Natural Antimicrobials: A Reservoir to Contrast Listeria monocytogenes.

Natural environments possess a reservoir of compounds exerting antimicrobial activity that are forms of defence for some organisms against others. Recently, they have become more and more attractive in the food sector due to the increasing demand for natural compounds that have the capacity to protect food from pathogenic microorganisms. Among foodborne pathogens, Listeria monocytogenes can contaminate food during production, distribution, or storage, and its presence is especially detected in fresh, raw food and ready-to-eat products. The interest in this microorganism is related to listeriosis, a severe disease with a high mortality rate that can occur after its ingestion. Starting from this premise, the present review aims to investigate plant extract and fermented plant matrices, as well as the compounds or mixtures of compounds produced during microbial fermentation processes that have anti-listeria activity.

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.

Valorization of orange peels exploiting fungal solid-state and lacto-fermentation.

BACKGROUND: Orange peels can serve as a cost-effective raw material for the production of lactic acid. Indeed, given their high concentration of carbohydrates and low content of lignin, they represent an important source of fermentable sugars, recoverable after a hydrolytic step. RESULTS: In the present article, the fermented solid, obtained after 5 days of Aspergillus awamori growth, was used as the only source of enzymes, mainly composed of xylanase (40.6 IU g-1 of dried washed orange peels) and exo-polygalacturonase (16.3 IU g-1 of dried washed orange peels) activities. After the hydrolysis, the highest concentration of reducing sugars (24.4 g L-1 ) was achieved with 20% fermented and 80% non-fermented orange peels. The hydrolysate was fermented with three lactic acid bacteria strains (Lacticaseibacillus casei 2246 and 2240 and Lacticaseibacillus rhamnosus 1019) which demonstrated good growth ability. The yeast extract supplementation increased the lactic acid production rate and yield. Overall, L. casei 2246 produced the highest concentration of lactic acid in mono-culture. CONCLUSION: To the best of our knowledge this is the first study exploiting orange peels as low-cost raw material for the production of lactic acid avoiding the employment of commercial enzymes. The enzymes necessary for the hydrolyses were directly produced during A. awamori fermentation and the reducing sugars obtained were fermented for lactic acid production. Despite this preliminary work carried out to study the feasibility of this approach, the concentrations of reducing sugars and lactic acid produced were encouraging, leaving open the possibility of other studies for the optimization of the strategy proposed here. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Dynamic changes in molecular composition of black soldier fly prepupae and derived biomasses with microbial fermentation.

Black soldier fly (BSF) is being increasingly used for agro-food by-products valorisation. Adult flies, puparia, and excess of prepupae are the by-products of this process, which could be further valorised. Lactic fermentation of BSF biomasses with two different strains (L. rhamnosus and L. plantarum) has been used for this purpose. Deep changes in the molecular composition were observed, without significant differences related to the different strains used. The lipid and protein fractions were the most impacted. Fermentation enriched the biomass in monounsaturated and polyunsaturated fatty acids and essential amino acids, significantly improving the nutritional properties of the substrates. Although not particularly marked, a proteolytic activity of lactobacilli was observed on the BSF muscular and cuticular proteins, especially in the samples of adult flies and puparia, where fermentation resulted more effective. Conversely, there was no evidence of chitinolytic activity.

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.

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.

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.

Fermentation of Agri-Food Waste: A Promising Route for the Production of Aroma Compounds.

Food waste and byproducts are generated along the entire food processing and storage chain. The large amount of waste deriving from the whole process represents not only a great economic loss but also an important ethical and environmental issue in terms of failure to recycle potentially reusable materials. New, clear strategies are needed to limit the amount of waste produced and, at the same time, promote its enhancement for further conversion and application to different industrial fields. This review gives an overview of the biological approaches used so far to exploit agri-food wastes and byproducts. The application of solid-state fermentation by different microorganisms (fungi, yeasts, bacteria) to produce several value-added products was analyzed, focusing on the exploitation of lactic acid bacteria as workhorses for the production of flavoring compounds.

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.

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.

Distribution of Antibiotic Resistance Genes in the Saliva of Healthy Omnivores, Ovo-Lacto-Vegetarians, and Vegans.

Food consumption allows the entrance of bacteria and their antibiotic resistance (AR) genes into the human oral cavity. To date, very few studies have examined the influence of diet on the composition of the salivary microbiota, and even fewer investigations have specifically aimed to assess the impact of different long-term diets on the salivary resistome. In this study, the saliva of 144 healthy omnivores, ovo-lacto-vegetarians, and vegans were screened by nested PCR for the occurrence of 12 genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, and ß-lactams. The tet(W), tet(M), and erm(B) genes occurred with the highest frequencies. Overall, no effect of diet on AR gene distribution was seen. Some differences emerged at the recruiting site level, such as the higher frequency of erm(C) in the saliva of the ovo-lacto-vegetarians and omnivores from Bologna and Turin, respectively, and the higher occurrence of tet(K) in the saliva of the omnivores from Bologna. A correlation of the intake of milk and cheese with the abundance of tet(K) and erm(C) genes was seen. Finally, when the occurrence of the 12 AR genes was evaluated along with geographical location, age, and sex as sources of variability, high similarity among the 144 volunteers was seen.

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.

Diet influences the functions of the human intestinal microbiome.

Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in 27 fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three interleukins (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.

Antimicrobial and Fermentation Potential of Himanthalia elongata in Food Applications.

Himanthalia elongata is a brown oceanic seaweed rich in bioactive compounds. It could play an important role in food production because of its antimicrobial and antioxidant properties. Three strains belonging to the Lactobacillus casei group (Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus) and a Bacillus subtilis strain were used for the solid-state fermentation of commercial seaweeds, and bacterial growth was monitored using the plate count method. High-pressure processing (HPP) was also employed (6000 bar, 5 min, 5 °C) before extraction. The antimicrobial activity of the extracts was tested in terms of the main food pathogenic bacteria (Salmonella spp., Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Bacillus cereus), and the phenolic content was estimated using the Folin-Ciocalteau method. In addition, targeted UHPLC-MS2 methods were used to unravel the profile of phlorotannins. H. elongata allowed the growth of the L. casei group strains and B. subtilis, showing the fermentability of this substrate. Significant antimicrobial activity toward L. monocytogenes was observed in the extracts obtained from unfermented samples, but neither fermentation nor HPP enhanced the natural antimicrobial activity of this seaweed species. The content in the phenolic compounds decreased because of the fermentation process, and the amount of phenolics in both the unfermented and fermented H. elongata extracts was very low. Despite phlorotannins being related to the natural antimicrobial activity of this brown seaweed, these results did not support this association. Even if fermentation and HPP were not proven to be effective tools for enhancing the useful compounds of H. elongata, the seaweed was shown to be a suitable substrate for L. casei group strains as well as for B. subtilis growth, and its extracts exhibited antimicrobial activity toward foodborne pathogens.

Dynamics of a natural bacterial community under technological and environmental pressures: The case of natural whey starter for Parmigiano Reggiano cheese.

Natural starter cultures are undefined multiple-strains culture communities of mostly thermophilic lactic acid bacteria (LAB), in association with minor amounts of mesophilic bacteria, which structure could be affected by small changes in the parameters of the cheese/whey-making process. This study aims to investigate the complex microbiota of natural whey starter (NWS) used in Parmigiano Reggiano (PR) cheese-making, focusing on both the absolute and relative abundance of bacterial species and on the modification of the bacterial community under environmental and technological pressures. To reach this purpose a combined approach, using quantitative PCR (qPCR) and High-Throughput Sequencing (HTS), was used to investigate the bacterial dynamics of 91 whey samples collected during different steps of PR cheese-making, in one dairy, through two different lines of production, one Conventional and one Organic, over a 10 weeks period. Our results highlighted that NWS used for the production of PR cheese is a dynamic microbial community, which adapts to the different technological parameters encountered in the cheese/NWS manufacturing process, while retaining a high level of resilience of the thermophilic LAB species mainly involved in the steps of curd acidification and the early maturation process. Differences were also observed in bacterial species diversity between samples from Conventional and Organic line but, in conclusion, NWS resulted to be shaped by technological treatments, regardless of its initial different composition.

Phenotypic Diversity of Lactobacillus casei Group Isolates as a Selection Criterion for Use as Secondary Adjunct Starters.

Autochthonous lactic acid bacteria (LAB) play a key role in the development of cheese flavor. As the pasteurization treatment on raw milk causes the elimination of LAB, secondary starter cultures are used in cheese manufacture to obtain cheeses with improved and standardized flavors. In this work, strains of the L. casei group isolated from traditional Italian cheeses were screened for their phenotypic features of technological interest for use as secondary starters. Their milk acidifying performance and the production of volatile compounds when grown in milk were evaluated. Simultaneously, the acetoin metabolic pathway presence was screened in the strains and assessed for its transcriptional activation. The results showed that the analyzed strains, despite belonging to taxonomically-related species, vary greatly according to the measured phenotypes. Four strains among the fourteen screened could be potentially used as adjunct cultures for cheese-making processes. The strain that showed the highest production of acetoin upregulated the aspartate pathway. An increased knowledge of volatile compounds' production and acidifying properties of LAB strains isolated from traditional dairy products might guide the selection of strains for industrial applications.