Lactic acid fermented microalgae and cyanobacteria as a new source of lipid reducing compounds: assessment through zebrafish Nile red fat metabolism assay and untargeted metabolomics.

Obesity is one of the most important threats to human health. Besides existing pharmacological or clinical interventions, novel effective and largely available solutions are still necessary. Among diverse natural resources, microalgae are well known for their complexity in the production of novel secondary metabolites. At the same time, lactic acid bacteria (LAB) are known for their capacity to metabolize, through fermentation, different matrices, and consequently to modify or produce new compounds with potential bioactivity. This work aimed to study the production of fermented microalgae and cyanobacteria, and to analyse their extracts in the zebrafish Nile red fat metabolism assay. Three microalgal species (Chlorella vulgaris, Chlorococcum sp. and Arthrospira platensis) were fermented with seven strains of LAB from 4 species (Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii bulgaricus and Lacticaseibacillus paracasei), derived from the UPCCO - University of Parma Culture Collection, Parma, Italy). All the selected strains were able to ferment the selected species of microalgae, and the most suitable substrate for LAB growth was Arthrospira platensis. Extracts from fermented Chlorella vulgaris and Chlorococcum sp. reduced significantly the neutral lipid reservoirs, which was not observed without fermentations. The strongest lipid reducing effect was obtained with Arthrospira platensis fermented with Lactobacillus delbrueckii bulgaricus 1932. Untargeted metabolomics identified some compound families, which could be related to the observed bioactivity, namely fatty acids, fatty amides, triterpene saponins, chlorophyll derivatives and purine nucleotides. This work opens up the possibility of developing novel functional foods or food supplements based on microalgae, since lactic acid fermentation enhanced the production of bioactive compounds with lipid reducing activities.

Lacticaseibacillus Strains Isolated from Raw Milk: Screening Strategy for Their Qualification as Adjunct Culture in Cheesemaking.

The microbial ecology fundamentals of raw milk and long-ripened cheeses consist of a complex interaction between starter lactic acid bacteria (SLAB) and non-starter LAB (NSLAB). Although NSLAB aromatic properties are paramount, other phenotypic traits need to be considered for their use as adjunct cultures, such as the capability to endure technological parameters encountered during cheesemaking. The present study focused on the isolation and characterization of NSLAB from spontaneously fermented raw cow's milk coming from 20 dairies that produce Grana Padano PDO cheese. From 122 isolates, the screening process selected the 10 most diverse strains belonging to Lacticaseibacillus spp. to be phenotypically characterized. The strains were tested for their growth performance in milk in combination with the application of technological stresses, for their ability to produce volatile compounds after their growth in milk, and for their ability to use different nutrient sources and resist chemicals. The complex characterization qualified the strains 5959_Lbparacasei and 5296_Lbparacasei as the best candidates to be used as adjunct strains in the production of raw milk and long-ripened cheeses, provided that antibiotic resistance is measured before their employment. Other strains with interesting aromatic capabilities but lower heat resistance were 5293_Lbparacasei, 5649_Lbparacasei and 5780_Lbparacasei, which could be candidates as adjunct strains for uncooked cheese production.

The microbiota of Mozzarella di Bufala Campana PDO cheese: a study across the manufacturing process.

Introduction: Mozzarella di Bufala Campana PDO cheese (MBC) is a globally esteemed Italian cheese. The traditional cheesemaking process of MBC relies on natural whey starter culture, water buffalo's milk, and the local agroecosystem. Methods: In this study, the microbial ecology of intermediate samples of MBC production, coming from two dairies with slightly different cheesemaking technology (dairy M large producer, and dairy C medium-small), was investigated using 16S rRNA amplicon sequencing. This research aimed to provide insights into the dynamics of microbial consortia involved in various cheesemaking steps. Results and discussion: All samples, except for raw buffalo milk, exhibited a core microbiome predominantly composed of Streptococcus spp. and Lactobacillus spp., albeit with different ratios between the two genera across the two MBC producers. Notably, the microbiota of the brine from both dairies, analyzed using 16S amplicon sequencing for the first time, was dominated by the Lactobacillus and Streptococcus genera, while only dairy C showed the presence of minor genera such as Pediococcus and Lentilactobacillus. Intriguingly, the final mozzarella samples from both producers displayed an inversion in the dominance of Lactobacillus spp. over Streptococcus spp. in the microbiota compared to curd samples, possibly attributable to the alleviation of thermal stress following the curd stretching step. In conclusion, the different samples from the two production facilities did not exhibit significant differences in terms of the species involved in MBC cheesemaking. This finding confirms that the key role in the MBC cheesemaking process lies with a small-sized microbiome primarily composed of Streptococcus and Lactobacillus spp.

Lactobacillus delbrueckii subsp. bulgaricus derivatives for 3D printed alginate/hyaluronic acid self-crosslinking hydrogels: Manufacturing and wound healing potential.

Derivatives [i.e. proteins and exopolysaccharides (EPS)] from Lactobacillus delbrueckii subsp. bulgaricus (LB) were extracted, characterized, and for the first time used in the production of novel self-crosslinking 3D printed alginate/hyaluronic acid (ALG/HA) hydrogels, as high-value functional biomaterials with therapeutic potentials in regenerative medicine applications. Derivatives coming from two different LB strains, LB1865 and LB1932, were tested in-vitro and compared for their cytotoxicity and effect on proliferation and migration on human fibroblast. EPS received particular attention as showing relevant dose-dependent cytocompatibility against the human fibroblast. The derivatives showed an ability to increase cell proliferation and migration, quantifiable between 10 and 20 % if compared to controls, with higher values for the derivatives obtained from the LB1932 strain. These were explained by liquid chromatography-mass spectrometry targeted protein biomarker analysis as a decrease in matrix-degrading and proapoptotic proteins, associated with an increase in collagen and antiapoptotic proteins production. LB1932 enriched hydrogel was found to be of benefit compared to control dressings, giving the more promising results as potential for in vivo skin wound healing tests.

Exploring the Potential of Lactic Acid Fermentation for the Recovery of Exhausted Vanilla Beans.

The market value of vanilla is constantly growing, as it is the aroma most appreciated by consumers worldwide. The key component of the aroma of vanilla beans is vanillin, which can be directly extracted from the plant, produced by chemical synthesis, or by bioconversion of natural precursors. Due to the increasing consumers' demand for products labeled as "natural," extraction from vanilla pods results in a more valuable aroma source. Once the extraction is completed, what remains are the exhausted beans that still contain small seeds and other compounds, including varying amounts of vanillin trapped in the cellular structures of the plant. The application of fermentation of exhausted vanilla beans is proposed here as a strategy to recover "natural" vanillin and other valuable aroma compounds as a result of the metabolic conversion by lactic acid bacteria (LAB). The aim of this study was to verify the fermentability of exhausted vanilla beans by-products for their valorization, allowing the recovery of high-value molecules or new applications in food products. Design of Experiment (DoE) was used to screen a library of LAB strains to identify the best condition of fermentation in response to varying cultivation conditions. A comparison between mono and co-culture of LAB was assessed. Moreover, sensory panel tests and the evaluation of the aromatic components by Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry analysis were carried out to better understand the modification of the aroma profile after fermentation. Fermentation with LAB changed the volatile profile and sensory characteristics of the exhausted vanilla beans and represents a promising method for the valorization of these by-products.

Influence of Processing Parameters and Natural Antimicrobial on Alicyclobacillus acidoterrestris and Clostridium pasteurianum Using Response Surface Methodology.

The food industry must ensure the stability of the products, and this is often achieved by exposing foods to heat treatments that are able to ensure the absence of pathogenic or spoilage microorganisms. These treatments are different in terms of temperature and duration and could lead to a loss in nutritional and sensory value. Moreover, some types of microorganisms manage to survive these treatments thanks to the sporification process. The addition of antimicrobials can become necessary, but at present, consumers are more inclined toward natural products, avoiding synthetic and chemical additives. Antimicrobials from plants could be a valuable option and, in this context, a patent concerning an antimicrobial extract from fermented plant substrate was recently tested against foodborne pathogens revealing high antimicrobial activity. The objective of this study was the creation of a model for the evaluation and subsequent prediction of the combined effect of different process and product variables, including antimicrobial addition, on the inhibition and reduction of spore germination of target microorganisms, Alicyclobacillus acidoterrestris and Clostridium pasteurianum, responsible for spoilage of tomato-based products.

Pseudomonas fluorescens and Escherichia coli in Fresh Mozzarella Cheese: Effect of Cellobiose Oxidase on Microbiological Stability during Refrigerated Shelf Life.

Background: Mozzarella cheese possesses a high moisture content (50−60%) and a relatively high pH (around 5.5) and is therefore considered a perishable food product characterized by high quality deterioration and the potential risk of microbial contamination. Moreover, it can be spoiled by Pseudomonas spp. and coliform bacteria, which may be involved in different negative phenomena, such as proteolysis, discolorations, pigmentation, and off-flavors. To prevent these, different methods were investigated. In this context, the present study aims to assess the antimicrobial effect of cellobiose oxidase on Pseudomonas fluorescens (5026) and Escherichia coli (k88, k99) in mozzarella cheese during refrigerated shelf life. Methods: microbiological challenge tests were designed by contaminating the mozzarella covering liquid containing different cellobiose oxidase concentrations with P. fluorescens (5026) and E. coli (k88, k99). The behavior of these microorganisms and the variation of hydrogen peroxide concentrations were then tested under refrigerated conditions for 20 days to simulate the mozzarella cheese shelf life. Results and Conclusions: The data obtained demonstrated the effect of cellobiose oxidase on microbial growth. In particular, E. coli (k88, k99) was inhibited over the entire shelf life, while P. fluorescens (5026) was only partially affected after a few days of refrigerated storage.

Eating Fermented: Health Benefits of LAB-Fermented Foods.

Lactic acid bacteria (LAB) are involved in producing a considerable number of fermented products consumed worldwide. Many of those LAB fermented foods are recognized as beneficial for human health due to probiotic LAB or their metabolites produced during food fermentation or after food digestion. In this review, we aim to gather and discuss available information on the health-related effects of LAB-fermented foods. In particular, we focused on the most widely consumed LAB-fermented foods such as yoghurt, kefir, cheese, and plant-based products such as sauerkrauts and kimchi.

Antimicrobial Activity of Fermented Vegetable Byproduct Extracts for Food Applications.

To prevent foodborne diseases and extend shelf-life, antimicrobial agents may be used in food to inhibit the growth of undesired microorganisms. In addition to the prevention of foodborne diseases, another huge concern of our time is the recovery of agri-food byproducts. In compliance with these challenges, the aim of this work was to more deeply investigate the antimicrobial activity of extracts derived from fermented tomato, melon, and carrot byproducts, previously studied. All the fermented extracts had antimicrobial activity both in vitro and in foodstuff, showing even higher activity than commercial preservatives, tested for comparison against spoilage microorganisms and foodborne pathogens such as Salmonella spp., L. monocytogenes, and B. cereus. These promising results highlight an unstudied aspect for the production of innovative natural preservatives, exploitable to improve the safety and shelf-life of various categories of foodstuff.

Arthrospira platensis Extract: A Non-Invasive Strategy to Obtain Adjunct Attenuated Cultures.

This study aims at proposing the use of Arthrospira platensis, commonly known as Spirulina, extract as a non-invasive method to attenuate the growth rate of non-starter adjunct cultures, thus preventing the over-acidification that may occur during cheese manufacturing. A preliminary screening using four different concentrations (0.20%, 0.30%, 0.50%, and 0.70%) of A. platensis extract and four starter and three non-starter lactic acid bacteria strains was performed by impedometric analysis. This allowed us to select one starter and one non-starter strain to be used in the in vitro simulation of a co-culture in milk with the best antimicrobial concentration (0.3%). The growth dynamics of the two selected strains, starter Lactococcus lactis 1426 and non-sarter Lacticaseibacillus rhamnosus 1473, co-cultured for 120 h was monitored by three different approaches: (i) plate counting on M17, for the enumeration of lactococci, and MRS for lactobacilli; (ii) fluorescence microscopic counting of viable and non-viable coccoid Lactococcus lactis 1426 and rod-shaped Lacticaseibacillus rhamnosus 1473 cells; (iii) the overall estimation of co-culture growth behavior by impedometric parameters Lag, Rate, and yEnd. All the data obtained from the in vitro simulation were in agreement, revealing that a slowdown of non-starter growth occurred, while the starter strain was not affected, or slightly stimulated, from the antimicrobial presence. In particular, the growth of Lb. rhamnosus 1473 was delayed without adversely compromise the cells' integrity, connected with metabolic functions, showing a great potential for use in cheese production.

Genome Sequencing of five Lacticaseibacillus Strains and Analysis of Type I and II Toxin-Antitoxin System Distribution.

The analysis of bacterial genomes is a potent tool to investigate the distribution of specific traits related to the ability of surviving in particular environments. Among the traits associated with the adaptation to hostile conditions, toxin-antitoxin (TA) systems have recently gained attention in lactic acid bacteria. In this work, genome sequences of Lacticaseibacillus strains of dairy origin were compared, focusing on the distribution of type I TA systems homologous to Lpt/RNAII and of the most common type II TA systems. A high number of TA systems have been identified spread in all the analyzed strains, with type I TA systems mainly located on plasmid DNA. The type II TA systems identified in these strains highlight the diversity of encoded toxins and antitoxins and their organization. This study opens future perspectives on the use of genomic data as a resource for the study of TA systems distribution and prevalence in microorganisms of industrial relevance.

Effect of fermentation with single and co-culture of lactic acid bacteria on okara: evaluation of bioactive compounds and volatile profiles.

Okara is the main soybean by-product resulting from the processing of soy milk and tofu. Despite being a product with a lot of potential and rich in many bioactive compounds such as polyphenols, it presents an unpleasant, rancid aroma. For this reason its use in the food industry is limited. In this study, we have reported the integral use of okara in a solid state fermentation process, conducted with wild strains of lactic acid bacteria, to evaluate the effect of bacterial metabolism on the volatile and polyphenolic profiles. Strains belonging to Lactobacillus acidophilus, Lacticaseibacillus rhamnosus and Pediococcus acidilactici species were used in monoculture and, for the first time, in co-culture. The results showed an improvement in the aromatic fraction showing a decrease in hexanal, responsible for off-flavour, and an increase in ketones with fruity and buttery notes in fermented okara. Polyphenols were also affected, and, in particular, a bioconversion of glucoside isoflavones to the aglycone forms was highlighted in all fermented substrates. In addition, the appearance of both phenyllactic and p-hydroxyphenyllactic acids as well as the increase in indole-3-lactic acid was observed for the first time upon okara fermentation. Overall, the co-culture appears to be the most promising for biovalorization of okara, thereby opening the possibility of its use in the development of functional ingredients.

Microbiological Contamination of Ready-to-Eat Algae and Evaluation of Bacillus cereus Behavior by Microbiological Challenge Test.

ABSTRACT: Consumption of seaweeds (forms of algae), often categorized as a superfood, is becoming popular in western countries. Algae can be marketed fresh, but are usually sold dehydrated to ensure longer shelf life. Their consumption, often as ready-to-eat, opens up possible risks for public health because of foodborne pathogens that can contaminate the raw material during harvesting or manipulation. In this study, 14 ready-to-eat foods based on dehydrated algae, representative of the most consumed species, were considered. The microbial content, with a focus on Listeria monocytogenes and Bacillus cereus, was investigated by plate counts, and B. cereus strains were isolated and identified by 16S rRNA gene sequencing. The microbiological quality was heterogeneous among the samples and, in particular, marine bacteria, Listeria spp., B. cereus, and coliforms were detected. To contribute to related risk assessment, the ability of B. cereus to grow during refrigerated storage was evaluated, to our knowledge for the first time, by a microbiological challenge test on two ready-to-eat foods based on Undaria pinnatifida and Palmaria palmata. Despite this study demonstrating the inability of B. cereus to proliferate in seaweed-based food, its presence in dehydrated foodstuffs cannot rule out replication after rehydration before consumption, making it necessary to elucidate the possible risks for consumers.

Probiotics and Covid-19.

Coronavirus disease 2019 (COVID-19) has become pandemic very rapidly at the beginning of 2020. In the rush to possible therapeutic options, probiotics administration has been proposed mainly based on indirect observation. Some evidence of COVID-19 effects on intestinal microbiota dysbiosis has been shown and probiotics have been considered for their efficacy in the management of respiratory tract viral infections. These observations could be reinforced by the more and more evident existence of a lung-gut axis, suggesting the modulation of gut microbiota among the approaches to the COVID-19 prevention and treatment. As different possible roles of probiotics in this extremely severe illness have been contemplated, the aim of this work is to collect all the currently available information related to this topic, providing a starting point for future studies focussing on it.

Antimicrobial Biomasses from Lactic Acid Fermentation of Black Soldier Fly Prepupae and Related By-Products.

Worldwide, thousands of insect species are consumed as food or are used as feed ingredients. Hermetia illucens, 'black soldier fly', is one of them, and a large amount of puparia and dead adults flies are accumulated during rearing. These materials represent important wastes but no studies are still present in the literature regarding their functional properties and potential reuse. Lactic acid bacteria (LAB) are a heterogeneous group of bacteria contributing to various industrial applications, ranging from food fermentation, chemicals production to pharmaceuticals manufacturing. A LAB feature of industrial interest is their ability to produce antimicrobial metabolites. Considering the scientific and commercial interest in discovering novel antimicrobials, this work will be direct towards fermentation of insect-derived biomasses: puparia and adults insect at the end of life cycle. To the best of our knowledge, the in vitro antimicrobial activity of fermented insects is tested for the first time. This study aimed also to evaluate differences in the composition between fermented and unfermented insects, and to study whether the fermentation and the type of LAB used played a crucial role in modifying the composition of the substrate. Results firstly highlighted fermentability of this species of insects, showed that fermented black soldier flies puparium possess a high antimicrobial activity against tested pathogens. Moreover, result of chemical composition showed that fermented biomass had a higher percentage of fat and a more complex fatty acids profile.

Edible Seaweeds and Spirulina Extracts for Food Application: In Vitro and In Situ Evaluation of Antimicrobial Activity towards Foodborne Pathogenic Bacteria.

Research is more and more focused on studying and selecting food preservatives of natural origin. In this scenario, algae are an excellent source of bioactive compounds, among which are antimicrobials, whose presence is variable depending on the algal species and environmental conditions. The aim of the present study was to obtain, by a food grade approach, antimicrobial extracts from five species already approved as foods and to test their efficacy in vitro (agar well diffusion assay) and in situ (microbial challenge test) towards foodborne pathogenic bacteria. Moreover, the total phenolic compounds of the extracts were determined in order to evaluate possible correlations with the antimicrobial activity. Strains belonging to Salmonella spp., Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Bacillus cereus were considered. Overall, the extracts showed a good antimicrobial activity in vitro towards all the tested microorganisms, especially L. monocytogenes (15 mm of inhibition diameter). The effect of inhibition was monitored during 24, 48 and 120 h showing a good persistence in time. Arthrospira platensis exerted the highest efficacy, further revealed towards L. monocytogenes on salmon tartare as bacteriostatic using 0.45% of the extract and bactericidal using 0.90%. The presence of phenolic compounds could be related to the antimicrobial activity but was not revealed as the main component of this activity. The extract with the highest phenolic content (18.79 ± 1.90 mg GAE/g) was obtained from Himanthalia elongata. The efficacy, confirmed also in a food matrix, might open perspectives for their application as food preservative.

Draft Genome Sequence of Lactobacillus helveticus Lh 23, Isolated from Natural Whey Starter.

Lactobacillus helveticus is a thermophilic lactic acid bacterium that is widely employed as a starter culture for manufacturing several Swiss and Italian hard-cooked cheeses. The sequencing of L. helveticus Lh 23, which consists of 2,100,230 bp with a GC content of 36.5%, reveals industrially useful traits and interesting metabolic pathways.

Development of Lactic Acid-Fermented Tomato Products.

BACKGROUND: lactic acid fermentation was recently proposed to produce fruit and vegetable beverages with high nutritional value. In this study, a wide screening of strains and fermentation parameters was carried out to develop fermented tomato-based drinks containing viable cells and potentially bioactive metabolites. METHODS: six different products (three extracts, two tomato juices and one tomato puree) were used as substrate for fermentation. After preliminary testing, eight fermentation conditions for each tested product were selected. The final products were stabilized with pasteurization or refrigeration and further characterized in terms of (i) antioxidant activity and (ii) total polyphenols. RESULTS: selected strains were able to grow in almost all tomato-based products except for one extract. Antioxidant activity and total phenolic content depend on products and fermentation conditions used and, except for tomato puree, an overall increase was observed. The best nutritional profile was reached in fermented samples stored at refrigerated temperature without thermal stabilization. CONCLUSION: an integrated data vision allowed to choose, for each substrate, the best combination of strains to produce novel fermented tomato-based products with different application perspectives.

Lactobacillus paracasei 4341 as adjunct culture to enhance flavor in short ripened Caciotta-type cheese.

Caciotta is the name used to define a type of Italian semi-hard cheese Caciotta-type cheese. Due to the short ripening time, pasteurization is necessary to eliminate the potential pathogenic bacteria, which may be present in raw milk, causing also the reduction of ripened cheese flavor. The purpose of this research was to evaluate the effect of a selected wild Lactobacillus paracasei strain experimentally used as adjunct culture to enhance the flavour formation in a short-ripened caciotta-type cheese. An integrated polyphasic approach was used to compare the experimental and control Caciotta produced in a company located in Emilia Romagna region (Italy). It was demonstrated how the L. paracasei 4341 was able to develop in curd and cheese interacting with the acidifying commercial starter. The main acidifying starter species, were differently affected by the presence of the adjunct culture. Streptococcus thermophilus shown comparable behavior in all cheese-making step of control and experimental Caciotta, while Lactobacillus delbrueckii subsp bulgaricus, growth was slowed down by the presence of the adjunct culture during the whole ripening time. The higher amount of volatile compounds and organic acids due to the adjunct L. paracasei 4341 lead to a clear differentiation of the experimental Caciotta respect to the control, in terms of aromatic profile, color, texture and sensorial perception.