Fruit juices and fruit-based drinks can support the growth of several types of microorganisms, such as bacteria, yeasts and molds that are primarily involved in causing the spoilage of these products. The contamination and/or growth of pathogenic bacteria such as Escherichia coli O157:H7 in acidic food products, fruit juices and fruit-based drinks has caused great concern. Several outbreaks of illness caused by the consumption of fruits or fruit juices contaminated with Salmonella, E. coli O157:H7, and Cryptosporidium have been reported in the U.S. by the Centers for Disease Control and Prevention (CDC).[1a-d] In the aftermath of these outbreaks, the U.S. Food and Drug Administration (FDA) issued regulations dealing with the warning label on unpasteurized juices and published the Juice Hazard Analysis & Critical Control Points (HACCP) final rule on Jan. 19, 2001.2

This article reviews the current literature published on some of the most common spoilage organisms found in fruit juices and fruit-based drinks, and details some of the most promising non-thermal alternative processing technologies used to control these bacteria in such products.

COMMON SPOILAGE BACTERIA
The most commonly encountered spoilage bacteria in fruit juices and beverages are the species of Acetobacter, Alycylobacillus, Bacillus, Clostridium, Gluconobacter, Lactobacillus, Leuconostoc, Saccharobacter, Zymomonas and Zymobacter (see Table 1).[3-10]

While the majority of bacteria do not grow in acidic conditions, many of them are able to survive at low pH. Acetobacter and Gluconobacter (Acetomonas) are acidophilic strictly aerobic bacteria. They can grow depending on the presence of free oxygen and cause spoilage of soft drinks, apple cider and fruit concentrates. Species of Lactobacillus and Leuconostoc causes spoilage of soft drinks and orange juice. Bacillus, Clostridium and Alycylobacillus are acidophilic spore-forming, heat-resistant organisms that may be found in fruit juices and beverages. Bacillus species, which most commonly occur in tomato juice and grow depending on the availability of oxygen, also are known to cause spoilage in soft drinks. Spoilage of fruit juices by Clostridia is characterized by production of gas, a strong butyric odor and increased acidity. However, spoilage organisms of particular interest to fruit juice and fruit-based beverage manufacturers are Alycylobacillus and yeasts and molds.

Alycylobacillus was first reported in 1982 as a cause of spoilage in apple juice in Germany.[11] When spoiled by Alycylobacillus, the juice appears normal. The characteristic spoilage is usually described as phenolic or antiseptic off-flavor or odor with or without cloudiness, and gas is generally not produced.[7,12] Compounds responsible for this off-flavor have been identified as guaiacol 2,6-dibromophenol and 2-methoxyphenol.[4-5,13] However, in a study of apple juice inoculated with A. acidoterrestris, the guaiacol content in apple juice did not always correlate with the number of cells.[14] This organism has generated much interest in the fruit juice processing industry, because of its heat-resistant characteristics.

Formerly known as members of genus Bacillus, Alycylobacillus are acidophilic spore-forming soil-borne bacteria that occur widely in nature. They can survive commercial pasteurization processes commonly applied to fruit juices, germinating, growing and causing spoilage.[7,12] The spoilage occurs seasonally, typically in the spring or summer, and occurs most commonly in apple juice and orange juice.[7-8,12,15] According to one 1998 study, contamination of fruit juices by Alycylobacillus was shown to occur via soil during the harvest, which entered through the surface of the fruits.[15] In the study, acidophilic, heat-resistant bacilli were found in five of 33 samples at one plant. Strains of acidophilic, heat-resistant bacilli were detected in seven of [18] soil samples from orange groves, on surfaces of unwashed oranges at eight of 10 processing plants, on surfaces of washed oranges at six of nine processing plants, and in condensate water used to wash fruit at six of seven test facilities. Two pear juice concentrates from 210-L drums, as well as retail packages of pear juice and orange juice nectar, also contained acidophilic, heat-resistant bacilli. The researchers suggested that because fruit surfaces may be continuously contaminated with spores from the condensate wash water, the extracted juice could very well contain spores, and theoretically, contaminate the evaporator.

Another study in 1999 examined 75 samples of concentrated orange juice and found that 11 (14.7%) of the samples tested positive for Alycylobacillus.[8] In 1998, Splittstoesser, et al, reported that white grape and tomato juices also are susceptible to spoilage by this bacteria. In a survey of the food industry, 35% of respondents had experienced spoilage of acidophilic spore-forming bacteria in their product. In addition to finding positive traces of this bacteria in apple and orange juices, it also has been found in apple-grape-raspberry juice and apple-pear juice blend beverages.[7,15]

The genus Alycylobacillus is comprised of three species: A. acidocaldarius, A. acidoterrestris and A. cycloheptanicus. Alycylobacillus spores are very heat-resistant. These bacteria can easily survive the typical heat treatment normally applied to pasteurize fruit Juices.[8,16] The elevated heat resistance shown by Alycylobacillus spores represents a potential risk for the deterioration of pasteurized, ultra-high temperature, or hot-fill orange juices when stored without refrigeration, because the spores of this organism are able to germinate and grow at temperatures below 35°C.

Researchers also have found that growth of Alycylobacillus is obtained over a pH range of 3.0 to 6.0 in an agar medium.[12] In the 1998 study, growth was inhibited when the ethanol concentration exceeded 6% and the sugar content exceeded 18 Brix. It has been indicated that raising the sugar content of juices increases the heat resistance of the bacteria. These results show that it would be more difficult to destroy the spores in a juice concentrate, as compared with a single-strength juice.

Additional research indicated that the complete elimination of these heat-resistant acidophilic bacteria from fruit juices would be difficult, but that improvement of fruit cleaning operations and condensate water systems may reduce the incidence of thermo-acidophilic bacilli in fruit juices.[15] Rinsing the sanitary surfaces of equipment and the evaporators with condensate water containing heat-resistant bacilli spores may contaminate the juice entering the evaporator or the final product. The study also suggested that heat treatment in the evaporator was not sufficient to kill the spores of these bacteria.

CONTAMINATION OF JUICES BY YEASTS AND MOLDS
Yeast and mold contamination also is a major cause of fruit and fruit juice spoilage. Yeasts predominate in the spoilage of acid fruit products, because they have a high acid tolerance and the ability to grow anaerobically. Yeasts most commonly associated with the spoilage of soft drinks and fruit juices include Brettanomyces intermedius, Saccharomyces bailii, S. bayanus, S. bisphorus, S. cerevisiae, S. rouxii, Schizosaccharomyces pombe, torulopsis holmii, Dekkera bruxellensis, Torulaspora delbruckii, and Zygosaccharomyces microellipisodes.[10,17] Parish and Higgins isolated species of yeast from commercially produced unpasteurized orange juice, including Candida maltosa, Candida sake, Hanseniaspora guilliermondii, Hanseniaspora sp., Pichia membranaefaciens, Saccharomyces cerevisiae, and Schwanniomyces occidentalis.[18]

Reportedly, 40% of commercial fruit juice is contaminated with yeast, which is primarily attributed to poor plant hygiene.[10] Fruit juices from healthy fruit may contain 1,000 to 100,000 yeast per mL, whereas those from damaged fruit can contain higher levels of yeast contamination.[18] Most spoilage yeasts are highly fermentative, forming ethanol and CO2 from sugar, which causes split cans and cartons, and explosions in glass or plastic bottles.

Many molds, which can be observed near the surface of contaminated product, are aerobic, their growth dependent on the presence of oxygen. However, some molds do grow well at low oxygen levels. For example, ascospores germinate and grow during storage of fruit based products at oxygen level as low as 0.1%.19 Heat-resistant genera of molds causing spoilage of fruit juices and soft drinks can include: Byssochlamys, Paecilomyces, Neosartorya, Talaromyces and some species of Eupenicillium. [10,19-20] Genera of Aureobasidium, Cladosporium, and Penicillium have been isolated from pasteurized orange juice.21 In another study, up to 27% of mango and tomato juices contained heat- resistant molds.[22] Heat-resistant molds that cause the spoilage of fruit juices, concentrates and soft drinks are shown in Table 2.

Mold growth can result in off-flavor or odor (i.e., “stale” or “old” flavors); development of mycelial mat; reduction in sugar content; and mycotoxin production in fruit juices and soft drinks.[19,23-26] Fungal growth does not appear be a problem in freshly squeezed orange juice if moldy or decomposed fruit is not used. As indicated in two research papers, mold growth in unpasteurized orange juice is of little concern due to the short refrigerated shelf life of this product.[21,24]

Because fruit juices usually are pasteurized, surviving organisms are predominantly heat-resistant molds or their ascospores, unless the finished product is contaminated after pasteurization due to inappropriate packaging. Narciso and Parish isolated fungi from citrus juice have not been heat resistant.[27] The authors suggested that fungi within the paperboard portion of gable-top cartons are a source of contamination for fruit juice and other foods. They also indicated that unskivved cartons (with raw paperboard edges exposed to juice) are more prone to mold contamination in a shorter time period than are skivved cartons (edges folded under and sealed), unless the paperboard is heavily contaminated. This demonstrates that the paper- board is involved with producing mold spoilage in these products. The researchers highlighted that adding to the problem of contaminated pulp in food grade paperboard is the increased use of recycled fibers, a move to conserve paper and reduce waste. It has become increasingly important for processors to use packaging that is free from contamination.

MICROBIAL CONTAMINATION AND CONTROL MEASURES
A wide range of methods for controlling the microbial contamination of fruit juice and beverages are available. These include: preventive measures at orchards, chemical washes, high-pressure sprays, brushing and good agricultural practices (CAPs) related with apple juice/cider production at orchards, mandatory pasteurization, and processing by alternative technologies such as high hydrostatic pressure (HHP) or ultra-high pressure (UHP), ultraviolet (UV), and pulsed electric field (PEF).[28] The design and implementation of HACCP systems to sup- port these processes for microbial control is a strategy now in effect.[29]

Washing. One of the main reasons for contamination is failure to wash fruits properly before processing. Washing and brushing fruit before the juicing step is common in juice processing. According to one industry survey, 98% of orchards surveyed washed apples before crushing, 18% used detergent-based fruit wash, 37% used sanitizer after washing, and 64% employed brushing in conjunction with washing.[30]

Winniczuk showed that the maximum cleaning efficacy of most fruit wash systems produces a 90-99% reduction in the population of microorganisms on a citrus fruit surface under optimum pilot plant situations, whereas less-than-optimum conditions may result in only a 60% reduction of fruit surface microflora.[31] However, washing trials using water showed only a one or two log reduction in many experimental research studies.[32,33] Conventional washing practices using chlorine and brushing only may be partially effective in controlling microbial contamination.[34]

Preservatives. Another approach for controlling contamination, especially in processed product, is the use of preservatives. In an industry survey, just 12% producers reported using preservatives; among them, 60% used potassium sorbate and 40% used sodium benzoate.[33] While preservatives may have some merit for extending product shelf life, they cannot be relied upon to eliminate pathogens from fruit juice or cider. In one study, researchers discovered that E. coli O157:H7 survived in refrigerated cider containing 0.l% sodium benzoate for 21 days.[33]

Non-thermal Alternative Processing Technologies. Pasteurization of juices and beverages is known to inactivate pathogenic bacteria, reduce microbial population and thus, extend the product shelf life. In fact, contemporary pasteurization processes are designed to inactivate 99.999% (5 log) organisms present in fruit juice. However, according to some surveys, thermal processing of apple juice or cider is not a popular option because of the perceived negative effects of pasteurization on natural flavor and color of the juice products. In a recent survey, the majority (78%) of cider producers in Virginia indicated that they do not pasteurize their cider.[30] In another survey, 88% of producers in Wisconsin did not heat pasteurize their apple juice or cider.[35] Also, mandatory pasteurization may be cost prohibitive for many smaller operations, because these costs increase sharply as production capacity and number of days per year of processing decrease.

Since traditional thermal processes, though effective in inactivating bacteria, can affect the quality of the finished product, the scientific community has stepped up efforts to identify and review the kinetics and use of non-thermal alternative processes.[28] Most notably, as part of the five-year contract between the Institute of Food Technologists (IFT) and the U.S. Food and Drug Administration (FDA), the ongoing scientific review of these alternative processing technologies includes the consideration of many questions, including: “What might be used to produce food products free from any public health hazard and what are their critical control points? Which organism(s) of public health concern is the most resistant to the process(es), since the mechanism of microbial inactivation for an alternative processing technology may not follow that of traditional thermal processing? How do factors such as growth phase and growth conditions of organism(s), processing substrate or food matrix, the pathogenic organisms associated with specific foods, processing conditions, storage conditions and potential storage abuse affect the determination of the most resistant organism(s) of concern for a each alternative processing technology, which may be different from that established for traditional thermal processing? And, how do users determine the effectiveness of an alternative processing technology? These are but a few of the significant issues being addressed.

The IFT/FDA scientific review of the different types of alternative processing technologies that might be used for both pasteurization and sterilization type processing includes: high pressure processing (HPP), pulsed electric field (PEF), pulsed x-ray or ultraviolet light (UV), ohmic heating, inductive heating, pulsed light, combined ultraviolet light and low concentration hydrogen peroxide, submegahertz ultrasound, filtration, and oscillating magnetic fields.[28] Here, we will review three of the most promising of these non-thermal alternative processing technologies as they apply specifically to the inactivation of bacteria in fruit juices and fruit beverages.

High Pressure Processing. The use of HPP, and/or ultra high pressure (UHP), as a food preservation technique is well documented. This type of non-thermal processing currently is used in various parts of the world in the manufacture of a number of products, including fruit juices, fruit purees and jams.[36] HPP involves subjecting both packaged and unpackaged foods and beverages to pressures between 100 and 800 Mpa within a cylindrical pressure vessel. The equipment used for a batch HPP system in which the foods to be treated go through also includes two end closures with restraints such as yoke threads, a low pressure pump, an intensifier that uses liquid from the low pressure pump to generate high pressure process fluid for system compression, and system controls and instrumentation.[28] These batch system steps are rearranged for use to treat unpackaged liquid foods; such as fruit juices, semi-continuously.

Recent studies suggest that this emerging alternative technology can offer food processors a viable non-thermal approach to ensuring food safety goals by inactivating bacteria. Several researchers have studied the efficacy of various HPP treatments in inactivating microorganisms, especially pathogens E. coli O157:H7 and Salmonella, in fruit juices.[36-39] Some of these studies have shown that the lower the food’s pH, the higher the number of microorganisms are inactivated by HPP and damaged cells do not resuscitate. This has been observed with the inactivation rates of E. coli O157:H7.[36] Similarly, spoilage organisms like yeast in fruits can be effectively inactivated by using HPP due to their inherent low pH. Parish targeted S. cerevisiae in a nonpasteurized low-pH (3.7) orange juice with HPP, and reported D-values of 76 seconds for ascospores treated at pressures between 500 and 350 MPa, respectively.[37] The D-values for native flora of the orange juice ranged from 3 to 74 seconds. Yeasts and gram-positive and gram-negative organisms were found to survive 1 to 300 seconds of HPP treatment.[28]

In addition, UHP extends the shelf life of refrigerated juice by up to 30 days if the pressures range from 44,000 to 73,500 psi for 20 seconds to one minute. One significant problem with fresh orange juice is limited shelf life due to cloud loss caused by the activity of several pectin methylesterase (PME) enzymes. High-pressure processing can inactivate spoilage microflora and reduce PME inactivity.[40]

Pulsed Electric Field. PEP processing involves the application of high voltage pulses for just a few microseconds to food placed or flowing between two electrodes. The process destroys both pathogens and spoilage organisms through breakdown or rupturing the cell membrane. Pores become permanent in most vegetative cells treated above 15,000 V/cm. PEF inactivates bacterial spores by reducing the dipicolinic acid needed for spore germination. The components of a PEP system essentially include: a high-voltage power supply, an energy storage capacitor, a treatment chamber(s), a pump to conduct food though the treatment chamber(s), a cooling device, voltage, current, temperature measurement devices, and a computer to control operations.[28,40]

Currently, there are two commercially available systems that have been used in pilot studies.28 The most common use of pulse electric field processing has focused on food preservation and product quality aims, including extending the shelf life of orange juice, apple juice, bread, milk, and liquid eggs. In fact, shelf-life studies show that the process can extend refrigerated shelf life of fresh citrus juice to beyond 60 days.[40]

In terms of inactivating spoilage microbes in fruit juices and fruit-based beverages, PEP has proved efficient in some research. In pilot experiments, researchers using PEP achieved a 5-log reduction of E. coli O157:H7 and its non-pathogenic surrogate E. coli 8739 in apple cider in 143 microseconds at a field strength of 30 kV/cm and average temperature of 25°C (near ambient). Spoilage organisms in orange juice were reduced by 5 logs at a peak field intensity of 40 kV/cm for 60 microseconds.[40] PEP technology also has been applied to process citrus juices in a slightly different, energy-efficient low-voltage electric pulse process in which electricity is directly pulsed into the juice. Less than 1 joule per mL is applied to process flow rate of 20 gpm, resulting in a 7-log reduction of Listeria monocytogenes and Salmonella typhimurium, and a 5-log reduction of E. coli O157:H7 in fresh orange juice.[40]

Acidic liquid products such as fruit juices and pumpable particulate containing liquids offer the best opportunity for commercialization of the PEP technology. A commercial system combining the PEP processing and aseptic packaging is being designed by a university - industry consortium.[40] This system will reportedly be capable of processing juice at flow rates of 2000 liters/h at 35 kV/cm for 50 microseconds.

Ultraviolet Light. Recently, the use of UV light as an alternative processing technique has focused almost exclusively on treating fruit juices, primarily apple juice and cider, to reduce microbial counts and inactivate pathogens such as E. coli O157:H7 and Cryptosporidium parvum. UV light processing involves the use of mercury lamps, which generate 90% of their energy at a wavelength of 253.7 nanometers. Exposure of bacteria to UV results in the cross-linking of the thymine dimers of the DNA of the organism, preventing repair of injury and reproduction.

Recently, California Day—Fresh Foods of Glendora, CA, filed a petition with PDA to allow the UV process in conjunction with HACCP to assure a 5-log reduction of pathogens in its fresh, refrigerated juices.[40] The system consists of modules enclosing a one-inch diameter Teflon tubing and has a process capacity of 420 gph at an exposure of UV at 253.7 nm for approximately one minute. A double pass through the module reportedly results in a S-log reduction of E. coli O157:H7, Salmonella and Listeria monocytogenes.

SUMMARY
The spoilage and microbial contamination of fruit juices and fruit-based drinks remains a concern for the industry. However, several approaches and processes are available to minimize the risk of pathogens and assure safety of fruit juice and fruit-based beverages. These include washing and surface decontamination treatments, good orchard practices and application of pasteurization processes. With the emerging use and availability of non-thermal alternative processing technologies, such as HPP/UHP, PEF and UV light, prospects for greater control look good. The successful application of these processes will depend on, among other things, the cost of equipment and effectiveness of the process.[41] While a few processes are at or near production scale, many are pilot scale and need further development. Also, most of these processes are new inventions and thus must be subjected to appropriate validation tests. Another complicating factor is the regulations dealing with labeling (e.g., designation as “fresh” for a juice “pasteurized” by a nonthermal process) and premarket approval. As juice HACCP gets underway, pinpointing the critical process hazards and identifying effective control measures will be more important than ever before. Stay tuned, there is more to come.

Purnendu C. Vasavada, Ph.D., is professor of food science at University of Wisconsin-River Falls. As an extension specialist in the area of food safety and microbiology, he has worked with rapid methods and been an instructor in programs the world over. Vasavada directs the three-day course, “Current Concepts in Foodborne Pathogen and Rapid Methods and Automation in Food Microbiology” designed to provide food processors with up-to-date information on rapid methods, testing protocols and technologies most suitable for their operations. The 2002 symposium will be held October 13-16 in River Falls, WI.
 
Dilek Heperkan, Ph.D., is professor of food engineering at Istanbul Technical University, Turkey She teaches food microbiology and hygiene and sanitation courses at the ITU and conducts research in food safety and quality assurance. Her research interests are food mycology probiotics, mycotoxins and risk assessment. Recently she spent part of her sabbatical leave at the University of Wisconsin-River Falls.

ACKNOWLEDGEMENT
This paper is a contribution from the College of Agriculture, Food and Environmental Sciences, UW River Falls and the cooperative extension service of the University of Wisconsin. The support and assistance provided to D.H. by the Animal and Food Science Department, UW River Falls during her sabbatical visit is gratefully acknowledged.

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