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.

Orange peels: from by-product to resource through lactic acid fermentation.

BACKGROUND: Considering the large amounts of by-products derived from orange processing, which are generally discarded, the present study aimed to explore the feasibility of using orange peel for lactic acid production in solid state fermentation. RESULTS: Different species of lactic acid bacteria were employed, singly and in co-culture, to evaluate their ability to ferment orange peel and produce lactic acid. Among the single cultures tested, Lactobacillus casei 2246 was the most efficient strain, reaching the highest concentration of lactic acid (209.65 g kg-1 ) and yield (0.88 g g-1 ). The use of Lactobacillus plantarum 285 and Lactobacillus paracasei 4186 in co-culture produced a comparable amount of lactic acid, showing a better performance than the same strains in single cultures. CONCLUSION: Orange peels represent a suitable raw material for solid state fermentation employing lactic acid bacteria. Lactic acid was obtained that consumed the most of sugars available, leading to high yields. Despite all the strains tested showing the same growth ability, different peculiarities in lactic acid production were revealed, dependent on the species/strains, suggesting the relevance of strain selection. © 2019 Society of Chemical Industry.

High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome.

OBJECTIVES: Habitual diet plays a major role in shaping the composition of the gut microbiota, and also determines the repertoire of microbial metabolites that can influence the host. The typical Western diet corresponds to that of an omnivore; however, the Mediterranean diet (MD), common in the Western Mediterranean culture, is to date a nutritionally recommended dietary pattern that includes high-level consumption of cereals, fruit, vegetables and legumes. To investigate the potential benefits of the MD in this cross-sectional survey, we assessed the gut microbiota and metabolome in a cohort of Italian individuals in relation to their habitual diets. DESIGN AND RESULTS: We retrieved daily dietary information and assessed gut microbiota and metabolome in 153 individuals habitually following omnivore, vegetarian or vegan diets. The majority of vegan and vegetarian subjects and 30% of omnivore subjects had a high adherence to the MD. We were able to stratify individuals according to both diet type and adherence to the MD on the basis of their dietary patterns and associated microbiota. We detected significant associations between consumption of vegetable-based diets and increased levels of faecal short-chain fatty acids, Prevotella and some fibre-degrading Firmicutes, whose role in human gut warrants further research. Conversely, we detected higher urinary trimethylamine oxide levels in individuals with lower adherence to the MD. CONCLUSIONS: High-level consumption of plant foodstuffs consistent with an MD is associated with beneficial microbiome-related metabolomic profiles in subjects ostensibly consuming a Western diet. TRIAL REGISTRATION NUMBER: This study was registered at clinical trials.gov as NCT02118857.

The spxB gene as a target to identify Lactobacillus casei group species in cheese.

This study focused on the spxB gene, which encodes for pyruvate oxidase. The presence of spxB in the genome and its transcription could be a way to produce energy and allow bacterial growth during carbohydrate starvation. In addition, the activity of pyruvate oxidase, which produces hydrogen peroxide, could be a mechanism for interspecies competition. Because this gene seems to provide advantages for the encoding species for adaptation in complex ecosystems, we studied spxB in a large set of cheese isolates belonging to the Lactobacillus casei group. Through this study, we demonstrated that this gene is widely found in the genomes of members of the L. casei group and shows variability useful for taxonomic studies. In particular, the HRM analysis method allowed for a specific discrimination between Lactobacillus rhamnosus, Lactobacillus paracasei and L. casei. Regarding the coding region, the spxB functionality in cheese was shown for the first time by real-time PCR, and by exploiting the heterogeneity between the L. casei group species, we identified the bacterial communities encoding the spxB gene in this ecosystem. This study allowed for monitoring of the active bacterial community involved in different stages of ripening by following the POX pathway.

Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese.

BACKGROUND: Lactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, leading to the formation of flavor. In long-ripened cheeses it persists throughout the whole time of ripening due to its capacity to adapt to changing environmental conditions. The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways. However, the molecular mechanisms allowing the growth of L. rhamnosus in the cheese dairy environment are still poorly understood. The aim of the present study was to identify genes potentially contributing to the growth ability of L. rhamnosus PR1019 in cheese-like medium (CB) using a transcriptomic approach, based on cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time reverse transcription-PCR (qPCR). RESULTS: Using three primer combinations, a total of 89 and 98 transcript-derived fragments were obtained for L. rhamnosus PR1019 grown in commercial MRS medium and CB, respectively. The cDNA-AFLP results were validated on selected regulated genes by qPCR. In order to investigate the main adaptations to growth in a cheese-mimicking system, we focused on 20 transcripts over-expressed in CB with respect to MRS. It is worth noting the presence of transcripts involved in the degradation of pyruvate and ribose. Pyruvate is a intracellular metabolite that can be produced through different metabolic routes starting from the carbon sources present in cheese, and can be released in the cheese matrix with the starter lysis. Similarly the ribonucleosides released with starter lysis could deliver ribose that represents a fermentable carbohydrate in environments, such as cheese, where free carbohydrates are lacking.Both pyruvate degradation and ribose catabolism induce a metabolite flux toward acetate, coupled with ATP production via acetate kinase. Taking into account these considerations, we suggest that the energy produced through these pathways may concur to explain the great ability of L. rhamnosus PR1019 to grow on CB. CONCLUSIONS: By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

Effect of protein hydrolysates on growth kinetics and aminopeptidase activities of Lactobacillus.

The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: ß-naphthyl amide derivatives of L-anomers of leucine, lysine, proline, glycine-proline, and phenilalanine-proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile.

Invited review: Microbial evolution in raw-milk, long-ripened cheeses produced using undefined natural whey starters.

The robustness of the starter culture during cheese fermentation is enhanced by the presence of a rich consortium of microbes. Natural starters are consortia of microbes undoubtedly richer than selected starters. Among natural starters, natural whey starters (NWS) are the most common cultures currently used to produce different varieties of cheeses. Undefined NWS are typically used for Italian cooked, long-ripened, extra-hard, raw milk cheeses, such as Parmigiano Reggiano and Grana Padano. Together with raw milk microbiota, NWS are responsible for most cheese characteristics. The microbial ecology of these 2 cheese varieties is based on a complex interaction among starter lactic acid bacteria (SLAB) and nonstarter lactic acid bacteria (NSLAB), which are characterized by their different abilities to grow in a changing substrate. This review aims to summarize the latest findings on Parmigiano Reggiano and Grana Padano to better understand the dynamics of SLAB, which mainly arise from NWS, and NSLAB, which mainly arise from raw milk, and their possible role in determining the characteristics of these cheeses. The review is presented in 4 main sections. The first summarizes the main microbiological and chemical properties of the ripened cheese as determined by cheese-making process variables, as these variables may affect microbial growth. The second describes the microbiota of raw milk as affected by specific milk treatments, from milking to the filling of the cheese milk vat. The third describes the microbiota of NWS, and the fourth reviews the knowledge available on microbial dynamics from curd to ripened cheese. As the dynamics and functionality of complex undefined NWS is one of the most important areas of focus in current food microbiology research, this review may serve as a good starting point for implementing future studies on microbial diversity and functionality of undefined cheese starter cultures.

Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter.

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA(Ala)-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1-V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.

Microbial origin of non proteolytic aminoacyl derivatives in long ripened cheeses.

Cheese ripening involves a complex series of biochemical events that contribute to the development of each cheese characteristic taste, aroma and texture. Proteolysis, which has been the subject of active research in the last decade, is the most complex of these biochemical events. However, also aminoacyl derivates of non-proteolytic origin (γ-glutamyl-amino acids and lactoyl-amino acids) with interesting sensory properties have been identified in cheeses. In the present work, an enzymatic activity producing γ-glutamyl-phenylalanine in Parmigiano-Reggiano water soluble extracts was observed. It was hypothesized that γ-glutamyl-amino acids and lactoyl-amino acids could be originated by enzymes of bacterial origin. In order to confirm this hypothesis, Lactobacillus helveticus and Lactobacillus rhamnosus were chosen as representative of starter and non starter microbiota of Parmigiano Reggiano cheese. They were used as model bacteria, in the presence of suitable precursors, to verify their ability to produce γ-glutamyl-phenylalanine and lactoyl-phenylalanine. The eventual abilities of these strains were tested both during growth and after cell lyses. While γ-glutamyl-phenylalanine was produced only by lysed cells, lactoyl-phenylalanine was produced either by growing or lysed cells in different amount depending on the species, the cells condition and the time of incubation.

Effect of protein hydrolizates on growth kinetics and aminopeptidase activities of some Bifidobacterium species.

This study evaluated the effect of a new hydrolizate from poultry feathers on growth kinetics and aminopeptidase activities of eight bifidobacteria compared with common peptones. The growth kinetics results suggest that the experimental hydrolizate could be a cheaper medium ingredient without affecting the modulation of common aminopeptidase activities.

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.

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.

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.

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.

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.

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.

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.