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This study focuses on the development of a functional yoghurt oral flm strip with antibacterial properties and probiotics [(Streptococcus thermophilus (S. thermophilus), Lactobacillus bulgaricus (L. bulgaricus), and Lactococcus cremoris (L. cremoris)] using a pectin/glycerol mixture. The tensile characters, colour, physiochemical characters, in vitro disintegration time, mouth dissolving time and in vitro dissolution test of the functional yoghurt oral strip was compared with commercial oral strips. The study examined the storage stability of functional yogurt oral strips over a period of 35 days at room temperature (25 °C) using blister packs made of polypropylene/aluminium and polypropylene/linear low-density polyethylene (LLDPE). Signifcant changes in mechanical properties, colour, surface pH, in vitro disintegration, and mouth dissolving time was observed with in vitro dissolution time of 6 min. Functional oral strip showed signifcant (p<0.05) reduction in surface pH and probiotic viability, along with a signifcant increase in mouth dissolving time during storage in both packaging materials. Developed functional yoghurt oral strip also demonstrated better antibacterial quality on both storage materials with similar in vitro dissolution time periods. A satisfactory probiotic viability (>6 log CFU/strip) was upheld for 28 days in a polypropylene/aluminium blister pack, resulting in an extended shelf life for the functional yogurt oral strip. Therefore, this functional yoghurt oral strip development can be considered as a promising production method to retain functional qualities with longer shelf life at room temperature.

Spray-drying transforms functional yogurt into yogurt powder quickly and efficiently, maintaining product quality and enhancing storage stability. This process also adds value to the product. The study aimed to develop spray dried functional yoghurt powder encapsulating probiotics (Streptococcus thermophilus (S. thermophilus), Lactobacillus bulgaricus (L. bulgaricus), and Lactococcus lactis (L. lactis)) with longer shelf life. The production of functional yoghurt powder was optimized with Box–Behnken design using independent variables inlet air temperature (110–170 °C), feed temperature (10–20 °C), and pump speed (10–20%). The optimum functional yoghurt powder was obtained at 164.04 (164) °C inlet air temperature, 20 °C feed temperature, and 10.72 (11) % pump speed, respectively. The functional yoghurt powder with amorphous nature had storage viability (log CFU g−1) of 8.88 ± 0.03, 6.71 ± 0.12 and 8.16 ± 0.10 for S. thermophilus, L. bulgaricus, and L. lactis correspondingly on 49th day at 4 °C. The functional yoghurt powder demonstrated antibacterial activity, resulting in inhibition zones measuring approximately 18.33 ± 1.15 mm and 11.00 ± 0.00 mm against Klebsiella pneumoniae and Pseudomonas aeruginosa, respectively. Functional yoghurt powder had water activity 0.35–0.52, moisture content 7.27–16.22 (%), process yield 2.29–5.33 (%), hygroscopicity 10.09–16.87 (g/100 g) and degree of caking as 37.46–59.02 (%). The bulk densities, tapped densities, particle density and porosity of yoghurt powder ranged from 242 to 425 (kg m−3), 301.9–485.7 (kg m−3), 1050–2500 (kg m−3), and 58.67–87.85 (%) respectively. Furthermore, the solubility, dispersibility, and wettability of the powder ranged from 33 to 60%, 46.86–86.37%, and 2.13–4.28 min, respectively. The current study successfully achieved optimization at an inlet temperature of 164.04 °C (164 °C), a feed temperature of 20 °C, and a pump speed of 10.72% (11%), with desirability of 72.6%. This study makes a substantial adv

Vandachostylis ‘Sri-Siam’ is a fragrant orchid, suitable as a potted plant for the floriculture and perfumery industries due to its sweet citrus-like fragrance. Fragrance from the flowers was captured using SPME and subjected to GC–MS analysis. A transcriptome library was constructed from the tepals for sequencing using the Illumina MiSeq Desktop Sequencer and for de novo assembly using the CLC Genomic Workbench version 6.0. All de novo assembly sequences were subjected to bioinformatics analyses including BLASTx analysis, functional annotations and mapping of the annotated genes/gene products to the KEGG database using the Blast2go Pro software. GC–MS analysis reveals the fragrance are dominated by 68.73% monoterpenes, 11.26% esters, 4.20% sesquiterpenes and traces of alcohols (1.20%), dissimilar to both its parents. Analysis of the de novo assembly sequences generated 45,585 contigs which identified 14 fragrance-related transcripts. Information on the fragrance constituents and fragrance-related transcripts is deemed important as a reference for current and future work of using this hybrid in the breeding process of commercially valuable fragrant orchids.

Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) Lactococcus lactis is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of Staphylococcus aureus. A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host.

Multi-drug resistance has called for a race to uncover alternatives to existing antibiotics. Phage therapy is one of the explored alternatives, including the use of endolysins, which are phage-encoded peptidoglycan hydrolases responsible for bacterial lysis. Endolysins have been extensively researched in different fields, including medicine, food, and agricultural applications. While the target specificity of various endolysins varies greatly between species, this current review focuses specifically on streptococcal endolysins. Streptococcus spp. causes numerous infections, from the common strep throat to much more serious life-threatening infections such as pneumonia and meningitis. It is reported as a major crisis in various industries, causing systemic infections associated with high mortality and morbidity, as well as economic losses, especially in the agricultural industry. This review highlights the types of catalytic and cell wall-binding domains found in streptococcal endolysins and gives a comprehensive account of the lytic ability of both native and engineered streptococcal endolysins studied thus far, as well as its potential application across different industries. Finally, it gives an overview of the advantages and limitations of these enzyme-based antibiotics, which has caused the term enzybiotics to be conferred to it.

Background. Staphylococcus aureus is an opportunistic Gram-positive bacterium that can form biofilm and become resistant to many types of antibiotics. The treatment of multi-drug resistant Staphylococcus aureus (MDRSA) infection is difficult since it possesses multiple antibiotic-resistant mechanisms. Endolysin and virion-associated peptidoglycan hydrolases (VAPGH) enzymes from bacteriophage have been identified as potential alternative antimicrobial agents. This study aimed to assess the ability of Lactococcus lactis NZ9000 secreting endolysin and VAPGH from S. aureus bacteriophage 88 to inhibit the growth of S. aureus PS 88, a MDRSA. Method. Endolysin and VAPGH genes were cloned and expressed in L. lactis NZ9000 after fusion with the SPK1 signal peptide for secretion. The recombinant proteins were expressed and purified, then analyzed for antimicrobial activity using plate assay and turbidity reduction assay. In addition, the spent media of the recombinant lactococcal culture was analyzed for its ability to inhibit the growth of the S. aureus PS 88. Results. Extracellular recombinant endolysin (Endo88) and VAPGH (VAH88) was successfully expressed and secreted from L. lactis which was able to inhibit S. aureus PS 88, as shown by halozone formation on plate assays as well as inhibition of growth in the turbidity reduction assay. Moreover, it was observed that the spent media from L. lactis NZ9000 expressing Endo88 and VAH88 reduced the viability of PS 88 by up to 3.5-log reduction with Endo88 being more efficacious than VAH88. In addition, Endo88 was able to lyse all MRSA strains tested and Staphylococcus epidermidis but not the other bacteria while VAH88 could only lyse S. aureus PS 88. Conclusion. Recombinant L. lactisNZ9000 expressing phage 88 endolysin may be potentially developed into a new antimicrobial agent for the treatment of MDRSA infection.

Limonene is a plant monoterpene which contributes significantly to the scent of most essential oils due to its pleasant fragrance. The compound had been reported to have anti-cancer properties against several types of cancer including colorectal cancer. However, the production of this compound in nature is limited because it is produced as a secondary metabolite. To overcome these challenges, Lactococcus lactis was developed as a heterologous host for the production of limonene. A synthesized limonene synthase (LS) from Mentha spicata (mint) was cloned into L. lactis NZ9000. Western blot analysis using mouse IgG His-Tag monoclonal antibody showed successful LS expression by L. lactis at the size of ~55 kDa. GC-MS analysis results showed that limonene production was optimum after 24 h of induction (~8.0 ppm). Metabolic engineering was attempted to enhance the limonene production by overexpression of lactococcal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and mevalonate kinase (mvk) genes in the bacterial host. The recombinant L. lactis carrying pNZ:LSMM plasmid successfully enhanced the limonene production to two-fold (~15.1 ppm) after 24 h of induction. The outcomes of this study show the potential of L. lactis to produce plant proteins and bioactive compounds production, which prospectively leads to an oral delivery system for anti-cancer compounds.

There is an increasing demand for natural food preservatives due to consumers’ concern on the negative effects of chemical preservatives in food products. Nisin (bacteriocin) is an effective food biopreservative that has been approved globally. However, its low yield proves to be a limiting factor and must be addressed to meet the increasingly high demand from the food industry. The present work thus investigated the effects of individual and combined fermentation factors on Lactococcus lactis ATCC 11454 growth and nisin activity using the one-factor-at-a-time (OFAT) method. The level of each factor that gave the highest nisin production was then selected and combined to further improve its activity. The best combined conditions for highest cell growth and nisin activity were 30 °C, pH 6.0, and mild agitation with the addition of 1.0% w/v glucose, 1.0% w/v skim milk, and 0.5% v/v Tween 20. This increased nisin production by 22.7% as compared to control (basic condition). The present work provided critical information on the relationship between fermentation conditions, growth, and nisin activity of L. lactis ATCC 11454 that could be explored to understand the potential and limitation of the strain. This fermentation strategy can also serve as a benchmark to further enhance the production of bacteriocin or other biopreservative compounds.

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 librar

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.