Studies on the storage stability of fermented red dragon fruit (Hylocereus polyrhizus) drink

The objective of this work was to study the effect of storage temperatures and duration on the stability of fermented red dragon fruit drink (FRDFD) on its betacyanins content, physicochemical and microbiological qualities (BPM) and determining sensory acceptability. Results showed that both storage temperatures and duration have a significant effect on betacyanins content and physicochemical properties of FRDFD. Aerobic mesophilic and yeast and mold counts were lower than 1 × 103 CFU/mL for FRDFD stored at both temperatures. The loss of betanin (16.53–13.93 g/L) at 4 °C was 15.73% with no significant changes in physicochemical properties from week two onwards compared to 56.32% (16.53–7.22 g/L) of betanin loss at 25 °C. At week eight, FRDFD stored at 4 °C still contained 13.93 g/L betanin with a pH value of 3.46, suggested its potential as a functional drink which is sensory acceptable (mean score > 80% using hedonic test) among consumers

Study on bioaccessibility of betacyanins from red dragon fruit (Hylocereus polyrhizus)

Betacyanins are bioactive dietary phytochemicals which can be found in red dragon fruit (RDF). Therefore, the bioaccessibility of betacyanins that present in fermented red dragon fruit drink (RDFD) and pressed red dragon fruit juice (RDFJ) was accessed in simulated gastric and intestinal digestion. Results disclosed that betacyanins from RDFD and RDFJ suffered minor loss (< 25%) at gastric-like environment but greater loss was observed during the intestinal phase digestion. After subjected to intestinal digestion, RDFD retained 46.42% of betanin while RDFJ retained 43.76%, with betanin concentration of 17.12 mM and 12.37 mM, respectively. Findings also revealed that RDFD exhibited higher antioxidant capacity compared to RDFJ after subjected to intestinal digestion, with values of 0.88 mM Trolox equivalent antioxidant capacity (TEAC) and 0.85 mM TEAC, respectively. The data suggests that betacyanins that present in RDF are bioaccessible while fermentation able to enhance the bioavailability with more betacyanins retained after intestinal digestion

Lactococcus lactis harbouring Ara h 2.02 alleviates allergen-specific Th2-associated responses in sensitized mice

Aim: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice. Methods and results: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group. Conclusions: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect. Significance and impact of the study: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

Background: KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies

In silico-guided sequence modifications of K-ras epitopes improve immunological outcome against G12V and G13D mutant KRAS antigens

Background: Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. Methods: In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. Results: In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. Discussion: In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives

Synergistic enhancing effect of xanthan gum, carboxymethyl cellulose and citric acid on the stability of betacyanins in fermented red dragon fruit (Hylocereus polyrhizus) drink during storage

Nowadays, the demand for using healthy natural pigments (betacyanins) in the food industry is increasing. The present study aimed to overcome the circumstances that render the betacyanins instability in the red dragon fruit drink using mild approaches. These included optimised fermentation, incorporation of anionic polysaccharide mixture solution [xanthan gum (XG, 0.30–0.40 %, w/v) and carboxymethyl cellulose (CMC, 0.50–0.90 %, w/v)] and also addition of citric acid (CA, 0.05–0.20 %, w/v). The results of this study showed that the hydrocolloid mixture solution of XG and CMC significantly increased the samples' viscosity, pH and °Brix but reduced the aw, while betacyanins concentration had no significant change. The incorporation of CA at increasing concentration only reduced the samples’ pH significantly without affecting the viscosity, aw and °Brix. Among all fermented samples, Formulation 3E (0.40 % XG + 0.50 % CMC + 0.20 % CA) had achieved the desired commercial reference viscosity while also successfully minimised betacyanins degradation from 60.18 % to 14.72 %, had the best pH stability and no significant change in viscosity, aw and °Brix values after 4-week storage at 25 °C. The fermented red dragon fruit drink with betacyanins stabilised by Formulation 3E can be produced and served as an independent functional drink product and as a stable, functional ingredient (natural colourant) for the food industry