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J. Dairy Sci. 88:3130-3138
© American Dairy Science Association, 2005.

Low Pressure CO2 Storage of Raw Milk: Microbiological Effects

M. Rajagopal, B. G. Werner and J. H. Hotchkiss

Department of Food Science, Cornell University, Ithaca, NY 14853

Corresponding author: Joseph H. Hotchkiss; e-mail: jhh3{at}cornell.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The effects of holding raw milk under carbon dioxide pressures of 68 to 689 kPa at temperatures of 5, 6.1, 10, and 20°C on the indigenous microbiota were investigated. These pressure-temperature combinations did not cause precipitation of proteins from the milk. Standard plate counts from treated milks demonstrated significantly lower growth rate compared with untreated controls at all temperatures, and in some cases, the treatment was microcidal. Raw milk treated with CO2 and held at 6.1°C for 4 d exhibited reduced bacterial growth rates at pressures of 68, 172, 344, and 516 kPa; and at 689 kPa, demonstrated a significant loss of viability in standard plate count assays. The 689-kPa treatment also reduced gram-negative bacteria and total Lactobacillus spp. The time required for raw milk treated at 689 kPa and held at 4°C to reach 4.30 log10 cfu/mL increased by 4 d compared with untreated controls. Total coliform counts in the treated milk were maintained at 1.95 log10 cfu/mL by d 9 of treatment, whereas counts in the control significantly increased to 2.61 log10 cfu/mL by d 4 and 2.89 log10 cfu/mL by d 9. At d 8, Escherichia coli counts had not significantly changed in treated milk, but significantly increased in the control milk. Thermoduric bacteria counts after 8 d were 1.32 log10 cfu/mL in treated milk and 1.98 log10 cfu/mL in control milk. These data indicated that holding raw milk at low CO2 pressure reduces bacterial growth rates without causing milk protein precipitation. Combining low CO2 pressure and refrigeration would improve the microbiological quality and safety of raw milk and may be an effective strategy for shipping raw single strength or concentrated milk over long distances.

Key Words: carbon dioxide • raw milk • microbiology • shelf life

Abbreviation key: SPC = standard plate count assay.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Raw milk serves as an excellent growth medium for microorganisms, and the numbers and types of organisms that can be found in milk directly affect safety and shelf life (Rowe, 1989; Espie and Madden, 1997). Gram-negative pseudomonads predominate in raw milk and are major contributors to milk spoilage; other types of bacteria, including pathogens, survive and grow (Muir et al., 1979; Griffiths et al., 1987). Most milk-borne vegetative organisms are generally inactivated by pasteurization but their lipolytic and proteolytic enzymes can survive and cause undesirable lipolysis and proteolysis. Additionally, thermoduric microorganisms that are potential pathogens or cause milk spoilage may survive the pasteurization process. The major strategy to extend shelf life of unpasteurized (raw) milk has been to provide rapid refrigeration. For example, decreasing the storage temperature from 6 to 2°C increases the time for the psychrotrophic count to reach 106 cfu/mL from 2.9 to 5 d (Griffiths et al., 1987). Benefits in distribution and pasteurized milk quality could be derived from reducing microbial growth in raw milk.

Several authors have reported on the use of CO2 as an antimicrobial agent in foods including dairy products (Dixon and Kell, 1989; Haas et al., 1989). In raw milk, bacterial growth was reduced by 50% after addition of CO2 and storage at 6.7°C for 48 h (Shipe et al., 1978). King and Mabbitt (1982) demonstrated an extension in storage life of both poor and good quality milks by the addition of 30 mM CO2. Carbon dioxide is effective at reducing the rate of growth of organisms detected in aerobic plate count assays (Roberts and Torrey, 1988). Compared with control milk, the standard plate count (SPC) of milk containing 20 to 30 mM dissolved CO2 was 3 log10 cfu/mL lower after 4 d of storage at 7°C (Mabbitt, 1982). In the presence of CO2, the time for SPC to reach 7 log10 cfu/mL was extended from 3 to 9 d at 7°C and 6 to 11 d at 4°C, whereas in the control milk, this level was reached in 5 d at 7°C and 8 d at 4°C (Hotchkiss, 1996). Coliforms and psychrotrophs were also significantly reduced compared with control milk under the same conditions (Roberts and Torrey, 1988). Generally, gram-negative psychrotrophs are more susceptible to the effects of CO2, whereas grampositive bacteria and spores are more resistant; Lactobacillus spp. are relatively CO2 resistant, and their growth may be enhanced by a CO2-enriched environment (Hendricks and Hotchkiss, 1997). Excessive growth of Lactobacillus spp. in raw milk may lead to spoilage or development of off-flavors due to fermentation. Treatments that reduce microbial populations may result in outgrowth of thermoduric spore-forming bacteria due to reduced competition, increasing the likelihood of postpasteurization spoilage or reduced food safety.

The addition of CO2 has been shown to increase the lag phase of growth and decrease the growth rate of microorganisms (Martin et al., 2003). In CO2-treated milk, extension of the lag phase increased the generation times of Pseudomonas spp. (Roberts and Torrey, 1988). Increasing concentrations of CO2 increased lag phases and extended growth rates. King and Mabbitt (1982) demonstrated an extension in storage life of poor quality milk (105 cfu/mL) by 1.2 d and of good quality milk (103 cfu/mL) by 3 d with the addition of 30 mM/L CO2. The extension of keeping quality of milk due to CO2 was maximized when the initial counts in the milk were low. Low-level carbonation of bulk tank milk inhibits the increase in microbiota for 3 to 4 d. The reduction in counts would thus reduce the thermotolerant lipases and proteases secreted into the milk postpasteurization (Espie and Madden, 1997).

Several theories explaining the mechanism of CO2 action on microorganisms have been proposed. The exclusion of oxygen by replacement with CO2 may contribute to the overall effect by slowing the growth rate of aerobic bacteria (Daniels et al., 1985). Carbon dioxide can readily pass through cell membranes and form carbonic acid within the cell with a resultant decrease in intracellular pH, which slows intracellular enzyme activities (Wolfe, 1980). Carbon dioxide has been demonstrated to be inhibitory of certain enzymes, especially decarboxylating enzymes (Gill and Tan, 1979). Carbon dioxide can also accumulate in membrane lipid bilayers, altering membrane properties and inhibiting membrane functions (Enfors and Molin, 1978). The effect of CO2 is enhanced at lower temperatures (Gill and Tan, 1979). The increasing solubility of CO2 at lower temperatures increased the relative inhibitory effect of CO2 on Pseudomonas fragi (Enfors and Molin, 1981).

Under specific combinations of pressure and temperature, CO2 effectively precipitates the proteins from milk. For example, at 38 and 50°C, and pressures above 5514 kPa, complete precipitation of casein from milk results (Tomasula and Boswell, 1999). Calvo and Balcones (2001) demonstrated that the amount of casein aggregation increased when increased pressure was applied to milk for increasing residence times. Carbon dioxide pressure treatments of 294 to 735 kPa applied at 20, 30, 40, and 50°C resulted in casein aggregation. Clearly, any storage strategy that uses CO2 must consider the potential adverse effects of protein precipitation.

Most of the work cited above has focused on the addition of low levels of dissolved CO2 into raw milk held at atmospheric pressure. Our goal was to investigate the effect on raw milk spoilage and pathogenic microbiota of holding raw milk under positive CO2 pressures that do not result in precipitation of milk solids. We examined changes in the following groups of typical milk microbiota as indicators of potential quality and safety: total Lactobacillus spp., lactose-fermenting, and nonlactose-fermenting gram-negative bacteria, Escherichia coli, thermoduric bacteria, and SPC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Test System Design
The apparatus for pressurizing and holding raw milk samples consisted of two 13-mL stainless steel 1.27-cm o.d. cylindrical vessels; one vessel was pressurized and the other served as a control (Figure 1, A and BGo). Compressed and filtered CO2 from a high-pressure tank (Figure 1CGo) was used (Empire Airgas, Inc., Elmira, NY). The system consisted of pressure regulator (Figure 1DGo), a fine metering valve (Figure 1EGo) (NUPRO Company, Willoughby, OH), an on-off valve (Figure 1FGo) (Circle Seal, Anaheim, CA), and a check valve (Figure 1GGo) (NUPRO Co.). The fine metering valve controlled gas flow such that the time to reach desired pressure was <5 s. The gas entered the vertically positioned treatment vessel from the bottom and was thus bubbled through the milk until the set pressure was reached. A check valve was placed immediately before the inlet to the pressure vessel to prevent the backward flow of the fluid milk into the gas inlet line. The outlet of the vessels consisted of a pressure gauge (Figure 1HGo) and a high-pressure release valve (Figure 1IGo) (High Pressure Equipment, Erie, PA). The release valve was kept tightly closed during treatment. The control vessel was closed off from both ends but not connected to the carbon dioxide line inlet and outlet lines.



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  		Figure 1. Carbon dioxide batch pressurization system.

 
The apparatus was cleaned and sanitized before and after each treatment as follows: water rinse, Conquest sodium hydroxide (Ecolab Inc., St. Paul MN) soak (20 min, 23°C), warm tap water rinse (50°C), Monarch CIP phosphoric acid bath immersion (Ecolab) (20 min, 23°C), warm tap water rinse (50°C), Trichloro-o-cide XP (Ecolab) soak (30 min, 23°C), and sterile water (50°C) rinse (3x). This protocol was validated by testing swab samples of critical control points in the dismantled apparatus for microbial load, and by testing equipment rinse water pH and residual chlorine content (Hach Company, Loveland, CO). All pH measurement was performed using an Accumet model 925 pH meter and Accumet polymer body combination electrode with silver/silver chloride reference (Fisher Scientific, Pittsburgh, PA). Temperature was controlled by a circulating water bath (Figure 1JGo) (VWR 1145 Refrigerated Temperature Constant Circulator, Westchester, PA), which circulated hot/cold water through copper coils (Figure 1KGo) immersed into water in a vacuum Dewar flask that held the treatment and control vessels. Copper-constantan thermocouples (Figure 1LGo) measured the temperature of the treatment and control vessels, and were continuously logged onto a temperature recorder (Figure 1MGo) (Omega Engineering Inc., Stamford, CT).

Milk Sampling, Treatment, and Analysis
Whole, unhomogenized, raw milk was obtained from 2 sources. Commingled milk samples were obtained from the Northeast DHIA (Ithaca, NY), a dairy analytical consulting laboratory. These samples were commingled bulk milks from 236 farms from New York, Pennsylvania, and New Jersey; and thus, could be considered representative of a wide range of milk flora. Milk was also obtained from the Cornell University Teaching and Research Center bovine herd (T&R Center; Dryden, NY). All milk was stored at 6°C until use. Raw milk from the T&R Center was received less than 12 h after milking in sterile bottles and held on ice until it could be moved to a 6°C cooler.

Milk samples were mixed and 5 mL added into the treatment and control vessels. The treatment vessel was connected to the apparatus and the control vessel closed off. Both vessels were placed in the waterbath. When the desired temperatures were attained in both treatment and control vessels, CO2 was introduced through the bottom of the treatment vessel until the set pressure was reached. The CO2 pressure was maintained throughout the test period. When the desired time was reached, the CO2 inlet was turned off, the pressure release valve on the outlet line opened, and the pressure released in <1 min. After depressurization, the treatment and control vessels were removed from the water bath and their external surfaces were wiped dry, sanitized with 95% (vol/vol) ethanol, detached from the apparatus, and the contents transferred into sterile containers for dilution and plating.

The effect of CO2 pressure/temperature combinations on protein precipitation was measured at CO2 pressures of 344, 689, 1378, 2067, 2757, and 3446 kPa at temperatures of 20, 10, and 5°C for 5, 15, 30, and 60 min. The amount of protein precipitation was quantified and expressed as percentage precipitated solids by the method of Tomasula (1995).

Short (<1 h) and long-term (1, 4, and 9 d) experiments were conducted. Raw milk (Northeast DHIA) in 5-mL aliquots was treated at each of the following combinations of CO2 pressure, temperature, and time (kPa/°C/min): 1378/5/15, 2757/5/5, 3446/5/5. In the long-term studies, raw milk from the T&R Center was first stored at 6°C/48 h so that the SPC were at detectable levels at treatment initiation. Five milliliters of milk was treated with CO2 pressures of 0 (control), 68, 172, 344, 516, and 689 kPa for 1 to 9 d at 4.1 to 10°C.

Raw milk from the T&R Center was monitored for changes in aerobic bacteria, gram-negative bacteria, and total Lactobacillus spp. as follows: CO2 pressures of 0 (control), 68, 172, 344, 516, and 689 kPa, at 6.1°C for 4 d. Standard plate count, gram-negative bacteria, and total Lactobacillus spp. were enumerated before (d 0) and after (d 4) treatment. The time to reach an SPC of 2 x 105 cfu/mL was determined using raw milk (T& R Center) without a 2-d storage time. Equal volumes were transferred into treatment and control vessels and held at 0 and 689 kPa CO2 and 4.1°C.

The progression of these counts (total, coliform/E. coli, and thermoduric bacteria) in the treatment and control samples was tracked by conducting checks on the total aerobic counts (SPC) on treatment d 4 and 6. Based on the levels of total counts on d 4 and 6, analyses of total coliforms/E. coli, and thermoduric bacteria after d 6 were conducted either at 1- or 2-d intervals. The control sample final count was measured on d 4 and 6.

Microbiological Methods
For all microbiological assays, milk sample aliquots of 1 mL were used in dilution series. Standard plate counts were performed by the method described in Standard Methods for the Examination of Dairy Products (Houghtby et al., 1992). Gram-negative bacteria were enumerated on MacConkey agar (Difco Manual, Becton Dickinson & Co., Sparks, MD) after spread plating and incubation at 30°C for 48 h. MacConkey agar (Difco Manual, Becton Dickinson & Co., Sparks, MD), a selective and differential media, can be used to discriminate between lactose-fermenting and nonlactose-fermenting gram-negative bacteria. Thus, use of this media allows a one-step method of obtaining estimates of both coliform and noncoliform gram-negative bacteria. Coliform bacteria may include species of Escherichia, Klebsiella, and Enterobacter, potential pathogens and spoilage organisms. Noncoliform gram-negative bacteria may include spoilage organisms such as pseudomonads or potential pathogens such as Salmonella spp. or Shigella spp. Lactobacillus spp. were estimated by pour plating in acidified (adjusted to pH 5.5 with glacial acetic acid) Lactobacillus de Man, Rogosa, and Sharpe agar (Difco Manual, Becton Dickinson & Co.), incubated at 32°C for 48 h under anaerobic conditions. Representative and distinctive suspect colonies were gram-stained, and confirmed gram-positive bacilli colonies were counted as an estimate of total Lactobacillus spp.

Initial total, coliform, and thermoduric counts were each determined for control and treated samples. Thermoduric organisms were enumerated by the laboratory pasteurization count method described in the Standard Methods for the Examination of Dairy Products (Houghtby et al., 1992). The 3M Petrifilm count plate (3M Microbiology Products, St. Paul, MN) was used to enumerate total coliforms and E. coli in the raw, treated, and control milk samples.

Statistical Analyses
MINITAB Release 13.1 (Minitab Inc., State College, PA) was used for statistical analyses of the data. Experiments were conducted in duplicate using 2 different milk samples (n = 2); each sample was tested in triplicate. Significant difference among samples was determined at P ≤ 0.05. Analysis of variance was used to determine the effect of CO2 pressure, and the interaction effects of pressure and temperature.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Effect of Low Pressure CO2 on Milk Protein Precipitation and pH
Application of CO2 pressures greater than 1378 kPa for 15 to 60 min resulted in more than 1% precipitation of milk solids at 20°C (data not shown). Treatment for 30 min at 2067 kPa resulted in 2.6% (wt/wt) solids, which approached the maximum (2.8%) produced by sulfuric acid precipitation (Southward, 1986). However, lowering the holding temperature reduced the amount of precipitation; at 5°C and pressures of less than 2067 kPa, precipitation could not be detected, even after 60 min. Treatment combinations of 689 kPa for 60 min, 1378 kPa for 30 min, 2757 kPa for 5 min, and 3446 kPa for 5 min did not cause detectable precipitation at 5°C.

The pH of the treated and control milk samples (as measured at atmospheric pressure) was 6.6 to 5.9 at CO2 pressures ≤516 kPa and 5.7 when treated at pressures ≥516 kPa and 20°C. The pH of the treated and control milk samples, when treated at 10°C, was 5.5 at CO2 pressures ≤516 kPa, and 5.8 when treated at pressures >516 kPa.

These results generally agree with previous reports including Jordan et al. (1987), Tomasula (1995), and Calvo and Balcones (2001), who independently investigated the precipitation of caseins from raw skim milk using pressurized CO2. Tomasula (1995) found that CO2 pressures between 2757 and 5514 kPa and temperatures between 38 and 49°C caused complete casein precipitation. Calvo and Balcones (2001) precipitated 85% of raw skim milk caseins by applying CO2 pressures above 1998 kPa for 3 h at 40°C. Jordan et al. (1987) obtained 99% precipitation of skim milk casein by treatment with 3515 kPa at 50°C.

Protein precipitation occurs when the pH of the milk has been reduced below the isoelectric point of the casein (pH 4.6). The addition of CO2 to milk leads to the formation of carbonic acid and a decrease in pH; however, pressurization with CO2 can cause precipitation of caseins at a pH higher than its isoelectric point (Tomasula and Boswell, 1999). Ma and Barbano (2003) found that increasing CO2 concentration and pressure decreased the pH of skim milk; the pressure effect was greater as CO2 concentrations increased. These researchers also determined that increasing temperature influenced the solubility of milk colloidal calcium phosphate, resulting in a decrease in milk pH. Jordan et al. (1987) found that precipitation of casein occurred between 40 and 70°C, and that the yield at any specific temperature was dependent upon a minimum pressure; this minimum pressure was inversely related to temperature. Thus, specific pressure/time/temperature treatment combinations can be manipulated that will not cause precipitation of proteins from raw milk.

Effect of Low Pressure CO2 on Raw Milk Microflora
Changes in gram-negative lactose-fermenting and nonlactose-fermenting bacteria, Lactobacillus spp., and SPC in raw milk treated under different temperatures and pressures is illustrated in Figure 2Go. All time/ pressure combinations significantly reduced the SPC of the raw milk compared with untreated controls, even at a low pressure/high temperature combination of 68 kPa and 20°C. At 1378 kPa, the control SPC was 7.89 log10 cfu/mL, whereas the treated milk SPC was reduced by 0.33 log10 after 15 min and 0.39 log10 after 30 min. Twenty-four-hour treatments at 20°C and pressures ≥344 kPa resulted in microbial inactivation. The SPC of milk treated at 344, 516, and 689 kPa was significantly reduced from initial SPC by 0.39, 0.62, and 0.82 log10, respectively, whereas the SPC of the control milks significantly (P < 0.05) increased by as much as 2.06 log10 cfu/mL. The SPC in milk held at 68 and 172 kPa significantly increased by 1.07 and 0.59 log10 cfu/mL, respectively; however, this population increase was significantly less than that exhibited by the control milk.



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  		Figure 2. Changes in gram-negative lactose fermenting ({square}) and nonlactose fermenting bacteria ({graysqu}), Lactobacillus spp. ({graysqu}), and standard plate count ({blacksquare}) in raw milk treated at (A) 68 kPa, (B) 172 kPa, (C) 344 kPa, (D) 516 kPa, and (E) 689 kPa CO2 pressure and 6.1°C for 4 d; n = 2, each sample plated in triplicate. a-lCounts with different letters are significantly different (P ≤ 0.05).

 
Carbon dioxide pressure treatments of 68 and 172 kPa at 10°C applied over 24 h were more effective at curtailing growth than similar pressure/time treatments at 20°C. As found at 20°C, there was a loss in viability at pressures ≥344 kPa, and the differences between control and test counts increased with increasing holding time; significant decreases in counts of 0.31, 0.56, and 0.71 log10 cfu/mL at 344, 516, and 689kPa CO2, respectively, were achieved. The difference in SPC between control and test milks at 689 kPa was 2.68 log10 cfu/mL. These data indicate that holding raw milk under CO2 not only slowed the growth of the microorganisms in the raw milk but also, in some cases, resulted in a loss in viability.

Others have shown inactivation of microbiota in raw and pasteurized milk with CO2 at significantly higher pressures (Erkman, 1997, 2000; Calvo and Balcones, 2001). Calvo and Balcones (2001) found a decrease in bulk raw milk microbiota of 2 log10 cfu/mL upon treatment with 3997 kPa CO2 at temperatures ≥40°C for 30 min. Erkman (2000) demonstrated a reduction in aerobic microorganisms in whole milk of 6 log10 cfu/mL after a 24-h treatment under 6044 kPa CO2 pressure at 45°C. Erkman (1997) also demonstrated a reduction of 8 log10 cfu/mL after a 5-h 14,598 kPa CO2 treatment at 25°C. However, the use of such pressures would, in our experience, result in complete precipitation of the caseins and would require the use of specially designed equipment. Calvo and Balcones (2001) reported that pressures of 3997 kPa caused precipitation, whereas Erkman (1997, 2000) made no mention of the state of the milk. We are not aware of any reports testing the effects of low pressures.

Lowering the holding temperature to 6.1°C significantly reduced microbial growth compared with control milks when CO2 pressures of 68, 172, 344, 516, and 689 kPa were applied for 4 d. For example, the SPC of milk held at 689 kPa was 0.89 log10 cfu/mL lower than initial counts and 3.48 log10 cfu/mL lower than controls. After 9 d storage under 689 kPa CO2 at 4°C, the ratios of treated to untreated SPC, thermoduric, coliform, and E. coli counts were consistently lower than the ratios of control to untreated counts for the comparable groups (log10 cfu/mL) (Table 1Go). Milks treated at 68, 172, 344, and 516 kPa significantly increased from an initial SPC of approximately 3.30 log10 cfu/mL by 1.28, 1.10, 0.94, and 0.82 log10 cfu/mL, respectively, whereas the control SPC increased by 2.86, 2.85, 2.86, and 2.93 log10 cfu/mL, respectively. Milk held at 689 kPa and 6.1°C for 4 d exhibited greater inactivation than that observed after treatments at 10 or 20°C for 24 h (P < 0.05). The pH decreased from 6.6 (before treatment) to 5.5 in milks treated at 516 kPa, to 5.8 at 344 kPa, and 5.9 at 68 kPa.


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  		Table 1. The effect of 689-kPa CO2 pressure at 4°C after 4-, 6-, 8-, and 9-d treatments on standard plate count, thermoduric bacteria, coliforms, and Escherichia coli counts in untreated, treated, and control raw milks.1
 
In addition to SPC, there were significant differences in gram-negative lactose-fermenting and nonlactose-fermenting bacteria and Lactobacillus spp. between CO2-treated and control milks (Figure 2Go). For example, levels of gram-negative fermenters and nonfermenters were reduced at all pressures compared with untreated controls. Likewise, Lactobacillus spp. counts were approximately 1 to 2 log10 cfu/mL lower in the test milks compared with control milk. At 689 kPa, gram-negative lactose-fermenting and nonlactose-fermenting bacteria exhibited significant decreases of 0.80 and 0.64 log10 cfu/mL, respectively, compared with initial counts. Under 516 kPa CO2 pressure, SPC of treated samples were not significantly different from initial untreated samples, whereas SPC of control samples increased by 2.95 log10 cfu/mL. Reductions in total microbial populations as well as reductions in gram-negative and Lactobacillus spp. populations would result in improved quality of the raw milk. Ruas-Madiedo et al. (1996) found that lower levels of volatile compounds (ethanol, 2-propanone, and 2-butanone, which are microbial metabolites) were produced in carbonated milk during storage and that higher sensory scores were achieved than in untreated milks. In a later study, Ruas-Madiedo et al. (2000) found a direct association between reduced microbial growth and reduced levels of microbial glucosidases in raw milk stored with CO2; degradation of milk glucose was subsequently reduced in the treated milks. It has also been found that levels of fat-soluble vitamins (retinol, ß-carotene, and {alpha}-tocopherol) in milk treated with CO2 and stored at 4°C for 7 d were higher than that measured for untreated raw and pasteurized milks (Ruas-Madiedo et al., 1998a,b).

In the current study, populations of Lactobacillus decreased after CO2 treatment. Others have found that treatment with CO2 at concentrations between 0 and 2000 mg/L had no impact on the lag phase of Lactobacillus sake when grown at 7°C, and influences on the maximum specific growth rate was least affected compared with species of Pseudomonas, Aeromonas, Bacillus, Brochothrix, and Shewanella (Devlieghere and Debevere, 2000). Espie and Madden (1997) reported no effect of 30 and 45 mM CO2 on the growth of Lactobacillus spp. Neither of these investigations, however, incorporated pressures above atmospheric in their treatments. Reductions in populations of Lactobacillus plantarum of more than 6 logs was achieved after treatment with CO2 pressures of 13 MPa at 30°C for 30 min (Hong et al., 1999). In subsequent studies, these researchers found that high-pressure CO2 treatment of L. plantarum resulted in irreversible cellular membrane damage and reduced activity of some intracellular enzymes, physiological changes that could result in microbial inactivation (Hong and Pyun, 2001). Combined or enhanced effects of low pressures and CO2 treatments could thus explain why we observed reductions in total Lactobacillus populations.

The effect of 689 kPa CO2 at 4°C on the time to reach an SPC of 1 x 105cfu/mL was investigated. Pasteurized Milk Ordinance Grade A regulations specify that the SPC for raw milk should be <1 x 105 cfu/mL before pasteurization (US Department of Health and Human Services, 1999). The treated milks reached 105 cfu/mL after 8 d of treatment, whereas the control milk reached this level after just 4 d (Figure 3Go). Treatment at 689 kPa and 4°C extended the treatment holding time by at least 4 d compared with the control. At the end of 4 d, treated milk SPC had decreased to 2.89 from 3.48 log10 cfu/mL, whereas control milk SPC increased by nearly 2 log10 cfu/mL. This reduction in SPC in treated milk agrees with the trend observed in the 4-d experiments conducted at 6.1°C (Figure 2Go). Milk SPC increased to 4.64, 4.99, and 5.37 log10 cfu/mL after 6, 8, and 9 d, respectively (Figure 3Go). Neither E. coli nor total thermoduric bacteria counts increased in the treated milk but both significantly increased in the controls. The pH of the treated milk samples changed from an initial value of 6.6 to 5.5 at the end of d 4, 6, 8, and 9 of treatment.



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  		Figure 3. Total counts ({square}), thermoduric bacteria ({graysqu}), total coliforms ({graysqu}), and Escherichia coli ({blacksquare}) counts in milk treated at 4°C and 689 kPa CO2 pressure after 4, 6, 8, and 9 d of storage. Experiments were conducted in duplicate, with 2 milk samples per experiment, and each sample plated in triplicate. a-rCounts with different letters are significantly different (P ≤ 0.05).

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Under selected pressure and temperature combinations, CO2 resulted in lethality of both total aerobic bacteria and several important classes of milk-borne bacteria, whereas milder conditions decreased the rate of growth of these organisms. After 4 d treatment at a CO2 pressure of 689 kPa and temperature of 6.1°C, a significant decrease in SPC was observed. Between 4 and 9 d, the SPC of the CO2-treated milk increased slowly to population levels that were always significantly lower than that observed for the control SPC. Carbon dioxide-treated milk SPC was within Pasteurized Milk Ordinance Grade A raw milk limits up to 8 d of storage, whereas untreated milk reached the Grade A limits before 4 d. Growth of spoilage organisms, coliforms, thermoduric bacteria, or E. coli was not enhanced by any pressurized CO2 treatment. These data suggest that low positive CO2 pressure might be an effective strategy for holding and shipping raw milk, providing shelf-life extension and maintenance of safety. Although CO2 would need to be removed before further processing of raw milk, such methodology and necessary equipment is standard and readily available in the dairy industry. Potential financial benefits of extended bulk storage and low associated costs make this application attractive to commercial use.

Received for publication May 11, 2004. Accepted for publication May 7, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


Calvo, M. M., and E. Balcones. 2001. Inactivation of microorganisms and changes of proteins during treatment of milk with subcritical carbon dioxide. Milchwissenschaft 56:366–369.

Daniels, J. A., R. Krishnamurthi, and S. S. H. Rizvi. 1985. A review of effects of carbon dioxide on microbial growth and food quality. J. Food Prot. 48:532–537.

Devlieghere, F., and J. Debevere. 2000. Influence of dissolved carbon dioxide on the growth of spoilage bacteria. Lebensm. Wiss. Technol. 33:531–537.

Dixon, N. M., and D. B. Kell. 1989. The inhibition by carbon dioxide of the growth and metabolism of microorganisms. J. Appl. Bacteriol. 67:109–136.[Medline]

Enfors, S. O., and G. Molin. 1978. Influence of high concentrations of carbon dioxide on germination of bacterial spores. J. Appl. Bacteriol. 45:279–285.[Medline]

Enfors, S. O., and G. Molin. 1981. The influence of temperature on the growth inhibitory effect of carbon dioxide on Pseudomonas fragi and Bacillus cereus. Can. J. Microbiol. 27:15–19.[Medline]

Erkman, O. 1997. Antimicrobial effect of pressurized carbon dioxide on Staphylococcus aureus in broth and milk. Lebensm. Wiss. Technol. 30:826–829.

Erkman, O. 2000. Antimicrobial effect of pressurized carbon dioxide on Enterococcus faecalis in physiological saline and foods. J. Sci. Food Agric. 80:465–470.

Espie, W. E., and R. H. Madden. 1997. The carbonation of chilled bulk milk. Milchwissenschaft 52:249–253.

Gill, C. O., and K. H. Tan. 1979. Effect of carbon dioxide on growth of Pseudomonas fluorescens. Food Microbiol. 4:285–291.

Griffiths, M. W., J. D. Phillips, and D. D. Muir. 1987. Effect of low temperature storage on the bacteriological quality of raw milk. Food Microbiol. 4:285–291.

Haas, G. J., H. E. Prescott, E. Dudley, R. Dik, C. Hintlain, and L. Keane. 1989. Inactivation of microorganisms by carbon dioxide under pressure. J. Food Safety 9:253–265.

Hendricks, M. T., and J. H. Hotchkiss. 1997. Effect of carbon dioxide on the growth of Pseudomonas fluorescens and Listeria monocytogenes in aerobic atmospheres. J. Food Prot. 60:1548–1552.

Hong, S. I., W. S. Park, and Y. R. Pyun. 1999. Non-thermal inactivation of Lactobacillus plantarum as influenced by pressure and temperature of pressurized carbon dioxide. Int. J. Food Sci. Technol. 34:125–130.

Hong, S. I., and Y. R. Pyun. 2001. Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment. Int. J. Food Microbiol. 63:19–28.[Medline]

Hotchkiss, J. H. 1996. Commitment to cottage cheese. Dairy Foods 29.

Houghtby, G. A., L. J. Maturin, and E. K. Koenig. 1992. Microbiological count methods. Pages 213–246 in Standard Methods for the Examination of Dairy Products. 16 ed. T. R. Marshall, ed. American Public Health Association, Washington, DC.

Jordan, P. J., K. Lay, N. Ngan, and G. F. Rodley. 1987. Casein precipitation using high pressure carbon dioxide. N.Z. J. Dairy Sci. Technol. 22:247–256.

King, J. S., and L. A. Mabbitt. 1982. Preservation of raw milk by the addition of carbon dioxide. J. Dairy Res. 49:439–447.

Ma, Y., and D. M. Barbano. 2003. Effect of temperature of CO2 injection on the pH and freezing point of milks and creams. J. Dairy Sci. 86:1578–1589.[Abstract/Free Full Text]

Mabbitt, L. A. 1982. Preservation of refrigerated milk. Kieler Milchw. Forsch. 34:28–31.

Martin, J. D., B. G. Werner, and J. H. Hotchkiss. 2003. Effects of carbon dioxide on bacterial growth parameters in milk as measured by conductivity. J. Dairy Sci. 86:1932–1940.[Abstract/Free Full Text]

Muir, D. D., J. D. Phillips, and D. G. Dalgleish. 1979. Lipolytic and proteolytic activity of bacteria isolated from blended raw milk. J. Soc. Dairy Technol. 32:19–23.

Roberts, R. F., and G. S. Torrey. 1988. Inhibition of psychrotrophic bacterial growth in refrigerated milk by addition of carbon dioxide. J. Dairy Sci. 71:52–60.[Abstract/Free Full Text]

Rowe, M. T. 1989. Carbon dioxide to prolong the safe storage of raw milk. Milk Ind. 91:17–19.

Ruas-Madiedo, P., J. C. Bada-Gancedo, E. Fernandez-Garcia, D. Gonzalez De Llano, and C. G. De Los Reyes-Gavilan. 1996. Preservation of the microbiological and biochemical quality of raw milk by carbon dioxide addition: A pilot-scale study. J. Food Prot. 59:502–508.

Ruas-Madiedo, P., V. Bascaran, A. F. Brana, J. C. Bada-Gancedo, and C. G. De Los Reyes-Gavilan. 1998a. Influence of carbon dioxide addition to raw milk on microbial levels and some fat-soluble vitamin contents of raw and pasteurized milk. J. Agric. Food Chem. 46:1552–1555.

Ruas-Madiedo, P., V. Bascaran, A. F. Brana, J. C. Bada-Gancedo, and C. G. De Los Reyes-Gavilan. 1998b. Influence of carbon dioxide addition to raw milk on microbial levels and some fat-soluble vitamin contents of raw and pasteurized milk (correction). J. Agric. Food Chem. 46:2894.

Ruas-Madiedo, P., C. G. De Los Reyes-Gavilan, A. Olano, and M. Villamiel. 2000. Influence of refrigeration and carbon dioxide addition to raw milk on microbial levels, free monosaccharides and myo-inositol content of raw and pasteurized milk. Eur. Food Res. Technol. 212:44–47.

Shipe, W. F., R. Bassette, D. D. Deane, W. L. Dunkley, E. G. Hammond, W. V. Harper, D. H. Kleyn, M. F. Morgan, J. H. Nelson, and R. A. Scalan. 1978. Off flavors of milk: Nomenclature standards and bibliography. J. Dairy Sci. 61:855–869.[Abstract/Free Full Text]

Southward, C. R. 1986. Utilization of milk components: Casein. Pages 317–368 in Modern Dairy Technology: Advances in Milk Processing. Vol. 1. R. K. Robinson, ed. Elsevier Applied Science Publishers, London, UK.

Tomasula, P. M. 1995. Preparation of casein using carbon dioxide. J. Dairy Sci. 78:506–514.[Abstract]

Tomasula, P. M., and R. T. Boswell. 1999. Measurement of the solubility of carbon dioxide in milk at high pressures. J. Supercrit. Fluid. 16:21–26.

U.S. Department of Health and Human Services. 1999. Grade "A" Pasteurized Milk Ordinance. Vol. Publication No. 229, rev. ed. U.S. Department of Health and Human Services, Public Health Service, Food and Drug Administration, Washington, DC.

Wolfe, S. K. 1980. Use of carbon monoxide and carbon dioxide enriched atmospheres for meats, fish and produce. Food Technol. 34:55–58.



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