Encapsulation in an alginate–goats’ milk–inulin matrix improves survival of probiotic Bifidobacterium in simulated gastrointestinal conditions and goats’ milk yoghurt

In this work, a new encapsulating matrix, alginate–goats’ milk–inulin, was used to encapsulate Bifidobacterium animalis subsp. lactis BB‐12. The addition of inulin resulted in capsules with a compact structure, and a higher probiotic cell count under simulated gastrointestinal conditions and in probiotic goats’ milk yoghurt during refrigerated storage. Encapsulation of the probiotic bacteria led to slower post‐acidification yoghurts. The results of this study showed that the alginate–goats’ milk–inulin matrix has potential to be used as a new encapsulation material to encapsulate probiotics for use in goats’ milk‐based probiotic fermented dairy products, avoiding the cross‐contamination caused by using capsules based on cows’ milk

Optimised production and extraction of astaxanthin from the yeast Xanthophyllomyces dendrorhous

Currently, astaxanthin demand is fulfilled by chemical synthesis using petroleum-based feedstocks. As such, alternative pathways of natural astaxanthin production attracts much research interest. This study aimed at optimising bioreactor operation parameters for astaxanthin production and evaluated strategies for its subsequent extraction. The effect of pH and agitation were evident as significant reduction in both biomass and astaxanthin production was observed when the culture pH was not controlled, and low agitation speed was applied. At controlled pH condition and high agitation speed, significant increase in biomass (16.4 g/l) and astaxanthin production (3.6 mg/l) were obtained. Enzymatic yeast cell lysis using two commercial enzymes (Accellerase 1500 and Glucanex) was optimised using central composite design of experiment (DoE). Accellerase 1500 led to mild cell disruption and only 9% (w/w) of astaxanthin extraction. However, glucanex treatment resulted in complete astaxanthin extractability, compared to standard extraction method (DMSO/acetone). When supercritical CO2 was employed as an extraction solvent in accellerace pre-treated Xanthophyllomyces dendrorhous cells, astaxanthin extraction increased 2.5-fold. Overall, the study showed that extraction conditions can be tailored towards targeted pigments present in complex mixtures, such as in microbial cells

Study on the effect of citric acid adaptation toward the subsequent survival of Lactobacillus plantarum NCIMB 8826 in low pH fruit juices during refrigerated storage

Pre-treatment of stationary phase cells of Lactobacillus plantarum NCMIB 8826 with citric acid (pH 3 to 6) for a short period of time significantly improved subsequent cell survival in several highly acidic fruit juices namely cranberry (pH 2.7), pomegranate (pH 3.5), and lemon & lime juices (pH 2.8). Although the mechanism for this adaptation is still unclear, the analysis of the cellular fatty acid content of acid adapted cells and the reverse transcription polymerase chain reaction (RT-PCR) showed a significant increase (by ~1.7 fold) of the cellular cyclopropane fatty acid, cis-11,12-methylene octadecanoic acid (C19:0cyclow7c) and a significant upregulation (~12 fold) of cyclopropane synthase (cfa) were observed, respectively, during acid adaptation. It is likely that these changes led to a decrease in membrane fluidity and to lower membrane permeability, which prevents the cells from proton influx during storage in these low pH fruit juices

Purification and polymerisation of microbial D-lactic acid from DDGS hydrolysates fermentation

A multi-step process was developed for microbial D-lactic acid purification, followed by poly-D-lactic acid (PDLA) synthesis via azeotropic polycondensation process. Several anion exchange resins were screened for their binding capacity using model lactic acid solutions. Amberlite® IRA67 (weak base anion exchange resin) showed the highest lactic acid adsorption, with maximum adsorption capacity, qmax, of 136.11 mg lactic acid / g of resin, and was further selected to purify D-lactic acid from DDGS hydrolysates through a three-step process; (1) treatment with 7% w/v activated carbon, (2) acidification of fermentation broth (Amberlite® IRA120) and (3) adsorption of lactic acid by anion exchange (Amberlite® IRA67). At the end of the purification process, 80.4% (w/w) D-lactic acid was recovered with 91.8% (w/w) purity, indicating the effectiveness of the developed downstream process. Furthermore, a clear yellowish solid polymer with a molecular weight of 3010 Da was obtained, suitable for applications in biomedical and agricultural sectors

Development of surfactant-coated alginate capsules containing Lactobacillus plantarum

A novel concept is proposed in which alginate capsules containing a model probiotic Lactobacillus plantarum strain are coated with different surfactants with the aim to enhance cell survival during passage initially through simulated gastric (SGF) and then intestinal (SIF) fluid. The surfactants investigated included the anionic sodium dodecyl sulphate (SDS) and ammonium lauryl sulphate (ALS), the cationic dimethyldioctadecylammonium chloride (DDAC), benzalkonium chloride (BZK) and hexadecyltrimethylammonium bromide (CTAB), and the zwitterionic lecithin. Coating the alginate capsules with CTAB, BZK, ALS and SDS resulted in worst survival (~ 4-9 log CFU/g decrease) compared to uncoated capsules (~3 log CFU/g decrease), after 1 hour exposure to SGF and two hours in SIF, which was most likely associated with their gradual penetration inside the microcapsules, as shown by confocal microscopy, and their antimicrobial effects. Coating the alginate capsules with DDAC improved cell survival compared to uncoated capsules (~1.2 CFU/g decrease), whereas coating with lecithin improved cell survival considerably, resulting in almost complete recovery of viable cells in SGF and SIF (~ 0.3 log CFU/g decrease). Although the interaction between alginate and lecithin was relatively weak as demonstrated by turbidity and contact angle measurements, it is likely that the protection was associated with the fact that lecithin was able to penetrate into the capsule rapidly, an observation that was supported by the fact that lecithin enhanced the viability of free cells in SGF and SIF. Lecithin has significant potential of being used as a coating material for probiotic containing capsules

Supercritical fluid extraction of carotenoids from vegetable waste matrices

The aim of this work was to evaluate a previously-developed model on supercritical fluid extraction (SFE) for carotenoid recovery from carrot peels on various carotenoid-rich fruit and vegetable wastes. To this end, 15 matrices, including flesh and peels of sweet potato, tomato, apricot, pumpkin and peach, as well as flesh and wastes of green, yellow and red peppers, were submitted to SFE under optimised conditions (59 °C, 350 bar, 15 g/min CO2, 15.5% (v/v) ethanol as co-solvent, 30 min of extraction time). The obtained extracts were characterised for their total carotenoid content, antioxidant activity and total carotenoid recovery (TCR). TCR values were greater than 90% w/w for most samples, with β-carotene being the most successfully extracted compound (TCRs 88–100% w/w). More polar carotenoids, such as lutein and lycopene, exhibited lower TCRs. A comparison with literature data suggested that carotenoid extraction is partially dependent on the composition of vegetable matrices, specifically on polysaccharide and moisture content. The results indicated that the optimised SFE conditions can be used as a general model for carotenoid extraction from various fruit and vegetable matrices and as a viable method for adding value to these waste streams by generating carotenoid-rich extracts

Supercritical carbon dioxide extraction of phenolic compounds from potato (Solanum tuberosum) peels

In the last three decades, greener technologies have been used, aiming at extracting phenolic compounds from vegetable matrices due to the inherent advantages compared to organic solventbased methodologies. In this work, supercritical CO2 was investigated for recovering phenolic acids from potato peels. Following screening runs for assessing the significant extraction parameters, a Central Composite Design of Experiments was carried out aiming at process optimization, with methanol concentration (MeOH, %) and CO2 flow rate (qCO2, g/min) as independent variables. Both parameters were deemed to impart a significant effect on the final response. Although the major phenolic acid in potato peels is chlorogenic acid (CGA), the main compound extracted was caffeic acid (CFA), present at a concentration of 0.75 mg/g dry peel in the extracts. The optimum extraction conditions were 80 �C, 350 bar, MeOH 20%, and flow rate of 18.0 g/min, which enabled a total phenolic recovery of 37% and a CFA recovery of 82%. The antioxidant activity of the supercritical fluid extraction (SFE) extracts was also measured, with the highest scavenging capacity reaching 73%. The need for using mixtures of water and organic solvents as co-solvents in SFE to enable CGA recovery seems necessary, possibly due to its better dissolution in aqueous solutions than in pure solvents

Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health

Adhesion ability to the host is a classical selection criterion for potential probiotic bacteria that could result in a transient colonisation that would help to promote immunomodulatory effects, as well as stimulate gut barrier and metabolic functions. In addition, probiotic bacteria has a potential protective role against enteropathogens through different mechanisms including production of antimicrobial compounds, reduction of pathogenic bacterial adhesion and competition for host cell binding sites. The competitive exclusion by probiotic bacteria not only has a beneficial effect on the gut but also in the urogenital tract and oral cavity. On the other hand, prebiotics may also act as barriers to pathogens and toxins by preventing their adhesion to epithelial receptors. In vitro studies with different intestinal cell lines has been widely used along the last decades to assess the adherence ability of probiotic bacteria and pathogen antagonism. However, extrapolation of these results to in vivo conditions still remain unclear, leading to the need of optimization of more complex in vitro approaches that includes interaction with the resident microbiota to address the current limitations. The aim of this mini-review is to provide a comprehensive overview on the potential effect of the adhesive properties of probiotics and prebiotics on the host by focusing on the most recent findings related with adhesion and immunomodulatory and antipathogenic effect on human health

Purification and characterization of microbial protease produced extracellularly from Bacillus subtilis FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca2+, Mg2+, Mn2+, Fe2+, Ca2+ and K+, but 10 mM Fe3+ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%)

Development of chitosan-coated agar-gelatin particles for probiotic delivery and targeted release in the gastrointestinal tract

This study reports the development of a novel and simple formulation for probiotic delivery using chitosan-coated agar-gelatin gel particles. This methodology involves the production of agar-gelatin particles by thermally treating a mixture of agar and gelatin solutions at high temperatures (121°C) and subsequently coating with chitosan. The particles were able to protect the probiotic strain Lactobacillus plantarum NCIMB 8826 during incubation for 2 hours in simulated gastric fluid (pH 2) as no statistically significant loss (P > 0.05) in cell concentration was observed, and also resist dissolution in simulated intestinal fluid (pH 7.2). Interestingly, this protection is related to the fact that the intense thermal treatment affected the physicochemical properties of agars, and resulted in the formation of a strong and tight polymer network, as indicated by the X-ray diffraction (XRD) analysis. Using an in vitro faecal batch fermentation model simulating the conditions of the distal part of the large intestine (pH 6.7-6.9), it was demonstrated by quantitative real time PCR that the majority of L. plantarum cells were released from the agar-gelatin particles within 30 to 48 hours. Overall, this work led to the development of a novel methodology for the production of probiotic containing particles which is simpler compared to current encapsulation technologies, and has a lot of potential to be used for the controlled release of probiotics and potentially other solid bioactives in the large intestine