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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 synergetic 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

The fermentation process of plant-based yoghurt (PBY)-like products must be followed for consistency by monitoring, e.g., the pH, temperature, and lactic acid concentration. Spectroscopy provides an efficient multivariate in situ quality monitoring method for tracking the process. Therefore, quality monitoring methods for pea- and oat-based yoghurt-like products using Fourier transform infrared (FT-IR) spectroscopy and high-performance liquid chromatography (HPLC) were developed and modeled. Plant-based yoghurt (PBY) was formulated by fermenting pea and oat plant drinks with a commercial starter culture based on Lactobacillus and Streptococcus strains. The main variance during fermentation was explained by spectral carbohydrate and protein bands with a notable shift in protein band peaks for the amide II band at 1548 cm−1 to 1576 cm−1. In addition to the identification of changed spectral bands during fermentation, FT-IR efficiently tracked the variation in oat and pea fermentation using pH as the main indicator. Prediction models with an R2 for the predicted value of pH as a fermentation indicator (R2 = 0.941) with a corresponding root-mean-squared error of prediction (RMSEP) of 0.247 was obtained when compared to the traditional pH method.

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.

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.

Brewer's spent grain (BSG) is an abundant brewing by-product, but a short shelf life limits its use. A rotary drum press can separate BSG into a solid and a liquid fraction (BSG liquid) that contains sugars. Propionic acid producing bacteria (PAB) strains from the family Propionibacteriaceae (DSM 20270, 20272, 20274, 106790) were screened for organic acids formation from BSG liquid in three fermentation setups: A single-step fermentation, a two-step fermentation with Lactiplantibacillus plantarum (NFICC 27) followed by PAB, and a co-culture of PAB with L. plantarum. The two-step fermentation produced 9.5 g/L propionic acid and 5.4 g/L acetic acid. GC-MS analysis of the fermented BSG liquid showed a decrease in off-flavours and an increase in compounds with fruity, chocolate and caramel notes. The fermented BSG liquid also had antimicrobial activity. Fermentation of BSG liquid may therefore be a suitable means of creating a clean label food ingredient with long shelf life.

In this study, a wide pool of lactic acid bacteria strains deposited in two recognized culture collections was tested against ropy bread spoilage bacteria, specifically belonging to Bacillus spp., Paenibacillus spp., and Lysinibacillus spp. High-throughput and ex vivo screening assays were performed to select the best candidates. They were further investigated to detect the production of active antimicrobial metabolites and bacteriocins. Moreover, technological and safety features were assessed to value their suitability as biocontrol agents for the production of clean-label bakery products. The most prominent inhibitory activities were shown by four strains of Lactiplantibacillus plantarum (NFICC19, NFICC 72, NFICC163, and NFICC 293), two strains of Pediococcus pentosaceus (NFICC10 and NFICC341), and Leuconostoc citreum NFICC28. Moreover, the whole genome sequencing of the selected LAB strains and the in silico analysis showed that some of the strains contain operons for bacteriocins; however, no significant evidence was observed phenotypically.

In this work, three lactic acid bacteria (LAB) strains, specifically, Lactiplantibacillus plantarum UMCC 2996, Furfurilactobacillus rossiae UMCC 3002, and Pediococcus pentosaceus UMCC 3010, were tested in new bread-whey media composed by wheat bread and sweet cheese whey, designed as an alternative to the conventional MRS medium. The medium resulting from hydrolysis with amylase and neutrase (AN) was considered the best for the growth of all the strains. This medium was particularly optimal for the strain F. rossiae UMCC 3002, which showed an increase in growth of 114% compared to that in MRS medium. Additionally, the bio-preservative ability of the selected LAB was assessed in pectin-based coated sliced bread inoculated with Aspergillus flavus ITEM 7828, Penicillium paneum ITEM 1381, and Aspergillus niger ITEM 7090. Different LAB strain behavior was observed towards the specific molds. A good bio-preservation activity was shown from F. rossiae UMCC 3002 against A. flavus ITEM 7828 with results compared to the protection by ethanol treatment. The results obtained in this study suggest a novel strategy for the cultivation of selected starters with a bio-protection activity by valorizing bread waste and cheese whey by-products, in a circular economy perspective.

Plant-based food is the new worldwide trend for healthier processed food to promote better human heath and implement ways to eliminate animals from the food chain equation. Our food system and society are already under tremendous pressure with a food production that is no longer sustainable and is part of the planetary crisis we are currently experiencing (The Eat-Lancet Commission, 2019). One of the big global challenges is to feed the fast-growing human population while reducing the impact of the food systems on the planet and preserve economic viability, social welfare, and healthy nutrition. Overall, the food sector accounts for about 26% of the global Greenhouse Gas (GHG) emissions (Poore & Nemecek, 2018). Livestock derived products alone accounts to 18% of the global GHG emission (Steinfeld et al., 2006). Therefore, there is a gradual shift towards plant based diet, which is evident in modern time due to consumer demands for a innumerable variety of foods that can reduce or replace meat consumption will help to address animal welfare as well. In addition, the recent COVID-19 crisis challenged the world population with regards to consumption of animal sourced food products (meat and processed meat etc.), which were linked with potential ability of zoonotic pathogens to infect humans (Mascaraque, 2020). During the pandemic and subsequent lockdown periods, a disruption in supply chain of meat products has been observed and reflects the consumers’ desire to embrace healthier dietary habits (FutureBridge, 2021). At the same time, multiple factors call for better utilization of food side streams generated by the food industry which is otherwise used as fodder and fertilizer or discarded due to their short shelf life. Improved sustainability by utilizing food grade biomass in the food value chains as new functional and nutritional ingredient for application in foods, is well documented and articulated among global authorities, experts, industry and is an increase

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 ma

Lactic acid bacteria (LAB) are a broad group of gram-positive bacteria that have actively been used worldwide in food productions due to their nutritional, technological, bioprotection and flavour-related characteristics, which define them as powerful biological tools to be used in fermented food applications. LAB are ubiquitous in nature and some species have been found in fructose-rich niches, such as insect microbiomes. Twenty-eight LAB species were isolated from Apis mellifera, Bombus spp. and Andrena nigroaenea insect sources. Lactococcus lactis was the most widespread species in all insect species. A phenotypical characterization analysis based on carbohydrate metabolism, subproduct and acid production, pectinolytic activity, citrate uptake, phytate degradation and diacetyl production was carried out. Based on this analysis, two strains belonging to Leuconostoc pseudomesenteroides and L.lactis species were found to be the most suitable strains for use in common food plant matrices. The former showed an ability to significantly catabolize raffinose, maltose and citrate, which are present in soy beverages, while the latter was able to produce high concentrations of diacetyl and lactic acid, which are relevant for the generation of plant dairy alternatives, and noticeably degraded phytic acid, pectin and sucrose mostly found in bean, cereal and fruit-based plant matrices.

Plant-based foods have risen in popularity in recent years including a number of dairy alternative products. Fermentation has the potential to support the development of innovative plant-based foods with enhanced flavour, texture and nutritional quality. Lactic acid bacteria (LAB) have been used for thousands of years to carry out fermentation of a wide variety of food substrates through production of organic acids and flavour compounds. However, LAB strains used in dairy fermentations are commonly found to be suboptimal in their metabolism of plant substrates, so efforts to identify alternative strains are needed. We provide an overview of the plant-based milk alternative category and explore screening approaches (including citizen-science efforts) to identify new LAB that hold potential in acidification and flavour formation of plant-based substrates.

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 β-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 β-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.

There is increasing interest in gluten degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten degrading enzymes are of great interest. We have identified a new thermostable gluten degrading proline specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterise the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The VmaxKm and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1 respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (alpha-gliadin) and SQQQFPQPQQPFPQQP (gamma-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up for new food processing possibilities and further the understanding of proline specific protease diversity.

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.

Nourishment of the growing human population requires new and alternative food sources, preferable produced without occupying new land areas. Cultivation of seaweed presents an opportunity, however, a major obstacle is sustainable preservation. Fermentation has been used for centuries to preserve vegetables, e.g., to produce kimchi based on cabbage. This study investigated changes in the microbiota, characteristics (pH, organic acids and water soluble carbohydrates) and food safety of raw shredded Alaria esculenta and Saccharina latissima during fermentation by the natural microbiota with or without addition of a Lactiplantibacillus plantarum starter culture. The Lb. plantarum fermented products retained a high Shannon diversity index, indicating a partially unsuccessful fermentation. Lb. plantarum performed better in A. esculenta causing pH to drop to below 4.6, a critical limit for control of growth of Clostridium botulinum, within 2 days compared to 7 days for S. latissima. Natural fermentation by the endogenous microbiota resulted in unsafe products with high final pH values (4.8–5.2), presence of unwanted organic acids, such as butyric acid, and in the case of A. esculenta sustenance of inoculated Listeria monocytogenes. Fermentation of A. esculenta and S. latissima by Lb. plantarum is a promising preservation method. However, future work is needed to optimise the process, by investigation of the use of different starter cultures, seaweed pre-treatments (blanching, freezing, etc.) and adjuvants (i.e., addition of sugars, minerals and similar) to promote growth of the starter culture and ensure the fermented products are safe to eat.

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-positives, 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, and a separately expressed mtlD. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements (cre). 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-positives, is repressed by phosphorylation by EIIAmtl, and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, 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/L mannitol with a 55% yield, which 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 s