Fourier transform infrared spectroscopy analysis of physicochemical changes in UHT milk during accelerated storage

The feasibility of using Fourier transform infrared spectroscopy (FTIR) to detect heat induced conformational rearrangements of proteins, protein–protein and protein–lipid interactions was studied with accelerated shelf-life protocols. Ultra-high temperature treated whole (WM) and skim milk (SM) were stored at 20, 30, 40 and 50 °C for 28 days. The changes leading to increased sedimentation in SM and WM at higher temperatures (≥40 °C) were observed during first 14 days of the storage period. Milk samples stored at 40 and 50 °C showed marked changes in the bands corresponding to conformations of milk lipids and formation of intermolecular β sheet of proteins, indicating protein–lipid interactions and aggregation. Dried sediment contained fat confirming protein–lipid participation in the sedimentation. FTIR was also able to detect changes that led to increased sedimentation in SM at temperatures lower than 40 °C, but only after 28 days.     

Predicting sediment formation in ultra high temperature-treated whole and skim milk using attenuated total reflectance-Fourier transform infrared spectroscopy

Establishing accelerated shelf-life testing using Fourier transform infrared spectroscopy (FTIR) as a tool for prediction of stability requires pre-assessment of correlations between spectral changes and instability development during storage at room temperature. Comparison of results with those at elevated temperatures would establish appropriateness of accelerated shelf-life testing. UHT skim milk (SM) and UHT whole milk (WM) were stored at 20 °C for 9 months to investigate the feasibility of identifying spectral markers to predict sedimentation (a measure of instability). Marker variables corresponding to changes in structure and interactions of lipids, proteins and carbohydrates successfully predicted sedimentation in SM (R² = 0.92) and WM (R² = 0.60). Low predictability in WM may be due to influence of fat. These markers were similar to those observed during accelerated shelf-life testing, hence affirming its application with further work required to develop a model able to forecast sedimentation and other instabilities in UHT milk.     

Effect of green tea catechins on physical stability and sensory quality of lactose-reduced UHT milk during storage for one year

Ultra-high temperature (UHT) processed lactose-reduced milk containing added green tea extract (GTE) at two concentrations (0.1% and 0.25%) was stored at 22 ± 2 °C for one year. The effect of GTE addition on physical stability, protein binding, and sensory quality was evaluated. Sedimentation in skim milk and creaming of full fat milk were inhibited by addition of GTE. The formation of Maillard-related flavour compounds was inhibited during storage as determined by dynamic headspace GC–MS. Using Western blot analysis, milk proteins were found to be highly conjugated to polyphenols. Addition of GTE before UHT treatment resulted in increased bitterness and astringency in UHT milk and this remained during storage. Even though GTE addition improved the physical stability and inhibited Maillard reactions in the milk, the taste and flavour contribution from GTE was dominating throughout storage, and alternative sources of polyphenols should be explored for increasing shelf-life stability of long-life milk.     

Relationship of Protease Activity to Shelf-Life of Skim and Whole Milk

This study was to investigate causes of a possible difference in shelf-life of pasteurized skim milk and whole milk. Samples of skim and whole milk were obtained the day of processing, in 235 ml containers, from commercial dairies throughout South Carolina. They were stored at 4.5°C for 0,4,8,10,12,14, and 16 days; 7°C for 0,4,6,8,10, and 12 days; and 10°C for 0,1,2,3,4,5, and 6 days. On each sampling day milks were tested for coliform count, psychrotrophic count, flavor score, and relative protease activity. The shelf-life of skim milk was significantly less than that of whole milk when both were stored at 4.5°C and 7°C, but not at 10 C. Bacteria counts were not significantly different; thus, they were of no predictive value as anticipated changes in flavor score. Relationship between flavor score and relative protease activity of skim and whole milk was linear. Also, relative protease activity was significantly higher in skim milk as compared to whole milk stored at 4.5 and 7° C. Therefore, a higher protease activity in skim milk may account partially for its decreased shelf-life.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

Plasmin activity in direct-steam-injection UHT-processed reconstituted milk: Effects of preheat treatment

Ultra-high-temperature (UHT)-processed reconstituted milk that is subjected to a minimal preheat treatment during the direct-steam-injection heating process may have a shortened shelf-life as a result of plasmin-mediated proteolysis. Some manufacturers apply a preheat treatment before UHT treatment (140 °C for 4 s) with the aim of prolonging the shelf-life. Preheat treatments are, however, often arbitrary in terms of temperature and holding time. The aim of the current work was to determine guidelines for the minimum preheat treatment that will effectively inhibit or prevent plasmin-type enzyme activity in UHT milk. A selected range of preheat treatments was applied to milk preparations reconstituted from several batches of low-heat skim milk powder. Increased plasmin-type proteolysis was observed after intermediate preheat treatments at ⩾80 and <90 °C. Effective inhibition of plasmin-type proteolysis was obtained by preheating at 90 °C for 30 or 60 s.     

Investigate the efficacy of UV pretreatment on thermal inactivation of Bacillus subtilis spores in different types of milk

Shortwave ultraviolet (UVC) radiation is commonly used for sterilization of drinking water. However, its low transmittance within opaque liquids limits its use for milk and other liquid food products. The objective of this study was to assess the efficacy of UV pretreatment on thermal inactivation of B. subtilis spores in skim cow milk, whole cow milk and whole sheep milk. In this work, UV treatment was applied by using a coiled tube UV unit with a perfluoroalkoxy tube around a quartz sleeve containing a 254-nm UV lamp. It was observed that UV pretreatment (D _Act 2.37 ± 0.126 J/ml) combined with thermal treatment at 110 °C for 30 s resulted in a reduction of approximately 6 log CFU/mL in bovine skim milk, 2.90 log CFU/ml in whole bovine milk and 1.1 log CFU/ml in ovine milk. The results showed that UV in combination with heat can possibly be an alternative to sterilization of skim milk at lower temperatures compared to ultra high temperature (UHT) treatment (135 °C, 3 s).     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

Simultaneous HPLC–DAD quantification of vitamins A and E content in raw,pasteurized, and UHT cow’s milk and their changes during storage

An improved extraction and HPLC method for the simultaneous extraction and quantitation of retinol, α-tocopherol, α-tocotrienol, and β-carotene was developed to analyze commercial whole/semi-skim/skim samples of raw/pasteurized/UHT milk in transparent plastic/glass bottles and Tetra Brik? containers. The sample preparation method required prior saponification at 40 °C for 15 min followed by n-hexane extraction. An isocratic acetonitrile/methanol (65:35 v/v) mobile phase, C₁₈ analytical column, and UV detector were chosen for HPLC quantification. The liposoluble vitamin content in raw, pasteurized conventional/organic, and UHT milk ranged 0.055–5.540 (retinol), 0.135–1.410 (α-tocopherol), and 0.040–0.850 mg/L (β-carotene). No significant differences (p > 0.05) were observed on losses of retinol, α-tocopherol, and β-carotene content in UHT whole milk after 5 days at 4 °C in the dark. After 14 days at 4 °C in the dark, the contents of retinol, α-tocopherol, and β-carotene remained higher in milk with higher fat content and were higher in unopened containers. In UHT whole milk, samples containing 0.02 % NaN₃, retinol (33 %), and α-tocopherol (11 %) but not β-carotene (2 %) decreased significantly (p < 0.05).     

Forward osmosis concentration of milk: Product quality and processing considerations

Concentration of milk in the dairy industry is typically achieved by thermal evaporation or reverse osmosis (RO). Heat concentration is energy intensive and leads to cooked flavor and color changes in the final product, and RO is affected by fouling, which limits the final achievable concentration of the product. The main objective of this work was to evaluate forward osmosis (FO) as an alternative method for concentrating milk. The effects of fat content and temperature on the process were evaluated, and the physicochemical properties and sensory qualities of the final product were assessed. Commercially pasteurized skim and whole milk samples were concentrated at 4, 15, and 25°C using a benchtop FO unit. The FO process was assessed by monitoring water flux and product concentration. The color of the milk concentrates was also evaluated. A sensory panel compared the FO concentrated and thermally concentrated milks, diluted to single strength, with high temperature, short time pasteurized milk. The FO experimental runs were conducted in triplicate, and data were analyzed by single-factor ANOVA. Water flux during FO decreased with time under all processing conditions. Higher temperatures led to faster concentration and higher concentration factors for both skim and whole milk. After 5.75 h of FO processing, the concentration factors achieved for skim milk were 2.68 ± 0.08 at 25°C, 2.68 ± 0.09 at 15°C, and 2.36 ± 0.08 at 4°C. For whole milk, after 5.75 h of FO processing, concentration factors of 2.32 ± 0.12 at 25°C, 2.12 ± 0.36 at 15°C, and 1.91 ± 0.15 at 4°C were obtained. Overall, maximum concentration levels of 40.15% total solids for skim milk and 40.94% total solids for whole milk were achieved. Additionally, a triangle sensory test showed no significant differences between regular milk and FO concentrated milk diluted to single strength. This work shows that FO is a viable nonthermal processing method for concentrating milk, but some technical challenges need to be overcome to facilitate commercial utilization.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ～4 log₁₀ spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ～4 log₁₀ cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.     

Calcium-Aggregated Milk: a Potential New Option for Improving the Viability of Lactic Acid Bacteria Under Heat Stress

Heat-induced inactivation of viable cells restricts a wide range of application of spray drying in producing dried lactic acid bacteria (LAB) products. In the present study, an effective method to enhance the stability of LAB under heat stress has been identified. This was done by enhancing the heat stability of skim milk as the carrier. Here, skim milk was supplemented with 10 mM CaCl₂ and heated to 90 °C for 10 min to induce protein aggregation. Using this Ca-aggregated skim milk as carrier, the survival of five LAB strains tested was found two orders of magnitudes higher than that of an untreated milk after the heating at a rising temperature from about 25 to 70 °C within 45 s. Possible mechanisms of the protection were explored by comparing the residual viability, microstructure of the cell-contained milk, and changes of suspension particle sizes caused by heat treatment of the four carriers, i.e., untreated milk, Ca-added milk, heat-treated milk, and Ca-aggregated milk. Ca-aggregated milk induced the highest microbial heat stability among them, providing a thick and compact encapsulation around LAB cells before the heat treatment for inactivation. The viable cells could stay in a comparatively more stable extracellular environment. This work reveals potentially a new option for using milk protein aggregates as a protectant of microorganisms. A series of calcium-enriched probiotic products may be developed based on the described principles of the finding of the Ca-aggregated milk.     

Changes in structure of the bovine milk fat globule membrane on heating whole milk.

The effects of heat-induced interactions between milk fat globule membrane components and skim milk proteins in whole milk on the structure of the membrane were examined by isopycnic sucrose density gradient centrifugation and by using Triton X-100 as a membrane probe. Skim milk components were incorporated into all the lipoprotein fractions separated by density gradient centrifugation. High density complexes, higher in density than those found in the natural milk fat globule membrane, were formed during the heat treatment. Losses of natural membrane polypeptides from the medium and low density lipoproteins were observed on heating. Heating whole milk also altered the rate of release of membrane components by detergent, with decreases in protein released and an increase in phospholipid constituents released. Studies on washed cream indicated that some of the changes in the membrane on heating whole milk occurred due to the heat treatment alone, independent of the interactions with skim milk proteins.     

Age gelation in UHT-processed reconstituted concentrated skim milk

UHT reconstituted concentrated skim milks made from high-heat powder had considerably longer gelation times than those made from medium- or low-heat powders. Addition of hexametaphosphate to the concentrated milk before UHT processing markedly delayed the onset of gelation during storage. Sediment formation was greatest in the UHT concentrated skim milk made using high-heat powder followed by samples made using medium- and low-heat powders, respectively. The extent of proteolysis, as measured by 12% TCA-soluble amino groups, increased at a faster rate in the UHT milks stored at 40°C than in those stored at 22°C but decreased with increasing heat treatment of the milk prior to powder manufacture. The electrophoretic patterns of samples stored at 22°C clearly showed the breakdown of β-casein with a corresponding increase in slower moving bands, presumably γ-casein and proteose-peptone components. However, storage of samples at 40°C resulted in diffused 'blurred' protein patterns with some protein material not entering the resolving gel. At 22°C there was some evidence of proteolysis but no evidence of high molecular weight polymer formation, while at 40°C both proteolysis and high molecular weight polymer formation increased with storage time. It appeared that both physico-chemical and proteolytic processes play some part in the mechanism of gelation in UHT reconstituted concentrated skim milk.     

Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.     

Microbiological safety of UHT milk treated at 120 °C for 2 s,as estimated from the distribution of high-heat-resistant Bacillus cereus in dairy environments

Unlike common ultra-high-temperature (UHT) milk, so-called “UHT milk” in Japan is typically pasteurised at 120–130 °C for 2 s and distributed at 10 °C or less, and there is a potential risk of Bacillus cereus. To estimate the microbiological safety of UHT milk, we surveyed the distribution of high-heat-resistant B. cereus strains (defined as showing <∼3 log reduction after treatment at 120 °C for 2 s) among 200 isolates from dairy environments. Only four strains, which were isolated from the milk plant environment, showed high-heat resistance. All of them were unable to grow at 10 °C but grew at 12 °C. In contrast, heat-labile strains grew well at 10 °C. Therefore, UHT milk pasteurised at 120 °C for 2 s can be microbiologically safe, provided it is kept at 10 °C or less, within a rational shelf-life and avoiding contamination with B. cereus, especially of milk-plant-environment origin.     

Stability of Membrane-Sterilized Bovine Immunoglobulins Aseptically Added to UHT Milk

Lyophilized immunoglobulin (IgG) of 56% purity isolated from Cheddar cheese whey was reconstituted with distilled water to a protein content of 60–70 mg/mL. The solution was membrane-sterilized (0.2 μ.m) and aseptically injected into cartons of UHT milk. This method could be used for fortification of UHT products without denaturing IgG. Concentration of IgG remained constant in milk stored at 4, 25 and 35°C over 5 mo. However, when stored at 25 and 35°C, the milk thinned probably due to residual enzymes in the IgG solution. D values measured between 62 and 80°C for thermal destruction of IgG in UHT milk appeared to confirm shelf life results.     

Seasonal variations in composition,properties, and heat-induced changes in bovine milk in a seasonal calving system

We determined seasonal variations in the composition and characteristics of bovine milk, as well as heat-induced changes in the physicochemical properties of the milk, in a typical seasonal-calving New Zealand herd over 2 full milking seasons. Fat, protein, and lactose contents varied consistently during the year in patterns similar to those of the lactation cycle. Seasonality also had significant effects on milk calcium, ionic calcium, fat globule size, buffering capacity, and ethanol stability, but not on casein micelle size. The ratio of casein to total protein did not vary significantly over the season, but late-season milk had the highest content of glycosylated κ-casein (G-κ-CN) and the lowest content of α-lactalbumin in both years. We observed significant between-year effects on protein, total calcium, ionic calcium, pH, and casein:total protein ratio, which might have resulted from different somatic cell counts in the 2 years. Compared with heating at 90°C for 6 min, UHT treatment (140°C for 5 s) induced greater dissociation of κ-casein, a similar extent of whey protein denaturation, a lower extent of whey protein–casein micelle association, and a larger increase in casein micelle size. Indeed, UHT treatment might have triggered significant dissociation of G-κ-CN, resulting in aggregation among the casein micelles and increased apparent mean casein micelle diameter. Seasonality had significant effects on the partitioning of G-κ-CN between the micelle and the serum phase, the extent of whey protein–casein micelle association under both heating conditions, and the casein micelle size of the UHT milk.