzymes secreted may be involved in biocontrol action. Indeed, the secretion of enzymes degrading cellular components such as chitinases, glucanases, or proteases is a common feature in all kinds of host–pathogen interactions and has been intensively studied. Chitinolytic enzymes allow degrading fungal cell walls [74]. Yeasts belonging to genera Aureobasidium, Candida, Debaryomyces, Metschnikowia, Meyerozyma, Pichia, Saccharomyces, Tilletiopsis, Wickerhamomyces and Saccharomycopsis, exhibited this enzymatic activity [16,39]. Chitinases from sources i.e. fungi and filamentous bacteria have demonstrated biocontrol activity against plant pathogenic fungi and chitinases are extensively studied as potential biopesticides, targets for resistance breeding or as transgenes in genetically modified plants. Chitinases, probably in indirect manner, influence biocontrol activity because Chito-oligosaccharides (CHOS) resulting by of chitin degradation, are potent inducers of plant immune responses [51].

Glucans are major cell wall components in fungi and exoglucanases are involved in cell wall modification, cell adhesion and resistance to mycocins [75]. A 1,3-β-glucanase from Candida oleophila was the first gene cloned in this organism and overexpression or deletion of this gene did not significantly affect Penicillium digitatum spore germination, but subsequent studies have documented a reduced inhibitory activity of the β-exoglucanase deletion mutant compared to the wild type and overexpressing strain [in vitro and in fruit], thus demonstrating the involvement of glucanases in the biocontrol activity of yeast [76].

In W. anomalus the deletion of two exo-β-glucanases (PaEXG1 and PaEXG2) significantly reduced the fruit biocontrol activity against B. cinerea [Friel et al. 2007], while the single deletion of PaEXG2 did not reduce biocontrol performance. Exoglucanase activity was also detected in several biocontrol yeasts and was linked to antagonist activity, but without demonstrating a causal involvement.

It has long been proven [77] that in Rhodotorula glutinis and Cryptococcus laurentii, β-1,3-glucanase activity did not correlate with their respective inhibitory activity against B. cinerea. The pathogenicity of yeast species belonging to Candida, Cryptococcus or Malassezia is related to lipase activity. Several studies have also correlated the role of lipases with the biocontrol action of fungi and bacteria against plate diseases. For this reason, the lipolytic activity of yeasts may represent an aspect to be investigated in relation to biological control [78].

The alkaline serine protease Alp5 of A. pullulans reduced spore germination and germ tube length of Penicillium expansum, B. cinerea, M. fructicola and Alternaria alternata in vitro and showed a concentration-dependent inhibitory effect on these pathogens on the apple tree [79]. Protease activity has been reported but not confirmed or investigated in the genera Metschnikowia, Pichia, and Wickerhamomyces, but has not been further studied or confirmed.

2.2.5. Mycoparasitism

Mycoparasitism is little studied in yeasts, some studies have shown that P. guilliermondii adheres to the hyphae of B. cinerea and causes the collapse of the hyphae, presumably due to the secretion of hydrolytic enzymes such as glucanases [80].

Species belonging to the genus Saccharomycopsis have been studied against biocontrol of several clinically relevant Penicillium species and yeasts [39].

2.2.6. Volatile organic compounds (VOCs)

Volatile organic compounds (VOCs) are the most relevant metabolites that show a biocontrol action. These metabolites are molecules <300 Da with a low solubility in water and a high vapor pressure and include molecules such as hydrocarbons, alcohols, thioalcohols, aldehydes, ketones, thioesters, cyclohexenes, heterocyclic compounds, phenols, and benzene derivatives. Volatiloma is specific for each yeast species as well as the spectrum of action against pathogenic microorganisms [81,82]. VOCs are species specific and are produced by fungi, bacteria and yeast during and their primary and secondary metabolism, limiting the growth of other microorganisms. The chemical composition of VOCs strongly depends on the environment and the pathogen being antagonized. The chemical composition includes alcohols, aldehydes, cyclohexenes, benzene derivatives, heterocyclic compounds, hydrocarbons, ketones, phenols, thioalcohols and thioesters. VOCs are produced by several yeasts species to reduce the growth of pathogens moulds. In this context, Di Francesco and co- workers [83] have demonstrated both in vitro and in vivo that the VOCs produced by A. pullulans reduced the growth and the infection of B. cinerea, Colletotrichum acutatum, P. expansum, P. digitatum and P. italicum.

Also VOCs formed by NSYs W. anomalus, M. pulcherrima and A. pullulans as well as S. cerevisiae showed a biocontrol action against B. cinerea on table grape berries [84]. Selected strains of Cyberlindnera jadinii, Candida friedrichii, C. intermedia, and L. thermotolerans inhibited the formation of both mycelial growth and ochratoxin A in Aspergillus carbonarius and Aspergillus ochraceus identifying β-phenyl ethanol as the active compound [85,86]. The VOCs by W. anomalus, preventing spore germination, mycelial growth and toxin production of Aspergillus flavus [87]. Similarly, VOCs released by Candida sake reduced the incidence of apple rot caused by P. expansum and B. cinerea [88]. The inhibitory activity of Sporidiobolus pararoseus on spore germination and mycelial growth of B. cinerea was mainly attributed to 2-ethyl-1-hexanol, whereas C. intermedia produced 1,3,5,7-cyclooctatetraene, 3-methyl1-butanol, 2-nonanone, and phenylethyl alcohol as the major components of its volatilome during the interaction with this pathogen. VOCs released by W. anomalus, Pichia kluyveri, and H. uvarum inhibited A. ochraceus growth and ochratoxin A production during the fermentation process of coffee [89].

Recently, Ruiz-Moyano et al., [90] screened yeasts isolated from figs producing antifungal VOCs. Eleven out of 34 yeasts, belonging to A. pullulans, Filobasidium oeirense, H. uvarum and Hanseniaspora opuntiae showed a reduction in the growth of B. cinerea correlated with the production of 10 volatile compounds: two acids [acetic acid and octanoic acid], 7 esters [ethyl propionate, n-propyl acetate, isobutyl acetate, 2-methylbutyl acetate, furfuryl acetate, phenylmethyl acetate, 2-phenylethyl acetate] and a ketone (heptane-2-one).

A strain Torulaspora spp. showed biological action against Alternaria arborescens causal agent of tomato fruits decay. The mechanisms involved were both the production of volatile compounds and the competition for nutrients [91]. In a screening of 147 yeasts and yeast-like fungi five strains belonging to Anthracocystis sp., two strains of Aureobasidium sp. Rhodotorula sp. and Solicococcus keelungensis produced VOCs and were active against Aspergillus flavus that produce aflatoxin B1. Alcohols, alkenes, aromatics, esters and furans. 2-phenyl ethanol and methyl benzene acetate were the most abundant compounds generated by Aureobasidium sp. On the other hand, 2-methyl-1-butanol and 3-methyl-1-butanol were significant compounds produced by the other three genera [92]. In another recent work, Candida pseudolambica showed a significant reduction such as 41.2 % of the disease incidence of gray mold in peach inoculated with B. cinerea [93]. Several modalities of action were identified: the growth dynamics, VOCs effects, parasitism, and biofilm formation. VOCs released from C. pseudolambica inhibited the mycelial growth and conidia germination of B. cinerea. Fourteen VOCs were identified with the main compounds being 3-methyl-1-butanol and 2-phenylethanol, which made up 85.90 % of the relative peak area.

2.3. Registered formulations of biocontrol NSYs species

Nowadays, there are few formulations based on yeasts suitable for plant protection that are registered and marketed such as C. oleophila, A. pullulans, Metschnikowia fructicola, C. albidus, and S. cerevisiae. However, selected NSYs are also used in postharvest to extend the shelf life of vegetable or in winery to limit the common use of conventional chemical antimicrobials, such as sulfur dioxide. For example, strains of M. pulcherrima and T. delbrueckii are commercialized as a natural biological control agents with potential to partially or fully replace sulphites. A brief description of the NSYs strains used in registred formulations present in the market is reported.

2.3.1. Candida oleophila

Species of the genus Candida strongly inhibit plant pathogens are: Candida diverse [94], Candida ernobii [95], Candida guillermondii [96], C. oleophila [37], Candida saitoana, C. sake [88,97], or Candida subhashii. These species are biocontrol agents against molds and post-harvest diseases of hazelnut and citrus fruits.

C. oleophila was the first yeast to be developed into a commercial plant protection agent and the basis for its antifungal activity several different mechanisms of action have been demonstrated. In addition to competition for nutrients and space, studies of several Candida species have identified hydrolytic enzymes such as proteases, chitinases and glucanases, as well as volatile compounds, which have been implicated in antifungal activity [98].

Furthermore, bioflm formation, high osmotolerance, induction of resistance in the plant/fruit, and direct parasitism of hyphae were shown to contribute to the biocontrol activity of Candida species [99]. To overcome the inconsistent performance of the initial Candida-based biocontrol products combinations with fungicides, different buffers [e.g., calcium chloride, bicarbonate], chitosan, or lysozyme were studied [100]. The C. oleophila strains I-182 and strains O have been developed into the biocontrol products Aspire® and Nexy®, respectively. The latter was the first biocontrol yeast to be registered against a postharvest disease [99] and C. oleophila strain O has been approved as a plant protection agent in Europe in 2015 (European Commission Health & Consumers Directorate-General 2013; European Food Safety Authority, EFSA) [101].

2.3.2. Aureobasidium pullulans

The biocontrol activity of A. pullulans has been documented in several different strains, but only DSM 14940 and DSM 14941 are registered, in admixture Botector-New (Manica), as active ingredients of plant protection products against the disease caused by the bacterium Erwinia amylovora and post-harvest diseases (European Food Safety Authority EFSA, 2013) [102]. These two strains of A. pullulans were selected based on their strong inhibition against E. amylovora. The two strains were formulated into the Blossom-Protect® product and tested over several years in different sites under field conditions [103] and also registered for post-harvest apple tree disease control as a Boni-Protect® product [104]. As with many other yeasts with a biocontrol action, the mode of action of A. pullulans involves competition for space and nutrients, but enzymatic activities such as proteases, chitinases or secreted molecules may also be involved (see above). Specific metabolites or enzymes and their contribution to the biocontrol activity of DSM 14940 and DSM 14941 have not been identified.

2.3.3. Metschnikowia spp.

M. fructicola and M. pulcherrima are the most studied yeast species regarding biocontrol action. Indeed, they can inhibit a range of postharvest and plant rot diseases, [84, 105,106]. The antifungal activity of Metschnikowia species is mediated by a range of mechanisms that involve competition for nutrients (e.g.; amino acids, iron), secretion of glucanases and chitinases, and the production of volatile organic compounds [38,74,79]. Originally, M. fructicola was isolated and discovered in Israel and developed and registered as a biocontrol product to prevent postharvest diseases, particularly in sweet potato and carrot [107]. M. fructicola has also been patented as an antagonist of plant pathogenic microorganisms [108].

2.3.4. Cryptococcus spp.

Basidiomycetes belonging to the genus Cryptococcus are isolated from water sources, soil and decomposing plant material. Criptococcus albidus, C. laurentii and Cryptococcus flavus strains have been shown to have a post-harvest protective action in peach, cherry, strawberry, tomato, citrus and pome fruit from post-harvest decay [109-111]. C. albidus was used in the preparation of Yieldplus® (Anchor Bio-Technologies in South Africa) against B. cinerea, P. expansum of pome and citrus friut and B. cinerea during post-harvest cold storage of strawberries [112]. The modality of action of this yeast is related to competition of nutrient and space. Moreover, C.albidus exhibits glucanase, chitinase and protease activity and produces unidentifed volatile compounds that inhibit fungal growth and can display killer activity against C. glabrata [113]. However, none of these mechanisms have been directly linked to the inhibitory activity of the target plant pathogens.

2.3.5. Torulaspora delbrueckii and Metschnikowia pulcherrima

A strain of M. pulcherrima registered as LEVEL2 INITIA™ is a biological tool selected with purpose of preserving the aromatic potential and protecting from oxidative phenomena in the pre-fermentation phases, limiting the use of SO₂. LEVEL2 GUARDIA™ is another strain of M. pulcherrima that added at very early stages of the production process, colonizes and grows rapidly in the medium ensuring prompt and efficient bioprotection of red musts. The mode of action is based on the ability to secrete high concentrations of pulcherriminic acid able of chelating the iron present in the medium which makes the environment unsuitable for the growth of the contaminating microbiota, facilitating the alcoholic fermentation driven by S. cerevisiae sequentially inoculated. A multistarter formulated based on two strains of the species T. delbrueckii and M. pulcherrima are marketed under the name of ZYMAFLORE® ÉGIDETDMP to limit the prevalence of unwanted microorganisms on the surface of the harvesting equipment in contact with the grapes.

2.4. Application of NSYs in biopackaging

In recent years, the need to reduce the production and therefore the disposal of plastic and other non-ecofriendly materials has led the world of research towards the development and application of natural systems as green packaging in agri-food industry [114]. The prefix bio- associated to packaging has a double meaning: biodegradable and/or coming from natural materials. In any case, the objective of food bio packaging is to protect and preserve food products in a sustainable way. Indeed, appropriate packaging has to be capable of maintaining food sensorial and nutritional quality and guaranteeing food safety both in international and national markets, for long time and even in complex conditions [115].

The observation that contaminating microorganisms in food can be controlled during their shelf-life by antimicrobial compounds release from packaging has led to focused efforts to active bio packaging systems that incorporate natural antimicrobial compounds. Castelan and coworkers [116] named this bioactive antimicrobial molecules NACs and reviewed the principal natural sources suitable for this purpose. The NACs are mainly represented by minerals, fruit extracts, essential oils, animal shells, but also some microorganisms or their active molecules. Regarding to this last group, there is a growing interest in the development of antimicrobial packaging materials containing natural antimicrobial agents [117]. The same interest has been driven by consumer concerns about health-related issues, such as the use of synthetic antimicrobial agents. Incorporating synthetic antimicrobial agents directly into foods can effectively inhibit the growth and survival of various microorganisms, but consumers demand minimally processed, preservative-free food products with a longer shelf life. Active antimicrobial bio packaging provides a suitable natural alternative to chemical additives as advanced barrier able to prevent the develop pf food pathogens.

Since 2012, many articles have appeared regarding the use of antimicrobials of bacterial origin [118] such as bacteriocins synthesized by Archaebacteria, Lactococcus, Streptococcus, and Lactobacillus. For example, the incorporation of nisin and pediocin produced by Lactococcus lactis and Pediococcus acidilactici, respectively, into polymer matrices were applied in bio packaging, exploiting their bactericidal and bacteriostatic efficacy against L. monocytogenes [119,120] for mozzarella preservation.

The effectiveness of yeast in preventing food decay has already been established over the last decades, particularly in postharvest treatments of fruits to control mould decay [21,121]. Recently, Zhang et al., [16] reviewed the recent literature about the antagonistic role of yeasts in food industry, highlighting their suitable role among various microbial antagonists. Indeed, yeast and yeast-like fungi as biocontrol tools against pathogens are environmentally friendly, possess greater stress tolerance then bacteria, and can potentially be genetically improved. Moreover, the utilization of yeasts is generally considered safe, and easily acceptable by market, and growing antagonistic yeasts with excellent biocontrol performance have been developed and registered as commercial products.

The incorporation of yeast into the packaging material could impart antimicrobial activity as well as enhancing its nutritional value and serving as the probiotic. For instance, the yeast Meyerozyma guilliermondii is non-pathogenic and demonstrates good antimicrobial activity and it is a rich source of vitamins and proteins [122]. In 2013, Coda and collegues [123] demonstrated the efficacy of M. guilliermondii strain LCF1353 for the effective antifungal activity on long-term storage in wheat bread. Therefore, the direct incorporation of M. guilliermondii or its metabolites to the packaging material could contribute to increasing the shelf life of food. On this, Atta at al., [124] published a study aimed to develop edible and bioactive food packaging films comprising yeast incorporated into bacterial cellulose in conjunction with carboxymethyl cellulose and glycerol to extend the shelf life of packaged food materials. M. guilliermondii strain MT502203.1 and Gluconacetobacter xylinus strain ATCC53582, biofilms were developed ex situ then incorporated into the fibrous cellulose matrix. The findings of this study indicate that the developed biofilm could be used as edible packaging material with high nutritional value and distinctive properties related to the film component, which would provide protection and extended shelf life of foods.

Recently, Guimaraes and colleagues [125] have provided a comprehensive review of the use of edible films and coatings for the incorporation of living microorganisms, aiming at the biopreservation and probiotic capacity of food products. They summarized six benefits and advantages of edible films and coatings containing living microorganisms: physical barrier to protect food, antimicrobial and/or probiotic add value, protection against mechanical damages, increasement of shelf-life and green approach.

The ability of two bio-based films of sodium alginate and locust bean gum to deliver the antimicrobial yeasts W. anomalus cells for the growth control of P. digitatum was investigated [126]. The authors confirmed the efficacy of yeasts incorporation in preserving the postharvest quality of 'Valencia' oranges artificially infected, where the reduction of green mold were more than 73% after 13 days of shelf-life. The edible coating also represents a strategy for conveying probiotic microorganisms and making them readily available in foods which would therefore have an improved nutraceutical value. Tripathi and Giri, [127] catalogued about 500 probiotic food products introduced in the market during the last years mainly yoghurt and other fermented dairy products, such as cheese, ice cream and fermented children formula. Since the criteria for a microbial strain to be used as a probiotic includes the ability of remaining viable at high cell count throughout the manufacture and storage of the product, conveying probiotic strains in non-fermented foods through edible coatings could represent a cutting-edge strategy. The first study of edible coating with yeast dates back to 1994 and concerns the use of C. oleophila in cellulose films to extend the storage of table grapefruits [128]. When C. oleophila was incorporated in methylcellulose or hydroxypropylcellulose the storage time was increased 11 days moreover, C. oleophila was not adversely affected by the incorporation of 0.15% potassium sorbate, the maximum concentration allowed in food products. In the following years, C. guillermondii and Debaryomyces spp. were applied in formulation to extend the shelflife of oranges. Sharma et al. [129] used chitosan films containing Candida utilis for the control of decay in tomatoes caused by Alternaria alternata and Geotrichum candidum, while Fan et al. [130] tested the incorporation of C. laurentii in alginate-based coatings to extend the shelf life of strawberries. These last results showed that the addition of C. laurentii had an antagonistic effect and inhibited the growth of molds as well as maintaining the overall qualities of the strawberries during prolonged storage. Furthermore, sodium alginate films incorporated with C. laurentii did not show any significant effect on color parameters or anthocyanin concentrations in strawberries, but were able to maintain fruit firmness during storage. More recently, Yinzhe and Shaoying [131] investigated the effect of carboxymethylcellulose and alginate-based coatings incorporating brewer yeast on grape preservation highlighting an effective reduced decay compared with uncoated control and an increase of general quality of the grape since, coatings decreased weight loss. Again, Parafati et al., [132] tested the survival and biocontrol ability of W. anomalus, M. pulcherrima, and A. pullulans in coated mandarins, where the incorporation of yeasts reduced the incidence of P. digitatum in mandarins.

Although many positive effects of active food packaging have been proven by research, there are still few applications. Theoretically, regulation has been well stablished in EU, where active packaging are considered as active materials and articles; they are framed in the Regulation 1935/2004/EC (European Commission, 2004) and Regulation 450/2009/EC (European Commission, 2009), however bioackaging materials are still limited in commercial applications if compared with traditional packaging applications reported in the literature. The main principle for the successful packaging of fresh and fresh-cut produce specific, since each produce varies and hence the requirements for packaging and storage vary.

3. NSYs as probiotic yeasts

Probiotics are defined, by an Expert Panel in 2001, as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”, according with the international organizations FAO (Food and Agriculture Organization of the United Nations) and WHO (World Health Organization). Since then, many researchers focused the attention on this topic, going from about 750 papers indexed prior to 2001, to more than 20,000 until early 2019 [133]. However, most of the papers use the general term “probiotics” and only a minor part (< 3 %) use the term probiotic yeasts [134]. Most of the studies conducted so far have been almost exclusively focused on the bacteria population as probiotics and only recently the scenario is opening to yeasts as new probiotics. Effectively, the subject of probiotic and potentially probiotic yeasts has been developing and arising potential for new probiotic products with novel properties, which are not offered by bacteria-based probiotics available on the current market, showing a lot of technological usefull traits (Figure 2).

Initial in vitro screening is necessary to propose possible new probiotic candidates. Mainly, the screening need to include the ability to survive in the host. Indeed generally probiotics are taken orally reaching the gastrointestinal tract, thus it is important to evaluate their resistance to gastrointestinal conditions (the presence of digestive enzymes, gastric and pancreatic juices, bile salts, pH and body temperature of host). To these conditions are added the ability to colonize intestinal mucosal surfaces [correlated to auto- and coaggregation capability and surface hydrophobicity], the interaction with the existent gut microbiota (antimicrobial activity toward pathogenic microorganisms) and the exibition of the antibiotic resistance [135,136]. No less important is also the assessment of the safety of the new candidate against the host: they must not produce toxins, be pathogenic or exhibit hemolytic and DNase activities and gelatinase production [137-139]. Lastly, the evaluation of technological features that include the ability to easily cultivation and biomass production, and resistance to preservation procedures such as lyophilisation, genetic stability and no deterioration, are also important [134].

Actually, the only recognized and commercialized yeast for the human applications, which fully satisfies the presence of probiotic characteristics, is S. cerevisiae var. boulardii [140]. According to the current literature and Index Fungrum, 2021, it is closely related to the S. cerevisiae wine strains [141], therefore currently it has been re-classified as S. cerevisiae var. boulardii, although it exhibits some unique properties. This yeast is commonly used as biotherapeutic in humans, especially for the treatment of gastrointestinal tract disorders [142] and, more recently, as starting-culture to produce functional and probiotic foods/beverages characterized by health-promoting properties [143].

The recent growing interest for functional and probiotic foods and beverages has prompted scientists to focalize the research on the selection of new strains with both technological and innovative traits such as functional and probiotic properties and bioactive compound production [144,145]. Yeasts are widespread in nature and their relatively easy availability and cultivation make them promising probiotic candidates, especially NSYs, still poorly investigated. However, a recent report from Li et al. [146], described the GRAS status of some NSYs, necessary prerequisite for their evaluation as probiotics. Generally, these yeasts are isolated from fermented foods and drinks, vegetables, fruit juices, fruits, dairy products, industrial food waste and grains [147,148]. The isolation of these yeasts from human-related food matrices could represent a way to guarantee their safety.

Un-anthropized natural environments and spontaneous processed foods could represent valid sources for the isolation of new strains with probiotic features. Thirteen out of 180 yeasts isolated from moss on oak, beech tree bark, wine, wineries and grapes, sugar cane juice and papaya leaves revealed the potential probiotic and antimicrobial activity against some pathogenic yeasts. The isolated yeasts were belonging to L. thermotolerans, Metschnikowia ziziphicola, S. cerevisiae and T. delbrueckii species [149]. Findings highlighted their promising probiotic characteristics, although these aptitudes were strictly strain-dependent [150,151]. Also naturally fermented table olives represented a source of potential probiotic yeasts: five NSYs belonging to Candida orthopsilosis, C. tropicalis, D. hansenii, P. guillermondii and Meyerozyma carribica showed resistance to 37 °C, pH 2.0, in presence of bile salts and antimicrobial activity against Salmonella enteritidis [136].

Several studies described similar properties in other different NSYs including H. osmophila, H. guilliermondii, H. uvarum, H. osmopholia, K. thermotolerans, K. marxianus, P. membranifaciens, P. kudriavzevii, Pichia masmurika, Pichia occidentalis, Candida quercitresa, C. intermedia, C. sake, T. delbrueckii, W. anomalus and M. guillermondii [140]. They were unaffected by conditions that mimicking a dynamic gastrointestinal system and, thus, could be proposed for new commercial functional foods [152,153]. In a study conducted to compare functional and biotechnological characteristics of potential probiotic yeast strains belonging to Saccharomyces and NSYs (genera Pichia, Lachancea, Hanseniaspora, Candida, and Zygosaccharomyces) it was reported that in aerobic conditions all NSYs showed higher capacity for adhesion to Caco-2/TC7 intestine-derived cell line and the higher assimilation for nine prebiotics (cellulose, inulin, melibiose, raffinose, xylan, trehalose, pectin, cellobiose and beta-glucans) than Saccharomyces strains. Furthermore, considering other properties, the presence of digestive enzymes, antioxidant activity, antifungal resistance and vitality after sonication treatment two strains of H. osmophila and one of L. thermotolerans were identified as health-promoting probiotics [154]. The evaluation of non-pathogenicity and safe for consumption aspectes excluded the strain H. osmophila 1094 for the high production of biogenic amines, especially tyramine [152].

In 2018 it was firstly described a strain of Cryptococcus (95% of ITS sequence similarity with C. albidus) from the Red Sea with probiotic traits: tolerance to low pH and gastric juice, resistance to bile salts, hydrophobicity, antimicrobial activity and ability to degrade cholesterol [155]. Other NSYs with probiotic properties was the strain of P. pastoris X-33 wild type that showed the ability to survive the stresses of the gastrointestinal tract and caused no lesions when provided to mice thorough the diet exibiting a high antibacterial activity against S. enterica serovar typhimurium[156]. Mice infected by a virulent strain of S. typhimurium showed high survival when supplemented with P. pastoris by gavage or via diet. These results concluded that the yeast P. pastoris X-33 has probiotic properties with considerable antibacterial activity against S. typhimurium. Also, P. kluyveri was described as potential probiotic yeast proposed in co-fermentation with S. cerevisiae strains and Lactobacillus paracasei to obtain a novel, functional fermented maize-based beverage. Similarly, Canonico and co-workers [157] proposed the application of wild non-Saccharomyces potential probiotic yeasts to produce a premium craft beer Kazachstania unispora, isolated from artisanal sourdough, capable of produce a craft beer with low ethanol content and distinctive aromatic notes. Still, P. kudriavzevii DCNa1 and Wickerhamomyces subpelliculosus DFNb6 were proposed to ferment cornelian cherry fruits puree with the aim to obtain a functional beverage characterized by low ethanol content, high amounts of alcohols and esters, and low levels of aldehydes and alkanes. Moreover, these yeasts remained alive in a dose recommended for a probiotic beverage after 21 days of cold storage and after in vitro simulated digestion system. They were able to modulate the intestinal microbiota composition, when they were ingested through this beverage, in an in vitro gastrointestinal simulator [158].

Retracing all the aspects here reported, a general reflection emerges: the long history of yeasts in science, their useful contributions to research and above all to human life, as well as the broad prospects for their use in new fields of application, lead us to assume that they will continue to accompany scientists for many years, contributing to the improvement of human life, as they have done since the beginning. The recently highlighted potential of NSYs, their specific contribution in biotechnological food matrices represent the central themes of this review, where the recently highlighted potential of NSYs, their specific contribution in biotechnological food matrices represent the central themes. In this regard NSYs constitute a great variety of species that have shown effective antagonistic capacity against several spoilage and pathogenic microorganisms, as attested by this review in which references related to more than 70 yeast species involved in the central topic of biocontrol or probiotic trait, were considered (Table 1).

4. Conclusion and perspectives

In the last two decades there was growing interest on NSYs with its improving application in agri-food industry with consequent commercial relevance. In addition to the features regarding quality of food and beverages, as the enhancement of flavor and aroma complexity or ethanol reduction, there are growing interest on NSYs concerning antimicrobial issue. Furthermore, the interest in NSYs, triggered by their rich and diverse reservoir of enzymes and secondary metabolites typically not produced by the conventional S. cerevisiae yeast, as well as their metabolic diversity, is now fundamental to meet consumer demands for novel sensory properties and health benefits. Effectively, NSYs can be used against pathogenic microorganisms in all producing chain step, starting to primary production in field, ongoing in postharvest or during the transformation processes and the packaging and storage phase of food and beverages. But also, enriched food with the addition of NSYs with probiotics traits represents a safe and easy way to bring to market fermented foods and beverages with health attributes as well, without increasing costs. Therefore, the consumer would have the possibility of simultaneously consuming a fermented food supplemented with a source of viable probiotics, bypassing the purchase of pharmaceutical formulations with high costs and dubious vitality. For these reasons, the metabolic diversity provided by NSYs promises to meet the consumer demands for new sensorial and beneficial health properties.

Moreover, the growing interest in ecological, economical and health sustainability of agri-food industry in reducing agrochemical treatments but also conventional synthetic additives and antibiotics or antiseptics during technological food transformation, represent the starting point to explore in this topic. On the other hand, this field of research is still little explored and deserves to be investigated. Little is known about the activities of yeasts and NSYs against very dangerous pathogenic bacteria involved in food processing and preservation. In this way, more in-depth knowledge on the spectrum of action of NSYs towards pathogenic or alterative bacteria together with the study of the mode of action could represent an opportunity to counteract the antibiotic resistance phenomenon, which afflicts not only the medical field, but also food sector. It would therefore be desirable to direct the research towards studies aimed at the interactions between the safe and easily handled NSYs and bacteria, even in complex fermentations involving the interaction of several microbial species, with the aim of clarifying the contribution of the antagonist and the sensitive microorganisms.

Finally integrated studies of applied microbiology and food technology would be desirable to increase the possibilities of supplying formulations in which the microorganisms remain alive for long time and in which the management of the inoculum and fermentation process is as flexible, economical and easy as possible.