Enzymatic hydrolysis of potato starches containing different amounts of phosphorus

The rapid hydrolysis of potato starches differing in phosphorus content, as well as sweet potato, cassava and yam starches, was accomplished by treatment of gelatinised starches with bacterial liquefying α-amylase at 50 °C for 1 h, followed by Bacillus licheniformis α-amylase at 55 °C up to 24 h, and then by glucoamylase at 40 °C for a further 24 h. Among the potato starches, the high-phosphorus starches showed higher starch resistant capacity than the medium-phosphorus starches, as well as other tuber and root starches. The hydrolysis rate of tuber and root starches was not greatly influenced by their amylose content and median granule size. Only glucose was detected in the almost completely hydrolysed tuber and root starch samples, indicating that the concomitant enzymes treatment could hydrolyse both the α-1,4 and α-1,6 linkages of the starches examined.     

A kinetic assessment of the enzymatic hydrolysis of potato (Solanum tuberosum)

The biotechnology industry demands raw materials to prepare growth media for fermentative processes. These media contain a carbon source which can be obtained from potato, an abundant source of starch. This work deals with the modelling of the enzymatic hydrolysis of potato using α -amylase and glucoamylase. Several strategies were evaluated, including the use of those enzymes alone, in a mixture and under different conditions of temperature, time and substrate concentration. Using α -amylase alone or glucoamylase alone, both enzymes reached the same hydrolysis yield (0.42 g/g). The hydrolysis using a mixture of both enzymes allowed obtaining hydrolysates with reducing sugar concentrations up to 14.1 g/L at 70 °C. In this case, the yield of hydrolysis was increased up to 0.82 g/g. Considering the important effect and interactions of time and temperature, a statistical Box-Behnken design was conducted including substrate concentration, time and temperature as operational variables and reducing sugar concentration released as dependent variable. The conditions to obtain the maximum response were 6% DM of substrate concentration, 68 °C and 180 min, where 32.62 g/L of reducing sugar concentration was predicted. Verification experiments gave a mean value of 33.57 g/L, confirming the accuracy of the model.     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Characteristics of native and enzymatically hydrolyzed Zea mays L., Fritillaria ussuriensis Maxim. and Dioscorea opposita Thunb. starches

Comparative studies on glucoamylase hydrolysis of A-type Zea mays L., B-type F. ussuriensis Maxim., and C-type Dioscorea opposita Thunb. were carried out by scanning electron microscope (SEM), X-ray diffractometer (XRD), Fourier transform infrared (FT-IR) spectra and differential scanning calorimetry (DSC). Maize, Fritillaria, Dioscorea starches were hydrolyzed with glucoamylase for 2, 4, 8, 12 and 24 h, respectively. The SEM and XRD results revealed that A-type, B-type starch and C-type starch displayed different hydrolysis mechanisms. A-type starch was digested with enzyme penetrating into starch granules through natural pores on the surface and disrupted the interior of the starch granules. The glucoamylase worked by attacking the surface of B-type starch and forming cracks. When endo-corrosion occurred, the internal part of the granule was corroded through small cracks. However, the glucoamylase primarily attacked the interior of the C-type starch granules and then the exterior of starch granules. FT-IR confirmed that the amorphous regions in the starch granules are firstly hydrolyzed and could be hydrolyzed completely as long as the hydrolysis time is sufficient. The transition temperatures and enthalpy of gelatinization (ΔH_gel) were determined using DSC. According to the gelatinization parameters, it could be further proved amorphous and crystalline structures were hydrolyzed.     

Inhibitor potential of protease and α-amylase inhibitors of sweet potato and taro on the digestive enzymes of root crop storage pests

The inhibitory potential of purified protease and α-amylase isoinhibitors of sweet potato and taro (5 accessions each) on the digestive enzymes of four major root crop pests viz, Araecerus fasciculatus, Sitophilus oryzae, Cylas formicarius elegantulus and Tribolium castaneum was studied under in vitro conditions. Wide differences in inhibitory potential were noticed among the isoinhibitors of a single accession as well as among the same isoinhibitor of the different accessions. The isoinhibitor SPAI₁ from Kanhangad was inhibitory to all the four insect α-amylases (25-58% inhibition), while only 0.8-15% inhibition was exerted by the isoinhibitor SPAI₁ from S 1195. Very high inhibition of A. fasciculatus and C. formicarius elegantulusα-amylases (73-94%) was caused by isoinhibitors SPAI₂ and SPAI₄ from the sweet potato accession S 56-2. Cylas formicarius elegantulusα-amylases were inhibited to a greater extent by the taro α-amylase inhibitor. Among the four insect proteases, those from A. fasciculatus and T. castaneum were not appreciably inhibited by the protease isoinhibitors of sweet potato and taro. The S. oryzae protease was inhibited by 51% by isoinhibitor SPI₂, while only 3% inhibition was caused by isoinhibitor SPI₄. The selective inhibitory potential of the isoinhibitors of sweet potato and taro on the digestive enzymes of root crop pests could be exploited for making transgenic plants with improved resistance against major pests.     

Structural and functional properties of starches from field peas

Starch was isolated from seven varieties of field peas (Pisumsativum L.) and characterised using a combination of physical, chemical and functional tests. The total starch content of the peas ranged between 34% and 42.7% of dry matter, and the amylose content of the starch was between 35% and 38%. Average particle diameter of the seven starches varied between 21.4 and 26.1 μm. All of the pea starches gave a typical C-type X-ray diffraction pattern, with relative crystallinity ranging between 36% and 55% and the proportion of B-type crystallites between 3.8% and 30.4%. Although there were only small differences between the starches in amylose content, they displayed significant variability in functional properties, including swelling power, pasting characteristics, thermal transition temperatures in the differential scanning calorimeter, and in susceptibility to invitro attack by α-amylase. The results indicate the importance of structural characteristics of starch molecules, particularly amylopectin, as determinants of the properties of native starch granules.     

Enzymatic hydrolysis of protein isolate from hull-less pumpkin oil cake: Application of response surface methodology

Enzymatic hydrolysis of protein isolate from hull-less pumpkin (Cucurbita pepo L.) oil cake was studied by response surface methodology, using a central-composite experimental design. The hydrolysis was carried out with an acid protease, at temperature of 30 °C and pH 3.00. Second-order polynomial model was proposed with regard to of effect of time, enzyme and NaCl concentration. The mathematical model showed good fit with the experimental data, since the R² of 0.946 indicated that 94.6% of the variability within the range of values studied could be explained by the model. A hydrolysis time of 32.5 h, enzyme concentration of 0.137% (v/v) and NaCl concentration of 0.84% (w/v) were found to be the optimal conditions to achieve the highest value of degree of hydrolysis (DH).     

Granola bars prepared with Agave tequilana ingredients: Chemical composition and in vitro starch hydrolysis

The stem of Agave tequilana is used to obtain: agave syrup (AS) and native agave fructans (NAF). Ground-agave-fiber is the by-product from fructans production. These ingredients were used to design a food ingredient: agave dietary fiber (ADF), containing NAF (30 g/100 g) as soluble dietary fiber (DF) and ground-agave-fiber (70 g/100 g) as an insoluble DF. The objective of this work was to evaluate the effect of the incorporation of A. tequilana ingredients (AS, NAF, ADF) on the proximate composition, in vitro starch hydrolysis (HI) and predicted glycemic index (pGI) of oat-based granola bars. Total DF (82.03 g/100 g) was the main component in ADF, with 22.8 g/100 g soluble DF. Granola bars were prepared by substituting honey and wheat flour by AS and ADF. A sensory test was used to select the level of sugar substitution by NAF, where 62 g NAF/100 g was the preferred one. The effect of each ingredient on the chemical composition was evaluated using a 2³⁻¹ fractional design. Soluble DF in a granola bar containing a combination of three agave ingredients (AS, NAF and ADF) was 23.35 g/100 g, with HI and pGI values of 74 and 72%, respectively, pointing this product as a moderate GI food.     

Crystallography,morphology and thermal properties of starches from four different medicinal plants of Fritillaria species

To fully understand the medicinal plant, Fritillaria, and its species, we investigated the physical properties of starch contained in four Fritillaria species, Fritillaria thunbergii Miq., Fritillaria ussurensis Maxim., Fritillaria pallidifloca Schrenk and Fritillaria cirrhosa D.Don, by means of various analytical methods. The crystal type of the former three kinds of Fritillaria starches was in characteristic B-type, which was in agreement with the crystal type of potato starch. However, the cirrhosa F. starch showed a typical C_B-type pattern. The degrees of crystallining of the four Fritillaria starches were about 43.2%, 40.5%, 44.8% and 41.8%, corresponding to thunbergii F. starch, ussurensis F. starch, pallidifloca F. starch and cirrhosa F. starch. The granule sizes of the former two Fritillaria starches ranged from 5 to 40 μm, and were cycloidal or elliptic-shaped. However, the latter two Fritillaria starch granules had granule sizes ranging from 5 to 50 μm, and the granule shape varied from oval to irregular or cuboidal. From the thermogravimetric analysis, it was concluded that the thermal stabilities of the four kinds of starch differed from each other, due to their different structures.     

Effects of azadirachtin on post-embryonic development, energy reserves and α-amylase activity of Plodia interpunctella Hübner (Lepidoptera: Pyralidae)

The effects of azadirachtin on the fourth instar larvae of Plodia interpunctella (Lepidoptera) were investigated. When incorporated into the diet at 2 and 4 ppm, azadirachtin provoked larval weight loss, developmental delay and high larval and pupal mortality. Spectrophotometric assays showed that azadirachtin caused a severe reduction in protein, glycogen and lipid contents 7 days after the beginning of the treatment. In addition, α-amylase activity was reduced in larvaefed azadirachtin.     

Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates

Enzymatic hydrolysis of hemp protein isolate (HPI) by six proteases (alcalase, flavourzyme, neutrase, protamex, pepsin and trypsin) and antioxidant activities of the resulting hydrolysates, obtained for 2 and 4 h were investigated. The yield of trichloroacetic acid (TCA)-soluble peptides (Y_sp), protein composition and surface hydrophobicity (H_o) of the hydrolysates were evaluated. The results showed that the hydrolysates exhibited varying DPPH radical scavenging (with lowest IC₅₀, ∼2.3 mg/mL) and Fe²⁺⁺ chelating (with lowest IC₅₀ of 1.6–1.7 mg/mL) abilities and reducing power (with highest absorbance at 700 nm of 0.31–0.35), depending on their Y_sp and H_o values. The DPPH radical scavenging and Fe²⁺⁺ chelating abilities of the hydrolysates were positively correlated with their Y_sp or H_o values. The results suggest that enzymatic hydrolysis can be used as an effective technique to produce high value-added products of hemp proteins.     

Chemical compositions,fine structure and physicochemical properties of kudzu (Pueraria lobata) starches from different regions

Three commercial kudzu starches from Vietnam, Japan and Korea were used to determine chemical compositions, isoflavone compounds, fine structure and physicochemical properties. The kudzu starch from Vietnam had polygonal granules, whereas the kudzu starches from Japan and Korea contained both polygonal and spherical granules. Total protein, lipid, ash and phosphorus contents present in these kudzu starches were less than 1% (starch basis). The kudzu starch from Vietnam and Korea contained both daidzein and daidzin, whereas the kudzu starch from Japan had only daidzein. These starches had similar actual amylose contents (22.2–22.9%). However, λ_max, blue value and apparent amylose contents of the kudzu starch from Vietnam were lower than those from Japan and Korea. Amylose molecules of the kudzu starch from Vietnam had the largest average degree of polymerization (DP_n) and number of chains (NC), followed by the kudzu starches from Japan and Korea. Amylopectin molecules of the kudzu starch from Vietnam also had the largest DP_n and NC, followed by the kudzu starches from Korea and Japan. X-ray diffraction patterns of the kudzu starches from Vietnam, Japan and Korea were A-type, C-type and B-type, respectively. The kudzu starch from Vietnam was found to have the specific characteristics such as significantly high gelatinization temperature, transition enthalpy and degree of crystallinity as compared to the kudzu starch from Korea and Japan.     

Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus

β-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9 h, respectively, and the K_m and V_max values were 2.96 mM and 6.62 μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe²⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Sn²⁺, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.     

Effect of genetic variation on the tryptic hydrolysis of bovine beta-lactoglobulin A, B, and C

The structure, stability, and hydrolysis characteristics of beta-lactoglobulin (LG) A are different from those of either beta-LG B or beta-LG C. Purified samples of these proteins were hydrolyzed with trypsin and the rates of loss of native monomeric beta-LG structure were measured using sodium dodecyl sulfate PAGE. At the same time, the appearance of many individual peptides were identified and followed in time by HPLC, measuring their concentration as a function of solution pH, temperature, protein concentration, and added urea or palmitate. The identity of the peptides was confirmed by liquid chromatography-mass spectrometry. This semiquantitative exploration showed that the rate of hydrolysis was in the order beta-LG A > beta-LG B > beta-LG C under most circumstances, and that 12 of the 18 trypsin-susceptible bonds were cleaved at very similar rates that were governed by the variant type. Consequently, the rate of hydrolysis of the intact protein was related to the overall structural stability of the individual proteins and the accessibility of certain peptide bonds to the enzyme. The hydrolysis of mixtures of 2 or more variants or of denatured beta-LG gave more heterogeneous peptide mixtures.     

Studies on the physicochemical,morphological, thermal and crystalline properties of starches separated from different Dioscorea opposita cultivars

The physicochemical, morphological, thermal and crystal properties of the starches separated from four different Dioscorea opposita Thunb. cultivars (Taigu, Ribenbai, Wenxi and Zhongbowen) were studied. Amylose contents of D. opposita Thunb. starches from different cultivars ranged from 21.17% to 25.00%. The shape of starch granules separated from different D. opposita Thunb. cultivars varied from round or oval to elliptic or caky. The surface of the starch granules appeared to be smooth without any fissures. The average particle diameter of starches from different D. opposita Thunb. cultivars ranged from 25.90 to 28.06 μm. The transition temperatures (T_o, T_p and T_c) and enthalpy of gelatinization (ΔH_gel) were determined using differential scanning calorimetry (DSC). T_o, T_p, T_c and ΔH_gel of D. opposita Thunb. starches ranged from 73.1 to 73.9, 77.6 to 80.4, 82.1 to 85.9 °C and 6.548 to 12.13 J/g, respectively. The crystal type of starches separated from different D. opposita Thunb. cultivars was a typical C-type pattern. The relative degree of crystallinity of the four D. opposita Thunb. cultivars starches were about 38.79%, 39.88%, 41.67% and 49.03%, respectively.     

pH stability and influence of salts on activity of a milk-clotting enzyme from Solanum dubium seeds and its enzymatic action on bovine caseins

In this study we investigated the pH stability and effect of salts on the activity of a partially purified enzyme from Solanum dubium seeds as well as its hydrolytic power on caseins and caseins components. The seeds of S. dubium were blended and extracted using 50 g/L NaCl in 50 mmol/L acetate buffer, pH 5.0. The enzyme was then partially purified using ammonium sulfate. The results obtained showed that both NaCl and CaCl₂ enhanced the proteolytic activity of the enzyme and the enhancement was found to be significant when NaCl was used. Moreover, the stimulatory effect was found to be concentration dependent. The proteolysis of bovine whole casein and casein subunits by the enzyme during incubation was studied by SDS-PAGE. The results obtained revealed that both κ-casein and β-casein are the most susceptible to hydrolysis than α-casein. The three main casein components α-, β-, and κ-caseins were sensitive to the action of the enzyme and the order of hydrolysis obtained was κ- casein, β- casein, and α- caseins.     

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25-45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca²⁺ and Mg²⁺ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L⁻¹, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.     

Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.     

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion.     

Microbiological responses to depuration and transport of native and exotic clams at optimal and stressful temperatures

The microbiological responses of two bivalves species from Tagus estuary, Venerupis pullastra (native clam) and Ruditapes philippinarum (exotic clam) were investigated during 48 h of depuration and subsequent simulated transport in semi-dry conditions at two temperatures (4 and 22 °C) until reaching 50% lethal time (LT50). Regardless of temperature and species, the maintenance of clams in water for 48 h (depuration period) did not affect LT50 during transport. R. philippinarum showed higher survival rates than V. pullastra, always reaching LT50 later, especially at 4 °C. Significant differences between clams' species were found in almost all microbiological parameters. This can be related with clams' biological activity and habitat environmental conditions since both clams do not coexist in Tagus estuary. Depuration was efficient to reduce the bacterial load, particularly Escherichia coli, but not efficient to remove Vibrio spp. In both species, the growth of Vibrio spp. was inhibited at 4 °C, whereas exponential growth occurred at 22 °C. Total viable counts significantly increased in most treatments, while E. coli counts significantly decreased to undetected levels, except for non-depurated R. philippinarum simulated transported at 4 °C. Thus, this study highlights the importance of clams depuration for at least 24 h in polluted estuarine areas, followed by transport at low temperatures (4 °C).