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.     

Survival analysis,consumer perception and physico-chemical analysis of low fat UHT milk stored for different time periods

Survival analysis based on consumers' acceptance or rejection of milk of different storage ages, was used to validate the shelf-life of low fat ultra-high temperature treated (UHT) milk in high density polyethylene bottles, as previously determined by a multivariate accelerated shelf-life test (MASLT). UHT milk between 120 and 290 d of storage were evaluated. Based on 50% of consumers rejecting the product, the shelf-life was estimated to be 214 d, validating the shelf-life of 211 d estimated by the MASLT. In addition, consumers completed check-all-that-apply attribute questions and rated the acceptability of the milk. The consumers noted positive sensory attributes more frequently in fresher milk samples with an increase in negative attributes with storage. Along with this, hedonic scores for the milk decreased and physicochemical and enzymatic reactions associated with the deterioration of UHT milk increased with storage.     

Growth of Pseudomonas weihenstephanensis,Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL⁻¹). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.     

应用Weibull Hazard Analysis方法预测食品货架寿命

本实验以市售灭菌乳为研究对象,应用Weibull模型通过对食品非感官指标的分析,预测食品的货架期。灭菌乳分别在25、30、35、40℃环境下恒温储藏,并定期随机抽样进行感官检验和测定酸度、细菌总数。应用Weibull Hazard Analysis(WHA)方法分析非感官实验数据来预测样品货架期,并与采用寿命加速方法ASLT和文献WHA方法得到的预测结果进行了比较。     

Electrophoretic characterization of protein interactions suggesting limited feasibility of accelerated shelf-life testing of ultra-high temperature milk

Accelerated shelf-life testing is applied to a variety of products to estimate keeping quality over a short period of time. The industry has not been successful in applying this approach to ultra-high temperature (UHT) milk because of chemical and physical changes in the milk proteins that take place during processing and storage. We investigated these protein changes, applying accelerated shelf-life principles to UHT milk samples with different fat levels and using native- and sodium dodecyl sulfate-PAGE. Samples of UHT skim and whole milk were stored at 20, 30, 40, and 50°C for 28 d. Irrespective of fat content, UHT treatment had a similar effect on the electrophoretic patterns of milk proteins. At the start of testing, proteins were bonded mainly through disulfide and noncovalent interactions. However, storage at and above 30°C enhanced protein aggregation via covalent interactions. The extent of aggregation appeared to be influenced by fat content; whole milk contained more fat than skim milk, implying aggregation via melted or oxidized fat, or both. Based on reduction in loss in absolute quantity of individual proteins, covalent crosslinking in whole milk was facilitated mainly by products of lipid oxidation and increased access to caseins for crosslinking reactions. Maillard and dehydroalanine products were the main contributors involved in protein changes in skim milk. Protein crosslinking appeared to follow a different pathway at higher temperatures (≥40°C) than at lower temperatures, making it very difficult to extrapolate these changes to protein interactions at lower temperatures.     

Evaluation of pulsed electric field and minimal heat treatments for inactivation of pseudomonads and enhancement of milk shelf-life

Pulsed Electric Field (PEF) treatment of milk provides the opportunity to increase the shelf-life of fresh milk for distribution to distant markets. PEF treatments were evaluated in sterile (UHT) milk to determine the inactivation of added spoilage Pseudomonas isolates and the subsequent gains in microbial shelf-life (time taken to reach 10⁷ CFU mL^− 1). Little inactivation of Pseudomonas was achieved at 15 or 40 °C compared with 50 or 55 °C. The greatest inactivation (> 5 logs) was achieved by processing at 55 °C with 31 kV cm^− 1 (139.4 kJ L^− 1). Heat treatment at the application temperature without PEF treatment caused minimal inactivation of Pseudomonas (only 0.2 logs), demonstrating that the inactivation of the Pseudomonas was due to the PEF treatment rather than the heat applied to the milk. At added Pseudomonas levels of 10³ and 10⁵ CFU mL^− 1, the microbial shelf-life of PEF-treated milk was extended by at least 8 days at 4 °C compared with untreated milk. The total microbial shelf-life of the PEF-treated milk was 13 and 11 days for inoculation levels of 10³ and 10⁵ CFU mL^− 1 respectively. The results indicate that PEF treatment is useful for the reduction of pseudomonads, the major spoilage bacteria of milk.Industrial relevancePseudomonads are the major psychrotrophic spoilage microflora of refrigerated, stored HTST pasteurised milk. Long-life (UHT) products are an important component of milk sales in South-East Asia, but in recent years there has been an increasing demand for less processed milk products with extended shelf-life. The recent practice of shipping fresh bulk milk from Australia to South-East Asian countries has necessitated additional heat treatment prior to export and on arrival, to achieve the required shelf-life. Pulsed electric field treatment of HTST milk, applied alone or in combination with mild heat under optimised conditions, offers the opportunity of shelf-life extension, while limiting the reduction in quality attributes of milk associated with more severe additional heat treatments.     

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.     

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.     

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.     

Proteolysis and storage stability of UHT milk produced in Turkey

The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 °C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 °C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 °C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 °C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 °C showed quite small number of peaks after 180 d of storage. Sterilization at 150 °C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness.     

Effect of feed supplementation based on extruded linseed meal and fish oil on composition and sensory properties of raw milk and ultra-high temperature treated milk

The objectives of this study were to test the influence of a recently developed rumen protected feed supplement containing extruded linseed meal and fish oil (LFO), which was fed to lactating Holstein-Friesian cows for 10 weeks at the rate of 800 g day⁻¹ per animal, on the chemical and fatty acid (FA) composition of raw and ultra-high temperature (UHT) treated milks and to evaluate changes in sensory properties of UHT milk by both instrumental analysis and a panel of human assessors. Inclusion of LFO in the diet did not affect milk yield or the protein and fat contents of raw and UHT milks; however, it improved the FA composition of the milk fat by increasing the concentrations of health-enhancing polyunsaturated FA and beneficially decreasing the n-6/n-3 FA ratio without adversely affecting the sensory properties of the final product (UHT milk).     

Short communication: Suitability of fluorescence spectroscopy for characterization of commercial milk of different composition and origin

Thirteen milk brands comprising 76 pasteurized and UHT milk samples of various compositions (whole, reduced fat, skimmed, low lactose, and high protein) were obtained from local supermarkets, and milk samples manufactured in various countries were discriminated using front-face fluorescence spectroscopy (FFFS) coupled with chemometric tools. The emission spectra of Maillard reaction products and riboflavin (MRP/RF; 400 to 600 nm) and tryptophan (300 to 400 nm) were recorded using FFFS, and the excitation wavelengths were set at 360 nm for MRP/RF and 290 nm for tryptophan. Principal component analysis (PCA) was applied to analyze the normalized spectra. The PCA of spectral information from MRP/RF discriminated the milk samples originating in different countries, and PCA of spectral information from tryptophan discriminated the samples according to composition. The fluorescence spectral data were compared with liquid chromatography-mass spectrometry results for the glycation extent of the milk samples, and a positive association (R² = 0.84) was found between the degree of glycation of α-lactalbumin and the MRP/RF spectral data. This study demonstrates the ability and sensitivity of FFFS to rapidly discriminate and classify commercial milk with various compositions and processing conditions.     

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.     

Correlation between sensory and chemical markers in the evaluation of Brazil nut oxidative shelf-life

Sensory analysis is one of the most suitable processes for measuring oxidative damage and determining the shelf-life of nuts, but it is an expensive and time-consuming methodology. Thus, our objective was to correlate sensory data and chemical markers obtained during the accelerated oxidation of Brazil nuts and to determine the chemical parameters values associated with the sensory shelf-life of the nuts as established by the consumers. Brazil nuts were kept at 80 °C for 21 days. At intervals of 2 days, the oxidized odor of the samples was analyzed by nine trained panelists using a discriminative scale, and the oil was extracted to quantify the chemical parameters. A high (r > 0.95) and significant correlation (p < 0.05) was observed between the sensory data and the hydroperoxide concentration (PV), para-anisidine value (pAV), hexanal content, and α- and γ-tocopherol concentrations. When compared with fresh samples, sensory identification of oxidized odor occurred on the 4th day, noticeably earlier than changes in chemical markers (12th day). Consumers rejected the nuts after 12 days of storage, which corresponded to PV = 18.8 meq kg⁻¹ oil, pAV = 7.68, hexanal = 48.95 μmol 100 g⁻¹ oil, α-tocopherol = 15.01 mg kg⁻¹ oil, and γ + β-tocopherol = 73.88 mg kg⁻¹ oil. Our study suggests that simple spectrometric methods, such as PV and pAV, can be used to estimate the oxidative shelf-life of nuts based on sensory analysis.     

Fluorometric determination of chemically available lysine: adaptation, validation and application to different milk products

A spectrophotometric method based on the reaction between available lysine and ortho-phthaldialdehyde (OPA) was adapted and validated for fluorometric determination of the chemically available lysine contents in milk matrices (UHT and conventional in-bottle sterilized cow milk, milk-based infant formulas and infant formula ingredients). The values of the analytical parameters show its usefulness as a routine method (linearity, r = 0.9992; detection limit, 0.0066 mg/mL assay; accuracy, 99-108%; precision, intra-day 2.1-5.9% and inter-day 3.5 10.2%). No statistically significant differences (p < 0.05) were found between the values obtained with the adapted method and those obtained applying the 1-fluoro-2,4-dinitrobenzene (FDNB) (Carpenter) technique. The OPA method was used to measure the chemically available lysine contents in UHT and sterilized milk marketed in Spain, to study the evolution of chemically available lysine during the shelf-life of UHT milks, and finally the quality of name- and store-brand UHT milks was also compared. No statistically significant differences (p < 0.05) were found between either the available lysine contents of the same type of UHT or sterilized milk or between store- and name-brand UHT milks. Statistically significant differences (p < 0.05) were found between the chemically available lysine contents in UHT and sterilized milk. Losses of chemically available lysine ranging from 2.7 to 29% were obtained during the shelf-life of UHT milk.     

Protein oxidative changes in whole and skim milk after ultraviolet or fluorescent light exposure

We investigated how protein changes occur, at the primary or higher structural levels, when proteins are exposed to UV or fluorescent (FL) light while in the complex matrix, milk. Whole milk (WM) or skim milk (SM) samples were exposed to FL or UV light from 0 to 24 h at 4°C. Protein oxidation was evaluated by the formation of protein carbonyls (PC), dityrosine bond (DiTyr), and changes in molecular weight (protein fragmentation and polymerization). Oxidative changes in AA residues were measured by PC. Dityrosine and N′-formylkynurenine (NFK), a carbonylation derivative of Trp, were measured by fluorometry. Protein carbonyls increased as a function of irradiation time for both WM and SM. The initial rate for PC formation by exposure to FL light (0.25 or 0.27 nmol/h for WM and SM, respectively) was slower than that following exposure to UV light (1.95 or 1.20 nmol/h, respectively). The time course of NFK formation resembled that of PC. After 24 h of UV exposure, SM had significantly higher levels of NFK than did WM. In contrast, WM samples irradiated with UV had higher levels of DiTyr than did SM samples, indicating different molecular pathways. The formation of intra- or intermolecular DiTyr bonds could be indicative of changes in the tertiary structure or oligomerization of proteins. The existence of NFK suggests the occurrence of protein fragmentation. Thus, proteolysis and oligomerization were analyzed by sodium dodecyl sulfate-PAGE. After 24 h of exposing WM to UV or FL light, all the proteins were affected by both types of light, as evidenced by loss of material in most of the bands. Aggregates were produced only by UV irradiation. Hydrolysis by pepsin and enzyme-induced coagulation by rennet were performed to evaluate altered biological properties of the oxidized proteins. No effect on pepsin digestion or rennet coagulation was found in irradiated SM or WM. The oxidative status of proteins in milk and dairy products is of interest to the dairy industry and consumers. These findings provide knowledge that could be useful in determining the optimal lighting conditions in the dairy industry in general and in cheese making in particular.     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

Age Gelation,Sedimentation, and Creaming in UHT Milk: A Review

Demand for ultra‐high‐temperature (UHT) milk and milk protein‐based beverages is growing. UHT milk is microbiologically stable. However, on storage, a number of chemical and physical changes occur and these can reduce the quality of the milk. These changes can be sufficiently undesirable so as to limit acceptance or shelf life of the milk. The most severe changes in UHT milk during storage are age gelation, with an irreversible three‐dimensional protein network forming throughout, excessive sedimentation with a compact layer of protein‐enriched material forming rapidly at the bottom of the pack, and creaming with excessive fat accumulating at the top. For age gelation, it is known that at least two mechanisms can lead to gelation during storage. One mechanism involves proteolytic degradation of the proteins through heat‐stable indigenous or exogenous enzymes, destabilizing milk and ultimately forming a gel. The other mechanism is referred to as a physico‐chemical mechanism. Several factors are known to affect the physico‐chemical age gelation, such as milk/protein concentration, heat load during processing (direct compared with indirect UHT processes), and milk composition. Similar factors to age gelation are known to affect sedimentation. There are relatively few studies on the creaming of UHT milk during storage, suggesting that this defect is less common or less detrimental compared with gelation and sedimentation. This review focuses on the current state of knowledge of age gelation, sedimentation, and creaming of UHT milks during storage, providing a critical evaluation of the available literature and, based on this, mechanisms for age gelation and sedimentation are proposed.     

Occurrence of aflatoxin M1 in raw, pasteurized and UHT milk commercialized in Esfahan and Shahr-e Kord, Iran

Between September 2006 and September 2007, 236 samples of raw (n = 140), pasteurized (n = 48) and UHT (n = 48) milk were collected from supermarkets and from bulk milk tanks of eight dairy plants in the cities of Esfahan and Shahr-e Kord, Iran. All samples were analyzed for aflatoxin M₁ (AFM₁) contamination by ELISA and 213 (90.3%) were positive with mean concentrations 65 ng.l⁻¹. These concentrations are lower than the standards of Codex Alimentarius and FDA (500 ng.l⁻¹), but 119 samples (55.9%) had higher concentrations than the maximum tolerance accepted by some European countries (50 ng.l⁻¹). Mean concentrations of AFM₁ in raw, pasteurized and UHT milk were 68, 56, and 65 ng.l⁻¹, respectively. Mean concentrations of AFM₁ in autumn and winter samples were significantly higher (P < 0.05) than those of spring and summer but differences between AFM₁ concentrations of spring and summer samples were not significantly different. Concentrations of AFM₁ in milk from Shahr-e Kord were significantly lower (P ≤ 0.05) than those from Esfahan.     

Proteolysis of casein micelles by heat-stable protease secreted by Serratia liquefaciens leads to the destabilisation of UHT milk during its storage

Serratia liquefaciens is a psychrotrophic species, frequently found in raw milk, which secretes Ser2, a heat-resistant protease. Involvement of this species in UHT milk destabilisation was investigated in the present study. Microfiltered milk was inoculated independently with strains S. liquefaciens L53 or L64. Then, UHT treatment was performed and stability of the corresponding UHT milk was investigated during three months of storage. The residual proteolytic activity of strain L53 led to destabilisation of UHT milk, with sedimentation and formation of aggregates. Hydrolysis of casein micelles was confirmed by the increase in the content of non-casein nitrogen and the identification of numerous peptides coming from the four caseins using mass spectrometry. For strain L64, no visual and biochemical alteration were found. This study showed that Ser2 resists UHT treatment and could be a cause of UHT milk destabilisation; however, this destabilisation by S. liquefaciens was strain-dependent.