Influence of temperature and pH on the nonenzymatic reduction of triphenyltetrazolium chloride

The effects of temperature and pH on the nonenzymatic (chemical) reduction of triphenyltetrazolium chloride (TTC) to triphenyl formazan (TF) in cheese whey and municipal solid waste compost samples were studied. Ten different incubation temperatures and 13 pH levels were tested. The study showed that the TTC could be reduced nonenzymatically at high temperatures and/or under alkaline pH conditions. The nonenzymatic TTC reduction was observed at pH values greater than 9.5 and 11.0 for the cheese whey and compost, respectively. The TTC chemical reduction rate followed the same trend in both media. The TF content increased with increasing the pH value, reaching its maximum at a pH of 12, then decreased and was not detected at a pH of 13. The TTC was also reduced nonenzymatically at temperatures higher than 70 and 85 degrees C for cheese whey and compost, respectively. Evaporation did not seem to have any significant effect on the TTC chemical reduction since less than 3% of water content was lost at a temperature of 100 degrees C. It was noticed that the TF yield in cheese whey samples was higher than that in compost samples. This was due to the higher moisture content of cheese whey and the presence of copper in the compost samples, which reacted chemically with the TF causing reduction in the red color. For a given incubation period, the effect of pH on the TTC chemical reduction was more significant than the effect of incubation temperature (at a 2 h incubation period, 57.5% and 17.9% of the TTC were chemically reduced at a pH of 12 compared to 10.9% and 7.7% at an incubation temperature of 100 degrees C, for cheese whey and compost, respectively). Among the six metals tested (Ca, Cu, K, Na, Ni, and Zn) only Cu affected the color intensity of the TF. The activation energy of the TTC chemical reduction was 168,808 and 239,102 J/mol in cheese whey and municipal solid compost, respectively. For dehydrogenase activity measurement, the pH of the samples and the incubation temperature should not be higher than 9 and 60 degrees C in order to ensure that the TTC reduction is caused only by the biochemical reaction. Measuring the color intensity of TF in waste samples that contain copper could give misleading results as a result of the formation of formazan copper complex, which reduces the red color.     

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.     

Effect of chitosan type on protein and water recovery efficiency from surimi wash water treated with chitosan-alginate complexes

Previous research has shown that soluble protein recovery by chitosan (Chi) complexes with polyanions such as alginate (Alg) is more effective than using chitosan alone. In this study, Chi-Alg complexes were used to recover soluble proteins from surimi wash water (SWW) slightly acidified to pH 6. Six Chi samples differing in molecular weight (MW) and degree of deacetylation (DD) were used at 20, 40 and 100mg/L SWW Chi-Alg complexes prepared with a Chi:Alg mixing ratio previously optimized (MR=0.2). FTIR analysis of the solids recovered revealed the three characteristic amide bands observed in the same region for untreated SWW confirming protein adsorption by Chi-Alg. The superior effectiveness of Chi complexes was confirmed but differences among chitosan types could not be correlated to MW and DD. Experimental Chi samples with 94%, 93%, 75% and 93% DD and 22, 47, 225 and 3404 x 10(3)Da, respectively, showed 73-76% protein adsorption while a commercial chitosan sample with 84% DD and 3832 x 10(3)Da had 74-83% protein adsorption. An experimental chitosan, SY-1000 with 94% DD and 1.5 x 10(6)Da, showed the highest protein adsorption (79-86%) and turbidity reduction (85-92%) when used at 20mg/L SWW.     

Simultaneous single-cell protein production and COD removal with characterization of residual protein and intermediate metabolites during whey fermentation by K. marxianus

Cheese whey fermentation with Kluyveromyces marxianus was carried out at 40 °C and pH 3.5 to examine simultaneous single-cell protein production and chemical oxygen demand (COD) removal, determine the fate of soluble whey protein and characterize intermediate metabolites. After 36 h of batch fermentation, the biomass concentration increased from 2.0 to 6.0 g/L with 55 % COD reduction (including protein), whereas soluble whey protein concentration decreased from 5.6 to 4.1 g/L. It was confirmed through electrophoresis (SDS-PAGE) that the fermented whey protein was different from native whey protein. HPLC and GC–MS analysis revealed a change in composition of organic compounds post-fermentation. High inoculum concentration in batch fermentation resulted in an increase in biomass concentration from 10.3 to 15.9 g/L with 80 % COD reduction (including protein) within 36 h with residual protein concentration of 4.5 g/L. In third batch fermentation, the biomass concentration increased from 7.3 to 12.4 g/L with 71 % of COD removal and residual protein concentration of 4.3 g/L after 22 h. After 22 h, the batch process was shifted to a continuous process with cell recycle, and the steady state was achieved after another 60 h with biomass yield of 0.19 g biomass/g lactose and productivity of 0.26 g/L h. COD removal efficiency was 78–79 % with residual protein concentration of 3.8–4.2 g/L. The aerobic continuous fermentation process with cell recycle could be applied to single-cell protein production with substantial COD removal at low pH and high temperature from cheese whey.     

Whey utilization in furrow irrigation: effects on aggregate stability and erosion

Improving soil structure often reduces furrow erosion and maintains adequate infiltration. Cottage cheese whey, the liquid byproduct from cottage cheese manufacture, was utilized to stabilize soil aggregates and reduce sediment losses from furrow irrigation. We applied either 2.4 or 1.9L of whey per meter of furrow (3.15 or 2.49Lm(-2), respectively) by gravity flow without incorporation to two fields of Portneuf silt loam (Durinodic Xeric Haplocalcid) near Kimberly, ID. Furrows were irrigated with water beginning four days later. We measured sediment losses with furrow flumes during each irrigation and measured aggregate stability by wet sieving about 10 days after the last irrigation. Overall, whey significantly increased aggregate stability 25% at the 0-15mm depth and 14% at 15-30mm, compared to controls. On average, whey reduced sediment losses by 75% from furrows sloped at 2.4%. Whey increased the aggregate stability of structurally degraded calcareous soil in irrigation furrows.     

Surimi wash water treatment for protein recovery: effect of chitosan-alginate complex concentration and treatment time on protein adsorption

Chitosan (Chi), a protein recovery agent for the treatment of aqueous food processing streams, appears to work by mechanical entrapment and electrostatic interaction of chitosan amino groups with anionic groups on proteins. Chitosan effectiveness for recovering soluble proteins from surimi wash water (SWW) is increased by complexation with alginate (Alg) and by adjusting complex concentration and treatment time. Flocculation at 20 degrees C with Chi-Alg at a 0.2 mixing ratio added as 20, 40, 100 and 150 mg/L SWW was aided by 5 min agitation at 130 rpm and then held at the same temperature for 30 min, 1 and 24 h. Turbidity measurements, protein determinations and qualitative FTIR analysis confirmed SWW protein adsorption which depended on Chi-Alg concentration and reaction time while turbidity reduction was affected by concentration only. No differences (p < 0.05) in protein adsorption were found between 1 and 24 h. Using 100 mg Chi-Alg complex/L SWW for 1 h achieved 83% protein adsorption and 97% turbidity reduction.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

On-line sterilization of cheese whey using ultraviolet radiation

The effectiveness of ultraviolet radiation for on-line sterilization of cheese whey was investigated. The effects of flow rate and residence time on the performance of three UV reactors having different gap sizes (18, 13, and 6 mm) were studied. Six flow rates and six residence times were tested with the three UV reactors. The cheese whey used in this study had a very high turbidity (4317 NTU), very poor transmittance in the UV radiation germicidal range ( approximately 0%), and high percentage of large solid particles ( approximately 20% > 100 microm). Although the cheese whey physical characteristics showed low probability of sterilization using UV radiation, the study showed that UV radiation can be used on-line to sterilize cheese whey if the proper reactor gap size and the appropriate residence time are used. There were combined effects of the flow rate and gap size. The cell removal efficiency increased with increases in residence time and decreases in the UV reactor gap size. Removal efficiency of 100% was not achieved in this study with the first UV reactor (18-mm gap size), whereas 100% removal efficiency was achieved with the second (13-mm gap size) and third (6-mm gap size) UV reactors at residence times of 2.0 and 0.5 h, respectively. The microbial decay rates achieved in this study were 4.94, 7.62, and 20.9 h(-)(1) using the first, second, and third UV reactor, respectively. Residence times of 3.3, 2.1, and 0.8 h would be required to completely destruct a microbial population of 5.95 x 10(6) cells/mL using the first, second, and third UV reactors, respectively. Although cheese whey sterilization using UV radiation seems to be a good alternative to pasteurization, increases in cheese whey temperature resulted in lamp fouling. If online sterilization is to be used, the fouling problem should be investigated and a maintenance scheme for the UV reactor should be developed.     

Fed-batch fermentation to produce oligonucleotides from Kluyveromyces marxianus grown on whey

Oligonucleotides (ON) extracted from yeasts are used as antiviral agents, immunostimulators, and flavour enhancers. Fed-batch fermentation of cheese whey by Kluyveromyces marxianus was carried out to produce high biomass yields to extract ON. K marxianus was grown for 20 h in medium containing 5% (w/v) dehydrated whey, at 30°C (pH 4.5), with agitation (350 rpm), and under aeration (1.0–2.0 vvm). After 20 h, media containing 10–15% (w/v) of dehydrated whey were added at different flow rates (180–230 ml/h). Samples were analyzed at 6–8 h intervals for cell count, lactose consumption, and ethanol production. Maximum production of biomass (28.13 g/l), yield (0.58 g/g), productivity (2.42 g/l per h), and specific growth rate (0.63 1/h) were obtained when medium containing 15% (w/v) of whey was added at 180 ml/h under 2 vvm aeration. Fed-batch fermentation converted 95% of whey lactose into biomass.     

Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches

This study focuses on the exploitation of cheese whey as a source for hydrogen and methane, in a two-stage continuous process. Mesophilic fermentative hydrogen production from undiluted cheese whey was investigated at a hydraulic retention time (HRT) of 24 h. Alkalinity addition (NaHCO₃) or an automatic pH controller were used, to maintain the pH culture at a constant value of 5.2. The hydrogen production rate was 2.9 ± 0.2 L/Lreactor/d, while the yield of hydrogen produced was approximately 0.78 ± 0.05 mol H₂/mol glucose consumed, with alkalinity addition, while the respective values when using pH control were 1.9 ± 0.1 L/Lreactor/d and 0.61 ± 0.04 mol H₂/mol glucose consumed. The corresponding yields of hydrogen produced were 2.9 L of H₂/L cheese whey and 1.9 L of H₂/L cheese whey, respectively. The effluent from the hydrogenogenic reactor was further digested to biogas in a continuous mesophilic anaerobic bioreactor. The anaerobic digester was operated at an HRT of 20d and produced approximately 1 L CH₄/d, corresponding to a yield of 6.7 L CH₄/L of influent. The chemical oxygen demand (COD) elimination reached 95.3% demonstrating that cheese whey could be efficiently used for hydrogen and methane production, in a two-stage process.     

Behaviour of Listeria monocytogenes during the traditional manufacture of water-buffalo Mozzarella cheese

F. VILLANI, O. PEPE, G. MAURIELLO, G. MOSCHETTI, L. SANNINO AND S. COPPOLA. 1996. The behaviour of a four-strains mixture of Listeria monocytogenes (strains Scott A, V7, OH and Cal) during the traditional manufacture of water-buffalo Mozzarella cheese was investigated at two levels of inoculation: ca 10⁵and 10³cfu ml^-1of vat milk. No significant change in Listeria counts was observed during the curd ripening (4.0–4.5 h), at the end of which the pH ranged between 4.83 and 4.91. A decrease of about 2 log was observed after stretching of the curd in hot water (95°C), followed by complete elimination of Listeria after 48 and 24 h of storage of the final cheese in the conditioning liquid (skim water resulting from the stretching, pH ca 4.0) with initial high and low contamination of the cheese milk respectively. Results also indicated that a 1.7 log reduction of L. monocytogenes could be achieved during the preparation of the natural whey culture utilized as starter in cheesemaking.     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. III. Coagulation of cheese whey solids

Ten chitosan products, prepared as described in part I of this study, were evaluated in jar tests that measured their effectiveness for coagulation of suspended solids and removing turbidity from cheese whey. A polynominal regression analysis was found to be useful for determining the optimal effectiveness of each chistosan preparation, and was expressed as the percent reduction on turbidity per unit concentration of chitosan added. The effectiveness of the chitosan products was found to be inversely related to their molecular-weight values. This situation was different from the findings described in part II of this study, in which the filterability of activated sludge was tested. Enzymatic deproteination yielded chitosan products that performed better than those produced by alkali deproteination. Demineralized products were also more effective than those that had not been demineralized. The preparations deacetylated under a nitrogen atmosphere were more effective than those deacetylated in air, but this was shown to be true only for the first 5 min of deacetylation. When deacetylated for 15 min, no differences were noted. In this study, differences in performance between the various products were largely due to the differing dosages required to achieve the maximum reduction in turbidity of cheese whey, while the maximum responses achieved by the various products tested were about the same. A commercial product, which was less effective as a sludge coagulating agent in part II of this study, was more effective for cheese whey coagulation and turbidity removal than the majority of the experimental chitosan preparations tested.     

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.     

Complex coacervation of whey proteins and gum arabic

Mixtures of gum arabic and whey protein (whey protein isolate, WP) form an electrostatic complex in a specific pH range. Three phase boundaries (pH(c), pHphi(1), pHphi(2)) have been determined using an original titration method, newly applied to complex coacervation. It consists of monitoring the turbidity and light scattering intensity under slow acidification in situ with glucono-delta-lactone. Furthermore, the particle size could also be measured in parallel by dynamic light scattering. When the pH is lowered, whey proteins and gum arabic first form soluble complexes. This boundary is designated as pH(c). When the interaction is stronger (at lower pH), phase separation takes place (at pHphi(1)). Finally, at pHphi(2) complexation was suppressed by the charge reduction of the gum arabic. The major constituent of the whey protein preparation used was beta-lactoglobulin (beta-lg), and it was shown that beta-lg was indeed the main complex-forming protein. Moreover, an increase of the ionic strength shifted the pH boundaries to lower pH values, which was summarized in a state diagram. The experimental pH(c) values were compared to a newly developed theory for polyelectrolyte adsorption on heterogeneous surfaces. Finally, the influence of the total biopolymer concentration (0-20% w/w) was represented in a phase diagram. For concentrations below 12%, the results are consistent with the theory on complex coacervation developed by Overbeek and Voorn. However, for concentrations above 12%, phase diagrams surprisingly revealed a "metastable" region delimited by a percolation line. Overall, a strong similarity is seen between the behavior of this system and a colloidal gas-liquid phase separation.     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

Production of β-carotene from deproteinized waste whey filtrate using Mucor azygosporus MTCC 414 in submerged fermentation

The cheese whey, a by-product of dairy industry proved to be an attractive substrate for production of β-carotene. The β-carotene production from Mucor azygosporus MTCC 414 by using deproteinized waste whey filtrate under submerged fermentation was investigated. Various fermentation variables, such as lactose content in whey, initial pH, production temperature, incubation time, and carbon and nitrogen sources played significant role on β-carotene production. Maximum β-carotene production (385 μg/g dcw) was obtained with the whey (pH 5.5) containing 3.5% (w/v) lactose supplemented with soluble starch at (1.0%, w/v) at 30°C after a 5 days incubation. Moreover, unlike other microorganisms which utilize pre-hydrolyzed lactose, this Mucor azygosporus MTCC 414 was found to be capable of utilizing unhydrolyzed lactose present in the whey.     

Genetic structure of a novel biofuel-producing microorganism community

Biofuels are an important alternative, renewable source of energy in the face of the ongoing depletion of fossil fuels. Cheese whey is a dairy industry waste characterized by high lactose concentration, which represents a significant environmental problem. Bio-ethanol production by cheese whey could be an effective nonvegetable source for renewable energy production. Here, we report the isolation of a mixed microbial population, able to produce ethanol as main fermentation product from fermenting whey. The microbial consortium has been used to perform a batch fermentation of crude whey in both anoxic and hypoxic conditions. Maximum ethanol concentrations achieved in this study was obtained using the mixed culture in hypoxic conditions, grown at pH 4 and 30°C, with ethanol production yield of 60 g/L. Our research has pointed out an alternative way to both dispose and valorize cheese whey, a dairy by-product that could cause water pollution and harm to the environment if not properly treated.     

Influence of growth conditions on bacteriocin production by Brevibacterium linens

The influence of temperature, NaCl concentration and cheese whey media on growth of Brevibacterium linens ATCC 9175 and production of bacteriocin-like antimicrobial activity was studied. Bacteriocin production and activity were higher at 25 degrees C than at 30 degrees C. No significant growth or production of bacteriocins was observed at 37 degrees C. When bacteriocin production was investigated in media containing different concentrations of NaCl, increased activity was observed in media containing 40 or 80 g l(-1), but not 120 g l(-1) NaCl. The addition of NaCl resulted in a significant increase in specific production rates of bacteriocin-like activity. Antimicrobial activity was also observed by cultivation of B. linens at 25 degrees C in cheese whey media.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.