Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry.

Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3' end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL-1) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol.

Design and development of a machine for continuous popping and puffing of grains.

Popping/puffing have been traditionally practiced for enhancing storage life, improving organoleptic properties and ease of incorporation in ready-to-eat-foods. Currently, batch type sand and electric popping/puffing machines involving conduction mode of heat transfer are employed. The major drawbacks of these methods are high-energy consumption, scorching of grains, non-uniform product quality, contamination (by sand/ash) and problems in scale-up. Since fluidization is known to increase heat and mass transfer, a continuous fluidized popping/puffing machine (capacity 10-20 kg/h) involving convective mode of heat transfer is designed/developed. Hot-flue gas generating from burning of LPG was used as the eco-friendly fuel. Process parameters such as expansion ratio, fluidization velocity, terminal velocity, carry over velocity, bulk density and voidage were estimated for un-popped and popped/puffed rice, maize, jowar (sorghum) and paddy. Fluidization and carry over velocities for these grains were in the range of 4.18-5.78 m/s and 2.15-6.18 m/s, respectively. Based on the terminal velocity of the grains and volumetric air flow rate of the blower, fluidization chamber diameter was arrived. Chamber diameter of 0.15 m was found to be sufficient to generate required air velocity of 6.89 m/s which met the fluidization and carry over velocities of popped/puffed grains. The designed fluidization chamber was analyzed for heat and mass transfer during popping/puffing. Convective heat and mass transfer coefficients were estimated to be in the range of 103-187 W/m2 °C and 0.124-0.162 m/s, respectively. Theoretical values for total heat and mass transfer were similar to the experimental values.

A novel method for double encapsulation of C-phycocyanin using aqueous two phase systems for extension of shelf life.

Spirulina platensis is having high nutritive value due to pigments such as chlorophyll-a, phycobiliproteins (especially phycocyanins) and carotenoids. In our present work, C-phycocyanin (C-PC) was extracted from dry biomass of Spirulina platensis. C-PC being heat sensitive, reiterates the need for additional protection during drying (micro encapsulation). Accordingly, a novel method employing aqueous two phase systems (ATPSs) as carrier materials to achieve double encapsulation was studied for the first time. PEG 4000/Potassium phosphate and PEG 6000/Dextran were used at already standardized tie line length, at different volume ratios (by varying the total phase composition). ATPS at each volume ratio acted as different carrier materials offering varied degree of heat protection during double encapsulation while maltodextrin, being the conventional carrier material, was used for comparison. The best results of spray dried powders using PEG (4% w/w)/Potassium phosphate salt (18%, w/w) and PEG (6%)/Dextran (10%, w/w) phase systems as carrier materials were compared with conventional encapsulation (MDX as a carrier material) and freeze dying as control. PEG/Dextran as a carrier material with volume ratio of 0.25 resulted in the highest retention of blue colour (b*value), purity (0.43) as well as yield (YEP) of 94.99% w/w of C-PC, which could be stored for 6 months without much reduction from initial powder characteristics. From the overall results, it can be concluded that ATPS can be used as an effective carrier material for double encapsulation of biomolecules such as C-PC with additional benefit of enhancing the purity.

Recovery of proteins from coconut milk whey employing ultrafiltration and spray drying.

The coconut whey (CW) (underutilized by-product of virgin coconut oil production, with 2-3% protein) was subjected to ultrafiltration for concentration of protein and sugar removal prior to spray drying. The process parameters of ultrafiltration were standardised with respect to transmembrane flux, protein retention efficiency and removal of sugar by using 300 kDa membrane cut off, feed pH 4 and 2 bar transmembrane pressure at temperature 25 ± 2 °C. The protein content was found to increase in the retentate after ultrafiltration (termed as concentrated coconut whey, CCW) (from 21 to 46%, w/w) and sugar content to reduce (from 59 to 34%, w/w). The color lightness (L*) values of the CW and CCW powders obtained by spray drying were found to be 83.81 ± 0.33 and 80.70 ± 0.47, respectively. Both the samples of coconut protein powders were found to be microbiologically safe having water activity index for CCWP of 0.27 and CWP of 0.26. Carr index values for CCW (32.95) and CW (33.14) powders indicate both of them to have fair flowability. The powders have high potential as new source of protein or as a functional ingredient in the food processing industries.

Recovery of chlorophylls from spent biomass of Arthrospira platensis obtained after extraction of phycobiliproteins.

Extraction of chlorophylls has received scant attention or priority over phycobiliproteins from Arthrospira platensis. In fact extraction of chlorophylls from spent biomass (left after extraction of phycobiliproteins which goes as waste or underutilized) on drying, will improve the economics of the overall downstream processing. Ethanol (yield 5.75 mg/g, db), being a food grade solvent, was preferred over acetone and dimethyl sulfoxide in spite of their slightly better yields (5.85 mg/g, db). The best conditions were 100% concentration of ethanol, 1:8 S/L ratio, pH 6, 50 °C temperature and 1 h extraction time. An increase of 125% in yield besides reduction of 83.3% in extraction time (from 6 to 1 h) could be achieved at standardized conditions. Low-Humidity drying was observed to be a possible alternative to freeze drying for drying of spent biomass. Ultrasonication as pre-treatment and ethanol as solvent were found effective for extraction of chlorophylls from dry spent biomass.

Ultrasound assisted methods for enhanced extraction of phycobiliproteins from marine macro-algae, Gelidium pusillum (Rhodophyta).

Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which offer major hindrance for cell disruption. The present study is aimed at developing most suitable methodology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum(Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication for different parameters such as ultrasonication amplitude (60, 90 and 120µm) and ultrasonication time (1, 2, 4, 6, 8 and 10mins) at different temperatures (30, 35 and 40°C) was carried out. Kinetic parameters were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power density were estimated to be 41.97W, 14.81W/cm2 and 0.419W/cm3, respectively. Synergistic effect of ultrasonication was observed when employed in combination with other conventional primary extraction methods. Homogenization in combination with ultrasonication resulted in an enhancement in efficiency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrasonication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE was present in the extract and remained intact even after processing. Microscopic studies indicated a clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a given primary extraction method. These combination methods were found to be effective for extraction of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for downstream processing of biomolecules also from other macro-algae.

Removal of toxic Congo red dye from water employing low-cost coconut residual fiber.

The coconut residual fiber (CRF) is the major byproduct obtained during production of virgin coconut oil. Its application as a biosorbent for adsorption of Congo red was investigated. The CRF was subjected to different pretreatments, namely, pressure cooking, hexane treatment, acid treatment and their combinations. The pretreatment of CRF with the combination of hexane, acid, and pressure cooking resulted in the highest degree of adsorption. The equilibrium data were analyzed and found to fit best to both Langmuir and Freundlich isotherms. Thermodynamic parameters such as standard free energy (ΔG0 kJ mol-1), standard enthalpy (ΔH0, kJ mol-1) and standard entropy (ΔS0, kJ mol-1 K-1) of the systems were calculated by using the Langmuir constant. The ΔG0, ΔH0 and ΔS0 were found to be 16.51 kJ mol-1, -19.39 kJ mol-1 and -0.12 kJ mol-1 K-1, respectively, at 300 K. These thermodynamic parameters suggest the present adsorption process to be non-spontaneous and exothermic. The adsorption process was observed to follow pseudo-second-order kinetics. The results suggest that CRF has potential to be a biosorbent for the removal of hazardous material (Congo red dye) with a maximum adsorption capacity of 128.94 mg g-1 at 300 K.

Efficacy of free and encapsulated Bacillus lichenformis strain SL10 on degradation of phenol: A comparative study of degradation kinetics.

The present study exemplifies phenol degradation efficacy of the free and encapsulated bacterial isolate, explored the degradation kinetics and storage stability in detail. In brief, isolation, identification and phenol degradation potential of the bacterial made from wastewater treated sludge samples. The organism identified as B. licheniformis demonstrates phenol degradation at a concentration more than 1500 ppm. Optimization of environmental parameters reduces the time taken for degradation considerably. The organism has further been encapsulated using whey protein and the efficacy of encapsulated species suggested that encapsulation protects the cells from high concentration of phenol and at the same time expedite the degradation of the chosen pollutant at appreciable level. The encapsulated species effectively degrade 3000 ppm concentration of phenol within 96 h of incubation. Both pH and temperature stability observed in the encapsulated species suggests the effectiveness of the encapsulation. The encapsulated cells displayed storage stability for a four week period at 4 C and reusability up to three exposures. Degradation effected through intracellular catechol 2,3 dioxygenase. In conclusion, encapsulation of B. licheniformis (i) protects the cells from direct exposure to toxic pollutants; (ii) facilitates the field scale application and (iii) eliminate the practical difficulties in handling wet biomass in field application and assures the best possible way of remediating the phenol contaminated soil.

Analysis of modes of heat transfer in baking Indian rice pan cake (Dosa,) a breakfast food.

Heat transfer by individual modes is estimated during baking of rice (Oryza sativa) pan cake (Dosa), a traditional food. The mathematical expressions proposed could be used to modify the baking oven for controlling the individual modes of heat transfer to obtain the desired product texture, colour and flavour. Conduction from the rotating hot plate is found to be the most prominent mode of heat transfer and is critical for obtaining the desired product characteristics such as texture and flavour. Temperature profiles along the thickness of Dosa are obtained and compared with those obtained experimentally. Heat transfer parameters such as thermal conductivity and emissivity of Dosa are determined (0.42 W/m K and 0.31, respectively). The effect of material of construction of the hot plate such as alloy steel, teflon coated aluminum, cast iron and stainless steel on product texture was studied and stainless steel was found to give good surface finish to the product, which was confirmed by scanning electron microscope. Sensory evaluation was carried out to evaluate the product acceptability. The thermal efficiency of the baking oven was 51.5%.

Optimization of pneumatic sheet extrusion of whole wheat flour poory dough using response surface methodology.

Pneumatic extrusion of whole wheat flour dough is a challenge in the preparation of Poory. In the present study, the pneumatic extrusion process variables (pneumatic pressure, rate of extrusion) and quality of deep fried product (oil uptake, frying time, puffed height) was evaluated to get Poory of maximum overall sensory quality, minimum shear and minimum oil uptake. These parameters depend on the moisture content of wheat dough. Response surface methodology was demonstrated to be an efficient tool for the optimization of process parameters of pneumatic extrusion. The results indicated that extrusion pressure ranging from 3 ~ 6 × 10(5) Pa for the whole wheat flour dough with added moisture of 56 ~ 60 % was found to give a uniform rate of extruded sheet. It was observed that submerged frying time for the extruded dough sheet was in the range of 35 ~ 40 s, with the temperature of the vegetable oil to be in the range of 180 ~ 185 °C. Oil uptake during frying was about 12 ± 1 % and the textural shear force was found to be 9.9 N with an overall sensory score of 7.2 ± 0.5 on nine point scale. The experimental errors for all attributes were non-significant (p > 0.05) and thus optimum variables predicted by the model are found suitable.

Recent advances in nanoparticle based aptasensors for food contaminants.

Food safety and hazard analysis is a prime concern of human life, thus quality assessment of food and water is the need of the day. Recent advances in nano-biotechnology play a significant role in providing possible solutions for developing highly sensitive and affordable detection tools for food analysis. Nanomaterials based aptasensors hold great potential to overcome the drawbacks of conventional analytical techniques. Aptamers comprise a novel class of highly specific bio-recognition elements which are produced by SELEX (systematic evolution of ligands by exponential enrichment) process. They bind to target molecules by folding into 3D structures that can discriminate different chiral compounds. The flexibility in making modifications in aptamers contribute to the design of biosensors, enabling the generation of bio-recognition elements for a wide variety of target molecules. Nanomaterials such as metal nanoparticles, metal nanoclusters, metal oxide nanoparticles, metal and carbon quantum dots, graphene, carbon nanotubes and nanocomposites enable higher sensitivity by signal amplification and introduce several novel transduction principles such as enhanced chemiluminescence, fluorescence, Raman signals, electrochemical signals, enhanced catalytic activity, and super-paramagnetic properties to the biosensor. Although there are a few reviews published recently which deal with the potential of aptamers in various fields, none are devoted exclusively to the potential of aptasensors based on nanomaterials for the analysis of food contaminants. Hence, the current review discusses several transduction systems and their principles used in aptamer based nanosensors which have been developed in the past five years, the challenges faced in their designing, along with their strengths and limitations.

Single step aqueous two-phase extraction for downstream processing of C-phycocyanin from Spirulina platensis.

C-phycocyanin, a natural food colorant, is gaining importance worldwide due to its several medical and pharmaceutical applications. In the present study, aqueous two-phase extraction was shown to be an attractive alternative for the downstream processing of C-phycocyanin from Spirulina platensis. By employing differential partitioning, C-phycocyanin selectively partitioned to the polymer rich (top) phase in concentrated form and contaminant proteins to the salt rich (bottom) phase. This resulted in an increase in the product purity (without losing much of the yield) in a single step without the need of multiple processing steps. Effect of process parameters such as molecular weight, tie line length, phase volume ratio, concentration of phase components on the partitioning behavior of C-phycocyanin was studied. The results were explained based on relative free volume of the phase systems. C-phycocyanin with a purity of 4.32 and yield of about 79 % was obtained at the standardized conditions.

Development of a lemon cutting machine.

Cutting of lemon and other similar fruits is conventionally done manually by sharp knife, which is labor intensive and often un-hygienic. In the present work, a device has been designed and developed for cutting of lemon hygienically into four pieces of similar shape based on stationery cutters and rotating centralizing/locating slit plate concept. Machine has a unique knife assembly consisting of two bird wing shaped knives, joined by welding perpendicularly to a vertical knife, so that the lemon can be cut into four pieces in a single sweep. Six numbers of rotating centralizing/locating slit plates are welded on to the side plates and the plates carry a groove on its inner face, to enable the wing shaped knife to complete the horizontal cut. The rotating slit plates, having centralizing angle of 90°, are rotated by an electric geared motor. The prototype machine has capacity of over 5,000 lemons/h with a power consumption of 0.11 kW.

Production of coconut protein powder from coconut wet processing waste and its characterization.

Virgin coconut oil (VCO) has been gaining popularity in recent times. During its production, byproducts such as coconut skim milk and insoluble protein are obtained which are underutilized or thrown away to the environment at present. This study deals with utilization of these byproducts to obtain a value-added product, namely, coconut protein powder. When coconut milk was subjected to centrifugation, three phases, namely, fat phase (coconut cream), aqueous phase (coconut skim milk), and solid phase (insoluble protein) were obtained. The coconut skim milk and insoluble protein were mixed and homogenized before spray drying to obtain a dehydrated protein powder. The proximate analysis of the powder showed high protein content (33 % w/w) and low fat content (3 % w/w). Protein solubility was studied as a function of pH and ionic content of solvent. Functional properties such as water hydration capacity, fat absorption capacity, emulsifying properties, wettability, and dispersibility of coconut protein powder were evaluated along with morphological characterization, polyphenol content, and color analysis. Coconut protein powder has shown to have good emulsifying properties and hence has potential to find applications in emulsified foods. Sensory analysis showed high overall quality of the product, indicating that coconut protein powder could be a useful food ingredient.

An investigation of bread-baking process in a pilot-scale electrical heating oven using computational fluid dynamics.

A computational fluid dynamics (CFD) model was developed for bread-baking process in a pilot-scale baking oven to find out the effect of hot air distribution and placement of bread on temperature and starch gelatinization index of bread. In this study, product (bread) simulation was carried out with different placements of bread. Simulation results were validated with experimental measurements of bread temperature. This study showed that nonuniform air flow pattern inside the oven cavity leads to uneven temperature distribution. The study with respect to placement of bread showed that baking of bread in upper trays required shorter baking time and gelatinization index compared to those in the bottom tray. The upper tray bread center reached 100 °C at 1200 s, whereas starch gelatinization completed within 900 s, which was the minimum baking index. Moreover, the heat penetration and starch gelatinization were higher along the sides of the bread as compared to the top and bottom portions of the bread.

Reverse micellar extraction of beta-galactosidase from barley (Hordeum vulgare).

The reverse micellar system of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane was used for the extraction and primary purification of beta-galactosidase (EC 3.2.1.23) from the aqueous extract of barley (Hordeum vulgare) for the first time. The process parameters such as the concentration of the surfactant, the volume of the sample injected, and its protein concentration, pH, and ionic strength of the initial aqueous phase for forward extraction, buffer pH, and salt concentration for back extraction are varied to optimize the extraction efficiency. Studies carried out with both phase transfer and injection mode of reverse micellar extraction confirmed the injection mode to be more suitable for beta-galactosidase extraction. The extent of reverse micellar solubilization of proteins increased with an increase in protein concentration of the feed sample. However, back extraction efficiency remained almost constant (13-14.4%), which indicates the selectivity of AOT reverse micelles for a particular protein under given experimental conditions. beta-Galactosidase was extracted with an activity recovery of 98.74% and a degree of purification of 7.2-fold.

Extraction and purification of Ipomoea peroxidase employing three-phase partitioning.

Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 degrees C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.

Fractionation and purification of the phycobiliproteins from Spirulina platensis.

C-Phycocyanin and allophycocyanin of Spirulina platensis are fractionated and purified using a non-chromatographic method namely, aqueous two phase extraction for the first time. Optimized process parameters of aqueous two phase extraction (PEG 4000/potassium phosphate of tie line length 18.64% with a phase volume ratio 1.45) resulted in pure C-phycocyanin and allophycocyanin with a purity of 3.23 and 0.74, respectively, in a single extraction. Multiple extractions (two) improved the purity of C-phycocyanin from 3.23 to 4.02. Integration of aqueous two phase extraction with membrane process not only facilitated the separation of phase forming components from the products and also increased the purity of allophycocyanin from 0.74 to 1.5.

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes.

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.

Opportunities and challenges in high pressure processing of foods.

Consumers increasingly demand convenience foods of the highest quality in terms of natural flavor and taste, and which are free from additives and preservatives. This demand has triggered the need for the development of a number of nonthermal approaches to food processing, of which high-pressure technology has proven to be very valuable. A number of recent publications have demonstrated novel and diverse uses of this technology. Its novel features, which include destruction of microorganisms at room temperature or lower, have made the technology commercially attractive. Enzymes and even spore forming bacteria can be inactivated by the application of pressure-thermal combinations, This review aims to identify the opportunities and challenges associated with this technology. In addition to discussing the effects of high pressure on food components, this review covers the combined effects of high pressure processing with: gamma irradiation, alternating current, ultrasound, and carbon dioxide or anti-microbial treatment. Further, the applications of this technology in various sectors - fruits and vegetables, dairy, and meat processing - have been dealt with extensively. The integration of high-pressure with other matured processing operations such as blanching, dehydration, osmotic dehydration, rehydration, frying, freezing / thawing and solid-liquid extraction has been shown to open up new processing options. The key challenges identified include: heat transfer problems and resulting non-uniformity in processing, obtaining reliable and reproducible data for process validation, lack of detailed knowledge about the interaction between high pressure, and a number of food constituents, packaging and statutory issues.