Chemical Profile Characterization of Fruit and Vegetable Juices after Fermentation with Probiotic Strains.

Lactic acid bacteria (LAB) are widely applied for fermentation purposes in dairy and non-dairy food matrices with beneficial technological and health-promoting properties. This study describes the effect of two lactic acid bacteria, namely, Lactiplantibacillus paracasei SP5 and Pediococcus pentosaceus SP2, on the phenolic profiles, antioxidant activities, total phenolic content (TPC), carotenoid content, and sensorial profile of two different mixed fruit juices. After 48 h of fermentation, both LABs retained viability over 9 Log CFU/mL in both juices. The TPC, zeaxanthin + lutein, ß-carotene content, and antioxidant activity (AA) were elevated for both LABs and mixed juices after 48 h of fermentation compared to control samples. Regarding the phenolic profile, both juices exhibited a significant decrease in chlorogenic acid levels, while quinic acid and tyrosol concentrations showed notable increases.

Novel Wild-Type Pediococcus and Lactiplantibacillus Strains as Probiotic Candidates to Manage Obesity-Associated Insulin Resistance.

As the food and pharmaceutical industry is continuously seeking new probiotic strains with unique health properties, the aim of the present study was to determine the impact of short-term dietary intervention with novel wild-type strains, isolated from various sources, on high-fat diet (HFD)-induced insulin resistance. Initially, the strains were evaluated in vitro for their ability to survive in simulated gastrointestinal (GI) conditions, for adhesion to Caco-2 cells, for bile salt hydrolase secretion, for cholesterol-lowering and cellular cholesterol-binding ability, and for growth inhibition of food-borne pathogens. In addition, safety criteria were assessed, including hemolytic activity and susceptibility to antibiotics. The in vivo test on insulin resistance showed that mice receiving the HFD supplemented with Pediococcus acidilactici SK (isolated from human feces) or P. acidilactici OLS3-1 strain (isolated from olive fruit) exhibited significantly improved insulin resistance compared to HFD-fed mice or to the normal diet (ND)-fed group.

Exploiting the beneficial effects of Salvia officinalis L. extracts in human health and assessing their activity as potent functional regulators of food microbiota.

Salvia officinalis L. has attracted scientific and industrial interest due to its pharmacological properties. However, its detailed phytochemical profile and its correlation with beneficial effects in the human microbiome and oxidative stress remained elusive. To unveil this, S. officinalis was collected from the region of Epirus and its molecular identity was verified with DNA barcoding. Phytochemical profile for both aqueous and ethanol-based extracts was determined by high-pressure liquid chromatography-tandem mass spectrometry and 103 phytochemicals were determined. The effect of S. officinalis extracts as functional regulators of food microbiota by stimulating the growth of Lacticaseibacillus rhamnosus strains and by suppressing evolution of pathogenic bacteria was verified. Furthermore, we recorded that both extracts exhibited a significant cellular protection against H2O2-induced DNA damage. Finally, both extracts exhibited strong inhibitory effect towards LDL oxidation. This study provides a comprehensive characterization of S. officinalis on its phytochemical components as also its potential impact in human microbiome and oxidative stress.

Pathogenic Biofilm Removal Potential of Wild-Type Lacticaseibacillus rhamnosus Strains.

The emergence of antimicrobial resistance remains one of the greatest public health concerns. Biofilm formation has been postulated as a mechanism of microbial pathogens to resist antimicrobial agents. Lactic Acid Bacteria (LAB) and their metabolites have been proposed to combat bacterial biofilms due to their antimicrobial activity. In this vein, the aim of the present study was to investigate the biofilm removal potential of cell-free supernatants (CFSs) of five wild-type Lacticaseibacillus rhamnosus strains, isolated from Greek natural products, in comparison to the commercially available L. rhamnosus GG strain, against biofilms formed by common foodborne pathogens (Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus). The biofilm removal activity of LAB was assessed on a two-day-old mature biofilm using a microtiter plate-based procedure. Both non-neutralized and neutralized CFSs removed biofilms in a concentration-dependent manner. The biofilm removal activity of the non-neutralized CFSs was significantly higher compared to the neutralized CFSs, as expected, with ranges of 60-89% and 30-80%, respectively. The biofilm removal efficiency of L. rhamnosus OLXAL-3 was significantly higher among the wild-type L. rhamnosus strains tested (20-100% v/v). In conclusion, our results suggest the great potential of the application of wild-type L. rhamnosus strain' CFSs as effective natural agents against pathogenic bacterial biofilms.

COVID-19 on the spectrum: a scoping review of hygienic standards.

The emergence of COVID-19 in Wuhan, China, rapidly escalated into a worldwide public health crisis. Despite numerous clinical treatment endeavors, initial defenses against the virus primarily relied on hygiene practices like mask-wearing, meticulous hand hygiene (using soap or antiseptic solutions), and maintaining social distancing. Even with the subsequent advent of vaccines and the commencement of mass vaccination campaigns, these hygiene measures persistently remain in effect, aiming to curb virus transmission until the achievement of herd immunity. In this scoping review, we delve into the effectiveness of these measures and the diverse transmission pathways, focusing on the intricate interplay within the food network. Furthermore, we explore the virus's pathophysiology, considering its survival on droplets of varying sizes, each endowed with distinct aerodynamic attributes that influence disease dispersion dynamics. While respiratory transmission remains the predominant route, the potential for oral-fecal transmission should not be disregarded, given the protracted presence of viral RNA in patients' feces after the infection period. Addressing concerns about food as a potential viral vector, uncertainties shroud the virus's survivability and potential to contaminate consumers indirectly. Hence, a meticulous and comprehensive hygienic strategy remains paramount in our collective efforts to combat this pandemic.

Sentiment analysis of epidemiological surveillance reports on COVID-19 in Greece using machine learning models.

The present research deals with sentiment analysis performed with Microsoft Azure Machine Learning Studio to classify Facebook posts on the Greek National Public Health Organization (EODY) from November 2021 to January 2022 during the pandemic. Positive, negative and neutral sentiments were included after processing 300 reviews. This approach involved analyzing the words appearing in the comments and exploring the sentiments related to daily surveillance reports of COVID-19 published on the EODY Facebook page. Moreover, machine learning algorithms were implemented to predict the classification of sentiments. This research assesses the efficiency of a few popular machine learning models, which is one of the initial efforts in Greece in this domain. People have negative sentiments toward COVID surveillance reports. Words with the highest frequency of occurrence include government, vaccinated people, unvaccinated, telephone communication, health measures, virus, COVID-19 rapid/molecular tests, and of course, COVID-19. The experimental results disclose additionally that two classifiers, namely two class Neural Network and two class Bayes Point Machine, achieved high sentiment analysis accuracy and F1 score, particularly 87% and over 35%. A significant limitation of this study may be the need for more comparison with other research attempts that identified the sentiments of the EODY surveillance reports of COVID in Greece. Machine learning models can provide critical information combating public health hazards and enrich communication strategies and proactive actions in public health issues and opinion management during the COVID-19 pandemic.

Chemical Composition, Antibacterial and Antibiofilm Actions of Oregano (Origanum vulgare subsp. hirtum) Essential Oil against Salmonella Typhimurium and Listeria monocytogenes.

Essential oils (EOs) are plant mixtures that are known to present strong bioactivities, including a wide antimicrobial action. Biofilms are microbial sessile structures that represent the default mode of growth of microorganisms in most environments. This study focused on the antimicrobial action of the EO extracted from one of the most representative oregano species, that is, Origanum vulgare (subsp. hirtum), against two important foodborne pathogens, Salmonella enterica (serovar Typhimurium) and Listeria monocytogenes. For this, the minimum inhibitory concentrations of the EO against the planktonic and biofilm growth of each bacterium were determined (MICs, MBICs), together with the minimum bactericidal and biofilm eradication concentrations (MBCs, MBECs). The EO was also analyzed for its chemical composition by gas chromatography-mass spectrometry analysis (GC-MS). The influence of EO exposure on the expression of some important virulence genes (hly, inlA, inlB and prfA) was also studied in L. monocytogenes. Results revealed a strong antibacterial and antibiofilm action with MICs and MBICs ranging from 0.03% to 0.06% (v/v) and from 0.06% to 0.13% (v/v), respectively. The application of the EO at 6.25% (v/v) for 15 min resulted in the total eradication of the biofilm cells of both pathogens. The EO was mainly composed of thymol, p-cymene, γ-terpinene and carvacrol. The 3 h exposure of L. monocytogenes planktonic cells to the EO at its MBIC (0.06% v/v) resulted in the significant downregulation of all the studied genes (p < 0.05). To sum, the results obtained advocate for the further exploitation of the antimicrobial action of oregano EO in food and health applications.

Insights into Antimicrobial and Anti-Inflammatory Applications of Plant Bioactive Compounds.

Plants have long been thought to contribute to health promotion due to their fiber and phenolic content, as well as their inherent biological potential. The bioactive derivatives of medicinal plants are a valuable resource in the fight against serious diseases all around the world. The present review focuses on the current state of knowledge on the usage and medicinal applications of plant bioactives. Issues concerning the effect of aromatic plant derivatives on human gut microbiota and their antimicrobial and anti-inflammatory potentials are discussed and worth further exploring.

Corinthian Currants Supplementation Restores Serum Polar Phenolic Compounds, Reduces IL-1beta, and Exerts Beneficial Effects on Gut Microbiota in the Streptozotocin-Induced Type-1 Diabetic Rat.

The present study aimed at investigating the possible benefits of a dietary intervention with Corinthian currants, a rich source of phenolic compounds, on type 1 diabetes (T1D) using the animal model of the streptozotocin-(STZ)-induced diabetic rat. Male Wistar rats were randomly assigned into four groups: control animals, which received a control diet (CD) or a diet supplemented with 10% w/w Corinthian currants (CCD), and diabetic animals, which received a control diet (DCD) or a currant diet (DCCD) for 4 weeks. Plasma biochemical parameters, insulin, polar phenolic compounds, and inflammatory factors were determined. Microbiota populations in tissue and intestinal fluid of the caecum, as well as fecal microbiota populations and short-chain fatty acids (SCFAs), were measured. Fecal microbiota was further analyzed by 16S rRNA sequencing. The results of the study showed that a Corinthian currant-supplemented diet restored serum polar phenolic compounds and decreased interleukin-1b (IL-1b) (p < 0.05) both in control and diabetic animals. Increased caecal lactobacilli counts (p < 0.05) and maintenance of enterococci levels within normal range were observed in the intestinal fluid of the DCCD group (p < 0.05 compared to DCD). Higher acetic acid levels were detected in the feces of diabetic rats that received the currant diet compared to the animals that received the control diet (p < 0.05). Corinthian currant could serve as a beneficial dietary component in the condition of T1D based on the results coming from the animal model of the STZ-induced T1D rat.

Novel Functional Grape Juices Fortified with Free or Immobilized Lacticaseibacillus rhamnosus OLXAL-1.

During the last decade, a rising interest in novel functional products containing probiotic microorganisms has been witnessed. As food processing and storage usually lead to a reduction of cell viability, freeze-dried cultures and immobilization are usually recommended in order to maintain adequate loads and deliver health benefits. In this study, freeze-dried (free and immobilized on apple pieces) Lacticaseibacillus rhamnosus OLXAL-1 cells were used to fortify grape juice. Juice storage at ambient temperature resulted in significantly higher (>7 log cfu/g) levels of immobilized L. rhamnosus cells compared to free cells after 4 days. On the other hand, refrigerated storage resulted in cell loads > 7 log cfu/g for both free and immobilized cells for up to 10 days, achieving populations > 109 cfu per share, with no spoilage noticed. The possible resistance of the novel fortified juice products to microbial spoilage (after deliberate spiking with Saccharomyces cerevisiae or Aspergillus niger) was also investigated. Significant growth limitation of both food-spoilage microorganisms was observed (both at 20 and 4 °C) when immobilized cells were contained compared to the unfortified juice. Keynote volatile compounds derived from the juice and the immobilization carrier were detected in all products by HS-SPME GC/MS analysis. PCA revealed that both the nature of the freeze-dried cells (free or immobilized), as well as storage temperature affected significantly the content of minor volatiles detected and resulted in significant differences in the total volatile concentration. Juices with freeze-dried immobilized cells were distinguished by the tasters and perceived as highly novel. Notably, all fortified juice products were accepted during the preliminary sensory evaluation.

Exploring the Potential of Sustainable Acid Whey Cheese Supplemented with Apple Pomace and GABA-Producing Indigenous Lactococcus lactis Strain.

This study aimed to utilize two by-products, acid whey and apple pomace, as well as an indigenous Lactococcus lactis LL16 strain with the probiotic potential to produce a sustainable cheese with functional properties. Acid whey protein cheese was made by thermocoagulation of fresh acid whey and enhancing the final product by adding apple pomace, L. lactis LL16 strain, or a mixture of both. The sensory, the physicochemical, the proteolytic, and the microbiological parameters were evaluated during 14 days of refrigerated storage. The supplementation of the cheese with apple pomace affected (p ≤ 0.05) the cheese composition (moisture, protein, fat, carbohydrate, and fiber), the texture, the color (lightness, redness, and yellowness), and the overall sensory acceptability. The addition of the presumptive probiotic L. lactis LL16 strain decreased (p ≤ 0.05) the concentration of glutamic acid, thus increasing γ-aminobutyric acid (GABA) significantly in the acid whey cheese. The supplementation with apple pomace resulted in slightly (p < 0.05) higher counts of L. lactis LL16 on day seven, suggesting a positive effect of apple pomace components on strain survival. The symbiotic effect of apple pomace and LL16 was noted on proteolysis (pH 4.6-soluble nitrogen and free amino acids) in the cheese on day one, which may have positively influenced the overall sensory acceptance.

Chemical Composition, Antioxidant, and Antibiofilm Properties of Essential Oil from Thymus capitatus Plants Organically Cultured on the Greek Island of Lemnos.

Essential oils (EOs) are mixtures of volatile plant secondary metabolites and have been exploited by humans for thousands of years for various purposes because of their many bioactivities. In this study, the EO from Thymus capitatus, a thyme species organically cultured on the Greek Island of Lemnos, was analyzed for its chemical composition (through GC-FID and GC-MS), antioxidant activity (AA), and total phenolic content (TPC), as well as its antimicrobial and antibiofilm actions against three important foodborne bacterial pathogens (Salmonella enterica ser. Typhimurium, Listeria monocytogenes, and Yersinia enterocolitica). For the latter investigations, the minimum inhibitory concentrations (MICs) and minimum biofilm inhibitory concentrations (MBICs) of the EO against the planktonic and biofilm growth of each pathogen were determined, together with the minimum biofilm eradication concentrations (MBECs). Results revealed that T. capitatus EO was rich in thymol, p-cymene, and carvacrol, presenting high AA and TPC (144.66 µmol TroloxTM equivalents and 231.32 mg gallic acid equivalents per g of EO, respectively), while its MICs and MBICs ranged from 0.03% to 0.06% v/v and 0.03% to 0.13% v/v, respectively, depending on the target pathogen. The EO was able to fully destroy preformed (mature) biofilms of all three pathogenic species upon application for 15 min, with MBECs ranging from 2.00 to 6.25% v/v. Overall, the results demonstrate that the EO of organically cultured T. capitatus presents strong antioxidant, antibacterial, and antibiofilm properties and could, therefore, be further exploited as a functional and antimicrobial natural formulation for food and health applications.

The Role of Probiotics in Inducing and Maintaining Remission in Crohn's Disease and Ulcerative Colitis: A Systematic Review of the Literature.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract affecting millions of patients worldwide. The gut microbiome partly determines the pathogenesis of both diseases. Even though probiotics have been widely used as a potential treatment, their efficacy in inducing and maintaining remission is still controversial. Our study aims to review the present-day literature about the possible role of probiotics in treating inflammatory bowel diseases in adults. This research was performed according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. We included studies concerning adult patients who compared probiotics with placebo or non-probiotic intervention. We identified thirty-three studies, including 2713 patients from fourteen countries. The role of probiotics in Crohn's disease was examined in eleven studies. Only four studies presented statistically significant results in the remission of disease, primarily when used for three to six months. On the other hand, in twenty-one out of twenty-five studies, probiotics proved effective in achieving or maintaining remission in ulcerative colitis. Supplementation with Bifidobacterium sp. or a combination of probiotics is the most effective intervention, especially when compared with a placebo. There is strong evidence supporting the usage of probiotic supplementation in patients with ulcerative colitis, yet more research is needed to justify their efficacy in Crohn's disease.

Torulaspora delbrueckii May Help Manage Total and Volatile Acidity of Santorini-Assyrtiko Wine in View of Global Warming.

Non-Saccharomyces (NS) yeasts are gaining popularity in modern winemaking for improving wine quality. Climate change is one of the biggest challenges winegrowing now faces in warm regions. Here, Lachancea thermotolerans LtS1 and Torulaspora delbrueckii TdS6 combined with Saccharomyces cerevisiae ScS13 isolated from Assyrtiko grapes from Santorini island were evaluated in grape must fermentation with the aim to mitigate major consequences of temperature rise. Different inoculation protocols were evaluated, including simultaneous and sequential mixed-strain inoculations, displaying significant variation in the chemical and kinetic characteristics. Both LtS1 and TdS6 could raise the titratable acidity (TA). TdS6 also reduced the volatile acidity (VA) and was thus chosen for further evaluation in microvinifications and pilot-scale fermentations. Consistent with lab-scale trials, sequential inoculation exhibited the longest persistence of TdS6 resulting in minimum VA levels. Diethyl succinate, ethyl propanoate, and ethyl isobutyrate were significantly increased in sequential inoculations, although a decline in the net total ester content was observed. On the other hand, significantly higher levels of TA, succinic acid, and 2-methylpropanoic were associated with sequential inoculation. The overall performance of TdS6 coupled with a high compatibility with S. cerevisiae suggests its use in the fermentation of Santorini-Assyrtiko or other high sugar musts for the production of structured dry or sweet wines.

Microbiological stability and sensorial valorization of thyme and oregano essential oils alone or combined with ethanolic pomegranate extracts in wine marinated pork meat.

In the present study, wine-based marinades containing ethanolic extract from pomegranate (Punica granatum L.), alone or in combination with two Essential Oils (Thyme & Oregano), were used for pork fillets marination and their antimicrobial activity, as well as their sensorial impact were evaluated. Likewise, the marinades exhibited promising results concerning their recorded antimicrobial activity versus Enterobacteriaceae, Total Mesophilic Bacteria, Yeasts/molds, Staphylococcus spp., Pseudomonas spp. & Lactic Acid Bacteria (LAB). The outcome demonstrated that pork fillets marinated with wine containing ethanolic extract of pomegranate and Oregano Essential Oil were more resistant to spoilage compared to all other samples; thus, their shelf-life was significantly extended (4 days in some cases). Triterpenes (maslinic, oleanolic and betulinic acid), monoterpenes (p-cymene, carvacrol, thymol, limonene), organic acids (citric & malic acid) and phenols, were the main constituents found in the plant extract, the wine and Essential Oils applied, as determined through LC-QTOF/MS and HPLC analysis. Additionally, the sensorial properties (color, tenderness, flavor and juiciness) of the marinated meat samples were not negatively influenced. Consequently, marinades of this type could be used as natural preservatives in meat products, with satisfying antimicrobial and organoleptic results.

Assessment of the Anti-Amyloidogenic Properties of Essential Oils and Their Constituents in Cells Using a Whole-Cell Recombinant Biosensor.

Essential oils exhibit numerous medicinal properties, including antimicrobial, anti-inflammatory and antioxidant effects. Recent studies also indicate that certain essential oils demonstrate anti-amyloidogenic activity against ß-amyloid, the protein implicated in Alzheimer's disease. To investigate whether the anti-aggregating properties of essential oils extend to α-synuclein, the protein involved in Parkinson's disease, we constructed and employed a whole-cell biosensor based on the split-luciferase complementation assay. We validated our biosensor by using baicalein, a known inhibitor of α-synuclein aggregation, and subsequently we tested eight essential oils commonly used in food and the hygienic industry. Two of them, citron and sage, along with their primary components, pure linalool (the main constituent in citron essential oil) and pure eucalyptol (1,8-cineole, the main constituent in sage essential oil), were able to reduce α-syn aggregation. These findings suggest that both essential oils and their main constituents could be regarded as potential components in functional foods or incorporated into complementary Parkinson's disease therapies.

Effect of Free or Immobilized Lactiplantibacillus plantarum T571 on Feta-Type Cheese Microbiome.

BACKGROUND: Cheese microbiome plays a key role in determining the organoleptic and physico-chemical properties and may be also used as an authenticity tool for distinguishing probiotic cultures. Due to significant reduction of cell viability often witnessed during food production processes and storage, immobilization is proposed to ascertain high probiotic cell loads required to confer the potential health benefits. Hence, the aim of the present study was to investigate the effect of free or immobilized Lactiplantibacillus plantarum T571 on whey protein on feta cheese microbiome. METHODS: Next-Generation Sequencing technology was used to investigate cheese microbiome. Cheese samples containing free or immobilized Lactiplantibacillus plantarum T571 (a wild type strain isolated from Feta cheese brine) on whey protein, along with products containing commercial starter culture, were analyzed. RESULTS: The results showed a great diversity of bacteria and fungi genera among the samples. An increased presence of Lactobacillus OTUs in cheese with immobilized cells on whey protein was witnessed, highlighting the survival of the strain in the final product. The immobilized culture had also a significant impact on other genera, such as Lactococcus, Leuconostoc and Debaryomyces, which are associated with improved technological characteristics and health benefits. CONCLUSIONS: Enrichment of feta cheese with immobilized potential probiotics to secure cell viability consists of an industrial challenge and leads to distinct microbiome composition that may be used as a valuable food authenticity tool.

Indigenous Lactococcus lactis with Probiotic Properties: Evaluation of Wet, Thermally- and Freeze-Dried Raisins as Supports for Cell Immobilization, Viability and Aromatic Profile in Fresh Curd Cheese.

Indigenous Lactococcus lactis enriched raisins were incorporated in fresh curd cheese in wet, thermally dried, and freeze-dried form to produce a novel probiotic dairy product. Symbiotic cheese represents a rising trend in the global market. The viability of L. lactis cells was assessed in the cheeses during storage at 4 °C for 14 days and the effect of the added enriched raisins on physicochemical parameters, microbiological characteristics, and sugar content, aromatic profile, and sensory acceptance of cheeses were evaluated. Immobilized L. lactis cells maintained viability at necessary levels (>6 log cfu/g) during storage and significantly increased the acceptability of cheese. The addition of raisins enhanced the volatile profile of cheeses with 2-furanmethanol, 1-octanol, 3-methylbutanal, 2-methylbutanal, 2-furancarboxaldehyde, 1-(2-furanyl)-ethanone, 5-methyl-2-furancarboxaldehyde. The obtained results are encouraging for the production of novel fresh cheeses with improved sensorial and nutritional characteristics on industrial and/or small industrial scale.

New Wild-Type Lacticaseibacillus rhamnosus Strains as Candidates to Manage Type 1 Diabetes.

The incidence of type 1 diabetes (T1D) has been dramatically increased in developed countries, and beyond the genetic impact, environmental factors, including diet, seem to play an important role in the onset and development of the disease. In this vein, five Lacticaseibacillus rhamnosus, isolated from traditional fermented Greek products, were screened for potential probiotic properties, aiming at maintaining gut homeostasis and antidiabetic capability to alleviate T1D symptoms. L. rhamnosus cell-free supernatants induced strong growth inhibitory activity against common food spoilage and foodborne pathogenic microorganisms, associated with several diseases, including T1D, and were also able to inhibit α-glucosidase activity (up to 44.87%), a promising property for alternatives to the antidiabetic drugs. In addition, survival rates up to 36.76% were recorded during the application of the static in vitro digestion model. The strains had no hemolytic activity and were sensitive to common antibiotics suggested by the European Food and Safety Association, apart from chloramphenicol. However, it is highly unlikely that the resistance has been acquired. In conclusion, our results suggest a great health-promoting potential of the newly isolated wild-type L. rhamnosus strains, but further confirmation of their efficiency in experimental animal models is considered an essential next research step.

Biotechnological potential of Zymotis-2 bioreactor for the cultivation of filamentous fungi.

BACKGROUND/AIM: A new prototype of Solid-State Fermentation Bioreactor, namely "Zymotis-2 ", was developed to produce fungal spores. MAIN METHODS AND MAJOR RESULTS: A fermentation process for fungal spores, and hydrolase enzymes (endo and exoglucanases, amylases) production by Trichoderma asperellum DWG3, Aspergillus niger G131 and Beauveria bassiana was scaled-up from flasks and glass Raimbault column packed with 20 g of solid substrates (dry weight) to 5 kg of solid substrate by using the new Zymotis-2 bioreactor. Fungi strains growth using a mix of vine shoots, wheat bran, and olive pomace was tested under similar experimental conditions in Zymotis-2 bioreactor, column bioreactor and flasks in a parallel fermentation system. Overall, significant spore production on Zymotis-2 bioreactor was obtained, achieving 22.01 ± 0.01×109 spores/g DM 16.30 ± 0.07 × 109 spores/g DM, and 3.30 ± 0.07 × 109 spores/g DM for B. bassiana, T. asperellum DWG3, and A. niger G131, respectively. Forced aeration increased the endoglucanases, exoglucanases and amylases activities for T. asperellum DWG3 but B. bassiana and A. niger G131 were affected negatively by the aerated process, showing the lowest enzyme activities. CONCLUSIONS AND IMPLICATIONS: In conclusion, a high yield of spores was obtained at 137 h of cultivation time, confirming the validity of the new Zymotis-2 bioreactor to produce virulent spores at low cost by T. asperellum, B. bassiana and A. niger G131.