Bio-Layer Interferometry-Based SELEX and Label-Free Detection of Patulin Using Generated Aptamer.

This study reports a novel bio-layer interferometry (BLI)-based SELEX for generation of high affinity aptamers against patulin. Unlike conventional SELEX, the present method enabled real-time monitoring of increasing affinity of the oligonucleotides to the toxin. After seven rounds of selection cycles, the enriched pool of aptamers was characterized by cloning and sequencing and clustered into two families based on similarity. Two sequences, PAT C3 and PAT C4, each belonging to different clades, were further evaluated for their binding affinity. SPR studies determined the dissociation constants (KD) of 8.2 × 10-8 and 1.9 × 10-7 M for aptamer PAT C3 and PAT C4, respectively. The highest affinity PAT C3 aptamer was used to develop a patulin BLI aptasensor, which indicated a linear detection range from 0.045 to 100 ng/mL [limit of detection (LOD) = 0.173 ng/mL; limit of quantification (LOQ) = 0.526 ng/mL]. The aptasensor displayed no cross-reactivity with its structural analogue isopatulin or any of the other mycotoxin groups tested. Spiking studies in simulated apple juice samples showed recoveries in the range of 82.11 to 100.23%, indicating good sensor performance. The study is the first report of BLI-based SELEX for a non-protein toxin, which resulted in the generation of high affinity aptamers and development of an aptasensor which can have wide application in the food industry for high throughput screening of samples for patulin contamination within a short span of time.

Physicochemical Characterization and In Vitro Digestibility Study of an In Silico Designed Recombinant Protein Enriched with Large Neutral Amino Acids and Lacking Phenylalanine for Phenylketonuria.

In our previous study, a 3D structure of LNAA66 model protein containing 4-5 α-helices, high large neutral amino acids (LNAA) and lacking phenylalanine was designed, refined, expressed in Pichia pastoris and confirmed by Western blotting. Here the study is focused on the characterization of the expressed and purified recombinant LNAA66 protein. The results revealed that the expressed protein had 68.59% of LNAA enrichment, containing 41.6% of α-helix, 50.4% turns and 8% ß-sheet, which are as per the in silico designed protein. The LC-ESI-MS/MS results confirmed the recombinant protein by identifying the first 30 N-terminal amino acids with a sequence coverage of ~ 29%. The protein was digested entirely into smaller molecular weight fragments when treated with digestive enzymes mimicking the human GI tract digestion, which indicated complete digestibility of the protein. These results suggest that the protein can be utilized for the envisioned application of dietary treatment for phenylketonuria.

Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris.

We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% ß-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of ~16% from N-terminal region. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. In vitro digestibility of the protein using SGF and SIF showed the disappearance of ~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. Further in vivo digestibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases.

Improvement, cloning, and expression of an in silico designed protein enriched with large neutral amino acids in Pichia pastoris for possible application in phenylketonuria.

Phenylketonuria (PKU) is an inborn disease caused by defective phenylalanine hydroxylase, which consequently results in the accumulation of phenylalanine in the brain leading to further complications. One of the promising approaches in dietary treatment is the supplementation of large neutral amino acid (LNAA). The LNAA compete with phenylalanine for the common L-type LNAA transporter across the blood-brain barrier, and decrease phenylalanine levels in the brain. In this study, the earlier LNAA-enriched protein model was improved (Protein Model-66) and validated in silico. The reverse translated and codon-optimized synthetic LNAA66 gene was cloned into pPICZαC and expressed in Pichia pastoris. The expressed protein was purified by His Select affinity chromatography. SDS-PAGE and Western blotting analysis showed a band at an expected molecular weight of 12 kDa, confirming the expression of the modeled protein. To our knowledge, this is the first report showing the cloning and expression of an in silico designed LNAA-enriched protein. PRACTICAL APPLICATIONS: One of the promising dietary treatment of phenylketonuria (PKU) is the supplementation of large neutral amino acid (LNAA), wherein high levels of LNAA compete with phenylalanine for the same L-type LNAA transporter, and consequently decrease phenylalanine accumulation in the brain, thereby decreasing neurological complications. For the first time, here, we are showing that an in silico designed and validated Protein Model-66, rich in LNAA, can be successfully cloned and expressed in Pichia pastoris. The complete biochemical and structural characterization of this protein will give a clear insight into its potential application for PKU treatment. The protein can be potentially used as a supplement to treat PKU to those who are non-adherent to the restricted, non-palatable, and expensive diet. Furthermore, this novel and effective strategy of in silico designing, cloning and expression can be exploited to develop proteins for various applications of industrial, food, medical, and academic relevance.

Physico-chemical characteristics and stability aspects of coconut water and kernel at different stages of maturity.

Coconut water and kernel are the edible portions of the coconut. A study was carried out to evaluate the physico-chemical characteristics, phytonutrients and stability of coconut water (CW), kernel (CK) at different stages of maturity and commercial coconut products (CCP). The moisture content of CW, CK and CCP were in the range of 95-97 g 100 g(-1), 50-85 g 100 g(-1) and 0.4-3 g 100 g(-1) respectively. Fat content in CW was low (4-115 mg 100 g(-1)) whereas in kernel it was high (37-56 g 100 g(-1)). The CW was acidic in nature (pH 4.5-5.2). Ash content of CK decreased with maturity (1.0-1.5 %) whereas that of CW remained steady (0.3-0.4 g 100 g(-1)) with maturity. The total sugar content (3.9-4.6 g 100 g(-1)) and acidity (0.3-0.4 g 100 g(-1)) of CW did not change with maturity. The phenolics content increased in water (1.4-4.3 mg 100 g(-1)) and kernel with maturity (18.5-24.8 mg 100 g(-1)). The fatty acid composition of the oil extracted from the CK had increased saturated fatty acids (C12:0) (38-48 g 100 g(-1)) and decreased monounsaturated fatty acid (C18:1) (13-5 g 100 g(-1)) with maturity. The percentage of medium chain fatty acids increased with different stages of maturity (47-78 g 100 g(-1)). The CW and CK contained higher amount of phenolics (1.4-4.3 mg 100 g(-1) and 18.5-24.8 mg 100 g(-1) respectively) and total tocopherols of CK (0.14-0.59 mg 100 g(-1)) when compared to CCP. This study indicated that CW and CK could serve as valuable raw materials for the preparation of functional food supplement.

Preparation of food supplements from oilseed cakes.

Oilseed cakes have been in use for feed preparation. Being rich in proteins, antioxidants, fibers, vitamins and minerals, oilseed cakes have been considered ideal for food supplementation. These oilseed cakes can be processed and made more palatable and edible by suitable treatments and then incorporated as food supplements for human consumption. Rice bran pellets (RBP), stabilized rice bran (SRB), coconut cake (CC) and sesame cake (SC) were taken up for the study. These were mixed with distilled water and cooked in such a way to separate the cooked solid residue and liquid extract followed by freeze drying to get two products from each. The raw, cooked dried residue and extract were analyzed for various parameters such as moisture (0.9-27.4 %), fat (2.1-16.1 %), ash (3.3-9.0 %), minerals (2.6-633.2 mg/100 g), total dietary fiber (23.2-58.2 %), crude fiber (2.7-10.5 %), protein (3.2-34.0 %), and the fat further analyzed for fatty acid composition, oryzanol (138-258 mg/100 g) and lignan (99-113 mg/100 g) contents and also evaluated sensory evaluation. Nutritional composition of products as affected by cooking was studied. The cooked products (residue and extract) showed changes in nutrients content and composition from that of the starting cakes and raw materials, but retained more nutrients in cooked residue than in the extract. The sensory evaluation of cooked residue and extract showed overall higher acceptability by the panelists than the starting cakes and raw materials. On the basis of these findings it can be concluded that these cooked residue and extract products are highly valuable for food supplementation than the raw ones.