Glucose Oxidase Assay Kit

Bee pollen (BP) and bee bread (BB) have attracted great attention due to their biological activities including antibacterial activity. However, the mechanism of antibacterial activity is largely unknown. Therefore, we aimed to characterise the antibacterial effect of BP and BB aqueous extracts against bacterial pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Enterococcus faecalis) and identify the key compound(s) responsible for this effect. Here, we demonstrate that BP and particularly BB extracts display antibacterial activity which is significantly increased in the presence of glucose. Immunoblot analysis of extracts revealed the presence of MRJP1 in all analysed BP and BB samples and the enzyme glucose oxidase (GOX) in the majority of BB samples. Treatment of extracts with catalase resulted in the restoration of bacterial growth but only in those samples where glucose supplementation caused the enhancement of antibacterial activity. Our findings provide a deeper understanding of antibacterial activity of BP/BB which is mediated by the enzymatic activity of bee-derived GOX.

A versatile sample pretreatment method for the honey matrix is still needed for any proteomic-based investigations. Invertase and diastase are the most important enzymes in the maturation of pine honey and the origin of these enzymes are attributed to the bee's hypopharyngeal glands (HPG). In our study, we aimed to isolate and enrich these enzymes as model proteins representing the honey proteome in an efficient and practical way. As authenticity comparison, isolating the same enzymes from HPG samples was also accomplished. For yielding pine honey crude protein isolate, as a tandem two-step approach, stirred cell ultrafiltration followed by centrifugal ultrafiltration (CUF) protocol was determined after experimental optimization. HPGs were dissected from the Apis mellifera L. and proteins were extracted by using a bead beater followed by concentration using CUF. Protein profiles of pine honey and HPG were compared by SDS-PAGE. The resulting protein concentrations, enzyme activities, and the cleaning efficiencies of the applied techniques were evaluated and optimized using the Bradford assay, modified enzyme activity assays, and sugar profiling method developed at HPLC-RID. The novel pretreatment method provided invertase at 1055.1 U/kg activity and diastase at 693.3 Shade U/g activity with yields of 900.9% and 2432.6%, respectively. The final crude protein isolate can be interpreted as reference material at any authenticity and quality assays of honey. The obtained crude protein extract will pave the way for high throughput proteomic investigations at the honey matrix. Furthermore, this template methodology could be scaled up in the industry for natural enzyme production.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H₂O₂ were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe⁺²/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

To date, how the pH conditions of thermal processing tailor the structure and digestibility of resulting starch-based complexes remains largely unclear. Here, indica rice starch (IRS), stearic acid (SA), and a whey protein isolate (WPI) were used as materials. Increasing the pH value from 4 to 8 during thermal processing (pasting) mainly suppressed the starch digestion of starch-WPI-SA complexes rather than starch-SA counterparts. The starch-SA complexes showed moderate structural changes as the pH value rose, and there was less rapidly digestible starch (RDS) only at pH 8. For the starch-WPI-SA complexes, an increased pH value allowed larger nonperiodic structures and more V-type starch crystallites, with almost unchanged short-range orders but apparently collapsed networks at pH 8. Such ternary complexes displayed more resistant starch (RS) as the pH value rose. The ternary sample at pH 8 contained ca. 29.87% of the RS fractions.

Resistant starch type 2 (RS) was isolated from lotus stem using enzymatic digestion method. The isolated RS was subjected to ultrasonication (US) at different sonication power (100–400 W). The US treated and untreated RS samples were characterized using dynamic light scattering (DLS), scanning electron microscopy (SEM), light microscopy and Fourier transform infrared spectroscopy (FT-IR). DLS revealed that particle size of RS decreased from 12.80 µm to 413.19 nm and zeta potential increased from −12.34 mV to −26.09 mV with the increase in sonication power. SEM revealed smaller, disintegrated and irregular shaped RS particles after ultrasonication. FT-IR showed the decreased the band intensity at 995 cm⁻¹ and 1047 cm⁻¹ signifying that US treatment decreased the crystallinity of RS and increased its amorphous character. The bile acid binding, anti-oxidant and pancreatic lipase inhibition activity of samples also increased significantly (p < 0.05) with the increase in sonication power. Increase in US power however increased the values of hydrolysis from 23.11 ± 1.09 to 36.06 ± 0.13% and gylcemic index from 52.39 ± 0.38 to 59.50 ± 0.11. Overall, the non-thermal process of ultrasonic treatment can be used to change the structural, morphological and nutraceutical profile of lotus stem resistant starch which can have great food and pharamaceutical applications.

Chestnut rose (Rosa Roxburghii) is an endemic fruit in China with many bioactivities. The present study investigated the effect of chestnut rose juice concentrate (CRJC) on the digestibility of normal wheat starch (NWS), with a focus on the interaction between polyphenols and amylose as well as that between polyphenols and porcine pancreatic α-amylase (PPA). NWS was mixed with CRJC and digestibility was determined using Englyst method. The composition of CRJC was analyzed by ultrahigh performance liquid chromatography-mass spectrometry. Molecular dynamics (MD) was used to study the binding between polyphenols and PPA and between polyphenols and amylose. The results showed that CRJC could significantly increase the resistant starch content. The MD results agreed with previous docking and experimental results regarding the inhibitory effects (IEs) of polyphenols (with galloyl and glycosyl group differences) against α-glucosidase. The MD results also showed that complex polyphenols with greater molecular weights or a high number of hydrogen bond donors (HBDs)/acceptors (HBAs) bind in a stronger and more lasting manner with amylose than simple polyphenols. CRJC has the potential to reduce the risk of developing type 2 diabetes and molecular simulation can be a supporting tool to study mechanisms in food system.

Enzyme immobilization is necessary process to improve the bioactivity and stability of the biocatalyst. In this study, glucose oxidase (GOx) enzyme was immobilized on plasma-treated fibrous carbon felt as a textile carrier to produce a heterogeneous catalyst. Genipin, as a naturally occurring crosslinker, that has less cytotoxicity than conventional crosslinkers, was used in the enzyme immobilization process. UV-Vis and FTIR spectra confirmed the crosslinking reaction between genipin and the primary amines of GOx enzyme, by forming blue-pigmented aggregates. GOx relative activity after crosslinking and immobilization on the carbon felt was maintained up to 40%, with stability in performance up to 6 cycles for the plasma treated carbon, while maintaining their bio-electro-activity as shown from cyclic voltammetry scans (CV). The obtained heterogeneous catalysts have been tested for use in sustainable wastewater treatment of Remazol Blue RR (RB) dyestuff by means of Bio-Fenton (BF) and enzymatic Bio-electro-Fenton (BEF) processes. The produced samples resulted in high color removal efficiency, up to 93% discoloration of (RB) for the first use in (BF) process in 3 h. Meanwhile, enzymatic (BEF) process resulted in up to 34% of COD removal, with simultaneous power density generation up to 0.16 ± 0.01 μW.cm⁻² at a current density of around 10 ± 2 μA.cm⁻² in 12 h. These results highlight the importance of genipin as a bio-based crosslinker for enzymes, and the potential use in both (BF) and (BEF) as sustainable approaches for wastewater treatment and as a step towards zero-energy degradation of organic matter.

We report a novel strategy to offer a high-performance biosensor platform by fabricating an electrically conductive and adhesive electrode composed of polypyrrole (PPy)/polydopamine (PDA) with enzymes (i.e., glucose oxidase (GOx) or lactate oxidase (LOx)) by one-step electrochemical coating. We focused on electrochemical glucose sensing using PDA/PPy and GOx as a model biosensor. PDA/PPy/GOx biosensor was highly sensitive (28 times higher) compared to the PPy/GOx, which appeared to result from enhanced GOx immobilization and efficient signal transduction by PDA/PPy. A selective layer of polyphenol (PPh) on the PDA/PPy/GOx successfully eliminated possible interference. Finally, the GOx biosensor displayed a good sensitivity of 22.15 μ A mM⁻¹ cm^-2, a short response time of 5-6 s, a linear range up to 5.0 mM, and a detection limit of 138 μM glucose (R² = 0.995). Human blood serum tests accurately measured glucose concentration. PPh-layered PDA/PPy/GOx biosensor maintained exceptional stability over 90 days (93.9%). Furthermore, we fabricated a lactate biosensor (PDA/PPy/LOx) by electrochemical PDA/PPy deposition with LOx and demonstrated the significant improvement of a lactate sensing ability compared to PPy/LOx. This facile fabrication strategy involving PDA/PPy and enzymes will serve as an attractive platform for highly sensitive and stable biosensors, such as urate and bilirubin detection.

Aims: This research is aimed at developing a method of processing to increase the quantity of resistant starch in garri and reduce its glycemic index using pullulanase-producing Bacillus subtilis organism. Place and Duration of Study: Department of applied microbiology and brewing, Nnnamdi Azikiwe University, Awka between January, 2018 and February, 2019. Methodology: The organism was isolated from different cassava processing sites in Anambra metropolis, Nigeria. It was then identified based on phenotypic, biochemical and molecular characteristics After which the pullulanase assay, the fermentation studies, resistant starch analysis and glycemic index was analysed. Results: Pullulanase assay result showed Bacillus subtilis as a very good pullulanase producing organism with a pullulanase quantity of. The resistant starch content was found to be higher for the samples fermented with the choice organism and retrograded at 10oC at 14.29%, than the control garri sample fermented without any organism and not retrograded at 4.73%. The glycemic index was relatively high in all the garri samples, however, the lowest glycemic index, 62% was observed in the garri sample produced with the choice organism. Conclusion: This research has been able to show that pullulanase enzyme from Bacillus subtilis is a very useful industrial raw material in production of functional foods with low glycemic index.

The objective of this study was to establish the impacts of supercritical fluid extraction (SFE) processing on the physicochemical properties of pea flour and the structure of isolated pea starch. A significant (p < 0.05) increase in protein content and reduction in several pasting and thermal parameters as measured by rapid visco-analyzer and differential scanning calorimeter were observed after SFE. Additionally, SFE increased starch digestibility as determined by an in vitro starch digestion assay. An increased amylopectin content and crystallinity along with the loss of double helix content was supported by size exclusion chromatography and FT-IR data, respectively. X-ray diffraction and scanning electron microscopy showed minimal alterations of starch, by SFE, in long-range crystalline and morphological structure of starch granules, respectively. The data demonstrated SFE influenced the physicochemical and structural characteristics of pea starch. These outcomes illustrated that SFE might be a green and novel technology for starch modification.

Sweet cassava and guinea corn were processed into flour using two different drying methods (sun and oven) to produce unfermented enriched fufu. Experimental design from which ten blends were selected for analyzes, sample F (100% traditional processed fufu) and sample C (commercial market sample - semolina) were used as control. In-vitro starch digestibility, glycemic index/load, antioxidant potential, and sensory attributes were evaluated, analyzed and mean were separated using Duncan New Multiple Range (DNMR) test at p ≤ 0.05. In-vitro starch digestibility of the experimental samples were significantly (p ≤ 0.05) higher than 8.91% obtained for sample F in both sun- and oven-dried samples. Results of the estimated glycemic index/load of the experimental samples showed that enriched fufu can be classified as low glycemic foods and are significantly (p ≤ 0.05) higher than sample F (55%) and glucose (100%). The samples also had better ability to scavenge free radicals against DPPH and ABTS.

Background and objectives: Consumption of pulses is recommended by health organizations. Pulses are usually soaked and cooked in water before consumption which alters their nutrient composition and ultimately health benefits. This study investigated the effect of four cooking methods (boiling, pressure, microwave, and slow) and four heating solutions (water, salt, sugar, and citric acid) on composition of nutritionally‐important starch fractions and flatus oligosaccharides in faba bean, lentil, and pea. Findings: The three pulses had slowly digestible starch (SDS) as the highest fraction with faba bean and lentil exhibited higher SDS than pea, but pea contained the highest level of resistant starch (RS). Rapidly digestible starch (RDS) was comparable among the three pulses. Stachyose and verbascose were the dominant oligosaccharides in faba bean, lentil, and pea. Regardless of the heating solution, slow cooking was more effective in improving starch nutritional fractions, i.e., producing lowest RDS and highest SDS in the three pulses with some exceptions. The RS fraction increased in the three pulses subject to cooking method and pulse type. Pressure and slow cooking methods were effective in reducing oligosaccharides in the three pulses. Conclusions: The results suggest that slow cooking in water or salt solution has great potential to improve starch nutritional fractions and diminish flatus oligo‐sugars. Significance and novelty: This is the first study to demonstrate the effectiveness of slow cooking in enhancing starch nutritional fractions and reducing flatus oligo‐sugars in pulses. The method could hold a promise for implementation especially small‐ and large‐volume slow cookers are commercially available.

The cereal grains such as wheat, barley, sorghum, millets were evaluated before and after germination (24 h, 48 h and 72 h) and compared for their proximate composition, antioxidant activity, total phenolic content, total flavonoid content, pasting properties, in vitro starch digestibility and FTIR spectroscopy. Germination inversely affected the protein, fat, and ash content of different cereal grains. The germinated flours have less water content and higher oil absorption capacities along with reduced starch content. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in the ungerminated cereal flours ranged from 20.7 to 32.1%, 26.9 to 38.0% and 6.2 to 17.6% respectively but after germination of 72 h, the RDS content increased from 26.5 to 36.2% while SDS and RS content decreased from 26.1% (sorghum) to 16% (barley) and 14.7% (barley) to 4.6% (wheat) respectively. The drought–tolerant crops (sorghum, millets and barley) are potential sources of antioxidants and phenolic content and yielded lower hydrolysis index and estimated glycaemic index upon germination. The highest section of antiparallal β-sheet, α-helix and β-turns were found in wheat flour followed by sorghum flour and their proportion decreased with continuous germination. The continuous reduction of viscosity was evaluated with the progress in germination. Overall, germination is a way to get health-promoting compounds from less utilizing cereal such as millets, sorghum and barley and enhance their uses to nourish the huge population with the aim to fulfill their nutritional requirements.

The study aims to evaluate the effects of xanthan gum (XG), inulin and defatted rice bran on the physicochemical, functional and sensory properties of the gluten-free noodle prepared from Pathumthani 80 rice (RD 31) native autoclaved resistant starch (NARS). The increase in concentration of XG (0.625-5%) in the formulation of gluten free noodles resulted a significant decrease (p ˂ 0.05) in resistant starch content whereas, the tensile strength was increased. RD 31-NARS gluten-free noodles with 2.5% XG received the high sensory scores and further selected for the addition of inulin and defatted rice bran (5%, w/w), separately. RD 31-NARS gluten free noodle with XG (2.5%) and defatted rice bran exhibited the highest protein (4.62%), fiber (0.35%) and ash content (1.45%). Further, addition of inulin and defatted rice bran significantly (p ˂ 0.05) decreased the glycemic index and increased the cooking time and firmness. The surface morphology of gluten free noodles showed relatively porous structure under the scanning electron microscope (SEM) after the addition of rice bran (5%) and inulin (5%). Thus, resistant starch along with gums and fibers can be used to develop gluten free functional foods with better acceptability.

There is scant information regarding the role of interleukin (IL)-6 in obesity-related metabolic dysfunction in humans. Thus, we studied the serum levels of IL-6 in normal weight, overweight, and obese subjects, and examined associations of IL-6 with hyperglycemia, insulin resistance, dyslipidemia, and systemic inflammation. One hundred three women and men were included in the study. Anthropometric parameters, blood glucose, insulin, total cholesterol, and triglycerides were measured. Serum levels of tumor necrosis factor-alpha (TNF-alpha), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance (ANOVA) showed a 2.5-fold significant decrease in serum IL-6 in overweight and obese individuals when compared with normal weight controls. Serum IL-6 exhibited significant inverse correlations with body mass index (r = −0.39/P < 0.0001), waist circumference (r = −0.42/P < 0.001), blood glucose (r = −0.40/P < 0.0001), triglycerides (r = −0.34/P < 0.0001), and TNF-alpha (r = −0.48/P < 0.0001), whereas a strongly positive correlation was found with IL-10 (r = 0.77/P < 0.0001). Multiple linear regression analysis revealed that behavior of IL-6 was mainly influenced by IL-10 (beta = 0.28/P = 1.95 × 10⁻⁶), TNF-alpha (beta = −0.67/P = 0.0017), and body fat percentage (beta = −5.95/P = 7.67 × 10⁻⁵) in women. In contrast, IL-10 (beta = 0.37/P = 1.34 × 10⁻⁹), TNF-alpha (beta = −0.85/P = 0.0005), and triglycerides (beta = 1.07/P = 0.0007) were major influencing factors of IL-6 in men. This study demonstrates that IL-6 is a marker of metabolic dysfunction that is differentially regulated in obese women and men.

Isomalto-oligosaccharides (IMOs) may promote health by modulating intestinal microbiota. We hypothesized that the proportion of α–(1 → 6) linkages in IMOs determines their digestibility. Ileal-cannulated pigs were fed diets containing IMO, IMO-DP3 with a greater DP and more α-(1 → 4) linkages, and digestible or resistant maltodextrins. Oligosaccharides were analysed by high-performance anion-exchange chromatography. Compared to IMO, IMO-DP3 contained more panose (18.6 vs. 10.3%) but less isomaltose (7.5 vs. 22.3%) and isomaltotriose (6.1 vs. 12.6%). The apparent ileal digestibility of dry matter were 3% greater for IMO-DP3 and digestible maltodextrin than resistant maltodextrin; the digestibility of IMO was not different from other oligosaccharides. Ileal propionate, isovalerate, and total SCFA was greater for IMO-DP3 and digestible maltodextrin than IMO. In conclusion, IMO was less digestible than IMO-DP3. Structural properties of IMOs are important determinants of their functional properties within the porcine digestive tract.

The effect of annealing on structure and physicochemical properties of four different waxy starches were investigated to understand the mechanism of annealing in the absence of amylose. Granule morphology, X-ray pattern, molecular order, and gelatinization enthalpy remained unchanged upon annealing. Relative crystallinity of waxy corn starch increased from 42.4 to 46.1% on annealing, while it remained unchanged in other starches. Annealing increased the gelatinization temperatures, while it decreased the temperature range. Waxy potato starch showed the highest increase in T_o, where it was 8.1°C after 72 h of annealing. Viscosity profiles were only slightly affected by the annealing treatment. Susceptibility towards amylolysis increased in waxy corn and waxy barley, while it decreased in waxy potato and waxy rice. This study demonstrated that even in the absence of amylose, annealing caused changes in the structure and properties, however, the extents of these changes varied depending on the botanical origin.

Increasing wettability of carbon felts is an important strategy to improve their efficiency in bio‐electrochemical applications. Herein, influence of cold remote plasma (N₂ + O₂) treatment on surface properties of carbon felts with poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) coating was tested, aiming to improve immobilizing of glucose oxidase enzyme (GOx). Spectra of N 1s and O 1s confirmed the integration of carbonyl and ether as well as amide and amine groups on bare carbon fiber surface, while on coated fibers, carbonyl groups were pre‐dominant. S 2p spectra confirmed oxidation of PEDOT:PSS coating with reduction of (S⁻) compared to (SO³⁻) group. GOx immobilized on different samples showed highest activity for PEDOT:PSS coating subjected to plasma with 2% O₂, maintaining up to 60% after immobilization, and 37% of its activity after six cycles for some samples. Enzymes immobilized on samples without plasma treatment lost their activity after four cycles.

This study investigated the protein fractions, in vitro protein digestibility (IVPD), in vitro starch digestibility (IVSD), antinutritional factors, hydrolysis index (HI), and estimated glycemic index (eGI) of the raw and cooked flours of a new sorghum inbred line (high β-glucan type I non-tannin sorghum) compared to two popular Sudanese sorghum cultivars (low β-glucan type 1 non-tannin and type II tannin sorghums). Significant differences (P < 0.05) were observed in protein fractions, antinutritional factors, IVPD, IVSD, rapidly digestible starch (RDS), resistant starch (RS), HI, and eGI. Low β-glucan type II tannin sorghum showed the highest antinutritional factors and RS values, whereas high β-glucan type I non-tannin and low β-glucan type 1 non-tannin sorghum cultivars showed the least values (P < 0.05). Low β-glucan type 1 non-tannin sorghum had the highest IVPD, RDS, HI, and eGI values. Cooking significantly (P < 0.05) reduced the protein fractions, antinutritional factors, IVPD, and RS and increased the IVSD, RDS, HI, and eGI of sorghums. Based on low eGI of high β-glucan type I non-tannin sorghum cultivar, it can be considered a low glycemic index sorghum and could thus be the best choice for diabetic people.

Although the effects of heat-moisture treatment on starch structures and digestibility have been evaluated, the changes in structures and digestion of starches in heat-moisture treated starch-based food systems with varying amylose contents are not well understood. In the present study, starches isolated from heat-moisture treated polished rice grains were characterized with multi-scale structures and digestibility to reveal the effect of amylose in rice grains on structures and digestibility of starch subjected to heat-moisture treatment. Results indicated that heat-moisture treatment contributed to the partial gelatinization of starch granules, but also significantly reinforced the molecular interactions between starch molecules and thus increased starch resistance to hydrothermal treatment and enzymes attack. The higher amylose content of the native starch was, the greater reduction in digestion was observed for heat-moisture treated starch. Pearson's correlation matrix showed that the amylose content highly correlated with the changes in structural features (e.g., molar mass, short-range ordered structure, crystalline structure, and lamellar structure) of starches followed by heat-moisture treatment. The amylose promoted the reinforcement of interactions between starch chains during heat-moisture treatment and then significantly contributed to the reduction in starch digestibility.