Production of lactic acid and probiotic biomass on distillery stillage

Mlečna kiselina je važna supstanca za prehrambenu, farmaceutsku i hemijsku industriju. Srbija trenutno uvozi mlečnu kiselinu pa bi korišćenje destilerijske džibre, otpadne vode iz procesa proizvodnje bioetanola, kao jeftinog supstrata u proizvodnji mlečne kiseline moglo biti efikasna i ekološki povoljna strategija. Osnovni cilj ove disertacije je bio da se ispita mogućnost integrisane proizvodnje mlečne kiseline i stočne hrane na industrijskoj destilerijskoj džibri iz proizvodnje bioetanola na skrobnim sirovinama. Hemijskom karakterizacijom džibre je pokazano da je džibra bogata proteinima i da sadrži odgovarajući sastav jona metala za rast bakterija mlečne kiseline. U toku selekcije mikroorganizama odabran je soj Lactobacillus rhamnosus ATCC 7469, kao odgovarajući soj za paralelnu proizvodnju mlečne kiseline i biomase pogodne za stočnu ishranu. U šaržnom sistemu je ispitan uticaj temperature, koncentracije inokuluma, mešanja i kontrole pH. Nakon optimizacije, odabrana je temperature od 41 °C, mešanje od 90 obrt/min, koncentracija inokuluma 5 % (v/v). Ispitana je mogućnost korišćenja CaCO3 i 30% NaOH kao sredstava za neutralizaciju i kontrolu pH vrednosti i odabran je dodatak 30% NaOH u četvorosatnim intervalima kao najpovoljniji za integrisani postupak proizvodnje mlečne kiseline i stočne hrane. Primenom NaOH je ostvarena visoka produktivnost i intenzivan rast Lb. rhamnosus ATCC 7469. Značajno unapređenje procesa je postignuto primenom dolivnog postupka gde je konačna koncentracija mlečne kiseline u medijumu povećana za 47,6% a zapreminska produktivnost za 21% u odnosu na šaržni postupak. Maksimalna postignuta produktivnost mlečne kiseline je iznosila 1,80 g L-1 h-1, sa prinosom mlečne kiseline od 0,87 g g-1 i koncentracijom mlečne kiseline od 97,1 g L-1. Broj vijabilnih ćelija po završetku fermentacije je bio 109 CFU ml-1. Recirkulacioni šaržni postupak sa imobilisanim Lb. rhamnosus ATCC 7469 na zeolitu je takođe isptivan. Snažna adsorpcija ćelija na površinu zeolita je omogućila lako odvajanje imobilizata od fermentisanog medijuma, gust rast ćelija u biofilmu i visoku stabilnost imobilisanih ćelija za recirkulaciju. Na kraju četvrtog ciklusa broj vijabilnih imobilisanih ćelija iznosio je više od 1010 CFU g-1 zeolitnog nosača. U šaržnom procesu sa recirkulacijom na tečnoj džibri bez dodatka izvora azota i minerla najviša postignuta produktivnost je iznosila 1,69 g L-1 h-1, uz maksimalnu koncentraciju mlečne kiseline 42,19 g L-1 i prosečni koeficijent prinosa od 0,96 g g-1. Ipak, u skladu sa potrebama Lb. rhamnosus ATCC 7469 i mineralnim sastavom džibre izvršena je i modifikacija zeolita, izmenom jona Na+ jonima Mg2+, što je rezultovalo povećanom adsorpcijom bakterija na površinu modifikovanog zeolita, kao i povećanjem proizvodnje mlečne kiseline za oko 10%...Lactic acid is a significant chemical for food, cosmetic, pharmaceutical and chemical industry. Currently, Serbia imports lactic acid and utilization of distillery stillage, a waste water from bioethanol production, as a cheap substrate for lactic acid fermentation could be an efficient and environmentally friendly approach. The main goal of this work was to investigate the possibilities of integrated production of lactic acid and animal feed on an industrial distillery stillage from bioethanol production on starch feedstock. Chemical analysis of the stillage has shown high content of proteins and valuable composition of minerals for the growth of LAB. In the selection process, Lactobacillus rhamnosus ATCC 7469 was chosen as the most promising strain for integrated production of lactic acid and biomass suitable for animal feed. The kinetics of lactic acid and biomass production in the batch fermentation and the impact of temperature, inoculum concentration, shaking and pH control were evaluated. The temperature of 41°C, shaking (90 rpm) and inoculum concentration of 5% (v/v) were selected for the fermentation. Among two different neutralizing agents (powdered CaCO3 and 30% solution of NaOH) a solution of 30% NaOH was selected for pH adjustment in four hour intervals during the fermentation. This system enabled the most optimal pH control and resulted in high lactic acid productivity and extensive growth of Lb. rhamnosus ATCC 7469. Significant improvements of the process were achieved in fed-batch fermentation where the final concentration of lactic acid was increased for 47.6 % and volumetric productivity for 21 % over the batch process. Maximal lactic acid productivity in fed-batch fermentation was 1.80 g L-1 h-1, with lactic acid yield of 0.87 g g-1 and lactic acid concentration of 97.1 g L-1. The number of viable cells at the end of fermentation was 109 CFU ml-1. The fermentation with a recirculation of immobilized Lb. rhamnosus ATCC 7469 onto zeolite was studied as a fermentation strategy. The strong adsorption of Lb. rhamnosus ATCC 7469 cells onto the zeolite surface allowed easy cell separation from the fermentation media, dense growth and high stability of immobilized cells for reuse in repeated batch cycles. A number of viable cells of over 1010 CFU per g of zeolite was achieved at the end of fourth fermentation cycle. A maximal process productivity of 1.69 g L-1, maximal lactic acid concentration of 42.19 g L-1 and average yield coefficient of 0.96 g g-1 were reached in the fermentation of stillage without mineral or nitrogen supplementation. However, in accordance to mineral requirements of Lb. rhamnosus ATCC 7469 and chemical content of minerals in stillage, exchange of Na+ ions in zeolite structure with selected Mg was performed. Exchange of Na+ with Mg2+ ions had improved immobilization of bacterial cells and increased lactic acid production for approximately 10%..

Intraoral administration of probiotics and postbiotics: An overview of microorganisms and formulation strategies

The last decade provided significant advances in the understanding of microbiota and its role in human health. Probiotics are live microorganisms with proven benefits for the host and were mostly studied in the context of gut health, but they can also confer significant benefits for oral health, mainly in the treatment of gingivitis. Postbiotics are cell-free extracts and metabolites of microorganisms which can provide additional preventive and therapeutic value for human health. This opens opportunities for new preventive or therapeutic formulations for oral administration. The microorganisms that colonize the oral cavity, their role in oral health and disease, as well as the probiotics and postbiotics which could have beneficial effects in this complex environment were discussed. The aim of this study was to review, analyse and discuss novel probiotic and postbiotic formulations intended for oral administration that could be of great preventive and therapeutic importance. A special attention has been put on the formulation of the pharmaceutical dosage forms that are expected to provide new benefits for the patients and technological advantages relevant for industry. An adequate dosage form could significantly enhance the efficiency of these products

Non-thermal plasma and lignocelullose susbstrates in biorefinery processes

Lignocelullosic substrates are abundant and available in almost all parts of the world. However, we are still lacking efficient ways to valorise it due to recalcitrant nature of lignocelullose and presence of lignin which prevents enzymatic hydrolisis of cellulose. The compact nature of lignocellulose biomass prevents its wider application in biorefineries. Effective way to separate lignin from cellulose or to make cellulose more accessible to enzymes remains challenge. Among lignocellulose biomass, agricultural by-products like corn stalks or husks are considered more convenient as substrates as they contain less lignin then wood biomass and therefore can be more easily degraded. However, acid or alkaline hydrolysis are the most common approach in pretreatment of lignocellulose substrates, but they are not selective, lead to loss of significant amount of sugars and cause generation of inhibitory substances in hydrolysate. Additionally, acid and alkaline pretreatments are performed at elevated temperatures and have significant environmental impact

Effect of non-thermal plasma on cellulose crystallinity and lignin content in corn stalks

Lignocellulosic biomass is a cheap raw material that, thanks to its high carbohydrate content, can be used in fermentation to produce biofuels, biogas and other compounds. Its complex structure, including cellulose, hemicellulose and lignin, requires prior treatment of the biomass to facilitate hydrolysis to simple sugars. Today, biomass is only partially utilized and generates about 14% of the world's energy. This is because the most commonly used physical, chemical and physicochemical treatments are not sustainable. They are energy-consuming but still low in productivity and toxic inhibitors formed during these treatments could hinder later steps of fermentation. Biomass treatment with advanced oxidation techniques has great potential as an environmentally friendly, so-called "green" treatment. These processes generate reactive species (radicals, electrons, ions and peroxides) that attack cellulose, hemicellulose, and lignin components. In this work, the effects of non-thermal plasma, the Fenton process, and the combined treatment of corn stalks with non-thermal plasma/Fenton were compared. Grounded biomass of corn stalks was mixed with Fenton reagent and hydrogen peroxide at different ratios and subjected to non-thermal plasma treatment. Carbohydrate content was decreased in non-thermal plasma treated samples both with and without Fe2+. However, a specific biomass: Fe2+:H2O2 ratio was required to achieve the highest rate of lignocellulose decomposition. The cellulose and hemicellulose fractions were affected and reduced by the treatments studied but resulted in almost no changes in the cellulose crystallinity index. The lower lignin content and cellulose crystallinity allow for more efficient enzyme hydrolysis of the treated lignocellulose and new options for valorization in fermentations

Advanced oxidation processes in treatment of agricultural biomass residues

Introduction Agricultural residues are abundant and cheap sources of lignocellulose widely available but with varying composition which strongly affects valorization routes. Valorization of agricultural biomass into bio-based chemicals is usually oriented towards treatments to release fermentable sugars from complex and strongly bonded molecules of cellulose, hemicellulose and lignin within lignocellulose. Treatments of lignocellulose were mostly studied to make it accessible to cellulases for hydrolisis of carbohydrates which could be then fermented into organic acid by lactic acid bacteria or bioetahnol by Saccharomyces sp., for example. In these processes valorization of lignin fraction was often neglected. However, lignin is among the most abundant aromatic polymers on planet with estimated amounts of over 300 billion tons and with annual increments estimated by around 20 billion tons (Smolarski, 2012). Lignin could be a raw material for numerous currently petrol-based chemicals like additives, thermoplastics and compounds used in pharmaceutical industry (Ponnusamy et al., 2019). Material and methods We studied the effects of advanced oxidation techniques, namely Fenton process, non-thermal plasma treatment and combination of these treatments on degradation of corn stalks as agroindustry based lignocellulosic substrate. Chemical composition was studied using FTIR, bioaccessibility and acetyl bromide soluble lignin content were determined by spectrophotometric methods while morphology of samples was examined by SEM. Different concentrations of Fe2+/Fe3+ and H2O2 were added to the grounded biomass of dried corn stalks and subjected to the plasma treatment. Results and Discussion The results showed that Fe/H2O2 ratio strongly affects degradation outcomes, where 1:5 Fe/H2O2 affects more carbohydrate fraction of substrate, while higher ratios are needed to affect significantly lignin fraction. The effect is potentiated with additional plasma treatment. These results are in agreement with recently reported study of Zhou et al (2020). Iodine sorption assey did not reveal significant changes in the bioaccesibility but FTIR spectra showed changes in the number of H-bonds in treated substrate. Increase in the relative content of lignin in plasma treated samples was probably a result of more prominent degradation of carbohydrate fraction, as confirmed by an increase in glucose content in liquid fraction of treatment media. Conclusion Shorter non-thermal plasma treatments (10 min) do not affect significantly lignin fraction of corn stalks per se, while addition of iron causes more significant changes in treated lignocellulose substrate under the milder conditions. Obtained liquid fraction could be potentially valorized in fermentations after removal of excess iron, while remaining lignin fraction could be modified or degraded by other means in biorefineries

Corn stalks as a lignocellulose substrate for biorefinery applications

We combined cold plasma treatment (CPT) with water, ethanol, Fenton and hydrogen peroxide media under acidic and basic conditions for treatment of corn stalks as significant lignocellulose agri-industrial waste. Chemical properties of treated samples were analyzed by FTIR and mercury porosimetry, acetyl bromide soluble lignin content was determined by spectrophotometric methods, while carbohydrate fractions were subjected to enzymatic hydrolysis followed by spectrophotometric analysis to assess its potential for fermentations. Lignin fraction which is often neglected (Ponnusamy et al., 2019) was also examined as a source of antioxidants

Development and validation of LC-MS/MS method for the citrinin determination in red rice

The liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the citrinin (CIN) determination in red rice was developed. The mycotoxin was extracted from red rice using anetonitrile/water/acetic acid mixture followed by the clean-up step on Captiva EMR cartridges. The developed method was validated according to the Commission Regulation No. 401/2006/EC. The validation data were evaluated in terms of recoveries, reproducibility, limits of quantification (LOQ), limit of detection (LOD), specificity, linearity and matrix effects for CIN in matrix using 13C20-OTA as an internal standard. The obtained method performance parameters indicate that the method is suitable for the CIN routine analysis