Preliminary study of L-lysine production by Bacillus species using various agricultural by-products

The production of lysine by Bacillus megaterium SP-14 and Bacillus circulans Tx-22 using agricultural by-products as carbon and nitrogen sources was assessed. Among the carbon substrates used were potato, sorghum, plantain, millet, yam, cassava, and corn starches, while the nitrogen sources include cowpea, bambara-nut, cotton seed, groundnut, soybean, and blood meals. The effect of natural nitrogen sources (1.0% w/v) and synthetic nitrogen source (4.0% w/v (NH4)2SO4) on lysine production by the Bacillus strains showed that natural nitrogen sources gave better lysine yields.     

Effect of Organic Nitrogen Sources on Raw Starch‐Digesting Glucoamylase Production of Rhizopus sp. MKU 40

In a liquid cultivation of Rhizopus sp. MKU 40, supplementation of the medium with 1.5% (w/v) organic nitrogen sources (neopeptone, casein from milk, and meat extract) had a slightly positive effect on glucoamylase (GA) (EC 3.2.1.3) activity compared with the medium lacking organic nitrogen sources. The addition of organic nitrogen sources induced production of protease. Supplementation of the medium with 1.5% (w/v) organic nitrogen sources resulted in an acid and neutral protease activity of 11 — 25 U/mL and 12 — 20 U/mL, respectively. The co‐existence of GA‐I [a highly raw starch‐digesting glucoamylase (RSDG)] and protease in the same medium leads to the production of Ga‐II (an extremely weak RSDG) from GA‐I. As a result the RSDG activity in the medium decreases. Raw starch adsorption rates of a medium without organic nitrogen sources were 100%, because the medium contained only GA‐I. In contrast, the media supplemented with organic nitrogen sources had low starch adsorption rates because the media contained both GA‐I and GA‐II. The results presented in this paper indicate that supplementation of the culture medium of Rhizopus strains with organic nitrogen sources negatively affects GA‐I production.     

Production of feed enzymes from citrus processing waste by solid‐state fermentation with Eupenicillium javanicum

Bingtang sweet orange processing waste was utilised to produce four feed enzymes (Endoglucanase, β‐glucosidase, pectinase and xylanase) by the solid‐state fermentation (SSF) with Eupenicillium javanicum. The factors related with SSF including moisture content, temperature, initial pH, time, carbon source (0.5 g), nitrogen sources (0.05 g), inorganic mineral salts (0.1 g) were investigated separately. The corresponding optimal condition was: moisture content 80% (w/w), temperature 30 °C, natural pH, time 96 h, wheat bran 0.5 g, (NH₄)₂SO₄ 0.05 g or NaNO₃ 0.05 g, CaCl₂ 0.1 g. The L₉(3⁴) orthogonal experiment results showed that the optimal condition for producing above multiple enzymes was: moisture content 80% (w/w), temperature 30 °C, wheat bran 1 g, (NH₄)₂SO₄ 0.05 g, NaNO₃ 0.05 g, CaCl₂ 0.1 g, fermentation time 96 h and natural pH. Under this condition, the average activity of Endoglucanase (CMCase), β‐glucosidase, pectinase and xylanase by E. javanicum could reach 46.80, 49.64, 51.87 and 106.42 U g^?1, respectively, which was significantly higher than those in single factor experiments. Our present results demonstrated that E. javanicum could also be an effective and useful fungus for multienzyme preparation especially for β‐glucosidase and xylanase from citrus processing wastes.     

Changing in processing yield and physical properties of frozen white shrimp (Penaeus vannamei) treated with lysine and sodium bicarbonate

The objective of this research was to investigate the phosphate alternative use of natural compound, lysine with sodium bicarbonate (NaHCO₃), at low concentration for freezing of white shrimp (Penaeus vannamei). Shrimp were treated with lysine, NaHCO₃ and lysine with NaHCO₃ at various concentrations and frozen in an air‐blast freezer. Thawing yield, cooking yield, colour, texture and nanostructure of the sample were studied compared to the control (nontreated sample) and sodium tri‐polyphosphate (STPP) treated one. Use of lysine/NaHCO₃ each at 1% (w/v) could improve water holding capacity (WHC) of the frozen shrimp, increasing cooking yield to 100.45% (w/w), comparable to the 101.73% (w/w) of STPP‐treated sample. The colour of the noncook‐thawed shrimp was also improved. Microstructure and lipid oxidation of the treated samples were also studied. The combination of lysine and NaHCO₃ indicated high potential use as phosphate alternative for frozen white shrimps.     

Citric acid production by Aspergillus foetidus in batch and fed‐batch cultures

Abstract

Sucrose and ammonium nitrate, respectively, were the preferred carbon and nitrogen sources for citric acid production by Aspergillis foetidus CCRC 30206. For a 7‐day fermentation, the maximal citric acid production of 7.2 ～ 28.4 g/l appeared at an initial sucrose concentration of 12 % under various initial levels of nitrogen and phosphorous sources. Citric acid production commenced and ceased earlier in either the nitrogen‐limited (NH₄NO₃, 0.5g/l) or the phosphorous‐limited (KH₂PO₄, 0.2 g/1) medium. The richer media delayed the onset of citric acid production. Nonetheless, 48.3 g/1 of citric acid was produced in the medium rich in nitrogen (NH₄NO₃, 2.0 g/1) and phosphorous (KH₂PO₄, 1.4 g/1) when the batch culture was extended to 16 days. The cultivation time of this rich medium was reduced to 12 days in a fed‐batch culture under dual limitation of both nitrogen and phosphorous.     

Milk‐clotting enzymes produced by Aspergillus flavo furcatis strains on Amazonic fruit waste

Six strains of Aspergillus flavo furcatis were screened to investigate milk‐clotting enzyme production by fermentation in natural liquid medium. The growth media comprised extracts of cupuaçu exocarp+rice bran [10% or 20% (v/v) CE+RB] and açai waste+rice bran [10% or 20% (v/v) AW+RB] with or without supplementation of 0.1% (w/v) yeast extract and 0.5% (w/v) gelatin. Significant values of milk‐clotting activity were determined by A. flavo furcatis DPUA 1461 and DPUA 1608, in the standard and natural media, respectively. According to criteria of clot and whey formations, 8.3% of the samples tested were classified as strong coagulation, 41.70% showed weak coagulation and in 50% was not observed milk coagulation. The enzyme optimal action of DPUA 1608, the selected strain, was at 40 °C and pH 7.0. Milk‐clotting proteases were inhibited by pepstatin (94.72%) and moderately inhibited by the others metal ions tested.     

Microbial and physicochemical modifications of hake (Merluccius merluccius) steaks stored under carbon dioxide enriched atmospheres

Microbial counts (total viable, Brochothrix thermosphacta, lactic acid bacteria and Enterobacteriaceae), biochemical parameters [pH, total volatile nitrogen, nucleotide breakdown products, non‐volatile amines, D (−) and L (+) lactic acid and short‐chain fatty acids] and sensory attributes (colour and odour) of refrigerated (2 °C) hake (Merluccius merluccius) steaks under carbon dioxide enriched air (CO₂ /air, 20/80 v/v and CO₂ /air, 40/60 v/v) and air atmospheres were determined. When compared with air, sensory results showed shelf‐life extension of 4 and 11 days for 20% and 40% CO₂‐enriched atmospheres, respectively. Microbial and biochemical results also revealed that the 40% CO₂‐enriched atmosphere was the most effective packaging type for refrigerated hake. © 2000 Society of Chemical Industry     

Production of L‐Lactic Acid from Spent Grain,a By‐Product of Beer Production

L‐lactic acid production from spent grain with immobilized lactic acid bacteria was investigated. Spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. When 1 kg of wet spent grain was treated under the 30 kg/cm²pressure for 1 min using a 5‐L steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. Furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. When batch L‐lactic acid production was carried out by Lactobacillus rhamnosus NBRC14710, 19.0 g/L L‐lactic acid was produced from the Tween 80 liquefied spent grain after 5 days. Furthermore, during repeated batch production with immobilized Lactobacillus rhamnosus NBRC14710 from Tween 80 liquefied spent grain at 37°C, the productivity of L‐lactic acid was maintained at a 10 time higher level over a period of 40 days.     

Decontamination of aflatoxin B1‐contaminated corn by ammonium persulphate during fermentation

The decontamination of aflatoxin B₁(AFB₁)‐contaminated corn, which is required if the corn is to be suitable for alternative use, by an ammoniation–fermentation integrated process was studied. This process could be used for the production of fuel ethanol from aflatoxin‐containing corn. Different concentrations (0.25, 0.50, 1.00, 1.5 and 2.0% w/w) of ammonium persulphate were tested in the detoxification of AFB₁‐contaminated corn during fermentation. In order to increase the decontamination of corn, 0.5 and 1.0% (w/w) azodicarbonamide, benzoyl peroxide and hydrogen peroxide were tested. Peroxides were added at three different stages of the fermentation process: liquefaction, saccharification and fermentation. Levels of AFB₁ and ethanol were determined after each fermentation process. Treated corn was tested for mutagenic potential using the Ames test with TA100 tester strain and pure AFB₁ as positive control. Addition of 2.0% (w/w) ammonium persulphate caused the highest level of decontamination without affecting ethanol production. Addition of peroxides did not significantly (P < 0.05) increase ethanol production or significantly (P < 0.05) improve the decontamination process. The best processes for decontamination of corn and for ethanol production included the addition of 2.0% (w/w) ammonium persulphate for both and of 1.0 and 0.5% (w/w) benzoyl peroxide respectively. All treated corn samples showed no mutagenic potential. Possible industrial use of these processes is discussed. © 2002 Society of Chemical Industry     

Alpha‐Cyclodextrin Production using Cyclodextrin Glycosyltransferase from Klebsiella pneumoniae AS‐22

Alpha‐cyclodextrin (α‐CD) production, using cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS‐22, was investigated in the presence of various complexing agents using raw wheat starch and dextrin as substrates. The addition of many alcohols resulted in increased conversion of both raw wheat starch and dextrin to α‐CD. With 125 g/L raw wheat starch, 42.5% (w/w) conversion to CDs was obtained in the presence of 2% (v/v) butan‐1‐ol. The ratio of α:β‐CD formed was 97:3, with negligible formation of γ‐CD and malto‐oligosaccharides. The production of α‐CD was optimized using two‐level factorial designs in three variables: dextrin, hexan‐1‐ol and enzyme concentrations. Under optimum conditions, 12.1% (w/w) conversion of 500 g/L dextrin to total‐CDs was achieved. The ratio of α:β:γ‐CD formed was 91:3:6, with negligible production of malto‐oligosaccharides. This CGTase was strongly inhibited by α:‐CD; 50% inhibition of reaction rate was observed at an initial concentration of 4 g/L α:‐CD. The effectiveness of an ultrafiltration membrane bioreactor for continuous removal of product was also tested.     

Optimization of Ultrasonic‐Assisted Aqueous Two‐Phase Extraction of Phloridzin from Malus Micromalus Makino with Ethanol/Ammonia Sulfate System

Application of an aqueous two‐phase system (ATPS) coupled with ultrasonic technology for the extraction of phloridzin from Malus micromalus Makino was evaluated and optimized by response surface methodology (RSM). The ethanol/ammonium sulfate ATPS was selected for detailed investigation, including the phase diagram, effect of phase composition and extract conditions on the partition of phloridzin, and the recycling of ammonium sulfate. In addition, the evaluation of extraction efficiency and the identification of phloridzin were investigated. The optimal partition coefficient (6.55) and recovery (92.86%) of phloridzin were obtained in a system composed of 35% ethanol (w/w) and 16% (NH₄)₂SO₄ (w/w), 51:1 liquid‐to‐solid ratio, and extraction temperature of 36 °C. Comparing with the traditional solvent extraction with respective 35% and 80% ethanol, ultrasonic‐assisted aqueous two‐phase extraction (UAATPE) strategy had significant advantages with lower ethanol consumption, less impurity of sugar and protein, and higher extracting efficiency of phloridzin. Our result indicated that UAATPE was a valuable method for the extraction and preliminary purification of phloridzin from the fruit of Malus micromalus Makino, which has great potential in the deep processing of Malus micromalus Makino industry to increase these fruits’ additional value and drive the local economic development.     

Improving 2‐phenylethanol and 6‐pentyl‐α‐pyrone production with fungi by microparticle‐enhanced cultivation (MPEC)

Trichoderma atroviride IMI 206040 synthesizes the coconut lactone 6‐pentyl‐α‐pyrone (6‐PAP) de novo and Aspergillus niger DSM 821 produces the rose‐like flavour compound 2‐phenylethanol (2‐PE) from the precursor l ‐phenylalanine. Here, microparticles of different chemical composition and nominal particle diameter in the range 5–250 µm were added to shake‐flask cultures of both fungi to investigate the particles' effect on product formation. Maximum 2‐PE concentration increased by a factor of 1.3 to 1430 mg/l with the addition of 2% w/v talc (40 µm diameter). Maximum 6‐PAP concentration increased by a factor of 2 to 40 mg/l with the addition of 2% w/v iron (II, III) oxide. The influence of ions leaching out of the particles was investigated by cultivating the fungi in leached particle medium. For the first time, the positive effect of the microparticle‐enhanced cultivation (MPEC) technique on the microbial production of volatile metabolites, here flavour compounds from submerged fungal cultures, is demonstrated. The effect is strain‐ and particle‐specific. Copyright © 2014 John Wiley & Sons, Ltd.     

Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. In this study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that a complex structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen sources, and mutual interaction between carbon and nitrogen sources, including the mechanisms involved in the regulation of aerobic/anaerobic metabolism, may play an important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen source, with a strong impact on fermentation performance.     

Optimising chitosan–pectin hydrogel beads containing combined garlic and holy basil essential oils and their application as antimicrobial inhibitor

Chitosan–pectin hydrogel beads that trap and release the maximal amount of combined garlic and holy basil essential oils to inhibit food microorganisms were developed based on the central composite design, with chitosan (0.2–0.7% w/v), pectin (3.5–5.5% w/v) and calcium chloride (CaCl₂) (5.0–20.0% w/v) contents. The optimal bead consisted of 0.3–0.6% w/v chitosan, 3.9–5.1% w/v pectin and 8.0–17.0% w/v CaCl₂, which had a high encapsulation efficiency (62.16–79.06%) and high cumulative release efficiency (31.55–37.81%) after storage at 5 °C for 15 days. Optimal hydrogel beads were packed into a cellulose bag to evaluate antimicrobial activity by the disc volatilisation method. The beads inhibited Bacillus cereus, Clostridium perfringens, Escherichia coli, Pseudomonas fluorescens, Listeria monocytogenes and Staphylococcus aureus but did not affect Lactobacillus plantarum and Salmonella Typhimurium. The oil-containing beads could potentially be applied in food packaging to inhibit the mentioned microorganisms.     

Formation of protein bound lysine‐derived galactosyl and glucosyl pyrroles in heated model systems

Residues of lysine‐substituted AGEs (advanced glycosylation end‐products) arising from the Maillard reaction and containing the carbon backbone of lactose or maltose, thus deriving from 1‐desoxy‐ketose degradation, were produced when casein was heated in the presence of the corresponding U¹⁴C labelled disaccharides. The enzymatic hydrolysates of the washed casein were purified by SPE and submitted to HPLC on a C18 column flushed with diluted acetic acid. A specific chromatographic peak (λ_max, 288.1 nm; MW, 416.2 Da) with a different retention time was obtained for each disaccharide reacted. On the basis of the value of the specific radioactivity, the two compounds appeared to contain the whole carbon backbone of the parent sugar. Analyses by MS/MS and NMR performed on the same two compounds extracted at preparative scale from lysine‐lactose and lysine‐maltose model systems allowed the structure assignment of 6‐[2‐acetyl‐3‐(β‐D‐galactopyranosyloxy)‐1‐pyrrolyl] 2‐amino hexanoic acid and6‐[2‐acetyl‐3‐(α‐D‐glucopyranosyloxy)‐1‐pyrrolyl] 2‐amino hexanoic acid, respectively. Both compounds submitted to enzymatic deglycosylation by specific α‐ or β‐ glucosidases produced the lysine‐derived acetyl pyrrole 6‐(2‐acetyl‐3‐hydroxy‐1‐pyrrolyl) 2‐amino hexanoic acid(λ_max, 288.1 nm; MW, 254.1 Da). Galactosyl‐ and glucosyl‐ isomaltoles, extracted from the lysine‐containing systems, identified with the reference molecules and heated in the presence of lysine under slightly alkaline conditions, gave the expected lysine‐derived glycosyl pyrroles as identified above. The HPLC conditions were optimized by adjusting the composition of the eluting solvent and temperature of the column to achieve the best separation and identification of the AGEs in mixtures such as foods with possible interfering molecules like Trp and lysyl pyrrole aldehyde. Because of the reported presence of the two precursors isomaltol glycosydes in some foods, the corresponding lysine‐derived glycosyl pyrroles can occur as both protein bound and in free form.     

FED-BATCH FERMENTATION FOR RAPID PRODUCTION OF XANTHAN BY XANTHOMONAS CAMPESTRIS

The object of the present work was to optimise inoculum size and nitrogen source for rapid production of xanthan by Xanthomonas campestris by fed-batch fermentation. The fermentation period for production of xanthan by X. campestris decreased from 72 h to 48 h by using 30% (v/v) inoculum instead of the usual 10% level. Among various nitrogenous substances tested in the medium having the C/N ratio of 28, the best concentration of 1.9% (w/v) of xanthan with a viscosity of 10,000 cP was obtained for KNO₃ medium. Intermittent harvests operated for three cycles of 48 h each, using 30% inoculum resulted in xanthan broth of high viscosity (9000 cP) with a concentration of 1.9% and a yield of 68% (w/w). Potassium nitrate was a better suited nitrogen source for the medium used for rapid production of xanthan.     

Physicochemical and sensory properties of milk fortified with iron microcapsules prepared with water‐in‐oil‐in‐water emulsion during storage

This study investigated the possibility of fortifying iron microcapsule powder into milk and the effects of the fortification on the physicochemical and sensory properties of the products during storage. The iron microcapsules were prepared by the water‐in‐oil‐in‐water (W/O/W) emulsion technique. Fortifying the lower concentrations (0.1–0.3%, w/v) of iron microcapsules into the milk samples did not significantly change thiobarbituric acid values. The L‐values for the milk samples were not significantly influenced by fortifying iron microcapsules (0.1–0.7%, w/v). The overall acceptability scores were not affected when the lowest concentration of iron microcapsules (0.1%, w/v) was fortified into the milk.     

Steady and Dynamic Shear Rheology of Rice Starch‐Galactomannan Mixtures

Rheological properties of rice starch‐galactomannan mixtures (5%, w/w) at different concentrations (0, 0.2, 0.4, 0.6 and 0.8%, w/w) of guar gum and locust bean gum (LBG) were investigated in steady and dynamic shear. Rice starch‐galactomannan mixtures showed high shear‐thinning flow behaviors with high Casson yield stress. Consistency index (K), apparent viscosity (η_a,100) and yield stress (σ_oc) increased with the increase in gum concentration. Over the temperature range of 20–65°C, the effect of temperature on apparent viscosity (η_a,100) was described by the Arrhenius equation. The activation energy values (E_a = 4.82–9.48 kJ/mol) of rice starch‐galactomannan mixtures (0.2–0.8% gum concentration) were much lower than that (E_a = 12.8 kJ/mol) of rice starch dispersion with no added gum. E_a values of rice starch‐LBG mixtures were lower in comparison to rice starch‐guar gum mixtures. Storage (G′) and loss (G′′) moduli of rice starch‐galactomannan mixtures increased with the increase in frequency (ω), while complex viscosity (η*) decreased. The magnitudes of G′ and G′′ increased with the increase in gum concentration. Dynamic rheological data of ln (G′, G′′) versus ln frequency (ω) of rice starch‐galactomannan mixtures have positive slopes with G′ greater than G′′ over most of the frequency range, indicating that their dynamic rheological behavior seems to be a weak gel‐like behavior.     

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o‐phthaldialdehyde reagent

The potential application of the o‐phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH₃, a by‐product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH₃ (at NH₃ concentrations > 10 mM ) was found to decrease during extended incubation at 23, 37 and 50°C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH₃ from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mM NH₃ at 23°C and pH 7.0 was found to be 88%. The release of NH₃ and the decrease in ε‐amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23°C resulted in progressive increases and decreases, respectively, in NH₃ and ε‐amino group concentration with increased incubation time. These changes were dependent on TGase : NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase‐catalysed cross‐links. However, the observed decrease in ε‐amino group concentration was higher than expected. This may be due to concealment of noncross‐linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.     

Rheology of Rice Starch‐Sucrose Composites

The effect of sucrose at different concentrations (0, 10, 20 and 30%) on rheological properties of rice starch pastes (5% w/w) was investigated in steady and dynamic shear. The steady shear properties of rice starch‐sucrose composites were determined from rheological parameters for power law and Casson flow models. At 25°C all the starch‐sucrose composites exhibited a shear‐thinning flow behavior (n=0.25–0.44). The presence of sucrose resulted in the decrease in consistency index (K), apparent viscosity (η_a,100) and yield stress (σ_oc). Dynamic frequency sweeps at 25°C indicated that starch‐sucrose composites exhibited weak gel‐like behavior with storage moduli (G′) higher than loss moduli (G′′). G′ and G′′ values decreased with the increase in sucrose concentration. The dynamic (η*) and steady‐shear (η_a) viscosities at various sucrose concentrations did not follow the Cox‐Merz superposition rule. G′ values as a function of aging time (10 h) at 4°C showed a pseudoplateau region at long aging times. In general, the values of G′ and G′′ in rice starch‐sucrose composites were reduced in the presence of sucrose and depended on sucrose concentration.