Synergistic effect of the coculture of Leuconostoc pseudomesenteroides and Lactococcus lactis, isolated from honeybees, on the generation of plant-based dairy alternatives based on soy, pea, oat, and potato drinks.

The production of plant-based dairy alternatives has been majorly focused on the improvement of sensorial, technological and nutritional properties, to be able to mimic and replace milk-based fermented products. The presence of off-flavours and antinutrients, the lack of production of dairy-like flavours or the metabolic inaccessibility of plant proteins are some of the challenges to overcome to generate plant-based dairy alternatives. However, in the present study, it is demonstrated how the synergistic effect of two LAB strains, when cocultured, can simultaneously solve those challenges when fermenting in four different plant-based raw materials: soy, pea, oat, and potato drinks (SPOP). The fermentation was performed through the mono- and co-culture of the two LAB strains isolated from Apis mellifera (honeybee): Leuconostoc pseudomesenteroides NFICC 2004 and Lactococcus lactis NFICC 2005. Firstly, the coculture of both strains demonstrated to increase the acidification rate of the four plant matrices. Moreover, L. pseudomesenteroides (LP) demonstrated to in situ produce high concentrations of mannitol when fructose was present as C-source. Furthermore, L. pseudomesenteroides, which encoded for PII-proteinase, demonstrated to break down SPOP proteins, releasing free amino acids that were used by L.lactis (LL) for growth and metabolism. Lastly, the analysis of their co-metabolic volatile performance showed the principal ability of removal of the main off-flavours found in SPOP, such as hexanal, 1-octen-3-ol, 2-pentylfuran, pentanal, octanal, heptanal, and nonanal, mainly led by L. pseudomesenteroides, as well as the production of dairy-like flavours, such as diacetyl and 3-methyl-1-butanol, triggered by L. lactis metabolism. Overall, these findings endorsed the use of honeybee isolated strains as starter cultures, demonstrated the potential of coupling genotypes and phenotypes of multiple strains to improve the organoleptic properties suggesting a potential of combining plant-based matrices for the generation of future high-quality plant-based dairy alternatives.

Evaluation of the fermentation potential of lactic acid bacteria isolated from herbs, fruits and vegetables as starter cultures in nut-based milk alternatives.

Fermentation of plant-based milk alternatives (PBMAs), including nut-based products, has the potential to generate new foods with improved sensorial properties. In this study, we screened 593 lactic acid bacteria (LAB) isolates from herbs, fruits and vegetables for their ability to acidify an almond-based milk alternative. The majority of the strongest acidifying plant-based isolates were identified as Lactococcus lactis, which were found to lower the pH of almond milk faster than dairy yoghurt cultures. Whole genome sequencing (WGS) of 18 plant-based Lc. lactis isolates revealed the presence of sucrose utilisation genes (sacR, sacA, sacB and sacK) in the strongly acidifying strains (n = 17), which were absent in one non-acidifying strain. To confirm the importance of Lc. lactis sucrose metabolism in efficient acidification of nut-based milk alternatives, we obtained spontaneous mutants defective in sucrose utilisation and confirmed their mutations by WGS. One mutant containing a sucrose-6-phosphate hydrolase gene (sacA) frameshift mutation was unable to efficiently acidify almond, cashew and macadamia nut milk alternatives. Plant-based Lc. lactis isolates were heterogeneous in their possession of the nisin gene operon near the sucrose gene cluster. The results of this work show that sucrose-utilising plant-based Lc. lactis have potential as starter cultures for nut-based milk alternatives.

Leuconostoc performance in soy-based fermentations - Survival, acidification, sugar metabolism, and flavor comparisons.

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in Lactococcus lactis.

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD, encoding the enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element (cre) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positive bacteria, is repressed by phosphorylation by EIIAmtl, and when we knocked out mtlF, we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

Production of allergen-specific immunotherapeutic agents for the treatment of food allergy.

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts.

miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

CTSH regulates ß-cell function and disease progression in newly diagnosed type 1 diabetes patients.

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.

A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells.

As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.

Regulation of microRNAs miR-30a and miR-143 in cerebral vasculature after experimental subarachnoid hemorrhage in rats.

BACKGROUND: microRNAs (miRNAs) are important regulators of translation and have been implicated in the pathogenesis of a number of cardiovascular diseases, including stroke, and suggested as possible prognostic biomarkers. Our aim was to identify miRNAs that are differentially regulated in cerebral arteries after subarachnoid hemorrhage (SAH), using a rat injection model of SAH and a qPCR-based screen of 728 rat miRNAs. Additionally, serum was analyzed for a possible spill-over to the circulation of regulated miRNAs from the vessel walls. RESULTS: We identified 482 different miRNAs expressed in cerebral arteries post-SAH. Two miRNAs, miR-30a and miR-143, were significantly upregulated in cerebral arteries after SAH when compared to sham-operated animals. However, none of these exhibited significantly altered serum levels after SAH versus post-sham surgery. The most robust upregulation was seen for miR-143, which has several predicted targets and is a strong regulator of vascular morphology. We hypothesize that miR-30a and miR-143 may play a role in the vascular wall changes seen after SAH. CONCLUSIONS: We report that miR-30a and miR-143 in the cerebral arteries show significant changes over time after SAH, but do not differ from sham-operated rats at 24 h post-SAH. Although this finding suggests interesting novel possible mechanisms involved in post-SAH cerebrovascular changes, the lack of regulation of these miRNAs in serum excludes their use as blood-borne biomarkers for cerebrovascular changes following SAH.

Exploring the Diversity and Potential Use of Flower-Derived Lactic Acid Bacteria in Plant-Based Fermentation: Insights into Exo-Cellular Polysaccharide Production.

Isolation of new plant-derived lactic acid bacteria (LAB) is highly prioritized in developing novel starter cultures for plant-based fermentation. This study explores the diversity of LAB in Danish flowers and their potential use for plant-based food fermentation. A total of 46 flower samples under 34 genera were collected for LAB isolation. By introducing an enrichment step, a total of 61 LAB strains were isolated and identified using MALDI-TOF and 16S rRNA sequencing. These strains represent 24 species across 9 genera, predominantly Leuconostoc mesenteroides, Fructobacillus fructosus, Apilactobacillus ozensis, and Apilactobacillus kunkeei. Phenotypic screening for exo-cellular polysaccharide production revealed that 40 strains exhibited sliminess or ropiness on sucrose-containing agar plates. HPLC analysis confirmed that all isolates produced exo-cellular polysaccharides containing glucose, fructose, or galactose as sugar monomers. Therefore, the strains were glucan, fructan, and galactan producers. The suitability of these strains for plant-based fermentation was characterized by using almond, oat, and soy milk. The results showed successful acidification in all three types of plant-based matrices but only observed texture development in soy by Leuconostoc, Weissella, Lactococcus, Apilactobacillus, and Fructobacillus. The findings highlight the potential of flower-derived LAB strains for texture development in soy-based dairy alternatives.

Using pre-fermented sugar beet pulp as a growth medium to produce Pleurotus ostreatus mycelium for meat alternatives.

This study aimed to determine the compatibility of pre-fermented sugar beet pulp to support the growth of Pleurotus ostreatus mycelium in submerged fermentation. The goal was to create a meat alternative based on mycelial-fermented pulp. It was further explored whether pre-fermentation with lactic acid bacteria (LAB) on the pulp increased meat-like properties, such as aroma, springiness, and hardness, in the final product. Three strains were selected from a high throughput screening of 105 plant-derived LAB based on their acidification and metabolite production in the pulp. Two homofermentative strains (Lactococcus lactis) and one heterofermentative strain (Levilactobacillus brevis) were selected based on their low ethanol production, high lactic acid production, and overall acidification of the pulp. Mycelium of P. ostreatus was grown in submerged fermentations on the pre-fermented pulp, and the biomass was removed by centrifugation. The fungal strain consumed all available sugars and acids and released arabinose to the media. Volatiles were detected using GC-MS, and a large increase in concentrations of hexanal, 1-octen-3-ol, and 2-octenal was measured. Concentration of 1-octen-3-ol was lower in the pre-fermented samples vs. the non-pre-fermented. LC-MS amino acid analysis showed the presence of all essential amino acids on day 0 and 7 of fermentation. The highest concentration of amino acids was for glutamic acid/glutamine and aspartic acid/asparagine. A decrease in all amino acids after 7 days of fungal fermentation was measured for all fermentations. The decrease was more significant for pre-fermented samples. This was also confirmed through a total protein determination, except for samples pre-fermented with Lactococcus lactis strain NFICC142 which increased in total protein content after fungal fermentation. The protein digestibility increased after fungal fermentation, and the highest increase was seen for non-pre-fermented samples. The springiness of the fermented product indicated similarities to meat alternatives, while the hardness was much lower than other meat alternatives. The results indicate that dried sugar beet pulp can be used for submerged cultivation of P. ostreatus, but that pre-fermentation does not improve the physical or nutritional properties of the end product significantly, except for an increased protein content for NFICC142 pre-fermented media. This is the first known attempt to use LAB and P. ostreatus in mixed fermentation to produce fungal mycelium, as well as the first attempt at using SBP in a liquid fermentation for mycelial production of P. ostreatus.

Do post-translational beta cell protein modifications trigger type 1 diabetes?

Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading to the T cell attack against beta cells is presented. In this model, PTM plays a prominent role in triggering beta cell destruction. We discuss literature of relevance and perform genetic and human islet gene expression analyses. Both direct and circumstantial support for the involvement of PTM in type 1 diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1 diabetes as a classical autoimmune disease is open for debate.

A family with ulcerative colitis maps to 7p21.1 and comprises a region with regulatory activity for the aryl hydrocarbon receptor gene.

We have mapped a locus on chromosome 7p22.3-7p15.3 spanning a 22.4 Mb region for ulcerative colitis (UC) by whole genome linkage analyses of a large Danish family. The family represent three generations with UC segregating as an autosomal dominant trait with variable expressivity. The whole-genome scan resulted in a logarithm of odds score (LOD score) of Z = 3.31, and a whole genome sequencing (WGS) of two affected excluded disease-causing mutations in the protein coding genes. Two rare heterozygote variants, rs182281985:G>A and rs541426369:G>A, both with low allele frequencies (MAF A:0.0001, gnomAD ver3.1.2), were found in clusters of ChiP-seq transcription factors binding sites close to the AHR (aryl hydrocarbon receptor) gene and the UC associated SNP rs1077773:G>A. Testing the two SNPs in a promoter reporter assay for regulatory activity revealed that rs182281985:G>A influenced the AHR promoter. These results suggest a regulatory region that include rs182281985:G>A close to the UC GWAS SNP rs1077773:G>A and further demonstrate evidence that the AHR gene on the 7p-tel region is a candidate susceptible gene for UC.

Comparative Structure Analysis of the Multi-Domain, Cell Envelope Proteases of Lactic Acid Bacteria.

Lactic acid bacteria (LAB) have an extracellular proteolytic system that includes a multi-domain, cell envelope protease (CEP) with a subtilisin homologous protease domain. These CEPs have different proteolytic activities despite having similar protein sequences. Structural characterization has previously been limited to CEP homologs of dairy- and human-derived LAB strains, excluding CEPs of plant-derived LAB strains. CEP structures are a challenge to determine experimentally due to their large size and attachment to the cell envelope. This study aims to clarify the prevalence and structural diversity of CEPs by using the structure prediction software AlphaFold 2. Domain boundaries are clarified based on a comparative analysis of 21 three-dimensional structures, revealing novel domain architectures of CEP homologs that are not necessarily restricted to specific LAB species or ecological niches. The C-terminal flanking region of the protease domain is divided into fibronectin type-III-like domains with various structural traits. The analysis also emphasizes the existence of two distinct domains for cell envelope attachment that are preceded by an intrinsically disordered cell wall spanning domain. The domain variants and their combinations provide CEPs with different stability, proteolytic activity, and potentially adhesive properties, making CEPs targets for steering proteolytic activity with relevance for both food development and human health.

Role of mesenchymal stem cells and short chain fatty acids in allergy: A prophylactic therapy for future.

Allergic diseases are broadly classified as IgE-mediated type-I hypersensitivity immune reactions due to exposure to typically harmless substances known as allergens. These allergenic substances activate antigen presenting cells, which further triggers T-helper 2 cells immune response and class switch B-cells for synthesis of allergen-specific IgE, followed by classical activation of inflammatory mast cells and eosinophils, which releases preformed mediators involved in the cascade of allergic symptoms. However, the role of Mesenchymal stem cells (MSCs) in tissue repair ability and immunomodulation, makes them as an appropriate tool for treatment of various allergic diseases. Several clinical and preclinical studies show that MSCs could be a promising alternative therapy to allergic diseases. Further, short chain fatty acids, produced from gut microbes by breaking down complex fibre-rich foods, acts through G-coupled receptor mediated activation of MSCs, and their role as key players involved in amelioration of allergic inflammation needs further investigation. Therefore, there is a need for understating the role of SCFAs on the activation of MSCs, which might shed light on the development of new therapeutic regime in allergy treatment. In summary, this review focuses on the underlying of therapeutic role of MSCs in different allergic diseases and the prospects of SCFA and MSC therapy.

Utilization of plant derived lactic acid bacteria for efficient bioconversion of brewers' spent grain into acetoin.

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive.

Allergenicity evaluation of quinoa proteins - A study in Brown Norway rats.

The popularity of quinoa seeds has increased in the last decade due to their high nutritional value and natural gluten-free composition. Consumption of new proteins may pose a risk of introducing new allergies. In the present study the immunogenicity and sensitising capacity of quinoa proteins were assessed in a dose-response experiment in Brown Norway rats in comparison to proteins from spinach and peanut. Cross-reactivity between quinoa proteins and known allergens was evaluated by in silico analyses followed by analyses with 11 selected protein extracts and their anti-sera by means of ELISAs and immunoblotting. Further, an in vitro simulated gastro-duodenal digestion was performed. Quinoa proteins were found to have an inherent medium to high immunogenicity and sensitising capacity, being able to induce specific IgG1 and IgE levels higher than spinach but lower than peanut and elicit reactions of clinical relevance similar to peanut. Quinoa proteins were generally shown to resist digestion and retain capacity to bind quinoa-specific antibodies. Quinoa proteins were shown to be cross-reactive with peanut and tree nut allergens as high sequence homology and antibody cross-binding were demonstrated. Present study suggests that quinoa pose a medium to high level of allergenicity that should be further investigated in human studies.

Extracellular microbial proteases with specificity for plant proteins in food fermentation.

Plant-based food products are generating a growing interest as part of the ongoing transition to a primarily plant-based diet, which makes demands to the quality, functionality, and health properties of plant proteins. Microbes used for traditional food fermentations such as lactic acid bacteria (LAB) and fungi (yeasts and molds) carry out enzymatic changes on their protein substrates by which technological and sensorial characteristics can be improved. The literature on extracellular proteases targeting plant proteins, on the other hand, is scattered with only a narrow representation of plants even for traditionally plant-based products. Therefore, this review aims to explore the current state of knowledge regarding the application potential of microbial extracellular proteases targeting plant proteins, with a focus on traditional applied food microbes. Plant proteins are targeted by proteolytic microbes of both animal and plant origins, and their proteases show a wide range of activities. Extracellular microbial proteases can hydrolyze specific protein-based allergens and even reduce the toxicity of plant proteins. Additionally, microbial assisted proteolysis can improve plant protein digestibility by increasing availability of peptides and amino acids. This catabolic process will change the organoleptic characteristics of fermented plant proteins, and the release of bioactive peptides can provide additional functionalities to the plant matrix. The proteolytic activity is determined by the microbial strain, and it can be quite substrate selective, which is why proteases may be overlooked by the prevalent use of casein as substrate in proteolytic screenings. The synergetic effects of LAB and fungal species consortia can facilitate and steer plant protein hydrolysis by which co-fermentation may increase or change the properties of plant protein hydrolysates. Microbes do not necessarily require extracellular proteases because endogenous proteases in a plant-matrix may meet the microbial amino acid requirements. However, extracellular proteases have the potential to provide central properties to diverse food-matrixes by which the full proteolytic potential of food microbes needs to be explored in order to facilitate the development of high-quality plant-based food products.

Droplet-Based Microfluidic High Throughput Screening of Corynebacterium glutamicum for Efficient Heterologous Protein Production and Secretion.

With emerging interests in heterologous production of proteins such as antibodies, growth factors, nanobodies, high-quality protein food ingredients, etc. the demand for efficient production hosts increases. Corynebacterium glutamicum is an attractive industrial host with great secretion capacity to produce therapeutics. It lacks extracellular protease and endotoxin activities and easily achieves high cell density. Therefore, this study focuses on improving protein production and secretion in C. glutamicum with the use of droplet-based microfluidic (DBM) high throughput screening. A library of C. glutamicum secreting ß-glucosidase was generated using chemical mutagenesis coupled with DBM screening of 200,000 mutants in just 20 min. Among 100 recovered mutants, 16 mutants exhibited enhanced enzyme secretion capacity, 13 of which had unique mutation profiles. Whole-genome analysis showed that approximately 50-150 SNVs had occurred on the chromosome per mutant. Functional enrichment analysis of genes with non-synonymous mutations showed overrepresentation of genes involved in protein synthesis and secretion relevant biological processes, such as DNA and ribosome RNA synthesis, protein secretion and energy turnover. Two mutants JCMT1 and JCMT8 exhibited the highest secretion with a six and a fivefold increase in the ß-glucosidase activity in the supernatant, respectively, relative to the reference strain JC0190. After plasmid curing, a new plasmid with the gene encoding α-amylase was cloned into these two mutants. The new strains SB024 and SB025 also exhibited a five and a sixfold increase in α-amylase activity in the supernatant, respectively, relative to the reference strain SB023. The results demonstrate how DBM screening can serve as a powerful development tool to improve cell factories for the production and secretion of heterologous proteins.

Chufa Drink: Potential in Developing a New Plant-Based Fermented Dessert.

Plant-based foods with desirable texture and nutritional value have attracted considerable interest from consumers. In order to meet the growing demand for more sustainable and health-focused products, new sources for plant-based products are needed. In this study, we aimed to develop an innovative plant-based dessert based on the underutilized crop chufa tubers (Cyperus esculentus). The chufa extract was fermented with plant-adapted lactic acid bacteria and formulated with the purpose of imitating the Danish summer dessert "cold butter-milk soup". The effect of various bacterial fermentations and formulations on steady and oscillatory rheology, stability, dry matter, pH, and sugar profile of the product were studied and compared to a commercial cold buttermilk soup sample. A strain of Leuconostoc mesenteroides was found to create the most similar taste to a commercial sample. By adding lemon juice, sucrose, xanthan gum, and vanilla to the fermented chufa drink, the drink was found to mimic the pH, texture, acid profile, and stability of a commercial dairy-based sample, while containing a lower concentration of carbohydrates.