Applications of Bio Luminescence in Dairy Industry

Applications of Bioluminescence in the Dairy Industry

ABSTRACT

Several applications of ATP bioluminescence of relevance to the dairy industry have been proposed. This paper reviews some of the major benefits of the technology. New developments in bioluminescence research have made the simple, rapid, sensitive detection of foodborne pathogens a distinct possibil- ity. (Key words: bioluminescence, food safety, evaluation, dairy products)

INTRODUCTION

Current trends in nutrition and food technology are increasing the demands on food microbiologists to ensure a safe food supply. The trend toward foods that are low in fat and high in fiber creates unknown pressures on food as an ecosystem. For example, increases in the unsaturated fatty acid concentration of foods can change the growth rates of bacteria (15, 49). The consumer is also demanding more variety, which means that the food processor is developing novel products con- tinuously. These involve, in some instances, bringing together ingredients with different associated microbiological problems, the result of which is uncertain. The concern over food additives has led to greater reliance on low temperatures to effect preservation and the emergence of minimally processed and cook- chill products; thus, the need for strict microbiological control during processing becomes evident. In addition, processors, retailers, and consumers are constantly making new demands for shelf-life of products and expect these to be met. To achieve this, microbial loads in foods must be minimal.

The consumer has become much more aware of food safety issues as a result of

Received August 19, 1993. Accepted January 8, 1993.

M. W. GRlFFlTHS Deparbnent of Food Science

University of Guelph Guelph, ON, Canada N1G 2W1

publicity given to foodborne disease in the media. In the past decade, headlines have been related to Listeria monocytogenes in soft cheese, Salmonella enteritidis in eggs, Es- cherichia coli 0157 in hamburgers, bovine spongiform encephalopathy in beef, and other problems. It sometimes appears that the food industry is lurching from one crisis to another. However, most foodborne illnesses were due to mishandling in food service establishments (46.8%) or at home (20.5%) (Table 1) (40, 41). Nevertheless, a significant proportion of illness was the result of mishandling by food processors (2.9%) and retailers (16.0%). This necessi- tates, in any food manufacturing operation, the provision for adequate microbiological testing to verify quality of raw materials, to instigate process controls such as Hazard Analysis Criti- cal Control Point (HACCP) procedures, and to monitor hygiene. For these measures to be effective, results must be obtained as quickly as possible.

New techniques have had greatest application in the dairy industry, but their usefulness transcends commodities. Of the emerging tech- nologies for rapid microbiological analysis, ar- guably the technique giving results in the shortest time is bioluminescence. Two distinct areas of bioluminescence are of use to the food industry: ATP bioluminescence and bacterial bioluminescence.

ATP BIOLUMINESCENCE

All living cells contain the molecule ATP. This molecule may be assayed simply using an enzyme and coenzyme complex (luciferase- luciferin) found in the tail of the firefly, Photi- nus pyralis. The reaction is essentially the stoichiometric conversion of ATP to photons of light (Figure 1). If the level of ATP in bacterial cells is assumed to be relatively constant, the amount of light emitted during the reaction is proportional to the numbers of bacteria present. The ATP bioluminescence assay has been adapted for a variety of applications

1993 J Dairy Sci 76:3118-3125 3118

SYMPOSIUM: EVALUATION OF MILK AND DAIRY PRODUCTS 3119

TABLE 1. Places of acquisition of incidents of foodborne illness of known microbiological origin in Canada between 1975 and 1986.1

Place Incidents

Food service establishment Home Food processing plant Retail outlet Farms and dairies Delicatessens Other Unknown

(W 46.8 20.5 2.9

16.0 5.0 .6

2.2 6.0

'Data of Todd (40, 41).

in the dairy industry (Table 2), been reviewed by Griffiths (11).

which have

Raw Mllk Quallty

Microbial Quality. Sharpe et al. (36) were the first to determine ATP concentrations in milk contaminated by bacteria. Since then,

luciferin + ATP + M#+

Q, luciferase

oxyluciferin + AMP + C q +

light 0 Figure 1. Schematic representation of the firefly lu-

ciferase reaction.

several workers (11) have used ATP bioluminescence to determine the microbial content of milk. In general, a bacterial concentration of 1 x 106 cfulml was required for detection, but results were obtained in about 5 min. This lack of sensitivity was mainly due to high concentrations of nonmicrobial ATP present in milk as free ATP associated with casein micelles (33) and ATP present in somatic cells (6). This nonmicrobial ATP was removed by hydrolysis with apyrase, an ATP-hydrolyzing enzyme, after addition of chelating agents to disrupt the casein micelle and detergent to lyse somatic cells. A second detergent was added to release the microbial ATP for assay.

Recent research has focused on ways to improve the sensitivity of the assay. The incorporation of a filtration step or other procedures to concentrate the bacteria following selective lysis of somatic cells with nonionic detergents has improved assay sensitivity (Figure 2). The ATP can then be extracted from the entrapped bacteria with a cationic detergent and assayed. Using this procedure, workers (4, 12, 14, 21, 26, 35,45) have claimed that ATP bioluminescence can be used to detect as few as 1 x 104 cWml of bacteria in milk within 5 to 10 min. This result makes the technique useful as a rejection test for incoming tanker milks at milk processing plants. These tests are available commercialiy and are marketed by Biotrace (Bridgend, Wales) and Lumac (Laandgraaf, The Netherlands).

Bacteria can also be concentrated from milk by centrifugation following addition of a ''con-

TABLE 2. Applications of ATP bioluminescence cur- m t l y available to the dairy industry.

Raw Inilk quality somatic cell count Microbial count

Rodua quality Pasteurized milk Pasteurized cream Milk powder

UHT sterility testing Starter culture activity Hygiene monitoring Enzyme assay Rotease Auraline phosphatase

Journal of Dairy Science Vol. 76, No. 10, 1993

3120 GRIFFITHS

centrating” agent, EnlitenTM (Promega, Madi- son, WI). The concentration of ATP in the bacterial pellet can be determined with luciferase-luciferin. The manufacturers claim that the technique can detect as few as 2 x 104 cfdml, and up to 24 samples can be processed in 1 to 2 h without culturing (27).

Somatic Cell Concentrations. An indication of somatic cell concentrations in milk can be obtained from the concentration of ATP in milk following treatment with a nonionic detergent such as Triton X-100 (6, 9, 10). Those researchers claim that the technique can be used as an index of mastitic infection.

An alternative method for detecting mastitis by bioluminescence has been proposed (22). When bacterial growth in milk was monitored by measuring ATP concentration, bacteria grew more rapidly in mastitic milk, and the increase in bacterial ATP correlated well with inflammatory markers for mastitis.

Pasteurized Milk Quality

The prime factor controlling the shelf-life of pasteurized dairy products is the reintroduction of Gram-negative, psychrotrophic bacteria into the milk after heat treatment (29). In addition to limiting shelf-life, postpasteurization contamination of milk may have important public health significance. A number of large outbreaks of foodborne illness attributed to the consumption of pasteurized milk were subse- quently shown to be due to postprocess contamination (29). These outbreaks included listeriosis and salmonellosis; of particular concern was an outbreak of salmonellosis in 11- linois involving over 16,000 cases (29).

A number of methods for detection of postpasteurization contamination of dairy products has been described (3. These methods invaria- bly involve a preincubation procedure in the presence of inhibitors for Gram-positive organisms, which is designed to select for the

Incubate at 3 f ~ for 5 min

Raw milk ma=+ Somatic cell lysing agent

ATP I A T P ( ATP

ATP A b )rTp ATP ATP ATP u

B r a c t bacterial ATP with bacterial Measure light emission

after addition of luciferaseluciferi n lysing agent

Syringa through bacteria retaining

filter

Figure 2. Schematic representation of the ATP bioluminescence assay for enumration of the microbial content of milk.


SYMPOSILTM: EVALUATION OF MILK AND DAIRY PRODUCTS 3121

growth of Gram-negative psychrotrophs. Waes and Bossuyt (46, 47) preincubated pasteurized milks at 30'C for 24 h in the presence of benzalkon A 50% (.06%) and crystal violet (.002%) to select for the growth of p s y c h - trophic, postpasteurization contaminants. Fol- lowing this preincubation, bacterial numbers were assessed using ATP bioluminescence. Those authors (46) claimed that they were able to detect initial contamination at 1 L of milk using this technique. The test could be made more quantitative by employing a most proba- ble number approach.

Phillips and Griffiths (28) claimed that growth of Gram-negative psychrotrophs was better when pasteurized milks were preincubated at 21'C for 25 h in the presence of crystal violet (20 pg/ml), penicillin (200 U/ml), and nisin (400 U/ml), added as inhibitors for Gram-positive bacterial growth. The shelf-life of pasteurized milk and cream could be ac- curately predicted when ATP counts were performed on the preincubated products. This test also gave good agreement with the recently introduced European Community keeping quality test.

An ATP bioluminescent method for prediction of the shelf-life of pasteurized milk has recently been described using the Charm II system (42).

Such tests can be useful during milk processing to detect sources of contamination within a reasonable time (25 to 26 h), espe- cially when used in conjunction with on-line sampling techniques (13).

Sterility Testing of UHT and Other Dairy Producta

The sterility of UHT products is generally determined by assessment of plate count or pH changes after incubation of the product at 30'C for 3 d. Incubation followed by plate count is time-consuming, yielding results after 5 to 8 d. Although the monitoring of pH changes gives more rapid results, those results are not always reliable, particularly when contamination is due to sporeformers (48). Waes et al. (48) described a bioluminescence method of monitoring growth after storage of the product at 30'C that involved treatment with apyrase (.01 U) to remove free ATP prior to extraction and assay of bacterial ATP with luciferase-

luciferin. All of the contamination caused by understerilization of the milk could be detected in 3 d by this ATP method. Significant detection of contamination of UHT milks by bacterial ATP determination after a I-d preincubation at 30'C has been documented (8). Testing of UHT products for sterility by bioluminescence may be particularly useful for chocolate milks for which the color precludes the use of dye reduction (31). Bioluminescence techniques have been used to determine the bacterial content of milk powder with varying success (17, 23).

Monitoring Starter Culture Activity

Because ATP is an integral part of the metabolism of bacterial cells, the concentrations of ATP may provide a better indication of the activity of lactic acid bacteria than measurement of pH changes. A strong correla- tion between acid production and ATP concentrations exists for a number of lactic acid bacteria, including Lactococcus lactis and Lac- tobacillus acidophilus, during growth in milk (7, 25). Monitoring of the changes in ATP during growth in milk rapidly indicated the presence of antibiotic residues or phage (32. 50.51). Concentrations as low as ,005 U/ml of penicillin could be detected in about 90 min (51).

Hygiene Monitoring

Probably the most widely used current a p plication of ATP bioluminescence in the food industry is the estimation of surface cleanli- ness. The total ATP present on a swabbed surface can be extracted and assayed extremely rapidly (i.e., within 5 min) with no less ac- curacy than that obtained using traditional techniques (16). The result indicates the overall contamination of the surface because ATP from food residues and from microbial sources will be detected (4, 20). The amount of contamination determined by ATP and plate count methods correlated in about 80% of samples. More surfaces were identified as being soiled by the ATP test than by plate count (Table 3). A number of bioluminescence-based hygiene monitoring kits are now commercially available and are used routinely to monitor critical control points in milk processing operations worldwide.


3122 GRIFFITHS

Enzyme Assays

An interesting application of luminescence is emerging that has implications for the dauy industry in the future: the development of bioluminescent or chemiluminescent assays for a variety of enzymes of importance in the dajl industry.

Profease. Rowe et al. (34) described a bioluminescent assay for proteases produced by psychrotrophic bacteria during growth in milk. These enzymes are heat-resistant and can sur- vive pasteurization and UHT processing, thus reducing the shelf-life of these products (29). The assay relies on rate of inactivation of the protein luciferase, because of proteolysis, being directly proportional to the protease concentration at a constant initial luciferase concentration. In 5 min, protease can be detected in milk at concentrations as low as .01 U/ml, which is well below concentrations found to limit shelf-life of UHT products.

Lipase. Thermostable lipases produced by psychrotrophic bacteria also limit the shelf-life of dairy products (29). Possibilities exist for development of bioluminescent lipase assays (ll), but, so far, these have not been widely applied to detection of the enzyme in dairy products.

Alkaline Phosphatase. A chemiluminescent substrate, AMPDD (4-methoxy-4-(3-phospha- phenyl)spiro( 1 ,2-deoxetane-3,2yl)phenylphos- phate), for alkaline phosphatase is available, and its application for detection of the enzyme following heat treatment of milk has been described (52).

fl-Galacrosidase. &Galactosidase is used to hydrolyze the lactose in milk so that it may be

consumed by the lactose-intolerant population. A bioluminescent substrate, D-luciferin-0-@- galactoside, for this enzyme has recently been synthesized (43) and may find application in the dairy industry.

Instrumentation

Stanley (37) has documented over 90 commercially available luminometers with varying degrees of sophistication, ranging from fully automated instruments to small, portable instruments. The latter have been particularly convenient for use in conjunction with hygiene monitoring kits; they are easily transported throughout the factory and are relatively inexpensive.

An automated instrument, BactoFoss (Foss Electric, H i l l e d Denmark), with a built-in filtration sample preparation unit and dedicated for food use, has been developed (26). This luminometer gave good correlations with plate counts for raw milks in the range 1 x 104 to 1 x 108 cfu/ml (21, 26).

Luminometers are also available that are designed to accept a microtiter plate format, which is particularly useful when large numbers of assays are performed.

BACTERIAL BIOLUMINESCENCE

Although ATP bioluminescent techniques offer fast enumeration of bacterial populations in a variety of products, these methods lack specificity. The need for rapid detection systems that are specific for certain bacterial spe- cies is highlighted by recent concerns over the presence of potential pathogens in dairy

TABLE 3. Comparison of hygiene monitoring &ed put by ATP bioluminescence and conventional meth0ds.l

Counts

Q LE'- <27 ATP3

2-3 LPC 2 8 4 9 ATP

3 4 LPC 50-149 ATP

>4 LPC >150 ATP

(no. samples) 121 31 16 70 101 23 12 . 38

lData of Bautista et al. (4). 2Log plate count (swab was placed in 5 ml of broth, and counts were determined as colony-forming units per milliliter

3ATP Count (relative light units per assay). of this diluent).

Journal of Dairy Science Vol. 76. No. 10, 1993

SYMPOSIUM: EVALUATION OF MILK AND DAIRY PRODUCTS 3 123

products. Advances in molecular genetics have made bioluminescent detection systems for specific types of bacteria a reality. The applications of this technology to the food industry have been reviewed (3, 38).

Detection of Pathogens

The genes responsible for bacterial bioluminescence (lux genes) have been identified and cloned (24). The DNA carrying these genes can be introduced into host-specific phages (44). The phages do not possess the intracellular biochemistry necessary to express these genes, and, therefore, phages remain "dark". However, on transfer of the lux genes to the host bacterium during infection, tran- scription results in light emission that can be easily detected with a luminometer. The amount of light emitted is proportional to the numbers of bacteria infected, and the specificity of the assay is dependent upon the specific-

@

ity of the phage (Figure 3). The bacteriophage P22 is essentially specific for SulmoneIZu typhimun'um This phage has been engineered to contain lux genes, and, upon infection of a culture of S. typhimurium with this modified phage, as few as 1 x 102 bacteria could be detected in 60 min (39). Kricka (19) has claimed that as few as 10 E. coli cells could be detected in milk within 1 h using this technol- ow.

Stewart (39) postulated that incorporation of lux genes into phages specific for enteric bacteria or even Pseudomonas spp. would result in an inexpensive, on-line hygiene test for dairy products that could be completed in 1 h or less. A near on-line detection of enteric bacteria using lux recombinant bacteriophage has been developed (18).

Research is ongoing at University of Guelph to produce bioluminescence-based assays for the detection of mastitis-causing organisms and foodborne pathogens.

Lux genesfrom luminescent bacteria

&- tnfection of host

by phage

Host-specific

phage

Phage transformation

Production of host with luminescent phenotype

Figure 3. Schematic representation of the lux gene-based bacteriophage assay for bacteria.


3124 GRIFFITHS

Monltorlng Starter Culture Activity

The lux genes have been introduced into strains of lactic acid bacteria, including Lc. lactis, Lactobacillus casei, and Lactobacillus pluntamm (1). In the presence of exogenous aldehyde, these gknetically modified organisms become luminescent. However, luminescence is curtailed in the presence of inhibitory agents such as antibiotics and bacteriophage. Lactic acid bacteria possessing a luminescent phenotype have been proposed for use to indicate the effective fermentation of dsllry products (39). Using a bioluminescent mutant of Lb. casei, Ahmad and Stewart (1) showed that concentrations of penicillin G as low as .03 pg/ml (.05 U/ml) could be detected in 30 min. The technique also enabled bacteriophage concentrations as low as 105/ml to be observed within 100 min.

Bacterial bioluminescence techniques can also be applied to evaluate biocidal efficiency (3).

Other Applications of Luminescence

Chemiluminescent-based probes are available for use with immunological and nucleic acid hybridization assays (2, 30). The sensitivity of these labeling methods in conjunction with low light level image analyzing systems offers exciting possibilities for the detection of pathogens in food samples.

CONCLUSIONS

The resurgence of interest in ATP bioluminescence and the dramatic improvements in reagent quality and instrument sensitivity have led to the availability of applications of direct relevance to the dairy industry (11). The most noteworthy are the hygiene monitoring assay and the new generation of milk bacteria enumeration kits. Developments in bacterial bioluminescence offer exciting possibilities for the detection of pathogens and spoilage organisms in foods (3, 38).

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