High-pressure homogenisation of raw bovine milk. Effects on fat globule size distribution and microbial inactivation

Whole raw milk was processed using a 15 L h⁻¹ homogeniser with a high-pressure (HP) valve immediately followed by a cooling heat exchanger. The influence of homogenisation pressure (100–300 MPa) and milk inlet temperature T_in (4°C, 14°C or 24°C) on milk temperature T₂ at the HP valve outlet, on fat globule size distribution and on the reduction of the endogenous flora were investigated. The T_in values of 4–24°C led to milk temperatures of 14–33°C before the HP valve, mainly because of compression heating. High T_in and/or homogenisation pressure decreased the fat globule size. At 200 MPa, the d_4.3 diameter of fat globules decreased from 3.8±0.2 (control milk) to 0.80±0.08 μm, 0.65±0.10 or 0.37±0.07 μm at T_in=4, 14°C or 24°C, respectively. A second homogenisation pass at 200 MPa (T_in=4°C, 14°C or 24°C) further decreased d_4.3 diameters to about 0.2 μm and narrowed the size distribution. At all T_in tested, an homogenisation pressure of 300 MPa induced clusters of fat globules, easily dissociated with SDS, and probably formed by sharing protein constituents adsorbed at the fat globule surface. The total endogenous flora of raw milk was reduced by more than 1 log cycle, provided homogenisation pressure was ⩾200 MPa at T_in=24°C (T₂∼60°C), 250 MPa at T_in=14°C (T₂∼62°C), or 300 MPa at T_in=4°C (T₂∼65°C). At all T_in tested, a second pass through the HP valve (200 MPa) doubled the inactivation ratio of the total flora. Microbial patterns of raw milk were also affected; Gram-negative bacteria were less resistant than Gram-positive bacteria.     

High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Relationship between pungency and food components – A comparison of chemical and sensory evaluations

The influence of food ingredients (water, starch, fat, and sugar), individually and in combination, on the sensory perception of the pungency of capsaicin was studied in model food systems using the time-intensity method. Furthermore, the transferability of the results obtained using model food matrices to complex matrices, such as convenience salsas, was investigated. Significant differences between the samples (p < 0.05) were examined by analysis of variance (ANOVA) followed by the Tukey-HSD post hoc test. A highly complex matrix and a high starch content reduced the perception of pungency in both the model food matrices and the convenience salsa products. Therefore, the time-related maximum intensity of pungency (I_max) was affected by other factors in addition to the capsaicin concentration.The total capsaicinoid content in the salsas was quantified by high-performance liquid chromatography–mass spectrometry (HPLC–MS) and correlated to the results of the sensory evaluation. The best correlation (R² = 0.8029) was observed for the total capsaicinoid content and the maximum intensity of pungency (I_max) in comparison to the duration of the total pungency perception T_Dur (R² = 0.5176) and the area under the curve (R² = 0.6898). The calculated regression models serve as an example of how empirical calculations can be generated and used in a specific context.     

Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100 kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45 °C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with k_cat/K_m of 8.1 × 10⁶ M^?1 s^?1, and the activation energy (E_a) of 72.5 kJ M^?1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn²⁺, Mn²⁺ and Co²⁺ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.     

The kinetics of colour and fluorescence development in concentrated milk systems

The kinetics of colour and fluorescence development were studied in aqueous systems containing whole milk powder (WMP) and sucrose (S) at three WMP/S ratios (R=0.30, 0.68, 1.06) and three temperatures (T=105, 122.5, 140 °C). At each testing time, the colour parameters Kubelka-Munk index (K/S), lightness (L), optical density (OD), absorption coefficient (K), dominant wavelength (DWL) and fluorescence (F) were determined. F and 100/L were the most appropriate parameters to study non-enzymatic browning in concentrated milk systems. Adequate models (r²>0.96) developed to analyse the effect of T and R on the rate constants, showed that colour parameters and their kinetic coefficients were mainly affected by temperature. The influence of R was more relevant at high temperatures. The activation energies and Q₁₀ values obtained for K/S, 100/L, F and OD were in the range 81–159 kJ mol⁻¹ and 1.8–3.8, respectively. F and reflectance properties showed different dependence on temperature and R, reflecting that although related, they do not have the same origin and significance.     

The influence of lactoperoxidase,heat and low pH on survival of acid-adapted and non-adapted Escherichia coli O157:H7 in goat milk

In hot climates where quality of milk is difficult to control, a lactoperoxidase (LP) system can be applied in combination with conventional preservation treatments at sub-lethal levels to inhibit pathogenic microbes. This study investigated the effect of combined heat treatments (55 °C, 60 °C and 72 °C) and milk acidification (pH 5.0) on survival of acid-adapted and non-adapted Escherichia coli O157:H7 strains UP10 and 1062 in activated LP goat milk. Heat treatment at 72 °C eliminated E. coli O157:H7. Acid-adapted strains UP10 and 1062 cells showed resistance to combined LP and heat at 60 °C in fresh milk. The inhibition of acid-adapted and non-adapted E. coli O157:H7 in milk following combined LP-activation, heat (60 °C) and milk acidification (pH 5.0) suggests that these treatments can be applied to reduce E. coli O157:H7 cells in milk when they occur at low numbers (<5 log₁₀ cfu mL^?1) but does not eliminate E. coli O157:H7 to produce a safe product.     

Stickiness of skim milk powder using the particle gun technique

The particle gun method for investigating the initiation of stickiness of dairy powders was examined using skim milk powder (SMP). The point at which significant stickiness occurred was at a constant temperature above the glass transition temperature (T_g) of amorphous lactose regardless of the temperature and relative humidity (RH) conditions of the air in the particle gun. This point has been called (T–T_g)_critical. The initial water activity of SMP did not significantly affect (T–T_g)_critical, confirming that stickiness is a surface phenomenon. Changing the particle feed rate did not effect (T–T_g)_critical or the rate at which the powder accumulated on the target plate. The errors in measuring the value of (T–T_g)_critical and the rate of stickiness development [slope of the deposition line versus (T–T_g)] for SMP were found to be 33.6 ± 0.8 °C and 3.1 ± 0.7% deposition °C^?1, respectively, after standardisation of the ambient air conditions around the powder feeder and the initial water activity of the SMP.     

Dual-enzymatic synthesis of lactulose in organic-aqueous two-phase media

Lactulose has been successfully synthesized by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials. Cyclohexane–buffer system C₆H₁₂:buffer = 95:5 (v/v) was employed as the organic-aqueous media for the reaction. The dual-enzymatic system was consisted of immobilized lactase (IL) and immobilized glucose isomerase (IGI). Immobilized lactase was prepared by cross-linking the free lactase into Fe₃O₄-chitosan magnetic microspheres. The main enzymatic reaction parameters were investigated, including reaction temperature (T), pH value and reaction time (t). Under the optimum reaction conditions, i.e., lactose 0.8 g mL^?1, fructose 0.1 g mL^?1, IL 0.1 g mL^?1, IGI 0.05 g mL^?1, T = 30 °C, pH = 8.0 and t = 2 h, the obtained highest lactulose yield was approximately 151 g L^?1 and the corresponding productivity was 75.5 g L^?1 h^?1. Experimental results indicated that the organic-aqueous media can significantly promoted the transglycosidation activity of lactase and therefore improve the lactulose yield. The possible reaction mechanism of the synthesis of lactulose using IL and IGI in two-phase system was also proposed.     

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R² = 0.95).     

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) with high activity in the hydrolysis of long chain peptide substrates: Purification and biochemical characterisation

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55 °C, respectively, using succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50 °C after 1 h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl-l-phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or N_α-tosyl-l-lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with k_cat 18 s^?1 and K_m 22 μM. However, the enzyme had a lower K_m for SAAPLpNA, 54 μM.The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV.     

Glycine max (L.) Merr., Vigna radiata L. and Medicago sativa L. sprouts: A natural source of bioactive compounds

The consumption of sprouts, common in Asia, has been growing in western countries, once they are a natural healthy food and considered as a valuable dietary supplement. Comparing with their mature counterparts, sprouts are usually richer in health-promoting phytochemicals. So, the nutritional composition and the biological potential of widely consumed sprouts of three species – Glycine max (L.) Merr., Vigna radiata L. and Medicago sativa L. – were compared for the first time. Phenolic compounds and phytosterols were analyzed by HPLC–DAD and organic acids by HPLC–UV. The volatile profile was determined by HS-SPME/GC–IT/MS. Fourteen phenolic compounds (including four isoflavones), three sterols one triterpene, sixteen fatty acids, seven organic acids and thirty volatile compounds were determined. The antioxidant activity was assessed against DPPH^?, superoxide and nitric oxide radicals. G. max sprouts were the most active against DPPH^? (IC₅₀ = 1.337 mg/mL), while those of M. sativa were the most effective against superoxide and nitric oxide radicals (IC₅₀ = 67 μg/mL and IC₅₀ = 426 μg/mL, respectively). Data provide evidence of great similarities between G. max and M. sativa sprouts, both being rich in phenolic compounds, fatty acids and volatiles, and exhibiting better antioxidant activity. On the other hand, V. radiata showed higher amounts of sterols, triterpenes and organic acids. In this study it was found that the sprouts are a good source of bioactive compounds in our diet with health-promoting antioxidants.     

Physicochemical properties of waxy and normal corn starches treated in different anhydrous alcohols with hydrochloric acid

Corn starches (25 g, d. b.) were treated in anhydrous methanol, ethanol, 2-propanol or 1-butanol (100 ml) with 1 ml 36% hydrochloric acid at 45 °C for 1 h, the molecular weight and chain length distributions of starch were examined by high-performance size-exclusion chromatography (HPSEC), and the granule size, granular structure, λ_max, blue value, solubility and gelatinization thermal properties of starch were also examined. Results showed the recovery yields of the treated starch were higher than 96%, and the granule sizes of treated starches were slightly lower than their counterpart native starch. Starches after acid-alcohol treated showed internal fissures or cavities in some granules, and the number of granule with fissures or cavities increased with the increasing carbon number of alcohol. The weight average degree of polymerization and relative content of F1 fraction of starch after treated profoundly decreased, and the amylose and long chain of amylopectin of starch were preferentially degraded. The degradation extent of molecules after acid-alcohol treatment was found directly related to the alternation of internal structure of starch granule. The λ_max and blue value of both waxy and normal corn starches after treated also obviously decreased with the increasing carbon number of alcohol, while the solubility of starch profoundly increased after treated. The gelatinization onset temperature (T_o) of acid-alcohol treated waxy corn starch decreased with the increasing carbon number of alcohol, but the treated normal corn starches showed similar T_o values. Despite the alcohol used, the solubility of treated waxy corn starch linearly correlated (r²=0.983) with T/T_o value (T was the measuring temperature used for solubility determination). Whereas, normal corn starch treated in different alcohols showed distinct relations between T/T_o and solubility.     

Microbiological and chemical changes during the manufacture of Kefir made from cows’ milk,using a commercial starter culture

The counts of total aerobic mesophilic bacteria, lactic acid bacteria (in three different culture media, M17 agar, MSE agar, and Rogosa agar) and yeasts, and some biochemical parameters (levels of lactose, glucose, galactose, L(+)- and D(−)-lactic acids, ethanol, titratable acidity and pH) were determined during 196 h of fermentation in five batches of Kefir made from cows’ milk using a commercial starter culture. Lactococcus spp. predominated during the first 48 h of fermentation (∼8 log₁₀ cfu g⁻¹); Lactobacillus spp. became the predominant species after 48 h (∼8.5 log₁₀ cfu g⁻¹). During the first 24 h of fermentation, the lactose content decreased from a mean value of 4.92% (w/w) to 4.02% (w/w); the concentration of L(+)-lactic acid increased from 0.01% to 0.76% (w/w) and the pH decreased to 4.24 over the same period. After 24 h of fermentation, the changes in the levels of lactose and L(+)-lactic acid, and in pH, occurred more slowly. Neither glucose nor galactose were detected during fermentation. The production of ethanol was limited, reaching a mean final value of 0.018% (w/w).     

Effect of tomato by-products in the diet of Comisana sheep on composition and conjugated linoleic acid content of milk fat

To evaluate the effect of supplementing the diet of Comisana sheep with by-products from industrial tomato manufacture on the fat composition and conjugated linoleic acid (CLA) content of milk fat, two groups of 50 ewes each were fed either total mixed ration standard (TMRS) or total mixed ration with added tomato by-products (TMRA). Milk fat composition was determined by high-resolution gas chromatography (HRGC). The milk fat content for the animals fed the TMRA diet increased by 6.41% (P < 0.01) after six weeks, compared with the animals fed the TMRS diet. The CLA content in the milk fat for the group of animals fed the TMRA diet was 19.8% (P < 0.05) higher than for those fed the TMRS diet, and reached 1.33 g 100 g^?1 fat, while the polyunsaturated fatty acid (PUFA) content increased by a 6.43% (P < 0.05) and reached 7.12 g 100 g^?1 fat. The fatty acid composition showed an increase in the amount of polyunsaturated fatty acids. The n ? 3:n ? 6 ratio increased by 13% in the milk from sheep fed with the TMRA diet. These observations were confirmed by triglyceride analysis, which showed a decrease in the amount of short-chain (C₂₈–C₃₂) and medium-chain (C₃₄–C₄₂) triglycerides after six weeks, while the opposite was observed for the long-chain triglycerides (C₄₄–C₅₄).     

Enrichment of the conjugated linoleic acid content of bovine milk fat by dry fractionation

This study investigated the effect of fat fractionation on the conjugated linoleic acid (cis-9, trans-11-C_18 : 2) content of bovine milk fat. Anhydrous milk fat was fractionated into hard and soft fractions using controlled cooling and agitation. Fractionation of milk fat pre-melted at 60°C using a temperature programme of 33–10°C and a cooling rate of 0.58°C h⁻¹ yielded a soft fraction containing 63.2% more conjugated linoleic acid (2.22 g 100 g⁻¹ FAME), which was also enriched in polyunsaturated fatty acids and vaccenic acid (trans-11-C_18 : 1) compared with the parent fat. Agitation following fractionation was found to have a negative effect on the conjugated linoleic acid content of the soft fraction. Refractionation of the soft fraction did not increase the yield of conjugated linoleic acid. The conjugated linoleic acid and trans fatty acid content of 26 selected food products ranging in milk fat content from 0 to 100% is reported. Conjugated linoleic acid concentrations ranged from 0 to 16.2 mg g⁻¹ fat and were generally lower than the trans fatty acid content which ranged from 0 to 155.7 mg g⁻¹ fat. Spreads containing vegetable oils contained higher trans fatty acid and lower conjugated linoleic acid contents than milk fat-containing products. This study highlights that a milk fat fraction enriched in conjugated linoleic acid may be achieved by dry fractionation.     

Effect of chemical phosphorylation on solubility of buffalo milk proteins

Buffalo milk proteins (casein, co-precipitate or whey protein concentrate) were phosphorylated with phosphorus oxychloride (POCl₃) at three different pH values (5.0, 7.0 and 9.0). The solubilities of phosphorylated milk proteins were examined over the pH range 3.0–9.0 in water and ionic (0.1 m NaCl or 10–70 mm Ca²⁺) systems. The solubilities of buffalo milk proteins decreased at pH 3.0, while there was an increase in the solubilities of casein and co-precipitate near their isoelectric points upon phosphorylation. Solubilities of these phosphorylated milk proteins were pH dependent in 0.1 m NaCl but there was a decrease in their solubilities with increase in calcium ion concentration. This alteration could be due to the shifting of isoionic points of phosphorylated buffalo milk proteins towards acidic pH.     

Effect of thermosonication,pulsed electric field and their combination on inactivation of Listeria innocua in milk

The impact of thermosonication (TS) and pulsed electric field (PEF), individually and combined, on the survival of Listeria innocua 11288 (NCTC) in milk was investigated. TS (400 W, 160 s) without pre-heating reduced L. innocua by 1.2 log₁₀ cfu mL^?1, while shorter treatment times produced negligible inactivation, suggesting TS to be a hurdle rather than an effective standalone treatment. PEF (30 and 40 kV cm^?1, 50 μs) at 10 °C caused a reduction of L. innocua of 1.1 and 3.3 log cycles, respectively. The highest field strength (40 kV cm^?1) combined with TS (80 s) led to 6.8 log₁₀ cfu mL^?1 inactivation. Milk pre-heated to 55 °C (over 60 s) prior to TS followed by PEF (30 and 40 kV cm^?1) showed inactivation between 4.5 and 6.9 log₁₀ cfu mL^?1, the latter being comparable (P > 0.05) with thermal pasteurisation. The data indicate that TS followed by PEF represents a valid alternative for L. innocua inactivation in milk.     

Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening

The effect of proteolytic enzymes from somatic cells on cheese quality was studied. In preliminary experiments, milk and two sodium caseinate systems (pH 6.5 and pH 5.2, the latter in the presence of 5% NaCl) were used as substrates to investigate the proteolytic activity of somatic cells recovered from mastitic milk. Urea-polyacrylamide gel electrophoretograms of hydrolysates suggested that somatic cell extracts contributed directly to proteolysis both in buffer and in milk, but that such activity was reduced by batch pasteurisation (63 °C for 30 min). Sodium caseinate was readily hydrolysed by somatic cell extracts; hydrolysis of α_s1-casein was greater at pH 5.2 and increased with level of somatic cells, suggesting that somatic cells contain proteolytic enzymes which are more active at acidic pH values. Subsequently, miniature Cheddar-type cheeses were made from batches of milk to which somatic cells were added (at levels of levels of 3×10⁵ or 6×10⁵ cells mL⁻¹), either before or after pasteurisation. Proteolysis during ripening of cheese (as measured by levels of pH 4.6-soluble nitrogen) increased with somatic cell addition, although this effect was reduced by pasteurisation after cell addition. Somatic cells may also have directly influenced cheese moisture content, which has been established as a principal indicator of quality of Cheddar-type cheese. Proteolytic enzymes of somatic cells from milk were shown to contribute directly to proteolysis in milk and cheese.     

Lipase-catalyzed synthesis and characterization of flaxseed oil-based structured lipids

The biosynthesis of structured lipids (SLs) was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC) in an organic solvent medium (OSM), using selected commercial lipases, including Amano DF, Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM. The fatty acyl chains of the synthesized triacylglycerols (TAGs) were identified by atmospheric pressure chemical ionization/mass spectrometric (APCI/MS) analysis, while the fatty acid positional distribution of the MLM- and MML-SLs (M-medium and L-long chain fatty acids) was determined by silver-ion high-performance liquid chromatographic (Ag⁺/HPLC) analysis. The effects of reaction temperature (T_r, 30–50 °C), enzyme concentration (E_c, 0.5–4%, w/v), initial water activity (a_w, 0.05–0.43) and reaction time (R_t, 0–72 h) on the efficiency of the enzymes, were studied. The bioconversion yield (%) of the synthesized MLM- and MML-SLs was monitored under the established reaction parameters for each lipase. The maximum yield of MLM-SLs was obtained in the order, of Novozym 435 > Lipozyme TL-IM > Lipozyme RM-IM > Amano DF. Moreover, considering the ratio of the MLM- to MML-SLs produced by each enzyme, Novozym 435 and Lipozyme TL-IM were selected as the most effective enzymes for interesterification of FO and TC.