Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.     

Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: analysis of their mechanical and barrier properties

Chitosan-whey protein edible films with different protein concentrations were prepared in the absence or presence of microbial transglutaminase as cross-linking agent. The films prepared in the presence of the enzyme showed low solubility at a wide range of pH, a lower degree of swelling, and good biodegradability following protease treatments. The presence of transglutaminase induced also an enhancement in film mechanical resistance and a reduction in their deformability. Finally, the barrier efficiency toward oxygen and carbon dioxide was found to be markedly improved in the cross-linked films which showed also a lower permeability to water vapor. Some potential practical applications of transglutaminase-treated chitosan-whey protein films are suggested.     

EFFICIENT PROTEOLYSIS AND APPLICATION OF AN ALKALINE PROTEASE FROM HALOPHILIC Bacillus sp. EMB9

A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca²⁺ and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.     

Effect of Cross-Linking with <Emphasis Type="Italic">Aloe vera</Emphasis> Gel on Surface and Physicochemical Properties of Edible Films Made from Plantain Flour

Films made from plantain flour with incorporation of different concentrations (0, 2, 4 and 6 %) of a natural filler (Aloe vera gel - Av gel) were obtained by the casting method. The aim of this paper was to characterize the surface and physicochemical properties on plantain flour edible films. The average molecular weight, moisture, infrared spectroscopy, contact angle and mechanical properties were determined. Also, microstructural characterization was performed by atomic force microscopy and scanning electron microscopy. The Av gel produced cross-linking on the starch that is found in plantain flour, resulting in films with the following characteristics: smoother, more transparent, more rigid and plastics, less moist and with more hydrophobic surfaces. Finally, the surface properties of these materials were defined by the surface energy of material, which depends on the intermolecular interactions such as van der Waals-type interactions (hydrogen bond) and new bonds (cross-linking) formed between biopolymeric chains (plantain flour).     

Microbial transglutaminase and its application in the food industry. A review

The extremely high costs of manufacturing transglutaminase from animal origin (EC 2.3.2.13) have prompted scientists to search for new sources of this enzyme. Interdisciplinary efforts have been aimed at producing enzymes synthesised by microorganisms which may have a wider scope of use. Transglutaminase is an enzyme that catalyses the formation of isopeptide bonds between proteins. Its cross-linking property is widely used in various processes: to manufacture cheese and other dairy products, in meat processing, to produce edible films and to manufacture bakery products. Transglutaminase has considerable potential to improve the firmness, viscosity, elasticity and water-binding capacity of food products. In 1989, microbial transglutaminase was isolated from Streptoverticillium sp. Its characterisation indicated that this isoform could be extremely useful as a biotechnological tool in the food industry. Currently, enzymatic preparations are used in almost all industrial branches because of their wide variety and low costs associated with their biotechnical production processes. This paper presents an overview of the literature addressing the characteristics and applications of transglutaminase.     

Nutrient-Enhanced Production of Remarkably High Concentrations of Ethanol by Saccharomyces bayanus through Soy Flour Supplementation

The supplementation of a simple medium with soy flour led to an increase in the specific growth rate and viable cell concentration of Saccharomyces bayanus during fermentation. Increasing the amount of soy flour led to an increase in the maximum number of viable yeast cells and the percentage of glucose fermented. It was possible in 64 h to reach 12.8% (wt/vol) ethanol by adding 4% soy flour (wt/vol) to a simple medium with 300 g of glucose per liter. The aqueous extract from soy flour was nearly as effective as whole-soy flour, whereas the lipidic fraction had no positive effect.     

Production of phenolic antioxidants by the solid-state bioconversion of pineapple waste mixed with soy flour using Rhizopus oligosporus

The ability of Rhizopus oligosporus to produce enhanced levels of free phenolics from pineapple residue mixed with soy flour as potential nitrogen source was investigated. Concurrently, phenolic-linked β-glucosidase and the antioxidant activity of the extracts were followed. Two treatments were studied: (A) 9 g of pineapple residue and 1 g of soy flour (P9); (B) 5 g of pineapple residue and 5 g of soy flour (P5). The increase of water extractable phenolics was 39.3% for P9 treatment and 79.4% for P5 treatment. During early stages of growth high antioxidant activity, low phenolic content and low β-glucosidase specific activity was observed. High antioxidant activity was likely due to the presence of insoluble polymeric phenolics, know for their high antioxidant activity. A marked decrease of the antioxidant activity of P5 treatment during late stages of growth was observed due to likely formation of free soluble phenolics. The moderate total phenolics content and high β-glucosidase specific activity of P9 treatment in late stages is likely the consequence of low nitrogen content in this treatment. The bioconversion of pineapple residue by R. oligosporus leads to enhanced levels of phenolic compounds, mainly for P5 treatment. This approach offers a novel strategy to enhance the value of pineapple wastes.     

Bacteriological survey of sixty health foods.

A bacteriological survey was performed on 1,960 food samples encompassing 60 types of health foods available in the Baltimore-Washington, D.C., metropolitan area. No consistent bacteriological distinction (aerobic plate counts, total coliform and fecal coliform most probable numbers) was observed between foods labeled as organic (raised on soil with compost or nonchemical fertilizer and without application of pesticides, fungicides, and herbicides) and their counterpart food types bearing no such label. Types and numbers of samples containing Salmonella were: sunflower seeds, 4; soy flour, 3; soy protein powder, 2; soy milk powder, 1; dried active yeast, 1; brewers' years, 1; rye flour, 1; brown rice, 1; and alfalfa seeds,1. The occurrence of this pathogen in three types of soybean products should warrant further investigation of soybean derivatives as potentially significant sources of Salmonella.     

Bacteriological survey of sixty health foods

A bacteriological survey was performed on 1,960 food samples encompassing 60 types of health foods available in the Baltimore-Washington, D.C., metropolitan area. No consistent bacteriological distinction (aerobic plate counts, total coliform and fecal coliform most probable numbers) was observed between foods labeled as organic (raised on soil with compost or nonchemical fertilizer and without application of pesticides, fungicides, and herbicides) and their counterpart food types bearing no such label. Types and numbers of samples containing Salmonella were: sunflower seeds, 4; soy flour, 3; soy protein powder, 2; soy milk powder, 1; dried active yeast, 1; brewers' years, 1; rye flour, 1; brown rice, 1; and alfalfa seeds,1. The occurrence of this pathogen in three types of soybean products should warrant further investigation of soybean derivatives as potentially significant sources of Salmonella.     

Improvements in ethanol concentration and fermentor ethanol productivity in yeast fermentations using whole soy flour in batch,and continuous recycle systems

Summary Ethanol concentrations and fermentor productivities were increased 20.2 and 15.5% at 90 and 95% recycle, respectively, when whole soy flour was added to the feed (2 g/ at 90% recycle, and 1 g/ at 95% recycle) of a continuous yeast fermentation system with recycle of cells and soy flour (soy flour concentrations were 2% at steady state in the fermentor) by UF membranes as compared to controls. The improvements were primarily due to increases in cell concentrations. Similar results were obtained for batch cultures.     

Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

A recombinant Thermotoga maritima β-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties K _m, k _cat, and k _cat/K _m of BglA towards isoflavone glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of BglA in hydrolyzing malonylglycosides than non-conjugated glycosides (daidzin and genistin). During hydrolysis of soy flour by BglA at 80°C, the isoflavone glycosides (soluble form) were extracted from soy flour (solid state) into the solution (liquid state) in thermal condition and converted to their aglycones (insoluble form), which mostly existed in the pellet to be separated from BglA in the reaction solution. The enzymatic hydrolysis in one-step and two-step approaches yielded 0.38 and 0.35 mg genistein and daidzein per gram of soy flour, respectively. The optimum conditions for conversion of isoflavone aglycones were 100 U per gram of soy flour, substrate concentration 25% (w/v), and incubation time 3 h for 80°C.     

Water-induced hydrophobicity of soy protein materials containing 2,2-diphenyl-2-hydroxyethanoic acid

Biodegradable soy protein isolate (SPI), containing 2,2-diphenyl-2-hydroxyethanoic acid, films (SB) were successfully prepared with bis-(2-hydroxyethyl)sulfide as a plasticizer by compression molding at 155 degrees C and 15 MPa. By immersing the SB in distilled water for 26 h, we prepared the films (coded as SB-WM) having good water resistance. The films were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, dynamic mechanical thermal analysis, and tensile testing to evaluate their structure and properties. Moreover, the surface of the SB-WM films was analyzed by X-ray photoelectron spectroscopy and contact angle measurement. SB-WM films exhibited significantly higher contact angle than SB. The results revealed that a lotus-like nanoscale structure was created in SB-WM films, with increased hydrophobicity, through the process of the solvent-induced microphase separation during the immersion in water. More stable compound diphenylhydroxymethane could form from 2,2-diphenyl-2-hydroxyethanoic acid of SB in water, leading to the hydrophobicity of the SB-WM materials. A "green" and easy method for fabricating hydrophobic materials from soy protein has been provided in this work.     

Characterization of Soy Polysaccharide and Its In Vitro and In Vivo Evaluation for Application in Colon Drug Delivery

The objective of the present investigation was to establish potential of commercially available soy polysaccharide (Emcosoy®) for colon drug delivery. The soy polysaccharide–ethyl cellulose films were fabricated and characterized. The effect of the pectinase enzyme on the tensile strength and surface morphology of the film was evaluated. The permeation of chlorpheniramine maleate (CPM), a model hydrophilic drug from pectinase enzyme treated and untreated films was measured in pH 7.4 buffer. The soy polysaccharide–ethyl cellulose films were also incubated with Lactobacillus sp. culture for a specific duration, and effect on the CPM permeation was evaluated. The CPM capsules were coated with the soy polysaccharide–ethyl cellulose mixture, and Eudragit S100 was applied as a secondary coat. The coated CPM capsules were radiolabelled, and their in vivo transit was evaluated in human volunteers on oral administration. The pectinase enzyme had a significant influence on the tensile strength and surface morphology of the soy polysaccharide–ethyl cellulose films. The permeability of pectinase enzyme-treated and Lactobacillus sp.-treated films was significantly higher than that of untreated films. The CPM capsules were coated with the soy polysaccharide–ethyl cellulose mixture and Eudragit S100 and were successfully radiolabelled by a simple method. Gamma scintigraphic studies in human volunteers showed that the radiolabelled capsules maintained integrity for at least 9 h after oral administration. Thus, the soy polysaccharide has a potential in colon drug delivery.     

Solvent extraction selection in the determination of isoflavones in soy foods

Acetonitrile is superior to acetone, ethanol and methanol in extracting the 12 phytoestrogenic soy isoflavone forms found in foods. At 53% organic solvent in water, raw soy flour, tofu, tempeh, textured vegetable protein and soy germ were evaluated for isoflavone extraction efficiency. The efficiency of acetonitrile extraction was demonstrated in mass balance evaluations of toasting of soy flour and soymilk heating.     

Fennel waste-based films suitable for protecting cultivations

Biodegradable, flexible, and moisture-resistant films were obtained by recycling fennel waste and adding to fennel homogenates the bean protein phaseolin that was modified or not modified by the enzyme transglutaminase. All films were analyzed for their morphology, mechanical properties, water vapor permeability, and susceptibility to biodegradation under soil-like conditions. Our experiments showed that transglutaminase treatment of the phaseolin-containing fennel waste homogenates allowed us to obtain films comparable in their mechanical properties and water vapor permeability to the commercial films Ecoflex and Mater-Bi. Furthermore, biodegradability tests demonstrated that the presence of the enzyme in the film-casting sample significantly influences the integrity of such a product that lasts longer than films obtained either with fennel waste alone or with a mixture of fennel waste and phaseolin. These findings indicate the fennel-phaseolin film prepared in the presence of transglutaminase to be a promising candidate for a new environmentally friendly mulching bioplastic.     

纤维素酶液体发酵最佳培养基的确定

用响应面法对里氏木霉WX—112液体发酵产纤维素酶的培养基进行了优化。首先用快速登高路径逼近最大产酶区域,然后根据快速登高法的实验结果进行响应面实验。运用逐步回归分析法,获得滤纸酶活与豆饼粉、麸皮、KH2PO4、微晶纤维素粉（Avicel）的最优回归方程,且分析了各因子间的交互效应。最后,通过岭脊分析确定了滤纸酶活达最大值10．53IU／mL时的最佳组合条件：豆饼粉3．18％、麸皮2．95％、KH2PO4 0．25％、Avicel 3．79％。     

Optimization of roller culturing of influenza virus vaccine strains in MDCK and Vero cell cultures using plant origin components

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).     

Improvement in ethanol production from beet molasses by soy flour supplementation

Summary Ethanol concentration and the rate of ethanol production were substantially increased when soy flour was added to the inoculum medium, which saved 95% added soy flour compared to supplementing fermentation medium. 11.7% ethanol was obtained by supplementing inoculum medium with soy flour and the fermentation time was reduced by more than 15%.     

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.     

Growth of Bacillus cereus in Media Containing Plant Seed Materials and Ingredients Used in Chinese Cookery

Growth and sporulation of enterotoxigenic strains of Bacillus cereus in media containing 20 different plant seed flours and meals, with and without added infusions of beef, pork, chicken and shrimp, monosodium glutamate (MSG), and soy sauce, were studied. Suspensions (2%; pH 7–1) of seed flours and meals from diverse botanical origins were found to be excellent sources of nutrients for growth. No correlations could be made between composition of seed materials and rate of cell division. Mean generation times of B. cereus cultured in soy, peanut and rice flour media supplemented with animal flesh infusions were significantly faster ( P ≤ 0.05) than those of respective controls. Monosodium glutamate (1–2%) and soy sauce (5–10%) stimulated the rate of growth of B. cereus in rice flour medium. Test flours supporting slower growth rates appeared generally to support higher rates of sporulation.