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.

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.

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.

Silicon-induced changes in antifungal phenolic acids, flavonoids, and key phenylpropanoid pathway genes during the interaction between miniature roses and the biotrophic pathogen Podosphaera pannosa.

Application of 3.6 mm silicon (Si+) to the rose (Rosa hybrida) cultivar Smart increased the concentration of antimicrobial phenolic acids and flavonoids in response to infection by rose powdery mildew (Podosphaera pannosa). Simultaneously, the expression of genes coding for key enzymes in the phenylpropanoid pathway (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and chalcone synthase) was up-regulated. The increase in phenolic compounds correlated with a 46% reduction in disease severity compared with inoculated leaves without Si application (Si-). Furthermore, Si application without pathogen inoculation induced gene expression and primed the accumulation of several phenolics compared with the uninoculated Si- control. Chlorogenic acid was the phenolic acid detected in the highest concentration, with an increase of more than 80% in Si+ inoculated compared with Si- uninoculated plants. Among the quantified flavonoids, rutin and quercitrin were detected in the highest concentrations, and the rutin concentration increased more than 20-fold in Si+ inoculated compared with Si- uninoculated plants. Both rutin and chlorogenic acid had antimicrobial effects on P. pannosa, evidenced by reduced conidial germination and appressorium formation of the pathogen, both after spray application and infiltration into leaves. The application of rutin and chlorogenic acid reduced powdery mildew severity by 40% to 50%, and observation of an effect after leaf infiltration indicated that these two phenolics can be transported to the epidermal surface. In conclusion, we provide evidence that Si plays an active role in disease reduction in rose by inducing the production of antifungal phenolic metabolites as a response to powdery mildew infection.

Harnessing cross-resistance - Sustainable nisin production from low-value food side streams using a Lactococcus lactis mutant with higher nisin-resistance obtained after prolonged chlorhexidine exposure.

Nisin has a tendency to associate with the cell wall of the producing strain, which inhibits growth and lowers the ceiling for nisin production. With the premise that resistance to the cationic chlorhexidine could reduce nisin binding, variants with higher tolerance to this compound were isolated. One of the resistant isolates, AT0606, had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Characterization revealed that AT0606 had an overall less negatively charged and thicker cell wall, and these changes appeared to be linked to a defect high-affinity phosphate uptake system, and a mutation inactivating the oleate hydratase. Subsequently, the potential of using AT0606 for cost efficient production of nisin was explored, and it was possible to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production.

Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis.

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 characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km 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 (α-gliadin) and SQQQFPQPQQPFPQQP (γ-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 new food processing possibilities and further the understanding of proline-specific protease diversity.

Site-specific, silicon-induced structural and molecular defence responses against powdery mildew infection in roses.

BACKGROUND: Silicon (Si) application to miniature potted roses can decrease severity of powdery mildew (Podosphaera pannosa) and this is associated with increased accumulation of callose and hydrogen peroxide (H2 O2 ) as well as hypersensitive (HR) cells. We used microscopy, gene expression and specific inhibitors of callose and H2 O2 to determine how effective these plant responses are in stopping infection. RESULTS: Pathogen arrest in Si-treated (Si+) plants was accompanied by increased accumulation of callose and H2 O2 in papillae and HR cells, respectively. These responses were reduced by application of specific inhibitors (2-deoxy-d-glucose for callose and catalase for H2 O2 ), which increased disease severity in Si+, but not in Si- plants. As markers for HR and callose, expression of the HR-specific gene hsr203J and the wound-related callose synthase GSL5, respectively, was studied. An up-regulation of expression was only seen after isolation of HR cells with laser capture microdissection. The up-regulation was higher in Si+ than in Si- plants and occurred concomitantly with more efficient photosynthesis in Si+ plants at high disease severity as compared to Si- plants. CONCLUSION: Silicon-mediated activation of callose and H2 O2 are decisive factors in the defence of rose against P. pannosa and these responses were accompanied with more efficient photosynthesis to strengthen the plant. Only by isolation of HR cells using laser capture microdissection as compared to analysis of whole leaf tissues allowed detection of elevated transcript levels of hsr203J and GSL5 at infection sites as markers for HR. © 2021 Society of Chemical Industry.

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles.

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co2+ and inhibited by Mg2+, K+ and Ca2+ followed by Zn2+, Na+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation.

Binary logistic regression analysis of hard palate dimensions for sexing human crania.

Sex determination is the preliminary step in every forensic investigation and the hard palate assumes significance in cranial sexing in cases involving burns and explosions due to its resistant nature and secluded location. This study analyzes the sexing potential of incisive foramen to posterior nasal spine length, palatine process of maxilla length, horizontal plate of palatine bone length and transverse length between the greater palatine foramina. The study deviates from the conventional method of measuring the maxillo-alveolar length and breadth as the dimensions considered in this study are more heat resistant and useful in situations with damaged alveolar margins. The study involves 50 male and 50 female adult dry skulls of Indian ethnic group. The dimensions measured were statistically analyzed using Student's t test, binary logistic regression and receiver operating characteristic curve. It was observed that the incisive foramen to posterior nasal spine length is a definite sex marker with sex predictability of 87.2%. The palatine process of maxilla length with 66.8% sex predictability and the horizontal plate of palatine bone length with 71.9% sex predictability cannot be relied upon as definite sex markers. The transverse length between the greater palatine foramina is statistically insignificant in sexing crania (P=0.318). Considering a significant overlap of values in both the sexes the palatal dimensions singularly cannot be relied upon for sexing. Nevertheless, considering the high sex predictability of incisive foramen to posterior nasal spine length this dimension can definitely be used to supplement other sexing evidence available to precisely conclude the cranial sex.

Binary Logistic Regression Analysis of Foramen Magnum Dimensions for Sex Determination.

Purpose. The structural integrity of foramen magnum is usually preserved in fire accidents and explosions due to its resistant nature and secluded anatomical position and this study attempts to determine its sexing potential. Methods. The sagittal and transverse diameters and area of foramen magnum of seventy-two skulls (41 male and 31 female) from south Indian population were measured. The analysis was done using Student's t-test, linear correlation, histogram, Q-Q plot, and Binary Logistic Regression (BLR) to obtain a model for sex determination. The predicted probabilities of BLR were analysed using Receiver Operating Characteristic (ROC) curve. Result. BLR analysis and ROC curve revealed that the predictability of the dimensions in sexing the crania was 69.6% for sagittal diameter, 66.4% for transverse diameter, and 70.3% for area of foramen. Conclusion. The sexual dimorphism of foramen magnum dimensions is established. However, due to considerable overlapping of male and female values, it is unwise to singularly rely on the foramen measurements. However, considering the high sex predictability percentage of its dimensions in the present study and the studies preceding it, the foramen measurements can be used to supplement other sexing evidence available so as to precisely ascertain the sex of the skeleton.