fermentation

Article Activity Interactions of Crude Biopreservatives against Spoilage Consortia

Maxwell Mewa-Ngongang 1,2,3,* , Heinrich W. du Plessis 1 , Edwin Hlangwani 1,2, Seteno K. O. Ntwampe 2,3 , Boredi S. Chidi 1,2 , Ucrecia F. Hutchinson 1,2,3 and Neil P. Jolly 1

1 Post-Harvest and Agro-Processing Technologies, ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Private Bag X5026, Stellenbosch 7599, South Africa 2 Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa 3 Department of Chemical Engineering, Cape Peninsula University of Technology, P.O. Box 652, Cape Town 8000, South Africa * Correspondence: [email protected]; Tel.: +27-021-809-3442

 Received: 29 March 2019; Accepted: 11 June 2019; Published: 29 June 2019 

Abstract: It is common to find different spoilage organisms occurring in the same food item, which usually requires food producers to utilize a mixture of synthetic preservatives to control spoilage. This study evaluated the interaction between mixtures of crude biopreservatives against consortia of common spoilage occurring in beverages. Crude biopreservatives produced from separate yeasts were formulated in different growth inhibition combinations (GICs), i.e., GIC1 (Candida pyralidae Y1117 and Pichia kluyveri Y1125), GIC 2 (C. pyralidae Y1117 and P. kluyveri Y1164), GIC3 (P. kluyveri Y1125 and P. kluyveri Y1164), and GIC4 (C. pyralidae, P. kluyveri Y1125 and P. kluyveri Y1164). The spoilage yeast consortia combinations, i.e., SC1 (Dekkera. anomala and D. bruxellensis), SC2 (D. anomala and ), SC3 (D. bruxellensis and Z. bailii), and SC4 (D. anomala, D. bruxellensis and Z. bailii), were also prepared. The highest growth inhibition activities of the crude biopreservatives were observed at a pH of 3.0 and 2.0 for C. pyralidae and P. kluyveri strains, respectively, while reduced activity was observed at a pH of 4.0 and 5.0. The growth inhibition proficiency depended on the spoilage yeast or the consortia of spoilage yeasts. Biocontrol agents from an individual yeast or mixtures can be used to prevent food and beverage spoilage.

Keywords: crude biopreservatives; grape pomace; microbial consortia; beverage spoilage yeasts; growth inhibition activity

1. Introduction Protection of food and beverages against microbiological spoilage is essential for maintaining an adequate food supply to growing world populations. Yeast species such as Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Zygosaccharomyces florentinus, Zygosaccharomyces microellipsoides, Zygosaccharomyces rouxii, Hanseniaspora uvarum, Candida guilliermondii, and even cerevisiae have been reported to cause spoilage in alcoholic and non-alcoholic beverages [1–3]. To curb microbial spoilage, synthetic chemicals and/or physical methods can be used [3–7]. Physical methods include pasteurization, cold processing, filtration, the control of water content, ultrasound processing, and irradiation. However, some of these physical methods can have a negative effect on the quality of food items, such as fruits, vegetables, and beverages [3,5–7]. Furthermore, thermophiles, spores, psychrophiles, and xerophiles can sometimes survive these methods [8,9].

Fermentation 2019, 5, 53; doi:10.3390/fermentation5030053 www.mdpi.com/journal/fermentation Fermentation 2019, 5, 53 2 of 12

Chemical preservatives such as SO2, dimethyl dicarbonate, benzoate, benzoic, lactic, sorbic and acetic acid, triazoles, hydroanilide fenhexamid, dicarboximides, and succinate dehydrogenase sorbate are commonly used to extend the shelf life and to improve the safety of food. However, in some cases, more than one chemical preservative is used to prevent spoilage from a consortium of spoilage microorganisms. This can lead to the presence of undesirably high levels of chemical preservatives in food. Increasingly, consumer fears about potential preservative toxicity and antimicrobial-resistant pathogens in food have led to strict regulations for the use of preservatives on fruit and in fruit-derived beverages. Some regulations also advocate the use of safer alternative preservatives [10–12]. The desire for a safe alternative to synthetic chemicals has led to the investigation of benign microorganisms, specifically yeasts and bacteria, with growth inhibition properties against spoilage microorganisms [1,2,13,14] From bioprospecting studies, some microorganism-derived biocontrol agents, such as killer toxins, have been identified [1,2,15]. Volatile organic compounds (VOCs) such as ethyl acetate, phenylethyl alcohol, 1,3,5,7-cyclooctatetraene, isobutyl acetate, 2-phenyl ethyl acetate, ethyl propionate, isoamyl acetate, and isoamyl alcohol have also been linked to growth inhibition properties [2,15–19]. In previous studies [18,20,21], yeast-derived crude biopreservatives from C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 were individually tested against selected spoilage fungi and yeasts. However, the growth inhibition efficiency of combining the different crude biopreservatives has not been investigated. The aim of this study was therefore to investigate the growth inhibition activity of individual and mixtures of crude biopreservatives produced by C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 against individual and consortia of spoilage yeasts.

2. Materials and Methods

2.1. Yeasts Selection, Growth Inhibition Activity (GIA), and Screening Assays (Plate Assays) Three strains of yeasts, Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164, were obtained from the culture collection of the Agricultural Research Council (ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa). Candida pyralidae Y1117 was previously isolated from grape must and Pichia kluyveri Y1125 and Pichia kluyveri Y1164 were isolated from “Marula” (Scelerocarya birrea) fruit juice. These yeasts were screened for growth inhibition activity against the beverage spoilage yeasts listed in Table1. Dekkera anomala, Dekkera bruxellensis, and Zygosaccharomyces bailii were used as spoilage yeasts and seeded into grape pomace extract agar (GEA) at the desired concentration, depending on the assay being carried out, as described by Mewa-Ngongang et al. [21]. All plate assays were performed in triplicate using 90 mm petri dishes. A cross-screening procedure was performed whereby the selected growth inhibiting yeasts were also screened against each other. The grape pomace extract was also tested for growth inhibition activity against the selected spoilage yeasts (Dekkera anomala, Dekkera bruxellensis, and Zygosaccharomyces bailii. Growth inhibition activity and quantification was carried out as described by Mewa-Ngongang et al. [18,20]. Growth inhibition activity was quantified using the concept of the volumetric zone of inhibition (VZI), with the units being reported as a liter contaminated solidified media per mL crude biopreservatives used (L. CSM/mL BCU), as described by Mewa-Ngongang et al. [20]. This approach was used to quantify what volume of crude biopreservatives would be needed to inhibit the growth of spoilage organisms at a specific concentration in a liter of contaminated solidified media or beverage. Fermentation 2019, 5, 53 3 of 12

Table 1. Screening for growth inhibition activity, including the cross screening of biocontrol yeasts against each other on grape pomace extract. All yeasts used in the study.

Biological control yeasts Candida pyralidae Pichia kluyveri P. kluyveri Grape Pomace Y1117 Y1125 Y1164 extract Dekkera anomala C96V37 - + + - D. anomala C96V38 + + + - D. bruxellensis C96V33 + + + - D. bruxellensis C96V30 - + + - Brettanomyces lambicus Y0106 - - - - B. lambicus Y0175 + + -- B. lambicus Y0191 + + -- B. lambicus Y0167 + + -- C. magnoliae Y1061 - - - - C. guilliermondii Y0848 + --- Spoilage yeasts Zygosaccharomyces bailii Y0070 + --- Z. bailii Y0891 + --- Z. bailii Y0186 + --- Z. rouxii Y0115 - - - - Z. rouxii Y0111 - - - - Z. microellipsoides Y0159 - - - - Z. cidri Y0169 - + + - Z. florentinus Y0277 - - - - Z. fermentati Y0182 - - - - Z. bisporus Y0288 - - - - Z. bisporus Y0113 - - - - C. pyralidae Y1117 - - - - Biological P. kluyveri Y1125 - - - - control yeasts P. kluyveri Y1164 - - - -

2.2. Media, Inoculum Preparations, and Crude Biopreservative Production Grape pomace juice extract was used as the primary fermentation medium in this study. The grape pomace from which the extract was obtained originated from the research cellar of the Agricultural Research Council (ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa). The extraction method used was similar to the normal white grape juice extraction procedure, but with an increased extraction pressure (1 to 2 bar) to allow the recovery of the remaining juice (extract) from the pomace. The 1 1 juice from the grape pomace was composed of 210 g.L− total , 185.12 mg L− yeast assimilable 1 nitrogen (YAN), and 34.13 mg L− ammonium-nitrogen. The extracted residual juice from Chenin 1 Blanc grape pomace was diluted to different sugar concentrations (150, 100, 75, and 50 g L− total sugar, equal ratio of glucose and fructose), and adjusted to pH 5.0 using 0.1 M NaOH, subsequent to autoclaving for 20 min at 120 ◦C, which resulted in grape pomace extract broth (GEB). The availability of yeast assimilable nitrogen (YAN) to support yeast growth was also quantified using an enzyme robot (Arena 20XT; Thermo Fisher Scientific, Vantaa, Finland). The autoclaved GEB was used for yeast growth and crude biopreservative production. Concurrently, the extracted juice from the pomace was supplemented with 2% agar bacteriological and autoclaved, which was then poured on to petri dishes. Hereafter, this medium will be referred to as grape pomace extract agar (GEA). Throughout the study, GEB with a suitable sugar concentration, which best supported microbial growth, was used to grow the inoculum of both the biopreservative-producing yeasts and the beverage spoilage yeasts. For the biopreservative-producing yeasts, the incubation temperature for the inoculum was 25 ◦C for 24 h in a 1 shaking incubator (150 rmin− ). Similar conditions to those described by Mewa-Ngongang et al. [2,20], using GEB as fermentation medium, were used for the production of the crude biopreservatives, whereby samples were withdrawn after 24 h of fermentation and tested for growth inhibition activity.

2.3. Preparation of Crude Growth Inhibition Mixtures and Spoilage Yeast Consortia

2.3.1. Mixtures of Crude Biopreservatives Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164 were grown separately in 1 ® GEB (75 g L− ) for 24 h at 25 ◦C in a shaking incubator (LM-53OR, RKC Instrument INC, Ohta-ku Fermentation 2019, 5, 53 4 of 12

1 Tokyo, Japan) at 150 r/min. The broths, after fermentation, were centrifuged for 5 min at 5000 r min− . The resulting supernatant from each of the yeast cultures was aliquoted into Eppendorf tubes (2 mL). The mixture of the crude biopreservatives was prepared as follows: a volume (50 µL) from each of the three cell free supernatants was mixed in a separate Eppendorf tube (2 mL) and stored at 4 ◦C for further use. The different growth inhibitor combinations (GICs) from the cell free supernatants were GIC1 (C. pyralidae Y1117 and P. kluyveri Y1125), GIC2 (C. pyralidae Y1117 and P. kluyveri Y1164), GIC3 (P. kluyveri Y1125 and Pichia kluyveri Y1164), and GIC4 (C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164).

2.3.2. Spoilage Yeast Consortia Preparation Three beverage spoilage yeast strains, Dekkera anomala, Dekkera bruxellensis, and Zygosaccharomyces bailii, were grown in GEB without agitation for 48 h at 25 ◦C prior to the growth inhibition assays. From the 48-h-old spoilage yeast cultures, a volume (2 mL) of the broth was centrifuged for 5 min 1 at 5000 r min− , after which the supernatant was discarded, with the resulting pellet from each of the spoilage yeast cultures being washed with sterile distilled water prior to storage for the assays. The mixtures of spoilage yeasts were prepared in different combinations, i.e., SC1 (D. anomala and D. bruxellensis), SC 2 (D. anomala and Z. bailii), SC3 (D. bruxellensis and Z. bailii), and SC4 (D. anomala, D. bruxellensis, and Z. bailii). For each consortium, the individual spoilage organism was at a final concentration of 103 cells/mL. The prepared mixtures were then used to seed the GEA plated for growth inhibition assays.

2.4. Effect of Proteolytic Enzymes on the Denaturation of Crude Biopreservatives To evaluate the nature of the crude biopreservatives, it was important to subject the crude samples to protease treatments in order to determine whether the crude biopreservatives responsible for growth inhibition activity were of a protein nature. The assay used was adapted from Mehlomakulu et al. [2]. The protease enzymes used were Proteinase K, pepsin, and proteases from Aspergillus saitoi and Rhizopus spp. (Sigma-Aldrich, Darmstadt, Germany). Thereafter, treated crude samples were tested for growth inhibition activity, using the method described by Mew-Ngongang et al. [20], with the positive control being used as the crude sample that was not subjected to any of the treatments. Furthermore, the negative control consisted of just the protease without the crude biopreservative, with all samples being prepared in three replicates.

2.5. Effect of pH and Temperature on Activity and Stability of Crude Biopreservatives The temperature activity study was performed by spotting three replicates, with 20 µL of the crude sample in a 5 mm diameter well created on GEA seeded with D. anomala as a spoilage organism. The plates were incubated at 5, 15, 20, 25, 30, and 40 ◦C, respectively. Additionally, the pH activity was also determined by spotting 20 µL on GEA adjusted to a pH of 2.0, 3.0, 4.0, and 5.0 respectively, which is the pH range of many foods and beverages. These GEA plates were then incubated at 25 ◦C until the volumetric zone of inhibition (VZI) was observed. Thereafter, the growth inhibition activity was measured. A stability test was also performed after confirming the temperature and pH optima. The stability test was carried out by storing the crude biopreservative mixtures at different temperatures ( 10, 5, 15, 20, 25, 30, and 40 C) for 16 weeks. A volume of 20 µL of the respective stored samples − ◦ was spotted onto three replicate plates prepared at the pH optima, subsequent to incubation at the temperature optima until the zone of inhibition was observed.

2.6. Growth Inhibition Study of Crude Biopreservative Mixtures against Spoilage Yeast Consortia

2.6.1. Effect of Cell Free Supernatants on Growth Inhibition Activity of Single Spoilage Organism The growth inhibition efficiency of C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 extracellular metabolites was tested by spotting 20 µL of the cell free supernatant in a 5 mm diameter Fermentation 2019, 5, 53 5 of 12 well on GEA seeded with individual spoilage yeasts at the concentration of 106 cells/m. All treatments had three replicates and the plates were incubated at 25 ◦C until zones of inhibition were observed around the wells of the GEA plates. The growth inhibition activity was quantified as described by Mewa-Ngongang et al. [20].

2.6.2. Effect of Cell Free Supernatants from Single Yeasts on Growth Inhibition Activity of Spoilage Yeast Consortia The ability of C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 extracellular metabolites to inhibit the consortia of spoilage yeasts was assessed by spotting 20 µL of the crude biopreservation samples in the GEA plate wells prepared according to the different spoilage combinations. The spoilage combination plates were also prepared with three replicates. The plates were incubated at 25 ◦C until the zone of inhibition was obtained. The growth inhibition quantification was also assessed by the method developed and reported by Mewa-Ngongang et al. [20].

2.6.3. Effect of Mixed Crude Biopreservatives on the Growth Inhibition of Single and Consortia of Spoilage Organisms A mixture of extracellular crude biopreservatives from the three producing yeasts was prepared by mixing an equivalent volume of the crude supernatants. The spoilage yeast consortia were prepared by mixing an equivalent volume of each culture (103 cells/mL) of the individual spoilage yeasts. From the mixture of the crude supernatants, 20 µL was spotted on different spoilage yeast consortia plates, prepared in three replicates, followed by incubation, until zones of inhibition were observed, i.e., when quantifiable.

3. Results and Discussion

3.1. Growth Inhibition Activity Screening and Crude Pounds Production The growth inhibition activity of C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 was screened against the yeasts listed in Table1, which included D. anomala (2), D. bruxellensis (2), Brettanomyces lambicus (4), C. magnoliae, C. guilliermondii, Z. baillii (3), Z. bisporus (2), Z. cidri, Z. fermentati, Z. florentinus, Z. microellipsoides, and Z. rouxii (2). A zone of inhibition around the sensitive yeast colony indicates growth inhibition as depicted in Figure1. In this study, in order to limit uncertainties in the origin of the growth inhibition activities, biopreservative-producing yeasts were screened against the spoilage yeasts and were cross-screened among themselves to see whether they displayed inhibition activity against each other (Table1). The results show that the biopreservative-producing yeasts exhibited no growth inhibition activity against each other. The GP extracts did not have any growth inhibition effect on any of the yeasts studied. Thereafter, grape pomace juice extract broth was used as the fermentation medium for the production of crude biopreservatives. This was confirmed by the zone of inhibition observed on the plates when 20 µL of the crude samples from each yeast culture was spotted in a 5 mm well on the GEA plates against spoilage organisms. The VZI was not quantified in this section because only visual inspections were required for further experiments.

1 Thereafter, grape pomace juice extract broth was used as the fermentation medium for the 2 production of crude biopreservatives. This was confirmed by the zone of inhibition observed on the 3 plates when 20 µL of the crude samples from each yeast culture was spotted in a 5 mm well on the 4 GEA plates against spoilage organisms. The VZI was not quantified in this section because only visual Fermentation 2019, 5, 53 6 of 12 5 inspections were required for further experiments.

6 Figure 1. Growth inhibition activity of Candida pyralidae Y1117 (1), Pichia kluyveri Y1125 (2), and Pichia 7 Figure 1. Growth inhibition activity of Candida pyralidae Y1117 (1), Pichia kluyveri Y1125 (2), and Pichia kluyveri Y1164 (3) against Dekkera anomala on grape pomace extract agar. One plate out of all the 8 kluyveri Y1164 (3) against Dekkera anomala on grape pomace extract agar. One plate out of all the different treatments was selected for a visual representation of growth inhibition activity. 9 different treatments was selected for a visual representation of growth inhibition activity. 3.2. Effect of Proteolytic Enzymes on the Denaturation of the Crude Biopreservatives 10 3.2. Effect of Proteolytic Enzymes on the Denaturation of the Crude Biopreservatives The denaturation ability of crude biopreservatives was investigated using a proteolytic enzymes 11 treatmentThe denaturation approach. Minimal ability of denaturationcrude biopreservatives of the crude was biopreservatives investigated using was a proteolytic observed (dataenzymes not 12 shown).treatment This approach suggests. M thatinimal the crudedenaturatio biopreservativesn of the crude responsible biopreserv for growthatives inhibitionwas observed activity (data are not 13 proteinaceousshown). This suggest compoundss that producedthe crude duringbiopreservatives fermentation. responsible A few Candida for growthspecies, inhibition including activityCandida are 14 pyralidaenot proteinaceous, have been compounds found to secrete produced non-proteinaceous during fermentation type killer. A toxins few thatCandida have species growth, inhibition including 15 activityCandida [pyralidae2]. This study, have therefore been found suggests to secret thate thesenon-proteinaceous yeasts could o fftypeer di killerfferent toxins types that of metabolites have growth for 16 growthinhibition inhibition, activity against[2]. This a study variety therefore of spoilage suggest organisms,s that these than thoseyeasts of could a proteomic offer diffe nature,rent whichtypes of is 17 inmetabolites agreement for with growth other inhibition reports [15, ,against22]. We a previously variety of reportedspoilage [organ18] thatism thes, than growth those inhibition of a proteomic activity 18 ofnature the crude, which biopreservatives is in agreement with produced other byreportsC. pyralidae [15,22]. Y1117,We previouslyP. kluyveri reportedY1125, [18] and thatPichia the kluyverigrowth 19 Y1164inhibition was activity possibly of due the to crude volatile biopreservatives organic compounds, produced i.e., isoamyl by C. pyralidae acetate, isoamylY1117, P. alcohol, kluyveri 2-phenyl Y1125, 20 ethylacetate,and Pichia kluyveri and 2-phenyl Y1164 ethanol.was possibly However, due the to volatile focus of organicthis study compounds was on investigating, i.e., isoamyl the acetate,growth 21 inhibitionisoamyl alcohol, activity 2 of-phenyl the crude ethylacetate biopreservatives,, and 2-pheny whichl canethanol. potentially However, be used the asfocus pre- of and this post-harvest study was 22 biocontrolon investigat agents.ing the growth inhibition activity of the crude biopreservatives, which can potentially 23 be used as pre- and post-harvest biocontrol agents. 3.3. Effect of pH and Temperature on Activity and Stability of Crude Biopreservatives 24 3.3. Effect of pH and Temperature on Activity and Stability of Crude Biopreservatives The effect of pH and temperature on activity was tested. Observations of C. pyralidae indicate that 25 the bestThe pH effect to retain of pH the and highest temperature growth inhibitionon activity activity was tested. was pH Observations 3.0; however, of for C. thepyralidae two P. indicate kluyveri 26 strains,that the thebest suitable pH to retain pH level the washighest 2.0, growth while a inhibition pH of 4.0 activity and 5.0 was showed pH 3.0 extremely; however reduced, for the growth two P. 27 inhibitionkluyveri strains activity, the for suitable these yeasts pH level (Figure was2 2)..0 Similar, while resultsa pH of were 4.0 and observed 5.0 show againsted extremely all three spoilagereduced 28 yeasts.growth Comparinginhibition activity these results for these to some yeast findingss (Figure reported 2). Similar in the res literature,ults were theseobserved pH values against are all within three 29 thespoilage range yeasts. previously Comparing reported these for otherresults yeasts to some [2,15 findin,22].gs The reported stability in of the crude literature biopreservatives, these pH values at the 30 pHare optima within observedthe range was previousl investigatedy reported further. for other yeasts [2,15,22]. The stability of crude 31 biopreservativesFor the stability at the test pH of optima the crude observed samples was atinvestigated different pH further levels,. it was noted that P. kluyveri samples were stable at a pH of 2.0 for 16 weeks and C. pyralidae samples was stable at a pH of 3.0 for the same period. Furthermore, temperature variation resulted in stability assessments for all the7 crude samples, which were found to be stable between - 10, 5, and 10 ◦C, respectively, where the activity was retained for more than 16 weeks. The pH and temperature assessments with regard to retaining growth inhibition activity of the crude biopreservatives were performed under different pH and temperature conditions that are usually used for consumer-packaged goods. The optimal activity and stability of the crude biopreservatives were obtained at a lower pH (2 and 3), which falls within

32 For the stability test of the crude samples at different pH levels, it was noted that P. kluyveri 33 samples were stable at a pH of 2.0 for 16 weeks and C. pyralidae samples was stable at a pH of 3.0 for 34 the same period. Furthermore, temperature variation resulted in stability assessments for all the 35 crude samples, which were found to be stable between - 10, 5, and 10 °C, respectively, where the 36 activity was retained for more than 16 weeks. The pH and temperature assessments with regard to 37 retaining growth inhibition activity of the crude biopreservatives were performed under different

38 pHFermentation and temperature2019, 5, 53 conditions that are usually used for consumer-packaged goods. The optimal7 of 12 39 activity and stability of the crude biopreservatives were obtained at a lower pH (2 and 3), which falls 40 within the pH ranges of most beverages. These findings further expand and elucidate the in-situ 41 applicationthe pH ranges of crude of most biopreservatives beverages. These in findingsother type furthers of food expand and and beverage elucidate items the with in-situ the application same pH 42 andof crude storage biopreservatives temperature conditions in other types. of food and beverage items with the same pH and storage 43 temperature conditions.

a (pH 2.0) b (pH 3.0)

c (pH 4.0) d (pH 5.0)

Figure 2. Growth inhibition activity of the crude biopreservatives from Candida pyralidae Y1117 (1), 44 Figure 2. Growth inhibition activity of the crude biopreservatives from Candida pyralidae Y1117 (1), Pichia kluyveri Y1125 (2), and Pichia kluyveri Y1164 (3) against Dekkera anomala on grape pomace extract 45 Pichia kluyveri Y1125 (2), and Pichia kluyveri Y1164 (3) against Dekkera anomala on grape pomace extract agar at different pH levels: (a) pH 2.0, (b) pH 3.0, (c) pH 4.0, and (d) pH 5.0. 46 agar at different pH levels: (a) pH 2.0, (b) pH 3.0, (c) pH 4.0, and (d) pH 5.0. 3.4. Growth Inhibition Interactions 47 3.4. Growth Inhibition Interactions 3.4.1. Effect of Single Isolates and Their Cell Free Supernatants on Growth Inhibition Activity of 48 3.4.1.Single Effect Spoilage of Single Isolates and Their Cell Free Supernatants on Growth Inhibition Activity of 49 Single Spoilage The quantification results of the effect of single crude versus single spoilage showed that C. pyralidae did not inhibit the growth of D. anomala, but inhibited the growth of D. bruxellensis and Z. bailii (Figure3a).8 Meanwhile, the P. kluyveri strains inhibited the growth of D. anomala and D. bruxellensis with minimal growth inhibition activity against Z. bailli. Growth inhibition activity (visually indicated VZI) of the three biopreservation yeasts against D. bruxellensis is shown in Figure3b.

50 The quantification results of the effect of single crude versus single spoilage showed that C. 51 pyralidae did not inhibit the growth of D. anomala, but inhibited the growth of D. bruxellensis and Z. 52 bailii (Figure 3a). Meanwhile, the P. kluyveri strains inhibited the growth of D. anomala and D. 53 bruxellensis with minimal growth inhibition activity against Z. bailli. Growth inhibition activity 54 (visually indicated VZI) of the three biopreservation yeasts against D. bruxellensis is shown in Figure 55 3b. 56 The results obtained were in agreement with what usually occurs industrially during food 57 preservation – microbial spoilage may still occur even though a specific food environment contains 58 a preservation agent. In this case, the occurrence of spoilage organisms could be attributed to the 59 resistance of spoilage organisms to the synthetic preservation agent used. Furthermore, a specific 60 preservation agent could be used in targeting specific spoilage organisms; however, it may happen 61 that a different spoilage organism contaminates the manufactured product. When this phenomenon 62 occurs, the efficacy of the preservation agent is retained, but the biocontrol efficiency may seem lost 63 due to the incorrect microbial target or microbial resistance to the crude biopreservatives used by the 64 spoilage organism. Based on these reasons, more than one preservation agent can be used in the same 65 food/beverage environment; hence the preference to use a mixture of preservation agents. In 66 combating beverage spoilage suspected to occur due to the presence of D. anomala, D. bruxellensis, 67 and Z. bailli, a combination of crude biopreservatives from Candida pyralidae Y1117 and Pichia kluyveri 68 Y1125 or Pichia kluyveri Y1164 is recommended. Furthermore, the high efficacy observed for P. kluyveri Fermentation 2019, 5, 53 8 of 12 69 against D. anomala and D. bruxellensis means that either P. kluyveri Y1125 or Y 1164 could be used in 70 combating spoilage for either single or mixed spoilage organisms. a b

2.5

2

1.5

1

VZI (L/mL) VZI 0.5

0 D. anomala D. bruxellensis Z. bailli

C. pyralidae Y1117 P. kluyveri Y1125 P. kluyveri Y1164

71 FigureFigure 3.3. ((aa)) Values Values of of the the volumetric volumetric zone zone of inhibitionof inhibition (VZI) (VZI) obtained obtained from from the single the single isolates isolates and their and 72 celltheir free cell supernatants free supernatants against spoilage against yeasts spoilage (Dekkera yeast anomala,s (Dekkera D. bruxellensis anomala,, and D. Zygosaccharomyces bruxellensis, and 73 bailiiZygosaccharomyces)(b) An example bailii of a) plate(b) A showingn example the of VZI a plate obtained showing for Candida the VZI pyralidae obtainY1117ed for (1),CandidaPichia pyralidae kluyveri 74 Y1125Y1117 (2),(1), andPichiaPichia kluyveri kluyveri Y1125Y1164 (2), and (3) against Pichia kluyveriDekkera Y1164 bruxellensis. (3) againstThe valuesDekkera presented bruxellensis are. The the meansvalues 75 ofpresented three replicates are the withmeans the of standard three replicates deviation with ranging the standard between deviation 0.004 and ranging 0.09. between 0.004 and 76 0.09. The results obtained were in agreement with what usually occurs industrially during food 77 preservation—microbial3.4.2. Growth Inhibition Activity spoilage of may Crude still B occuriopreservatives even though From a specific Individual food Yeasts environment against contains 78 aConsortia preservation of Spoilage agent. Yeast In this case, the occurrence of spoilage organisms could be attributed to the resistance of spoilage organisms to the synthetic preservation agent used. Furthermore, a specific 79 Since there was a broad-spectrum growth inhibition activity from single yeasts, the efficacy of preservation agent could be used in targeting specific spoilage organisms; however, it may happen 80 the biopreservation extracts against the consortia of spoilage yeasts was assessed. All crude that a different spoilage organism contaminates the manufactured product. When this phenomenon 81 biopreservation samples showed growth inhibition activity against the spoilage yeast consortia, SC1- occurs, the efficacy of the preservation agent is retained, but the biocontrol efficiency may seem lost 82 SC4, albeit, at different efficacy levels (Figure 4a), proving the hypothesis that crude biopreservatives due to the incorrect microbial target or microbial resistance to the crude biopreservatives used by 83 from a single yeast can act against a consortium of spoilage organisms. However, the highest growth the spoilage organism. Based on these reasons, more than one preservation agent can be used in the same food/beverage environment; hence the preference to use a mixture of preservation agents.9 In combating beverage spoilage suspected to occur due to the presence of D. anomala, D. bruxellensis, and Z. bailli, a combination of crude biopreservatives from Candida pyralidae Y1117 and Pichia kluyveri Y1125 or Pichia kluyveri Y1164 is recommended. Furthermore, the high efficacy observed for P. kluyveri against D. anomala and D. bruxellensis means that either P. kluyveri Y1125 or Y 1164 could be used in combating spoilage for either single or mixed spoilage organisms.

3.4.2. Growth Inhibition Activity of Crude Biopreservatives from Individual Yeasts against Consortia of Spoilage Yeast Since there was a broad-spectrum growth inhibition activity from single yeasts, the efficacy of the biopreservation extracts against the consortia of spoilage yeasts was assessed. All crude biopreservation samples showed growth inhibition activity against the spoilage yeast consortia, SC1-SC4, albeit, at different efficacy levels (Figure4a), proving the hypothesis that crude biopreservatives from a single yeast can act against a consortium of spoilage organisms. However, the highest growth inhibition was observed with C. pyralidae and P. kluyveri Y1164 against SC1 and SC3 (Figure4b). Figure4b shows the clear zones of inhibition obtained. This observation suggested that a beverage that is contaminated with a mixture of D. anomala and D. bruxellensis including a mixture of D. bruxellensis and Z. bailli, can be preserved using biopreservative from either C. pyralidae or P. kluyveri Y1164. Although the biopreservatives from P. kluyveri Y1125 can also be used in all spoilage organism combinations, a higher dose might be required compared to the quantity used if C. pyralidae or P. kluyveri Y1164 are to be used as fermenters of the crude biopreservatives of interest. Overall, it can be mentioned that each of the

84 inhibition was observed with C. pyralidae and P. kluyveri Y1164 against SC1 and SC3 (Figure 4b). 85 Figure 4b shows the clear zones of inhibition obtained. This observation suggested that a beverage 86 that is contaminated with a mixture of D. anomala and D. bruxellensis including a mixture of D. 87 bruxellensis and Z. bailli, can be preserved using biopreservative from either C. pyralidae or P. kluyveri 88 Y1164. Although the biopreservatives from P. kluyveri Y1125 can also be used in all spoilage organism Fermentation 2019, 5, 53 9 of 12 89 combinations, a higher dose might be required compared to the quantity used if C. pyralidae or P. 90 kluyveri Y1164 are to be used as fermenters of the crude biopreservatives of interest. Overall, it can be 91 mentionedbiopreservative that crude each ofsamples the biopreservative was able to inhibit crude the samples growth ofwasD. anomalaable to, D. inhibit bruxellensis the growth, and Z. of bailli D. 92 anomalasimultaneously, D. bruxellensis under the, and same Z. medium bailli simultaneo and conditions.usly under Previously, the sameC. pyralidae mediumyielded and conditions a relatively. 1 −1 93 Previously,lower growth C. inhibitionpyralidae yielded activity a (VZI)relatively of 1.05 lower L mL growth− when inhibition Yeast Peptone activity Dextrose (VZI) of (YPD) 1.05 L wasmL usedwhen as 94 Yeasta fermentation Peptone Dextrose media by (YPD Mewa-Ngongang) was used as a et fermentation al. [20]. This media study suggestsby Mewa that-Ngongang utilizing et a al. combination [20]. This 95 studyof crude suggest biopreservativess that utilizing from a dicombinationfferent yeasts of couldcrude improve biopreservatives the inhibition from activity. different yeasts could 96 improve the inhibition activity. a b

3 2.5 2 1.5 1

VZI (L/mL) VZI 0.5 0 SC1 SC2 SC3 SC4

C. pyralidae Y1117 P. kluyveri Y1125 P. kluyveri Y1164

97 FigureFigure 4. 4. ((aa)) Growth Growth inhibition inhibition activity, activity, expressed expressed as as the the volumetric volumetric zone zone of of inhibition inhibition (VZI), (VZI), of of the the 98 crudecrude biopreservatives biopreservatives from from single single yeasts yeasts against against spoilage spoilage yeast yeast consortia consortia (SC) (SC) on on grape grape extract extract agar. agar. 99 ((bb)) A Ann example example of of a a plate showing th thee VZI VZI obtained for for Candida pyralidae pyralidae Y1117Y1117 ( (1),1), PichiaPichia kluyveri kluyveri 100 Y1125Y1125 (2) (2),, and and PichiaPichia kluyveri kluyveri Y1164Y1164 (3) (3).. SC SC11 (D. (D. anomala anomala andand D. D.bruxellensis bruxellensis), SC), SC22 (D. (D.anomala anomala andand Z. 101 bailiiZ. bailii), SC),3 SC3 (D. ( D.bruxellensis bruxellensis andand Z. Z.bailii bailii), and), and SC SC44 (D. (D. anomala anomala, D., D. bruxellensis bruxellensis, ,and and Z.Z. bailii bailii).). The The values values 102 areare the the means means of of three three replicates replicates with the standard deviation ranging between 0.004 and 0.09.0.09.

103 3.4.3.3.4.3. Effect Effect of of Crude Crude Biopreservative Biopreservative Mixtures Mixtures on on the the Growth Growth Inhibition of Single Spoilage Yeasts 104 AA further further interest interest in in assess assessinging the the efficacy efficacy of of mixed mixed crude crude biopreservative biopreservativess and and their their ability ability to to 105 inhibitinhibit single single spoilage spoilage yeast yeast was was developed developed.. It It was was obser observedved that that the the spoilage spoilage yeast yeast consortia consortia were were a a 106 lotlot more more sensitive sensitive to to the the mixed mixed crude crude biopreservative biopreservative mixtures mixtures.. It It was was found found that, that, in in all all the the growth growth 107 inhibitioninhibition combination combinationss (GIC) (GIC) studied studied,, DD.. anomala anomala waswas the the most most sensitive sensitive spoilage spoilage yeast yeast,, sy symbolizedmbolized 1 108 byby a a bigger bigger VZI VZI of of 2.5 2.5 L mL −1 (Figure(Figure 5a)5a),, while while D.D. bruxellensis bruxellensis, ,and and Z.Z. bailli bailli hadhad the the least least sensitivity, sensitivity, 1 1 109 withwith a a VZI VZI of of 0.17 0.17 L LmL mL−1 −forfor GIC3 GIC3 (Figure (Figure 5a,5b).a,b). A VZI A VZI of 2.5 of L 2.5 mL L− mL1 suggests− suggests that 1 thatmL of 1 mLthe crude of the 110 biopreservativescrude biopreservatives inhibits inhibits the growth the growth of D. of anomalaD. anomala in ain 2.5 a 2.5 L of L of beverage beverage contaminated contaminated at at a a cell cell 6 1 1 111 concentrationconcentration of of 10 6 cellscells mL mL−−1. .In In addition, addition, the the least least sensitive sensitive,, i.e. i.e.,, with with a a VZI VZI of 0.17 L mL −1,, mean meantt 112 ththatat the the same same volume volume ( (11 mL mL)) of of crude crude biopreservatives biopreservatives can can only only inhibit inhibit the the growth growth of of D.D. bruxellensis bruxellensis 6 1 113 andand Z.Z. bailli bailli inin a a 0.17 0.17 L L volume volume of beverage contaminated withwith 10106 cells mL−−1.. The The model model of of the the current current 114 studystudy therefore therefore showed showed that that crude crude volumes volumes are are depende dependentnt on on the the sensitivity sensitivity of of the the spoilage spoilage organism organism 115 towardstowards the the biopreservation biopreservation agents. agents. Although Although different different levels levels of of growth growth inhibition inhibition were were observed, observed, it it 116 waswas interesting interesting to to note note that, that, if if the the mixed mixed crude crude biopreservative biopreservativess from from all all yeasts yeasts are are used, used, the the growth growth 117 inhibitioninhibition of of spoilage spoilage will will occur occur under under a a minimal minimal biopreservative biopreservative dosage. dosage. When When processing processing food food or or 118 beverages,beverages, the the addition addition of of preservative preservativess should should always always be be minimal minimal,, while while considering considering an an optimum optimum efficacy. This is employed to limit the negative impact of large quantities of the crude biopreservatives in the final product that could affect the taste, texture, and possibly the appearance of the final product.10 However, based on our observations, a mixture of crude biopreservatives can also be used to target a single spoilage organism.

119 efficacy. This is employed to limit the negative impact of large quantities of the crude 120Fermentation biopreservatives2019, 5, 53 in the final product that could affect the taste, texture, and possibly the appearance 10 of 12 121 of the final product. However, based on our observations, a mixture of crude biopreservatives can 122 also be used to target a single spoilage organism. a b

3 2.5 2 1.5

1 VZI (L/mL) VZI 0.5 0 GIC1 GIC2 GIC3 GIC4

D. anomala D. bruxellensis Z. bailli

Figure 5. (a) Values of the volumetric zone of inhibition (VZI) obtained from mixtures of 123 growthFigure inhibition 5. (a) Values combinations of the volumetric (GICs) zone against of inhibi spoilagetion (VZI) yeasts obtained (Dekkera from mixtures anomala, of growthD. bruxellensis , and 124 inhibition combinations (GICs) against spoilage yeasts (Dekkera anomala, D. bruxellensis, and Zygosaccharomyces bailii). (b) An example of a plate showing the VZI obtained for GIC1 (C. pyralidae 125 Zygosaccharomyces bailii). (b) An example of a plate showing the VZI obtained for GIC1 (C. pyralidae 126 Y1117Y1117 and andP. P. kluyveri kluyveri Y1125Y1125)) (1), (1), GIC2 GIC2 (C. pyralidae (C. pyralidae Y1117 Y1117and P. kluyveri and P. Y1164 kluyveri) (2),Y1164) GIC3 (P. (2), kluyveri GIC3 (P. kluyveri 127 Y1125Y1125 and andPichia Pichia kluyverikluyveri Y1164Y1164)) (3), (3), and and GIC4 GIC4 (C. pyralidae (C. pyralidae Y1117, P.Y1117 kluyveri, P. Y1125 kluyveri, and Y1125,P. kluyveri and P. kluyveri 128 Y1164)Y1164 (4)) (4) against against ZygosaccharomycesZygosaccharomyces bailli bailli on grapeon grape pomace pomace extract agar extract. Theagar. valuesThe presented values are presented are 129 meansmeans of of three three replicates replicates with with the the standard standard deviation deviation ranging ranging between between 0.004 and 0.0040.09. and 0.09.

1303.4.4. 3.4.4. Eff Effectect of of Crude Crude BiopreservativeBiopreservative Mixtures Mixtures on the on Growth the Growth of Spoilage of Spoilage Yeast Consortia Yeast Consortia 131 In foodIn food spoilage, spoilage, it is is reasonable reasonable to hypothesi to hypothesizeze that different that di spoilagefferent spoilageorganisms organismscan be within can be within 132 the same beverage. The efficacy of the crude biopreservative mixture was assessed against consortia the same beverage. The efficacy of the crude biopreservative mixture was assessed against consortia of 133 of spoilage yeasts. Therefore, it was observed that all growth inhibition combinations (GICs) showed 134spoilage growth yeasts. inhibition Therefore, activity at itdifferent was observed levels, depending that all on growth the spoilage inhibition combination combinationss assessed. The (GICs) showed 135growth highest inhibition growth inhibition activity activity at diff, erentindicated levels, as the depending volumetric zone on theof inhibition spoilage (VZI) combinations, was observed assessed. The 136highest for GIC2 growth (2.27 inhibitionL mL−1) and activity,GIC4 (2.21 indicated L mL−1) against as the SC3 volumetric (Figure 6a). zone All GICs of inhibition showed low (VZI), growth was observed 137 inhibition activity against1 SC4, with values of 1.55, 1.41,1 1.34, and 1.36 for GIC1, GIC2, GIC3, and for GIC2 (2.27 L mL− ) and GIC4 (2.21 L mL− ) against SC3 (Figure6a). All GICs showed low 138 GIC4, respectively. This could be due to crude biopreservative interactions in the crude growth inhibition activity against SC4, with values of 1.55, 1.41, 1.34, and 1.36 for GIC1, GIC2, 139 biopreservation mixtures. GIC3, and GIC4, respectively. This could be due to crude biopreservative interactions in the crude biopreservation mixtures. a b

a b

2.5

2

1.5

1 VZI (L/mL) VZI 0.5 11 0 SC1 SC2 SC3 SC4 GIC 1 GIC 2 GIC 3 GIC 4

FigureFigure 6. ( 6.a )(a Values) Values of of the the volumetric zone zone of inhibition of inhibition (VZI) (VZI) obtained obtained from growth from inhibition growth inhibition combinationscombinations (GICs), (GICs), i.e., i.e. GIC1,, GIC1,C. C. pyralidaepyralidae Y1117Y1117 and and P. P.kluyveri kluyveri Y1125Y1125 (1); GIC2, (1); GIC2, C. pyralidaeC. pyralidae Y1117 Y1117 and P. kluyveriand P.Y1164 kluyveri (2); Y1164 GIC3, (2); GIC3,P. kluyveri P. kluyveriY1125 Y1125 and andPichia Pichia kluyveri kluyveri Y1164 (3) (3);; and and GIC4, GIC4, C. pyralidaeC. pyralidae Y1117, Y1117, P. kluyveri Y1125, and P. kluyveri Y1164 (4) against spoilage yeast consortia (SC), i.e., SC1 (D. P. kluyveri Y1125, and P. kluyveri Y1164 (4) against spoilage yeast consortia (SC), i.e., SC1 (D. anomala anomala and D. bruxellensis), SC2 (D. anomala and Z. bailii), SC3 (D. bruxellensis and Z. bailii), and SC4 D. bruxellensis D. anomala Z. bailii D. bruxellensis Z. bailii D. anomala and (D. anomala, D. ),bruxellensis SC2 ( , and Z. bailiiand). (b) An example), SC3 (of a plate showingand the VZI obtained), and from SC4 ( , D. bruxellensisthe GICs against, and Z. SCs. bailii The). (b values) An examplepresented of are a the plate means showing of three the replicates VZI obtained with the from standard the GICs against SCs.deviation The values ranging presented between 0.004 are theand 0.09. means of three replicates with the standard deviation ranging between 0.004 and 0.09. In a review by Kumar and Jagadeesh [23], several microbial consortia against phytopathogens were highlighted. The microbial consortia reported thus far against Botrytis, Colletotrichum, Rhizoctoniasolani, and Pyriculariaoryzae species were composed of two or more species of yeasts and/or bacteria acting on wounded fruits [23–27]. Comparing the results obtained from this work with those reported on phytopathogens, it could be suggested that microbial consortia cannot only be composed of two strains of microorganisms. Furthermore, this study also suggests that crude biopreservatives from yeasts, without supplementation by chemical preservatives, could be effective against beverage spoilage yeasts, even when they are in a consortium.

4. Conclusion and Recommendations Subsequent to finding the conditions where the crude biopreservatives were stable, the activity of the individual, as well as GICs, was dependent on the composition of the SCs. Our findings showed the potential of GICs from C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164, as an alternative to synthetic chemicals as preservatives in food, including beverages susceptible to contamination by D. bruxellensis, Z. baillii, and D. anomala. Future research should focus on testing the reported GICs against other spoilage microorganisms and toxicological studies should be performed to assess the impact of these GICs on human health, as well as the microorganisms associated with the human gastrointestinal tract.

Author Contributions: Conceptualization, M.M.N. and H.W.d.P.; methodology and software, M.M.N., S.K.O. Ntwampe, and B.S.C..; validation, S.K.O. Ntwampe, H.W.d. P., B.S. C., and N.P.J.; formal analysis, M.M.N., E.H., and U.F. Hutchinson; investigation, M.M.N. and U.F.H.; resources, N.P.J., H.W.d. P., and S.K.O. Ntwampe; data curation, B.S.C., M.M.N., and E.H.; writing—original draft preparation, M.M.N.; visualization, review and editing, S.K.O. Ntwampe, H.W.d. P., N.P.J., B.S.C., E.H., and U.F.H.; supervision, S.K.O. Ntwampe, H.W.d. P., and N.P.J.; project administration, N.P.J., H.W.d. P., and S.K.O. Ntwampe; funding acquisition, N.P.J., H.W.d. P., and S.K.O. Ntwampe.

12

Fermentation 2019, 5, 53 11 of 12

In a review by Kumar and Jagadeesh [23], several microbial consortia against phytopathogens were highlighted. The microbial consortia reported thus far against Botrytis, Colletotrichum, Rhizoctoniasolani, and Pyriculariaoryzae species were composed of two or more species of yeasts and/or bacteria acting on wounded fruits [23–27]. Comparing the results obtained from this work with those reported on phytopathogens, it could be suggested that microbial consortia cannot only be composed of two strains of microorganisms. Furthermore, this study also suggests that crude biopreservatives from yeasts, without supplementation by chemical preservatives, could be effective against beverage spoilage yeasts, even when they are in a consortium.

4. Conclusions and Recommendations Subsequent to finding the conditions where the crude biopreservatives were stable, the activity of the individual, as well as GICs, was dependent on the composition of the SCs. Our findings showed the potential of GICs from C. pyralidae Y1117, P. kluyveri Y1125, and P. kluyveri Y1164, as an alternative to synthetic chemicals as preservatives in food, including beverages susceptible to contamination by D. bruxellensis, Z. baillii, and D. anomala. Future research should focus on testing the reported GICs against other spoilage microorganisms and toxicological studies should be performed to assess the impact of these GICs on human health, as well as the microorganisms associated with the human gastrointestinal tract.

Author Contributions: Conceptualization, M.M.-N. and H.W.d.P.; methodology and software, M.M.-N., S.K.O.N., and B.S.C.; validation, S.K.O.N., H.W.d.P., B.S.C., and N.P.J.; formal analysis, M.M.-N., E.H., and U.F. Hutchinson; investigation, M.M.-N. and U.F.H.; resources, N.P.J., H.W.d.P., and S.K.O.N.; data curation, B.S.C., M.M.-N., and E.H.; writing—original draft preparation, M.M.-N.; visualization, review and editing, S.K.O.N., H.W.d.P., N.P.J., B.S.C., E.H., and U.F.H.; supervision, S.K.O.N., H.W.d.P., and N.P.J.; project administration, N.P.J., H.W.d.P., and S.K.O.N.; funding acquisition, N.P.J., H.W.d.P., and S.K.O.N. Funding: Financial support was provided by the National Research Foundation (NRF) and the Agricultural Research Council (ARC) of South Africa. Acknowledgments: The authors would like to thank the Agricultural Research Council, the National Research Foundation (NRF), and the Bioresource Engineering Research Group (BioERG), Department of Biotechnology, Cape Peninsula University of Technology for funding and infrastructure. The authors thank all students, interns, technicians, and research assistants for their contribution. Conflicts of Interest: The authors hereby declare that they have no conflict of interest.

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