Microbiology of Paperboard

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Chapter 2: Migration of Penicillium spinulosum from Paperboard Packaging to Extended Shelf Life Milk

Introduction

In the United States, all milk and milk products are subject to regulation by the Grade A Pasteurized Milk Ordinance (PMO). Under these guidelines all milk and milk products must be pasteurized except for a few specific exceptions. Conventionally pasteurized milk, at time and temperature combinations defined in the PMO, has been packaged with approximately a two-week code date. Two recent innovations, “ultra-pasteurized” and “ultra-high temperature” processed milk have provided additional options to consumers. Ultra-pasteurized milk is a refrigerated product with an extended shelf life when compared to the prevailing High Temperature, Short Time (HTST) pasteurization products. UHT milk is an aseptically packaged product requiring no refrigeration with a shelf life of several months. UHT milk is very popular internationally, because of the convenience of its long shelf life, but it has not found widespread acceptance in the United States.
The increased shelf life of ultra-pasteurized milk, while convenient for the consumer, has also increased the time available for growth of psychrotrophic spoilage microorganisms. Therefore, precautions must be taken to assure adequate thermal processing and to avoid post-pasteurization contamination. Contamination after pasteurization can occur from many sources including air, machinery, workers or packaging.
Most commercial packaging of milk is either in high density polyethylene (HDPE) jugs or gable-top paperboard cartons coated on both sides with polyethylene. In the United States, the Department of Health and Human Services sets the standards for milk packaging including bacterial quality of paperboard in “Fabrication of Single Service Containers for Milk and Milk Products”. Later revisions of this publication include the use of reclaimed fiber for the production of paperboard milk cartons (FDA, 1993). While the use of recycled fiber is desirable for both environmental and economic reasons, paper products from recycled sources often contain more microorganisms than virgin paperboard.
Because of the extended shelf life of ultra-pasteurized milk, every effort must be taken to prevent contamination from paperboard packaging in order to ensure a high quality product. This research aims to examine the possibility of fungal contamination of milk by inoculating Penicillium spinulosum in paperboard packaging and investigating its ability to survive and grow in paperboard under refrigeration conditions.

Section I: Review of Literature

Ultra-Pasteurization

In order to increase the shelf life of milk, higher time-temperature combinations are used beyond that of normal pasteurization, but below that of ultra-high temperature (UHT) processing (Blake, et al. 1995). According to the Code of Federal Regulations 21, 131.3 (FDA, 1997b), and the Pasteurized Milk Ordinance (FDA, 1995) ultra-pasteurized milk is created by pasteurization at 138°C (280°F) or above for 2 or more seconds. This product must still be refrigerated and is referred to as extended shelf life milk (ESL). The extended shelf life allows longer shipping times and wider distribution of milk. The longer shelf life allows more time for growth of psychrotrophic microorganisms (Blake et, al. 1995).
ESL milk provides an intermediate shelf life product between HTST and UHT milk with greater consumer acceptability than aseptically packaged UHT milk. Blake et al. in 1995 evaluated the sensory characteristics of milk pasteurized by these different processes. They found that ESL milk was preferred over UHT processed milk, but conventional HTST milk was still preferred over both ESL and UHT milk.

Shelf Life of Milk

Because milk and other liquid dairy products are highly perishable, very stringent standards are enforced to ensure a safe product with a predictable shelf life. Many factors affect the shelf life of pasteurized milk including the quality of milk before processing, adequate heat processing, post-pasteurization contamination and storage temperature.
High quality milk prior to pasteurization is important in order to limit the number of thermoduric microorganisms capable of surviving pasteurization. Also, due to the ability of some organisms to produce enzymes that affect the character of the milk even after the organisms are eliminated, it is crucial to limit the levels of microorganisms as much as possible (Harding, 1995a; Nelson, 1981). Fecal contamination and mastitis infections seriously reduce the quality of milk and introduce not only potential spoilage organisms, but also pathogens (Harding, 1995a).
Pasteurization is intended to rid milk of possible pathogens, but not eliminate all spoilage organisms. Heat processing significantly reduces the number of psychrotrophic gram-negative bacteria in the milk, which are capable of rapidly causing spoilage, but leaves some of the more heat resistant gram-positive microorganisms that slowly grow to spoilage levels when milk is stored at proper temperatures (Harding, 1995b; Nelson, 1981). In addition to reducing microbial populations, pasteurization also inactivates the enzyme lipase naturally found in milk, which would otherwise result in rancidity of homogenized milk (Nelson, 1981).
Because pasteurization so significantly alters the microflora of milk, it becomes extremely important to prevent post-pasteurization contamination. Psychrotrophic spoilage organisms and pathogens capable of growth at low temperature can multiply with very little competition (Harding, 1995b; Nelson, 1981). Listeria monocytogenes is of great concern as an organism capable of contaminating milk after pasteurization because of its widespread occurrence and pathogenicity (Harding, 1995b).
In the United States the Pasteurized Milk Ordinance (FDA, 1997a) sets the highest allowable temperature for storage of pasteurized milk as 7°C or 45°F. The aim of this temperature is to significantly lengthen the generation time of most microorganisms to ensure a predictable shelf life for milk. Thermoduric organisms that may survive pasteurization are not usually well adapted to growth at low temperatures; therefore, proper storage can be a very effective means of inhibiting their growth (Harding, 1995b). Maintenance of temperature is important not only after pasteurization, but also before because of the ability of some bacteria to produce heat stable enterotoxins in raw milk and the general reduction of organisms in milk prior to processing (Nelson, 1981).

Contamination of Milk

A survey of spoilage microorganisms in dairy products by Walker in 1988 found that milk obtained directly from a healthy cow under hygienic conditions contains very few bacteria, yeasts or molds and generally is contaminated through handling and processing. This article states the four basic principles to prevent spoilage of dairy products as: (1) high quality raw materials, (2) consistent refrigeration at all stages, (3) monitoring critical processing points, and (4) good hygiene throughout. A study by Ren and Frank (1992) investigated aerosols at milk and ice cream plants as a source of contamination of milk. This study found a correlation between high levels of microbial aerosols and processing activity in the plant. To further examine microbial spoilage of milk, it is helpful to classify the contamination by type of organism: bacteria, yeast or mold.

Spoilage of Milk by Bacteria

Spoilage of milk by bacteria is particularly well documented. The bacterial contamination of pasteurized milk occurs by two methods when heat processing is properly performed, (1) by the ability to survive pasteurization or (2) by post-pasteurization contamination. Bacteria able to survive pasteurization and frequently isolated from pasteurized milk include the genera Alacaligenes, Bacillus, Clostridium, Microbacterium, Micrococcus, and Streptococcus (Stadhouders, 1975). In a spoilage incident of UHT chocolate milk in Trinidad, Antoine and Donawa (1990) attributed the cause to an Enterobacter and Micrococcus species, which were able to withstand high temperatures. It was concluded that these thermoduric organisms might have been able to withstand the UHT pasteurization process.
Of particular concern among heat resistant bacteria are those of the sporeforming, gram-positive Bacillus genus. Some of the Bacillus species pose a threat in pasteurized milk due to their ubiquitous nature, heat resistance, psychrotrophic growth and enterotoxin production. Additionally, trends toward higher heat treatments and extended shelf life in pasteurized milk may tend to eliminate competition and select for Bacillus sp. (Meer et al., 1991). Several studies have shown that a low percentage of B. cereus isolates from pasteurized dairy products have the ability to both grow at refrigeration temperatures and produce toxins necessary for foodborne illness (Christiansson et al., 1989; Granum et al., 1993). Other Bacillus species of concern in pasteurized milk include B. circulans, B. licheniformis, B. megaterium , B. pumilis and B. subtilis (Meer et al., 1991; Stadhouders, 1975). Cromie et al. (1989) concluded that B. circulans posed a particular threat since it was isolated from milk pasteurized and stored at a variety of temperatures even when packaged aseptically.
Important gram-negative spoilage organisms of pasteurized milk include the genera Achromobacter, Alcaligenes, Flavobacterium and Pseudomonas due to the psychrotrophic nature of these organisms (Stadhouders, 1975). Other significant, but less frequently encountered gram-negative organisms in milk and dairy products include those of the genera Acinetobacter, Aeromonas, Altermonas, Brucella, Campylobacter, Chromobacterium, Coxiella, Escherichia, Enterobacter, Hafnia, Klebsiella, Leptospira, Salmonella, Serratia, Xanthomonas and Yersenia (Vasavada and Cousin, 1993). A study of Swedish and Norwegian pasteurized milk found the spoilage flora of pasteurized milk stored at 5°C for 3 weeks was 65% gram-negative bacteria (Ternström et al., 1993).

Spoilage of Milk by Yeasts

A variety of yeasts are frequently isolated from raw milk and raw milk products such as cheese, but are not considered to be a threat in pasteurized milk because of their susceptibility to heat processing (Cooke and Brazis, 1968). A study by Vadillo Machota et al. (1987) found that depending on the season, between 95 and 100 % of raw milk samples from a Spanish milk cooperative were positive for yeasts. Interestingly, a 1987 survey of pasteurized milk by Fleet and Mian found only a slightly reduced occurrence of yeasts with approximately 80% of samples positive for the presence of yeasts.
In pasteurized milk yeasts are often reported, but at low levels because the organisms usually are outcompeted by psychrotrophic bacteria at refrigeration temperatures (Fleet, 1990). The ability of fungi and yeasts to outgrow bacteria at lower temperatures was examined by Beuchat (1983). In circumstances where yeasts are not in competition with psychrotrophic bacteria for nutrients in pasteurized milk, yeasts are able to easily grow to large numbers and spoil the product (Fleet et al., 1987).

Spoilage of Milk by Fungi

Like yeasts, fungi do not compete well with bacteria in liquid products, such as pasteurized milk, unless the product contains high levels of sugar, salt or a high acidity which limits the growth of bacteria (Walker, 1988). However, the neutral pH of pasteurized milk provides an ideal environment for the rapid growth of psychrotrophic bacteria (Pitt et al., 1997). Mold spoilage of liquid dairy products in particular is not generally regarded as a critical concern; but is more predominant in solid and semi-solid dairy products such as cheese and cottage cheese (Walker, 1988). Molds and yeasts were implicated as a major factor in reducing the shelf life of pasteurized milk in Saudi Arabia in addition to psychrotrophic bacteria. In this study, molds, yeasts and psychrotrophic bacteria in combination were found to be the main cause of pasteurized milk reaching unacceptable sensory and microbiological health limits as set by the Saudi Arabian Standard Organization (SASO) (Salji, et al., 1988).
Isolation of fungi in raw milk has produced a variety of results. Vadillo Machota et al. (1987) isolated a variety of molds from raw milk with the most frequent genera in order of prevalence being Geotrichum, Cladosporium, Penicillium, Aureobasidium and Aspergillus. Comparatively, a similar study by Frevel et al. (1985) determined the most frequent genus of mold isolated from raw milk to be Phoma, followed by Mucor, Fusarium, Penicillium and Aureobasidium. In agreement with Frevel et al. (1985), Cooke and Brazis (1968) found Phoma herbarum , a plant pathogen (Samson et al., 1996), to be the most frequently isolated species of fungi from raw milk.
Major fungal spoilers of dairy products in general include the genera Geotrichum, Aspergillus, Penicillium, Mucor and Alternaria according to a study by Walker in 1988. Due to storage conditions, the fungi that are found spoiling dairy products are generally psychrotrophic. Mold growth on dairy products results in spoilage because of off-flavors and aromas and/or visible growth on the products. These off-flavors and aromas are described by Walker (1988) as “ammoniacal, fruity or musty.”
The occurrence of fungi in milk is reason for concern beyond loss of product due to spoilage; the potential for the production of mycotoxins must also be considered especially due to the high consumption rates of milk among youth. For pasteurized milk, most consideration is given to aflatoxin M1 and M2, a metabolized product of aflatoxin B1 and B2 produced by Aspergillus flavus and A. parasiticus. This is a secondary form of mycotoxin contamination resulting from aflatoxin B1 and B2 in the feed of dairy cows, which is metabolized to M1 and M2 in the cow’s body and then excreted in the milk (Lück and Wehner, 1979; van Egmond, 1989b). In a 1976 study by Paul et al., 6.2% of milk samples were contaminated with aflatoxin M, but no aflatoxin B or G was detected in any of the 81 samples. A study of 60 milk samples by Fritz and Engst (1981) found detectable levels of aflatoxin M1 in milk produced during winter months when herds are fed stored grains as opposed to grazing. Ochratoxin A and sterigmatocystin are two mycotoxins that present potential carry-over effects in lactating cows from contaminated feeds, but are not considered a reason for serious concern (Lück and Wehner, 1979; van Egmond, 1989a).

ABSTRACT
ACKNOWLEDGEMENTS
INTRODUCTION
SECTION I: REVIEW OF LITERATURE
ULTRA-PASTEURIZATION
SHELF LIFE OF MILK3
CONTAMINATION OF MILK
MILK PACKAGING
Guidelines for Paperboard Packaging of Milk
Microbiology of Paperboard
MICROBIOLOGY OF WOOD
PENICILLIUM SPINULOSUM
CONTAMINATION OF CITRUS JUICES
REFERENCES
SECTION II: MIGRATION OF PENICILLIUM SPINULOSUM FROM PAPERBOARD PACKAGING TO EXTENDED SHELF LIFE MILK
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
APPENDIX
GET THE COMPLETE PROJECT
Migration of Penicillium spinulosum from Paperboard Packaging to Extended Shelf Life Milk

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