Retention of β-galactosidase activity in crude cellular extracts from Lactobacillus delbrueckii ssp. bulgaricus 11842 upon drying

Crude cellular extracts (CCEs) containing active β-galactosidase from Lactobacillus delbrueckii ssp. bulgaricus 11842 were spray-dried at three different outlet air temperatures (45, 55 or 65°C) or freeze-dried, with or without whey proteins, casein, whey or skim milk as drying adjuncts. The use of whey or skim milk resulted in significantly ( P   <  0.05) higher β-galactosidase activity retention in comparison to all other CCEs. This effect was not related to the initial total solids (TS) content (4–10%) of the feedstock solutions, but was presumably caused by the presence of lactose in the whey or skim milk CCE preparations.     

Proposing sequences for peptides derived from whey fermentation with potential bioactive sites

In fed-batch fermentation by Kluyveromyces marxianus var. marxianus, whey-soluble proteins were converted into oligopeptides. To assess whether bioactive peptides could be produced during whey fermentation, K. marxianus was cultured in batch in deproteinized media containing 5 or 15% (wt/vol) dehydrated whey for 20 h and then was in fed-batch mode for 50 h. After harvesting the biomass (25,000 x g, 15 min), at 6-h intervals, the wort was analyzed to determine protein consumption and oligopeptide production by HPLC. The proteins in the wort showed an oscillatory degradation with a constant increase in the production of oligopeptides. Four major peaks were collected and were analyzed by API mass spectroscopy. Sequences of fermented peptides were compared with sequences of known bioactive peptides. On the basis of their molecular weights, two amino acid sequences were proposed. The presence of sites containing the peptide sequence of beta-lactorphin (YLLF) suggests that these oligopeptides may have antihypertensive properties.     

Formulation of Culture Medium with Agroindustrial Waste for β-Galactosidase Production from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> ATCC 16045

Seven strains of the genus Kluyveromyces were screened for β-galactosidase activity and Kluyveromyces marxianus ATCC 16045 was selected as the best enzyme producer for culture medium optimization. The production of β-galactosidase by submerged cultivation was evaluated using a factorial design and response surface methodology. The culture medium containing whey and parboiled rice effluent was formulated to maximize the production of β-galactosidase. The effects of the initial pH and the concentrations of whey lactose, peptone, (NH₄)₂SO₄, yeast extract, and parboiled rice effluent on enzyme production were studied using a 2 _IV ^6-2 fractional design. A CCRD (24 trials plus axial and central points) was used for the four variables selected from the fractional design (lactose, peptone, (NH₄)₂SO₄ and yeast extract), with β-galactosidase activity as the response. The optimum conditions established for production were a whey (lactose) concentration of 120 g/L, a yeast extract concentration of 5 g/L, a peptone concentration of 15 g/L, a (NH₄)₂SO₄ concentration of 15 g/L, a parboiled rice effluent concentration of 30 g/L, and a pH value of 4.0. Under these conditions, the highest enzymatic activity of 10.4 U/mL was measured, being 9.5–9.7 as the values predicted by the proposed model, showing an enzymatic activity increase of 30% using alternative sources of lactose and nitrogen for β-galactosidase production.     

Selection of starters and a starter-mediated novel procedure for production of wara, a West African soft cheese

Summary Wara serves as an important protein source for people in West Africa. The ten most frequently isolated lactic acid bacteria from abomasum-soured milk samples were screened singly or in combinations as starter organisms for the production of wara . The screening was based on the bacteria's ability to produce lactic acid, diacetyl, β-galactosidase and an average weighted firm curd at 30 °C (6 h, 72 h). With lactic acid concentration of 10.0 g l⁻¹, β-galactosidase of 25.2 U mL⁻¹ and a firm curd weight of 16.0 g l⁻¹ milk at 30 °C, pH 5.1 after 6 h, a single-organism starter culture of Lactococcus lactis was chosen as the best suited. Compared with the commercial market samples, wara produced by this starter culture showed a 54% increase in percentage protein, 17.9% increase in ash, 23.6% increase in iron and a 150% increase in vitamin A content. Statistical analysis of sensory evaluation results showed a preference for the market sample only in terms of appearance and texture, while the starter-produced wara matched the market sample in terms of flavour and palatability. Treatment with 3% brine reduced microbial counts although dipping in 5% NaCl-in-whey gave better results in terms of the total viable count of mesophiles, psychrotrophs and coliforms compared with the untreated sample.     

Comparison of Galactooligosaccharide Production in Free-Enzyme Ultrafiltration and in Immobilized-Enzyme Systems

ABSTRACT:  Galactooligosaccharides (GOSs) are prebiotics that have been shown to reduce colon cancer risk and enhance immunity. The GOSs can be formed enzymatically from whey lactose using β-galactosidase, but commercial application has been limited. Free- and immobilized-enzyme recycle batch processes were investigated and compared in this study. Optimum initial conditions were estimated using batch solutions. Using these optimum conditions, an ultrafiltration (UF) free-enzyme system was developed, and UF fluid pressure (100 to 400 p.s.i.) effect on enzyme performance was studied and compared with a 0 p.s.i. batch control. The optimum conditions were also used to develop an immobilized-enzyme process using polyethyleneimine (PEI), glutaraldehyde (GA), and cotton cloth. The PEI and GA immobilized agents were studied for their effect on enzyme inactivation. Finally, compatible free- and immobilized-enzyme recycle batch systems were compared for GOS production. Optimum initial enzyme conditions were estimated as 270 g/L initial lactose, 4.5 g/L initial enzyme ( Aspergillus oryzae, 9400 U/g), and 30-min incubation. Fluid pressure within the free-enzyme UF membrane system had no significant effect on enzyme performance. The highly agitated UF enzyme system was found to be superior to the less agitated 0 p.s.i batch control. In the immobilization process, approximately 50% to approximately 90% enzyme inactivation was found with the combination of PEI and GA. Equivalent free- and immobilized-enzyme systems showed very similar maximum GOS production of approximately 22% and approximately 20% (w/v) at approximately 15 to 17 min, or 50% conversion for free- and immobilized-systems, respectively.     

Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl₂, and CaCl₂ than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl₂ was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl₂. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl₂, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K_iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.     

Production of α-Galactosidase from Aspergilus oryzae Grown in Solid State Culture

Aspergillus oryzae QM 6737 was grown in solid state culture to produce α-galactosidase extracellularly. An optimum enzyme yield of ～ 3 units/g solid was obtained on wheat bran, 35% initial moisture, after 5 days at 32°C, with agitation. Higher initial moisture levels reduced enzyme production. Higher yields were obtained by adding glucose (73% increase), sucrose (39% increase), or melibiose (39% increase) to the bran, but raffinose and stachyose had little or no effect. Enzyme yield increased three times when soy flour or soy beans was used as substrate but no enzyme production was observed using rice. Enzyme production on soy beans decreased with increased initial moisture levels and with decreased particle size.     

Rennin-like milk coagulant enzyme produced by a local isolate of Mucor

Among 20 isolates of Mucor isolated from various environments in Jordan and found to produce a rennin-like acid protease, known as Mucor rennin-like enzyme (MRE), Mucor J20 was found to produce the highest level of MRE. The optimum incubation conditions for enzyme production in a fortified wheat bran mixture using solid-state fermentation were 3–4 days at 30°C. The highest MRE activity (185–200 rennin units or RU) was produced in a medium containing wheat bran and lentil straw (1 : 1 w/w) moistened with whey, and incubated in clay pots at 30°C for 4 days. A slightly lower activity value (178 RU) was found when using a mineral salt solution or distilled water instead of whey, or when using wheat bran alone with whey. At pH 4, the MRE retained its complete activity (100%) for 6 weeks at 5°C and 10°C, and for 3 and 2 weeks at 20°C and 30°C, respectively. After heating at 60°C for 10 min, the enzyme lost its activity at all pH levels used (pH 2–8). The crude extract of MRE was successfully applied in the manufacture of a cheese curd.     

OPTIMIZATION OF IMMOBILIZATION PROCESS ON CRAB SHELL CHITOSAN AND ITS APPLICATION IN FOOD PROCESSING

Optimization of immobilization process on crab shell chitosan was carried out. The chitosan purified from the crab shell was used as the matrix for the immobilization of α-galactosidase. The prepared matrix was activated with glutaraldehyde at different concentrations and different time intervals and coupling time was determined. Immobilization of α-galactosidase on crab shell chitosan resulted in 72% immobilization yield. The parameters like the effect of pH, temperature, thermal stability and storage stability were determined. The study revealed that immobilized enzyme shows better thermal and storage stability than the free enzyme. The performance of the free and immobilized α-galactosidase was tested in continuous stirred batch reactor to hydrolyze raffinose family oligosaccharides in soymilk. The oligosaccharide content of the soymilk was reduced by 77% in continuous reaction by immobilized α-galactosidase.

PRACTICAL APPLICATIONS

Chitosan used for the immobilization of α-galactosidase offers several advantages for enzyme immobilization and it contains all characteristic features for use as industrial material. Immobilization of one of the industrial important enzyme α-galactosidase, as it has many potential application in hydrolyzing raffinose series of oligosaccharides. The hydrolyzed soymilk after processing by immobilized α-galactosidase is free from flatus-inducing factors like raffinose and stachyose. It can be used as an alternative means for cow's milk for lactose intolerance, particularly among individuals in developing countries. As chitosan used is from the crustacean waste from the crab shell, the production and utilization of chitosan provides an economical alternative means of crustacean shell waste disposal sought worldwide.     

HYDROLYSIS OF LACTOSE IN ACID WHEY BY LACTASE BOUND TO POROUS GLASS PARTICLES IN TUBULAR REACTORS

Partially purified lactases (β-galactosidase, EC3.2.1.23) from Aspergillus niger were covalently bound to acetone-silanized, diazotized porous glass particles (mean pore diam, 86.5 nm; particle diam, 75–125 μm). The temperature (∼55°C) and pH (3.5–4.0) optima were established in acid whey containing 5% total whey solids. Lactose hydrolyzing activity was stable during 43 days of semicontinuous operation at 55°C with reconstituted acid whey (pH = 4.5) at total solids (TS) concentrations varied between 4 and 25% and to which 5 ml/liter toluene had been added to retard microbiological contamination. Kinetic experiments with acid wheys gave results reproducible when assayed by both thin layer chromatography (TLC) and glucose oxidase (GS) procedures, although the TLC method gave systematically higher Values at intermediate conversions and high TS concentrations. The kinetics of lactose hydrolysis by columns of lactase bound to porous glass (LBG) of 1.6 cm diam and lengths of 1, 5 and 10.5 cm showed some evidence for reduction of the rate of lactose hydrolysis by film diffusion resistances. Calculations using correlations for packed beds also suggest the presence of diffusional effects. Lactose was hydrolyzed slightly more rapidly in whey than in deproteinized whey. Lactose hydrolysis rates in both types of reconstituted whey increased as the TS concentrations increased from 4 to 25%. The data did not obey any of a number of integrated reaction rate equations, including a rate equation which accounted for competitive product inhibition of Michaelis enzyme kinetics. Failure of simple models is due in part to diffusional resistances and in part to the large range of concentrations studied. The LBG preparation retained appreciable activity after more than 8 months of frequent use at a wide variety of conditions.     

Optimizing the use of power ultrasound to decrease turbidity in whey protein suspensions

This research employed power ultrasound (US) to decrease the turbidity of whey suspensions. US was applied using a 20 kHz generator and different power levels (3 and 15 W) for different application times (5 and 15 min) at different temperatures (20, 60 °C, and no temperature control [NTC]) to whey suspensions obtained at 4 different steps in a commercial process. The whey suspensions varied from 6.9 to 30.2% solids and 13.5 to 88% protein. This research shows an approximately 90% decrease in turbidity when US was applied to a whey suspension of 28.2% of solids containing 35.6% of protein on a dry basis. The greatest decrease in turbidity was observed when US was applied for 15 min using 15 W of electrical power at 60 °C and NTC conditions. Surprisingly, when US was used in whey suspensions containing 88.0% protein an increase in turbidity of samples was observed.     

Response surface methodology applied to the enzymatic synthesis of galacto-oligosaccharides from cheese whey

In this study, a strategy was proposed for making galacto-oligossaccharides (GOS), a high valueadded product, from a byproduct of the dairy industry, cheese whey, using a commercial β-galactosidase from Kluyveromyces lactis (Lactozym® 3000L). The effects of the substrate concentration, temperature, and enzyme dosage were statistically studied and their optimum combinations were determined using response surface methodology. The increase in lactose concentration, temperature, and enzyme concentration favored a transgalactosylation reaction. The maximum values for GOS concentration (119.8 mg/mL) and yield (29.9%) in a 4 h process were obtained in the reaction system, composed of 400 mg/mL of lactose and 10 U/mL of enzyme at 40°C. Under these conditions, the lactose conversion was 68.7%. The maximum value for lactose conversion (87.8%) was observed at the same temperature and enzyme concentration, although the lactose level was 20%.     

Apparent chemical composition of nine commercial or semi-commercial whey protein concentrates, isolates and fractions

Summary Analytical results are given for whey powders prepared on a commercial or semi-commercial scale by three companies. Altogether, five preparations enriched in β-lactoglobulin, four whey protein isolates and a fraction enriched in α-lactalbumin were analyzed for protein composition, including %β-lactoglobulin, α-lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native β-lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true β-lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimated true β-lactoglobulin weight % of 70.9 ± 1.1, 62.0 ± 3.4 and 39.5 ± 4.9. Physico-chemical properties of the group of samples are reported elsewhere (Holt et al ., 1999).     

Immobilization of <Emphasis Type="BoldItalic">Aspergillus oryzae</Emphasis> β-Galactosidase onto Duolite A568 Resin via Simple Adsorption Mechanism

In this study, a rapid, simple and economic method of enzyme immobilization was developed to hydrolyze lactose. Duolite A568 resin was used for the immobilization of β-galactosidase via simple adsorption mechanism. The effects of immobilization parameters such as time, pH, and temperature were studied. Immobilization parameters for maximum enzyme activity were estimated at 35 °C temperature, pH 4.5, 5 mg/mL enzyme concentration, and approximately 60 min immobilization time. A significant amount of enzyme was immobilized with high catalytic activity. Enzyme immobilization procedure explained in this study slightly affected the enzyme kinetic. The value of Michaelis constant K _m for immobilized enzyme was significantly larger, indicating decreased affinity by the enzyme for its substrate. It was observed that both free and immobilized enzyme showed maximum activity at 65 °C reaction temperature. Immobilized β-galactosidase was significantly more active at all temperatures as compared to its free form. However, optimal pH of immobilized enzyme was slightly affected by immobilization procedure. The optimum pH of immobilized enzyme was shifted up 0.5 unit to a more alkaline value of 6.0 compared to the free enzyme.     

Optimization of Thermal Pretreatment Conditions for the Separation of Native α-Lactalbumin from Whey Protein Concentrates by Means of Selective Denaturation of β-Lactoglobulin

ABSTRACT: In this study a method to obtain native α-lactalbumin with a high degree of purity of 98% (m/m) and recovery of 75% (m/m) by selective denaturation of β-lactoglobulin was developed. To achieve this goal, the thermal pretreatment of whey protein concentrate was optimized varying the composition of the liquid whey protein concentrate in terms of total protein, lactose and calcium content, and pH value. The kinetics of the thermal denaturation of α-la and β-lg were then investigated at predetermined optimal composition (protein content 5 to 20 g/L, lactose content 0.5 g/L, calcium content 0.55 g/L, and pH 7.5). Using the activation energies and reaction rate constants obtained, lines of equal effects for targeted denaturation degrees of α-la and β-lg were calculated. Depending on total protein content, an area of optimal heating temperature/time conditions was identified for each protein concentration level.     

The use of lactoperoxidase for the bleaching of fluid whey

Lactoperoxidase (LP) is the second most abundant enzyme in bovine milk and has been used in conjunction with hydrogen peroxide (H₂O₂) and thiocyanate (SCN^–) to work as an antimicrobial in raw milk where pasteurization is not feasible. Thiocyanate is naturally present and the lactoperoxidase system purportedly can be used to bleach dairy products, such as whey, with the addition of very little H₂O₂ to the system. This study had 3 objectives: 1) to quantify the amount of H₂O₂ necessary for bleaching of fluid whey using the LP system, 2) to monitor LP activity from raw milk through manufacture of liquid whey, and 3) to compare the flavor of whey protein concentrate 80% (WPC80) bleached by the LP system to that bleached by traditional H₂O₂ bleaching. Cheddar cheese whey with annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a standard Cheddar cheesemaking procedure. Various levels of H₂O₂ (5–100 mg/kg) were added to fluid whey to determine the optimum concentration of H₂O₂ for LP activity, which was measured using an established colorimetric method. In subsequent experiments, fat-separated whey was bleached for 1 h with 250 mg of H₂O₂/kg (traditional) or 20 mg of H₂O₂/kg (LP system). The WPC80 was manufactured from whey bleached with 250 mg of H₂O₂/kg or 20 mg of H₂O₂/kg. All samples were subjected to color analysis (Hunter color values and norbixin extraction) and proximate analysis (fat, protein, and moisture). Sensory and instrumental volatile analyses were conducted on WPC80. Optimal LP bleaching in fluid whey occurred with the addition of 20 mg of H₂O₂/kg. Bleaching of fluid whey at either 35 or 50°C for 1 h with LP resulted in >99% norbixin destruction compared with 32 or 47% destruction from bleaching with 250 mg of H₂O₂/kg, at 35 or 50°C for 1 h, respectively. Higher aroma intensity and increased lipid oxidation compounds were documented in WPC80 from bleached whey compared with WPC80 from unbleached whey. Monitoring of LP activity throughout cheese and whey manufacture showed that LP activity sharply decreased after 30 min of bleaching (17.01 ± 1.4 to <1 U/mL), suggesting that sufficient bleaching takes place in a very short amount of time. Lactoperoxidase averaged 13.01 ± 0.7 U/mL in unpasteurized, fat-separated liquid whey and 138.6 ± 11.9 U/mL in concentrated retentate (11% solids). Lactoperoxidase may be a viable alternative for chemical whey bleaching.     

Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Comparison of bioethanol and beta‐galactosidase production by Kluyveromyces and Saccharomyces strains grown in cheese whey

The production of ethanol and beta‐galactosidase by Kluyveromyces marxianus and Saccharomyces fragilis strains grown in cheese whey was evaluated. The conditions for fermentation in 50 g/L (3.3% lactose) and 150 g/L (8.8% lactose) cheese whey were 100 rpm for 24 h at 30, 35 and 40 °C. Saccharomyces fragilis IZ 275 in 8.8% lactose at 40 °C resulted in 3.90% ethanol. Kluyveromyces marxianus CCT 3172 showed higher beta‐galactosidase, 1.10 U/mg, at 30 °C. Therefore, the choice of cultivation conditions and the most suitable species is important for obtaining high yields of the products of interest.     

Peptide enriched functional food adjunct from soy whey: A statistical optimization study

Soy whey is generated as a process waste while preparing soy based food products tofu, causing environmental pollution and also representing an economic penalty against the industrial process. Therefore, its valorization is of prime importance to the industry. The present investigation aims to convert this proteinaceous waste into bioactive peptide enriched hydrolysate. Soy whey protein was enzymatically treated with the Aspergillus awamori nakazawa protease. Respective protease was efficient to produce antioxidant peptide beholding radical scavenging ability of 40–50% at normal conditions. Remarkable increase in the radical scavenging activity upto 70% was noticed at the response surface methodology (RSM) based optimized condition: temperature 40°C, salt concentration (NaCl) 0.05 M, surfactant concentration (Triton-X 100) 0.0075%, hydrolysis time 80 min, and enzyme to substrate concentration 164 IU/g of soy whey protein. The present study emphasizes the biotransformation of proteineceous waste into antioxidant peptide rich soy whey protein hydrolysate to be considered as additives for food preparation and formulation.     

Optimization of β-carotene production by a mutant of the lactose-positive yeast Rhodotorula acheniorum from whey ultrafiltrate

The present work investigated on carotenogenesis with high β-carotene content by a new isolated high-activity strain-producer Rhodotorula acheniorum mutant MRN in cheese whey ultrafiltrate. After a serial of UV, ethymethanesurfonate (EMS), and nitrosoguanidine (NTG) mutagenesis, a mutant named MRN of the red lactose-positive yeast strain R. acheniorum was obtained. Then, the effects of different growth medium factors on carotenoid production by this mutant at batch-scale level were identified and optimized by means of response surface methodology (RSM) in order to achieve high-level production of β-carotene. The optimum conditions required to achieve the highest level of β-carotene (262.12±1.01 mg/L) were determined as follows: whey ultrafiltrate (WU) lactose concentration 55 g/L, pH 5.85, ammonium sulfate concentration 3.5 g/L, temperature 23°C, and aeration rate 1.56 vvm. The medium optimization resulted in a 6.45-fold increase in volumetric production (262.12±1.01 mg/L) and a 4.62-fold increase in the cellular accumulation (10.69±0.19 mg/g) of β-carotene.