Viscosity changes and gel formation during storage of liquid micellar casein concentrates

Our objective was to determine the effects of temperature and protein concentration on viscosity increase and gelation of liquid micellar casein concentrate (MCC) at protein concentrations from 6 to 20% during refrigerated storage. Skim milk (∼350 kg) was pasteurized (72°C for 16 s) and filtered through a ceramic microfiltration system to make MCC and replicated 3 times. The liquid MCC was immediately concentrated via a plate ultrafiltration system to 18% protein (wt/wt). The MCC was then diluted to various protein concentrations (6–18%, wt/wt). The highest protein concentrations of MCC formed gels almost immediately on cooling to 4°C, whereas lower concentrations of MCC were viscous liquids. Apparent viscosity (AV) determination using a rotational viscometer, gel strength using a compression test, and protein analysis of supernatants from ultracentrifugation by the Kjeldahl method were performed. The AV data were collected from MCC (6.54, 8.75, 10.66, and 13.21% protein) at 4, 20, and 37°C, and compression force test data were collected for MCC (15.6, 17.9, and 20.3% protein) over a period of 2-wk storage at 4°C. The maximum compressive load was compared at each time point to determine the changes in gel strength over time. Supernatants from MCC of 6.96 and 11.61% protein were collected after ultracentrifugation (100,605 × g for 2 h at 4, 20, and 37°C) and the nitrogen distributions (total, noncasein, casein, and nonprotein nitrogen) were determined. The protein and casein as a percent of true protein concentration in the liquid phase around casein micelles in MCC increased with increasing total MCC protein concentration and with decreasing temperature. Casein as a percent of true protein at 4°C in the liquid phase around casein micelles increased from about 16% for skim milk to about 78% for an MCC containing 11.6% protein. This increase was larger than expected, and this may promote increased viscosity. The AV of MCC solutions in the range of 6 to 13% casein increased with increasing casein concentration and decreasing temperature. We observed a temperature by protein concentration interaction, with AV increasing more rapidly with decreasing temperature at high protein concentration. The increase in AV with decreasing temperature may be due to the increase in protein concentration in the aqueous phase around the casein micelles. The MCC containing about 16 and 18% casein gelled upon cooling to form a gel that was likely a particle jamming gel. These gels increased in strength over 10 d of storage at 4°C, likely due either to the migration of casein (CN) out of the micelles and interaction of the nonmicellar CN to form a network that further strengthened the random loose jamming gel structure or to a gradual increase in voluminosity of the casein micelles during storage at 4°C.     

Effect of manufacture and reconstitution of milk protein concentrate powder on the size and rennet gelation behaviour of casein micelles

Samples of raw skim milk, ultrafiltration/diafiltration retentate, concentrated retentate and milk protein concentrate powder (MPC80) from a single commercial production run were analysed using photon correlation spectroscopy. Measurements revealed insignificant differences in casein micelle size between the samples. In addition, there was no discernable difference between raw skim milk and MPC powder dissolved at 60 °C in the amount of casein remaining in supernatants from centrifugation at either 25,000 × g or 174,200 × g. Casein micelles did not appear to be altered during manufacture of MPC. The rennet gelation behaviour of reconstituted MPC was compared with raw skim milk. Reconstituted MPC did not coagulate unless supplemented with approximately 2 mm calcium chloride, which was attributed to the mineral removal during ultrafiltration/diafiltration. Addition of sufficient calcium could restore rennet coagulation kinetics and gel strength of reconstituted MPC to approximately that of raw skim milk.     

Use of Electrodialysis to Improve the Protein Stability of Frozen Skim Milks and Milk Concentrates

Removal of calcium from skim milk by electrodialysis was logarithmically related to the extent of demineralization (log% calcium removed = 0.18% ash removed + 0.53). This removal of calcium caused a logarithmic dissociation of micellar to serum (sub-micellar) casein (log% total casein in serum = 0.020% calcium removed + 0.70). Increases in protein stability for skim milk and concentrated skim milk were related to this dissociation of micellar casein, and the initial degree of dissociation persisted throughout frozen storage. Protein stability was > 53 wk at –8°C when 80% of the casein in unconcentrated skim milk was dissociated. Concentrated skim milk was stable for > 30 wk at –8°C when 45% of the casein was dissociated. Control samples (7–9% casein dissociated) were stable for only 4–8 wk at –8°C.     

Deamidation Kinetics of Precipitated by Carbon Dioxide Compared with Commercial Caseinates

Casein precipitated by carbon dioxide (CO₂-casein), and commercial sodium and calcium caseinates (1% w/v in water) were thermally deamidated at pH 8 for up to 96h at 85,100, and 115°C. CO₂-casein displayed the highest overall extent of deamidation at 100 and 115°C after 32 and 24h, respectively, while there was no difference at 85°C. Structural differences between CO₂-casein and the commercial caseinates may account for the difference in deamidation. Deamidation kinetics were apparent first-order with respect to concentrations of the amides, asparagine and glutamine. Reaction rate constants and Arrhenius parameters were comparable from regression modeling.     

Casein Hydrolysate Produced Using a Formed-in-Place Membrane Reactor

Casein hydrolysate was continuously produced by hydrolysis of bovine casein with Protease Type XXIII (from Aspergillus oryzue) in a pilotscale formed-in-place membrane reactor. A high percentage (>99%) of TCA soluble nitrogen in the hydrolysate (product) was achieved after 45 min at 37°C and pH 7. The product was completely soluble over pH range 2–9. Water sorption increased 4 to 6.5 times at water activity of 0.35–0.95 as compared to intact casein. The immunologically active casein and immunologically active whey protein in the product were reduced 99% and 97%, respectively. Long term operation showed that the membrane reactor maintained steady production of casein hydrolysate longer than 17 hr.     

Continuous Hydrolysis of Milk Protein in a Membrane Reactor

Casein was hydrolyzed using Alcalase in a continuous stirred tank membrane reactor (CSTMR) using a hollow fiber ultrafiltration membrane. A design of experiments methodology was used to optimize the four performance variables: enzyme concentration, substrate concentration, permeate flux and reactor volume. High conversion (>90%) of the casein could be achieved after one hour at 50°C and pH 8. Reactor activity was most affected by the enzyme-to-substrate ratio. The stability of the reactor (i.e., decay of activity with time) was a function of enzyme leakage through the membrane, thermal inactivation and enzyme-membrane interactions.     

Chemical Properties of Cryocasein

Some chemical properties of cryocasein were examined and compared to those of commercial casein and caseinates. Cryocasein contained more ash and lactose than commercial casein and caseinates. Compared to acid casein, cryocasein had slightly less β-casein due to the preparation procedures involving low temperature and ultrafiltration. Retainment of micellar structure was verified by electron microscopy and permeation chromatography on CPG-10. An increase in aggregation of casein miscelles was observed during frozen storage (?8°C) of diluted ultrafiltration retentate. Similar to the casein in skim milk, this intact micellar structure may be responsible for properties such as solvation and stability towards addition of ethanol and Ca²⁺.     

Effects of Frozen Storage on Survival of Staphylococcus aureus and Enterotoxin Production in Precooked Tuna Meat

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (?20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin‐producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum‐packed samples were stored in a freezer (?20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at ?20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat.     

Protein Quality of Fish in Modified Atmospheres as Predicted by the C-PER Assay

The nutritional quality of proteins from fillets of fresh rockfish (Sebastes spp) stored in air or in a modified atmosphere (MA) of 80% CO₂ and 20% O₂ at 4°C was evaluated by the Computed Protein Efficiency Ratio (C-PER) method. This procedure involves determination of in vitro digestibility and the amino acid composition of the samples prior to computation of the C-PER. Casein was used as a standard. Both air-held and MA-held fish showed similar amino acid profiles, but the air-held fish showed lower in vitro digestibility. The C-PER values of both types of samples were higher than the C-PER of 2.50 for standard casein. These results suggest that storage of fish in a modified atmosphere of 80% CO₂ and 20% O₂ does not result in deterioration in the nutritional quality of the fish proteins.     

Autoxidizing Process Interaction of Linoleic Acid with Casein

Autoxidized linoleic acid was classified to linoleic acid (LA), its hydroperoxide (LAHPO) and secondary autoxidation products (SP), and interactions of these products with casein were investigated. Casein was antioxidative to the autoxidation of LA. The products were readily bound to casein at 37°C within 1 hr in order of SP > LAHPO > LA. SP and LAHPO facilitated the access of fluorescent probes to casein and made casein insoluble. Casein was subsequently became undigestible during 1 day incubation with SP or after 2 days with LAHPO. Incubation with SP for 4 days or more caused casein to lose its amino acids and to polymerize.     

Effects of Silage Fermentation and Post-ruminal Casein Supplementation in Lactating Dairy Cows: 1—Diet Digestion and Milk Production

Two silages were prepared from the first-cut sward of timothy-meadow fescue and wilted to a dry matter (DM) content of 300 g kg^-1. One was ensiled with the addition of a formic-acid-based additive (4 litres formic acid (FA) per tonne) and the other with the addition of a bacterial inoculant (LAB) at a rate of 5×10⁶ colony forming units g^-1. Both silages were well preserved, but the extent of fermentation was greater in LAB-silage than in FA-silage as indicated by a lower concentration of water soluble carbohydrates (68 vs 177 g kg^-1 DM) and a higher concentration of lactic acid (147 vs 32 g kg^-1 DM). Four Ayrshire cows were used in a 4×4 Latin square experiment with 21-day periods to study the effects of silage fermentation and postruminal casein supplementation on silage intake, nutrient supply and milk production. The four treatments were FA-silage without casein (FA-0), LAB-silage without casein (LAB-0), FA-silage with casein (FA-C) and LAB-silage with casein (LAB-C). Both silages were given ad libitum with 8 kg day^-1 of barley without or with 400 g day^-1 of casein infused into the duodenum. Organic matter digestibility was lower (0·723 vs 0·753; P<0·01) for FA-silage than for LAB-silage. Cows offered FA-silage had a higher molar proportion of acetate and a lower proportion of propionate in ruminal fluid than cows offered LAB-silage. Microbial protein synthesis estimated from the output of purine derivatives in urine was greater (288 vs 260 g N day^-1; P<0·05) for cows given FA-silage compared with LAB-silage. Feeding LAB-treated silage tended (P<0·10) to decrease silage DM intake compared with FA-treated silage (10·61 vs 11·77 kg DM day^-1). Silage composition did not affect significantly milk yield or milk composition. Casein infusion increased milk yield (25·1 vs 27·1 kg day^-1; P<0·05), milk protein content (32·4 vs 33·8 g kg^-1; P<0·05) and protein yield (808 vs 905 g day^-1; P<0·01). The responses were similar for both silages. © 1997 SCI.     

Effect of temperature on initial bacteria of prawn

This study was carried out to evaluate the effect of heat and cold on the microorganisms existing in prawn. Roasting for 5 min, boiling for 10 min and storage at ? 18 °C for 10, 20 and 30 days were applied on raw prawn to study the effect of each treatment on the bacterial load. The average counts of APC (aerobic plate count) at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci were 1.3 × 10⁷, 6 × 10⁴, 3 × 10⁴, 2 × 10² and 3 × 10³ organisms per gram raw prawn sample, respectively, then reduced to 10⁴, 8 × 10², 3 × 10³, < 3 and 3 × 10² organisms per gram roasted prawn in shell, respectively. Such counts were more reduced in roasted peeled samples. Boiling reduced the average counts of APC at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci from 8 × 10⁸, 2 × 10⁵ 2 × 10⁷, 3 × 10 and 3 × 10⁴ organisms per gram raw prawn sample to 2 × 10⁵, 3 × 10³, 2 × 10², < 3 and 4 × 10² organisms, respectively. Concerning freezing storage, it could be observed that the average counts of APC at 25 °C and 0 °C, Enterobacteriaceae, Coliforms and Staphylococci were reduced from 4 × 10⁷, 5 × 10⁵, 3 × 10⁵, 10³ and 3 × 10² organisms per gram raw sample to 5 × 10⁴, 10³, 3 × 10², 10 and < 2 × 10² organisms per gram after freezing storage for 30 days.     

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.     

Production and storage stability of concentrated micellar casein

Concentrated micellar casein (CMC) is a high-protein ingredient that can be used in process cheese product formulations. The objectives of this study were to develop a process to produce CMC and to evaluate the effect of sodium chloride and sodium citrate on its storage stability. Skim milk was pasteurized at 76°C for 16 s and cooled to ≤4°C. The skim milk was heated to 50°C using a plate heat exchanger and microfiltered with a graded permeability (GP) ceramic microfiltration (MF) membrane system (0.1 μm) in a continuous feed-and-bleed mode (flux of 71.43 L/m² per hour) using a 3× concentration factor (CF) to produce a 3× MF retentate. Subsequently, the retentate of the first stage was diluted 2× with soft water (2 kg of water: 1 kg of retentate) and again MF at 50°C using a 3× CF. The retentate of the second stage was then cooled to 4°C and stored overnight. The following day, the retentate was heated to 63°C and MF in a recirculation mode until the total solids (TS) reached approximately 22% (wt/wt). Subsequently, the MF system temperature was increased to 74°C and MF until the permeate flux was <3 L/m² per hour. The CMC was then divided into 3 aliquots (approximately 10 kg each) at 74°C. The first portion was a control, whereas 1% of sodium chloride was added to the second portion (T1), and 1% of sodium chloride plus 1% of sodium citrate were added to the third portion (T2). The CMC retentates were transferred hot to sterilized vials and stored at 4°C. This trial was repeated 3 times using separate lots of skim milk. The CMC at d 0 (immediately after manufacturing) contained 25.41% TS, 21.65% true protein (TP), 0.09% nonprotein nitrogen (NPN), and 0.55% noncasein nitrogen (NCN). Mean total aerobic bacterial counts (TBC) in control, T1, and T2 at d 0 were 2.6, 2.5, and 2.8 log cfu/mL, respectively. The level of proteolysis (NCN and NPN values) increased with increasing TBC during 60 d of storage at 4°C. This study determined that CMC with >25% TS and >95% casein as percentage of TP can be manufactured using GP MF ceramic membranes and could be stored up to 60 d at 4°C. The effects of the small increase in NCN and NPN, as well as the addition of sodium chloride or sodium citrate in CMC during 60 d of storage on process cheese characteristics, will be evaluated in subsequent studies.     

Transglutaminase Catalyzed Cross-Linking of Myosin to Soya Protein, Casein and Gluten

The covalent linkage of nonmeat proteins to myosin at temperatures and pH's common in meat product processing was investigated. Iodinated proteins were cross-linked to immobilized myosin and then quantitated by gamma counting after noncross-linked material was removed by washing with 6M guanidine hydrochloride. Bovine plasma transglutaminase catalyzed the formation of ε- (γ-glutamyl) lysine cross-links between myosin and soya protein, casein or gluten at 4-5°C for 16 hr and at pH's from 5.5-7.0. Casein was the best substrate with approximately 0.4g of casein cross-linked to 1.0g myosin.     

Characteristics of mince from pond-bred silvercarp (Hypophthalmichthys molitrix) and preliminary experiments on its use in sausages

The quality of fresh, chilled and frozen mince made from the flesh of pond-bred silvercarp (Hypophthalmichthys molitrix (Val)) was evaluated. Flavour panel scores for all minces were acceptable and this quality did not deteriorate during storage for one week in a domestic refrigerator (5–6°C) or for one year at ?20°C. The levels of oxidative rancidity (2-thiobarbituric acid analysis) in the minces were consistent with this maintenance of quality. Aerobic plate counts at 25°C of fresh mince rose from 7–9 × 10⁴ to 1.1 × 10⁵ after 6 days' storage at 5–6°C. Coliform and psychrotroph counts rose from 1.0 × 10¹ and 7–8 × 10³ to 5–4 × 10² and 1.3 × 10⁴, respectively. The functional and textural properties of pond-bred silvercarp mince in terms of salt-soluble protein content, water binding capacity and penetrometer values were measured on fresh, refrigerated and frozen samples. All-fish sausages and frankfurters developed from pond-bred silvercarp mince were assessed in laboratory and consumer tests against commercial beef sausages and frankfurters. The fish products competed well and achieved levels of acceptance similar to those of the beef-containing products. Texture measurements revealed slight changes in the fish products during storage for 7 days at 5–6°C. No spoilage was detected in any of the products after a week at 5–6°C. The nutritional advantages of the fish sausages and frankfurters are discussed.     

On the influence of non‐enzymatic crosslinking of caseins on the gel strength of yoghurt

After storage of UHT milk at 37°C resp. 50°C, yoghurt was prepared. For a storage temperature of 37°C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non‐reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50°C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.     

Effect of Nitrogen Flushing on the Production of Proteinase by Psychrotrophic Bacteria in Raw Milk

Raw milk was flushed with 100 ml N₂ min ^-1 during storage at 4°C. Microflora (total psychrotrophs, proteolytic psychrotrophs, lactic acid bacteria) in nitrogen-flushed milk exhibited a longer lag phase and slower growth rates than those in milk stored aerobically at 4°C. Although proteolytic psychrotrophs grew in nitrogen-flushed milk, proteinases could not be detected in these milk samples. Proteinase assays and electrophoresis showed extensive proteolytic activity and hydrolysis of β-casein in control milk but no detectable casein degradation in nitrogen-flushed milk, even after storage for 18 days at 4°C. This study shows the potential of controlled atmosphere storage of raw milk for inhibition of the accumulation of proteolytic enzymes from psychrotrophic bacteria.     

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and β-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.     

High-Resolution NMR and Magnetic Resonance Imaging (MRI) Studies on Fresh and Frozen Cod ( Gadus morhua) and Haddock (Melanogrammus aeglefinus)

Changes in the fish muscle from cod ( Gadus morhua ) and haddock ( Melanogrammus aeglefinus ) were investigated by high-resolution NMR and magnetic resonance imaging (MRI). Water- and salt-soluble extracts from fish stored at −20°C and −30°C were analysed by high-resolution proton NMR and enabled the identification of metabolites including trimethylamine oxide, trimethylamine (TMA) and dimethylamine. It was not possible to detect formaldehyde by NMR either in the stored fish samples or in spiked water or salt extracts even at high levels of formaldehyde addition, probably due to polymerisation. Systematic and controlled storage trials indicated the presence of dimethylamine at around 9 months for samples stored at −20°C, whereas no changes were detected at the control storage temperature of −30°C. A comparison of cod and haddock fillets stored for 1 year at −20 and −30°C confirmed the production of dimethylamine only in cod stored at −20°C. It was interesting to note that ‘fresh’ cod and haddock samples purchased from a local supermarket showed high levels of TMA indicating a breakdown of trimethylamine oxide to TMA by bacteria. TMA was not detected in the fish fillets especially obtained for the storage trials. MRI of fresh cod and fish stored at −8 and −30°C indicated that the fish half stored at −8°C exhibited dense lines or arches which are indicative of gaps in the tissue due to possible breakdown of the connective tissue. The images of fish stored at −30°C did not indicate any differences compared with the fresh fish. MRI also showed the presence of frozen and unfrozen areas in the fish non-destructively.