Immobilization of recombinant Escherichia coli cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor.

To improve the operational stability of glucose isomerase in E. coli TEGI-W139F/V186T, the immobilized cells were prepared with modified diatomite as a carrier and 74.1% activity of free cells was recovered after immobilization. Results showed that the immobilized cells still retained 86.2% of the initial transformational activity after intermittent reused 40 cycles and the yield of D-fructose reached above 42% yield at 60 °C. Moreover, the immobilized cells were employed in the continuous production of High Fructose Corn Syrup (HFCS) in a recirculating packed bed reactor for 603 h at a constant flow rate. It showed that the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603 h. The space-time yield of high fructose corn syrup reached 3.84 kg L-1 day-1. The investigation provided an efficient immobilization method for recombinant cells expressing glucose isomerase with higher stability, and the immobilized cells are a promising biocatalyst for HFCS production.

Systemic Amyloidosis Model on Young Mice.

Subcutaneous daily injection (with neglect of aseptics) of 0.5 ml solution of soybean cream substitute (10% volume in distilled water) during 30 days caused systemic amyloidosis in 30-day-old mice. All the known methods for induction of systemic amyloidosis are based on the use of old animals, as senile tissue bradytrophy allows effective simulation of amyloidosis.

Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate, soy protein isolate and fermentable sugar syrup.

Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process.

Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup.

High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k cat/K m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.

Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

Exploiting agarose gel modified with glucose-fructose syrup as a green sorbent in rotating-disk sorptive extraction technique for the determination of trace malondialdehyde in biological and food samples.

This research reports for the first time on the application of agarose gel impregnated with high fructose corn syrup (AG/HFCS) as a biodegradable and eco-friendly extraction phase in rotating-disk sorptive extraction (RDSE). The gel disk is driven by a rotary rod attached to an electric stirrer during extraction. Malondialdehyde (MDA) was chosen as a model analyte, and was extracted from biological and food samples using the proposed technique after derivatization with 2-thiobarbituric acid (TBA). Due to the hydrophilic nature of the sorbent phase, MDA was concentrated efficiently. After extraction, MDA was quantified directly in the gel disk by solid phase visible spectrophotometry and smartphone-based Red-Green-Blue (RGB) detection. The procedure used no organic solvents, showed clear advantages in terms of simplicity and short analysis time (5 min), and showed potential as a green analytical method. The extraction procedure was studied and optimized to maximize the partition of MDA into the gel. Under optimized conditions, the method provided linear dynamic ranges of 5.5-1000 ng mL-1 for biological samples and 62.5-12500 ng g-1 for food samples with correlation coefficients (R2) higher than 0.9975, relative recoveries between 88.3 and 103.3% along with relative standard deviation (RSD) values less than 3.5%. Accordingly, the proposed method can be employed by analytical laboratories for the rapid determination of MDA in complex matrix of body fluids and food samples under the principles of green chemistry.

The effect of corn syrup and whey on the conversion process of CO to ethanol using Clostridium ljungdahlii.

CO is one of the toxic components of syngas, which is the major source of air pollution. Syngas fermentation technology has the ability to convert toxic gases into valuable biofuels, such as ethanol. Fermentative ethanol production is an important method that can be used to promote environmental protection. CO can be converted into ethanol, via the Wood-Ljungdahl pathway, using Clostridium ljungdahlii. The components of the growing medium--especially the trace-element solution and yeast extract--are the main reasons for the high costs associated with this process, however, and this especially impacts scaled-up operations. In this study, cheaper substitutes for these components were used in order to determine their effect on ethanol production. The study comprised three main parts--the optimization of CO concentration, and the substitution of corn syrup and whey powder in the process. The optimum volume of CO for ethanol production was found to be 10 mL. Corn syrup can be used instead of trace-element solution, but the use of yeast extract with the corn syrup was determined to be essential. Up to 1.4 g/L ethanol production was observed with the addition of 15 mL corn syrup. Whey powder had the advantage of being usable without yeast extract, with up to 2.5 g/L ethanol being produced from a 30-g/L concentration. The main finding was that either corn syrup or whey powder can be used as substitutes for expensive basal-medium components.

A Novel Glucose Isomerase from Caldicellulosiruptor bescii with Great Potentials in the Production of High-Fructose Corn Syrup.

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli. The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity (Km = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS.

Impact of formulation on high-protein bar rheological and wear behaviors.

Due to the popularity of high-protein bars, many new formulations are being generated to meet consumer preferences. New formulations may have different mechanical behaviors that can negatively impact processing ability, which makes determining the effect of ingredients on processing ability important. Thus, the objective of this study was to determine the effects of major ingredients in high-protein bars on their rheological and tribological behaviors. Two response surface designs of model high-protein bars comprising whey protein isolate (WPI), high-fructose corn syrup (HFCS), and either canola oil (first design) or vegetable shortening (second design) were evaluated. Rheological tests, including adhesion, strain and frequency sweeps, large amplitude oscillatory shear, and wear testing, were conducted to determine the impact of individual ingredients on high-protein bar mechanical behaviors. Oil-based formulations had greater adhesion at higher levels of HFCS, while shortening-based formulations were affected by WPI more than HFCS, resulting in lower overall adhesive maximum forces. Formulas with higher levels of WPI had lower phase angles and greater extent of nonlinear viscoelastic and strain-hardening behaviors, while formulas with higher lipid and HFCS levels had higher phase angles. Overall, ingredient ratios had a notable impact on both oil- and shortening-based high-protein bar rheological and wear behaviors, suggesting that rheological and tribological testing could be useful for indicating processing ability of high-protein bars. The information gained in this study can be used by food manufacturers that produce cold-extruded or laminated food products. The results can help predict the ability of various formulations to be successfully processed, decreasing product development, and reformulation time and expense.

The retrogradation kinetics of starches of different botanical origin in the presence of glucose syrup.

The influence of glucose syrup on the retrogradation of cereal starches was investigated. Laboratory isolated starches from wheat (WS) and oats (OS - oat starch and ROS - residual oat starch) were used in this research. ROS was isolated from the flour left after the industrial separation of ß-glucans. Gelatinization temperature of oat starches (63.82°C and 64.01°C for OS and ROS, respectively) was higher than for WS (62.26°C), whereas gelatinization enthalpy for oat starches (8.87J/g and 9.09J/g for OS and ROS, respectively) was lower than for WS (9.99J/g). Moreover, retrogradation percentage (%R) was similar for both oat starches (29.76% and 27.72% for OS and ROS, respectively), and was substantially lower than for WS (42.04%). The introduction of glucose syrup into system reduced the extent of the retrogradation. Rate of the process was suppressed for WS and ROS, whereas for OS it was increased. ß-Glucan production process had no significant effect on the gelatinization and retrogradation of oat starch.

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration.

A validated fast difference spectrophotometric method for 5-hydroxymethyl-2-furfural (HMF) determination in corn syrups.

Corn syrups, important ingredients used in food and beverage industries, often contain high levels of 5-hydroxymethyl-2-furfural (HMF), a toxic contaminant. In this work, an in house validation of a difference spectrophotometric method for HMF analysis in corn syrups was developed using sophisticated statistical tools by the first time. The methodology showed excellent analytical performance with good selectivity, linearity (R2=99.9%, r>0.99), accuracy and low limits (LOD=0.10mgL-1 and LOQ=0.34mgL-1). An excellent precision was confirmed by repeatability (RSD (%)=0.30) and intermediate precision (RSD (%)=0.36) estimates and by Horrat value (0.07). A detailed study of method precision using a nested design demonstrated that variation sources such as instruments, operators and time did not interfere in the variability of results within laboratory and consequently in its intermediate precision. The developed method is environmentally friendly, fast, cheap and easy to implement resulting in an attractive alternative for corn syrups quality control in industries and official laboratories.

Qualitative and quantitative detection of honey adulterated with high-fructose corn syrup and maltose syrup by using near-infrared spectroscopy.

Near-infrared spectroscopy (NIR) was used for qualitative and quantitative detection of honey adulterated with high-fructose corn syrup (HFCS) or maltose syrup (MS). Competitive adaptive reweighted sampling (CARS) was employed to select key variables. Partial least squares linear discriminant analysis (PLS-LDA) was adopted to classify the adulterated honey samples. The CARS-PLS-LDA models showed an accuracy of 86.3% (honey vs. adulterated honey with HFCS) and 96.1% (honey vs. adulterated honey with MS), respectively. PLS regression (PLSR) was used to predict the extent of adulteration in the honeys. The results showed that NIR combined with PLSR could not be used to quantify adulteration with HFCS, but could be used to quantify adulteration with MS: coefficient (Rp2) and root mean square of prediction (RMSEP) were 0.901 and 4.041 for MS-adulterated samples from different floral origins, and 0.981 and 1.786 for MS-adulterated samples from the same floral origin (Brassica spp.), respectively.

Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min-1. The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.

Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw . The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA-hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.

Impact of AFEX™ Pretreatment and Extrusion Pelleting on Pellet Physical Properties and Sugar Recovery from Corn Stover, Prairie Cord Grass, and Switchgrass.

The effects of AFEX™ pretreatment, feedstock moisture content (5,10, and 15 % wb), particle size (screen sizes of 2, 4, and 8 mm), and extrusion temperature (75, 100, and 125 °C) on pellet bulk density, pellet hardness, and sugar recovery from corn stover, prairie cord grass, and switchgrass were investigated. Pellets were produced from untreated and AFEX™ pretreated feedstocks using a laboratory-scale extruder. AFEX™ pretreatment increased subsequent pellet bulk density from 453.0 to 650.6 kg m(-3) for corn stover from 463.2 to 680.1 kg m(-3) for prairie cord grass, and from 433.9 to 627.7 kg m(-3) for switchgrass. Maximum pellet hardness of 2342.8, 2424.3, and 1298.6 N was recorded for AFEX™ pretreated corn stover, prairie cord grass, and switchgrass, respectively. Glucose yields of AFEX™ corn stover pellets, prairie cord grass, and switchgrass pellets varied from 88.9 to 94.9 %, 90.1 to 94.9 %, and 87.0 to 92.9 %, respectively. Glucose and xylose yields of AFEX™ pellets were not affected by the extruder barrel temperature and the hammer mill screen size. The results obtained showed that low temperature and large particle size during the extrusion pelleting process can be employed for AFEX™-treated biomass without compromising sugar yields.

NIR detection of honey adulteration reveals differences in water spectral pattern.

High fructose corn syrup (HFCS) was mixed with four artisanal Robinia honeys at various ratios (0-40%) and near infrared (NIR) spectra were recorded with a fiber optic immersion probe. Levels of HFCS adulteration could be detected accurately using leave-one-honey-out cross-validation (RMSECV=1.48; R(2)CV=0.987), partial least squares regression and the 1300-1800nm spectral interval containing absorption bands related to both water and carbohydrates. Aquaphotomics-based evaluations showed that unifloral honeys contained more highly organized water than the industrial sugar syrup, supposedly because of the greater variety of molecules dissolved in the multi-component honeys. Adulteration with HFCS caused a gradual reduction of water molecular structures, especially water trimers, which facilitate interaction with other molecules. Quick, non-destructive NIR spectroscopy combined with aquaphotomics could be used to describe water molecular structures in honey and to detect a rather common form of adulteration.

Utilization of High-Fructose Corn Syrup for Biomass Production Containing High Levels of Docosahexaenoic Acid by a Newly Isolated Aurantiochytrium sp. YLH70.

High-fructose corn syrup (HFCS) is an agro-source product and has been the most commonly used substitute for sugar as sweetener in food industry due to its low price and high solution property. In this study, the F55 HFCS, rich in fructose and glucose, was first tested for biomass and docosahexaenoic acid productions as a mixed carbon source by a newly isolated Aurantiochytrium sp.YLH70. After the compositions of the HFCS media were optimized, the results showed that the HFCS with additions of metal ion and vitamin at low concentrations was suitable for biomass and docosahexaenoic acid productions and the metal ion and sea salt had the most significant effects on biomass production. During the 5-l fed-batch fermentation, total HFCS containing 180 g l(-1) reducing sugar was consumed and yields of biomass, lipid, and DHA could reach 78.5, 51, and 20.1 g l(-1), respectively, at 114 h. Meanwhile, the daily productivity and the reducing sugar conversion yield for docosahexaenoic acid were up to 4.23 g l(-1)day(-1) and 0.11 g g(-1). The fatty acid profile of Aurantiochytrium sp.YLH70 showed that 46.4% of total fatty acid was docosahexaenoic acid, suggesting that Aurantiochytrium sp.YLH70 was a promising DHA producer.