Flavor, Enzymatic and Microbiological Profiles of Pressure-Assisted Thermal Processed (PATP) Milk
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Francisco Parada-Rabell
Graduate Program in Food Science and Technology
The Ohio State University
2009
Dissertation Committee:
Valente B. Alvarez, Advisor
David B. Min
Ahmed E. Yousef
V. M. Balasubramaniam
Copyright by
Francisco Parada-Rabell
2009
Abstract
The main objective of this study was to evaluate the application of high pressure processing (HPP) and pressure-assisted thermal processing (PATP) as an alternative technology to process high quality fluid milk. The specific objectives of this work were to compare the microbial load, chemical stability and flavor profiles of HPP and PATP milk to that of HTST pasteurized and ultra high temperature (UHT) processed milk. Milk
(2% milkfat) was subjected to combined pressure-heat treatment using a factorial 3x1x3 model at temperature (32, 72 and 105 oC), pressure (650 MPa), and time (0, 1, and 5 min).
Milk samples were processed within 72 hr, packed in light – protected polyethylene teraphtalane (PET) bottles without head space, and stored at either room temperature
(25±1 oC) or refrigeration (4±1 oC) conditions depending on the treatment applied. The shelf life of pressure treated milk samples was examined over a period of 20, 45 and 90 d.
Additionally, pasteurized (78±0.8 oC for 18 sec) and UHT processed (138±1 oC for 2 sec) milk samples obtained from commercial source were analyzed along with pressure treated milk. The quality of milk samples was analyzed by the following tests: (1)
Microbiological tests: Total plate count (TPC) and spore-forming bacteria survival analyses ( Bacillus stearothermophilus and B. amyloliquefaciens) . (2) Residual Plasmin activity in milk samples was measured using a BODIPY FL-Casein
ii spectrofluorometric assay. (3) The chemical stability of milk samples was assessed by measuring the extent of proteolysis and lipolysis. Proteolysis was evaluated by SDS-
PAGE analysis and lipolysis was measured by a modified copper soap method. (4) The flavor profile of milk samples was evaluated using selected ion flow tube mass spectrometry (SIFT-MS) to identify and quantify volatile organics compounds in milk on real time basis. Preliminary data showed that the application of pressure treatment is capable of rendering milk with longer shelf life than HTST pasteurized and UHT milk.
6 Up to 6-log reduction was obtained when a suspension (N 0 = 7x10 spore/mL) of B. stearothermophilus was inoculated in UHT milk treated at 700 MPa for 3 min at 105 oC.
7 Similarly, a 7-log reduction was achieved when a suspension (N 0 = 1x10 spore/mL) of
B. amyloliquefaciens was inoculated in UHT milk treated at the conditions described above. Pressure treatments at room temperature (32±1 oC) delivered milk with similar microbial load than pasteurized milk. The microbial population of milk further decreased
o 4 with increasing temperature (72 and 105 C). Up to 4 log reduction (N 0 = 2x10 CFU/mL) was obtained in PATP milk samples processed at 650 MPa and 105 oC for 0, 1 and 5 min, respectively. Storage temperature had an effect on the microbial growth rate in PATP milk. Microorganisms recovered in pressure treated milk processed at 650 MPa and 72 oC for 0, 1 and 5 min stored at refrigeration conditions showed slower growth rates than pressure-treated samples stored at room temperature conditions. No additional efforts were made to characterize the microorganisms recovered during storage. No significant proteolysis was observed in pressure-treated samples at the end of their shelf life. Plasmin inactivation rates increased with increasing temperature. Although the enzyme was not
iii completely inactivated, combinations of mild temperature with ultra high pressure were sufficient to inactivate the enzyme to levels similar to UHT processes. However, lipolysis was enhanced by pressure, holding time during pressurization and storage temperature.
The formation of volatile aroma compounds in HPP and PATP milk was different than in milk processed with traditional heat treatments. Pressure treatments enhanced only the formation of straight-chain aldehydes and sulfur compounds; whereas higher concentrations of methyl ketones and straight-chain aldehydes were observed in pasteurized and UHT milk samples. These results indicate that pressure treatment is capable of rendering milk with quality characteristics close to pasteurized and UHT milk.
However, processing temperature and storage conditions have a significant role on the quality of milk.
iv Dedication
Dedicated to Toñe
Thank you for your unconditional love and always believe in me.
v Acknowledgements
a) The author particularly acknowledges the support given by CONACyT – México for scholarship # 196181. b) I would like to show my most sincere gratitude to my advisor Dr. Valente B. Alvarez for his unconditional support during my graduate studies. Thank you for sharing with me your advice, your friendship and the work ethics that changed my life completely. c) I would like to particularly thank my committee members Dr. David Min, Dr. Ahmed Yousef and Dr. V. M. Balasubramaniam. This thesis would not have been possible unless your valuable suggestions and guidance. d) I would also like to thank Dr. Luis Rodriguez-Saona, Dr. James Harper and Dr. Sheryl Barringer for their always available and helpful advice. e) I owe my deepest gratitude to my parents (Dr. Francisco Parada Aguirre and Dr. Ana T. Rabell Urbiola) and my siblings (Rodolfo, Andres, Ana Giselle and Pablo Alonso) for their unconditional love and guide me to make these lines possible today. f) It is an honor for me to particularly acknowledge Bob, Paula and Meghan Quinn. Thank you for your selfless support. g) I am indebted to Mr. Gary Wenneker. Most of the research data presented in this work would not have been possible without your help. h) Lastly, I would like to kindly acknowledge the assistance provided by Dr. Balasubramaniam’s PATP laboratory staff with the processing of pressure treated milk samples
vi Vita
2004 ...... B.S. Food Engineering, National Autonomous
University (Mexico City, Mexico)
(Best Student of the 2000 – 2004 Food
Engineering Class)
2005 to Present ...... Graduate Research Associate, Department of
Food Science and Technology, The Ohio State
University
Publications
Parada-Rabell, F. and V. B. Alvarez. 2007. Food allergens: Status, risks and implications in
the food industry. Industria Alimentaria 29 (2): 28 – 33.
Fields of Study
Major Field: Food Science and Technology
vii Table of Contents
Abstract...... ii
Dedication...... v
Acknowledgments ...... vi
Vita ...... vii
List of Tables...... xii
List of Figures ...... xiii
Chapter 1: Literature Review ...... 1
1.1 Introduction ...... 1
1.2 Pasteurized Milk...... 4
1.2.1 Characteristics of fresh milk ...... 4
1.2.2 Chemistry of milk ...... 6
1.2.3 Milk Production ...... 9
1.2.4 Microbiology of pasteurized milk ...... 11
1.2.5 Chemical characteristics of pasteurized milk ...... 13
1.3 UHT Milk...... 15
1.3.1 Milk processing ...... 15
1.3.2 Microbiology of UHT milk ...... 16
viii 1.3.3 Chemical characteristics of UHT milk ...... 19
1.4 High Pressure Processed (HPP) Milk ...... 22
1.4.1 High pressure processing ...... 23
1.4.2 Microbiology of high pressure processed milk ...... 24
1.4.3 Effects of high pressure on milk constituents ...... 26
1.4.3.1 Effects on milk proteins...... 27
1.4.3.2 Effects on milkfat...... 30
1.4.3.3 Effects on the mineral balance of milk...... 30
1.4.3.4 Effects on lactose ...... 31
1.4.3.5 Effects on pH and appereance...... 31
1.4.3.6 Effects on flavor of milk...... 32
1.5 Pressure-Assisted Thermal Processed (PATP) milk...... 32
1.5.1 Microbiology of pressure-assisted thermal processed milk ...... 34
1.5.2 Chemical characteristics of pressure-assisted thermal processed milk ...... 36
1.6 Statement of the Problem...... 37
1.7 Rationale ...... 38
1.8 Hypothesis...... 39
Chapter 2: Microbial Load of Milk ...... 40
2.1 Abstract ...... 40
2.2 Introduction ...... 41
2.3 Material and Methods ...... 47
2.3.1 Pressure-heat treatments validation ...... 47
ix 2.3.2 Milk preparation ...... 48
2.3.3 High pressure processing ...... 52
2.3.4 Thermal treatments ...... 53
2.3.5 Microbial load of milk samples ...... 53
2.3.6 Data analysis ...... 54
2.4 Results and Discussion ...... 54
2.4.1 Pressure-heat treatments validation ...... 55
2.4.2 Pressure-temperature profiles of milk samples ...... 61
2.4.3 Microbial load of milk ...... 63
2.5 Conclusion...... 80
Chapter 3: Chemical Stability and Residual Plasmin Activity ...... 82
3.1 Abstract ...... 82
3.2 Introduction ...... 83
3.3 Materials and Methods...... 87
3.3.1 Milk preparation ...... 87
3.3.2 High pressure processing ...... 90
3.3.3 Thermal treatments ...... 91
3.3.4 Milk analyses ...... 91
3.3.5 Proteolysis ...... 92
3.3.6 Plasmin activity ...... 94
3.3.7 Lipolysis ...... 97
3.3.8 Data analysis ...... 99
x 3.4 Results and Discussion ...... 99
3.4.1 Pressure-temperature profiles of milk samples ...... 100
3.4.2 Proteolysis ...... 101
3.4.3 Plasmin activity ...... 106
3.4.4 Lipolysis ...... 112
3.5 Conclusion...... 120
Chapter 4: Flavor Profile of Milk...... 121
4.1 Abstract ...... 121
4.2 Introduction ...... 122
4.3 Materials and Methods...... 126
4.3.1 Milk preparation ...... 126
4.3.2 High pressure processing ...... 129
4.3.3 Thermal treatments ...... 129
4.3.4 SIFT-MS analysis ...... 130
4.3.5 Data analysis ...... 131
4.4 Results and Discussion ...... 131
4.4.1 Pressure-temperature profiles of milk samples ...... 132
4.4.2 Flavor analyses of milk ...... 134
4.5 Conclusion...... 164
References...... 166
xi List of Tables
Table 2.1 Longitudinal scheme of milk analyses...... 54
Table 2.2 Logarithmic reduction of bacterial spores suspended in milk subjected to different pressure – temperature combinations ...... 57
Table 3.1 Longitudinal scheme of milk analyses...... 92
xii List of Figures
Figure 2.1 Schematic diagram of milk processing and storage conditions ...... 51
Figure 2.2 Temperature profile of UHT milk during preheating time ...... 56
Figure 2.3 Pressure-temperature profiles observed during high pressure treatments of milk...... 62
Figure 2.4 Microbial population recovered in pasteurized and HPP milk samples subjected to different pressure and heating profiles ...... 66
Figure 2.5 Microbial population recovered in HPP milk samples subjected to different pressure and heating profiles...... 70
Figure 2.6 Microbial population recovered in UHT and PATP milk samples subjected to different pressure and heating profiles ...... 73
Figure 3.1 Schematic diagram of milk processing and storage condtions ...... 89
Figure 3.2 SDS-PAGE of milk subjected to HPP treatment...... 94
Figure 3.3 Standard curve for plasmin ...... 96
Figure 3.4 Standard curve for palmitic acid...... 98
Figure 3.5 Pressure-temperature profiles observed during high pressure treatments of milk...... 101
Figure 3.6 Relative percentage decrease of casein bands area as a result of protease activity in milk ...... 103
xiii Figure 3.7 Total plasmin concentration in milk ...... 108
Figure 3.8 Free fatty acids content in pasteurized and HPP milk treated with different pressure and temperature profiles...... 113
Figure 3.9 Free fatty acids content in HPP milk treated with different pressure and temperature profiles...... 115
Figure 3.10 Free fatty acids content in UHT and PATP milk...... 116
Figure 4.1 Schematic diagram of milk processing and storage conditions ...... 128
Figure 4.2 Pressure-temperature profiles observed during high pressure treatments of milk...... 134
Figure 4.3 Total concentration of aldehydes in milk samples ...... 135
Figure 4.4 Total concentration of methyl ketones in milk samples ...... 137
Figure 4.5 Total concentration of sulfur-containing compounds in milk samples...... 140
Figure 4.6 Total concentration of alcohol in milk samples ...... 143
Figure 4.7 Flavor profile of pasteurized milk ...... 146
Figure 4.8 Flavor profile of HPP milk processed at 32 oC ...... 149
Figure 4.9 Flavor profile of HPP milk processed at 72 oC ...... 152
Figure 4.10 Flavor profile of UHT milk...... 156
Figure 4.11 Flavor profile of PATP milk ...... 158
xiv Chapter 1: Literature Review
1.1 Introduction
Emerging food processing technologies are being investigated in recent years to inactivate microorganisms and enzymes as well as to retain more flavor, color and nutrients in foods. Among these, manothermosonication, high intensity magnetic field pulses and high pressure processing (HPP) have shown to be capable to process foods with reduced thermal damage (Alvarez, 2006). High pressure processing is a modern processing technique, which potentially offers distinctive advantages over traditional thermal treatments as it exerts microbial inactivation effects with minimal quality changes in foods (Huppertz et al., 2002). Since the early 90’s, high-pressure processed foods have been brought into the market. In the U.S., oysters, deli meat, seafood, orange juice and guacamole are now commercially available; whereas in Europe and Japan jams, juices, yogurt and some meat products have been successfully accepted in these markets.
These products are foods which their shelf life can be extended if stored under refrigeration conditions (Balasubramaniam, 2003). For low-acid foods, such as milk, mild temperatures in combination with high pressure are necessary to ensure extended shelf life products with higher quality characteristics (Balasubramaniam, 2003; Paredes-Sabja et al., 2007).
The quality standards of milk treated under elevated pressure conditions should be
1 on par or higher than those of milk processed with traditional thermal treatments. High temperature short time (HTST) pasteurization of fluid milk normally delivers a product with a shelf life no longer than 14 to 20 d, depending on the processing conditions and standard sanitary procedures at the dairy plant. After this point, the product becomes unacceptable as undesirable off-flavors start to develop due to bacterial growth and physical instability of the components of milk (Burton, 1986). More aggressive thermal treatments are capable of inactivate bacterial spores and their enzymes, and therefore increase the shelf life of milk. Among these, UHT processing is capable of extending the shelf life of milk up to 6 months at room temperature conditions
(Burton, 1986; Tetra Pak, 2003). However, the milk acquires a characteristic
“cooked” flavor not appealing to most consumers in the U. S. Pressure in combination with heat can be used for sterilization of milk (Matser et al., 2004;
Patterson et al., 2006; Paredes-Sabja et al., 2007). Researchers have shown that t he application of ultra high pressure combined with mild temperatures can extend the shelf life of milk up to 45 d. However, the milk acquires a different flavor profile than pasteurized and UHT milk (Vazquez-Landaverde et al., 2006a). The actual mechanisms of flavor formation in milk during pressure treatment are still unknown. Further research is necessary to fully understand the mechanisms of flavor formation during combined pressure-thermal treatment.
Enzymes are also responsible for undesirable changes in milk. In addition to bacterial enzymes, the activity of natural occurring enzymes is detrimental of milk’s quality. Lipoprotein lipase (LPL) and plasmin are probably the most
2 challenging enzymes to control in dairy operations due to their high temperature stability (Chen et al., 2003; Fox and Kelly, 2006). Lipoprotein lipase is the most abundant lipase in milk; and it is responsible for hydrolytic rancidity of milkfat.
The enzyme is relatively heat stable and it is naturally found in low concentrations in milk (Chen et al., 2003). However, serious flavor defects might develop when thermoresistant lipases are secreted by bacterial spores. These enzymes are significantly more heat resistant than indigenous lipolytic enzymes; and show activity at temperatures ranging from 60 to 75 oC (Chen et al., 2003). The resistance of lipases to high pressure processing is related to the pressure applied, time of exposure, temperature and the structure of the enzyme. Pressures ranging from 300 to 400 MPa at 3 oC have shown to enhance lipolytic activity in milk
(Lopez-Fandiño, 2006). The mechanisms of lipase inactivation under high pressure are not fully understood, yet. However, higher lipase inactivation rates are observed with increasing holding time and temperature ( Noel and Combes, 2003).
Proteases are responsible for degradation of proteins or proteolysis in milk.
Plasmin (EC 3.4.21.7) is the principal indigenous protease in milk (Chen et al.,
2003; Fox and Kelly, 2006). The enzyme is intimately associated with milk caseins and it is responsible for gelation in UHT milk (Politis et al., 1993) through disruption of hydrophobic interactions of αs- and β-caseins. It has optimal activity at pH 7.6 and 37 oC (Bastian and Brown, 1996; Fox and Kelly, 2006). The plasmin system in milk undergoes reversible denaturation with increasing temperature; and then refolds to its active form upon cooling. Temperatures up to 120 oC for 15 min
3 are necessary to ensure absence of plasmin in milk (Chen et al., 2003). Proteolysis in milk is also enhanced by HPP. Scollard and others, (2000) reported that conformational changes in the casein micelle and whey proteins structure during pressurization increase availability of substrate to plasmin. However, there is a synergistic effect of pressure (300-600 MPa) and temperature (40-60 oC) on inactivation of the plasmin system in milk (Lopez-Fandiño, 2006).
Information regarding the physical and chemical stability of milk processed with different combinations of pressure and temperature over its shelf life is limited.
Therefore, the objectives of this study were to compare the microbiological quality, chemical stability and flavor profile of milk subjected to different combinations of pressure and heat to that of thermally processed (HTST and UHT) milk. The quality of milk was observed over a period of 20, 60 and 90 d, respectively; and milk samples were stored under refrigeration or room temperature conditions depending on the treatment applied.
1.2 Pasteurized Milk
1.2.1 Characteristics of fresh milk
Milk is lacteal secretion, practically free from colostrum, obtained by the complete milking of one or more healthy cows, and contains no less than 8.25% milk- solids-non-fat and no less than 3.25% milkfat (PMO, 2005). Milk intended for human consumption may be homogenized and shall be pasteurized or ultra-pasteurized before retail (Alvarez, 2009).
4 The milk system consists of a true solution, a colloidal suspension and an emulsion. The most important mineral salts truly diluted in the solution are calcium phosphate, potassium citrate and sodium chloride (Tetra Pak, 2003). Milk is also a colloidal suspension because of the presence of casein micelles suspended as a colloid within the system. The milkfat emulsion is stabilized by proteins in the continuous aqueous phase of the system (Alvarez, 2009). These characteristics plus the ease of handle, natural over excess production, and the composition of milk make up an ideal food system that readily matches the grow needs of humans. Fresh milk is composed of water (85.5 – 89.5 %), fat (2.5 – 6.0 %), proteins (2.9 – 5.0 %), lactose (3.6 – 5.5 %), and a small fraction (0.6 – 0.9 %) of minerals (Tetra Pak, 2003; Alvarez, 2009). The composition of milk is directly affected by the type of feed, region, cow’s breed, season and overall health of the cow. Changes in the composition of milk due to variations in ruminant’s diet have been reviewed extensively (Palmquist et al., 1993; Baumgard et al.,
2002; Todd et al., 2006). The work of these authors suggest that diets rich in unsaturated fat causes an overall milkfat depression due to ruminal synthesis of conjugated linolenic acid (CLA). Some CLA isomers inhibit the abundance of acetyl-CoA carboxylase and fatty acid synthase mRNAs in the mammary gland, key enzymes necessary for the synthesis of short and medium chain fatty acids (Baumgard et al., 2002). The composition of milk also depends on the region it is being produced. United States is the largest milk producer in the world with over 189 billion pounds per year (Gould, 2008).
U. S. milk producers commonly prefer Hosltein cows because of the high milk yield obtained during the first five years of lactation. For this breed, milk’s yield can go as high
5 as ~ 16, 300 lb per lactation period (Tetra Pak, 2003). Cows typically breed in the fall and calve in spring. Hence, an overall increase in fat and protein content of milk is observed in September – November; and high yields are obtained in March – May; having the highest yields (40 – 50 L/day) 2 – 3 months post-parturition (Tetra Pak, 2003).
1.2.2 Chemistry of milk
The chemistry of milk is unique among food systems. Fat exists in milk as small globules with an average diameter ranging from 3 to 4 µm. The milkfat droplets are stabilized by a complex thin membrane composed of phospholipids, lipoproteins, cerebosides, proteins, enzymes and water. Also, fat soluble vitamins (A, D, E and K), sterols and carotenoids are found inside the milkfat globule (Tetra Pak, 2003). Milkfat is a mixture of different fatty acids (saturated and unsaturated) attached to a molecule of glycerol. Among saturated fatty acids, palmitic acid is the most abundant free fatty acid in milk. Butyric and caproic acids are exclusive of milk. These fatty acids are present in relatively larger amounts in the milkfat system. Oleic and linoleic acids are the most abundant unsaturated fatty acids present in milk (Tetra Pak, 2003). In addition to their health benefits, these unsaturated fatty acids in combination with other key components give the milk its characteristic mouthfeel (Alvarez, 2009). Milk also contains a complex protein system responsible for the stability of the 3.5% fat that occurs in the aqueous phase of milk (Mangino, 1994). Three major groups of proteins exist in the milk system namely caseins, whey proteins and enzymes. Caseins occur in milk as large colloidal particles called micelles with diameters ranging from 90 to 150 nm. These molecules are composed of smaller units called submicelles stabilized by hydrophobic interactions and
6 colloidal calcium phosphate bridges. Caseins are a diverse group of phosphoproteins that comprise about 80% of the total milk protein. Three main groups of caseins have been identified, namely αs-, β- and κ-caseins. The α- and β-caseins are found in the core of the casein micelle; whereas κ-caseins stabilize the system on the surface of the molecule through hydrophobic and hydrophilic interactions (Tetra Pak, 2003). αs-caseins are the most prevalent milk protein. These molecules contain 199 amino acids and 8 phosphate groups that are sterified into serum groups (McMahon and Brown, 1984). One segment of the molecule is highly charged. This segment contains all phosphate groups, 12 carboxyl groups and 2 ε amino groups. αs-casein has also three very hydrophobic regions giving rise to a largely apolar end of the molecule (McMahon and Brown, 1984). Isolate with αs1 –caseins are the αs2 – family of caseins. These caseins contain 8 more amino acids; have from 10 to 13 phosphate groups and also 2 cystein groups. β-casein comprises from 25 to 35% of the total casein. It is made up of 209 amino acids and contains 5 phosphate groups each one as esterified into serine phosphate esters. β-casein contains no cystein residues and has a net charge of -13 at pH 6.7. The N terminal segment of the molecule contains all the phosphate groups, and therefore the entire molecule’s net charge. The remaining segments of the molecule are hydrophobic and contain no net charge. There are five genetic variants of the β-casein, all of which are the result of one amino acid change. γ-casein comprises 5% of the total casein (McMahon and Brown, 1984). These molecules are the result of limited proteolysis of β-caseins.
Proteolysis results mainly from the activity of plasmin, which is transmitted from blood
7 plasma into the milk (Kelly et al., 2006). κ-casein consists of 169 amino acids and cotains from 0 to 5 trisaccharide units. The molecule contains one serine phosphate group and two cystein residues. Unlike αs- and β-caseins, κ-casein has charged sections at both ends of the molecule. The 53 c-terminal of the molecule has a net charge of -11, contain one phosphate group and all of the carbohydrates associated with κ-casein. The rest of the molecule is very hydrophobic and contains net positive charge at pH 6.7. κ-casein is
2+ soluble in the presence of Ca ions. This protein can also interact with αs- and β-caseins and stabilize them within the milk system in the presence of Ca 2+ ions (Mangino, 1994).
Whey proteins like caseins exist in the soluble phase of milk. There are least five main groups identified as whey proteins, namely: β-lactoglobulin, α-lactoalbumin, Bovine
Serum Albumin, Immunoglobulins and Proteose-peptones. β-lactoglobulin is the most prevalent whey protein in milk. The molecule contains two disulfide bridges and one free sylphydryl group and no phosphate groups. At the pH of milk, β-lactoglobulin is mainly found as a dimer (Liao and Mangino, 1987). α-lactoalbumin is the second most prevalent protein in whey. The molecule contains 4 disulfide linkages and no phosphate groups.
The molecule is remarkably stable to heat in the presence of Ca 2+ ions. α-lactoalbumin forms intramolecular ionic bonds with calcium that make the molecule more resistant to thermal unfolding (Liao and Mangino, 1987). Bovine serum albumin is identical to blood serum albumin. The molecule has specific binding sites for hydrophobic molecules and might serve as a carrier of fatty acids in milk. Immunoglobulins supply passive immunity to the calf when supplied in the colostrum. Proteose-peptones are defined as those proteins that remain in solution after milk has been heated at 95 oC for 20 min and then
8 acidified to pH 4.7. These proteins are derived from proteolysis of β-caseins. They are very surface active due to their low molecular weight and their improved capacity to form associations with carbohydrates in milk (Liao and Mangino, 1987). In addition to bacterial enzymes, up to seventy different indigenous enzymes have been identified in milk. From these, only twenty have been fully characterized. Lipoprotein lipase (LPL), proteinases, acid phosphatase and xanthine oxidoreductase (XOR) are probably the most significant enzymes in milk (Fox and Kelly, 2006). However, from a technological point of view LPL and plasmin are probably the most challenging enzymes in many dairy processes due to their high temperature stability (Chen et al., 2003). LPL hydrolyses milkfat giving rise to rancid off – flavors in milk; whereas plasmin is responsible of hydrolysis of milk proteins (Fox and Kelly, 2006). The specific characteristics of these enzymes are further discussed in subsequent chapters. Lactose is the carbohydrate found exclusively in milk. It is a disaccharide formed by glucose and galactose linked by β 1 –
4 bonds. Lactose exists as a true solution in milk in equilibrium between its α and β forms. The proportion of β-lactose is higher than α-lactose and the equilibrium between these forms is dependant of the temperature of the system (Rosenthal, 1991). Lactose is a key component responsible for Maillard reactions in milk. During the heat treatment, lactose can react with essential amino acids from caseins and form protein-carbohydrate complexes responsible for heat-induced browning reactions in milk. However, these reactions can be controlled by adequate temperature/time combinations during the processing of milk (Rosenthal, 1991).
1.2.3 Milk production
9 Processing of fluid milk combines at least five operations, namely: clarification, separation, standardization, homogenization and pasteurization. While raw milk contains
~ 4% milkfat, the fat content of fresh pasteurized milk in the U.S. is standardized to
3.25% milkfat. Lower fat alternatives such as 2%, 1% or skim milk (0.5% milkfat) are also available. Vitamins A and D are often added in the form of water soluble emulsions to offset their loss during separation (Tetra Pak, 2003).
Once the product has been standardized to the desired milkfat content, raw milk
(4 oC) is drawn into the regeneration section of the pasteurizer. In this section, milk is warmed up to 57 – 68 oC by heat given up by hot pasteurized milk flowing in a counter current direction on the opposite side of the plate heat exchanger. At this point, raw milk
(still under suction) passes through a positive displacement timing pump which delivers it under positive pressure through the rest of the HTST system. For continuous pasteurization processes, it is important to maintain a higher pressure on the pasteurized side of the heat exchanger. This pressure differential (of at least 1 psi) prevents that raw milk goes into the pasteurized milk side in event that a pin-hole leak develops in the thin stainless steel plates of the heat exchanger. The position of the timing pump is therefore crucial so that there is suction on the raw regenerator side and a positive pressure on the pasteurized side that pushes milk to the rest of the system. The positive displacement pump is usually built in the homogenizer. Homogenization causes disruption of fat globules from 3-4 µm to ~ 0.1 µm. This stabilizes the milkfat emulsion preventing separation in the aqueous phase of milk. Homogenization is commonly a two stage process where milk is forced through a narrow gap (0.1 mm) at high pressure. In the first
10 stage, the fat globules are split and then dispersed homogeneously as milk passes through a second gap in the homogenizer. Thereafter, milk is pumped through the heating section of the HTST system where it is heated up to 72 oC. Milk, still under pressure, flows through the holding tube and it is held for at least 16 sec. After passing through temperature sensors of the indicating thermometer, at the end of the holding tube, milk passes into the flow diversion valve. This valve assumes a positive forward-flow position if the milk passes through the recorder/controller at or above the preset pasteurization temperature (>72 oC). If the recorded temperature falls below this set value, the valve diverts the milk back to the constant level tank. Properly heated pasteurized milk flows to the regeneration section and gives up heat to the raw product and in turn it cooled to ~
32 oC. The warm milk passes through the cooling section where it is cooled to 4 oC or below. The cold pasteurized milk passes through a vacuum breaker at least 12 inches above the highest raw milk level in the HTST system; and then it is drawn into a storage tank/filler for packaging (Tetra Pak, 2003).
1.2.4 Microbiology of pasteurized milk
HTST pasteurization (72 oC for 16 s) normally delivers a product with a shelf life between 14 to 20 d, depending on the processing conditions and standard operating procedures at the dairy plant. High quality pasteurized milk intended for human consumption should have a microbial load not exceeding 20,000 CFU/mL and less than
10 CFU/mL for coliforms (PMO, 2005). However, these standards are the maximum allowed in the U.S.; and most milk producers provide a finished product with microbial loads considerably less than the maximum allowable limits (Alvarez and Parada-Rabell,
11 2005). Pasteurization of milk was designed to render a safe product intended for consumption and free of non-spore forming pathogens of human concern likely to be present in raw milk (Grant et al., 1996; Jay, 2000). The temperature and time combinations used during the pasteurization process (63 oC for 30 min – LTLT or 72 oC for 15 s – HTST) are sufficient to destroy the most heat resistant pathogens in milk.
Mycobacterium tuberculosis and Coxiella burnetti are considered the most heat resistant pathogenic microorganisms likely to be present in milk (Jay, 2000). A study assessing the thermal inactivation of eleven strains of M. paratuberculosis reported that HTST pasteurization (72±0.1 oC for 15 s) is sufficient to inactivate up to 5-6 log reductions of the initial bacterial load (10 7 CFU/mL) of the microorganism initially present in milk
(Grant et al., 1996). Other pathogens of concern in milk include Salmonella spp.,
Staphylococcus aureus, Listeria monocytogenes and Escherichia coli 0157:H7. These microorganisms are associated with samonellosis, hemorrhagic diarrhea, listeriosis, among other diseases if present in milk (Alvarez and Parada-Rabell, 2005). A recent study assessed the D value, which is the time required to kill 90% of the initial microbial count at a given temperature, of bacteria with similar heat resistance characteristics than
o the microorganisms mentioned above. Listeria innocua had a D54 C of 4.07 min and a z
o o value of 5.72 C. Salmonella Enteritidis showed D60 C value of 0.49 min and a z value of
6.53 oC (Aguirre et al., 2009). The information provided by D and z values can be used to obtain up to 5 log reductions of the initial population of each particular bacterium in milk
o (Aguirre et al., 2009). The reported D 71.7 C value for S. Senftenberg 775W is 1.2 s in milk
o (Jay, 2000). Strains of S. aureus have D 65.5 C values between 0.20 to 2.2 min; whereas C.
12 o burnetti and M. tuberculosis have D 65.5 C values of 0.5-0.6 min and 0.2-0.3 min, respectively (Jay, 2000). Pasteurization is also sufficient to kill yeast, molds, Gram- negative and many Gram-positive bacteria. Only those microorganisms that survived the heat treatment (thermoduric bacteria) and spores will be present in pasteurized milk (Jay,
2000). Therefore, the final product must be stored under refrigeration conditions to delay spoilage (Tetra Pak, 2003). Bacterial growth and their enzymes are responsible for undesirable changes that affect the quality of pasteurized milk. Pseudomonas spp . are the most important microorganisms responsible for spoilage in pasteurized milk (Ledford,
1998). These microorganisms are psychrotrophs that multiply slowly at refrigeration conditions (4±1 oC); however, they are capable of producing heat-stable proteases and lipases responsible bitter and rancid flavors in milk, respectively. Other microorganisms associated with pasteurized milk include Bacillus spp., Micrococcus sp., Alcaligenes sp.,
Flavobacterium sp. and other Lactic acid bacteria capable of standing the time and temperature combinations typically applied during pasteurization. Occasionally, thermoduric strains of Enterobacteriaceae family are also present in pasteurized milk
(Ledford, 1998).
1.2.5 Chemical characteristics of pasteurized milk
Pasteurized milk has a characteristic flavor profile which is pleasant slightly sweet without aftertaste (Alvarez, 2009). Any deviations from this flavor profile represent a major control issue in dairy operations as consumers readily perceive these changes. Off-flavors in pasteurized milk are mainly associated with bacterial growth, enzymatic activity and other are classified as “acquired” from the environment. Flavor
13 defects derived from bacterial growth include acid, malty and fruity flavors (Shipe et al.,
1978; Alvarez, 2009). Acid flavor results because of the growth of lactic acid producing bacteria in milk. Lactic acid bacteria are generally destroyed at pasteurization conditions.
However, if acid was produced in raw milk, pasteurization will not improve the flavor profile of milk. Malty flavors in milk develop as a result of the metabolism of
Streptococcus lactis subsp. maltigenes . The organism is associated with improper cleaning at the dairy plant; and the flavor defect is noticeable only when high bacterial counts are present in milk. The growth and metabolism of Pseudomonas fragi is responsible for fruity flavors in milk. This is a psychrotrophic ubiquitous microorganism and its presence in milk is an indicator of postpasteurization contamination. Other off- flavors associated with the growth of bacteria in milk include unclean, bitter and putrid flavors (Shipe et al., 1978). Flavor defects also occur in milk as a result of enzymatic activity. From a technological standpoint, proteases and lipases are the most challenging enzymes to control in dairy operations. These enzymes can be intrinsic to the milk system
(indigenous) or from bacterial origin. In either case, most proteases and lipases are known for their high temperature stability (Chen et al., 2003). Proteases breakdown milk proteins and produce bitter flavors in milk. Lipases are responsible for disrupting the ester bond between the glycerol and fatty acids in the milkfat. This causes serious defects as rancid flavors develop in milk (Shipe et al, 1978). Off-flavors derived from chemical sources are probably the most common flavor defects acquired from the environment.
These flavor defects are caused by contamination of milk with cleaners, sanitizers and disinfectants used in the dairy plant. Chlorine is probably the most common contaminant
14 (Shipe et al., 1978). Sulfur-containing compounds can also be present as a result of the cow’s metabolism or the heat treatment of milk. These compounds exist at low concentrations and dissipate upon storage of milk (Shipe et al., 1978; Vazquez-
Landaverde et al., 2006b).
1.3 UHT Milk
1.3.1 Milk processing
Milk subjected to ultra high temperature (UHT) treatment is considerably more stable than HTST processed milk. Such stability is achieved by heating the milk in relatively thin layers in a continuous heat exchanger, with close control over the sterilization temperature and holding time (Tetra Pak, 2003). Milk is first pumped into a continuous system where the product is pre-heated (70 – 80 oC), and then heated up to the desired process temperature (127 – 150 oC) for a few seconds (< 4 s). The product is homogenized and then cooled at room temperature. A pressurization device, the backpressure valve, is needed to maintain the milk liquid. In the end of the sterilization process, milk is aseptically filled into previously hydrogen-peroxide-sanitized multiple- layer containers that protect the product from light and oxidation reactions (Tetra Pak,
2003). Most UHT plants nowadays are designed to continuously process milk. The UHT process can be carried out by application of direct and indirect heating systems. In direct systems, the product comes in contact with steam by injection or infusion. Direct injection systems rely on direct steam addition to the milk; whereas in infusion systems milk is added to food – grade steam. The water added to the product, during heating, is
15 then evaporated by flash cooling under vacuum conditions (Scott, 2008). Indirect heating is probably the most cost-effective system to UHT-process milk. With this type of systems the product never comes into direct contact with the heating medium, as heat is transferred convectively to the product through a concurrent system of heat exchangers.
For continuous UHT milk processing, plate and tubular heat exchangers are normally preferred for heating and cooling applications (Tetra Pak, 2003). Heating occur at slower rates during indirect processing than in steam injection or infusion systems. Therefore, more pronounced chemical changes are commonly observed in UHT milk processed with indirect heating, as the processing times for this product are longer (Elliot et al., 2005;
Scott, 2008). In addition to adequate time/temperature combinations, the aseptic filling of milk represents a critical stage of the UHT processing system. This step of the process is designed to ensure that integrity and quality of the finished product are optimal during its shelf life (typically 6 months). Tetra Pak (2003) defines the aseptic process as “a procedure consisting of sterilization of packaging material or container, filling with a commercially sterile product in an aseptic environment, and producing hermetically sealed containers”. The combination of all these factors plus the application of such high temperatures for shorter time renders a commercially sterile product. UHT-treated milk is therefore a product free of those pathogens capable of growing at conditions in which the product is held during its distribution and storage and before its expiration date (Burton,
1988; Tetra Pak, 2003).
1.3.2 Microbiology of UHT milk
The shelf life of UHT milk is influenced not only by the initial bacterial count, but
16 more importantly the type of microorganisms capable of surviving the aseptic process.
When milk is exposed to severe heat treatments, such as UHT, not all microorganisms are killed. Instead, a proportion of the original bacterial count left in the product determines the sterilizing efficiency of the UHT treatment (Tetra Pak, 2003). Once milk surpasses its expiration date, microorganisms that survived the heat treatment and their metabolic enzymes cause the product to spoil (Burton, 1988). For UHT milk, the European Union
Hygiene directive 92/46 EEC has established a maximum of 10 CFU/0.1 mL of spore- forming bacteria in sealed packages after 15 days of incubation at 30 oC (Anonymous,
1992). The presence of bacterial spores in milk is a growing concern in the dairy industry. The ubiquitous nature of these microorganisms makes it difficult to prevent their presence in soil, silage, barn utensils, processing lines and equipment, and ultimately in milk. Furthermore, the ability of these microorganisms to adapt to extreme environments and their increased tolerance against severe heat treatments, such as UHT processing, represents a major challenge in any dairy operation (Te Giffel et al., 2002;
Scheldeman et al., 2006). Bacterial spores are also capable of producing heat resistant proteases and lipases which adversatively affect the quality of milk upon storage at room temperature conditions (~25 oC) (Burton, 1988; Chen et al, 2004). The resistance of bacterial enzymes to the thermal treatment applied (140-150oC for a few seconds) is associated with the optimum growth temperature of the microorganism present in milk. A study assessing the activity of proteases and lipases from seven Bacillus strains in milk powders reported that these enzymes survived UHT process and remained active even in milk powder products after 6 months of storage at room temperature conditions (Chen et
17 al., 2004). Fortunately, the incidence of spore-forming bacteria in milk and dairy products is relatively low. As with total bacterial count, the presence of spores in milk is subjected to seasonal variation (Verdi et al., 1986; Burton, 1988). Relatively low counts have been reported during summer; whereas higher counts prevail during the winter season (Burton,
1988). The average total count of spore formers reported for raw milk is in the range of
10 0 – 10 2 spores/mL (Te Giffel et al., 2002; Scheldeman et al., 2006). Bacillius spp. are the most predominant spore-forming bacteria found in milk. Among Bacillus species, B. cereus is a major spoilage microorganism that may grow in UHT milk (Te Giffel et al.,
2002). The microorganism is a toxin-producer, Gram-positive, sporeformer, rod that has been identified as facultative anaerobe with optimal growth temperatures between 30 to
35 oC. Bacillus cereus is also psychrotroph (Yousef, 2006); and thus its ability to grow in refrigerated milk upon storage. Spores of B. sporothermodurans have been reported to be major cause spoilage in UHT milk. This mesophilic, aerobic, sporeformer microorganism was first isolated from indirect UHT processing lines in Italy in 1985 (Scheldeman et al.,
2006). During its dormant stage, spores of this microorganism are exceptionally heat resistant. Spores of B. sporothermodurans isolated from UHT milk have decimal
o o reduction time values at 140 C (D 140 C) between 3.4 and 7.9 sec with reported z-values of
13.1–14.2 oC. This data suggest the significant thermal stability of B. sporothermodurans spores against severe heat treatments, such UHT processing (142 - 144 oC for < 4 sec).
Likewise, Bacillus stearothermophilus has been recognized for their significant temperature stability during processing of milk and dairy products. B. stearothermophilus has been used as a test microorganism to verify the efficacy of the UHT process (Tetra
18 o Pak, 2003; Scheldeman et al., 2006). Decimal reduction time (D121 C) values for spores of
B. stearothermophilus are reported in the range of 200–500 sec with corresponding z- values between 9.0-9.3 oC (Burton, 1988; Scheldeman et al., 2006). These values are 10 times higher than those reported for Clostridium botulinum spores (Burton, 1988; Jay,
2000). B. stearothermophilus is a spore forming, facultative anaerobe, thermophilic microorganism capable of growing at 40 to 70 oC in its vegetative form. This microorganism is responsible of flat sour spoilage in low-acid foods (Yousef, 2006).
However, the incidence of B. stearothermophilus spores is relatively small in milk. In fact, this microorganism corresponds to an insignificant portion of total bacterial count in raw milk (Burton, 1988).
1.3.3 Chemical characteristics of UHT milk
Color and flavor changes also occur in milk as a result of thermal processing.
Among these changes, Maillard reactions, sugar isomerization, spontaneous lipid oxidation and protein denaturation can be detrimental of milk’s quality (Morales and
Jimenez-Perez, 1999; Min and Boff, 2002; Cais-Sokoli ńska et al., 2004; Gliguem and
Bilouez-Aragon, 2005). Maillard reactions or non-enzymatic browning reactions in milk represent a major concern in the dairy industry. Along with a decrease on the nutritional value and food safety concern, formation of Maillard end products limits the shelf life of milk. Chemical and physical factors influence the extent of formation of Maillard end products in milk systems. Among these factors, initial concentration and type of reactants, pH, water content, processing and storage temperature, oxygen availability, and packaging conditions are responsible for formation of off-flavors, changes in color,
19 decrease in solubility and texture changes in milk and dairy products (Morales and
Jiménez-Pérez, 1999; Sithole et al., 2005). Milk is, by far, suitable to undergo Maillard reactions. The presence of a reducing sugar (lactose) and amino acid residues, pH value
(6.6 – 6.8), oxygen and common heat processing conditions are factors influencing development of off-flavors and color changes in milk (Cais-Sokoli ńska et al., 2004). In dairy products, the primary source of lysine free ε-amino residues comes from β- lactoglobulin; whereas the natural source of reducing sugar is lactose (Sithole et al.,
2005; Gliguem and Bilouez-Aragon, 2005). At the pH of milk, β-lactoglobulin is found as a dimmer, with a molecular weight of approximately 36,000 Da (Tetra Pak, 2003); and therefore, the availability of lysine reacting residues associated with lactose increases significantly in milk. Jones and others (1998) studied the extent of Maillard reaction between β-lactoglobulin and lactose in skim milk systems. The authors reported that the interaction between the free ε-amino group of lysine with lactose is responsible for the formation of lactuloselysine (galactose-fructose-lysine), at the initial stage of the reaction.
The compound subsequently degrades into deoxyosones which lead to the formation of high molecular mass materials. These compounds ultimately give rise to brown nitrogen- based polymers and copolymers called “melanoidins”, at the final stage of the Maillard reaction (Jones et al., 1998). Time and temperature during processing, and storage time are also critical factors affecting formation of brown-colored polymers during Maillard reactions in milk. With increasing temperature formation of Maillard intermediate compounds, such as lactuloselysine increases (Jones et al., 1998). In addition to temperature, pH and moisture content also influence the extent of formation of Maillard
20 end products. A recent study assessing the influence of pH and moisture content on the extent of Maillard reactions reported that an increase in pH on heated aqueous model systems led to a significant increase of degradation of initial reactants (lysine and lactose)
(Ajandouz et al., 2001). The authors also reported that as the pH value increased, the formation of brown colored polymers further increased. At pH values ranging from 8 to
12, brown color formation, in model systems of lysine containing reducing sugars, increased significantly at the intermediate stage of the Maillard reaction. Furthermore, depending on the acidity or alkalinity of the system, different end Maillard products can be formed, at the final stages of the reaction. Furfural and derivates, including hydroxymethylfurfural (HMF), are more likely to form at pH values below neutrality; whereas reductones and highly reactive dicarbonyls are mainly form, under alkaline conditions, after either Amadori’s or Heyn’s rearrangement (Ajandouz et al., 2001).
Maillard reactions must be considered carefully as they exert undesirable changes in milk. The presence of lactose as the source of reducing sugar, amino acid residues, pH and oxygen influence widely development of off-flavors and brown color in milk during processing. Time and temperature during processing and storage conditions must be controlled carefully to prevent formation of Maillard end products in milk.
Off-flavors also occur in milk as a result of the heat treatment. UHT milk has a characteristic “cooked” flavor or strong sulfurous note that comes from sulfur-containing amino acids from whey proteins that can be released upon heat treatment of milk (Shipe et al., 1978; Solano-Lopez et al., 2005). Sulfur-containing compounds associated with this off-flavor in UHT milk include hydrogen sulfide, methanethiol, carbon disulfide,
21 dimethyl sulfide, dimethyl disulfide and dimethyl trisulfide (Vazquez-Landaverde et al.,
2005). The concentration of these volatile compounds depends on the severity of the heat treatment applied; and it decreases after several days of storage due to their high reactivity and subsequent conversion to other volatile compounds (Vazquez-Landaverde et al., 2006b). Other compounds responsible for the heat-induced flavor of milk include diacetyl, aldehydes, methyl ketones, lactones and maltol (Shipe et al., 1978; Vazquez-
Landaverde et al., 2005). As with sulfur-containing volatiles, the concentration of these thermally induced off-flavors in milk depend on the severity of the heat treatment applied. Moreover, not all aroma compounds equally contribute to the heated flavor of
UHT milk. The sensory threshold or ability of consumers to perceive these off-flavors in milk is different for each compound. Vazquez-Landaverde et al., (2005) reported that 2- pentanone, 2-heptanone, 2-nonanone, benzaldehyde, and 2-undecanone are strongly correlated with the cooked flavor in UHT milk. Also, 2,3 butanedione, 2-methylpropanal,
3-methylbutanal, nonanal, decanal and dimethyl sulfide are important contributors to the heat-induced off-flavor of UHT milk. The activity of proteases and lipases from thermally resistant spores can form rancid and bitter flavors in UHT milk (Shipe et al.,
1978). However, the activity of heat-resistant enzymes in aseptically processed milk is relatively low as compared to HTST pasteurized milk.
1.4 High Pressure Processed (HPP) Milk
For the last two decades the food industry has been interested in exploring potential applications for HPP. The technique has worked successfully on acid foods; and
22 high pressure processed products such as oysters, meat, smoothies, fruit juices and guacamole are now commercially available in the U. S. market (Patterson et al., 2006). In
Europe and Japan jams, assorted juices, yogurt and some meat products have been successfully accepted in these markets. Pressure treatments (up to 600 MPa) at or near ambient temperature are effective in inactivating most vegetative bacteria. However, bacterial spores are not inactivated. Therefore, storage under refrigeration conditions is required (Balasubramaniam, 2003; Patterson et al., 2006).
During PATP, both pressure and heat can influence various reactions. The influence of pressure and heat on the reactions can be additive, synergistic or antogonistics. The Le Chatelier’s and isostatic principles along with the Arrhenius relationship are important to understand the behavior that biological systems, such as milk, undergo during HPP. The Le Chatelier’s principle states that with increasing pressure, molecules undergo a reduction in volume under equilibrium conditions. This phenomenon is an inherit thermodynamic effect of most biological systems. The isostatic principle explains that the volume of the sample is independent to the pressure applied because pressure is transmitted equally and almost immediately throughout the sample.
Lastly, the Arrhenius equation asserts that temperature determines reaction rates of various inorganic and organic substances. That is that all reactions under high pressure are also temperature dependent (Adams, 2003; Balasubramaniam, 2003).
1.4.1 High pressure processing
Batch and semi-continuous HPP systems are commercially available to process both liquid and solid food products (Patterson et al., 2006). Typical batch systems can
23 hold volumes ranging from 40 to 950 L (Balasubramaniam, 2003), and are suitable to process either solid or liquid products (Patterson et al., 2006). Before processing, the product is prepackaged, in a flexible or semi-rigid container, and then load into the vessel
(Schauwecker et al., 2002). The vessel is subsequently filled with a pressure transmitting fluid (typically water and/or glycol) and the product is processed at the desired target pressure. The processing pressure is achieved by using a piston that compresses the pressure transmitting fluid for the established processing time. Finally, the pressure vessel is depressurized and the product is unloaded (Balasubramaniam, 2003). Semi- continuous systems are typically designed to process liquid foods. These systems are arranged into three or more parallel vessels (Balasubramaniam, 2003; Patterson et al.,
2006). A typical semi-continuous process cycle consists in pumping the liquid product into the vessel at low pressure to force the floating piston down. Once all valves are closed, the pressure transmitting fluid is high-pressure pumped underneath the piston and lifts it to compress the food up to the desire process pressure and time. The product is then pumped out to the aseptic filler by means of low-pressure water pushing the piston to the top of the vessel (Patterson et al., 2006).
1.4.2 Microbiology of high pressure processed milk
High pressure at or near ambient temperature can result in pasteurization type effects. The treatment can inactivate variety of vegetative bacteria, but bacterial spores are not inactivated under these conditions. The resistance of microorganisms to pressure in foods is variable and depends on several process conditions, such as pressure, time and temperature. The specific mechanisms of bacterial inactivation under high pressure are
24 not clear; and further research is necessary to fully understand the effects of HPP on the resistance of microorganisms to ultra high pressure treatments (Patterson et al., 2006).
However, researchers have shown that damage to the cell membranes, enzymes or DNA are common causes of bacterial inactivation by emerging processing technologies (Lado and Yousef, 2002). Ultra high pressure treatments induce structural changes to the cell membrane and there are signs of cell contents leakage at pressures > 200 MPa. The rate of bacterial inactivation significantly increases with increasing pressure. Irreversible protein denaturation and leakage of cell contents occur at pressures > 300 MPa (Lado and
Yousef, 2002). The characteristics of the food system and the type of microorganism therein are also key elements to explain how microorganisms are inactivated during HPP.
Water activity (a w), pH and the stage of growth of microorganisms also influence the extent at which bacteria are inactivated during physical treatments (Jay, 2000). Gram- positive microorganisms tend to be more pressure-resistant than Gram-negative microorganisms. However, a considerable variation in pressure resistance within strains of the same species has been demonstrated in both Gram-positive and Gram-negative microorganisms. Inactivation of Gram-positive microorganisms has been reported at pressures ranging from 500 to 600 MPa at 25 oC during 10 min; whereas treatments of
300 to 400 MPa at 25 oC during 10 min are necessary to inactivate Gram-negative microorganisms (Trujillo et al., 2002). Yeast and mold are more sensitive to physical inactivation than bacteria. Pressures between 300-600 MPa have been reported to be effective yeast and mold in food (Rastogi et al., 2007). The effects of HPP on inactivation of microorganisms exponentially increase when the technology is applied in combination
25 with temperature. Temperatures up to 50 oC in combination with pressure increase the rate of microbial inactivation during HPP (Rastogi et al., 2007). Trujillo et al., (2002) reported that P. fluorescens , L. innocua and L. helveticus show higher resistance to pressure treatment at room temperature (25 oC) than at low temperature (4 oC). Similarly, E. coli and S. aureus showed less resistance to HPP at room temperature than at low temperature
(Trujillo et al., 2002). High pressure treatments have been shown to be effective on inactivation of pathogens in milk and dairy products. A recent review on the advantages and limitations of high pressure processing of foods reported that E. coli can be completely inactivated in goat’s milk after pressure treatments at 400-500 MPa for 5-15 min at temperatures up to 25 oC. Higher sensitivity to physical inactivation of E. coli is observed in cheddar cheese at pressures above 200 MPa. High pressure treatments are effective against L. monocytogenes at pressures ranging from 400 to 700 MPa for 1- 15 min (Rastogi et al., 2007). Similarly, a study assessing the effects of combined thermal and high pressure processing on the microbial stability of milk during refrigerated storage reported that combinations of pressure (586 MPa), time (1, 3 and 5 min) and temperature
(40 and 55 oC) were sufficient to inactivate mesophiles, Pseudomonas spp., psychrotrophs and coliforms counts. Moreover, the microbial load of pressure-treated samples remained lower for all treatments during refrigerated storage up to 45 days (Tovar-Hernández et al.,
2005).
1.4.3 Effects of high pressure on milk constituents
High pressure processing causes reversible and irreversible changes in foods. The effects of HPP on the functional properties of milk depend on the initial temperature of
26 the product, pressure applied, temperature, treatment time and cooling rate (Matser et al.,
2004). High pressure processing has the ability to instantaneously increase the temperature of the product due to the adiabatic heating effect during pressurization. This effect offers unique advantages over traditional thermal treatments. Not only the temperature of the product is evenly distributed during pressurization; but also relatively short treatment times are observed during HPP as compared to thermal treatments.
Therefore, the components of the food that are more susceptible to heat damage are less affected by high pressure processing. This results in food products with higher quality characteristics in terms of flavor, texture, color and nutrients (Matser et al., 2004).
However, the effects of HPP on product quality also depend on the type of food. Milk is a unique food system consisting of a true solution, a colloidal suspension and an emulsion.
The components of milk exist in perfect balance within the system and give the product its characteristic flavor and nutritional characteristics. Any deviations from this balance represent a major control issue in dairy operations as consumers readily sense these changes. Changes in the components of milk are associated with processing and handling, bacterial growth and their metabolism. In general, pressure induces changes in the proteins, some polysaccharides and lipids. These changes depend on the pressure and temperature applied during high pressure processing.
1.4.3.1 Effects of pressure treatment on milk proteins
The functional properties of proteins are altered by pressure treatment through disruption of protein-water interactions and protein-protein interactions. However, covalent bonds are not affected by high pressure processing (Alvarez and Ji, 2003).High
27 pressure treatments ranging from 250 to 600 MPa reduce the micelle size by disrupting it into small fragments in raw or reconstituted skim milk. At low pressure (< 300 MPa), the effects of HPP on micelle size are temperature dependent. High pressure treatments at
40 oC increase micelle size and treatments at 4 oC reduce it. However, there is a temperature independent decrease in micelle size at pressures > 450MPa (Huppertz et al.,
2004). Changes in micelle size promote changes in the physical appearance of milk. The increase or decrease in the size of milk’s micelle increases turbidity in milk. The turbidity increase during high pressure treatments at low temperature (5 – 10 oC) is related to solubilization of colloidal calcium phosphate (CCP); whereas the increase in turbidity during HPP at higher temperatures (>40 oC) may be due to interactions between fragments of casein micelles and denatured whey proteins. This suggests that high pressure treatments at higher temperatures also induce denaturation of whey proteins (Huppertz et al., 2004). The extent of whey protein denaturation during HPP depends on the structure of the protein. α-lactalbumin ( α-la) is more pressure resistant than β-lactoglobulin ( β-lg)
(Trujillo et al., 2002). The former is remarkably stable to denaturation because the presence of four disulfide linkages within its structure and its ability to form ionic bonds with calcium in milk (Liao and Mangino, 1987). Pressure treatments at 400MPa are sufficient to denture β-lg by 70–80%; whereas studies on raw and skim milk have shown that is resistant to denaturation at pressures up to 500MPa. However, with increasing temperature (50–60 oC) the extent of α-la denaturation significantly increases (Trujillo et al., 2002). Small molecules with little secondary, tertiary and quaternary structure such as amino acids, vitamins, flavor, and aroma compounds remain unaffected during HPP
28 (Huppertz et al., 2002).
The effects of HPP on inactivation of enzymes depend on the structure of the enzyme and the processing conditions applied. Alkaline phosphatase appears quite pressure-resistant, with no inactivation in raw milk after treatment at 400MPa for 60 min at 20 oC. The enzyme is completely inactivated at 800 MPa for 8 min. Indigenous milk lactoperoxidase, phosphohexoseisomerase and γ-glutamyltransferase are also resistant to pressures up to 400MPa at 20–25 oC. Pressure treatments at 400 MPa for 15min and 40 –
60 oC reduce the proteolytic activity in milk. The reduced proteolytic activity or proteolysis is probably due to inactivation of the plasmin system in milk at higher temperatures. There is also a synergistic effect of pressure and temperature on the extent of proteolytic activity in milk. Proteolysis in milk during incubation at 37 oC is more extensive in milk treated at 300MPa and room temperature (25 oC) than at 400 MPa and
60 oC. The reasons for this proteolytic activity increase are related to a combined effect of more disruption of casein micelles under pressure and little inactivation of plasmin at room temperature, resulting in more surface area of exposure to proteolytic enzymes in pressure treated milk (Huppertz et al., 2002). Researchers have indicated that lipoprotein lipase (LPL) is pressure stable. In fact, the activity of lipases in milk is enhanced by high pressure processing (Buffa et al., 2001; Pandey and Ramaswamy, 2004). The activity of lipase is enhanced after pressure treatments at 300 – 400 MPa; and holding time (0 – 180 min) has no effect on inactivation of the enzyme (Pandey and Ramaswamy, 2004).
Combinations of higher pressure and temperature are necessary to inactive lipases in milk. A study assessing the effects of temperature and high pressure processing on
29 Rhizomucor miehei lipase reported that combinations of pressure (300 to 500 MPa) and temperature (40 to 60 oC) can be used to completely inactivate the activity of the enzyme
(Noel and Combes, 2003).
1.4.3.2 Pressure effects on milkfat
Pressures up to 400 MPa at ambient temperatures do not destabilize the milkfat in milk. This is probably due to the fact that the phase transition from solid to liquid of milkfat is shifted to higher values under pressure (15.5 oC/100 MPa). In addition, lower degree of milkfat crystallization at higher pressures (>350 MPa) may be due to a reduced molecular mobility of this lipids under high pressure conditions (Huppertz et al., 2002).
The diameter of milkfat globules is not affected at pressures < 400 MPa.
However, at higher pressures (400 - 800MPa) the size of milkfat globules increases and become more broadened (Huppertz et al., 2002). Studies on free fatty acids content in ewe's milk have shown that HPP treatments 100-500 MPa at 4, 25, and 50 oC do not increase free fatty acids content. In fact, high pressure treatments at 50 oC showed lower free fatty acids concentration than fresh raw milk (Trujillo et al., 2002).
1.4.3.3 High pressure effects on the mineral balance of milk
The effects of HPP on minerals in milk can be enumerated as follow: (1) effects on the distribution between the colloidal and diffusible phases of calcium, and (2) effects on ionization calcium. Huppertz et al., (2002) reported that either minimal or no effects of pressure < 600 MPa on the concentration of ionic calcium in milk occur during HPP.
The diffusible level of calcium increased following treatments at 200 – 600 MPa
(Huppertz et al., 2002).
30 1.4.3.4 Pressure effects on lactose
In milk and other dairy products no changes in lactose have been observed at pressures ranging from 100 to 400 MPa, for 10 to 60 min at room temperature. In addition, no significant Maillard reaction products occur in milk after HPP. However, combinations of pressure and temperature may influence the rate of lactose isomerization and further degradation into acids and other sugars (Trujillo et al., 2002).
1.4.3.5 Pressure effects on pH and appearance
High pressure processing has little or no effect on the pH of milk (Huppertz et al.,
2002). However, some studies have shown that treatments at high pressures (> 600 MPa) can reversely increase the pH of milk from 6.6 – 6.7 to about 0.05 units more. This shift may be related to dissolution of CCP. In addition, milk’s pH is transiently reduced under pressure by about 1 unit at 1000 MPa due to an increase of water dissociation (Huppertz et al., 2002).
High pressure processing also affects the physical appearance of milk. As mentioned above, changes in the structure of the casein micelle lead to an increase in turbidity of milk. In turn, whiteness or the Hunter L-value of milk is affected by HPP.
Treatments at 200 MPa and 20 oC have little, or no, effect on the L-values in milk.
However, high pressure treatments between 250 and 450 MPa significantly decreased the
L-value in milk. Higher pressure conditions (> 450 MPa) have little effect on the L- values of milk (Huppertz et al., 2002). A study assessing the effects of high pressure processing on the color of milk reported that the greenness (-a*) and yellowness (+b*) values are visually negligible affected at pressures < 600 MPa. However, significant
31 changes in b*, a* and L values were observed in milk treated at 600 MPa for 15min
(Trujillo et al., 2002).
1.4.3.6 High pressure effects on flavor of milk
Information on the effects high pressure processing on the flavor of milk is still very limited. A recent study assessing the effects of pressure and thermal processing in the quality of milk reported that pressure treatments enhance the formation of volatile compounds differently than traditional heat treatments. Pressures ranging from 482 to
620 MPa in combination with temperatures between 25 and 60oC enhanced the formation of hydrogen sulfide and straight-chain aldehydes; unlike heat treatments, which favored the formation of sulfur compounds, methyl ketones and aldehydes. The authors suggested that higher oxygen solubilization under high pressure leads to the formation of more aldehydes through lipid oxidation patterns (Vazquez-Landaverde et al., 2006a). However, the actual formation mechanisms of flavor compounds under high pressure are yet unknown. Further research is necessary to fully understand the mechanisms of flavor formation during high pressure processing.
1.5 Pressure-Assisted Thermal Processed (PATP) Milk
High pressure processing has shown to be effective on inactivation of vegetative bacteria in acid foods. However, for low-acid foods, such as milk, temperatures (90-
121 oC) in combination with high pressure (500-700 MPa) are necessary to ensure inactivation of bacterial spores (Balasubramaniam, 2003; Paredes-Sabja et al., 2007).
Pressure treatment in combination with heat is commonly referred to as pressure-assisted
32 thermal processing (PATP), pressure-assisted thermal sterilization (PATS) or high- pressure temperature sterilization (HPHT) in the literature (Nguyen et al., 2009). As with
HPP, the Le Chatelier’s principle, the isostatic principle, the principle of microscopic order and the Arrhenius equation are important to understand the net effect of pressure and temperature on foods during PATP (Balasubramaniam et al., 2008). At a given pressure, the temperature of foods rapidly increases as a result of physical compression during pressurization. This temperature increase is independent of the heat exchange between the product and the surroundings because of the adiabatic nature of the process.
A rapid cooling effect is subsequently observed as the product returns to a temperature slightly less than its initial temperature value upon decompression. This temperature difference is due to heat loss of the product during pressurization at elevated temperatures
(Balasubramaniam et al., 2008). The rapid heating and cooling effects observed during processing give unique advantages over traditional thermal treatments as less heat damage is observed on the components of foods during PATP (Nguyen et al., 2009). The temperature increase of foods during pressurization depends on several factors, including target pressure, food composition, initial temperature of the product and its specific heat
(Rastogi et al., 2007; Balasubramaniam et al., 2008). Milk behaves similar to water under high pressure (Balasubramaniam et al., 2004). Therefore, the heat of compression value for milk is very similar to the one of water; and it is in the order of 3 oC for every 100
MPa of pressure increase at room temperature conditions (25 oC) (Balasubramaniam et al., 2008). It is extremely important that the temperature of the pressure transmitting fluid and the pressure vessel are equilibrated at the same value of the initial temperature of the
33 product. Therefore, close control over the preheating temperature of the sample is necessary to ensure that the desired initial temperature of the product is achieved
(Balasubramaniam et al., 2004).
1.5.1 Microbiology of pressure-assisted thermal processed milk
The inactivation of bacterial spores has been a major challenge to HPP, as spores are more resistant to physical treatments than vegetative bacteria. Combinations of ultra high pressure and high temperature are necessary to inactivate bacterial spores in foods
(Paredes-Sabja et al., 2007). With increasing pressure and temperature the mechanisms of bacterial spore inactivation are related to the release of dipicolinic acid (DPA) from the spore core. The release of DPA induces germination of spores and further activation of lytic enzymes that degrade the spore cortex, promote cell contents leakage during PATP treatment with subsequent bacteria lethality (Paidhungat et al., 2002). Research data has shown that combinations of pressure with increasing temperature are necessary to inactivate bacterial spores. In fact, the rate of bacterial spore inactivation during PATP increases with increasing temperature (Balasubramanian and Balasubramaniam, 2003;
Patazca et al., 2006). Similarly, inactivation of spores is more effective in processes with two or more pressurization stages. Meyer et al., (2000) reported that high pressure treatments using multiple high-pressure pulses and temperatures above 105°C produces sterility with minimal impact on flavor, texture, and color on foods. More recently, thermal treatments (90 to 121 oC) combined with elevated-pressure processing (600 to
900 MPa) have shown unique advantages to process low-acid food products, such as milk, over traditional HPP methods (Rajan et al., 2006a). As mentioned above, the
34 technology has the ability to instantaneously increase the temperature of the sample due to the heat compression associated with the food components and the pressurization rate during processing. Therefore, the severity of the thermal treatment is more effective as compared to traditional thermal sterilization processes (Ahn et al., 2007). Up to date, kinetic studies on spore inactivation during PATP have been limited for low-acid foods, such as milk (Ahn et al., 2007). Matser et al., (2004) recommended that a two-pulse high pressure process of 700 MPa and an initial product temperature of 60 oC are necessary to inactivate spores and achieve commercial sterility in milk. However, for commercial applications pressures up to 600 MPa are currently available to process foods (Patterson et al., 2006). The resistance of various bacterial spores to PATP has been assessed in model foods over the last decade. A recent study on inactivation of selected aerobic and anaerobic bacterial spores by PATP reported that spores of Thermoanaerobacterium thermosaccharolyticum and Bacillus amyloliquefaciens are the most PATP-resistant microorganisms among surrogate bacteria of Clostridium botulinum . Pressure-assisted thermal processing treatments at 700 MPa and 121 oC for less than 1 min were necessary to inactivate up to 7 to 8 log reductions of the initial spore load of T. themosaccharolyticum and B. amyloliquefaciens (Ahn et al., 2007). Similarly, inactivation studies in egg patties showed that spores of B. amyloliquefaciens , B. stearothermophilus , Clostridium botulinum , and T. thermosaccharolyticum have a rapid initial inactivation rate after the pressure come-up time, followed by a characteristic tailing effect with different pressure-holding times (Rajan et al., 2006a,b). C. botulinum has been recognized as heat and pressure resistant microorganism. However, this spore-
35 former is capable of producing a harmful neurotoxin; and therefore nonpathogenic surrogates with similar characteristics have been chosen to evaluate the resistance of C. botulinum to pressure and temperature. Since spores of B. amyloliquefaciens exhibit higher resistance to pressure and temperature treatments than C. botulinum , this microorganism has been proposed as a surrogate for C. botulinum in pressure-heat treatment validation studies (Margosch et al., 2004; Rajan et al., 2006b). B. amyloliquefaciens has also been chosen over traditional thermal sterilization surrogates, such as B. stearothermophilus , because the later can be easily inactivated by combined pressure-heat treatment. Up to 4 log reduction of the initial spore count of B. stearothermophilus can be achieved after PATP treatment at 700 MPa and 105 oC for 5 min, as compared to the 1.5 log reduction of the initial spore count of the bacterium obtained after thermal treatments at 121 oC for 15 min (Rajan et al., 2006a).
1.5.2 Chemical characteristics of pressure-assisted thermal processed milk
Up to date, the quality characteristics of PATP treated food are a topic of ongoing research. Recent studies have indicated that the texture, color and flavor of foods can be improved by PATP over traditional retort systems (Nguyen et al., 2009). Carrots, red radishes and jicamas show less textural damage after PATP treatments (600 MPa, 105 oC for 5 min) as compared to thermal treatments (105 oC for 5 min). The effects of PATP treatment on color of these foods was product dependent. However, PATP treated samples retained more color than thermal treated samples (Nguyen et al., 2009). The flavor profile of chicken, salmon, eggs, potatoes and green beans is closer to the flavor of untreated samples after two pressure pulses at 700 MPa, 105 oC for 1 min holding time for
36 each pressure pulse. These results are attributed to less thermal exposure during PATP as compared to traditional retort operations (Nguyen et al., 2009). Research data on the flavor and chemical characteristics of PATP milk is limited. Researchers have reported that the flavor profile of pressure treated milk is different than that of thermally treated milk. The specific formation mechanisms of aroma volatiles during pressure treatments are still unknown. However, the formation of selected sulfur-containing compounds
(methyl mercaptan, dimethyl sulfide and dimethyl disulfide) and aldehydes (2- methylpropanal, acetaldehyde and hexanal) seem to be enhanced by combinations of increasing pressure and temperature (Vazquez-Landaverde et al., 2006a).
1.6 Statement of the Problem
Fresh fluid milk has typically a shelf life between 14 to 20 d at refrigeration conditions. After this point, the product becomes unacceptable to consumers due to formation of off-flavors as a result of bacterial metabolism. More aggressive thermal treatments (such as UHT and aseptic filling) are capable of inactivate bacterial enzymes; and therefore increase the shelf life of milk (up to 6 months).
However, adverse chemical reactions take place in milk during thermal processing.
As a result, undesirable color and flavor changes occur in milk affecting its acceptability among consumers. The quality of UHT-treated milk has been discussed since its introduction in 1960. Since then, there have been considerable efforts to obtain a flavor profile closer to that clean, pleasant and slightly sweet flavor of pasteurized milk. Recently, high pressure processing has gained attention
37 as an alternative technology to process foods without compromising their nutritional and quality characteristics. Research data suggests that high pressure processing at ambient temperature is sufficient to render refrigerated foods with similar characteristics to thermally pasteurized foods. However, for milk combinations of ultra high pressure (up to 500-700 MPa) and temperature (90-121 oC) are necessary to render a shelf stable food product. Up to date, data available for milk treated with different combinations of pressure and temperature conditions is limited. Therefore, the main objective of this work was to evaluate the application of HPP and PATP as alternative technologies to process high quality fluid milk. The specific objectives of this work were to compare the microbial load, chemical stability and flavor profile of HPP and PATP milk to that of HTST pasteurized and UHT processed milk.
1.7 Rationale
High-temperature-short time (HTST) pasteurization (72 oC for 16 sec) is the preferred method to process fluid milk sold in the U.S. This thermal treatment renders a product with a shelf life typically of 14 - 20 days at refrigeration conditions. More aggressive thermal methods are capable of render milk with longer shelf life. Among these, ultra high temperature (UHT) pasteurization (142-144 oC < 4 sec) delivers a product with a shelf life of up to 6 months at room temperature conditions. However, the milk acquires a “cooked” flavor after processing, not appealing to most consumers in the
United States and other countries. Consequently, the market of pasteurized fluid milk in
38 the U.S. is limited to interstate sales, and the opportunity to export high-quality milk to other countries has not been met yet.
High pressure processing can be used for pasteurization of foods. PATP can be used for the inactivation of bacterial spores resulting in extended shelf life products with higher quality characteristics. The application of temperature-assisted ultra high pressure processing (PATP) seems a promising alternative to render extended shelf-stable milk without compromising its nutritional and quality characteristics.
Up to date, data available on HPP and PATP-treated milk is limited. Some researchers have reported the efficacy of high pressure in combination with mild temperatures, on inactivation of vegetative bacteria. The results of this work have shown that moderate temperatures in combination with pressure can significantly extend the shelf life of milk (up to 45 days). Additionally, milk subjected to pressure-temperature combinations has a different flavor profile than pasteurized and UHT milk. Information on spore inactivation, chemical stability and residual enzymatic activity of milk subjected to PATP conditions is still very limited.
1.8 Hypothesis
Combinations of mild heat with pressure are capable of rendering shelf stable milk with quality characteristics similar to pasteurized milk. The flavor profile of pressure treated milk is influenced not only by pressure–temperature combinations, but also by residual enzymatic activity present in the system.
39 Chapter 2: Microbial Load of Milk
2.1 Abstract
The application of high pressure and pressure–assisted thermal processing as an alternative technology to pasteurization and UHT treatment was evaluated to process high quality fluid milk. The microbial load of HPP and PATP milk was compared to that of milk processed with traditional thermal treatments. Milk (2% milkfat) was HPP and
PATP treated using a factorial 3x1x3 model at temperature (32, 72 and 105 oC), pressure
(650 MPa), and time (0, 1, and 5 min). Milk samples were processed within 72 hr; and stored at either room temperature (25 ± 1oC) or refrigeration (4 ± 1oC) conditions depending on the treatment applied. The shelf life of milk samples was examined over a period of 20, 45 and 90 d. Additionally, pasteurized (77 ± 0.8 oC for 18 sec) and UHT processed (138 ± 1 oC for 2 sec) milk samples were analyzed along with pressure–treated milk samples. High pressure and pressure–assisted thermal processing validation studies were conducted by spore–forming bacteria survival analyses of Bacillus
6 stearothermophilus ATCC 7953 (N 0 = 7x10 spore/mL) and B. amyloliquefaciens Fad 82
7 (N 0 = 1x10 spore/mL). The microbial index of milk samples was analyzed by total plate count analyses (TPC). Up to 6-log reduction was obtained when a suspension of B. stearothermophilus ATCC 7953 was inoculated in UHT milk treated at 700 MPa for 3
40 min at 105 oC. Similarly, a 7-log reduction was achieved when a suspension of B. amyloliquefaciens Fad 82 was inoculated in UHT milk treated at the conditions described above. Within the experimental range of this research, the shelf life of pressure treated milk was extended during regrigerated (HPP) and ambient temperature (PATP) storage.
Pressure treatments at 32 ± 1 oC delivered milk with similar microbial load than pasteurized milk. The microbial population of milk further decreased with increasing
o 4 temperature (72 and 105 C). Up to 4-log reduction (N 0 = 2 x 10 CFU/mL) was obtained in milk samples processed at 650 MPa and 105 oC for 0, 1 and 5 min. Even though milk samples had similar microbial loads on 0 d, there were large changes in the microflora of milk samples during their shelf life. Storage temperature had a significant effect on the microbial growth rate in HPP and PATP milk. Microorganisms recovered in HPP milk processed at 650 MPa and 72 oC for 0, 1 and 5 min stored at refrigeration conditions (4 ±
1oC) showed slower growth rates than PATP milk samples stored at room temperature
(25 ± 1 oC) conditions. These results indicate that the pressure–temperature combinations used in this study are potentially capable of rendering milk with quality characteristics close to pasteurized and UHT milk. However, this study did not characterize the type of microorganisms present in the milk during extended storage. Similarly, no additional tests on extend of bacterial injury were carried out. The type of microorganisms present in milk before and/or after processing and the storage conditions had a significant role on the quality of pressure–treated milk.
2.2 Introduction
41 The microbiological quality of milk is probably the most limiting factor in extending the shelf life of the final product. Upon secretion by the cow, milk (free of microorganisms) is first contaminated with bacteria associated with the environment of the dairy farm (Alvarez, 2009). Sources of this diverse microflora include soil, bedding, manure and feeds (Ledford, 1998). Milk can be further contaminated as a result of improper handling and sanitary operations at the farm. The level of contamination is directly related to the cleanliness, automation of handling equipment, cooling rates and storage temperature of the milk (Alvarez, 2009; Johnson, 1998). According to the PMO
(2005), the microbial load of high quality grade “A” raw milk must not exceed 1 x 10 5
CFU/mL for a single producer; and no more than 3 x 10 5 CFU/mL for commingled milk.
However, improvements in handling and milking operations at the farm have brought milk initial microbial counts to limits below regulatory standards. A study assessing the bacterial quality of milk in three different fluid milk processing plants reported that the average raw milk bacterial counts ranged between 12,000 and 66,000 CFU/mL (Fromm and Boor, 2004). Regardless of the initial microbial load, the unique microflora associated with the product is ultimately responsible of the quality and shelf life of milk.
Microorganisms associated with raw milk include Gram-negative and Gram-positive bacteria (Ledford, 1998). Among these, species of Pseudomonas, Achromobacter,
Aeromonas, Alcaligenes, Chromobacterium, Flavobacterium and Bacillus are commonly present in raw milk. Additional biota includes Enterococcus spp. , Lactococcus spp. ,
Leuconostoc spp. , Lactobacillus spp. , Propionibacterium spp. , Acinetobacter spp. ,
42 Staphylococcus spp. , Micrococcus spp. and species from the coliform family (Ledford,
1998; Yousef, 2006).
HTST Pasteurization normally delivers a product with a shelf life between 14 to
20 d. After this point, the quality of pasteurized milk is compromised due to development of undesirable changes as a result of bacterial growth and their metabolism. The bacteriological standards for pasteurized milk are 20000 CFU/mL or less, and <10
CFU/mL for coliforms (PMO, 2005). However, the type of microorganisms that survived the heat treatment or those present in the milk as a result of post processing contamination ultimately define the quality of the finished product. Microorganisms that can survive pasteurization treatment include bacterial spores, thermoduric bacteria and psychrotrophs that contaminate the milk after processing (Fromm and Boor, 2004;
Yousef, 2006). From these, Pseudomonas spp. are probably the most significant microorganisms that limit the shelf life of pasteurized milk (Ledford, 1998).
Pseudomonas spp. are aerobic, rod-shaped, Gram-negative, psychrotrophic microorganisms with motile polar flagella; and they are capable of producing heat-stable enzymes responsible for bitter and rancid flavors in milk (Chen et al., 2003; Yousef,
2006). The presence of these microorganisms in milk is an indicator of post processing contamination as Pseudomonas spp. are not capable of surviving the pasteurization
o o process. The reported D 53 C value for P. fluorescens is 1.16 min with a z value of 4.74 C.
Thermoduric bacteria are those microorganisms capable of surviving the pasteurization process but do not necessarily grow at this temperature (Jay, 2000). These microorganisms belong to the genera Arthrobacter, Microbacterium, Corynebacterium
43 (Collins, 1981; Johnston and Bruce, 1982; Fromm and Boor, 2004); Staphylococcus spp. and Enterobactericeae (Johnston and Bruce, 1982; Ledford, 1998); and Streptococcus and
Lactobacillus (Jay, 2000). Species of the genus Bacillus , however, are more commonly isolated from pasteurized milk (Collins, 1981; Johnston and Bruce, 1982; Huck et al.,
2008). Bacillus spp. are spore-forming, thermoduric bacteria that can grow under refrigeration conditions. These microorganisms can survive not only HTST pasteurization
(72 oC for 16 s) but also UHT processing (140-150 oC < 4 s) (Huck et al., 2008). A recent a study assessing the identification points of bacteria entry from raw milk tanker trucks to pasteurized milk reported that Bacillus spp. and Paenibacillus spp. are commonly present in the dairy farm environment. The ability of these microorganisms to form endospores represents a major control issue in any dairy operation, as these bacteria are consistently present in pasteurized milk (Huck et al., 2008). The presence of bacterial spores in milk is a growing concern in the dairy industry. These microorganism are ubiquitous in nature; and therefore this characteristic makes it difficult to prevent their presence in soil, silage, barn utensils, processing lines and equipment, and ultimately in milk. Also, the ability of these microorganisms to adapt to extreme environments, such as UHT processing, represents a major challenge in any dairy operation. Bacilli associated with UHT milk include Bacillus cereus, B. licheniformis, B. Stearothermophilus and B. sporothermodurans (Crielly et al., 1994; Scheldeman et al., 2006). These microorganisms are spore formers with exceptional heat resistance characteristics; and they have been
o identified as major cause of spoilage in UHT milk. The reported D95 C value for spores of
B. cereus at a w > 0.95 and a pH value close to that of milk (6.5) is 5.01 min; whereas the
44 o D95 C value for spores of B. licheniformis is 5.1 min (Jay, 2000). The heat resistance of
o spores of B. stearothermophilus can be estimated at D 140 F values between 0.9 s (Huemer et al., 1998). A study assessing the heat resistance of spores of B. sporothermodurans isolated from UHT milk reported that this microorganism can survive thermal treatments
o o in the range from 110 to 145 C. The reported D 140 F values for spores of B. sporothermodurans are in the range of 3.4-7.9 s (Huemer et al., 1998). The incidence of bacterial spores is relatively low in UHT milk (Burton, 1988). The average total count of spore formers reported for milk ranges from 10 to 10 2 spores/mL (Te Giffel et al., 2002;
Scheldeman et al., 2006).
For more than 20 years, high pressure processing has been used to process foods.
The technique has worked well on acid foods. However, for low–acid foods combinations of ultra high pressure with temperature are necessary to inactivate bacterial spores and their enzymes. High pressure processing at ambient temperatures is sufficient to inactivate pathogens such as Listeria monocytogenes , Salmonella spp. Escherichia coli and Staphylococcus aureus (Trujillo et al., 2002; Torres and Velazquez, 2005). Up to 5- log reductions (CFU/mL) of the initial population of these microorganisms can be obtained after HPP (Torres and Velazquez, 2005). However, the resistance of bacteria to high pressure treatments also depends on the type of microorganisms therein the food system. Bacterial spores represent a major challenge for HPP due their high tolerance to physical inactivation. The rate of bacterial spore inactivation during HPP increases at higher temperatures (Patazca et al., 2006). Over the last decade, the resistance of various bacterial spores to different pressure–temperature combinations or pressure-assisted
45 thermal processing (PATP) has been assessed in model foods. A recent study assessing the research challenges of high pressure processing of foods reported that spores of B. stearothermophilus, B. subtilis, C. perfringens and C. sporogenes are also considerably pressure resistant. Combinations of pressures ranging from 500 to 700 MPa, holding times from 5 to 30 min and temperatures between 50 and 80 oC are necessary to achieve inactivation of up to 4 to 7-log reductions (spore/mL) of these microorganisms (Ahn et al., 2007). PATP treatments at 700 MPa and 121 oC for less than 1 min are sufficient to inactivate spores up to 7 to 8 – log reductions (spore/mL) of Thermoanaerobacterium thermosaccharolyticum and Bacillus amyloliquefaciens (Ahn et al., 2007). These microorganisms are the most PATP resistant surrogate bacteria of Clostridium botulinum .
The efficacy of the high pressure process depends on the initial temperature, adiabatic heating, pressure applied and processing time (Ahn et al., 2007). Additionally, the resistance of microorganisms to pressure varies with pH, water activity, and food composition (Torres and Velazquez, 2005).
The objectives of this study were to validate a pressure-assisted thermal process of milk consisting of different combinations pressure, holding time and temperature; and to assess the feasibility of PATP as an alternative technology to process high quality fluid milk. Similarly, the overall microbial load of HPP and PATP-treated milk was also observed over a period of 20, 60 or 90 d, depending on the treatment applied. The microbial characteristics of pressure-treated milk samples were compared to those of
HTST pasteurized and UHT milk.
46 2.3 Materials and Methods
2.3.1 Pressure-heat treatments validation
The resistance of Bacillus stearothermophilus ATCC 7953 and B. amyloliquefaciens Fad 82 to different pressure–temperature combinations was assessed after PATP treatment. Spore suspensions were prepared as described by Rajan et al.,
(2006a,b). Stock cultures of B. stearothermophilus were spread-plated onto nutrient agar plates supplemented with 500 mg/kg of dextrose and 3 mg/kg of manganese sulfate.
Inoculated plates were incubated for 10 d at 55 oC; and spores were harvested when approximately 97% sporulation was achieved. The plates were flooded with deionized water and washed several times by differential centrifugation (2,000 to 8,000 g for 20 min at 4 oC). The spore suspension was sonicated for 10 min and subsequently heated at
80 oC for 10 min to remove any remaining sporangia and vegetative cell debris. The final spore suspension was washed again; and the spore pellet was diluted to a known concentration (~10 7 spores/mL). The suspension was stored at 4 oC until use. Similarly, spore suspensions of B. amyloliquefaciens obtained from OSU high pressure processing laboratory were spread-plated onto Trypticase soy agar (TSA) supplemented with 10 mg/kg of manganese sulfate. Inoculated plates were incubated at 32 oC for 10 d or when >
95% of sporulation was observed. Spores suspensions were collected by flooding the plates with deionized water and washed several times by differential centrifugation
(2,000 to 8,000 g for 20 min at 4 oC). Interfering sporangia was removed as explained before. Finally, spore suspensions of B. amyloliquefaciens were washed with deionized water and re-suspended to a known concentration (~10 7 spores/mL). The suspension was
47 stored at 4 oC until used.
Spore suspensions of these aerobic, spore-forming bacteria were inoculated in commercial UHT milk (2% milkfat) in order to validate the PATP process. Milk was aseptically filled into 8 oz amber polyethylene teraphtalane (PET) bottles without headspace and inoculated with 7 x 10 6 spore/mL of B. stearothermophilus and 1 x 10 7 spore/mL of B. amyloliquefaciens, respectively, by duplicate. Milk samples were pre- heated to 70, 77 and 83 oC, respectively, depending on the final processing temperature applied. Immediately after, milk samples were PATP treated using an ABB Quintus Food
Processor QFP-6 cold isostatic press (ABB Autoclave Systems, Columbus, Ohio) at 700
MPa and 90, 100, and 105 oC for 3 min. After decompression, milk bottles were immediately placed in an ice bath to stop further thermal damage; and stored at refrigeration conditions (4±1 oC) until analysis. Milk samples inoculated with spore suspensions of B. stearothermophilus were spread-plated onto nutrient agar (peptone 5 g/l, beef extract 3 g/l, agar15 g/l, pH 6.8), and incubated at 55oC for 48 h. Similarly, samples inoculated with spore suspensions of B. amyloliquefaciens were spread-plated onto trypticase soy agar at 32 oC for 48 h. After incubation, plates were examined for typical Bacillus colonies and the spore-log reduction in milk samples was determined following standard colony-counting rules as described by Yousef and Calrstrom (2003).
2.3.2 Milk preparation
Raw milk was obtained from a commercial dairy plant in Ohio (Orrville, OH) and transported to the OSU dairy pilot plant. The temperature of milk was kept at ~ 4 oC at all times during transportation (approximately 2 hr). Milk was standardized to 2% milkfat
48 and two-stage homogenized using a Lab 100 M-G homogenizer (Lubeck-Schlutut,
Germany). Homogenized milk was immediately cooled to 7±1 oC and stored under refrigeration conditions (4±1 oC) until processing by HPP and PATP as shown in Figure
2.1. All milk samples were subjected to various pressure-heat conditions within 72 hrs.
On the day of processing, milk samples were preheated to their corresponding initial temperature (Ti) using an UHT/HTST Lab-25HV Hybrid unit (Micro Thermics Inc.,
Raleigh, NC). The initial temperature of milk samples was adjusted as a function of the final target pressure during processing. This temperature was estimated based on the heat of compression of water and skim milk for various temperatures (Balasubramaniam et al.,
2004). The preheating time for milk to achieve the desired initial temperature was 2.5 min. The temperature of milk samples during preheating was set at values slightly higher than their required initial temperature (Ti) to compensate for the heat loss during filling and transportation of milk from the OSU dairy pilot plant to the high pressure processing laboratory. The initial temperature of milk samples was estimated based on the following equation:
CH Ti = T max − x∆P + ∆TH 100
Where, Ti = initial temperature before pressurization; Tmax = final processing temperature during pressurization; CH = heat of compression; and ∆P = applied pressure,
∆TH = Heat loss during pressurization; and Ti accounts for heat loss during preheating until pressure treatment (1±0.5 oC). The preheating temperature of milk samples was set at
4, 53 and 78±1 oC, respectively, depending on the pressure-heat treatment applied. Milk
49 was filled into light-protected 8 oz polyethylene teraphtalane (PET) bottles without head space and manually capped. Immediately after, milk samples were transported from the
OSU dairy pilot plant to the high pressure processing laboratory. The total heat loss during preheating of milk until pressure treatments was 1 ± 0.5 oC. This minimal heat loss was achieved by placing milk bottles in a water bath set at the same temperature of milk samples.
50 Raw milk UHT milk (Orrville, OH) (138±1 oC; 2 s) (New Jersey, NY)
Clarification Milk Hauling (~4 oC, 2hr) (Orrville, OH) OSU (Columbus, OH)
Separation Clarification (7±1 oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Separation 2% milkfat (60±1 oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Homogenization 2% milkfat OSU (Columbus, OH) (Orrville, OH)
Homogenization 500/1000 psi Pasteurization OSU (Columbus, OH) (77±0.8 oC) (Orrville, OH) Milk Storage ≤ 72 hr (4±1 oC) OSU (Columbus, OH)
HPP and PATP (650 MPa; 32, 72 & 105 oC; 0, 1 & 5 min) OSU (Columbus, OH)
Microbial Index Analysis
HPP & PM (4 oC); PATP & UHT (25 oC)
Figure 2.1 Schematic diagram of milk processing and storage conditions
51 2.3.3 High pressure processing
An S-IL-110-625-08-W cold isostatic press system (Stansted Fluid Power Ltd.,
Essex, UK) was used to high pressure processed (HPP; 650 MPa, 32 & 72 oC) and pressure-assisted thermal processed (PATP; 650 MPa, 105 oC) milk samples. A 1:1 ratio propylene glycol, water mixture was used as the pressure-transmitting fluid. Milk samples were processed using a factorial 3x1x3 model at temperature (32, 72, and
105 oC), pressure (650 MPa) and time (0, 1 and 5 min) by duplicate. Pressure come-up time ranged between 1.5 to 3 min depending on the treatment applied, and decompression time was 1.2 min. The temperature of the vessel and pressure-transmitting fluid was thermostatically adjusted to the desired initial processing temperature by circulating propylene glycol through the external jacket of the pressure chamber. This temperature was set at the same initial temperature of milk samples (6, 48 and 73 oC, respectively).
Milk samples were filled into a cylindrical sample basket (102 mm dia x 559 mm height)
(Stansted Fluid Power Ltd., Essex, UK) and loaded into high-pressure equipment using a mechanical lift mechanism. The temperature of milk samples during various pressure treatments was monitored at the center of the carrier basket using T-type thermocouples
(Omega engineering, CT, USA) placed inside of a milk sample bottle designated for temperature recording purposes. After decompression, milk bottles were immediately placed in an ice-water bath to stop further thermal damage; and stored at either room temperature (25 ± 1 oC) or refrigeration (4 ± 1 oC) conditions depending on the final pressure/temperature treatment applied. All pressure-heat treated milk samples used in this study were processed with these conditions unless noted below.
52 2.3.4 Thermal treatments
High-temperature-short-time pasteurized milk (2% milkfat), obtained from the same batch of raw milk used for pressure-heat treatments, was processed in a commercial dairy operation (Orrville, OH) at 77±0.8 oC for 18 s. Additionally, ultra high temperature
(UHT) treated (138±1 oC for 2 s) milk samples were obtained commercially (New Jersey,
NY). HTST pasteurized and UHT milk samples were stored under refrigeration condition
(4±1 oC) and room temperature conditions (25±1 oC), respectively, and analyzed for their overall microbial load along with pressure-treated milk.
2.3.5 Microbial load of milk samples
The overall microbial load of milk samples was evaluated using total plate count
(TPC) analysis. Decimal dilutions (up to 10 -6) were prepared from milk samples according to their expected microbiological load with 0.1% peptone water (1% peptone +
0.5% NaCl), by duplicate. Trypticase soy agar plates (BD Difco; Becton, Dickinson and
Co., Sparks, MD) were inoculated with 0.1 mL of each milk dilution and incubated at
32 oC for 48 h. After incubation, plates were evaluated for colony forming units
(CFU/mL) to determine their overall microbial population following standard colony- counting rules as described previously. The microbial load of HTST pasteurized, UHT,
HPP and PATP-treated milk samples was evaluated over a period of 20, 45 and 90 d, respectively, depending on the treatment applied (Table 2.1).
53 Time (days) Group a 0 2 4 6 10 15 20 30 45 60 75 90 A ●b ● ● ● ● ● ● B ● ● ● ● ● ● ● C ● ● ● ● ● ● ● Table 2.1 Longitudinal scheme of milk analyses a Group A: HTST pasteurized (78±1 oC for 18 s) and HPP milk (650 MPa, 25 oC for 0, 1 and 5 min, respectively). Group B: HPP milk (650 MPa, 72 oC for 0, 1 and 5 min). Group C: UHT (138±1 oC for 2 s) and PATP milk (650 MPa 105 oC for 0, 1 and 5 min) b Denotes day of analysis.
2.3.6 Data analysis
All experiments were conducted in duplicate on individual milk samples; and analyzed using crossed and nested ANOVA general linear model, with Tukey’s pairwise comparisons at 95% confidence level. Data analysis was performed using Minitab v. 15.2
(Minitab Inc., State College, PA).
2.4 Results and Discussion
The overall microbial load of HTST pasteurized, UHT, HPP and PATP milk samples was evaluated in this work. Due to equipment limitations, milk samples were obtained from two different sources and variations in the overall microbial load of milk samples were expected. However, the intent of this study was to evaluate the effects of different combinations of pressure-heat treatments on the overall bacterial load of milk samples. Milk was exposed to similar temperature histories with or without pressure than traditional thermal treatments. And, HTST pasteurized (77±0.8 oC for 18 s) and UHT
(138±1 oC for 2 s) milk samples were chosen as controls. Researchers have reported that
54 high pressure processing without temperature is sufficient to deliver a product with similar shelf life than pasteurized foods (Balasubramaniam, 2003). Therefore, combinations of high pressure (650 MPa) with mild temperature (32 oC) seemed appropriate to evaluate the effects of HPP to those of commercial pasteurization of milk.
Moreover, the shelf life of milk can be increased up to 45 d with combinations of pressure (586 MPa) and increasing temperature (40-55 oC) (Tovar-Hernandez et al.,
2005). Thermal treatments at elevated temperatures (90-121 oC) in combination with pressure (500-700 MPa) have shown to be sufficient to extend the shelf life of foods with less thermal damage (Rajan et al., 2006a). Therefore, the application of combined pressure (650 MPa) with temperature (HPP, 72 oC; PATP, 105 oC) seemed an excellent alternative to process extended shelf life milk with higher quality characteristics than
HTST pasteurized and UHT milk.
2.4.1 Pressure-heat treatments validation
Figure 2.2 shows the temperature profile of UHT milk samples during preheating time in a water bath set up at 85 oC. The initial temperature (Ti) was estimated based on previously reported heat of compression values for water at various temperatures, since milk behaves similarly to water under high pressure (Balasubramaniam et al., 2004). The preheating time for milk samples to achieve the desired Ti of 70, 77 and 83 oC was 6, 9 and 14 min, respectively. These initial temperatures were necessary to achieve final processing temperatures of 90, 100 and 105 oC, respectively, during pressure treatment at
700 MPa for 3 min. The long preheating times observed in this study represented a challenge as adverse effects on the appearance, flavor and nutritional value of milk can
55 occur at such high temperatures. Therefore, the combination of time and temperature during preheating of milk was optimized taking into account the desired microbiological effect and quality aspects of milk. Faster heating and cooling rates of milk were achieved using a UHT/HTST Lab-25HV Hybrid unit. This equipment allowed heating the milk in relatively thin layers in a continuous heat exchanger, with close control over the temperature and holding time. With this modification the preheating time for milk samples to achieve the desired initial temperature was 2.5 min; and the heat loss during preheating until pressure treatment was 1 ± 0.5 oC. This minimal heat loss was achieved by placing milk bottles in a water bath set at the same temperature of milk before loading the samples into the pressure vessel.
90 80
C) 70 o 60 50 40 30 20 Temperature ( Temperature 10 0 0 5 10 15 20 Time (min)
Figure 2.2 Temperature profile of UHT milk during preheating time a a Water bath temperature = 85 oC *Data shown are means of two independent samples
56 Table 2.2 shows the effects of preheating time, HPP and PATP on inactivation of bacterial spores of B. stearothermophilus and B. amyloliquefaciens . As expected, temperature alone did not exert a significant effect on the initial spore load of milk samples subjected to different preheating times. The spore load of milk samples, however, was significantly affected by different combinations of pressure (700 MPa), time (3 min) and temperature (90, 100 and 105 oC).
Treatment 70 oC 90 o C 77 o C 100 o C 83 o C 105 o C Spore 6 min 3 min 9 min 3 min 14 min 3 min 0.1 MPa 700 MPa 0.1 MPa 700 MPa 0.1 MPa 700 MPa B. stearothermophilus 0.1±0.1 a 5.9±0.0 b 0.3±0.2 a 5.9±0.0 b 0.1±0.1 a 5.6±0.0 b ATCC 7953 B. amyloliquefaciens 0.1±0.1 a 2.2±0.8 c 0.1±0.1 a 4.8±0.0 d 0.4±0.2 e 6.8±0.0 f Fad 82 Table 2.2 Logarithmic reduction of bacterial spores suspended in milk subjected to different pressure–temperature combinations. a-fMeans ± Std. dev. With a different superscript are significantly different *Initial population of B stearothermophilus was 6.9 log spore/mL **Initial population of B. amyloliquefaciens was 7 log spore/mL
Spores of B. stearothermophilus ATCC 7953 showed similar inactivation patterns in milk after PATP treatments at 90, 100, and 105 oC, respectively. Up to 5.9-log reductions of the initial B. stearothermophilus spore count was achieved after PATP in all milk samples as shown in Table 2.2. These observations suggest that combinations of increasing pressure and temperature have a synergistic effect on inactivation of the bacterium in milk. Similar results have been reported in previously reported work on
57 inactivation of B. stearothermophilus during PATP treatments. A recent study assessing the inactivation of spores of B. stearothermophilus ACTT 7953 by pressure-assisted thermal processing reported that the application of 700 MPa and 105 oC for 3 min is necessary to achieve up to 6-log reductions of the initial spore load in deionized water
(Rajan et al., 2006b). The inactivation rate of spores of B. stearothermophilus during
PATP follows different inactivation patterns than thermal treatments. The spore inactivation rate of spores decreased at all pressure levels tested after a pressure holding time of 1.5 min. The authors associated this tailing effect to stress adaptation of the bacterium and to a decrease in the processing temperature at longer pressure holding times (Rajan et al., 2006b). In this study, spores of B. stearothermophilus were not completely inactivated after pressure treatments at 700 MPa and 105 oC for 3 min. Under this conditions, up to 5.6-log reductions of the initial spore load (6.9 log spore/mL) were obtained after PATP treatment. These observation might be related to not only stress adaptation of the bacterium to physical inactivation (Rajan et al., 2006b), but also to the intrinsic heat resistance capability of B. stearothermophilus (Burton, 1988).
Combinations of higher temperatures for longer holding times might be necessary to completely inactivate spores of the bacterium during PATP treatments. Matser et al.,
(2004) reported that it is also possible to inactivate spores of B. stearothermophilus by two-pulse high pressure processing at 700 MPa in combination temperatures up to 90 oC.
The authors reported that careful selection of pressure and temperature is necessary to achieve inactivation values comparable to commercial sterility (Matser et al., 2004). B. stearothermophilus is a spore-forming, heat-resistant bacterium commonly found milk.
58 The microorganism is responsible for the flat sour spoilage of milk. Due to its remarkable heat resistance properties, this microorganism has been traditionally used to validate thermal process specifications (Burton, 1988; Scheldeman et al., 2006). The incidence of this spore–former bacterium in milk is well documented. Although the total spore counts of B. stearothermophilus is relatively low (10 0 to 10 2 spore/mL), the bacterium is commonly isolated from UHT milk along with B. licheniformis , B. subtilis and B.cereus
(Burton, 1988). There is also a seasonal effect on the incidence of spore-forming bacteria in milk. Relatively low counts have been reported during summer; whereas higher counts prevail during the winter season (Burton, 1988). Common sources of contamination for these microorganisms include feed concentrate, compost, silage and milk processing lines when reprocessing of contaminated milk occurs in UHT dairy plants (Scheldeman et al,
2006).
Reduction, up to 6.8-log of the initial spore load of B. amyloliquefaciens Fad 82 was achieved in milk samples after PATP treatment at 700MPa at 105 oC for 3 min. The log–reduction of spores followed different inactivation patterns in pressure-treated milk samples processed at 90 and 100 oC, respectively. There was a synergistic effect of pressure and temperature on inactivation of spores of the bacterium with increasing temperature. A 4.8-log reduction of the initial spore load of B. amyloliquefaciens was achieved in pressure-treated samples at 100 oC for 3 min; whereas pressure-treated samples processed at 90 oC showed inactivation of only 2.3-log reduction. As with spores of B. stearothermophilus , preheating time did not have any significant effect on inactivation of B. amyloliquefaciens . Similar observations were reported in recent studies
59 assessing the inactivation rates of spores of B. amyloliquefaciens Fad 82 by pressure- assisted thermal processing. Rajan et al., (2006a) reported that 700 MPa at 105 oC for 3 min are necessary to achieve up to 3 log-reductions of the bacterium in egg patties.
Similarly to spores of B. stearothermophilus , there is a tailing effect on inactivation of B. amyloliquefaciens with increasing holding time (Rajan et al., 2006a). In this study, similar combinations of pressure (700 MPa), temperature (105 oC) and holing time (3 min) were sufficient to inactivate spores of B. amyloliquefaciens up to 6.8-log reductions in milk samples. Higher inactivation rates in milk samples might be related to the composition of the food system. The presence of fat and proteins in complex food systems is known to have a protective effect and it is presume to increase the heat resistance of microorganisms with increasing concentration. Also, a w has an important role on the heat resistance of microorganisms. The sensitivity to heat of microorganisms increases with increasing a w values (Jay, 2000). The lethality of B. amyloliquefaciens spores also increases with pressure and temperature (Rajan et al., 2006). Lower pressure– temperature combinations (500 MPa at 95 oC) result in limited inactivation of spores of B. amyloliquefaciens (1 log-reduction). However, the inactivation rates of the bacterium significantly increase with increasing processing pressure and temperature. The authors reported that up to 7 log-reductions of the initial spore load (10 8 spore/g) is achieved at
700 MPa and 121 oC for 1 min (Rajan et al., 2006a). Similarly, Ahn et al., (2007) reported that PATP treatments at 700 MPa and 121 oC for less than 1 min are sufficient to inactivate up to 7 to 8-log reductions of spores of B. amyloliquefaciens Fad 82 and B. amyloliquefaciens Fad 11/2. The authors concluded that these microorganisms along with
60 T. thermosaccharolyticum are the most resistant spores to combinations of high pressure
(700 MPa) and elevated temperatures (121 oC) (Ahn et al., 2007). B. amyloliquefaciens is a mesophilic, aerobic, spore former microorganism extremely resistant to pressure stress conditions (Margosch et al., 2004). It has been originally isolated from ropy bread. Due to its high pressure resistance and non-toxic characteristics, it has been proposed as a surrogate microorganism of C. botulinum for PATP processes validation (Margosch et al., 2004; Ahn et al., 2007). In fact, some strains of B. amyloliquefaciens are more resistant to pressure and temperature than spores of C. Botulinum (Margosch et al., 2004).
Spores of B. amyloliquefaciens TMW 2.479 show up to 2-log reductions after treatment at 800 MPa at 80 oC for 64 min, as compared to 5-log reductions of C. botulinum TMW
2.357 at the same processing conditions. Although there is a synergistic effect of pressure and temperature on inactivation of spores, the medium in which the microorganism is therein and the type of microorganism will ultimately define the pressure–temperature combinations necessary to achieve commercial sterility in low-acid foods (Margosch et al., 2004).
2.4.2 Pressure-temperature profiles of milk samples
Figure 2.3 shows the temperature and pressure history of HPP and PATP milk samples processed at 650 MPa; 32, 72 and 105 oC with holding time of 5 min. The initial temperature of HPP milk samples (650 MPa; 32 oC for 5 min) was 7±1 oC and reached the target processing temperature (31±1 oC) after 1.5 min of compression. No significant heat loss was observed in these samples as a result of adiabatic compression during pressurization. Similarly, no significant heat loss was observed in HPP milk samples with
61 pressure holding times of 0 and 1 min, respectively (data not shown). Pressure treated samples processed at 650 MPa and 72 oC for 5 min were preheated at 48±1 oC before pressurization. This temperature allowed milk to reach a target processing temperature of
77±1 oC during compression. Unlike HPP milk samples processed at 32 oC, pressure treated milk samples processed at 72 oC attained higher processing temperatures possibly due to heat gain experienced as a result of compression during pressurization. The final processing temperature achieved during pressurization (5 min) for PATP milk samples was 104±1 oC. The temperature gradient between the top and bottom of the pressure vessel was assumed to be ~ 10 oC due to lack of research samples designated to thermal studies. The current study, therefore, did not consider the impact of the temperature gradient on the microbial load of HPP and PATP milk samples.
125 700
C) 600 o 100 500 75 400 50 300 200
)Temperature ( )Temperature 25 100 (---) Pressure (MPa) (---) Pressure ( ( 0 0 0 2 4 6 8 10 12 Tim e (min)
Pressure Pressure Depressurization come-up time holding time time
Figure 2.3 Pressure–temperature profiles observed during high pressure treatments of milk a a PATP milk processed at 650 MPa and 32, 72 and 105 oC for 5 min *Data shown are means of two independent samples
62 2.4.3 Microbial load of milk
The microbial population recovered in milk samples processed with different combinations of high pressure and temperature is shown in figures 2.4 – 2.6. Raw milk had a total plate count of 5 x 10 4 CFU/mL (4.7 ± 0.1 log CFU/mL) on 0 d (data not shown). This number was below the established regulatory microbial standards for raw milk (5–5.5 log CFU/mL) intended for human consumption (PMO, 2005). Low microbial loads in raw milk are associated with improvements in handling and milking operations at the dairy farm (Fromm and Boor, 2004). The outstanding progress achieved in milking and transportation of milk has caused the microflora present in raw milk to change from predominantly from Gram-positive to Gram-negative microorganisms (Ledford, 1998).
Due to the complexity of this study, no further efforts were made to identify the microorganisms likely to be present in milk samples. However, it is possible that
Pseudomonas spp. and species of Bacillus such as B. cereus and B. licheniformis might have been present. Pseudomonas spp are probably the most important bacteria responsible for spoilage of milk (Desmasures and Gueguen, 1997; Dogan and Boor,
2003; Fromm and Boor, 2004). These microorganisms are psychrotophs meaning they are capable of growing at refrigeration conditions (4 ± 1 oC) or less regardless of their optimal growth temperature (Ledford, 1998). Some species of Pseudomonas also produce heat resistant proteases and lipases; and therefore, can contribute to spoilage of pasteurized milk. A study assessing the genetic diversity and spoilage potential among
Pseudomonas spp. isolated from fluid milk reported that P. fluorescens is the microorganism with the highest incidence (51%) in processed milk. Most strains of this
63 microorganism (69%) tested positive for all enzymatic activities studied (i.e. protease, lipase and lecithinase activity). The presence of P. flurorescens in milk represents a challenge in dairy operations due to its short generation time at refrigeration conditions.
o However, the microorganism is easily killed by HTST pasteurization (D53 C = 1.16 min); and it only contaminates the milk during postprocessing operations (Jay, 2000; Dogan and Boor, 2003). Other Gram-negative bacteria associated with raw milk include
Achromobacter, Aeromonas, Alcaligenes, Chromobacterium , Flavobacterium and
Lactococcus . Also, Lactobacillus, Acinetobacter, Staphylococcus, Micrococcus and species of coliforms are likely to be present in milk (Ledford, 1998). From these,
Lactococcus sp . and Lactobacillus sp. are commonly found in milk regardless of the level of contamination. Staphylococcus aureus can also be sporadically present in milk
(Desmasures and Gueguen, 1997).
In this study, microorganisms recovered from decimal dilutions of milk samples were inoculated in trypticase soy agar (TSA) plates and evaluated for their total plate count after incubation at 32 oC for 48 h. This media might have contributed to not capturing the above mentioned microorganisms in milk samples. TSA media may not support the growth of fastidious microorganisms such as lactic acid bacteria; and therefore, these microorganisms may not be identify in total plate count analysis. Only those microorganisms capable of growing at incubation temperatures between 32-37 oC will be enumerated after incubation for 48 h. Therefore, microorganisms with different optimal growing conditions may be overlooked during total plate count analysis (Yousef and Calrstrom 2003). Standard plate count was used in this study as an indicator of the
64 microbiological condition of milk samples. However, for a complete evaluation of the microbiological quality of milk other techniques such as laboratory pasteurized count, coliform count, and somatic cell count might be necessary (Fromm and Boor, 2004).
Laboratory pasteurized count is used to estimate those microorganisms present in raw milk that survived pasteurization. The technique is performed by heating raw milk to
63 oC for 30 min, immediately cooling to 10 oC, and plating in plate count agar. This technique can be modified to recover thermoduric bacteria that are able to grow under refrigeration conditions (phsychrotrophic bacteria) by incubating milk samples at 7 oC for
10 d before plating onto standard plate count agar (Marshall, 1992). Suitable media to recover coliforms from milk samples is violet red bile agar and incubating plates between
32 to 37 oC (Fromm and Boor, 2004). The somatic cell count can be measured by performing direct microscopic cell count analysis. This technique is based on the estimation of the total number of somatic cells in a single strip of field diameters under the microscope multiplied by the single strip factor (SSF) (Marshall, 1992).
65 9 8
a 7 6 5 4 3
Log N (CFU/mL) (CFU/mL) N Log 2 1 0 0 5 10 15 20 Time (days)
Figure 2.4 Microbial population recovered in pasteurized and HPP milk samples subjected to different pressure and heating profiles b a Tryptic soy agar (TSA); 30 oC for 48h incubation b Treatments were: A ( ) Pasteurized milk (78 oC, 18 s); ( ) HPP milk (650 MPa, 32 oC for 0 min); ( x ) HPP milk (650 MPa, 32 oC for 1 min); ( x ) HPP milk (650 MPa, 32 oC for 5 min). *Data shown are means ± std. dev. of two independent samples
Figure 2.4 shows colony forming units (CFU/mL) recovered in HTST pasteurized and HPP milk samples processed at 650 MPa and 32 oC for 0, 1 and 5 min, respectively.
The total plate count in preheated milk samples was not determined. Pressure treatments delivered a microbial load in milk on par or below pasteurization (3.8 ± 0.0 log
CFU/mL). The microbial growth rate in pasteurized milk remained to some extent constant throughout its shelf life; and reached up to 4.2 ± 0.2 log CFU/mL on 20 d.
Pressure treatments at room temperature (32±1 oC) delivered milk with similar microbial load than pasteurized milk (2.8 – 3.3 ± 0.14 log CFU/mL) on 0 d; and pressure holding time did not exert any significant effect on the microbial load of these samples. The microbial population in HPP milk samples treated at 32 oC did not significantly differed
66 among milk samples with different holding times (0, 1 and 5 min) on 0 d and remained constant until 4 d. However, significant differences on the microbial load of pressure- treated samples at 32 oC for 0, 1 and 5 min, respectively, were observed on 6 d and throughout the rest of their shelf life (20 d). On 20 d, the microbial population for these samples ranged from 5.5 to 7.0 ± 0.27 log CFU/mL. Even though milk samples had similar microbial loads on the initial day of analysis, there were large changes in the microflora of milk samples during their shelf life. These results suggest that microorganisms that survived the thermal process or those present as a result of post processing contamination might have contaminated the milk. It is also extremely important that the equipment in the dairy plant is properly cleaned and sanitized to avoid contamination with microorganisms (Ledford, 1998). The shelf life of HPP milk samples processed at 32 oC was approximately of 14 d (Figure 2.4). This value was 6 d shorter than the typical shelf life observed in HTST pasteurized milk (Fromm and Boor, 2004).
Reduction of the shelf life of HPP milk samples treated at 32 oC is associated with the growth of bacteria. The shelf life of processed milk is influenced not only by the initial bacterial count, but more importantly by the type of microorganisms capable of surviving the heat and/or (in this case) the high pressure process. The presence of these microorganisms in milk ultimately defines the quality of the final product.
Microorganisms capable of surviving heat processes include thermoduric bacteria, bacterial spores and psychrotrophs that contaminate the milk post processing.
Thermoduric bacteria are those microorganisms capable of surviving the pasteurization process. These are usually mesophilic microorganisms that grow in the range of 20 to
67 45 oC; but they generally grow at a lower rate in refrigerated milk. Species of these microorganisms include Micrococcus, Bacillus and Lactobacillus. Also, some thermoduric strains of Enterococcus fecalis and Enterococcus faecium are sporadically present in pasteurized milk (Ledford, 1998). The incidence of thermoduric bacteria is relatively high as compared to other type of microorganisms isolated from milk. About
27% incidence of psychrotrophic termoduric bacteria has been reported in pasteurized milk. From these, Bacillus sp. has been recognized as one of the microorganisms with the highest incidence in milk (86%). Other bacteria of significance previously reported in pasteurized milk include Micrococcus spp., and those belonging to the coryneform group
(Johnston and Bruce, 1982). A study assessing the incidence of thermoduric bacteria in milk sampled from three processing plants during four months reported that species of
Bacillus and Microbacterium are likely to be present in milk. The authors reported that
42 % of the isolates from pasteurized milk were identified as Gram-positive cocci, 38 % as Gram-positive rods and 19 % as Gram-variable rods. And, the most frequent genera were Paenibacillus spp. (39%), Bacillus spp. (32%), and Microbacterium spp. (14%).
Other microorganisms found in pasteurized milk included Kocuria varians (formely
Micrococcus varians ) (5%), Pseudomonas (3%) and Acinetobacter (1%) (Fromm and
Boor, 2004). Paenibacillus spp. seems to be the genus with the highest incidence in milk from farm to packaged product. Even though research data indicates that the microflora of raw is considerably diverse, this microorganism has been consistently isolated from milk in all points of the processing chain in recent years. Common sources of contamination by Paenibacillus spp. include tank trucks (91.2%), dairy plant storage silos
68 (51.8%) and pasteurized milk in processing lines and packaged product (76.6%).
Paenibacillus spp. is a spore former psychrotrophic bacteria capable of surviving the pasteurization process and grow under refrigeration conditions (Huck et al., 2008). This
Gram-positive aerobic microorganism is closely related to the genera Bacillus and
Clostridium (Jay, 2000). The microorganism is responsible for degradation of proteins and polysaccharides in milk. Moreover, some Paenibacillus strains produce antibacterial and antifungal compounds; and therefore their ability to outcompete other microorganisms in milk (Scheldeman et al., 2006). In addition to Paenibacillus spp.,
Bacillus cereus and B. licheniformis are the microorganisms with the highest incidence in pasteurized milk (Crielly et al., 1994; Janstova et al., 2006; Huck et al., 2008). B. licheniformis has been more commonly isolated from dairy plants (Fromm and Boor,
2004). The incidence of this microorganism in milk has been reported in more recent studies. From all Bacillus species isolated from milk, the incidence of B. licheniformis can be as high as 42.5% as compared to the 15.5% incidence of B. cereus (Banyko and
Vyletelova, 2009). Microbacterium lacticum can also contribute to the total number of isolates collected from pasteurized milk. Fromm and Boor (2004) reported that the incidence of this microorganism in milk can be as high as 27%, followed by Kocuria spp.
(27%) and Streptococcus spp. (12%). B. cereus and B. licheniformis are ubiquitous microorganisms; and therefore, they can be isolated from soil, feed, manure and the farm environment. These microorganisms are also spore formers; and therefore, they represent a challenge during processing of milk. The resistance of these microorganisms in their
o dormant state to temperature is very similar. The reported D 95 C values for spores of B.
69 cereus and B. licheniformis are 5.01 and 5.1 min, respectively (Jay, 2000). The heat resistance of spores of the two species has been established above 100 oC; and the growth rate of these microorganisms is faster at room temperature than under refrigeration conditions (Crielly et al., 1994). The above published information clearly indicates that the sources of milk contamination by microorganisms can occur from the farm environment to the finished product. Therefore, the total bacterial count and the microorganisms recovered in milk samples of this study were significantly influenced by the processing plant environment and specific standard operational procedures.
9.00 8.00 7.00 a 6.00 5.00 4.00 3.00 Log (CFU/mL) Log 2.00 1.00 0.00 0 10 20 30 40 50 60 Time (days)
Figure 2.5 Microbial population recovered in HPP milk samples subjected to different pressure and heating profiles b a Tryptic soy agar (TSA); 30 oC for 48h incubation b Treatments were: ( ) HPP milk (650 MPa, 72 oC for 0 min); ( ) HPP milk (650 MPa, 72 oC for 1 min); ( x ) HPP milk (650 MPa, 72 oC for 5 min). *Data shown are means ± std. dev. of two independent samples
The microbial load in milk further decreased with increasing temperature.
70 Pressure treatments at 72 oC were sufficient to achieve up to 3-log reductions from the initial microbial load in milk as shown in Figure 2.5. Pressure holding time had a significant effect on the initial microbial load of pressure–treated samples processed at
72 oC. At this temperature, the microbial population of milk samples further decreased with increasing holding time. Milk samples processed at 72 oC for 0 min had an initial microbial load of 2.2 ± 0.1 log CFU/mL; whereas the initial microbial load in pressure– treated samples processed at 72 oC for 1 and 5 min was 1.6 and 1.5 ± 0.15 log CFU/mL, respectively. Pressure holding time had also a significant effect on the microflora of pressure treated milk samples processed at 72 oC during their shelf life. The growth rate of microorganism was significantly lower in milk samples processed at higher holding times
(1 and 5 min) than that of milk samples processed without pressure holding time (0 min).
With exception of HPP milk samples processed at 72 oC for 0 min, the exponential growth phase of microorganisms in milk samples processed with holding times of 1 and 5 min was observed on 10 d. On 20 d, the microbial population in HPP (72 oC) milk samples processed for 5 min (4.4 ± 0.8 log CFU/mL) was not significantly different from that of pasteurized milk (4.2 log CFU/mL). These results indicate that combinations of high pressure (650 MPa) and pasteurization temperature (72 oC) under refrigeration conditions are sufficient to increase the shelf life of milk up to 45 d. However, the microbial load in milk further increased during the shelf life studied. On 60 d, the microbial population in pressure–treated milk samples processed at 72 oC ranged from 6.7 to 7.2 ± 0.47 log
CFU/mL. These results indicate that more aggressive combinations of temperature and pressure might be necessary to extend the shelf life of milk. However, the type of
71 microorganisms present in milk, as discussed earlier, ultimately defined the quality of the product. Similar results have been reported previously. High pressure processing in combination with mild temperatures is effective on inactivation of vegetative bacteria
(Garriga et al., 2004). Up to 5–log reductions of the initial bacterial count of Salmonella spp., Staphylococcus aureus and Vibrio parahaemolyticus can be achieved by HPP
(Torres and Velazquez, 2005). Combinations of ~ 600 MPa with temperatures ranging form 40 to 55 oC have been also shown to be effective on inactivation of vegetative bacteria in milk. A recent study assessing the effects of HPP on the microbiological quality of milk reported that Pseudomonas spp. and members of the enterobactericeae family can be inactivated by combinations of 586 MPa and 55 oC for 5 min. Furthermore, the authors reported that the microbial load in pressure–treated milk samples remained lower than that of commercial pasteurized milk samples for up to 45 d (Tovar-Hernandez et al., 2005). Similar inactivation effects have been shown in Escherichia coli , P. fluorescens , Listeria innocua , L. monocytogenes and L. helveticus by HPP in combination with either low (4 oC) or mild temperatures ( > 25 oC) (Trujillo et al., 2002; Lado and
Yousef, 2002; Garriga et al., 2004). These inactivation patterns are equivalent to pasteurization processes; and therefore storage under refrigeration conditions, low a w or low pH are necessary to extend the shelf life of HPP foods (Lado and Yousef, 2002;
Torres and Velazquez, 2005).
72 8 7 6 a 5 4 3 2 Log (CFU/mL) Log 1 0 -1 0 20 40 60 80 100 Time (days)
Figure 2.6 Microbial population recovered in UHT and PATP milk samples subjected to different pressure and heating profiles b a Tryptic soy agar (TSA); 30 oC for 48h incubation b Treatments were: ( ) PATP milk (650 MPa, 105 oC for 0 min); ( ) PATP milk (650 MPa, 105 oC for 1 min); ( x ) PATP milk (650 MPa, 105 oC for 5 min); ( x ) UHT milk (139 oC for 2 s). *Data shown are means ± std. dev. of two independent samples
As expected, UHT treatment delivered milk with no significant microbial load on
0 d; and remained constant throughout the shelf life period studied (90 d) as shown in
Figure 2.6. These results are in agreement with the microbiological standards established for UHT milk. According to the European Union Hygiene directive 92/46 EEC the maximum microbial load in UHT milk should be < 10 CFU/0.1 mL in sealed packages after 15 days of incubation at 30 oC (Anonymous, 1992). PATP milk showed similar microbial load to UHT milk samples on 0 d. As with HPP milk samples treated at 32 and
72 oC, there was a synergistic effect of pressure, temperature and holding time on inactivation of microorganisms in PATP milk processed at 105oC. Up to 4-log reductions
(CFU/mL) of the initial microbial load of milk were obtained in milk samples processed at 650 MPa and 105 oC for 0, 1 and 5 min. There were no significant differences in the
73 total microbial load of PATP milk with increasing holding time on 0 d. However, the microflora of PATP milk significantly changed during the shelf life period studied. PATP processing conditions were expected to have similar microbial inactivation effect than
UHT processing. However, the microbial growth rate in PATP milk (650 MPa and 105 oC for 0, 1 and 5 min) was faster than pasteurized, UHT and HPP milk samples (650 MPa,
32 and 72 oC for 0, 1 and min). Holding time during pressurization and storage temperature had a significant role on the growth rate of microorganisms in PATP milk samples. The microbial growth rate in PATP milk stored at room temperature (25 ± 1 oC) was significantly faster than that of HPP milk samples stored at refrigeration conditions
(4 ± 1 oC). PATP milk samples processed at 105 oC for 0 min showed no lag phase of growth; and they were noticeably spoiled and therefore discarded on 30 d. The maximum microbial load observed in these samples was 5.5 ± 0.23 log CFU/mL on 15 d. Similarly,
PATP milk samples processed at 105 oC for 1 min showed a maximum microbial load of
5.7 ± 0.27 log CFU/mL on 30 d; and on 45 d these samples were discarded. Pressure– treated milk samples processed at 105 oC for 5 min resulted with the slowest microbial growth rate among PATP milk samples. These samples showed a maximum microbial load of 6.5 ± 1 log CFU/mL on 45 d. These results suggest that bacterial spores might have been present in milk samples and therefore, shortened their shelf life. Even though initial bacterial counts were not detected in PATP milk on the initial day of analysis, significant growth of psychrotropic spore forming bacteria might have influenced the shelf life of milk. Similar observations have been reported previously. The growth of bacterial spores in milk can be observed after 7 to 14 d of storage (Huck et al., 2008). A
74 recent study assessing the sources of contamination by B. cereus and B. licheniformis in milk reported that there are different points of entry of these microorganisms into the product. Genomic fingerprinting analyses (BOX – PCR) revealed that Bacillus spp. isolated from raw milk were not the same strains found in pasteurized milk. These results indicate that not only the farm environment is a source of contamination; but also processing lines can be additional sources of contamination of pasteurized milk.
Therefore, these microorganisms are not found exclusively in raw and/or processed milk but also result from post processing contamination and propagation during milk processing (Banyko and Vyletelova, 2009). The identification of points of entry of bacteria gives reliable insights to reduce or eliminate their presence in the milk processing chain; and therefore extend the shelf life of milk (Huck et al., 2008). HPP and
PATP treatment conditions used in the study were not optimized for extended milk storage. More systematic studies are needed to identify optimal process conditions that can ensure desired shelf-life extension without compromising milk quality.
In addition to their toxin–producing characteristics (i.e. B. cereus ), the presence of bacterial spores can be detrimental of milk’s quality. These microorganisms are capable of producing extracellular proteases and lipases which degrade caseins (mainly αs – and
β-caseins) and lipids giving rise to bitter and rancid off-flavors in milk (Janstova et al.,
2006). These enzymes are generally produced during the late exponential and early stationary phases of growth (Chen et al., 2003). A recent study assessing the effects of
Bacillus cereus enzymes in the quality of UHT milk reported that defects caused by bacterial enzymes were observed in milk samples with microbial counts of 1 x 10 4 – 10 5
75 CFU/mL. Moreover, these defects were more pronounced in milk samples stored at room temperature (25 oC) than those observed in samples stored under refrigeration conditions
(4 oC). The extent of proteolysis in milk samples was less than lipolysis caused by bacterial lipases over a period of 3 weeks. This was attributed to the fact that B. cereus strains have higher lipolytic activity than proteolytic capability. High concentrations of
FFA were found in milk samples even with low initial spore counts. The authors reported that processing and storage conditions directly affected the extent of proteolysis and lipolysis in milk samples. However, there were significant differences in production of bacterial enzymes even within the same bacterium species (Janstova et al., 2006). In addition to Bacillus spp., Pseudomonas spp. are lipolytic psychrotrophic bacteria commonly found in processed milk (48% incidence); and they have been recognized as major contributors to lipolyzed off-flavors in the product (Chen et al., 2003; Fromm and
Boor, 2004). Lipases from Pseudomonas spp. have shown thermal stability at temperatures up to 120 oC for 4.2 min, with optimum pH values ranging from 7.5 to 9
(Chen et al., 2003). Bacterial enzymes have different substrate specificities and characteristics that ultimately determine the extent of proteolysis and lipolysis in milk.
These characteristics are further discussed in chapter 3. Processing and handling of milk also have a significant role on the enzymatic activity in milk. Not only close control over the temperature–time combinations is desirable during processing, but also unit operations such as agitation, cooling and warming rates and homogenization are key factors that influence the activity of enzymes in milk.
The efficacy of the high pressure process on inactivation of bacteria depends on
76 initial temperature, heat of compression value, pressure applied, and processing time and temperature (Garriga et al., 2004; Ahn et al., 2007). However, the food matrix in which microorganisms are therein and environmental parameters are key factors that influence the extent of bacterial inactivation in foods (Torres and Velazquez, 2005). The resistance of microorganisms to physical treatments increases with decreasing water activity (a w) values. The reasons for this increased resistance at lower humidity values are not clear.
However, researchers have suggested that the rate of protein denaturation of cell contents occurs faster in water than in air. The presence of proteins, fats and carbohydrates in the food matrix also has a protective effect on the lethality of microorganisms. Foods with high concentration of these compounds must be processed at significantly higher temperatures than foods with low protein, carbohydrate and/or fat content. The resistance of microorganisms to processing is also pH dependent. At a constant temperature, the heat sensitivity of microorganisms increases if the pH of the system is raised or lowered from their optimum pH of growth (Jay, 2000). More importantly, the type of microorganism therein the food system will ultimately determine the microbial inactivation values. Bacterial spores are generally more resistant to physical inactivation than vegetative microorganisms (Lado and Yousef, 2002). The increased resistance of spores is associated with the spore structure and the chemical composition of its components. The cortex is the thickest membrane layer exclusively found in spores. This membrane consists mainly of peptidoglycan; and it is protected by an inner and outer coat consisting of a thick layer of cystein–rich proteins. These components have a significant protective effect against heat, lytic enzymes and hydrogen peroxide (Setlow and Johnson,
77 2007). The spore DNA is protected against physical damage by a group of proteins called small–acid–soluble proteins (SASP). SASP proteins and dipicolinic acid (DPA) are also associated with the increased resistance of bacterial spores to physical treatments (Lado and Yousef, 2002; Setlow and Johnson, 2007). Gram-positive bacteria are more resistant to physical inactivation than Gram-negative. As with bacterial spores, the reason of the increased resistance of Gram-positive microorganisms is associated with the rigidity of their cell wall. The characteristics of the cell membrane influences, therefore, the extent of bacterial inactivation by physical treatments. Cell membranes with higher concentration of unsaturated fatty acids have increased resistance to pressure and thermal treatments. These membranes have also low concentrations of diphosphatidylglycerol which is associated with higher pressure resistance capabilities (Lado and Yousef, 2002).
Up to date, thermal sterilization is still the most effective method to ensure absence of bacterial spores in foods. Even though combinations of ultra high pressure
(~700 MPa) with higher processing temperatures (105 – 121 oC) have shown to be effective on inactivation of bacterial spores, researchers have reported that the patterns of microbial inactivation during PATP treatments show a tailing or shoulder effect (Lado and Yousef, 2002; Margosch et al., 2004; Rajan et al., 2006a; Rajan et al., 2006b; Ahn et al., 2007). This shoulder effect, or the percentage of survivors after a given period of time during physical treatment, can be explained by variations in the degree of resistance of strains within the same population or by the fact that only a fraction of the population was affected by the treatment at a given time. The later theory is associated with sublethal injury, cell clumping and non-uniform treatment effects. Therefore, microorganisms are
78 more likely injured and not completely inactivated during PATP (Lado and Yousef,
2002). More aggressive thermal and high pressure treatments are, therefore, necessary to ensure that the target microorganisms exist in the food system to undetectable levels during the shelf life of the product. Ahn et al., (2007) reported that combinations of pressures ranging from 500 to 700 MPa, holding times from 5 to 30 min and temperatures between 50 and 80 oC are necessary to achieve spore inactivation of up to 4 to 7-log reductions (spore/mL) of the initial spore count. Similarly, inactivation of spores is more effective in processes with two or more pressurization stages. The first stage involves a high-pressure induced germination step; whereas in the second and subsequent stages germinated spores are killed (Patterson et al., 2006). Moreover, sterility in foods can be achieved with minimal impact on their nutritional and quality characteristics if multi– pulse pressure treatments are applied in combination with temperatures above 105 oC
(Meyer et al., 2000).
The mechanisms of bacterial inactivation by ultra high pressure in combination with temperature are associated to accumulated damage of the microbial cell (Garriga et al., 2004). The inactivation effects include changes to the cellular structure and/or physiological functions of the cell. Inhibition of protein synthesis by inactivation of key enzymes occurs at pressures < 200 MPa. Also, membrane nutrient transport is disabled and reversible protein denaturation occurs at this pressure level. The rate of microbial inactivation increases exponentially with increasing pressure. Breakage of the cell membrane, irreversible protein denaturation and leakage of cell contents is observed at pressures > 300 MPa (Lado and Yousef, 2002). Torres and Velazquez (2005) reported
79 that HPP treatments at 345 MPa and 25 oC for 5 min are sufficient to disrupt the membrane cell wall and make it permeable. This damage prevented cells from synthesizing ATP, which activated an autolytic enzyme degradation of cell walls.
Limited information is available on the mechanisms of spore inactivation by PATP treatments. However, some researchers have pointed out that pressure treatments ranging from 50 – 300 MPa induce a germination step followed by inactivation of bacteria at higher pressure and temperature combinations (Lado and Yousef, 2002). Additionally, spore inactivation patterns are related to denaturation of SASP in the spore core and subsequent DPA leakage with increasing pressure (Torres and Velazquez, 2005). The specific role of SASP on inactivation of bacterial spores is unknown up to date, and further research is necessary to fully understand the mechanisms of bacterial spore inactivation under PATP treatments.
2.5 Conclusion
Milk was successfully treated by high pressure and pressure–assisted thermal processing. Close control over the preheating time and temperature was critical to develop an integrated process taking into account the desired microbiological effects and quality characteristics of milk. There was a synergistic effect of pressure, temperature and holding time on inactivation of heat– and pressure–resistant spores in milk. Up to 6 log– reductions of the initial spore load of Bacillus stearothermophilus ATCC 7953 were obtained in milk PATP treated at 700 MPa and 90, 100 and 105 oC for 3 min. The lethality of B. amyloliquefaciens Fad 82 spores increased with pressure and temperature.
80 Up to 6.8 log-reductions of spores of the bacterium were achieved in milk samples after
PATP treatment at 700 MPa at 105 oC for 3 min. Pressure treatments at 650 MPa and 25,
72 and 105 oC for 0, 1 and 5 min, respectively, delivered a microbial load in milk on par or below pasteurization and UHT treatments. Even though milk samples had similar microbial loads on 0 d, there were large changes in the microflora of pressure–treated milk samples as compared to pasteurized and UHT milk during the shelf life studied.
Pressure, temperature, holding time and storage temperature significantly influenced the rate of microbial growth in milk. In addition, the batch–system nature of HPP did not allow for a continuous aseptic operation allowing more points of entry for bacteria during milk processing. The type of microorganisms present in milk ultimately defined the shelf life of the product. Thermoduric microorganisms, bacterial spores and psychrotrophs that contaminate the milk post–processing are commonly associated with the product. From these, Pseudomonas fluorescens , Bacillus licheniformis and Paenibacillus spp. are microorganisms with the highest incidence in processed milk. In addition to their toxin– producing capabilities, these microorganisms are produce heat–resistant proteases and lipases that further shorten the shelf life of the product. These results indicate that HPP and PATP are capable of rendering milk with a microbial load close to pasteurized and
UHT milk, respectively. However, not only pressure–temperature combinations during processing, but sanitary standard operational procedures at the dairy plant and storage temperature will ultimately define the quality and shelf life of milk.
81 Chapter 3: Chemical Stability and Residual Plasmin Activity
3.1 Abstract
The effects of different pressure–temperature combinations on the chemical stability and residual plasmin activity in milk were studied and compared to those of
HTST pasteurized (77 ± 0.8 oC for 18 s) and ultra high temperature (138 ± 1 oC for 2 s) processed (UHT) milk obtained from commercial source. Milk was high pressure processed (HPP) and pressure-assisted thermal processed (PATP) using a factorial 3x1x3 model at temperature (32, 72 and 105 oC), pressure (650 MPa), and time (0, 1, and 5 min).
The chemical stability of milk samples was evaluated based on their proteolysis and lipolysis values over a period of 20, 45 and 90 d depending on the treatment applied.
Proteolysis was assessed by SDS-PAGE analysis; and lipolysis was measured by a modified copper soap method. Total plasmin activity was evaluated after extraction from milk samples using BODIPY FL-Casein as a fluorogenic substrate for plasmin. With exception of milk samples processed at 650 MPa and 105 oC for 0 min, no significant proteolysis was observed in pressure-treated samples at the end of their shelf life.
Pressure-treated samples processed at 72 oC for 0, 1 and 5 min, respectively, showed the least extent of relative proteolysis among milk samples. Lipolysis was enhanced by pressure, holding time during pressurization and storage temperature. Pressure-treated samples showed higher concentration (0.5 meq FFA/kg of milk) of free fatty acids (FFA)
82 on 0 d than pasteurized and UHT milk samples. Plasmin inactivation rates increased with increasing temperature. Although the enzyme was not completely inactivated, combinations of temperatures (105 oC) with ultra high pressure (650 MPa) were sufficient to inactivate the enzyme to levels similar to UHT processes. These results indicate that the pressure–temperature combinations used in this study were capable of rendering milk with enzymatic activity profiles close to pasteurized and UHT milk. However, processing temperature and storage conditions had a significant role on the extent of protease and lipase activity in milk.
3.2 Introduction
Chemical changes can occur in dairy products as a result of enzymatic activity.
Such changes are not desirable in fresh milk as they contribute to adverse organoleptic effects, and thus limit its shelf life. In addition to bacterial enzymes, the activity of some naturally occurring or indigenous enzymes is detrimental of milk’s quality. Up to seventy different indigenous enzymes have been identified in fluid milk. And, only twenty have been fully characterized (Fox and Kelly, 2006a). Lipoprotein lipase (LPL), proteinases, acid phosphatase and xanthine oxidoreductase (XOR) are probably the most significant enzymes responsible for deterioration of milk constituents (Fox and Kelly, 2006a,b).
From a technological standpoint, LPL and plasmin are probably the most challenging enzymes in many dairy processes due to their high temperature stability (Chen et al.,
2003).
83 Milk contains relatively low levels of lipases. From these, LPL is the most abundant indigenous lipase found in milk. The enzyme has an optimum activity at pH 9 and 37 oC (Fox and Kelly, 2006a). LPL is responsible for hydrolytic rancidity in fresh raw milk, with activity values between 0.8 and 1 IU/mL at pH 7 and 37 oC (Chen et al., 2003).
An increase in LPL activity is observed, however, in pasteurized milk due to temperature changes and shear forces encountered during the pasteurization process (Chen et al, 2003;
Fox and Kelly, 2006a). Homogenization of milk damages the milkfat globule membrane
(MFGM), making fat globules prone to the action of lipases. Also, the balance between
LPL activators and inhibitors determines the level of lipolysis in milk. LPL is a relatively heat resistant enzyme compared to plasmin. HTST pasteurization (72 oC, 15sec) is sufficient to almost completely inactivate the enzyme in milk. Temperatures up to 65 oC for 20 min are necessary to completely inactivate LPL (Chen et al., 2003).
Plasmin (EC 3.4.21.7) is the principal indigenous proteinase in milk (Bastian and
Brown, 1996; Saint-Denis et al., 2001; Chen et al., 2003; Kelly et al., 2006a; Fox and
Kelly, 2006; Pereda et al., 2008). It is found in blood plasma and secreted into the milk through intersections between mammary cells (Fox and Kelly, 2006a). The enzyme is an alkaline trypsin-like proteinase with optimal activity at pH 7.6 and 37 oC (Kelly et al.,
2006). In milk and blood plasma, plasmin consists of a complex system formed by plasmin, its inactive form plasminogen, plasmin/plasminogen inhibitors and plasminogen activators (PAs). Plasmin intimately associates with casein micelles at pH values between
6.6 and 4.8. Plasminogen and PAs are also associated with casein micelles; while inhibitors of plasmin and plasminogen are mainly found in the serum phase of milk
84 (Bastian and Brown, 1996; Fox and Kelly, 2006a). Plasminogen consists of a glycoprotein with five disulfide bridges. Upon storage of milk (Bastian and Brown,
1996), plasminogen is converted into active plasmin by cleavage of the Arg 557 – Ile 558 bond by two types of proteinases: urokinase and tissue-type PAs (Fox and Kelly, 2006a).
Once it is activated, plasmin specifically hydrolyzes Lys and Arg peptide bonds of αs1 -,
αs2 - and β-caseins. Κ-casein; β-lactoglobulin and α-lactoalbumin are not affected by plasmin activity in milk (Richardson and Pearce, 1981; Fox and Kelly, 2006a).
Plasmin is partially resistant to UHT processes (>138 oC for less than 4 sec)
(Prado et al., 2006; Newstead et al., 2006). Longer periods of time at high temperatures are necessary to completely inactivate the enzyme. Temperatures, up to 120 oC for 15 min, are necessary to ensure absence of plasmin activity in milk
(Chen et al., 2003). Borda et al., (2004) observed that the inactivation pattern of the enzyme follows first order reaction kinetics, with irreversible denaturation between 65 –
85 oC (Borda et al., 2004; Pereda et al., 2008). At such temperatures, interference of sulfhydryl groups from partially denatured β-lactoglobulin makes plasmin prone to heat inactivation (Kelly and Foley, 1997). Upon storage, sulfhydryl groups become unavailable as the protein refolds in an attempt to minimize its free energy. Therefore, in absence of SH- groups both plasmin and plasminogen are very heat stable and are responsible for gelation of UHT milk (Bastian and Brown, 1996; Fox and Kelly, 2006a;
Pereda et al., 2008). Plasmin activity is also enhanced by pasteurization. Inhibitors of
PAs are inactivated at temperatures close to HTST conditions (72 oC for 15 sec), and thus there is an increase in plasmin activity in pasteurized milk (Kelly et al., 2006a). Prado et
85 al., (2006) concluded that inhibitors of plasmin and PAs have different thermal stabilities.
The activity of plasminogen inhibitors significantly decreases (90%) in buffer samples subjected to 74.5 oC for 15 sec. At such temperatures, plasmin inhibitors retain more activity (64%) compared to that of PAs inhibitors (Prado et al., 2006).
Some researchers have studied the effects of high pressure homogenization (HPH) on inactivation of the plasmin system (Hayes and Kelly, 2003; Sandra and Dalgleish,
2005; Pereda et al., 2008). The results of this work showed that plasmin and plasminogen inactivation is influenced by homogenization pressure, shear forces and inlet temperature
(Hayes and Kelly, 2003). Data available on inactivation of lipases and proteases by high pressure processing (HPP) is fairly limited (Scollard et al., 2000; Garcia-Risco et al.,
2003; Borda et al., 2004; Huppertz et al., 2004a; López-Fandiño, 2006). The application of high pressure in combination with temperature, or pressure-assisted thermal processing
(PATP), seems an excellent alternative to process extended shelf life milk. By choosing adequate process combinations of high pressure and temperature, bacterial spores and enzymes can be inactivated without compromising the quality characteristics of milk
(Balasubramaniam, 2003; Paredes-Sabja et al., 2007). Borda et al., (2004) reported pressure stability of plasmin when the enzyme is exposed to hydrostatic pressures ranging from 100 to 800 MPa for 60 min at room temperature. However, with increasing temperature a synergistic effect of temperature and pressure is observed on inactivation of plasmin. The authors reported an increase on the rate of thermal inactivation of plasmin with pressures ranging from 100 to 800 MPa and temperatures between 30 and
65 oC. The application of higher temperatures in combination with pressure could
86 significantly extend the shelf life of milk. Therefore, the objectives of this study were to evaluate the chemical stability of HPP, PATP, HTST pasteurized and UHT milk by comparing their proteolysis and lipolysis values over time. A sensitive protease assay was also developed to measure residual plasmin activity in milk samples.
3.3 Materials and Methods
3.3.1 Milk preparation
Raw milk was obtained from a commercial dairy plant in Ohio (Orrville, OH) and transported to the OSU dairy pilot plant. The temperature of milk was kept at ~ 4 oC at all times during transportation (approximately 2 hr). Milk was standardized to 2% milkfat and two-stage homogenized using a Lab 100 M-G homogenizer (Lubeck-Schlutut,
Germany). Homogenized milk was immediately cooled to 7±1 oC and stored under refrigeration conditions (4±1 oC) until HPP and PATP processing as shown in Figure 3.1.
All milk samples were subjected to various pressure-heat conditions within 72 hrs. On the day of processing, milk samples were preheated to their corresponding initial temperature
(Ti) using an UHT/HTST Lab-25HV Hybrid unit (Micro Thermics Inc., Raleigh, NC).
The initial temperature of milk samples was adjusted as a function of the final target pressure during processing. This temperature was estimated based on the heat of compression of water and skim milk for various temperatures (Balasubramaniam et al.,
2004). The preheating time for milk to achieve the desired initial temperature was 2.5 min. The temperature of milk samples during preheating was set at values slightly higher than their required initial temperature (Ti) to compensate for the heat loss during filling
87 and transportation of milk from the OSU dairy pilot plant to the high pressure processing laboratory. The initial temperature of milk samples was estimated based on the following equation:
CH Ti = T max − x∆P + ∆TH 100
Where, Ti = initial temperature before pressurization; Tmax = final processing temperature during pressurization; CH = heat of compression; and ∆P = applied pressure,
∆TH = Heat loss during pressurization; and Ti accounts for heat loss during preheating until pressure treatment (1±0.5 oC). The preheating temperature of milk samples was set at
4, 53 and 78±1 oC, respectively, depending on the pressure-heat treatment applied. Milk was filled into light-protected 8 oz polyethylene teraphtalane (PET) bottles without head space and manually capped. Immediately after, milk samples were transported from the
OSU dairy pilot plant to the high pressure processing laboratory. The total heat loss during preheating of milk until pressure treatments was 1 ± 0.5 oC. This minimal heat loss was achieved by placing milk bottles in a water bath set at the same temperature of milk samples.
88 Raw milk UHT milk (Orrville, OH) Commercial source (138±1 oC; 2 s) (New Jersey, NY) Clarification Milk Hauling (~4 oC, 2hr) (Orrville, OH) OSU (Columbus, OH)
Separation Clarification (7±1oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Separation 2% milkfat (60±1 oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Homogenization 2% milkfat OSU (Columbus, OH) (Orrville, OH)
Homogenization 500/1000 psi Pasteurization OSU (Columbus, OH) (77±0.8 oC) (Orrville, OH) Milk Storage ≤ 72 hr (4±1 oC) OSU (Columbus, OH)
HPP and PATP (650 MPa; 32, 72 & 105 oC; 0, 1 & 5 min) OSU (Columbus, OH)
Proteolysis and Lipolysis Total Plasmin Activity 20, 45 and 90 d 0 d HPP,PM, PATP & UHT HPP & PM (4 oC); PATP & UHT (25 oC) (-40 oC, until analysis) Figure 3.1 Schematic diagram of milk processing and storage conditions
89 3.3.2 High pressure processing
An S-IL-110-625-08-W cold isostatic press system (Stansted Fluid Power Ltd.,
Essex, UK) was used to high pressure processed (HPP; 650 MPa, 32 & 72 oC) and pressure-assisted thermal processed (PATP; 650 MPa, 105 oC) milk samples. A 1:1 ratio propylene glycol, water mixture was used as the pressure-transmitting fluid. Milk samples were processed using a factorial 3x1x3 model at temperature (32, 72, and
105 oC), pressure (650 MPa) and time (0, 1 and 5 min) by duplicate. Pressure come-up time ranged between 1.5 to 3 min depending on the treatment applied, and decompression time was 1.2 min. The temperature of the vessel and pressure-transmitting fluid was thermostatically adjusted to the desired initial processing temperature by circulating propylene glycol through the external jacket of the pressure chamber. This temperature was set at the same initial temperature of milk samples (6, 48 and 73 oC, respectively).
Milk samples were filled into a cylindrical sample basket (102 mm dia x 559 mm height)
(Stansted Fluid Power Ltd., Essex, UK) and loaded into high-pressure equipment using a mechanical lift mechanism. The temperature of milk samples during various pressure treatments was monitored at the center of the carrier basket using T-type thermocouples
(Omega engineering, CT, USA) placed inside of a milk sample bottle designated for temperature recording purposes. After decompression, pressure treated milk bottles were immediately placed in an ice-water bath to stop further thermal damage; and stored at either room temperature (25 ± 1 oC) or refrigeration (4 ± 1 oC) conditions depending on the final pressure/temperature treatment applied. Extra aliquots (n = 4) of each milk sample were taken on 0 d and quick frozen (-40 oC) until analyzed for their total plasmin activity.
90 All pressure-heat treated milk samples used in this study were processed with these conditions unless noted below.
3.3.3 Thermal treatments
High-temperature-short-time pasteurized milk (2% milkfat), obtained from the same batch of raw milk used for pressure-heat treatments, was processed in a commercial dairy operation (Orrville, OH) at 77±0.8 oC for 18 s. Additionally, ultra high temperature
(UHT) treated (138±1 oC for 2 s) milk samples were obtained commercially (New Jersey,
NY). HTST pasteurized and UHT milk samples were stored under refrigeration condition
(4±1 oC) and room temperature conditions (25±1 oC), respectively, and analyzed for their proteolysis and lipolysis values over a period of 20, 45 and 90 d, depending on the treatment applied. HTST pasteurized and UHT milk samples were also analyzed for their total plasmin activities values on 0 d.
3.3.4 Milk analyses
Chemical stability analyses of HTST pasteurized, UHT and pressure-treated milk samples were assessed by measuring their proteolysis and lipolysis values. Milk was sampled at regular intervals over a period of 20, 45 and 90 d, as shown in Table 3.1. The effect of processing conditions on the residual plasmin activity of milk was measured on the initial day of analysis (0 d).
91 Time (days) Group a 0 2 4 6 10 15 20 30 45 60 75 90 A ●b ● ● ● ● ● ● B ● ● ● ● ● ● ● C ● ● ● ● ● ● ● Table 3.1 Longitudinal scheme of milk analyses a Group A: HTST pasteurized (78±1 oC for 18 s) and HPP milk (650 MPa, 25 oC for 0, 1 and 5 min, respectively). Group B: HPP milk (650 MPa, 72 oC for 0, 1 and 5 min). Group C: UHT (138±1 oC for 2 s) and PATP milk (650 MPa 105 oC for 0, 1 and 5 min) b Denotes day of analysis.
3.3.5 Proteolysis
Proteolysis in milk samples was evaluated using sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) analysis according to the method described by Verdi et al., (1987), with modifications. Milk (2 mL) was centrifuged (4600 rpm, 15 min) to separate milkfat before electrophoresis run. Skim milk was diluted 1:10
(vol/vol) in sample buffer (35.5% deionized water, 62.5 mM Tris-HCl pH 6.8, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue and 0.5% β- mercaptoethanol); and vials were immersed in boiling water for 4 min. Aliquots (8.5 µL) of milk samples were then loaded into 12% resolving Tris-HCl gels (Bio-Rad
Laboratories Inc., Hercules, CA). Electrophoresis was conducted at a constant current of
145 V using a Bio-Rad Mini Protean Tetra Cell system (Hercules, CA) until dye reached the bottom edge of the gel (approximately 2 hr). Gels were maintained at 4±1 oC during the electrophoresis run. Subsequently, gels were washed three times with deionized water and copper-stained with 0.3 M CuCl 2 (Fisher Science Education, Rochester, NY) following the procedure described by Lee et al., (1987). A broad range pre-stained SDS-
92 PAGE standard (Bio-Rad Laboratories Inc., Hercules, CA) was run along milk samples to identify electrophoretic bands based on their molecular weight. Gels were scanned using a Bio-Rad GS-800 densitometer (Hercules, CA). The proportion of proteolysis products formed as a result of protease activity was determined by measuring the absolute decrease in area of known casein bands ( αs-casein, β-casein, and κ-casein). This decrease in area was proportional to the absolute increase in area of bands identified as proteolysis products as shown in Figure 3.2. Any increase of intensity in electrophoretic bands (proteolysis products) was also considered an index of proteolysis. Results were expressed as the sum of relative percentage of proteolysis of casein bands (intact minus enzymatically damaged) at the end of the shelf life of milk samples Results were expressed as the sum of relative percentage of proteolysis of casein bands (intact minus enzymatically damaged) at the end of the shelf life of milk samples and according to the following equation adapted from Verdi et al., (1987):
2 (Int × mm ) f
Relative Proteolysi s (%) = 100 − 2 ()Int × mm 0
2 Where, (Int x mm )f is the intensity x area of casein electrophoretic bands at the
2 end of the shelf life of milk. And, (Int x mm )0 is the intensity x area of casein electrophoretic bands on 0 d.
93 α-Casein β-Casein κ-Casein Unk −−− 111
β-Lactoglobulin
α-Lactoalbumin Unk - 2 Unk - 3 1 2 3 4 5 6
Figure 3.2 SDS-PAGE of milk subjected to HPP a treatment a HPP treated milk at 650 MPa, 32 oC for 5 min. *Lane 1: SDS electrophotogram of milk on 0 d; Lane 2: SDS electrophotogram of milk on 2 d; Lane 3: SDS electrophotogram of milk on 4 d; Lane 4: SDS electrophotogram of milk on 6 d; Lane 5: SDS electrophotogram of milk on 10 d; Lane 6 SDS electrophotogram of milk on 15 d. **Unk 1 – 3: major casein proteolysis products identified as unknown
3.3.6 Plasmin Activity
Residual plasmin activity was evaluated in HTST pasteurized, UHT and pressure- treated milk samples. A sensitive protease assay was developed using 4, 4-Difluoro-5, 7 – dimethyl -4-bora-3a, 4a – diaza-s-indacene-3-propionic acid, succinimidyl ester
(BODIPY FL, SE)-labeled casein (Invitrogen Corp., Carlsbad, CA) as a fluorogenic substrate for plasmin as described by Jones et al., (1997). This substrate is cleaved by a wide variety of proteases, including plasmin, into small fluorescent peptides in a linear time-dependent manner during the enzymatic reaction. The resulting increase in fluorescence upon hydrolysis is proportional to the enzymatic activity.
94 A standard curve was constructed with known concentrations (0, 0.5, 1, 1.5, 2 and
3 µg/mL) of purified plasmin (Sigma-Aldrich, St. Louis, MO) in phosphate buffer, pH
7.6. A 10 µg/mL substrate solution (BODIPY FL-Casein) was also prepared in 20 mM phosphate buffer, pH 7.6 + 150 mM NaCl (Fig. 4). The range of enzyme response was determined by mixing equal aliquots (375 µL) of each enzyme dilution with BODIPY
FL-Casein substrate solution. Samples were incubated for 1 hr at 37 oC, protected from light. Fluorescence intensity was measured in 1-cm path length cuvettes at 485 nm excitation and 530 nm emission filters, slits opened at 10/10, 1s average reading time and voltage between 590-610 V using a Varian Cary Eclipse Fluorescence spectrophotometer
(Walnut Creek, CA). Fluorescence emission intensities versus enzyme concentration were plotted; and limits of detection were determined as shown in Figure 3.3.
95 250
200
150
100
50
0 Fluorescence Intensity (a.u.) 0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014 0.0016 Plasmin (mg/mL)
Figure 3.3 Standard curve for plasmin * y = 52615x + 106.23; R 2 = 0.997 **Results are expressed as fluorescence intensity vs. plasmin concentration (mg/mL) measured at 485 nm excitation; 530 nm emission; slits open at 10/10, respectively; 1 s average reading time; 590 – 610 V ***Data shown are means ± std. dev. of 4 independent samples
Total plasmin activity was determined in milk samples after extraction of plasmin and plasminogen fractions as described by Politis et al., (1993) and Richardson and
Pearce, (1981). Milk (5 mL) was centrifuged at 4600 rpm for 15 min and the cream layer on the surface was discarded. Skim milk was then centrifuged at 32700 rpm for 1 hr at
4oC. The supernatant (whey fraction) was discarded; and the pellet (casein fraction) was reconstituted in 20 mM phosphate buffer, pH 7.6 + 50 mM ε-aminocaproic acid (EACA)
(Sigma-Aldrich, St. Louis, MO). The mixture was incubated for 2 hr at room temperature
(25±1 oC); and then centrifuged at 32700 rpm for 1 hr at 4 oC to allow dissociation and transfer of the plasmin system from the casein micelle into the buffer. Plasmin and plasminogen content was determined after activation with 1600 IU/mg of urokinase
(American Diagnostica Inc., Stamford, CT). Equal aliquots (0.5 mL) of each buffer
96 samples and urokinase solution were mixed and incubated for 1 hr at 37 oC under dark conditions. After incubation, total plasmin activity was measured upon hydrolysis of
BODIPY FL-Casein as described above.
3.3.7 Lipolysis
Free fatty acids (FFA) present in milk were measured by the modified copper- soap method reported by Ma et al., (2003). The increase in FFA as a result of lipase activity was used as an index of lipolysis in milk samples. A standard curve was constructed with known concentrations (0, 20, 40, 60, 120, 180 µg/mL) of palmitic acid
(Sigma-Aldrich, St. Louis, MO) in chloroform-heptane-methanol (CHM [49:49:2, vol:vol:vol]). Deionized water (0.5 mL) and 0.7N HCl (0.1 mL) were added to each tube; and the mixture was shaken vigorously for 30 min in a horizontal position using a
Babcock shaker at 470 rpm. Subsequently, 2 mL of the copper soap reagent (Shipe et al.,
1980) was added and the mixture was shaken again for 10 min. The tubes were centrifuged at 3250 rpm for 10 min; and 3 mL of the supernatant were transferred into acid-washed glass tubes containing 0.5 mL of the color reagent. Samples were diluted
(1:1, vol:vol) with CHM to ensure that detection limits fall within the linear range of the curve. Absorbance was read immediately after at 440 nm using a single-cell Hewlett
Packard 8453 UV spectrophotometer (Palo Alto, CA). Results were expressed as [ µg of palmitc acid / mL of CHM] as shown in Figure 3.4.
97 1.000
0.800
0.600
0.400 Absorbance 0.200
0.000 0 50 100 150 200
Palmitic Acid [ µµµg / mL CHM ]
Figure 3.4 Standard curve a for palmitic acid a y = 0.005x + (-0.049); R 2 = 0.995 *Results are expressed in µg Palmitic acid/mL solvent (choloroform – heptane – methanol; 49:49:2; vol:vol:vol) **Absorbance measured at 440 nm ***Data shown are means ± std. dev. of 3 independent samples
On the day of analysis, milk samples were thawed keeping the temperature below
7±1 oC at all times. An aliquot of milk (0.5 mL) was mixed with 0.1 mL of 0.7N HCl to prevent further lipolysis. Subsequently, 0.1 mL of 1% (vol/vol) Triton-X solution and 2 mL of the copper soap reagent were added to the milk-acid mixture. The CMH solvent (6 mL) was then added; and the mixture was shaken in a horizontal position for 30 min.
Samples were centrifuged at 5,000 rpm for 10 min. After centrifugation, the top colorless layer (3.5 mL) was mixed with 0.1 mL of the color reagent. Absorbance was measured immediately after at 440nm. Two blanks were prepared with 0.5 mL of deionized water and analyzed with milk samples. Results were expressed as [meq of FFA / kg of milk] according to the formula described by Shipe et al., (1980) considering a 1:1 dilution factor.
98 3.3.8 Data Analysis
All experiments were conducted in quadruplicate on individual milk samples; and analyzed using crossed and nested ANOVA general linear model, with Tukey’s pairwise comparisons at 95% confidence level. Data analysis was performed using Minitab v. 15.2
(Minitab Inc., State College, PA).
3.4 Results and Discussion
The extent of proteolysis as a result of protease activity in milk samples is shown in Figure 3.6. The formation of proteolysis products in pressure-treated samples was dependent of processing temperature, holding time during pressurization and storage temperature. With exception of milk samples processed at 650 MPa and 105 oC for 0 min, no significant proteolysis was observed in pressure-treated samples as compared to pasteurized and UHT milk. Further analysis revealed that plasmin activity decreases with increasing temperature during high pressure treatment (Fig. 3.7). Even though the enzyme was not completely inactivated, combinations of high pressure (650 MPa) and temperature (105 oC) were sufficient to inactivate the enzyme to levels similar to UHT processing. The enzyme level in the preheated milk before PATP was not estimated.
Formation of FFA as a result of lipase activity is shown in Figures 3.8 to 3.10. High pressure treatments enhanced formation of FFA in milk samples on 0 d; and the concentration of FFA in milk further increased with increasing storage time. Storage temperature had also a significant effect on formation of FFA in milk. These results suggest that not only high pressure-temperature combinations but also storage conditions
99 at low temperature are necessary to avoid significant proteolysis and lipolysis in milk.
3.4.1 Pressure-temperature profiles of milk samples
Figure 3.5 shows the temperature and pressure history of HPP and PATP milk samples processed at 650 MPa; 32, 72 and 105 oC with holding time of 5 min. The initial temperature of HPP milk samples (650 MPa; 32 oC for 5 min) was 7±1 oC and reached the target processing temperature (31±1 oC) after 1.5 min of compression. No significant heat loss was observed in these samples as a result of adiabatic compression during pressurization. Similarly, no significant heat loss was observed in HPP milk samples with pressure holding times of 0 and 1 min, respectively (data not shown). Pressure treated samples processed at 650 MPa and 72 oC for 5 min were preheated at 48±1 oC before pressurization. This temperature allowed milk to reach a target processing temperature of
77±1 oC during compression. Unlike HPP milk samples processed at 32 oC, pressure treated milk samples processed at 72 oC attained higher processing temperatures possibly due to heat gain experienced during processing. The final processing temperature achieved during pressurization (5 min) for PATP milk samples was 104±1 oC. Prior studies indicate that a temperature gradient about 7 oC exist during PATP in the sample basket. However, the current study, did not consider the impact of the temperature gradient on the chemical stability and residual plasmin activity of HPP and PATP milk samples.
100 125 700
C) 600 o 100 500 75 400 50 300 200
)Temperature ( )Temperature 25 100 (---) Pressure (MPa) (---) Pressure ( ( 0 0 0 2 4 6 8 10 12 Tim e (min)
Pressure Pressure Depressurization come-up time holding time time
Figure 3.5 Pressure–temperature profiles observed during high pressure treatments of milk a a PATP milk processed at 650 MPa and 32, 72 and 105 oC for 5 min *Data shown are means of two independent samples
3.4.2 Proteolysis
.The extent of relative proteolysis in milk samples at the end of their shelf life is shown in Fig. 3.6. The area of casein bands ( αs-casein, β-casein, and κ-casein) as percent of total casein in pasteurized milk decreased by 37.2% after 20 d of storage under refrigeration conditions (4 ± 1 oC). This value was proportional to the total area increase of proteolysis products formed (36.5%) on 20 d (data not shown). The increased extent of proteolysis observed in pasteurized milk samples was probably due to the activity of plasmin. As mentioned in the introduction section, the enzyme has optimal activity at a pH close to that of milk (6.6 -6.7) and has remarkable heat resistance characteristics
(Kelly et al., 2006). In fact, pasteurization enhances plasmin activity in milk as inhibitors of PA’s are inactivated at temperatures close to HTST conditions (72 oC for 15 sec).
101 Therefore, there is an observed increase of plasmin activity in milk over time (Kelly et al., 2006). These results are in agreement with previous work assessing proteolytic activity in milk. Verdi et al., (1987) reported that 30% of the initial casein gives rise to formation of proteolysis products in milk after incubation at 37 oC for 24 h. The authors reported that relative percentages of κ-casein did not significantly change over time.
However, αs-casein and β-casein were rapidly hydrolyzed possibly into γ-casein residues over the period of time studied (Verdi et al., 1987). γ-casein results from the limited proteolysis of β-caseins due to the activity of plasmin in milk (Pereda et al., 2008; Kelly and Fox, 2006a). The enzyme also hydrolyzes αs2 -casein in milk. However, κ-casein shows almost no proteolytic breakdown as a result of plasmin activity (Pereda et al.,
2008; Verdi et al., 1987).
102 100 90 80 b b b a 70 a a a a a 60 d 50
40 c 30
Relative Proteolysis (%) Proteolysis Relative 20 10 0 1 2 3 4 5 6 7 8 9 1011 Treatments f
Figure 3.6 Relative percentage decrease e of casein bands area as a result of protease activity in milk a-d Means ± std. dev. with different superscript are significantly different (n = 4) Int × mm 2 e Relative Proteolysi s (%) = 100 − ( ) 2 ()Int × mm 0 f Treatments were: (1) Pasteurized milk (78 oC, 18 s); (2-4) HPP milk (650 MPa, 32 oC for 0, 1 and 5 min); (5-7) HPP milk (650 MPa; 72 oC for 0, 1 and 5 min); (8-10) PATP milk (650 MPa, 105 oC for 0, 1 and 5 min); (11) UHT milk (139 oC for 2 s).
The extent of proteolysis in pressure-treated samples processed at room temperature (32 ± 1 oC) was not significantly different than that of pasteurized milk samples. The total area decrease of casein bands for milk samples processed at 650 MPa and 32 oC for 0, 1 and 5 min, respectively, ranged from 36.7 to 38.4% at the end of their shelf life (20 d). And, the area of proteolysis products formed as a result of proteolysis for these samples ranged from 33.4 to 40.7% (data not shown). No significant differences
103 were found among pressure-treated milk samples processed at room temperature with increasing holding time ( p = 0.09). These results suggest that pressure treatments at 650
MPa and 32 oC induced conformational changes in milk proteins making them more susceptible to undergo proteolysis patterns. The effects of HPP on the functionality of milk proteins depend on the type of protein, pH, ionic strength, solvent composition and protein concentration (Lopez-Fandiño, 2006). A recent study assessing the effects of HPP on casein micelles and whey proteins reported that pressure treatments at > 250 MPa enhanced the disruption of casein micelles in milk (Lopez- Fandiño, 2006). Denaturation of casein micelles is attributed to solubilization of colloidal calcium phosphate and disruption of hydrophobic and electrostatic interactions at higher pressures (Huppertz et al., 2004a; Huppertz et al., 2004b; Lopez-Fandiño, 2006).
There was a synergistic effect of pressure and temperature on the extent of proteolyis with increasing processing temperature. Pressure-treated samples processed at
650 MPa and 72 oC showed the least extent of proteolysis among milk samples. Relative proteolysis values for these samples ranged from 31.2 to 33.6%; and the total area increase of proteolysis products ranged from 33.7 to 35.4% (data not shown) at the end of their shelf life (60 d). There were no significant differences ( p = 0.114) between pressure- treated processed at 72 oC. The lesser extent of proteolysis in pressure treated samples at
72 oC can be attributed to the fact that partially denatured β-lactoglobulin at this temperature can form complexes with caseins through disulphide interactions which obstruct the access of proteases to its substrate (Tetra Pak, 2003). It is also possible that pressure treatment also induced conformational changes in proteins; and therefore
104 changes in the configuration of the substrate’s receptor sites impeded the action of the enzyme active site (Pereda et al, 2008). Similar observations have been reported previously (Huppertz et al., 2002; Huppertz et al., 2004; Lopez-Fandiño, 2006).
Depending on the pressure, time and temperature applied, high pressure can induce conformational changes by affecting hydrogen bonds, hydrophobic interactions and the ionic strength of proteins (Lopez-Fandiño, 2006). Also, the extent of protein denaturation by HPP occurs at different rates; and it is the dependent on the type of protein. β- lactoglobulin appears to be less pressure resistant than α-lactoalbumin during high pressure treatments from 100 to 800 MPa at 20 oC. Complete denaturation of β- lactoglobulin occurs at 600 MPa; whereas α-lactoalbumin is partially denatured (72 %) at
800 MPa for 30 min (Huppertz et al., 2004a). Combinations of pressure with temperature also have an effect on the extent of milk protein denaturation. High pressure treatments at
400 or 600 MPa reduce the casein micelle size regardless of the temperature applied.
However, treatments at lower pressure (250 MPa) at 20 or 40 oC increase the micelle size by approximately 25 % compared to the initial size of the casein micelle (Huppertz et al.,
2004b). Similarly, there is a synergistic effect on denaturation of whey proteins during high pressure treatments with increasing temperature. There is no significant denaturation of β-lactoglobulin at treatments < 400 MPa. However, pressures up to 600 MPa at 40 oC are sufficient to denature the protein by 78% (Huppertz et al., 2004a).
Pressure-treated milk processed at 105 oC for 0 min showed the highest proteolysis values among milk samples (68.9% total casein area decrease) at the end of its shelf life
(30 d). With increasing holding time, the extent of proteolysis in pressure-treated milk
105 processed at 105 oC was similar to that of pasteurized and UHT milk. The total casein area decrease in PATP milk samples treated at 105 oC for 1 min was 40.06% at the end of their shelf life. However, pressure treated samples at 105 oC for 5 min showed more proteolysis than pasteurized and UHT milk. The extent of proteolysis for these samples was 46.8% on 90 d. The increased proteolysis for these samples might be related to the increased activity of plasmin at temperatures closer to the optimal temperature at which the enzyme is most active (37 oC) (Kelly et al., 2006). Even though there was a synergistic effect of pressure and temperature on inactivation of the plasmin system in milk, the pressure– temperature conditions used in this study were not sufficient to completely inactivate the enzyme as explained in the next section.
Plasmin is a heat-resistant proteinase capable of standing UHT processes
(Newstead et al., 2006). As expected, relative proteolysis values in UHT milk was similar to pasteurized milk samples. The total casein area decrease for these samples was 34.8%; and the area of proteolysis products formed was 32.9% at the end of its shelf life (90 d).
These observations might be related to more inactivation of plasmin at UHT processing temperatures. Even though plasmin is capable of standing UHT processes, the enzyme is partially inactivated to levels considerably higher than HTST pasteurization conditions
(Newstead et al., 2006).
3.4.3 Plasmin Activity
A sensitive spectrofluorimetric assay was developed using BODIPY FL-Casein as a substrate for plasmin, as described by Jones et al., (1997). Unlike other spectrofluorometric assays, the use of BODIPY FL-Casein did not involve any separation
106 steps; and allowed for a direct, continuous fluorescent measurement upon incubation with plasmin. BODIPY FL-Casein belongs to a family of boron-containing flourophores with emission spectra between 510 to 610 nm (Lakowicz, 1999; Jones et al., 1997). The substrate has the advantage of having high quantum yields, extinction coefficients near
80 000 M -1 cm -1, and it resists changes in the ionic strength of the sample over a wide range of pH. A disadvantage of the substrate is its very small Stoke’s shift; and therefore a higher tendency to shelf quenching with increasing protein labeling (Lakowicz, 1999).
BODIPY FL labeled casein has approximately 5 mole of dye/mole of protein (Molecular
Probes, 2004); and the highest fluorescence intensity values were observed using a substrate solution of 10 µg/ml (data not shown). Since equal amounts (375 µL) of substrate and enzyme solution were added to the mixture, a 1:1 (v/v) dilution ratio was considered in the final calculations. At higher concentrations of plasmin an inner filtering or shelf-quenching effect was observed possibly due to interactions between excited molecules and molecules in the non-excited state (Lakowicz, 1999). However, the above serial dilutions accounted for plasmin concentration values previously reported in milk
(Richardson and Pearce, 1981). Plasmin plus plasminogen activities were expressed as total plamin activity ( µg/mL) following the procedure of Politis et al., (1993). This method has the advantage of eliminating inhibitors of plasmin in the serum phase of milk through a series of ultracentrifugation and reconstitution steps. Optimal reaction conditions for conversion of plasminogen into active plasmin were established by mixing equal aliquots (0.5 mL) of the plasmin solution and urokinase (1600 IU/mg) and incubating the solution for 1 hr at 37 oC under dark conditions (data not shown). This
107 method allows for a more reliable measurement of the total plasmin activity as compared to other methods previously reported for milk (Politis et al, 1993). Total plasmin activity was measured immediately after incubation with urokinase; and final results were expressed as fluorescence change (a.u.) per unit of plasmin.
0.9 b b b 0.8 b b b b 0.7 a 0.6 a a a a 0.5
g/mL) 0.4 µ µ µ µ ( 0.3 Total Total Plasmin 0.2 0.1 0 1 2 3 4 5 6 7 8 9 101112 Treatments f
Figure 3.7 Total plasmin concentration c in milk a-b Means ± std. dev. with different superscript are significantly different (n = 4) c Total Plasmin = Plasmin + Plasminogen ( µg/mL); 1 hr incubation at 37 oC; 485 nm excitation; 530 nm emission; slits open at 10/10, respectively; 1 s average reading time; 590 – 610 V f Treatments were: (1) Raw milk (2% milkfat, homogenized); (2) Pasteurized milk (78 oC, 18 s); (3-5) HPP milk (650 MPa, 32 oC for 0, 1 and 5 min); (6-8) HPP milk (650 MPa; 72 oC for 0, 1 and 5 min); (9- 11) PATP milk (650 MPa, 105 oC for 0, 1 and 5 min); (12) UHT milk (139 oC for 2 s).
Figure 3.7 shows the total concentration of plasmin in milk samples subjected to different pressure and temperature profiles. The concentration of plasmin in raw milk was
108 0.6 ± 0.03 µg/mL. Pasteurized milk and pressure-treated samples processed at 32 oC for 0,
1 and 5 min showed the highest plasmin activity values among milk samples. Total plasmin (plasmin + plasminogen) concentration in pasteurized milk samples were 0.74 ±
0.01 µg/mL; whereas the concentration of the enzyme in pressure-treated samples processed at 32 oC ranged from 0.68 to 0.77 µg/mL. Although no significant, there was an observed synergistic effect of pressure holding time during pressurization. With increasing holding time (0 to 5 min), plasmin inactivation rates increased in pressure- treated samples processed at room temperature. Similar inactivation rates were observed for pressure-treated samples processed at 72 oC for 0, 1 and 5 min. The concentration of plasmin in these samples ranged from 0.66 to 0.68 µg/mL. And, no synergistic effect of pressure holding time was observed for these samples. The concentration of plasmin in pasteurized milk samples was not significantly different ( p > 0.05) from that of pressure- treated samples processed at 32 and 72 oC for 0, 1 and 5 min, respectively. These results are in agreement with previously reported studies on plasmin inactivation rates in the milk system. The concentrations of plasmin in pasteurized skim milk ranged from 0.14 to
0.73 µg/mL; and plasminogen concentrations ranged from 0.55 to 2.75 µg/mL (Bastian and Brown, 1996; Richardson and Pearce, 1981). Thermal pasteurization enhances plasmin activity in milk. In addition to the considerably high thermal stability of the enzyme, inhibitors of plasminogen (PAI) have different thermal inactivation rates than plasmin and plasminogen (Prado et al., 2006). Thermal inactivation of PAI is 91% in buffer and 80% in milk after heat treatment at 74.5 oC for 15 s (Prado et al., 2006).
Therefore, more plasminogen is converted into active plasmin in pasteurized milk and the
109 total plasmin activity is increased upon storage. Similarly, Borda et al., (2004) observed an increase of plasmin activity at temperatures between 50 to 64 oC, with a maximum increase in activity at 61 oC. The authors reported that 85 oC for 5 min were necessary to completely inactivate plasmin in phosphate buffer (pH 6.6). Plasmin inactivation rates vary depending on the system the enzyme is therein. In milk, temperatures up 120 oC for
15 min are necessary to completely inactivate the enzyme (Chen et al., 2003).
Plasmin showed inactivation levels similar to those found in UHT milk with increasing temperature after PATP treatment. No significant differences were found between pressure-treated samples processed at 105 oC for 0, 1 and 5 min and UHT milk ( p
< 0.01). The concentration of plasmin in the former samples ranged from 0.53 to 0.56
µg/mL; whereas the concentration of plasmin in UHT milk was 0.54 µg/mL. These observations might be related to more inactivation of the enzyme during PATP treatment at higher temperatures (105 oC). The mechanisms of plasmin inactivation under high pressure are not fully understood yet. Plasmin shows pressure stability between 100 – 800
MPa at room temperature conditions; and there is an increase of stability at pressures >
600 MPa (Borda et al., 2004). The extent of plasmin inactivation under pressure increases in the presence of β-lactoglobulin. As with heat treatments, interference of sulfhydryl groups from partially denatured β-lactoglobulin makes plasmin prone to inactivation during HPP (Kelly and Foley, 1997; Lopez-Fandiño, 2006). There is also a synergistic effect of pressure and temperature on inactivation of the plasmin system in milk (Borda et al., 2004). These results are in agreement with previously reported work on inactivation of plasmin in UHT milk systems. The rate of formation of plasmin-type proteolysis
110 products from hydrolysis of β-casein was higher in UHT milk samples subjected to preheat treatments of 75 to 85 oC for 15 to 30s (Newstead et al., 2006). Storage temperature also enhanced plasmin activity in UHT milk. Plasmin activity was higher in milk samples stored at 30 oC that at 20 oC after 6 months of storage. The rate of formation of plasmin-type proteolysis products formed was directly correlated to the preheat treatment and storage temperature (Newstead et al., 2006). Even though in this study plasmin activity was only measured after processing, higher proteolysis values were observed in milk samples stored at room temperature (25 ± 1 oC) than those of milk samples stored under refrigeration conditions (4 ± 1 oC). Moreover, pressure holding time had also a significant effect ( p < 0.05) on inactivation of the enzyme in PATP milk. A pressure holding time of 5 min gave the lowest plasmin concentration value (0.53 ± 0.07
µg/mL) among milk samples processed at 650 MPa for 105 oC. These results are in agreement with previous studies on high pressure inactivation of plasmin at higher temperatures. A synergistic effect of temperature, pressure and holding time during pressurization on plasmin inactivation has been observed at pressures ranging from 300 to 600 MPa. The enzyme can be inactivated up to 86.5% after treatments at 400 MPa and
60 oC for 15 min (Lopez-Fandiño, 2006). However, at pressures > 600 MPa plasmin appears to be more stable. This increased stability is attributed to structural changes that stabilize the plasmin system in milk. At higher pressures the energy of activation decreases; and therefore, interactions between newly formed subgroups from partially denatured whey proteins and the enzyme are favored giving plasmin an increased stability (Borda et al., 2004; Lopez-Fandiño, 2006).
111 3.4.4 Lipolysis
The increase of FFA as a result of lipase activity in milk samples was studied. A sensitive spectrophotometric assay was developed to measure the concentration of FFA in milk samples as described by Shipe et al., (1980). Figure 3.8 shows the concentration of FFA in pasteurized and milk pressure processed at 650 MPa and 32 oC for 0, 1 and 5 min. The concentration of FFA in HTST pasteurized milk was 0.23 ± 0.12 on 0 d; and
0.88 ± 0.15 meq FFA/kg of milk after 20 d of storage under refrigeration conditions (4 ±
1 oC). Although FFA content of pasteurized milk samples significantly increased throughout its shelf life, the rate of FFA formation was slower in pasteurized milk than pressure-treated milk samples processed at room temperature conditions (32 oC). Pressure enhanced the formation of FFA in PATP treated milk. On 0 d, the concentration of FFA in pressure-treated samples processed at room temperature (32oC) for 0, 1 and 5 min ranged from 0.69 to 0.76 meq FFA/kg of milk. No significant differences ( p = 0.289) on formation of FFA were observed among pressure-treated samples with increasing holding time. However, storage time had a significant effect on the extent of lipolysis in pressure- treated samples processed at room temperature. The concentration of FFA in these samples was in the order of 3 ± 0.18 meq FFA /kg of milk, at the end of their shelf life
(20 d).
112 4 3.5 b 3 b b 2.5 2 1.5 1 a
meq FFA/kg milk of 0.5 0 0 5 10 15 20 Time (d)
Figure 3.8 Free fatty acids content in pasteurized and HPP milk treated with different pressure and temperature profiles c a-b Means ± std. dev. with different superscript are significantly different (n = 4) c Treatments were: A ( ) Pasteurized milk (78 oC, 18 s); ( ) HPP milk (650 MPa, 32 oC for 0 min); ( ) HPP milk (650 MPa, 32 oC for 1 min); ( X ) HPP milk (650 MPa, 32 oC for 5 min). *Results are expressed in meq of FFA/kg of milk; absorbance measured at 440 nm
The concentration increase of FFA in pressure treated milk at 32 oC was probably due to the activity of lipases. In addition to bacterial enzymes, LPL is probably the most abundant enzyme responsible for development of rancid flavors in milk. The enzyme is considered one of the most challenging enzymes to control in dairy operations due to its high temperature resistance (Fox and Kelly, 2006a; Chen et al., 2003). It is generally accepted that LPL is almost completely inactivated at HTST pasteurization conditions
(Chen et al., 2003). However, recent studies indicate that LPL is pressure stable. A study assessing the flavor of cheese made from pasteurized and high pressure processed milk
(500 MPa, 20 oC for 15 min) reported that the extent of lipolysis in cheeses made from
HPP milk was considerably higher than that of cheeses made with pasteurized milk
113 (Buffa et al., 2001). Similarly, Pandey and Ramaswamy (2004) reported that the activity of lipase in milk was enhanced after pressure treatments at 300 – 400 MPa. Holding time during pressurization (0 – 180 min) had no effect on inactivation of the enzyme. The authors concluded that lipase activity might be reduced by increasing holding time or at higher pressures. In this study, the concentration of FFA in pressure-treated samples processed at room temperature (32 ± 1 oC) was above the off-flavor sensory threshold value, caused by lipolysis, previously established for milk (Santos et al., 2003). The sensory threshold value determines the minimum concentration of a compound necessary to be detected by consumers in milk. With increasing lipolytic activity, the concentration of FFA increases giving rise to rancid off-flavors in milk. The detection values for off- flavors caused by lipolyis in high quality milk have been established in the range of 0.33 meq of FFA/kg of milk (Santos et al., 2003). Therefore, based on the conditions present in this study combinations of ultra high pressure and ambient temperature may not be sufficient to inactivate lipase activity in pressure-treated milk.
The extent of lipolysis was also different in pressure-treated milk samples processed at 650 MPa and 72 oC for 0, 1 and 5 min than pasteurized milk (78 ± 1 oC for 18 s). Figure 3.9 shows the concentration of FFA in pressure-treated samples processed at
72 oC. FFA content for these samples ranged from 0.65 to 0.80 meq FFA/kg of milk on 0 d. Significant differences in FFA content were found between pasteurized and these samples tested on the initial day of analysis. However, FFA content for pressure-treated milk samples did not significantly differ at 4 d and the concentration of FFA in pressure treated milk at 72 oC remained constant throughout their shelf life. Also, pressure holding
114 time did not exert any effect on lipolysis for these samples.
2.5
2
1.5
1 a a 0.5 a meq FFA/kg milk of
0 0 10 20 30 40 50 60 Time (d)
Figure 3.9 Free fatty acids content in HPP milk treated with different pressure and temperature profiles b a Means ± std. dev. with different superscript are significantly different (n = 4) b Treatments were: ( ) HPP milk (650 MPa, 72 oC for 0 min); ( ) HPP milk (650 MPa, 72 oC for 1 min); ( ) HPP milk (650 MPa, 72 oC for 5 min). *Results are expressed in meq of FFA/kg of milk; absorbance measured at 440 nm
These results might be related to an increased inactivation of lipolytic enzymes at higher temperatures (72 oC) and pressure. Also, storage under refrigeration conditions (4
± 1 oC) might have delayed the activity of lipases in HPP milk. Lipoprotein lipase has an optimal pH and temperature of 9 and 37 oC, respectively. Under optimal conditions, the catalytic rate of LPL is ~3000 s -1 (Fox and Kelly, 2006a). Therefore, a higher lipase activity is observed at room temperature than under refrigeration conditions. The activity of lipases is controlled by the concentration of lipase activators (apolipoprotein C-II) and the level of inhibitors (PP8) in milk (Fox and Kelly, 2006a). The presence of lipase
115 activators can cause spontaneous lipolysis in fresh milk. Fortunately, the activity of apolipoprotein C-II in refrigerated milk is low, as the optimal conditions for lipase activation are at 37 oC and pH 7 (Chen et al., 2003).
0.9 0.75 0.6 0.45 c a 0.3 b d
meq FFA/kg milk of 0.15 0 0 10 20 30 40 50 60 70 80 90 Time (d)
Figure 3.10 Free fatty acids content in UHT and PATP milk e a-d Means ± std. dev. with different superscript are significantly different (n = 4) e Treatments were: ( ) PATP milk (650 MPa, 105 oC for 0 min); ( ) PATP milk (650 MPa, 105 oC for 1 min); ( X ) PATP milk (650 MPa, 105 oC for 5 min); ( ) UHT milk (139 oC for 2 s). *Results are expressed in meq of FFA/kg of milk; absorbance measured at 440 nm
Figure 3.10 shows the concentration of FFA in UHT and PATP milk samples.
UHT processing aversively affected the activity of lipases in milk. UHT milk resulted with the lowest FFA content among milk samples. The FFA concentration for these samples was 0.12 ± 0.04 meq FFA/kg of milk on the initial day; and remained constant until 45 d. A gradual formation of FFA was observed in UHT milk from 45 to 90 d. The
116 FFA content in UHT milk was 0.29 ± 0.05 meq FFA/kg of milk at the end of its shelf life.
The formation of FFA in pressure-treated samples processed at 105 oC followed different patterns than the rest of milk samples. The FFA content for these samples on the initial day (0.23 – 0.28 meq FFA/kg of milk) was similar to that of pasteurized milk.
However, the rate of FFA formation was significantly faster than that of pasteurized and
UHT milk samples. Milk samples processed at 650 MPa and 105 oC for 0 and 1 min, respectively, showed a dramatic increase in FFA content on 15 d followed by a decrease to levels similar to those on the initial day of analysis. The decrease in FFA concentration can be attributed to the formation of volatile organic compounds such as ketones and aldehydes resulting from milkfat oxidation after hydrolysis of the ester bond between glycerol and fatty acids (Nawar, 1996). These aroma compounds are associated with common flavor defects in milk from lipase activity. Major contributors of lipolysis in milk include butyric, caproic, caprylic, capric and lauric fatty acids. Long-chain fatty acids and very short-chain fatty acids do not play a significant role in lipolysis of milk
(Shipe et al., 1978). On 30 d, PATP milk samples processed at 105 oC for 0 min were noticeably spoiled and therefore discarded. On 45 d, PATP milk samples processed at
105 oC for 1 min were discarded for similar reasons. Pressure-treated samples processed at 105 oC for 5 min resulted with the least FFA content among PATP milk samples. The
FFA concentration for these samples ranged from 0.21 to 0.38 meq FFA/kg of milk and remained constant through the rest of their shelf life (90 d); probably due to more inactivation of lipase activity with increased holding time at higher temperatures. It was observed that pressure treatments at 105 oC also enhanced the formation of FFA in milk
117 on 0 d. There is no much information about the mechanisms of lipase inactivation under high pressure treatments. However, the structure of the enzyme may have significant role on how lipases are affected by HPP. Changes in the tertiary and quaternary structure of the enzyme during are associated with a reduction in volume during the pressure treatment (Seyderhelm et al., 1996; Lopez-Fandiño, 2006) Also, the energy of activation decreases under pressure; and therefore enzymatic activity is favored as substrate and enzyme are in close proximity to each other. These results indicate that the high pressure
– temperature conditions used in this study are sufficient to inactivate bacterial lipases to a certain degree. However, the criteria of lipase inactivation based on the application of
PATP are different from that of thermal treatments (Lopez-Fandiño, 2006). A recent study assessing the effects of temperature and high pressure processing on Rhizomucor miehei lipase reported that combinations of pressure (300 to 500 MPa) and temperature
(40 to 60 oC) can be used to inactivate the enzyme. The authors reported that irreversible inactivation of Rhizomucor miehei lipase occurs at temperatures between 50 to 55 oC; and the rate of inactivation of the enzyme was equivalent to pressure treatments at 450 – 500
MPa at room temperature conditions. However, the mechanisms of enzyme inactivation are different in both thermal and high pressure processes. High pressure processing induced more aggregation and unfolding of the enzyme than thermal treatments with the same residual activity of the enzyme (Noel and Combes, 2003).
The activity of lipolytic enzymes in milk depends on several factors, such as source of the enzyme, substrate specificity, processing of milk and storage conditions. In addition to indigenous LPL, bacterial lipases can be present in milk. These enzymes are
118 generally produced during the late exponential and early stationary phases of growth; and they have different properties and substrate specificities (Chen et al., 2003). In fluid milk, the incidence of bacterial lipases is relatively high. Bacillus spp. and Pseudomonas spp. are lipolytic psychrotrophic bacteria commonly found in processed milk (48% incidence); and they have been recognized as major contributors to lipolyzed off-flavors in the product (Fromm and Boor, 2004; Chen et al., 2003). Lipases from Pseudomonas spp. have optimum pH values ranging from 7.5 to 9, with molecular weights from 50 to 130 kDa. The enzymes are quite heat resistant. Lipases from Pseudomonas spp. have shown thermal stability at temperatures up to 120 oC for 4.2 min. Similarly, lipases secreted by
Bacillus spp. are considerably heat stable. The optimum pH values for these enzymes range from 7 – 10; and they have shown heat stability at temperatures up to 75 oC for 186 min. These processing temperatures are, by far, sufficient to inactivate LPL in milk.
However, most bacterial lipases have shown highest hydrolytic activity at temperatures ranging from 60 to 75 oC (Chen et al., 2003). The substrate specificity of lipolytic enzymes also determines the extent of lipolysis in milk. Most bacterial lipases have higher affinities for fatty acids in the sn-1 and sn-3 positions of triacylglycerols; and some lipases have higher affinity toward diacylglycerols and monacylglycerols (Chen et al., 2003). Processing and handling of milk have also an important role on the activity of lipases. Unit operations such as agitation, cooling and warming and homogenization damage the milkfat globule membrane (MFGM); and therefore the risk of lipolysis increases during processing of fluid milk (Fox and Kelly, 2006a).
119 3.5 Conclusion
Pressure–temperature combinations used in this study are sufficient to retard the extent of proteolysis in milk. However, storage conditions have a significant role on the activity of proteases. There is a synergistic effect of pressure and processing temperature on the inactivation of the plasmin system in milk. Although, the enzyme was not completely inactivated, combinations of pressure (650 MPa) with temperature (105 oC) were sufficient to inactivate plasmin to levels similar to UHT processes. Lipolysis was enhanced by pressure, holding time during pressurization and storage temperature. These results suggest that the flavor of pressure-treated milk samples could be compromised, as higher concentrations of FFA are associated with rancid flavors in milk. More research is needed to investigate pressure-temperature-holding time combinations that may be necessary to inactivate lipases in milk.
120 Chapter 4: Flavor Profile of Milk
4.1 Abstract
The flavor profile of high pressure processed (HPP) and pressure-assisted thermal processed (PATP) milk was compared to that of thermally pasteurized and ultra high temperature (UHT) processed milk using selected ion flow tube mass spectrometry analysis (SIFT-MS). Milk was HPP and PATP treated using a factorial 3x1x3 model at temperature (32, 72 and 105 oC), pressure (650 MPa), and time (0, 1, and 5 min). Milk samples were processed within 72 hr; and stored at either room temperature (25 ± 1oC) or refrigeration (4 ± 1oC) conditions depending on the treatment applied. Additionally, pasteurized (77 ± 0.8 oC for 18 sec) and UHT processed (138 ± 1 oC for 2 sec) milk samples were analyzed along with pressure–treated milk samples. The flavor profile of milk was evaluated using SIFT-MS analysis over a period of 20, 60 and 90 d. This relatively new technique was capable of not only identify but also quantify volatile organics compounds in the headspace of milk samples in real time basis. The formation of volatile aroma compounds in HPP and PATP milk was different than pasteurized and
UHT milk. Pressure treatments enhanced the formation straight–chain aldehydes and sulfur compounds; whereas thermal treatments enhanced the formation of methyl ketones, aldehydes and sulfur–containing compounds in milk on 0 d. The formation of volatile aroma compounds in pressure–treated milk samples was influenced not only by
121 pressure, temperature and holding time, but also storage temperature. Major contributors to the overall flavor profile of pressure–treated milk samples were hexanal, acetaldehyde, dimethyl sulfide and dimethyl disulfide. To a lesser extent, thermal treatments enhanced only the formation of 2-pentanone, 2-heptanone and dimethyl sulfide. These results indicate that HPP and PATP influenced the formation volatile aroma compounds in milk differently than traditional thermal treatments. In addition to the pressure effect, processing temperature and storage conditions had a significant role on the overall flavor profile of pressure–treated milk.
4.2 Introduction
Fresh milk has a characteristic aroma profile which is neutral, pleasant and slightly sweet. The components of milk including lactose, proteins, milkfat, salts and minor flavor compounds are responsible for the bland flavor of milk (Alvarez, 2009).
Active volatiles associated with the flavor profile of fresh and of good quality milk include dimethylsulphone, ethyl butanoate, ethyl hexanoate, heptanal, indole, nonanal, and 1-octen-3-ol (Friedrich and Acree, 1998). Any deviations from this flavor profile represent a major control issue in dairy operations as consumers can readily perceive these changes. Off-flavors in milk are associated with the cow’s diet, processing and handling, bacterial metabolism, enzymatic activity and others are defined as acquired from the environment (Shipe et al., 1978). From a technological standpoint, off-flavors derived from processing, bacterial metabolism and enzymatic activity are probably the most challenging aroma deviations to control at the dairy plant.
122 Up to date, high-temperature-short-time (HTST) pasteurization is the preferred method to process milk in the U.S. Pasteurized milk typically has a shelf life between 14 to 20 d depending on the processing conditions and handling procedures at the dairy plant. After this point, the product becomes unacceptable as off-flavors develop as a result of bacterial metabolism and enzymatic activity (Fromm and Boor, 2004). Up to 71 different volatile compounds have been associated with fresh pasteurized milk. These compounds include straight-chain aldehydes, ketones, sulfur compounds, lactones, terpenes and esters (Bendall, 2001). The concentration of these volatiles in fresh milk is relatively low; and not all aroma compounds contribute to the flavor profile of milk as the sensory threshold is different for each compound (Bendall, 2001; Van Aardt et al., 2005;
Vazquez-Landaverde et al., 2005). Only few active compounds are responsible for the characteristic flavor of milk. And, hexanal, 2-nonanone, benzothiazole, and δ-decalactone are potent active flavor volatiles commonly found in pasteurized milk (Friedrich and
Acree, 1998). Also, dimethylsulfide, 2-3 butanedione (diacetyl), 2-methylbutanal, 4- heptanal, butenylisothicyanate and 2-nonanal are important flavor compounds responsible for the characteristic flavor of pasteurized milk (Belitz et al., 2004). More aggressive thermal treatments are available to process fluid milk with longer shelf life (up to 6 months). Such is the case of ultra high temperature (UHT) processed milk. However, the product has a characteristic cooked flavor not appealing to most consumers in the
U.S. The effect of the heat treatment on the flavor of UHT milk is directly related to the time and temperature combinations used during processing (up to 150oC for < 4 s) (Shipe et al., 1978). The process gives the milk a distinguishing sulfurous note which comes
123 from partially denatured β-lactoglobulin and other sulfur-containing volatiles (Shipe et al., 1978; Solano-Lopez et al., 2005). Volatile compounds associated with the heated flavor of UHT milk include lactones, aldehydes, methyl ketones and maltol (Shipe et al.,
1978; Vazquez-Landaverde et al., 2005). These volatiles actively contribute to the development of heated off-flavor in milk. The quality of UHT milk has been discussed since its introduction in the early 60’s. Since then, there have been considerable efforts to obtain a flavor profile closer to the clean, neutral flavor of pasteurized milk (Tetra Pak, 2003) .
Recently, high pressure processing has gained attention as an alternative technology to traditional thermal treatments to process foods without compromising their nutritional and quality characteristics. The technique has successfully worked on acid foods. However, for low-acid foods, such as milk, combinations of high pressure with temperature are necessary to render extended shelf life products with high quality and flavor characteristics (Balasubramaniam and Balasubramanian, 2003; Patterson et al., 2006; Rajan et al., 2006a; Rajan et al., 2006b; Vazquez-Landaverde et al., 2006a) . A recent study assessing the effects of pressure and thermal processing in the quality of milk reported that pressure treatments enhanced the formation of different volatile organic compounds than those commonly found in traditional heat treatments. Pressure (482 – 620 MPa) in combination with mild temperatures (25 and 60 oC) enhanced the formation of hydrogen sulfide and straight- chain aldehydes; unlike heat treatment which favored the formation of methanethiol, hydrogen sulfide, methyl ketones and aldehydes. The authors suggested that higher
124 oxygen solubilization under high pressure leads to the formation of more aldehydes through lipid oxidation patterns. However, the actual formation mechanisms of flavor compounds under high pressure are yet unknown (Vazquez-Landaverde et al., 2006a).
Current methods to measure aroma compounds in milk include extraction techniques such as static headspace, purge and trap and solvent-assisted extraction techniques which are time consuming, labor intensive and consist of multi-stage operations. Therefore, measurement errors or analyte losses can occur as new compounds might be formed during the extraction process (Vazquez-Landavarde et al., 2005).
Selected ion flow tube mass spectrometry (SIFT-MS) is a relatively new technology that has been successfully used in clinical, microbial and more recently in food applications
(Spanel and Smith, 1999; Smith and Spanel, 2005; Allardyce et al., 2006; Scotter et al.,
2006). The technology uses soft chemical ionization reactions coupled with mass spectrometric detection to identify and quantify volatile organic compounds in real time basis. Volatile compounds are identified and quantified based on the known rate
+ + + coefficient for reactions between the analytes and the reagent ions H 3O , NO and O 2 .
SIFT-MS is a powerful analytical tool to measure the release of volatile aroma compounds from food matrixes in the headspace of the sample.
The objectives of this study were to compare the flavor profiles of thermal pasteurized, UHT and pressure-treated milk samples subjected to different pressure-heat combinations. The formation of volatile aroma compounds in milk samples was monitored over a period of 20, 60 and 90 d, respectively, depending on the treatment applied.
125 4.3 Materials and Methods
4.3.1 Milk preparation
Raw milk was obtained from a commercial dairy plant in Ohio (Orrville, OH) and transported to the OSU dairy pilot plant. The temperature of milk was kept at ~ 4 oC at all times during transportation (approximately 2 hr). Milk was standardized to 2% milkfat and two-stage homogenized using a Lab 100 M-G homogenizer (Lubeck-Schlutut,
Germany). Homogenized milk was immediately cooled to 7±1 oC and stored under refrigeration conditions (4±1 oC) until HPP and PATP processing as shown in Figure 4.1.
All milk samples were subjected to various pressure-heat conditions within 72 hrs. On the day of processing, milk samples were preheated to their corresponding initial temperature
(Ti) using an UHT/HTST Lab-25HV Hybrid unit (Micro Thermics Inc., Raleigh, NC).
The initial temperature of milk samples was adjusted as a function of the final target pressure during processing. This temperature was estimated based on the heat of compression of water and skim milk for various temperatures (Balasubramaniam et al.,
2004). The preheating time for milk to achieve the desired initial temperature was 2.5 min. The temperature of milk samples during preheating was set at values slightly higher than their required initial temperature (Ti) to compensate for the heat loss during filling and transportation of milk from the OSU dairy pilot plant to the high pressure processing laboratory. The initial temperature of milk samples was estimated based on the following equation:
CH Ti = T max − x∆P + ∆TH 100
126 Where, Ti = initial temperature before pressurization; Tmax = final processing temperature during pressurization; CH = heat of compression; and ∆P = applied pressure,
∆TH = Heat loss during pressurization; and Ti accounts for heat loss during preheating until pressure treatment (1±0.5 oC). The preheating temperature of milk samples was set at
4, 53 and 78±1 oC, respectively, depending on the pressure-heat treatment applied. Milk was filled into light-protected 8 oz polyethylene teraphtalane (PET) bottles without head space and manually capped. Immediately after, milk samples were transported from the
OSU dairy pilot plant to the high pressure processing laboratory. The total heat loss during preheating of milk until pressure treatments was 1 ± 0.5 oC. This minimal heat loss was achieved by placing milk bottles in a water bath set at the same temperature of milk samples.
127 Raw milk UHT milk (Orrville, OH) Commercial source (138±1 oC; 2 s) (New Jersey, NY) Clarification Milk Hauling (~4 oC, 2hr) (Orrville, OH) OSU (Columbus, OH)
Separation Clarification (7±1 oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Separation 2% milkfat (60±1 oC) (Orrville, OH) OSU (Columbus, OH)
Standardization Homogenization 2% milkfat OSU (Columbus, OH) (Orrville, OH)
Homogenization 500/1000 psi Pasteurization OSU (Columbus, OH) (77±0.8 oC) (Orrville, OH) Milk Storage ≤ 72 hr (4±1 oC) OSU (Columbus, OH)
HPP and PATP (650 MPa; 32, 72 & 105 oC; 0, 1 & 5 min) OSU (Columbus, OH)
SIFT-MS Analysis HPP &PM (4 oC); PATP & UHT (25 oC) 20, 60 and 90 d
Figure 4.1 Schematic diagram of milk processing and storage conditions
128 4.3.2 High pressure processing
An S-IL-110-625-08-W cold isostatic press system (Stansted Fluid Power Ltd .,
Essex, UK) was used to high pressure processed (HPP; 650 MPa, 32 & 72 oC) and pressure-assisted thermal processed (PATP; 650 MPa, 105 oC) milk samples. A 1:1 ratio propylene glycol, water mixture was used as the pressure-transmitting fluid. Milk samples were processed using a factorial 3x1x3 model at temperature (32, 72, and
105 oC), pressure (650 MPa) and time (0, 1 and 5 min) by duplicate. Pressure come-up time ranged between 1.5 to 3 min depending on the treatment applied, and decompression time was 1.2 min. The temperature of the vessel and pressure-transmitting fluid was thermostatically adjusted to the desired initial processing temperature by circulating propylene glycol through the external jacket of the pressure chamber. This temperature was set at the same initial temperature of milk samples (6, 48 and 73 oC, respectively).
Milk samples were filled into a cylindrical sample basket (102 mm dia x 559 mm height)
(Stansted Fluid Power Ltd., Essex, UK) and loaded into high-pressure equipment using a mechanical lift mechanism. The temperature of milk samples during various pressure treatments was monitored at the center of the carrier basket using T-type thermocouples
(Omega engineering, CT, USA) placed inside of a milk sample bottle designated for temperature recording purposes. After decompression, milk bottles were immediately placed in an ice bath to stop further thermal damage; and stored at either room temperature (25 ± 1 oC) or refrigeration (4 ± 1 oC) conditions depending on the final pressure/temperature treatment applied
4.3.3 Thermal treatments
129 High-temperature-short-time pasteurized milk (2% milkfat), obtained from the same batch of raw milk used for pressure-heat treatments, was processed in a commercial dairy operation (Orrville, OH) at 77±0.8 oC for 18 s. Additionally, ultra high temperature
(UHT) treated (138±1 oC for 2 s) milk samples were obtained commercially (New Jersey,
NY). HTST pasteurized and UHT milk samples were stored under refrigeration condition
(4±1 oC) and room temperature conditions (25±1 oC), respectively, and their flavor profiles were analyzed over a period of 20, 60 and 90 d, depending on the treatment applied.
4.3.4 SIFT-MS analysis
The concentration of 24 different volatile compounds commonly found in heat- treated milk was evaluated using selected ion flow tube mass spectrometry (SIFT-MS) analysis. This technology allowed the identification and quantification of volatile compounds in milk in real time basis. A Voice 100 mass spectrometer (Syft Technologies
Ltd., Christchurch, New Zealand) was used to analyze the concentration of 2- methylpropanal, 3-methylbutanal, hexanal, heptanal, octanal, nonanal, formaldehyde, methional, acetaldehyde, 2-pentanone, 2-heptanone, 2-nonanone, 2-decanone, 2- undecanone, 2,3-butanedione, propanone, methyl mercaptan, ethyl mercaptan, dimethyl sulfide, dimethyl disulfide dimethyl trisulfide, hydrogen sulfide, methanol and ethanol in milk samples.
Milk (50mL) was placed into 500 mL pyrex glass bottles (Fisher scientific,
Waltham, MA), closed by a septum cap and equilibrated to 55oC for 15 min. This temperature and time combination released the highest concentration of volatile compounds into the headspace of the sample without affecting the components of milk
130 (data not shown). Volatile aroma compounds in the headspace of milk bottles were sampled through the sample inlet needle connected to the entry port or capillary inlet of the SIFT-MS instrument. Helium was used as the carrier gas at a flow tube pressure of
0.556 Torr; the ion source was air and the average scan time was 120 s. Once the sample
+ + + reacted with the reagent ions H 3O , NO and O 2 , the resulting product ions were mass- selected by a second mass filter quadrupole and detected using a particle multiplier. The detection system was run in selected ion mode (SIM) scan. This mode allowed the identification of volatile compounds present in the sample by measuring target compounds at their specific product-ion masses. Volatile compounds in milk samples were identified in real time based on known molecular-ion reactions and a rate coefficient database (Spanel and Smith, 1999). Results were expressed in µg/kg of milk (ppb).
4.3.5 Data analysis
All experiments were conducted in quadruplicate on individual milk samples; and analyzed using crossed and nested ANOVA general linear model, with Tukey’s pairwise comparisons at 95% confidence level. Data analysis was performed using Minitab v. 15.2
(Minitab Inc., State College, PA).
4.4 Results and Discussion
The concentration of volatile aroma compounds in milk samples processed with different pressure and temperature conditions was measured using selected ion flow tube
– mass spectrometry (SIFT-MS) analysis.
131 SIFT-MS is a relatively new technology that has been successfully used in clinical, microbial and more recently in food applications (Spanel and Smith, 1999; Smith and Spanel, 2005; Allardyce et al., 2006; Scotter et al., 2006). The technology uses soft chemical ionization reactions coupled with mass spectrometric detection to identify and quantify volatile organic compounds in real time basis. Volatile aroma compounds were identified and quantified based on the known rate coefficient for reactions between the
+ + + analytes and the reagent ions H 3O , NO and O 2 .. These reagent ions do not react with the major components of air, but they do react with most volatiles organic compounds and many inorganic molecules (Spanel and Smith, 1999). As a result, product ions are generated and further selected using a second quadrupole mass spectrometer detection system; and volatiles were determined based on their characteristic ion masses using an extensive reference molecular-ion reaction and rate coefficient database (Spanel and
Smith, 1999; Scotter et al., 2006). The carrier was helium at a pressure of 0.558 Torr. .
SIFT-MS was a powerful analytical tool to measure the release of volatile aroma compounds in the headspace of milk samples. Only relative concentrations of volatile compounds in milk were quantified. Absolute concentrations of volatile compounds in foods cannot be measure by SIFT-MS as these depend on the flow rate of the sampled air and the exposed surface areas of the sample (Spanel and Smith, 1999). However, consistent comparisons between volatile concentrations in the headspace of samples using SIFT-MS were possible as these were measured by the constant vapor pressure partition coefficient of each sample (Smith and Spanel, 2005).
4.4.1 Pressure-temperature profiles of milk samples
132 Figure 4.2 shows the temperature and pressure history of HPP and PATP milk samples processed at 650 MPa; 32, 72 and 105 oC with 5 min holding time. The initial temperature of HPP milk samples (650 MPa; 32 oC for 5 min) was 7±1 oC and reached the target processing temperature (31±1 oC) after 1.5 min of compression. No significant heat loss was observed in these samples as a result of adiabatic compression during pressurization. Similarly, no significant heat loss was observed in HPP milk samples with pressure holding times of 0 and 1 min, respectively (data not shown). Pressure treated samples processed at 650 MPa and 72 oC for 5 min were preheated at 48±1 oC before pressurization. This temperature allowed milk to reach a target processing temperature of
77±1 oC during compression. Unlike HPP milk samples processed at 32 oC, pressure treated milk samples processed at 72 oC attained higher processing temperatures possibly due to heat gain experienced as a result of compression during pressurization. The final processing temperature achieved during pressurization (5 min) for PATP milk samples was 104±1 oC. The temperature gradient between the top and bottom of the pressure vessel was assumed to be ~ 10 oC due to lack of research samples designated to thermal studies. The current study, therefore, did not consider the impact of the temperature gradient on the chemical stability and residual plasmin activity of HPP and PATP milk samples.
133 125 700
C) 600 o 100 500 75 400 50 300 200
)Temperature ( )Temperature 25 100 (---) Pressure (MPa) (---) Pressure ( ( 0 0 0 2 4 6 8 10 12 Tim e (min)
Pressure Pressure Depressurization come-up time holding time time
Figure 4.2 Pressure–temperature profiles observed during high pressure treatments of milk a a PATP milk processed at 650 MPa and 32, 72 and 105 oC for 5 min *Data shown are means of two independent samples
4.4.2 Flavor analyses of milk
Up to 24 different volatile compounds commonly present in heat-treated milk were studied. Figure 4.3 shows the total concentration of aldehydes in milk samples subjected to different pressure and temperature profile on 0 d. With exception of PATP milk (105 oC), the total aldehyde content in pressure-treated samples processed at 32 and
72 oC, respectively, was not significantly different than pasteurized and UHT milk, on 0 d.
However, holding time during pressurization and temperature had a significant effect on the formation of aldehydes in pressure-treated samples. With increasing pressure holding time (0 – 5 min) and temperature (105 oC) the concentration of aldehydes further increased in milk. The total aldehyde concentration for PATP milk samples processed at
105 oC for 0, 1 and 5 min ranged from 228 to 367 ± 36 µg/kg, on 0 d. Major contributors
134 to the total aldehyde content in milk samples were acetaldehyde, 2-methylpropanal and hexanal. Among these flavor compounds, the concentration of acetaldehyde and 2- methylpropanal were higher in pasteurized (71 and 31 µg/kg, respectively) and UHT (51 and 45 µg/kg, respectively) milk samples; whereas the concentration of hexanal was consistently higher in all pressure-treated samples regardless of the processing temperature (79 to 91 ± 5.6 µg/kg).
450 c 400
g/kg) 350 0 0 0 0 b 300 a
250 a a a a a 200 a a a 150 100 Concentration ( Concentration 50 0 1 2 3 4 5 6 7 8 9 1011 Treatments b
Figure 4.3 Total concentration of aldehydes a in milk samples 1 Total aldehydes: 2-methylpropanal, 3-methylbutanal, hexanal, heptanal, octanal, nonanal, formaldehyde, methional and acetaldehyde 2 Treatments were: (1) Pasteurized milk (78 oC, 18 s); (2) HPP milk (650 MPa, 32 oC for 0 min); (3) HPP milk (650 MPa, 32 oC for 1 min); (4) HPP milk (650 MPa, 32 oC for 5 min); (5) HPP milk (650 MPa, 72 oC for 0 min); (6) HPP milk (650 MPa, 72 oC for 1 min); (7) HPP milk (650 MPa, 72 oC for 5 min); (8) PATP milk (650 MPa, 105 oC for 0 min); (9) PATP milk (650 MPa, 105 oC for 1 min); (10) PATP milk (650 MPa, 105 oC for 5 min); (11) UHT milk (139 oC for 2 s). a-cMeans ± std. dev. with different superscript are significantly different (n = 4)
135 These results suggest that the concentration of selected aldehydes (i.e. hexanal) is enhanced by pressure treatment with increasing temperature during processing. The formation of aldehydes during HPP and PATP might be related to lipid oxidation patterns of milkfat during processing as oxygen becomes more soluble under ultra high pressure
(Vazquez-Landaverde et al., 2006a). These observations are in agreement with previous work on the flavor profile of HPP milk. A study assessing the effect of high pressure – moderate temperature processing on the volatile profile of milk reported that treatments at 482 – 620 MPa and 25 and 60 oC for 1, 3 and 5 min enhanced the formation of straight– chain aldehydes in milk. Regardless of the holding time during pressurization, pressure treatments at 25 oC did not have a significant effect on formation of aldehydes in milk samples. The total aldehyde content in pressure–treated milk samples was very similar to that of raw milk. However, increasing processing temperature and holding time enhanced the formation of straight–chain aldehydes in pressure–treated samples processed at 60 oC.
The concentration of hexanal in these samples was significantly higher (284%) than heat- treated samples at atmospheric temperature. The authors reported that both pressure and holding time enhanced the formation of aldehydes in milk samples at higher temperatures
(Vazquez-Landaverde et al., 2006a).
Figure 4.4 shows the total concentration of methyl ketones in milk samples on 0 d. The concentration of methyl ketones in pressure – treated and heat – treated only milk samples followed similar formation patterns than aldehydes on 0 d.
136 200 180 c 160 g/kg)
140 120 b b 100 80 a 60 a a a a a a 40 a
Concentration ( Concentration 20 0 1 2 3 4 5 6 7 8 9 1011
Treatments b
Figure 4.4 Total concentration of methyl ketones a in milk samples 1 Total methyl ketones: 2-pentanone, 2-heptanone, 2-nonanone, 2-decanone, 2-undecanone, 2,3 butanedione, propanone 2 Treatments were: (1) Pasteurized milk (78 oC, 18 s); (2) HPP milk (650 MPa, 32 oC for 0 min); (3) HPP milk (650 MPa, 32 oC for 1 min); (4) HPP milk (650 MPa, 32 oC for 5 min); (5) HPP milk (650 MPa, 72 oC for 0 min); (6) HPP milk (650 MPa, 72 oC for 1 min); (7) HPP milk (650 MPa, 72 oC for 5 min); (8) PATP milk (650 MPa, 105 oC for 0 min); (9) PATP milk (650 MPa, 105 oC for 1 min); (10) PATP milk (650 MPa, 105 oC for 5 min); (11) UHT milk (139 oC for 2 s). a-cMeans ± std. dev. with different superscript are significantly different (n = 4)
The total methyl ketones content in HPP milk treated at 32oC did not significantly differ from pasteurized milk samples. And, holding time during pressurization had no significant effect on formation of methyl ketones in pressure–treated samples processed at room temperature (32 oC). With exception of HPPmilk processed at 72 oC for 0 min,
HPP milk samples treated at 72 oC showed similar total methyl ketones content than pasteurized milk. The concentration of methyl ketones in HPP milk samples treated at
72 oC without pressure holding time (0 min) was 44 ± 2 µg/kg; whereas the total content of these compounds in HPP milk treated at 72 oC for 1 and 5 min was 54 and 59 ± 8
µg/kg, respectively. Temperature and holding time had a significant effect on formation of methyl ketones in PATP milk samples. The total concentration of methyl ketones in
137 PATP milk samples (650 MPa and 105 oC for 0, 1 and 5 min) ranged from 71 to 161 ± 19
µg/kg on 0 d. And, the concentration of ketones in these samples significantly increased with increasing holding time. UHT milk samples showed higher concentrations of methyl ketones than pasteurized milk (186% increase). On 0 d, the total content of methyl ketones for UHT milk was 115 ± 14 µg/kg. This value was similar to that of PATP milk samples processed 1 min; but 29% lower than that observed in PATP milk processed for
5 min. Major contributors to the total methyl ketones content in milk samples were 2- pentanone, 2-heptanone and 2,3-butanedione. Among these volatile aroma compounds,
2,3-butanedione was the major contributor to total methyl ketones content in milk samples. The concentration of this volatile compound increased in HPP and PATP milk samples with increasing temperature and holding time. Vazquez-Landaverde et al.,
(2006a) reported similar formation patterns of methyl ketones in milk samples processed with different combinations of pressure and temperature. The authors reported that the formation of methyl ketones is affected by temperature. At 586 and 620 MPa at 60 oC the concentration of methyl ketones significantly increased as compared to the concentration observed in pressure – treated samples processed at 25 oC. Major contributors to the total methyl ketones content in their study were 2-pentanone and 2-hexanone. The authors concluded that at the pressures and temperatures studied, high pressure processing has no significant effect on the formation of methyl ketones; and attributed their formation to autoxidation and thermal oxidation patterns in milk samples (Vazquez-Landaverde et al.,
2006a).
138 The formation of sulfur-containing compounds in pressure–treated samples was enhanced by pressure, holding time and temperature (Figure 4.5). The concentration of these compounds in HPP milk samples treated at 32 oC ranged from 41 to 50 ± 4 µg/kg.
Pasteurized milk samples showed similar total content of sulfur compounds (52 ± 3.7
µg/kg) on 0 d. With increasing temperature, the concentration of sulfur – containing compounds further increased in pressure–treated milk samples. The total content of sulfur compounds in pressure treated foods at 72 oC ranged from 47 to 56 ± 5 µg/kg, on 0 d.
Holding time during pressure treatment had a significant effect on formation of sulfur compounds in HPP milk treated at 72 oC. With increasing holding time (0 – 5 min) the concentration of sulfur compounds significantly increased in these samples. Similar to aldehydes and methyl ketones, pressure treatments at 105 oC further enhanced the formation of sulfur–containing compounds in milk. The total concentration of sulfur compounds in these samples ranged from 67 to 120 ± 9 µg/kg on 0 d. Pressure holding time had also a significant effect on the concentration of these volatile aroma compounds.
The total concentration of sulfur compounds in PATP milk processed for 5 min was the highest among pressure treatments; and 42% higher than the total concentration of these compounds in UHT milk. Surprisingly, the concentration of sulfur compounds in UHT milk was not significantly different from that of pasteurized milk. On 0 d, the total concentration of sulfur compounds in UHT milk was only 50 ± 5 µg/kg.
139 160 c 140
g/kg) 120 ° ° ° ° b 100 80 a 60 a a a a a a a a 40
Concentration ( Concentration 20 0 1 2 3 4 5 6 7 8 91011
Treatments b
Figure 4.5 Total concentration of sulfur-containing compounds a in milk samples 1 Total sulfur compounds: Methyl mercaptan, ethyl mercaptan, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide and hydrogen sulfide 2 Treatments were: (1) Pasteurized milk (78 oC, 18 s); (2) HPP milk (650 MPa, 32 oC for 0 min); (3) HPP milk (650 MPa, 32 oC for 1 min); (4) HPP milk (650 MPa, 32 oC for 5 min); (5) HPP milk (650 MPa, 72 oC for 0 min); (6) HPP milk (650 MPa, 72 oC for 1 min); (7) HPP milk (650 MPa, 72 oC for 5 min); (8) PATP milk (650 MPa, 105 oC for 0 min); (9) PATP milk (650 MPa, 105 oC for 1 min); (10) PATP milk (650 MPa, 105 oC for 5 min); (11) UHT milk (139 oC for 2 s). a-cMeans ± std. dev. with different superscript are significantly different (n = 4)
The low content of sulfur compounds in UHT milk might be attributed to the high reactivity of these compounds. Flavor analyses in UHT milk were performed in commercial samples and analyses on these samples were not performed after processing.
Therefore, the concentration of sulfur–containing compounds might have decreased over this period of time. This phenomenon has been previously reported in milk. Depending on the severity of the heat treatment applied, the concentration of sulfur compounds in milk dissipates over time. The total content of these compounds in pasteurized milk can significantly decrease after 2 or 3 days of processing (Shipe et al., 1978). Also, most sulfur volatiles have flash vaporization values below UHT temperatures; and therefore,
140 they are easily oxidized into a variety of odorless end products during processing
(Christensen and Reineccius, 1992). In this study, major contributors to the total sulfurous note in milk samples were dimethyl sulfide and dimethyl disulfide. With exception of dimethyl trisulfide, methanethiol, ethanethiol and hydrogen sulfide did not play a significant role in the total concentration of sulfur compounds in milk samples.
Heat treatments enhanced only the formation of dimethyl sulfide; whereas pressure treatments at room temperature enhanced the formation of dimethyl disulfide. There was a synergistic effect of pressure and temperature on formation of these compounds in milk.
With increasing temperature, pressure treatments enhanced the formation of dimethyl sulfide, dimethyl disulfide and to a lesser extent dimethyl trisulfide. Vazquez-Landaverde et al., (2006a) reported similar formation patterns of sulfur compounds in pressure– treated milk processed at different temperatures. The authors reported that the concentration of selected sulfur compounds was affected by pressure and holding time.
The concentration of hydrogen sulfide significantly increased with increasing temperature (60 oC). However, the concentration was not consistent for all pressure treatments. In this study, the concentration of hydrogen sulfide was not significantly affected by pressure, temperature and/or holding time. Instead, dimethyl sulfide and dimethyl disulfide were major contributors to the sulfurous note in pressure – treated milk samples. Dimethyl sulfide is probably one of the most active sulfur compounds in milk. The concentration level at which this compound can be perceived by consumers indicates is an important contributor to the heat flavor of milk. The odor activity value
(OAV = concentration/sensory threshold) previously reported for this compound in milk
141 is 3 to 11, indicating that its concentration in heated milk (pasteurized and UHT) is much higher than its sensory threshold (Vazquez-Landaverde et al., 2006b). The concentration of dimethyl sulfide in the pressure – treated milk samples processed at 105 oC for 5 min was 49 and 59% higher than the concentration of this compound in pasteurized and UHT milk, respectively, on 0 d.
Figure 4.6 shows the total alcohol content in milk samples. Pressure treatments enhanced the formation of methanol and ethanol in milk samples regardless of processing temperature the temperature and holding time during pressurization. The total concentration of methanol and ethanol in pressure–treated samples was 59 and 81%, respectively, higher than the total content of these compounds in pasteurized and UHT milk on 0 d. No significant differences on the total alcohol concentration were found among pressure–treated samples with increasing holding time and temperature. The total alcohol content for these samples ranged from 1277 to 1390 ± 80 µg/kg on the initial day of analysis. Ethanol was the major contributor to the total alcohol content in pressure– treated samples; whereas, methanol was found in higher concentrations in heat–treated only milk samples
142 1600 b b b b b b b b b 1400 1200 g/kg) 1000 800 a 600 400 c
Concentration ( Concentration 200 0 1 2 3 4 5 6 7 8 9 10 11
Treatments b
Figure 4.6 Total concentration of alcohol a in milk samples 1 Total alcohol: methanol and ethanol 2 Treatments were: (1) Pasteurized milk (78 oC, 18 s); (2) HPP milk (650 MPa, 32 oC for 0 min); (3) HPP milk (650 MPa, 32 oC for 1 min); (4) HPP milk (650 MPa, 32 oC for 5 min); (5) HPP milk (650 MPa, 72 oC for 0 min); (6) HPP milk (650 MPa, 72 oC for 1 min); (7) HPP milk (650 MPa, 72 oC for 5 min); (8) PATP milk (650 MPa, 105 oC for 0 min); (9) PATP milk (650 MPa, 105 oC for 1 min); (10) PATP milk (650 MPa, 105 oC for 5 min); (11) UHT milk (139 oC for 2 s). a-cMeans ± std. dev. with different superscript are significantly different (n = 4)
High concentration of ethanol in milk is attributed to bacterial metabolism. Lactic acid bacteria (LAB) can be present in milk as a result of improper handling and sanitary operations at the dairy plant. In the proper environment, such as milk, these microorganisms follow heterofermentative and/or homofermentative patterns giving rise to fermentation compounds which can affect the overall flavor profile of milk (Lindsay,
1996). The main products formed by heterofermentative LAB are lactate, carbon dioxide and ethanol; whereas homofermentative bacteria mainly produce lactate from hexoses.
Genus of the heterofermentative LAB group include Leuconostoc, Oenococcus,
Weissella, Carnobacterium, Lactosphaera and some species of Lactobacillus . These microorganisms use half of the energy (1 ATP) required by homofermentative LAB to
143 produce the end fermentation products mentioned above; and they can grow well at the pH and temperature of refrigerated milk (Jay, 2000). Other end products of LAB metabolism include acetaldehyde, acetic acid and diacetyl (Lindsay, 1996).
Higher concentrations of methanol and ethanol in milk samples are also associated with reduction of aldehydes to primary alcohols via catalytic hydrogenation
(Morrison and Boyd, 1998). Unsaturated ketones are also reduced to alcohols via chemoselective hydrogenation in the presence of metals, such as copper. However, under optimized conditions the catalytic hydrogenation of aldehydes is favored as these molecules are less thermodynamically stable than ketones (Chen et al., 2000). The catalytic hydrogenation reaction is favored by temperature and pressure; and selective conversion of unsaturated aldehydes to saturated alcohols can also be possible in the presence of the conventional group VIII metal hydrogenation catalysts (Claus, 1998).
High pressure processing might have influenced the total alcohol content in milk samples. Under pressure, the pH of milk is reduced by about 1 unit / 1000MPa due an increase of water dissociation (Huppertz et al., 2002). Therefore, more availability of H 2 groups plus the thermodynamically favored hydrogenation reaction at higher pressures may have contributed to the total increase of alcohol content in milk samples as compared to pasteurized and UHT milk. Furthermore, a generalized increase in the total alcohol content of pressure–treated samples was observed with increasing temperature.
In this study, dimethyl sulfide, dimethyl disulfide, 2-methylpropanal, hexanal, acetaldehyde, 2-pentanone, 2-heptanone and 2,3 butanedione were identified as major contributors to the flavor profile of milk samples. These results are in agreement with
144 previous work on formation of volatile aroma compounds in pressure – and/or heat – treated milk samples (Contarini et al., 1997; Solano-Lopez et al., 2005; Van Aardt et al.,
2005; Vazquez-Landaverde et al., 2005; Vazquez-Landaverde et al., 2006a). Contarini et al., (1997) reported that major contributors to the heated flavor of milk include hexanal, heptanal, 2-pentanone and 2-heptanone. Also, 2,3-butanedione, pentanal, dimethyl disulfide, 1-hexanol, 1-heptanol and nonanal are active volatile compounds associated with light induced oxidation in UHT milk (van Ardt et al., 2005). Similar findings were reported by Solano-Lopez et al., (2005). In addition to methyl ketones and aldehydes, the authors identified the presence of selected hydrocarbons, fatty acids and alcohols in ultrapasteurized milk, including 2-pentanone, 2-heptanone, D-limonene, hexanal, octanal and nonanal. Vazquez-Landaverde et al., (2005) also suggested that 2-heptanone, 2- nonanone, 2-methylpropanal, 3-methylbutanal, decanal and dimethyl sulfide are important contributors to the off-flavor of UHT milk. These findings suggest that heat treatments enhance the formation methyl ketones, straight–chain aldehydes and selected sulfur compounds in milk. However, the overall flavor profile of milk is influenced not only by processing conditions, but also by storage conditions (van Aardt et al., 2005).
High pressure treatments influence the formation of volatile compounds in milk differently than heat treatments. Combinations of high pressure and temperature enhance only the formation of selected sulfur compounds and aldehydes (Vazquez-Landaverde et al., 2006a). The specific mechanisms of flavor formation under pressure are not fully understood yet. However, researchers have suggested that the solubility of oxygen available for chemical reactions increases under high pressure; and therefore there is an
145 increase in hydroperoxides formation leading to more aldehyde formation through lipid oxidation patterns. Additionally, the rate of volatiles formation could be pH dependent.
However, the effect of pH on volatile formation has not been assessed yet because there are currently no pressure-resistant probes available in the market (Vazquez-Landaverde,
2006a).
The formation of major contributors to the aroma profile of milk samples was monitored over a shelf life period of 20, 60 and 90 d, depending on the treatment applied.
Figure 4.7 shows the concentration of selected volatile compounds in pasteurized milk during its shelf life.
200 180 160
g/kg) 140 µ µ µ µ 120 100 80 0 60 2 40 6 Time (d) Concentration ( Concentration 10 20 15 0 20 l e e e a al e ne id o ion pan an ex ed l sulf ro h ptan e an yl disulfid ylp etaldehyd h th c 2-pentanone2-h but a 3- dimethy -me 2, dimet 2
Figure 4.7 Flavor profile a of pasteurized milk b a Volatile aroma compounds (ppb) on 0 – 20 d. b HTST pasteurized milk (78oC, 18 s). *Data shown are means ± std. dev. of 4 independent samples.
146 The total concentration of volatile compounds in pasteurized milk ranged from
1.3 to 65 µg/kg on 0 d; and with exception of 2-methypropanal, their concentration remained constant throughout its shelf life. Pasteurization enhanced the formation of 2- methylpropanal, acetaldehyde, 2,3-butanedione and to some extent hexanal. The concentration of dimethyl sulfide and dimethyl disulfide was 39 ± 3 and 3.4 ± 0.9, respectively, on 0 d; and did not significantly changed over the shelf life period studied.
This can be attributed to the fact that flavor analyses of milk were conducted after 4 days of processing; and therefore, the concentration of sulfur – containing compounds decreased before flavor analyses were performed. A possible explanation for this decrease in concentration is that sulfur compounds were participating in further reactions with Maillard reactions products producing non – volatile end products (Christensen and
Reineccius, 1992).
An increase of straight-chain aldehydes (2-methylpropanal and hexanal) and sulfur compounds (dimethyl disulfide) as compared to pasteurized milk were observed in pressure treated milk samples at 32 oC (Fig. 4.7 A-C). Pressure holding time did not exert any effect on the formation of volatile compounds; and therefore, similar flavor profiles were observed in these samples.
147 A
200 175 150 g/kg) µ µ µ µ 125 100
75 0 2 50 6 10 Concentration ( Concentration 25 15 Time (d) 0 20 e e e n lfide anal yd ione u p ulfid eh tano ed hexanal n yl s tald yl dis ylpro utan h eth h ace 2-pe 2-heptanone-b im imet d -met 2,3 d 2
B
200 175 150 g/kg) µ µ µ µ 125 100
75 0 2 50 6 10
Concentration ( Concentration Time (d) 25 15 0 20 l l ne ne ana na o o one ulfide p xa hyde di s e he ntan ptan lpro tald e e ane thyl e ut e hy 2-p 2-h ac 3-b imethyl disulfidedim met 2, d 2-
148 C
200 175
g/kg) 150 µ µ µ µ 125 100 75 0 2 50 6 10
Concentration ( Concentration Time (d) 25 15 0 20 l e e a n ne fide fide ion l pan no ul o tano ta ed disu l s hexanal ep tan hy ylpr u et th -pen acetaldehyde2 2-h im 3-b d me 2, dimethyl 2-
Figure 4.8 Flavor profile a of HPP milk processed at 32 oCb a Volatile aroma compounds (ppb) on 0 – 20 d. b Treatments were: (A) HPP milk (650 MPa, 32 oC for 0 min); (B) HPP milk (650 MPa, 32 oC for 1 min); (C) HPP milk (650 MPa, 32 oC for 5 min) *Data shown are means ± std. dev. of 4 independent samples.
The concentration of hexanal was 84.6% higher in pressure treated milk samples at 32 oC than in thermally pasteurized milk. Total acetaldehyde content was 39% lower in pressure–treated samples processed at 32 oC than pasteurized milk on 0 d. However, the concentration of this compound increased during the shelf life of milk; and ranged from
34 to 44 ± 8 µg/kg for all HPP treatments at room temperature, on 20 d. These values were 30 to 45 times higher than pasteurized milk. The increase in acetaldehyde content was holding time dependent; and highest concentration values were observed in HPP treated at 32 oC for 5 min. 2-methylpropanal followed different formation patterns in HPP milk than pasteurized milk. On 0 d, the total concentration of this compound in HPP milk
149 samples treated at 32 oC was 58 – 81 % lower than in pasteurized milk. A 65% increase in the concentration of this compound as compared to pasteurized milk was observed on 15 d, followed by a dramatic decrease on 20 d. The total content decrease of 2- methylpropanal can attributed to reduction to alcohols via catalytic hydrogenation as explained above. Flavors associated with aldehydes and ketones in milk include buttery from 2,3-butanedione, stale from saturated aldehydes, cooked from methyl ketones and benzaldehyde, light oxidized from pentanal, hexanal and methional, and malty from 3- methylbutanal (Shipe et al., 1978; Vazquez-Landaverde et al., 2005). The concentration of sulfur containing compounds in this study was higher in pressure treated milk samples at 32 oC than in thermally pasteurized milk samples. The total concentration of dimethyl disulfide gradually decreased after 20 d of storage; whereas the concentration of dimethyl sulfide increased to levels similar in pasteurized milk (35 – 40 µg/kg). The presence of sulfur compounds in milk is associated with cooked and oxidized flavors in milk (Shipe et al., 1978). It is generally accepted that hydrogen sulfide is major contributor to the cooked flavor in milk followed by dimethyl sulfide, and methyl mercaptan (Christensen and Reineccius, 1991; Vazquez-Landaverde et al., 2006b). Alcohols do not influence the overall flavor profile of milk if produced in relatively low quantities (Vazquez-
Landaverde et al., 2005). In most cases, these compounds are the result of bacterial metabolism and flavor defects may not be evident after 10 to 14 d of storage (Shipe et al.,
1978).
Figure 4.9 (A – C) shows the flavor profile of pressure treated milk samples at
72 oC over a period of 60 d stored under refrigeration conditions (7 ± 1 oC). Higher
150 concentrations of straight-chain aldehydes (hexanal and acetaldehyde) and sulfur compounds (dimethyl sulfide and dimethyl disulfide) were observed in pressure treated milk samples at 72 oC than in pasteurized milk. On 0 d, the concentration of hexanal was
83% higher than in pasteurized milk. Total acetaldehyde content on 0 d was 15.6 ± 3
µg/kg; and the concentration of dimethyl sulfide and dimethyl disulfide was 15.5 and
22.5 ± 3 and 1.4 µg/kg, respectively. A pronounced concentration increase of these compounds was observed at the end of the shelf life of milk samples. The concentration of hexanal, acetaldehyde, dimethyl sulfide and dimethyl disulfide in HPP milk processed at 72 oC for 0 min was 1372, 2561, 1680 and 378 µg/kg, respectively, on 60 d.
A
200 175
g/kg) 150 µ µ µ µ 125 100 0 75 6 10 50 15 20 Time (d) 30 Concentration ( Concentration 25 45 0 60 e l al e ide n lf lfide na on one u hyd n dione e tan ta e l s ld p disu y a hexa en yl th et p he h e - butan t ac 2- 2 - e ,3 dim -methylpropa 2 dim 2
151 B
200 175
g/kg) 150 µ µ µ µ 125 100 0 75 6 10 50 15 20 Time (d) 30 Concentration ( Concentration 25 45 0 60 e e al ne ide de d nal a pan no non sulfi ehy o a a l d hex nt pt l disulf thy etal ylpr pe he utanedione e h 2- 2- b ethy m ac m di -met 2,3- di 2
C
200 175
g/kg) 150 µ µ µ µ 125 100 0 75 6 10 50 15 20 30 Time (d) Concentration ( Concentration 25 45 0 60 l e e l a e d a n n fid yde n a o h a p n sulfi e o ta isul l d hex p d y al lpr e tanedione h t h u thy - b thyl ace 2-pentanone2 e imet ,3- d -me 2 dim 2
Figure 4.9 Flavor profile a of HPP milk processed at 72 oCb a Volatile aroma compounds (ppb) on 0 – 60 d. b Treatments were: (A) HPP milk (650 MPa, 72 oC for 0 min); (B) HPP milk (650 MPa, 72 oC for 1 min); (C) HPP milk (650 MPa, 72 oC for 5 min) *Data shown are means ± std. dev. of 4 independent samples.
152 Unlike pressure treated milk at 32oC, pressure holding time had a significant effect on formation of some aroma compounds in milk pressure processed at 72 oC. The extent of formation of acetaldehyde, hexanal, dimethyl sulfide and dimethyl disulfide decreased with increasing holding time. The concentration of hexanal, acetaldehyde and dimethyl sulfide was between 70 – 75 times lower in HPP milk processed at 72 oC for 5 min on 60 d, than that of HPP milk processed for 0 and 1 min, respectively. The concentration of dimethyl disulfide was only 34% lower in HPP milk treated at 72 oC for
5 min as compared to milk processed with lower pressure holding times. No significant increased in methyl ketones content was observed in pressure–treated samples processed at 72 oC during their shelf life. Higher concentrations of straight chain aldehydes might be associated with volatile compounds derived from lipid oxidation patterns of milkfat.
Hydrolytic rancidity and oxidation reactions are common in milk flavor development
(Alvarez, 2009). These reactions involve liberation of fatty acids responsible for rancid flavors in milk and oxidation of free saturated or unsaturated fatty acids with subsequent formation of volatile compounds. And, liberation of fatty acids from the glycerol bound can be associated with activity of lipases in milk. A study assessing the effect of heat– resistant lipases on flavor of UHT milk reported that milk samples with high lipolytic activity had flavor profiles described as cardboardy, oxidized or metallic at the end of their shelf life. The authors concluded that this was probably due to oxidation of unsaturated fatty acids and the subsequent formation of aldehydes and ketones in milk.
The presence of aldehydes and ketones gives rise to an off-flavor defect in milk called oxidized and it is favored by the liberation of fatty acids (Andersson et al., 1981). Scanlan
153 et al., (1965) reported that major contributors to the rancid flavor of milk are free fatty acids from the series butyric acid (4:0) to oleic acid (18:1). However, no particular acid in this series has predominant effect on the rancid flavor of milk. Instead, each fatty acid equally contributes to this flavor defect in milk (Scanlan et al., 1965). Later, Shipe et al.,
(1978) reported that major contributors to lipolysis in milk include butyric, caproic, caprylic, capric and lauric fatty acids. Long–chain and very short–chain fatty acids do not play a significant role in lipolysis of milk (Shipe et al., 1978). Once the fatty acid is released from the glyceride bound, oxidation reactions result from interactions between reactive oxygen species and lipids. Triplet oxygen and singlet oxygen have been identified as main compounds involved in oxidative changes of milk. Singlet oxygen is the electron–rich reactive specie of oxygen formed in the presence of light–induced photosensitizers, such as riboflavin in milk. Triplet oxygen is a diradical and it is considered the most stable form of oxygen. Oxidative changes in milk occur faster via singlet oxygen than reactions with triplet oxygen (Min and Boff, 2002). Oxidation reactions consist of initiation, propagation and termination steps. Light, heat, presence of metals, enzymatic activity and some chemicals catalyze the formation of radicals in milk during the initiation step. Newly–formed radicals can readily react with reactive species of oxygen and give rise to hydroperoxides; which upon cleavage of the hydroxyl group these compounds form peroxy radicals. Subsequent cleavage and molecular rearrangement of these compounds lead to formation of hydrocarbons, alcohols, acids, aldehydes and ketones responsible for oxidized flavors (Min and Boff, 2002). Aldehyde formation is favored over ketone formation during oxidation reactions. Cleavage on the
154 carboxyl group side results in the formation of an aldehyde and an acid ester; whereas formation of hydrocarbons and/or oxiacids are favored if cleavage of the hydroperoxide occurs on the hydrocarbon side of the molecule (Nawar, 1996). By definition, aldehydes are more reactive molecules than ketones. Aldehydes contain hydrogen attached to their carbonyl group; whereas ketones contain two aryl groups. This structural difference makes ketones more stable molecules; and therefore, less prone to undergo oxidation reactions. Instead, aldehydes are easily oxidized to carboxylic acids as they are more reactive than ketones during nucleophilic additions. The hydrogen attached to the carbonyl group of aldehydes is what causes the high reactivity of these molecules. At a constant temperature, the energy of activation (Ea) to subtract the hydrogen from the carbonyl group of aldehydes is significantly less than that of separating the aryl group from ketones during oxidation reactions (Morrison and Boyd, 1998).
Figure 4.10 shows the flavor profile of UHT milk over a shelf life period of 90 d.
UHT treatment enhanced the formation of methyl ketones (2-pentanone and 2-heptanone) and acetaldehyde. On 0 d, the concentration of 2-pentanone and 2-heptanone were 73 and
96 times higher, respectively, than pasteurized milk. The concentration of these compounds gradually increased with increasing storage time. On 90 d, the total content of
2-pentanone and 2-heptanone was 46 and 54 µg/kg, respectively. The concentration of acetaldehyde also gradually increased during the shelf life of UHT milk. On 0 d, the total content of this compound was 41 ± 3 µg/kg; and reached a maximum value of 77 ± 6
µg/kg at the end of the shelf life of milk. UHT treatment enhanced the formation of 2- methylpropanal. The concentration of this compound was 46 ± 9 µg/kg on 0 d. However,
155 the total content of 2-methylpropanal rapidly decreased after 15 d of storage and remained constant throughout the shelf life of milk. On 90 d, the concentration of this compound was 3 ± 0.7 µg/kg. The formation of hexanal was not enhanced by the UHT treatment. The total content of hexanal in UHT milk ranged 2 to 4.7 µg/kg after 90 d of storage.
200 175
g/kg) 150 µ µ µ µ 125 100 0 75 15 30 50 45 60 Concentration ( Concentration 25 75 Time (d) 0 90 l e e a e e e id d lfi non non su a ta edion nt n disulf yl hexan e ep l h ylpropanal ta hy h -p -h bu t acetaldehyde 2 2 - e imet 3 im d 2, d 2-met
Figure 4.10 Flavor profile a of UHT milk b a Volatile aroma compounds (ppb) on 0 –90 d. b UHT milk (139 oC for 2 s) *Data shown are means ± std. dev. of 4 independent samples.
Formation of sulfur-containing compounds was also enhanced as a result of UHT processing. The concentration of dimethyl sulfide increased during the shelf life of milk
(90 d) and reached a maximum value of 59 ± 4 µg/kg. The highest concentration of
156 dimethyl disulfide was observed on 30 d (26 ± 2.6 µg/kg) and it gradually decreased with increasing storage time. On 90 d, the total concentration of this compound was 9 ± 1.4
µg/kg. These results indicate that more aggressive thermal treatments enhance the formation of methyl ketones, sulfur-containing compounds and to some extent the formation of aldehydes. These compounds are responsible for the non – appealing cooked flavor of UHT milk and are associated with the Maillard reaction and to a much smaller extent to lipid oxidation (Colahan-Sederstrom and Peterson, 2005).
PATP treatments enhanced the formation of volatile compounds differently than
UHT processing (Fig. 4.11 A – B). The flavor profile of PATP milk samples was also different from that of HPP milk processed at 32 and 72 oC, respectively.
A
200 175 g/kg) µ µ µ µ 150 125 100 75 50 0 25 15 Time Concentration ( Concentration 30 0 (d) l e l e e e de a fid ide na on on l lf non i ehy a an isu su d exan ropa t ed l h p nt yl a l tan h et pe hep thy - - bu ac e 2 2 - imet 3 d -m 2, dimethyl d 2
157 B
200 175
g/kg) 150 µ µ µ µ 125 100 75 50 0 15 25 30 Concentration ( Concentration 0 45 Time (d) e e d lfi nal one u xa n is e d h l tanedione y u th 2-pentanone2-hepta e acetaldehyde im dimethyl sulfid 2,3-b d 2-methylpropanal
C
200 175
g/kg) 150 µ µ µ µ 125 100 0 75 15 30 50 45 60 Time (d) Concentration ( Concentration 25 75 0 90 e l e e e de de lfid fi anal on ul xana non su s ehy op e a tan ld h nt nedion yl l di a a th ylpr pe hep ut e hy cet h - b a 2- 2 met ,3- dim imet - 2 d 2
Figure 4.11 Flavor profile a of PATP milk b a Volatile aroma compounds (ppb) on 0 –90 d. b Treatments were: (A) HPP milk (650 MPa, 105 oC for 0 min); (B) HPP milk (650 MPa, 105 oC for 1 min); (C) HPP milk (650 MPa, 105 oC for 5 min) *Data shown are means ± std. dev. of 4 independent samples.
158 PATP milk samples processed without pressure holding showed a dramatic increase in concentration of methyl ketones, aldehydes and sulfur–containing compounds during the shelf life of milk (Fig. 4.11-A). On 0 d, the concentration of aldehydes
(acetaldehyde, hexanal and 2-methylpropanal) in PATP-0 min milk samples ranged from
37 – 85 µg/kg. Hexanal content in these samples was 95% higher than in UHT milk. On
30 d, the concentration of acetaldehyde, hexanal and 2-methylpropanal was 6425, 4362 and 9746 µg/kg, respectively. PATP treatments enhanced the formation of aldehydes in milk with increasing holding time. The total concentration of acetaldehyde was 30 and
64% higher in PATP milk processed for 1 and 5 min, respectively, than that of PATP milk processed without pressure holding time. Holding time during pressurization did not have a significant effect on the total concentration of hexanal in PATP milk on 0 d.
However, the concentration of 2-methylpropanal in PATP milk processed for 1 and 5 min was 42 and 52% higher than the total content of this compound in PATP-0 min milk.
With exception of 2-methylpropanal, the concentration of acetaldehyde and hexanal in
PATP-1 min milk showed highest values on 30 d (1074 and 2857 µg/kg), followed by a dramatic concentration decrease on 45 d (Fig. 4.11-B). This was not the case in PATP milk with a pressure holding time of 5 min (Fig. 4.11-C). The concentration of these compounds on 45 d reached maximum values of 313 and 342 µg/kg, respectively, and remained constant throughout the shelf life of PATP milk.
Unlike aldehydes, the concentration of methyl ketones was not enhanced by the
PATP process. On 0 d, the concentration of 2,3-butanedione, 2-pentanone and 2- heptanone was 17, 9 and 19 µg/kg in PATP milk without pressure holding time.
159 However, a significant increase in the concentration of these compounds was observed at the end of the shelf life of milk. Total 2,3-butanedione content in PATP milk with 0 min pressure holding time reached a maximum value of 626 ± 95 µg/kg on 15 d; whereas 2- pentanone and 2-heptanone showed highest values on 30 d (1650 and 1540 µg/kg, respectively). The concentration of dimethyl sulfide was similar to that of UHT milk; whereas PATP enhanced the formation of dimethyl disulfide on 0 d (81% higher).
Contrary to PATP milk without any pressure holding time, increasing pressure holding times enhanced the formation of methyl ketones in milk. On 0 d, the concentration of 2,3- butanedione, 2- pentanone and 2- heptanone in PATP milk held for 1 min was 29, 26 and
16 µg/kg, respectively (Fig. 4.11-B); whereas the total content of these compounds in
PATP milk with a holding time of 5 min was 37, 46 and 44 µg/kg, respectively, on the initial day of analysis (Fig. 4.11-C). The formation of methyl ketones gradually increased during the shelf life of milk samples. On 45 d, the concentration of 2,3-butanedione, 2- pentanone and 2- heptanone in PATP milk with 1 min pressure holding time was 30, 50 and 32 µg/kg, respectively; whereas the total content of these compounds in PATP milk with 5 min pressure holding time was 27, 146 and 56 µg/kg, respectively, on 90 d. Even though PATP treatments enhanced the formation of methyl ketones in milk, the total concentration of these compounds was significantly less as compared to that of aldehydes. These results indicate that formation of methyl ketones is enhanced not only by pressure, but more importantly by a temperature increase. Methyl ketones naturally exist in milk. However, these compounds are also produced as a result of the heat treatment of milk by β-oxidation of fatty acids, followed by decarboxylation (Solano-
160 Lopez et al., 2005). A study assessing the development and flavor properties of methyl ketones in milkfat reported that a large increase of ketone formation is observed at temperatures above 100 oC; and becomes constant at temperatures of 140 oC. The maximum ketone formation was observed after heating milkfat at 140 oC for 3 hr. This temperature-time combination enhanced the hydrolysis of fatty acids milkfat; and therefore, more formation methyl ketones was observed in milkfat samples treated under the conditions described above (Langler and Day, 1964). In this study, we observed an increase in the concentration of methyl ketones with increasing processing temperature
(i.e. 105 oC). The precursors of methyl ketones are β-keto esters; and therefore, heat– induced ketone formation is enhanced in foods with high a w values such as milk (Langler and Day, 1964). The authors also reported that although the concentration of individual ketones varied with the particular heat treatment applied, the concentration of 2- heptanone was the highest in all milk samples. There was no correlation between the relative amount of particular fatty acids present in milk and the content of ketones formed as a result of lipid oxidation patterns (Langler and Day, 1964). The formation of 2,3- butanedione in milk has been attributed to the intensity of the heat treatment of milk and to microbial activity (Vazquez-Landaverde et al., 2005). A recent study, however, linked the formation of 2,3-butanedione to the reaction between riboflavin and singlet oxygen in milk. Riboflavin is as an important photosensitizer in foods; and milk is the most important source of riboflavin with concentrations ranging from 1.5 to 2 µg/mL. When exposed to light, riboflavin produces singlet oxygen by the annihilation mechanism of triplet riboflavin and triplet oxygen. This reaction is extremely fast (1 x 10 10 M -1 s -1); and
161 causes a significant decrease of riboflavin content in foods. The authors proposed that the mechanisms of 2,3-butanedione formation involve the reaction between electron–rich singlet oxygen and riboflavin to form riboflavin endoperoxide through 6,9 addition.
Dioxetane is subsequently formed by 7,8 – cycloaddition; and 2,3-butanedione is formed after cleavage of the oxygen bound at the 6 and 9 position of the molecule. The rate of
2,3-butanedione formation was enhanced by exposure to light and final product had a buttery odor (Jung et al., 2007).
Sulfur-containing compounds in PATP milk without pressure holding time showed a dramatic increase in their concentration on 15 d (Fig. 4.11-A). The total concentration of dimethyl sulfide and dimethyl disulfide was 8564 and 318510 µg/kg at the end of the shelf life of PATP milk with 0 min pressure holding time (30 d). The formation of sulfur-containing compounds was different in PATP milk samples processed for 1 and 5 min (Fig. 4.11 B-C). On 0 d, the concentration of dimethyl sulfide in PATP-1 min was 25% higher than UHT milk. This compound had a maximum value of 1235 ± 58
µg/kg in PATP milk with 1 min pressure holding time on 30 d; and followed a dramatic decrease in concentration after 45 d of storage. The extent of formation of dimethyl sulfide in PATP milk with a pressure holding time of 5 min was significantly less compared to that of PATP milk with a pressure holding time of 1 min. This compound reached a highest concentration value of 342 ± 83 µg/kg on 45 d, and remained constant throughout the rest of the shelf life of milk. PATP treatments also enhanced formation of dimethyl disulfide in milk. On 0 d, the concentration of this compound in PATP- milk with 1 min holding time was 81% higher than in UHT milk. The total content of dimethyl
162 disulfide in these samples reached a maximum value of 2549 ± 65 µg/kg on 30 d; followed by a significant decrease in concentration after 45 d of storage. As with dimethyl sulfide, the extent of formation of dimethyl disulfide in PATP with 5 min holding time was significantly less than PATP milk with 1 min holding time. The total concentration of this compound in PATP-5 min gradually increased with storage time; and reached a maximum value of 55 ± 9.5 µg/kg after 90 d of storage. Sulfur-containing compounds are formed in milk as a result of the thermal process, pH, light exposure, oxygen level and presence of photosensitizers (Christensen and Reineccius, 1992; Jung et al., 1998; Vazquez-Landaverde et al., 2006b). These compounds are extremely difficult to measure because of their high reactivity and subsequent conversion to other compounds. The formation of dimethyl sulfide and dimethyl disulfide has been linked to heat-denatured whey proteins in milk (Vazquez-Landaverde et al., 2006b). Researchers have postulated that oxidation of sulfur-containing amino acids such as cysteine and methionine gives rise to formation of mercaptans, sulfides and disulfides in milk (Jung et al., 1998). Methanetiol has been proposed as precursor of dimethyl sulfide and dimethyl disulfide formation in the presence of light and oxygen. The specific formation mechanisms of methanetiol are not clear. However, this compound appears to be generated from methionine during heat treatment of milk by denaturation of β- lactoglobulin. In the presence of water, oxygen, metals or free radicals, methanetiol further oxidizes to form dimethyl disulfide and dimethyl trisulfide in milk (Vazquez-
Landaverde et al., 2006b). Other authors have postulated that dimethyl disulfide can also be formed as a result of singlet oxygen oxidation of methionine in the presence of
163 riboflavin. Jung et al., (1998) reported that methionine is oxidized to methionine sulfoxide by singlet oxygen at a rate of 5 x 10 6 M -1 s -1 in the presence of light. Dimethyl disulfide is formed subsequently after cleavage of the methylthiol radical (CH 3S•) and addition of a second radical. The authors reported that electron – rich (nucleophilic) sulfur amino acids such as cysteine and methionine can also react with singlet oxygen and give rise to hydrogen sulfide, methyl sulfide, dimethyl disulfide and other sulfur containing compounds in milk (Jung et al., 1998).
4.5 Conclusion
Selected ion flow tube mass spectrometry was successfully used to compare the flavor profile of HPP, PATP, pasteurized and UHT milk. There was a synergistic effect of pressure, temperature and pressure holding time on the formation of volatile aroma compounds in pressure – treated milk samples. With increasing holding time (0 to 5 min) and temperature (32 to 105 oC) the concentration of aldehydes, ketones and sulfur – containing compounds significantly increased in pressure – treated milk samples as compared to pasteurized and UHT milk on the initial day of analysis. However, significance differences on the flavor profile of milk were found during the shelf life period studied. Pressure treatments enhanced the formation of straight – chain aldehydes and sulfur compounds in milk; whereas pasteurization and UHT treatments enhanced the formation of methyl ketones, straight – chain aldehydes and selected sulfur compounds.
The formation of volatile aroma compounds was enhanced not only by processing conditions, but also storage temperature of milk. Major contributors to the overall flavor
164 profile of pressure – treated milk samples were hexanal, acetaldehyde, dimethyl sulfide and dimethyl disulfide. Based on these results and research data reported in previous chapters, the flavor profile of pressure – treated milk might be influenced not only by pressure, temperature and pressure holding time, but also other factors including bacterial metabolism and enzymatic activity.
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