Propionibacteria as promising tools for the production of pro-bioactive scotta: a proof-of-concept study.

Dairy propionibacteria are Gram positive Actinomycetota, routinely utilized as starters in Swiss type cheese making and highly appreciated for their probiotic properties and health promoting effects. In this work, within the frame of a circular economy approach, 47 Propionibacterium and Acidipropionibacterium spp. were isolated from goat cheese and milk, and ewe rumen liquor, and characterized in view of their possible utilization for the production of novel pro-bioactive food and feed on scotta, a lactose rich substrate and one of the main by-products of the dairy industry. The evaluation of the Minimum Inhibitory Concentration (MIC) of 13 among the most common antibiotics in clinical practice revealed a general susceptibility to ampicillin, gentamycin, streptomycin, vancomycin, chloramphenicol, and clindamycin while confirming a lower susceptibility to aminoglycosides and ciprofloxacin. Twenty-five isolates, that proved capable of lactose utilization as the sole carbon source, were then characterized for functional and biotechnological properties. Four of them, ascribed to Propionibacterium freudenreichii species, and harboring resistance to bile salts (growth at 0.7-1.56 mM of unconjugated bile salts), acid stress (>80% survival after 1 h at pH 2), osmostress (growth at up to 6.5% NaCl) and lyophilization (survival rate > 80%), were selected and inoculated in scotta. On this substrate the four isolates reached cell densities ranging from 8.11 ± 0.14 to 9.45 ± 0.06 Log CFU mL-1 and proved capable of producing different vitamin B9 vitamers after 72 h incubation at 30°C. In addition, the semi-quantitative analysis following the metabolomics profiling revealed a total production of cobalamin derivatives (vitamin B12) in the range 0.49-1.31 mg L-1, thus suggesting a full activity of the corresponding biosynthetic pathways, likely involving a complex interplay between folate cycle and methylation cycle required in vitamin B12 biosynthesis. These isolates appear interesting candidates for further ad-hoc investigation regarding the production of pro-bioactive scotta.

Waste Citrus limon Leaves as Source of Essential Oil Rich in Limonene and Citral: Chemical Characterization, Antimicrobial and Antioxidant Properties, and Effects on Cancer Cell Viability.

This study investigated chemical composition, cytotoxicity in normal and cancer cells, and antimicrobial and antioxidant activity of the essential oil (EO) isolated by hydrodistillation from the discarded leaves of lemon (Citrus limon) plants cultivated in Sardinia (Italy). The volatile chemical composition of lemon leaf EO (LLEO) was analyzed with gas chromatography-mass spectrometry combined with flame ionization detection (GC/MS and GC/FID). The most abundant component of LLEO was limonene (260.7 mg/mL), followed by geranial (102.6 mg/mL) and neral (88.3 mg/mL). The antimicrobial activity of LLEO was tested using eight bacterial strains and two types of yeasts by a microdilution broth test. Candida albicans showed the greatest susceptibility (MIC = 0.625 µL/mL) and Listeria monocytogenes and Staphylococcus aureus were inhibited at low LLEO concentration (MIC values from 2.5 to 5 µL/mL). The C. limon leaf EO displayed radical scavenging ability (IC50 value of 10.24 mg/mL) in the 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) assay. Furthermore, the LLEO impact on cell viability was explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in cancer HeLa cells, A375 melanoma cell line, normal fibroblasts (3T3 cells), and keratinocytes (HaCaT cells). LLEO, at 24 h of incubation, significantly reduced viability from 25 µM in Hela cells (33% reduction) and A375 cells (27%), greatly affecting cell morphology, whereas this effect was found from 50 µM on 3T3 fibroblasts and keratinocytes. LLEO's pro-oxidant effect was also established in HeLa cells by 2',7'-dichlorodihydrofluorescein diacetate assay.

Effect of Gaseous Citral on Table Grapes Contaminated by Rhizopus oryzae ITEM 18876.

Rhizopus oryzae is responsible for rapidly producing a deliquescent appearance in grape berries, generally favoured by cold chain interruptions. To counteract fruit spoilage and to meet consumer acceptance, innovative strategies based on the application of natural compounds are ongoing. Due to their biological activities, including antimicrobial ones, natural flavour compounds extend the shelf life and improve the nutritional value as well as the organoleptic properties of foods. Thus, in this work, the application of the antimicrobial citral, a flavor component of monoterpenes identified in plant and fruit essential oils, was developed and validated against one spoiler of R. oryzae. Citral, as pure compound, was first investigated in vitro against R. oryzae ITEM 18876; then, concentrations equal to the minimal inhibitory concentration (MIC) and 4-fold MIC (4MIC) value were applied on the table grape cv Italia infected with this strain and stored. The MIC value was equal to 0.0125 µL/cm3; both citral concentrations (0.0125 and 0.05 µL/cm3) were effective in counteracting the microbial decay of infected table grapes over the storage period. The HS-SPME/GC-MS method showed citral persistence in the head space of plastic trays with the infected samples; as expected, a higher content of citral isomers was found in the sample treated with 4MIC value. In conclusion, citral revealed its efficacy to counteract the onset of soft rot by R. oryzae ITEM 18876 under storage conditions. Thus, it could be successfully exploited to develop an active packaging or natural preservatives to extend table grape shelf life without affecting its quality and sensory characteristics, whilst also satisfying the consumer demand for natural preservative agents.

Co-Occurrence of Regulated and Emerging Mycotoxins in Corn Silage: Relationships with Fermentation Quality and Bacterial Communities.

Sixty-four corn silages were characterized for chemicals, bacterial community, and concentrations of several fungal metabolites. Silages were grouped in five clusters, based on detected mycotoxins, and they were characterized for being contaminated by (1) low levels of Aspergillus- and Penicillium-mycotoxins; (2) low levels of fumonisins and other Fusarium-mycotoxins; (3) high levels of Aspergillus-mycotoxins; (4) high levels of non-regulated Fusarium-mycotoxins; (5) high levels of fumonisins and their metabolites. Altersetin was detected in clusters 1, 3, and 5. Rugulusovin or brevianamide F were detected in several samples, with the highest concentration in cluster 3. Emodin was detected in more than 50.0% of samples of clusters 1, 3 and 5, respectively. Kojic acid occurred mainly in clusters 1 and 2 at very low concentrations. Regarding Fusarium mycotoxins, high occurrences were observed for FB3, FB4, FA1, whereas the average concentrations of FB6 and FA2 were lower than 12.4 µg/kg dry matter. Emerging Fusarium-produced mycotoxins, such as siccanol, moniliformin, equisetin, epiequisetin and bikaverin were detected in the majority of analyzed corn silages. Pestalotin, oxaline, phenopirrozin and questiomycin A were detected at high incidences. Concluding, this work highlighted that corn silages could be contaminated by a high number of regulated and emerging mycotoxins.

Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins.

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows ß-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25-2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.

Antimicrobial Activity and Chemical Characterization of a Non-Polar Extract of Saffron Stamens in Food Matrix.

The production of saffron spice generates large quantities of plant by-products: over 90% of the plant material collected is discarded, and a consideration fraction of this waste is plant stamens. This work investigated the chemical composition and the antimicrobial activities of the non-polar fraction extracted from four different saffron flower stamens. The chemical composition of ethereal extracts of the saffron stamens was qualitatively assessed by means of gas-chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses. These analyses revealed ethereal extracts to possess a high polyunsaturated fatty acid content. In vitro antibacterial activity of stamen extracts showed no large differences between Gram-positive and Gram-negative bacteria in terms of minimal inhibitory concentration (MIC). In food matrix microbial analysis of the bacterial strains belonging to the main foodborne pathogen species, including Staphylococcus aureus DSM 20231, Escherichia coli DSM 30083, and Listeria monocytogenes DSM 20600, using low-fat UHT milk, revealed a statistically significant reduction in the number of cells (particularly for E. coli and S. aureus with a complete elimination of the population of the two target bacteria following incubation in diethyl ether extracts of saffron stamen (DES) at high concentrations tested, both at 37 °C and 6 °C (for 48 h and 7 days, respectively). A synergic effect was observed when the pathogens were incubated at 6 °C with DES. This work shows these by-products to be excellent sources of bioactive compounds, which could be exploited in high-added-value products, such as food, cosmetics, and drugs.

Preservation, Characterization and Exploitation of Microbial Biodiversity of Agri-Food and Environmental Interest.

This Special Issue collects original contributions in the form of review or research articles, dealing with different aspects of the preservation, characterization and exploitation of the biodiversity of bacteria, yeast, algae and filamentous fungi of different origins [...].

Production of a lyophilized ready-to-use yeast killer toxin with possible applications in the wine and food industries.

Kpkt is a yeast killer toxin, naturally produced by Tetrapisispora phaffii, with possible applications in winemaking due to its antimicrobial activity on wine-related yeasts including Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. Here, Kpkt coding gene was expressed in Komagataella phaffii (formerly Pichia pastoris) and the bioreactor production of the recombinant toxin (rKpkt) was obtained. Moreover, to produce a ready-to-use preparation of rKpkt, the cell-free supernatant of the K. phaffii recombinant killer clone was 80-fold concentrated and lyophilized. The resulting preparation could be easily solubilized in sterile distilled water and maintained its killer activity for up to six months at 4 °C. When applied to grape must, it exerted an extensive killer activity on wild wine-related yeasts while proving compatible with the fermentative activity of actively growing Saccharomyces cerevisiae starter strains. Moreover, it displayed a strong microbicidal effect on a variety of bacterial species including lactic acid bacteria and food-borne pathogens. On the contrary it showed no lethal effect on filamentous fungi and on Ceratitis capitata and Musca domestica, two insect species that may serve as non-mammalian model for biomedical research. Based on these results, bioreactor production and lyophilization represent an interesting option for the exploitation of this killer toxin that, due to its spectrum of action, may find application in the control of microbial contaminations in the wine and food industries.

The role of microorganisms on biotransformation of brewers' spent grain.

Brewers' spent grain (BSG) is the most abundant by-product of brewing. Due to its microbiological instability and high perishability, fresh BSG is currently disposed of as low-cost cattle feed. However, BSG is an appealing source of nutrients to obtain products with high added value through microbial-based transformation. As such, BSG could become a potential source of income for the brewery itself. While recent studies have covered the relevance of BSG chemical composition in detail, this review aims to underline the importance of microorganisms from the stabilization/contamination of fresh BSG to its biotechnological exploitation. Indeed, the evaluation of BSG-associated microorganisms, which include yeast, fungi, and bacteria, can allow their safe use and the best methods for their exploitation. This bibliographical examination is particularly focused on the role of microorganisms in BSG exploitation to (1) produce enzymes and metabolites of industrial interest, (2) supplement human and animal diets, and (3) improve soil fertility. Emerging safety issues in the use of BSG as a food and feed additive is also considered, particularly considering the presence of mycotoxins.Key points• Microorganisms are used to enhance brewers' spent grain nutritional value.• Knowledge of brewers' spent grain microbiota allows the reduction of health risks. Graphical abstract.

Yeast biofilm in food realms: occurrence and control.

In natural environments, microorganisms form microbial aggregates called biofilms able to adhere to a multitude of different surfaces. Yeasts make no exception to this rule, being able to form biofilms in a plethora of environmental niches. In food realms, yeast biofilms may cause major problems due to their alterative activities. In addition, yeast biofilms are tenacious structures difficult to eradicate or treat with the current arsenal of antifungal agents. Thus, much effort is being made to develop novel approaches to prevent and disrupt yeast biofilms, for example through the use of natural antimicrobials or small molecules with both inhibiting and dispersing properties. The aim of this review is to provide a synopsis of the most recent literature on yeast biofilms regarding: (i) biofilm formation mechanisms; (ii) occurrence in food and in food-related environments; and (iii) inhibition and dispersal using natural compounds, in particular.

Composition and functional profiling of the microbiota in the casts of Eisenia fetida during vermicomposting of brewers' spent grains.

Vermicomposting is a cost-effective biotechnology for the management of organic wastes that relies on the activity of earthworms and their associated microbiota. Here, the microbiotas of the earthworm Eisenia fetida fed with brewers' spent grains (FBSG), cow manure (FCM) and a mix of brewers' spent grains/cow manure (FMIX), were identified by high-throughput DNA sequencing (16S rRNA). Bacterial community variance was correlated with the pH and the organic carbon content of the rearing substrates. FBSG microbiota was enriched in Paenibacillaceae, Enterobacteriaceae, Chitinophagaceae and Comamonadaceae. In addition, FBSG microbiota had a predicted higher abundance of genes involved in cellulose degradation as well as in the nitrogen cycle and showed higher utilization of ammonia and nitrate. Results obtained will allow to optimize the vermicomposting of brewers' spent grains and to evaluate the effect of vermicompost addition on nutrient dynamics in soil.

Antimicrobial activity of gaseous Citrus limon var pompia leaf essential oil against Listeria monocytogenes on ricotta salata cheese.

Contamination by Listeria monocytogenes is a particularly challenging problem in the food industry due to the ability of the bacterium to develop under conditions normally used for food preservation. Here, we show that the gaseous phase of Citrus limon var pompia leaf essential oil (hereafter PLEO) exerts specific anti-Listeria activity on ricotta salata cheese stored at 5 °C. The synergic effect of gaseous PLEO treatment and refrigeration was first confirmed in vitro on L. monocytogenes strains treated for 3 h with gaseous PLEO and then stored at 5 °C. Ricotta cheese was then inoculated with L. monocytogenes strains and subjected to hurdle technology with different concentrations of gaseous PLEO. Cell counts revealed gaseous PLEO to exert a bactericidal effect on L. monocytogenes 20600 DSMZ and a bacteriostatic effect on a mix of L. monocytogenes strains. Scanning and transmission electron microscopy analyses of L. monocytogenes cells suggested that gaseous PLEO targets the bacterial cell wall and plasma membrane. Chemical analyses of the liquid and vapor phases of PLEO indicated linalyl acetate to be the predominant compound, followed by limonene and the two isomers of citral, whereas EO composition analysis, although generally in line with previous findings, showed the presence of linalyl acetate for the first time. Solid-phase microextraction coupled with gas chromatography confirmed the presence of all crude oil components in the headspace of the box.

Bacterial Biodiversity of Extra Virgin Olive Oils and Their Potential Biotechnological Exploitation.

Bacterial diversity of 15 extra virgin olive oils, obtained from different Italian varieties, including Frantoio, Coratina, Bosana, and Semidana, was analyzed in this study. All bacterial isolates were genotyped using RAPD and REP-PCR method and grouped by means of cluster analyses. Sequencing of 16S rDNA of 51 isolates, representative of 36 clusters, led to the identification of Bacillus spp., Brevibacillus spp., Micrococcus spp., Staphylococcus spp., Pantoea spp., Kocuria spp., Lysinbacillus spp., and Lactobacillus spp., most of which reported for first time in olive oils. Phenotypic characterization of the 51 isolates, some of which ascribed to potentially probiotic species, indicate that two of them have beta-glucosidase activity while 37% present lipolytic activity. Preliminary evaluation of probiotic potential indicates that 31% of the isolates show biofilm formation ability, 29% acidic pH resistance, and 25% bile salt resistance. Finally, 29% of the isolates were sensitive to antibiotics while the remaining 71%, that include bacterial species well-recognized for their ability to disseminate resistance genes in the environment, showed a variable pattern of antibiotic resistance. The results obtained underline that microbial diversity of extra virgin olive oils represents an unexpected sink of microbial diversity and poses safety issues on the possible biotechnological exploitation of this microbial biodiversity.

Rhodotorula mucilaginosa C2.5t1 Modulates Carotenoid Content and CAR Genes Transcript Levels to Counteract the Pro-Oxidant Effect of Hydrogen Peroxide.

In order to contribute to the elucidation of the biological role of carotenoids, the cellular response to hydrogen peroxide was analyzed in the red yeast R. mucilaginosa. For that, the wild strain C2.5t1, that produces ß-carotene, torulene, and torularhodin, and the albino mutant 200A6 that is incapable of producing detectable amounts of these carotenoids, were grown in the presence of increasing concentrations of hydrogen peroxide. In spite of the difference in carotenoid content, the two strains presented comparable resistance to the pro-oxidant that showed a minimum inhibitory concentration of 6 mM. When subject to 1 h treatment with 16 mM hydrogen peroxide the two strains increased catalase but not superoxide activity, suggesting that catalase plays a major role in cell protection in both the wild strain and the albino mutant. Moreover, C2.5t1 reduced its carotenoid content by about 40% upon hydrogen peroxide treatment. This reduction in carotenoids was in agreement with a significant decrease of the transcript levels of genes involved in carotenoid biosynthesis. Since an excess of ß-carotene may enhance reactive oxygen species toxicity, these results suggest that C2.5t1 modulates carotenoid content to counteract the pro-oxidant effect of hydrogen peroxide.

The administration of L-cysteine and L-arginine inhibits biofilm formation in wild-type biofilm-forming yeast by modulating FLO11 gene expression.

Microbial biofilms are undesired in food manufacturing, drinking water distribution systems, and clinical realms. Yeast biofilms are particularly problematic because of the strong capacity of yeast cells to adhere to abiotic surfaces, cells, and tissues. Novel approaches have been developed over recent years to prevent the establishment of microbial biofilms, such as through the use of small molecules with inhibiting and dispersing properties. Here, we studied the inhibitory activity of 11 different amino acids on the biofilm formation ability of three wild-type Saccharomyces cerevisiae strains and the reference strain ∑1278b. Subsequent evaluation of different concentrations of the two most effective amino acids, namely, arginine and cysteine, revealed that they acted in different ways. Arginine prevented biofilm formation by reducing FLO11 gene expression; its addition did not affect cell viability and was even found to enhance cell metabolism (vitality marker) as determined by phenotype microarray (PM) analysis. On the contrary, the addition of cysteine reduced both cell viability and vitality as well as FLO11 expression. Thus, the use of cysteine and arginine as agents against biofilm formation can be diversified depending on the most desired action towards yeast growth.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.

Green pesticides based on cinnamate anion incorporated in layered double hydroxides and dispersed in pectin matrix.

This paper reports the preparation of green pesticides based on nano-hybrids composed of a Layered Double Hydroxide (LDH) with cinnamate anion. The dispersion into a pectin matrix was obtained using high energy ball milling in wet conditions. Structure and physical properties of the fillers and the composites films were evaluated. Controlled release of cinnamate was followed using UV spectrophotometry and the release kinetics were found to be dependent on the filler loading. The experimental results were analyzed by the Gallagher-Corrigan model. Antimicrobial activity was evaluated on different bacterial strains, as well as plant pathogens belonging to the genus Phytophthora using modified agar diffusion, broth microdilution and dual culture methods, respectively. Experimental results suggested the possibility to use the analyzed composites as green protective coatings for crops' protection.

Isolation and characterization of microorganisms and volatiles associated with Moroccan saffron during different processing treatments.

Saffron may be spoiled by a variety of microorganisms during cultivation, harvesting, and post harvesting. As saffron can be dried and stored in different ways, this preliminary study explored the natural microbiota present in Moroccan saffron when subjected to different drying techniques. An analysis of the carotenoid-derived volatiles present in the saffron was also carried out. The culturable microbiota of the saffron samples dried using different methods, namely in the shade (also called natural), in the sun, or in the oven, were studied using classical and molecular approaches. The effect of the drying methods on head-space chemical volatiles was also determined. Eighty-two isolates grown in the different culture media were chosen from the colonies, and genotype analysis grouped the microorganisms into 58 clusters, revealing a wide diversity. Out of the 82 isolates, 75 belonged to the Bacillaceae family. The other isolates were distributed within the Dietziaceae, Paenibacillaceae and Carnobacteriaceae families. The dominant species was Bacillus simplex, which was detected in all samples, regardless of the drying method used. Lysinibacillus macroides was dominant in the sun-dried saffron. No pathogens were isolated, but an isolate belonging to Dietzia maris, a potential human pathogenic species, was detected. The biodiversity indexes were linked to the drying method and generally decreased as the intensity of the treatment increased. The results of this preliminary work show that the different drying methods strongly influenced the microbiota and affect the saffron volatile profile. Further analysis will be needed to determine possible effects of selected microbiota on saffron volatiles.

Microbial biodiversity of Sardinian oleic ecosystems.

The olives are rich in microorganisms that, during the extraction process may persist in the oils and can influence their physicochemical and sensory characteristics. In this work, and for the first time, we isolated and identified microbial species, yeast and bacteria, present during the production process in four Sardinian (Italy) oleic ecosystems. Among these varieties, we found that Nera di Gonnos was associated to the highest microbial biodiversity, which was followed by Bosana, Nocellara del Belice and Semidana. Among the different microbial species isolated, some are specific of olive ecological niches, such as Cryptococcus spp and Serratia spp; and others to olive oils such as Candida spp and Saccharomyces. Some other species identified in this work were not found before in oleic ecosystems. The enzymatic analyses of yeast and bacteria showed that they have good ß-glucosidase activity and yeast also showed good ß-glucanase activity. The majority of bacteria presented lipolytic and catalase activities while in yeast were species-specific. Interestingly, yeast and bacteria isolates presented a high resistance to bile acid, and about 65% of the yeast were able to resist at pH 2.5 for 2 h. Finally, bacteria showed no biofilm activity compared to yeast.

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris).

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with ß-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has ß-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.