Some of the most common yeasts often found in grape, musts and wines that can be considered spoilage yeast species in a wide range of food products.
Traditionally in winemaking, sulphur dioxide (SO2) is chemically the most widely used for microflora control as antimicrobial preservative. Other tested compounds for selective yeast control are sorbic and benzoic acids. Herein, we discuss the effectiveness and the application of traditional and novel treatments and biotechnologies for chemical and biological control of wine spoilage yeasts. The versatility of the killer toxins and the antimicrobial properties of natural compounds such as carvacrol, essential oils and bioactive peptides will be considered. Some of the wine spoilage yeasts that are intended to control belong to the genera Zygosaccharomyces, Saccharomycodes and Dekkera/Brettanomyces, but also the non-Saccharomyces yeasts species dominating the first phase of fermentation (Hanseniaspora uvarum, Hansenula anomala, Metschnikowia pulcherrima, Wickerhamomyces anomalus) and some others, such as Schizosaccharomyces pombe, depending on the kind of wine to be produced.
- spoilage yeasts
- essential oils
- bioactive peptides
- emerging biotechnologies
- killer toxins
- monitoring techniques
Ever since the wild yeast colonies are controlled in grape musts, the fermentation has produced wine with differentiated organoleptic attributes. The control over yeast colonies during the entire winemaking process has given the winemaker the possibility of moulding these characteristics towards producing wine with better quality parameters.
Even though yeasts are responsible for transforming grape must into wine through fermentation, there are yeasts capable of spoiling it (Figure 1). Spoiling yeasts are, in most cases, resistant to harsh conditions such as high ethanol concentration, relatively low pH and lethal concentration of sulphites (SO2) or dimethyl dicarbonate (DMDC) used as antimicrobial agents.
Some yeast genera can be considered as spoilage microorganisms due to their undesirable implantation in food in which they can cause nutritional and sensory quality degradation and consequently lead to major economic losses. Even their implication in relation to public health is currently under suspicion . Table 1 summarizes some yeast genera known as wine spoilage that also spoils certain food products, particularly specifying the main compounds affecting quality and the effect produced together with potential health hazard.
|Yeast species||Food product||Spoilage compounds||Effect observed||Health hazard|
|Products with 50% sugar [2, 3]||Alcohol, esters ||Gas production: bubbling and package expansion |
|Sweet wines ||Refermentation and CO2 production |
|Mould-ripened soft cheeses |
|Fruit juices, sauces, carbonated soft drinks, salad dressings, ketchup [3, 6]||Alcohol, esters ||Gas production: bubbling and package expansion |
|Bulk, barrel matured and bottled wines [7, 8]||4-ethylphenol,|
4-ethylguaiacol, acetic acid 
|Off aromas, cloudiness formation in sparkling wine, mousy aroma |
Unpleasant mousy and medicinal taints 
|Dairy and baking products, beer, high salt environments and silage ||No restriction on handling and no risk to healthy persons |
|Lactic acid-rich products |
|Wine, winemaking [7, 8]||Ethyl acetate |
Acetaldehyde, esters, acetic acid 
|Oxidation of ethanol |
|Grape juice ||4-ethylphenol ||Off-odours barnyard-like or horsey |
|Soft drinks |
|Mould-ripened soft cheeses ||Acetaldehyde ||Chalky film layer |
|Food products with SO2 as antiseptic |
|Bottled wines ||Spoilage by sediment or cloudiness formation |
|Wine ||High acetoin level ||Flocculent sediment |
|Fresh fruits: orange (||Candidiasis has not been transmitted by food products |
|Grated raw carrots ||CO2 ||Increase in exudate and softening |
|Fresh must |
|Various storage products ||Low ethanol concentration ||Undesirable fermentation products |
|Must under fermentation ||Acetate production ||Aroma modification at early fermentation stage in winemaking |
The uncontrolled use and misuse of antibiotics has caused increasing resistance in a broad group of pathogenic microorganisms, including food-borne pathogens, which, in addition to resisting the effect of antibiotics, are able to survive the processes of food preservation .
In this chapter, different technologies and treatments for the control of spoilage yeasts have been revised. These techniques were split into early and emerging technologies in accordance with the novelty of their application in winemaking industry. A brief review of monitoring techniques as a tool for improving quality control in the winery is also included.
2. Technologies for spoilage yeast control
2.1. Early technologies
In the food industry, the control of the spoilage and pathogenic microorganisms was traditionally carried out by means of using thermal processes, to ensure the partial or total elimination of the microflora present [17, 18]. Together with an aseptic and hermetic packaging, it was possible to effectively extend the shelf life of the food products ensuring at the same time its microbiological safety [19, 20]. The main drawback of this kind of inactivation processes is the damage in organoleptic quality due to high processing temperatures. Another traditional way to fight against unwanted microorganisms is the addition of natural or chemically synthesized preservatives, such as organic acids (ascorbic, citric, benzoic, sorbic, etc.) and salts (potassium sorbate, sodium benzoate, sodium metabisulphite, etc.) [21, 22]. It is also possible to limit undesirable microbial development by modifying certain environmental parameters (temperature, pH, water activity, nutrient availability, toxic compounds, etc.) during the production process in order to hinder its growth. In the field of oenology, the ethanol tolerance is believed to be one of the main factors limiting yeast growth . In addition, some of the antimicrobials most commonly used in winemaking are sulphur dioxide (SO2), dimethyl dicarbonate and sorbic acid.
Yeast species resistant to one preservative also tend to be resistant to others with similar chemical composition. Such is the case of benzoic acid, sorbic acid and sulphur dioxide . Also, sorbic acid resistance has demonstrated to be highly correlated to ethanol resistance . In general, yeast resistance to preservatives seems to be strain dependent and also dependent on the physiological characteristics of the cells [6, 26].
Sulphur dioxide (SO2) is the chemical additive mainly used in wineries as antioxidant and preservative to control bacteria, moulds and spoilage yeasts [11, 27] considering that its antiseptic property depends on the pH of the media . However, in the last decades, its use is being reconsidered due to increasing allergic concerns. Researchers are looking for alternative methods to reduce the doses commonly added to grape juice and wine [29, 30].
High doses of SO2 are needed to control the growth of
Due to long-term exposure to SO2, some wine yeasts have developed certain defence mechanisms to fight against this antimicrobial . The ability of
Dimethyl dicarbonate (DMDC) is a dimethyl ester of dicarbonic acid used as cold sterilant in food industry. It is legally authorized in the USA , Australia  and Europe  as chemical preservative for ensuring the microbiological stability of wines. DMDC antimicrobial effectiveness is maximal at low pH, high ethanol content and low microbial population . At permissible usage levels, DMDC is more effective against yeast than bacteria or moulds . The main mechanism of action of DMDC is to inactivate cellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase through reaction with nucleophilic groups (imidazoles, amines and thiols) . DMDC can be used to prevent spoilage yeasts growth in wines  as well as to stop alcoholic fermentation in the production of sweet wines  or to disinfect musts by removing native flora present .
In relation to their specific effect on spoilage yeasts,
Its use before the end of fermentations is not recommended since the antimicrobial effect of DMDC can be also exerted against the fermenting species such as
There also exists a synergistic activity to increase the inactivation effect against wine yeast and bacteria between DMDC and sulphur dioxide in both potassium and sodium metabisulphite salts [40, 43]. In this regard, the use of DMDC allows a significant reduction of sulphur content in grape juice and semi-sweet wines .
Regarding health issues, DMDC does not represent any threat since it is naturally, rapidly and completely hydrolysed to methanol and carbon dioxide in aqueous solutions . Notwithstanding, a disadvantage of the use of DMDC in winemaking is its potential toxicity during handling .
With relation to the use of weak acids, benzoic and sorbic acids are able to control spoilage yeasts in wines when used in certain concentrations. The lipophilic character of benzoic and sorbic acids allows their diffusion in its undissociated form through cell membranes . However, so far, benzoic acid is not authorized for use in wine .
Among the physicochemical parameters that can be handled in the cellar, the storage temperature and the level of ethanol in wine together can exert a synergistic limiting effect on the growth of the wine spoilage yeast
2.2. Emerging technologies
2.2.1. High hydrostatic pressure
The high hydrostatic pressure (HHP) treatment is a non-thermal process used to inhibit pathogenic microorganisms including spoilage microbes as well as enzymes . According to these authors, the food products that are treated do not change nutritional or modify their sensory quality. The HHP uses pressure-transmitting liquids (water, ethanol solutions, sodium benzoate solutions, etc.) to homogeneously transfer pressure to the food sample ; the process may be batch, semi-continuous or continuous. The use of HHP is also expected to increase the shelf life  of a wide range of food products able to be treated including meat, eggs, vegetables, seafood , fruits such as sweet cherries  or mango pulp , juices [55, 58] and Serrano ham , among others.
In the winemaking industry, the HHPs have been used to reduce or inhibit the presence of spoilage yeasts such as
2.2.2. Pulsed light irradiation
The pulsed light irradiation is a technique used to inactivate microorganisms in food products. The pulses are stored electricity in UV lamps that do not contain mercury but xenon , released in very short periods of time (fractions of millionths or thousandths of a second) that are rich in UV-C light (from 200 to 280 nm in the spectrum) . This sterilization technique can cause DNA damage in the same way that continuous UV light does, nonetheless irradiation with pulsed light may also induce distortion in cell membranes and vacuoles ; therefore, the pulsed light irradiation may be considered an improved version of the UV treatment .
Pulsed light has been evaluated in the last decade as an efficient technique against foodborne spoilage microorganisms in commercial and fresh fruit juices using pulsed light against
2.2.3. Electric pulses
The electric pulses (EPs) are a method used for food preservation that avoids the use of chemical compounds as well as the use of thermal treatments . Thus, it can be considered a cold temperature treatment that may preserve foodborne properties.
Hülsheger et al.  demonstrated that EPs have different effect on microorganisms. Yeasts and Gram-positive bacteria are more resistant than Gram-negative bacteria to the disruption caused by cellular structures by low-energy pulses while most of the microorganisms (>99%) die with high-energy pulses. The colonies were also more susceptible to die at logarithmic growth phase than when in steady state. Besides the growth phase, Gáskovà et al.  have shown that there are other factors involved in the efficiency of electric pulses for killing yeasts; such factors include the amplitude and the duration of the pulses, the size of the yeast cells and the temperature and conductivity of the media where the yeasts grow.
Electric pulses have been used lately in the production of fruit juices for its efficacy in microbial reduction and for keeping their sensorial and nutritional properties . The use of EP to eliminate spoilage yeasts such as
The effect of EP could be combined with the use of other antimicrobial technology like the lyticase digestion; the combined effect of both techniques may be a biotechnological application for spoilage yeast control since the use of mild EP (2–4.5 kV/cm) affects the cell-wall porosity and therefore lower doses of lyticase are needed to effectively inactive
Biotechnology applications of the electric pulses or the electroporation, other than spoilage yeasts control, may include genetic transformation of cells by incorporation of foreign DNA, extraction of intracellular metabolites and biomass drying .
2.2.4. Natural extracts
The use of antimicrobials extracted from nature such as chitosan, essential oils (active ingredients: eugenol, allicin, carvacrol, thymol and limonene), spices or nisin is very widespread in food preservation , but the limits of its antifungal activity and their potential applications as winemaking additives remain to be further explored.
Among the essential oils extracted from plants, those from cinnamon, clove, garlic, onion, oregano and thyme are the inhibitoriest against food spoilage yeasts . Antimicrobial activity of these compounds is well documented .
Eugenol is the main component of clove oil (≈85%). So far, eugenol has proven to be effective as antibacterial and antifungal in several food products . Both eugenol and thymol act on the membrane and cell wall, causing cell lysis . These same authors suggested the use of eugenol and thymol as lysing agents to extract the genomic DNA from yeast cell instead of using zymolyase and sodium dodecyl sulphate (SDS). However, according to Kubo et al.  eugenol alone may not respond as effective antimicrobial against
Carvacrol and thymol are phenolic compounds being part of the essential oil from oregano and thyme . According to Chavan and Tupe , low concentrations of carvacrol and thymol (≤64 mg/L; used independently) are effective in limiting the growth of several wine spoilage yeasts, including
As for allicin (diallyl thiosulphinate), it is the main bioactive component of garlic extract . It is well known for its antioxidant, antibacterial and antifungal activities  and also for its anticancer activity [96, 97]. The main mechanism involved in the antimicrobial effect of allicin is based on its rapid reaction with thiol-containing proteins . Thus, allicin can regulate the activity of enzymes containing very reactive or unshielded SH groups. With relation to its potent antifungal properties, allicin is effective against a wide range of yeasts, among them
Despite their interesting antimicrobial properties, there still exist some limitations to the use of some of these natural extracts such as their solubility in water, their susceptibility to oxidation (part of its effectiveness is lost) and the impact on sensory attributes . A possible solution to the low water solubility of some natural extracts is their transformation into nanoemulsions [104, 105] or the use of nanoencapsulation techniques to protect their antimicrobial properties .
The inhibition activity on the growth of some wine spoilage yeasts using different antimicrobial compounds was measured by agar diffusion tests (Figure 2). The dark areas around the disks suggest the inhibitory effect in growing colonies, thus demonstrating their effectiveness. This assay can be used to assess the susceptibility of the yeast species to each of the antimicrobials, taking into account the limitations due to the diffusion properties of the antifungal compounds in solid media.
Despite their interesting antimicrobial properties, there still exist some limitations to the use of some of these natural extracts such as their solubility in water and the impact on sensory attributes .
2.2.5. Antimicrobial peptides
Antimicrobial peptides are important molecules naturally occurring in the immune system  and may have different amino acids conformation. Their importance as antimicrobial agents increased when bacteria and other pathogens became more resistant to drugs . Among the most studied peptides are tritrpticin and indolicidin, related cathelicidins  rich in Arg and Trp residues  as well as the lactoferricin. Other peptides having microbicidal activity are cecropins, defensins, magainins, melittin and alamethicin .
Tritrpticin is a positively charged peptide with tryptophan (Trp) residues  that has antifungal activity besides having antibacterial activity against Gram-positive and Gram-negative bacteria [113, 114]; it is naturally found in granules of bovine and porcine neutrophils  as well as in insects [107, 116, 117]. The electrostatic interaction between the cationic residue and the negative charge of phospholipids in the membrane has been recognized as an important factor in the microbicidal action found in these biomolecules .
Indolicidin is another peptide with antimicrobial activity against both fungi and bacteria (Gram-positive and Gram-negative) . Similar to tritrpticin, indolicidin is a tryptophan (Trp)-rich peptide  that also contains proline (Pro) residues in its configuration ; its structure resembles that of detergent micelles and of phospholipid vesicles . It has been isolated from cytoplasmic granules of bovine neutrophils  but it is also present in humans and other mammals as well as in some primitive vertebrates . It acts directly on the lipidic bilayer  causing disruption in the cell membrane.
Lactoferricin B (Lfcin B) is a peptide obtained during the gastric digestion of the bovine protein lactoferrin . Despite the fact that other mammals including humans also produce lactoferricins, the Lfcin B has higher antimicrobial potency . Lfcin B shows different properties including antibacterial, antifungal, antiviral, antitumour, anti-inflammatory and immunoregulatory .
Antimicrobial peptides are important for the control of spoilage yeasts in food products. Lfcin B derivatives reduce spoilage yeasts such as
In medical applications, indolicidin has antimicrobial activity against different pathogens such as yeasts, viruses, bacteria, fungi and protozoa . Indolicidin has been tested against candidiasis produced by fungi
A drawback in the use of some antimicrobial peptides like tritrpticin and indolicidin for treatment of infectious diseases produced by pathogen agents is the hemolytic activity observed against blood hematocytes [113, 115, 132]; therefore, it is important to use alternative antimicrobial peptides with less toxicity in immunocompromised patients .
2.2.6. Killer toxins and
Killer toxins are known as pore proteins produced by yeast metabolism and are able to kill sensitive yeast cells by forming cell wall or cell membrane disruptions . There are different killer toxins produced by either
Among the toxins produced by non-
Even though both compounds have same disruptive inhibition mechanism, the activity of the
In the case of
The yeasts could be neutral or sensitive to toxins produced by killer yeasts. In this matter,  have shown that the yeast species
A summary of certain emerging antifungal compounds is presented in Figure 5.
3. Yeast populations monitoring techniques
Parallel to the use of technologies that reduce the colonization of spoilage yeasts in food products, there are technologies able to monitor the yeasts and bacteria populations and their nature in the alcoholic and malolactic fermentations during wine production. One of these techniques is the flow cytometry (FCM) ; other techniques are fluorescence-activated cell sorting (FACS), quantum dots (QDs) and emerging monitoring technologies such as mass cytometry (Cy-TOF), imaging flow cytometry and, recently, spectral cytometry.
FCM, used for counts of microorganism populations in wine, allowed the simultaneous determination of yeasts for as low as 103 cells/mL and, despite its size, populations of malolactic bacteria higher than 104 cells/mL . FCM is able to show real-time situation of microorganisms in different matrices although it is considered complicated implementation due to the cost of reagents and the need of recruiting trained staff. FCM that relies on the use of complementary fluorescence dying to selectively determine specific type of microorganisms  through, for example, the metabolic enzyme activity or antigen expression is known as fluorescence-activated cell sorting (FACS). This technology is useful in monitoring changes in yeast cellular organelles’ biogenesis such as the mitochondria .
The QD is a technology based on the production of semiconductor nanoparticles made from cadmium salts crystals with chloride (Cl), tellurium (Te) and sulphide (S) by target microbial cells such as spoilage yeast . These nanoparticles are used as biomarkers of nucleic acids and proteins and can be detected visually when they are excited by a light source. QDs are photostable and they have a wide range of absorption while they have a narrow emission peak .
Imaging flow cytometry has been used to compare the mode of action in which different killer toxins affect cell structures. Comitini et al.  saw that the toxin Kpkt from
The spectral cytometry was developed as to increase the accuracy and the precision of flow cytometry results by a higher resolution obtained with spectral analysis from more discrete bands of emission of multiple stained samples  as in the case of microbial counts in foodborne matrices stained with PI. Spectral measurements combined with flow cytometry technique allow obtaining fluorescence and RAMAN spectra analysis of large particles in chemical and biological processes .
4. Future trends
Several antimicrobial techniques have been developed to control the presence of spoilage yeasts in food products through the years. These techniques aim to diminish the negative economic impact of having contaminated/spoiled products as well as the potential health threat that this may represent. Some of these techniques are already in use by the winemaking industry and others may be explored by the different process of stages from vine to bottled wine.
The trend observed is that the winemaking industry is targeting the use of innocuous control techniques to avoid spoilage yeasts during the entire process in order to preserve varietal aromas from grapes, to protect and to extend the anthocyanins extraction yield and, in the best-case scenario, to improve the overall quality of wines.
This work was funded by the INNTER WINARGAL 2015 project (CDTI-MINECO).