Response of Spores to High-Pressure Processing

Authors

  • Elaine P. Black,

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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  • Peter Setlow,

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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  • Ailsa D. Hocking,

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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  • Cynthia M. Stewart,

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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  • Alan L. Kelly,

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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  • Dallas G. Hoover

    1. Authors Black and Hoover are with Dept. of Animal and Food Sciences, Univ. of Delaware, Newark, DE 19716-2150, U.S.A. Author Setlow is with Dept. of Molecular, Microbial and Structural Biology, Univ. of Connecticut Health Center, Farmington, CT 06030-3305, U.S.A. Author Hocking is with Food Science Australia, P.O. Box 52, North Ryde, NSW 2113, Australia. Author Stewart is with National Center for Food Safety and Technology, 6502 S. Archer Rd., Summit-Argo, IL 60501, U.S.A. Author Kelly is with Dept. of Food and Nutrition Sciences, Univ. College Cork, Ireland. Direct inquiries to author Hoover (E-mail: dgh@udel.edu).
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Abstract

ABSTRACT:  This review focuses on the responses of microbial spores to food processes that incorporate high hydrostatic pressures. While the majority of information deals with spores of Bacillus species, spores of Clostridium and Alicyclobacillus species are also discussed, and a section of the review surveys the responses of fungal spores to high-pressure processing. The mechanisms of the germination of bacterial spores are outlined in detail with regard to spore physiology and structure, along with molecular aspects of germinants and the interaction with spore receptors. Use of treatments combining pressure and temperature for a range of different food products is reviewed, including examples of hurdle technology employing high hydrostatic pressure. Pressure-assisted thermal sterilization is also discussed.

Introduction

Thermal processing possesses many advantages and applications in food manufacturing, but the use of heat has its drawbacks. Processed foods are normally changed in numerous ways from their fresh counterparts with regard to appearance, flavor, texture, nutrient content, and microbiota. Consumers prefer some foods raw or minimally cooked for consumption; however, these foods are often perishable and suffer a limited shelf-life unless frozen; such foods may harbor additional safety risks. Nonthermal treatments (often used in combination) can be attractive alternatives to traditional heat treatments for manufacturing minimally processed, high quality, preservative-free, convenient, and safe food products. Among the nonthermal food processes, high-pressure processing (HPP) is often the premier example of a new, commercially successful nonthermal process; however, one of the deficiencies of HPP remains its limited success in the inactivation of spores, primarily bacterial endospores in low-acid foods where survival and growth of Clostridium botulinum is a risk. For this situation, thermal processing is the traditional answer for preserving shelf-stable low-acid foods, and its most familiar application is commonly called canning (even though canning can also be called pressure cooking).

Two general principles underlie the antimicrobial effects of high pressure. First, Le Chatelier's principle states that any phenomenon (phase transition, change in molecular configuration, chemical reaction) accompanied by a decrease in volume is enhanced by pressure (Rovere and others 1998). Thus, pressure shifts the system to that of lowest volume. Second, the isostatic principle states that pressure is uniformly distributed throughout the entire sample, whether in direct contact or in a flexible container (Rovere and others 1998). Pressurization process time is therefore independent of sample size. This is in contrast to thermal processing.

High pressure can have a variety of effects on food and its constituents. Foods with high water content behave much like water itself. Water has a compressibility of around 15% at 600 MPa (Farkas and Hoover 2000). The ionic dissociation of water is enhanced under pressure, which can create a decrease in pH (Rovere and others 1998). Foods with a low pH have a desirable effect on the inactivation of microorganisms and promote product safety. Lipids under high pressure exhibit an increase in melting temperature; at room temperature, lipids can crystallize under pressure. It is also known that high pressure causes the dissociation of salt bonds and the strengthening of hydrogen bonds, and has minimal effects on covalent bonds within the product.

HPP has also been found to have an effect on the aggregation of proteins in microorganisms, most likely due to the unfolding of proteins under pressure (Rovere and others 1998). Protein denaturation becomes irreversible at higher-pressure thresholds and/or high protein concentrations, which promote aggregation. The inactivation of bacterial spores through the use of heat is directly associated with protein denaturation and enzyme inactivation (Rodriguez and others 2004).

The favored mechanism for explaining bacterial spore inactivation by HPP is pressure/temperature-induced spore germination in which the spores lose their inherent resistance and are inactivated by subsequent treatment conditions due to acquired sensitivity. Since not all germinated spores are inactivated by pressure and not all spores are germinated by exposure to conditions of HPP, pressure sterilization of low-acid foods is a fundamentally unreliable process (Sale and others 1970). This review surveys the current state of knowledge with regard to the primary problem of spore inactivation using HPP, an issue that presents particular challenges at mild HPP treatment temperatures. Bacterial spores in addition to spores from filamentous fungi are discussed.

Food Safety and Spores

The extreme resistance of bacterial endospores to physical and chemical treatments makes them a significant problem for the food industry. Applications of aggressive and quality-impairing preservation treatments are often needed to ensure food safety. Spores found in food, typically from species of Bacillus and Clostridium, are common agents of spoilage, causing detrimental changes to sensory quality (Brown 2000). Of health significance are spores from species of Bacillus and Clostridium that cause foodborne illnesses, ranging from mild emetic-type illness to life-threatening botulism. The major bacterial sporeforming foodborne pathogens are (1) Clostridium botulinum, which can produce one of the most lethal toxins known to humans; it is the causative agent of foodborne botulism, a rare but potentially fatal illness (Lund 1990); botulism is caused by ingestion of a neurotoxin produced by the bacterium in food, most commonly home-canned products, and is characterized by muscle paralysis that can lead to asphyxiation (Shapiro and others 1998); (2) Clostridium perfringens, which is responsible for foodborne illness with symptoms of acute abdominal pain, diarrhea, and nausea, and is often associated with cooked meat products (Smith and Williams 1984); and (3) Bacillus cereus, a ubiquitous microorganism that has been implicated in outbreaks involving many food products, especially those associated with soil (Borge and others 2001). The exact incidence of foodborne illness caused by B. cereus is difficult to estimate, as the illness is often self-limiting and it is not a reportable disease (Granum and Baird-Parker 2000). Bacillus licheniformis and Bacillus subtilis have also been occasionally implicated in outbreaks of foodborne illness (Salkinoja-Salonen and others 1999; Brown 2000).

Some of the sporeforming bacteria that cause food spoilage are listed in Table 1. Spoilage can cause substantial economic losses in the food industry, and the presence of spoilage agents can be indicative of conditions suitable for the proliferation of more serious disease-causing agents (Brown 2000). The effects of growth of some sporeforming bacteria include butyric acid production, burst packages, “gas-blowing” of cheeses, putrefactive odors, and “flat-sour” spoilage, which is acid production without the production of gas (Table 1). B. cereus not only can cause foodborne illness, but can affect the quality of dairy products, for example, “bitty cream” (aggregation of the cream layer by the action of lecithinase produced by bacteria) and “sweet-curdling” (coagulation without pH reduction which is mediated by bacterial proteases) (Andersson and others 1995). Most sporeforming bacteria cannot germinate at low pH, and therefore pose little threat of spoilage or foodborne illness in acidic foods; however, spores of Alicyclobacillus acidoterrestris are problematic in fruit juices due to their ability to germinate and grow in the pH range 2 to 6 (Splittstoesser and others 1994).

Table 1—.  Food spoilage caused by sporeforming bacteria
Sporeforming bacteriumType of spoilageReference
B. cereusBitty cream and sweet curdlingAndersson and others (1995)
B. subtilisSpoilage of pasteurized milkBrown (2000)
B. sporothermoduransSpoilage of UHT milkWesthoff and others (1996)
B. stearothermophilusFlat-sour spoilageBrown (2000)
B. coagulansFlat-sour spoilageBrown (2000)
C. butyricumGas and butyric odorDasgupta and Hull (1989)
C. tyrobutyricumGas and butyric odorDasgupta and Hull (1989)
C. sporogenesGas and putrefactive odorBrown (2000)
C. thermosaccharolyticumGas and cheesy odorBrown (2000)
C. putrefaciensSpoilage of cooked hamRoberts and Derrick (1975)
A. acidoterrestrisSpoilage of fruit juicesSplittstoesser and others (1994)

Inactivation of spores by high pressure

The complete inactivation of spores using high pressure is the “holy grail” of high-pressure food processors. Even at the beginning of pioneering work applying high hydrostatic pressure to food processing there were indications that spores would be difficult to eradicate with HPP. In the 19th century, Bert Hite subjected milk to high hydrostatic pressure, as opposed to high temperature which causes undesirable burnt flavor notes, in order to prevent it from turning sour (Hite 1899). By using pressures ranging from 400 to 700 MPa at room temperature, a 4-log10 reduction in microbial counts was achieved, while maintaining product freshness. Hite had some success in creating shelf-stable HPP-treated fruit products with a pH that prevented growth of spores, but he never could achieve shelf-stability of HPP-treated milk due to its neutral pH and presence of spores (Hite 1899; Hite and others 1914).

The ability to make low-acid pressure-treated products as safe as foods subjected to a botulinum cook (thermal sterilization equivalent to treatment at 121 °C for 3 min) by pressure alone has not yet been possible. For example, an early study by Timson and Short (1965) showed that spores of B. subtilis and Bacillus alvei survived in milk treated at 1034 MPa for 90 min at 35 °C. Hence, a number of coactive preservative factors (hurdle technology) to enhance or accompany the effects of high pressure on spores to achieve inactivation have been studied; these include pressure cycling and combinations of pressure, high and low temperatures, and antimicrobial agents.

Physiology and Genetics of Spores of Bacillus Species

Dormant spores of Bacillus and Clostridium species and their close relatives are of major concern in food processing since such spores (1) are present in soils, and are thus commonly found associated with foods; (2) are among the microorganisms most resistant to treatments commonly used in food processing; and (3) can become metabolically active with germination and outgrowth; the resultant replicating cells can cause food spoilage or foodborne disease. Since HPP has the potential to inactivate spores in foods (Gould and Sale 1970; Heinz and Knorr 2001), it is important to understand spore physiology, especially factors that are pertinent to spore inactivation by HPP.

Spores are formed in the sporulation process that is initiated in response to harsh environmental conditions. In the Bacillus species this signal is generally the starvation of one or more nutrients (Driks 2002a). The resultant spore is metabolically dormant and contains little to none of the high-energy compounds such as ATP that are present at high levels in growing cells (Setlow 1994). Spores are also extremely resistant to potentially lethal treatments such as exposures to moist and dry heat, UV- and γ-radiation, and toxic chemicals that rapidly kill growing cells (Nicholson and others 2000; Setlow 2006). Because of their dormancy and resistance, spores can survive for extremely long periods, certainly hundreds of years, and there are claims that spores can survive for tens to hundreds of millions of years (Kennedy and others 1994; Cano and Borucki 1995; Vreeland and others 2000). Because of their survival ability and since many sporeformers are soil microorganisms, spores are present at high levels in soils and are commonly associated with most foods.

Spore structure

In addition to different physiological properties, the dormant spore has a very different structure than that of a growing cell, as the spore has a number of layers and constituents not present in growing cells (Figure 1). The exosporium is the outermost layer of the spore. The size of the exosporium varies greatly between spores of different species, being very large in spores of B. cereus and its close relatives and extremely small, if present at all, in B. subtilis spores (Driks 1999, 2002b). The exosporium is composed of carbohydrate and protein, and the macromolecules in the exosporium are unique to spores. The precise function of the exosporium is not clear, and this structure is not known to play any role in spore resistance properties (Setlow 2006). Below the exosporium is the coat layer, which itself may be composed of several different layers. The coats are composed largely of proteins; more than 30 different proteins have been identified in B. subtilis spore coats, and these proteins are unique to spores (Driks 1999, 2002b; Kim and others 2006). The coat layer is important in spore resistance to some chemicals, perhaps by reacting with and detoxifying these chemicals before they damage targets deeper within the spore (Setlow 2006). The coats also protect spores from lytic (lysozyme-like) enzyme attack, by restricting access of such enzymes to peptidoglycan (PG) deeper further within the spore. Much of the coat protein can be removed by extraction with detergents without killing the spore, and B. subtilis spores are also available that have defective coats due to the lack of a key protein or proteins needed for coat assembly (Driks 1999). Spores with defective coats retain their resistance to high hydrostatic pressure (Paidhungat and others 2002), suggesting that the coat is not important in spore resistance to pressure; however, recent work has shown that coat-defective spores retain the great resistance of the dormant spore to mechanical disruption (Jones and others 2005) and still contain an extremely resistant and rigid coat layer, termed a “rind” (Klobutcher and others 2006). The role of this rind in pressure resistance has not yet been investigated. During spore outgrowth, the spore coat is shed in a process that is poorly understood.

Figure 1—.

(A) Schematic diagram of a dormant spore structure. The various layers of the spore are not necessarily drawn to scale, in particular the exosporium varies in size considerably in spores of different species. (B) Transmission electron micrograph of spores of Bacillus subtilis.

Beneath the spore coats is the outer membrane. This structure is a functional membrane in developing spores, but it is not clear if it is a complete membrane in dormant spores; it can be removed with only minimal effects on spore dormancy and most resistance properties (Setlow 2006). The spore cortex is the next layer, and is composed largely of PG with a structure similar to that of vegetative cell PG, but with some key differences (Popham 2002). Spore cortex PG invariably contains diaminopimelic acid, even if this is replaced by lysine in growing cell PG. In addition, much of the muramic acid in cortex PG is present as muramic acid-δ-lactam (MAL), with smaller amounts of muramic acid linked only to L-alanine. The cortex appears to be extremely important in establishing and maintaining spore dormancy, most likely by causing and maintaining the low water content in the central region or core of the spore (see below). The cortex PG is degraded early in spore germination, and this cortex hydrolysis is essential for spores to “return to life” (Paidhungat and Setlow 2002; Setlow 2003). Immediately beneath the cortex is the germ cell wall, also composed of PG. In contrast to cortex PG, the structure of germ cell wall PG appears to be identical to that of growing cell PG and lacks MAL (Popham 2002). The germ cell wall is not degraded during spore germination and becomes the cell wall of the outgrowing spore.

The next spore layer is the inner membrane. This is a complete membrane in dormant spores and has several unusual properties. These include an extremely low permeability to small molecules, whether these be charged or hydrophobic, including water (Westphal and others 2003; Cortezzo and others 2004b; Cortezzo and Setlow 2005), and immobility of the lipids in this membrane (Cowan and others 2004). Despite its unusual properties, the phospholipid and fatty acid composition of this membrane is not markedly different from that of the plasma membrane of growing cells (Cortezzo and others 2004b; Setlow 2006). This membrane also appears to be compressed significantly in the dormant spore as following cortex hydrolysis during spore germination, the volume encompassed by the inner membrane increases 2- to 3-fold in the absence of new lipid production and ATP synthesis (Cowan and others 2004). The low permeability of the inner membrane appears to be extremely important in restricting access of DNA-damaging chemicals to the spore core, the site of spore DNA (Cortezzo and Setlow 2005). A number of proteins that are essential for spore germination are also present and must function in the inner membrane (Setlow 2003). The unusual structure of this membrane undoubtedly influences the behavior of these proteins; however, the structure of the inner membrane is not known.

The final and central spore layer is the core that contains most spore enzymes, as well as the protein synthetic machinery and DNA. The core also has 2 unique constituents and 2 important biochemical properties (Gerhardt and Marquis 1989; Driks 2002b; Setlow 2006). One of the latter is that the water content of the spore core is extremely low. In contrast to growing cells, where about 80% of the wet weight is water, this value in the spore core ranges from 25% to 55% depending on the species. This low core water content is a major contributor for spore resistance to moist heat, since spores with higher core water contents are less resistant to moist heat. The low core water content is also undoubtedly a major reason for spore dormancy, as normally soluble and freely mobile proteins are immobile in the core (Cowan and others 2003). It has even been suggested that the spore core is in a glass-like state, although this suggestion has been disputed (Ablett and others 1999; Leuschner and Lillford 2003). In contrast to the low water content in the spore core, other spore layers have the normal high water content expected of growing cells.

The second novel biochemical property of the spore core is that its pH is 1.0 to 1.5 units lower than that of a growing cell, which is generally pH 7.5 to 8.0 (Setlow 1994; Paidhungat and Setlow 2002). The low core pH is attained late in sporulation and is important in modulating the activity of several key enzymes in the developing spore at this time. The pH of the spore core rises to that of the growing cell in the first minutes of germination through the excretion of protons.

As noted above, the cortex of the spore is involved in some fashion in bringing about the low water content of the core. Also involved in reducing the water content is a unique small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA)(Figure 2). DPA is likely present in the core as a 1:1 chelate with divalent cations, predominantly Ca2+, forming Ca-DPA (Gerhardt and Marquis 1989). DPA is made late in sporulation and accumulates in the spore core to about 20% of core dry weight. Spores of mutant strains that do not synthesize or accumulate DPA invariably have higher core water contents and, as a consequence, are less moist heat-resistant than are their wild-type counterparts (Paidhungat and others 2000; Setlow and others 2006). DPA is also important in protecting spore DNA from a variety of types of damage, although it generally plays a more minor role in protection of spore DNA than a second type of molecule unique to spores (Douki and others 2005; Setlow and others 2006).

Figure 2—.

DPA structure. The structure shown is that of the dianion, which is the form likely to exist within the spore core.

The α/β-type small, acid-soluble proteins (SASP) are this latter second type of unique molecules present in the spore core (Setlow 1994, 2006; Driks 2002b; Paidhungat and Setlow 2002). The α/β-type SASP are the products of a multigene family expressed only in the developing spore late in sporulation. These proteins are small (65 to 75 amino acids), comprise about 5% of total spore protein, and are located only in the spore core. In the spore the α/β-type SASP are associated only with spore DNA, and there is a sufficient amount of these proteins to saturate the spore's DNA. The binding of the α/β-type SASP to DNA changes the DNA structure and, although the specific changes in DNA structure caused by α/β-type SASP binding are not known, this binding protects spore DNA against a variety of agents, including moist and dry heat and oxidizing agents, and drastically alters the effects of ultraviolet radiation on spore DNA. Consequently, the α/β- type SASP are major factors in spore resistance to all of these agents; however, shortly after the completion of spore germination, the α/β-type SASP dissociate from spore DNA and are then rapidly degraded with this degradation being initiated by a SASP-specific protease termed the germination protease, GPR.

Spore germination

While dormant spores are extremely resistant to heat, radiation, and toxic chemicals, as noted above, germinated spores lose much of this extreme resistance. This latter feature appears to be the major reason that HPP kills spores, since HPP causes spore germination. The germinated spores are subsequently killed by exposure to pressure and elevated treatment temperatures; however, these treatment temperatures are not lethal to dormant spores. In view of the importance of spore germination in spore killing by HPP, it is important to understand the process of spore germination (Paidhungat and Setlow 2002; Setlow 2003) (Figure 3 and 4). Spore germination has been most studied with spores of B. subtilis because of the wealth of genetic information available for this species and the ease with which mutant strains can be generated; however, where it has been studied in other Bacillus species, the mechanism of spore germination appears similar to that for B. subtilis. The general features of germination of clostridial spores are also similar to those of B. subtilis, and clostridial species have been found to have many homologues of proteins shown to be important in B. subtilis spore germination. Unfortunately, the lack of good methods for genetic manipulation of clostridia has greatly limited detailed analysis of the mechanism of germination of clostridial spores.

Figure 3—.

Events in nutrient-triggered spore germination

Figure 4—.

Signal transduction pathways in spore germination triggered by different agents. Arrows denote the signal transduction pathway. Chemicals or treatments that can trigger germination are in color. The SpoVA proteins are likely involved in release of Ca-DPA, but this has not yet been definitively proven. The question marks adjacent to the arrow leading from cortex hydrolysis to release of Ca-DPA indicate that there is no indication of how one event triggers the other.

Spore germination has been defined in a number of ways over the years. In this section we will use the recent definition of spore germination as events that take place upon the addition of a germinant that requires no enzymatic action in the spore core and thus no metabolic reactions (Setlow 2003). In nature, spore germination is undoubtedly triggered by the presence of nutrients. The identity of these nutrients varies with the species and strain of sporeformer, but common nutrient germinants are L-amino acids, D-sugars, and purine nucleosides. These nutrients bind in a stereo-specific manner to germinant receptors that are located in the inner membrane of the spore. There are multiple germinant receptors in spores that have been examined in detail (three have been identified in B. subtilis spores) with different germinant receptors responding to different nutrients. The germinant receptors are encoded by tricistronic operons that are expressed only in the developing spore. The proteins encoded by all 3 cistrons are essential for germinant receptor function, and these 3 proteins probably interact physically to form the functional receptor (Igarashi and Setlow 2005). There also appears to be some cooperation between different germinant receptors (Igarashi and Setlow 2005; Atluri and others 2006), but levels of the germinant receptors in spores are very low (10 to 100 molecules/spore) (Setlow 2003; Igarashi and Setlow 2006). Metabolism of nutrients that bind to their cognate germinant receptors is not needed to trigger germination. Rather, nutrient-germinant receptor-binding leads to the release of ions from the spore core, including loss of K+, H+, and Ca-DPA. The mechanism whereby these ions leave the spore core is not clear, although the proteins encoded by the spoVA operon have been suggested to be involved in Ca-DPA release (Tovar-Rojo and others 2002; Vepachedu and Setlow 2004, 2005, 2007). The mechanism by which nutrient binding to the germinant receptors triggers ion release is also not known. Along with release of ions, in particular Ca-DPA, there is also net water movement into the core, presumably into space created by the release of the large amount of Ca-DPA. The events to this point are termed Stage I of germination (Figure 3). At the end of Stage I the spore has lost some of its resistance properties, in particular resistance to moist heat, due to the increased water content of the core. However, the Stage I-germinated spore remains metabolically dormant, and enzymes in the core are still not active and proteins do not diffuse readily (Paidhungat and Setlow 2002; Cowan and others 2003; Setlow 2003). Indeed, α/β-type SASP are not degraded in Stage I and, consequently, Stage I-germinated spores remain resistant to UV-radiation and hydrogen peroxide.

The events in Stage I of germination trigger progression into Stage II (Figure 3). The hallmark event in Stage II germination is the hydrolysis of the cortex. This hydrolysis allows the core volume to expand 2- to 3-fold, bringing the core water content to about 80% of wet weight, similar to the water content in growing cells. As a consequence, proteins in the core become mobile (Cowan and others 2003) and core enzymes begin to operate, leading to degradation of α/β-type SASP and initiation of metabolism followed by macromolecular synthesis (Paidhungat and Setlow 2002). The precise method for core expansion is not known, although there is significant information available on the mechanism of cortex degradation. In B. subtilis spores, either of 2 redundant cortex lytic enzymes (CLEs), CwlJ and SleB, is sufficient for cortex degradation. CwlJ and SleB are active only on PG that contains MAL and are present in the dormant spore in a mature form that could be active but is not. Thus, events in Stage I of germination somehow activate CwlJ and SleB. The mechanism of SleB activation is not known, but is probably due to some physical change to the cortex PG caused by release of Ca-DPA and attendant water uptake. In contrast, CwlJ is probably activated directly by the Ca-DPA released from the spore in Stage I (Paidhungat and others 2001).

In addition to nutrients, there are a number of other agents that can trigger spore germination (Figure 4). One such molecule is Ca-DPA, as exposures to high levels of Ca-DPA (tens of mM concentrations) effectively trigger germination of spores of many species. Ca-DPA appears to trigger spore germination by directly activating CwlJ (as noted previously), thus leading to cortex hydrolysis. Cortex hydrolysis then leads to release of endogenous Ca-DPA, although it is not known how this release is triggered or takes place. Hydrolysis of the cortex by exogenous lytic enzymes such as lysozyme can also lead to spore germination, but this can be achieved only in spores with a defective coat allowing enzymes to penetrate to the cortex. Again, it is not known how cortex lysis by lysozyme leads to release of Ca-DPA. Spore germination with exogenous lysozyme must also be carried out in a hypertonic medium, since lysozyme degrades not only cortex PG but also the PG of the germ cell wall.

The 3rd type of molecules that trigger spore germination are cationic surfactants, which are effective at 100 μM to 1 mM. Dodecylamine is the best studied member of this type of germinant (Setlow and others 2003). Triggering of spore germination by dodecylamine requires neither the germinant receptors nor any CLE, although completion of dodecylamine germination requires at least 1 molecule of CLE. Dodecylamine triggers release of Ca-DPA directly, and this release leads to subsequent events in germination. The details of how dodecylamine triggers release of Ca-DPA, whether by activating a normal channel for DPA present in the inner membrane or by the creation of such a channel, are not known; however, recent work is consistent with dodecylamine directly activating a Ca-DPA channel that may be composed of SpoVA proteins (Vepachedu and Setlow 2007).

An additional agent that triggers spore germination is mechanical abrasion (Jones and others 2005). With B. subtilis spores, germination by mechanical abrasion is not by activation of the germinant receptors; rather, abrasion appears to damage the spore cortex in some fashion, and this in turn leads to the activation of either of the CLEs of the spore.

With completion of Stage II of germination and initiation of enzyme activity in the spore core, spore germination is complete and leads to spore outgrowth that eventually converts the germinated spore into a growing cell. In contrast to spore germination which requires no exogenous or endogenous nutrients or even any energy metabolism, outgrowth eventually requires exogenous nutrients; however, energy reserves stored in the dormant spore, in particular the amino acids derived from SASP degradation, can support some metabolism early in outgrowth.

Pressure-induced germination

As noted previously, high pressure can also trigger spore germination, and this appears to be the major reason that HPP can result in spore killing. The mechanism whereby high pressure triggers spore germination varies depending on the exact pressure used. Moderately high pressures (MHP, 50 to 300 MPa) trigger germination by activating the nutrient receptors of the spore, setting in motion subsequent events proceeding via the nutrient-triggered germination pathway (Wuytack and others 2000; Paidhungat and others 2002; Black and others 2005b). In contrast, very high pressures (VHP, 400 to 800 MPa) do not trigger germination via the nutrient receptors (Wuytack and others 2000; Paidhungat and others 2002; Black and others 2007b). Rather, VHP appears to directly cause Ca-DPA release with this release of Ca-DPA, then triggering subsequent events in germination. For either pressure range, the spores whose germination has been triggered by pressure may continue through Stage II of germination; however, if conditions during HPP preclude or inhibit action of CLEs, the spores may only complete Stage I of germination rapidly and then only slowly, if at all, go through Stage II (Black and others 2007b). While Stage I-germinated spores are not as sensitive to agents such as moist heat as Stage II-germinated spores, they are more sensitive than dormant spores due to the increased core water content of Stage I-germinated spores. VHP treatment may also result in spores that have completed germination but go through outgrowth only slowly (Wuytack and others 1998). These spores will again be sensitive to moist heat, but can be as UV- and hydrogen peroxide-resistant as the dormant spores due to the persistence of α/β-type SASP in these germinated spores (Wuytack and others 1998). As is the case with spore germination by other agents, most detailed studies of the mechanisms of pressure-induced germination, whether MHP or VHP, have been carried out with B. subtilis spores. Consequently, in the discussion below, conclusions are generally based on work with B. subtilis spores unless noted otherwise.

Moderately high pressure-induced germination

MHP-induced germination proceeds via activation of any of the nutrient germinant receptors, as noted above, since spores lacking either all nutrient germinant receptors or the GerD protein essential for spore germination via the germinant receptors do not germinate with MHP (Wuytack and others 2000; Paidhungat and others 2002; Black and others 2005b; Pelczar and others 2007). In addition, spores with elevated levels of germinant receptors germinate more rapidly with MHP. Stimulation of different individual germinant receptors by MHP also results in different rates of spore germination, although the reason for these different rates is not known. Activation of germinant receptors by MHP then leads to further events in the pathway of nutrient-induced germination, including release of Ca-DPA followed by cortex hydrolysis. The overlap in the pathways of nutrient- and MHP-induced germination is also indicated by the isolation of a B. subtilis mutant that makes pressure-resistant spores defective not only in MHP-induced germination but also in nutrient-induced germination (Aertsen and others 2005).

Release of Ca-DPA is essential for triggering later events in MHP-induced germination, since DPA-less and demineralized spores do not germinate well if at all with MHP (Black and others 2005b). With B. subtilis spores, either CwlJ or SleB can cause cortex lysis in MHP-induced germination. Generally the rates of initiation of MHP-induced germination of mesophilic spores increase with temperature, but only up to about 40 °C (Black and others 2007b), presumably because either the germinant receptors or the Ca-DPA channel proteins or both types of proteins do not function well at higher temperatures.

A large number of compounds have been shown to inhibit spore germination with nutrients, with some such as D-alanine blocking the function of individual germinant receptors (Cortezzo and others 2004a); however, many strong inhibitors of nutrient-induced germination, including D-alanine, phenols, and alkyl alcohols, inhibit MHP-induced germination poorly if at all (Wuytack and others 1998, 2000; Black and others 2005b). Spore germination, in particular the initial release of DPA triggered by MHP, is inhibited by Hg2+, a compound that also inhibits DPA release in nutrient-triggered germination.

Another parameter that can affect the rate of germination of spores by MHP is the precise sporulation conditions. B. subtilis spores prepared at higher temperatures generally germinate more slowly with MHP than do those made at lower temperatures, but the opposite is the case for B. cereus spores (Raso and others 1998a, Black and others 2005b). It is typical for cell membrane fluidity measured at one temperature to be lower in cells grown at higher temperatures. This is likely also the case for the inner membrane of the spore, if its fluidity is reflected in its passive permeability, since this permeability decreases significantly as sporulation temperature increases (Cortezzo and Setlow 2005). B. subtilis spores prepared in media with high sodium chloride content also germinate slower with MHP than spores made in media containing low levels of salt. These differences in germination rates of MHP are significant and may be important considerations in designing HPP regimens as a function of sporulation temperature and salt concentration; however, the mechanistic basis for these effects of sporulation conditions on spore germination involving MHP is not known. One possible explanation is that sporulation temperatures and salt concentrations influence the fatty acid composition (especially the unsaturated fatty acid composition) of the inner membrane where the germinant receptors are located; however, large differences in the unsaturated fatty acid composition of the inner membrane of B. subtilis have no effect on rates of spore germination with MHP (Black and others 2005b). Spore germination by MHP is also not potentiated by prior treatment of spores with either of several oxidizing agents that can inactivate spores at levels of about 95% (Black and others 2005b). In contrast, the rate of DPA loss from spores upon moderate heat treatment is increased substantially by prior treatment of the spores with oxidizing agents, as are the rates of passive permeation across the inner membrane and spore germination with dodecylamine (Cortezzo and others 2004b).

Despite knowledge of many factors that influence the rate of MHP-induced germination, precisely how MHP triggers spore germination is not understood. Presumably MHP alters the structure of the germinant receptors or the membrane in which these receptors reside, or possibly both components are altered. Certainly, high pressure can cause structural changes in both proteins or membranes, but the nature of these changes in spores is not known (Braganza and Worcester 1986; Bartlett 2002); however, how nutrients activate the germinant receptors is also not understood. Perhaps when the latter process is better understood it will give some insight into the mechanism of spore germination by MHP.

VHP-induced germination

In contrast to MHP-induced germination, spores that lack all nutrient receptors or the GerD protein germinate as well as wild-type spores with VHP (Wuytack and others 2000; Paidhungat and others 2002; Black and others 2007b; Pelczar and others 2007). Consequently, VHP does not trigger germination by activation of the nutrient receptors; however, completion of germination triggered by VHP does require at least 1 CLE (Paidhungat and others 2002; Black and others 2007b). Although VHP-induced germination does not proceed via activation of the nutrient receptors, the first major event detected in VHP-induced germination is release of Ca-DPA (Paidhungat and others 2002; Margosch and others 2004a, b; Black and others 2007b), just as it is in nutrient-induced germination. Release of DPA in VHP-induced germination does not require the CLEs (Black and others 2007b), so it is not by activation of one of these enzymes that VHP triggers spore germination. Rather, Ca-DPA release triggered by VHP appears to lead to cortex lysis, and as expected DPA-less spores do not germinate with VHP (Black and others 2007b); however, the mechanism of DPA release triggered by VHP is not known and could be due to effects on DPA channels in the inner membrane or on the membrane itself. Interestingly, the pressure-resistant mutant spores of B. subtilis noted above that are defective in MHP-induced germination as well as nutrient-induced germination (Aertsen and others 2005) are also defective in VHP-induced germination and dodecylamine-induced germination. Again, the structure of the inner membrane of spores is not known, although it is clearly different from that of the plasma membrane of a growing cell.

In common with MHP-induced germination, VHP-induced germination is also not inhibited strongly, if at all, by compounds that strongly inhibit nutrient-induced germination, such as D-alanine, alkyl alcohols, and phenols (Wuytack and others 2000; Cortezzo and others 2004a; Black and others 2007b); however, VHP-induced germination, as measured by release of DPA, is also not inhibited by Hg2+, although this ion does inhibit the completion of VHP-induced germination, presumably by inhibiting CLEs (Black and others 2007b). In contrast to rates of MHP-induced germination, rates of VHP-induced germination as measured by release of DPA increase with temperature up to at least 60 °C and perhaps even slightly higher (Black and others 2007b). Spores that have released Ca-DPA in response to VHP treatment also appear to go through Stage II germination or outgrowth, or both processes slowly, as these spores degrade α/β-type SASP and synthesize ATP slowly (Wuytack and others 1998); however, the reason(s) for the slow rates of these processes in VHP-germinated spores is (are) not clear.

As found with MHP-induced germination, sporulation conditions also affect the VHP-induced germination of the resultant spores (Raso and others 1998a, 1998b; Black and others 2007b). Thus, rates of VHP-induced germination of B. cereus and B. subtilis spores decrease markedly with decreasing sporulation temperature and also with increasing salt concentration in the sporulation medium. With B. subtilis spores these effects are opposite to those on rates of MHP-induced germination; however, B. subtilis spores killed by 95% with either of several oxidizing agents germinate fairly normally with VHP, and the level of unsaturated fatty acids in the inner membrane does not influence the rate of VHP-induced germination as also found with MHP (Black and others 2007b).

In some respects, VHP-induced germination resembles spore germination induced by dodecylamine, as the first event that appears to be caused by both agents is release of Ca-DPA; however, there are a number of differences in the germination triggered by these 2 agents, including (1) rates of spore germination with dodecylamine are faster with spores made at lower temperatures, that is, spores made at 23 °C as compared to those made at 44 °C (Cortezzo and Setlow 2005); (2) rates of spore germination with dodecylamine increase markedly if the spores have been inactivated by about 95% by any of a number of oxidizing agents, for example, betadine, chlorine dioxide, H2O2, ozone, or sodium hypochlorite (Cortezzo and others 2004b); and (3) DPA release triggered by dodecylamine is inhibited by Hg2+ (Vepachedu and Setlow 2007). Most surprisingly, dodecylamine strongly inhibits VHP-induced germination (Black and others 2007b), suggesting that VHP and dodecylamine trigger spore germination by different mechanisms; however, these mechanisms remain unclear.

Overall, much has been learned about pressure-induced germination of spores in recent years, but there are still many unknowns and uncertainties about this process. One of these unknowns concerns the problem of a small percentage of superdormant spores in spore populations that are germinated only extremely slowly by various germination agents, including pressure, much more slowly than the great majority of spores in a population. Consequently, these superdormant spores can survive treatments such as HPP that rely on spore germination in order to obtain spore killing. Survival of a small percentage of superdormant spores can make achieving food sterility by HPP alone problematic at present. Unfortunately, a major hurdle in dealing with the problem of superdormant spores in HPP is that factors making spores superdormant are not understood. Consequently, the phenomenon of superdormancy is an area of spore biology that deserves further study, because if superdormant spores could somehow be made just “dormant”, this would have major implications for potential applications of HPP in the food industry.

A 2nd major unknown is that although MHP and VHP trigger spore germination by different mechanisms, the precise mechanism for spore germination by either magnitude of pressure is unknown. This is again unfortunate, since knowledge of these mechanisms might allow more efficient HPP regimens to be designed. It is possible that the elucidation of the mechanisms of spore germination by agents such as nutrients and dodecylamine will facilitate our understanding of the mechanisms of pressure-induced germination. Conversely, elucidation of the mechanisms of pressure-induced germination may also be extremely helpful in furthering the understanding of spore germination by other agents.

Effects of heat and high pressure

Resistance of bacterial endospores to pressure and heat appears to be unrelated (Nakayama and others 1996; Margosch and others 2004a, 2004b). From the study of a number of Bacillus species, Nayakawa and others (1996) showed that B. subtilis IAM12118 spores were very pressure-resistant, but their heat resistance was much lower than that of B. stearothermophilus spores, which have similar pressure resistance. B. stearothermophilus IAM1101 spores were the second most heat-resistant spores in the latter study, but were the least pressure-resistant. A number of studies have compared the efficacy of inactivation of bacterial spores by pressure at ambient temperature with that at higher temperatures (Table 2). Elevation of the pressure-processing temperature from ambient to > 50 °C enhances inactivation of spores of both Bacillus and Clostridium species in buffer and meat (Reddy and others 2003; Moerman 2005). Stewart and others (2000) found that spore numbers of B. subtilis in McIlvaine citrate phosphate buffer were reduced more effectively at high temperatures than at ambient temperature; a 5-log10 reduction in spores was achieved at 404 MPa at 70 °C, whereas only a 0.5-log10 reduction could be achieved at 25 °C at the same pressure. Mills and others (1998) reported little or no inactivation of spores of C. sporogenes following treatment at 600 MPa for 30 min at 20 °C; however, combining heat and pressure simultaneously (400 MPa for 30 min at 60 °C) or sequentially (heating at 80 °C for 10 min at ambient pressure followed by treatment at 400 MPa for 30 min at 40 or 60 °C) was more effective, resulting in a 3-log10 reduction.

Table 2—.  Effect of elevated temperatures on pressure-induced inactivation of spores of Bacillus and Clostridium
BacteriumAmbient temp.a HPP treatment/elevated temp. HPP treatmentLog reduction lower temp./higher temp.SubstrateReference
  1. aProcess temperatures.

C. sporogenes20 °C and 600 Mpa for 30 min/60 °C and 400 MPa for 30 minNo inactivation/3-log10Distilled waterMills and others 1998
Bacillus and Clostridium20 °C and 400 Mpa for 30 min/50 °C and 400 MPa for 30 min<1.0 log10/1- to 4-log10Minced porkMoerman and others 2001
C. sporogenes25 °C and 404 Mpa for 30 min/70 °C and 404 MPa for 30 min2.5-log10/6.0-log10Citrate phosphate bufferStewart and others 2000
B. subtilis25 °C and 404 Mpa for 15 min/70 °C and 404 MPa for 15 min<0.5-log10/5-log10Citrate phosphate bufferStewart and others 2000
B. anthracis20 °C and 500 Mpa for 360 min/75 °C and 500 MPa for 190 min<2-log10/9.0-log10Distilled waterClery-Barraud and others 2004

While the combination of high pressure with moderate to high temperatures negates the “nonthermal” designation of HPP, pressure-assisted thermal sterilization (PATS) treatments are still attractive alternatives to conventional thermal sterilization or retorting, as this type of process has been proven to be just as effective and less damaging to flavor, texture, and nutritional attributes of food than thermal processes (Meyer and others 2000; Ananta and others 2001). PATS will be discussed in greater detail in a later section of this review.

Toxins of B. cereus

Bacterial toxins, such as those produced by B. cereus, can either be heat-labile (the diarrheal toxins) or heat-stable (cereulide, the emetic toxin); however, relatively little is known about their pressure resistance. Recently, Margosch and others (2005) demonstrated some loss in cytotoxity of the B. cereus hemolysin BL toxin, a heat-labile diarrheal toxin, following treatment at 800 MPa for 30 min at 30 °C. The other toxins studied from nonsporulating bacteria, cholera toxin, staphylococcal enterotoxins A-E, and E. coli enterotoxin (STa), were found to be relatively pressure-stable, but combined application of high pressure with high temperature increased their inactivation.

Combination of High-Pressure and Antimicrobial Compounds

A wide range of antimicrobial compounds have been studied in combination with HPP to enhance the efficacy of pressure-induced inactivation of both vegetative bacteria and bacterial endospores. Application of one or more additional hurdles with HPP has the advantage of allowing less severe pressure treatments, thus allowing a higher probability of maintaining the nutritional and sensory qualities of the food. Roberts and Hoover (1996) investigated the use of a combination of citric acid addition (to pH 4.0), pressure, and heat to inactivate B. coagulans; a 6-log10 reduction was achieved following treatment at 400 MPa for 30 min at 70 °C. Shearer and others (2000) applied a treatment of 392 MPa for 10 to 15 min at 45 °C in the presence of the emulsifier, sucrose laurate (<1%), which resulted in 3.0- to 5.5-log10 reductions in numbers of B. cereus and B. coagulans spores in milk and beef, and Alicyclobacillus in fruit juices, as compared to 1-log10 reductions at the same pressure at 45 °C in the absence of the emulsifier. The difference in numbers plated on nutrient agar and agar supplemented with sucrose laurate suggested that the emulsifier inhibited spore outgrowth rather than delivering a lethal effect to the spores. Similar processing conditions (0.1% sucrose laurate, treatment at 404 MPa at pH < 6.0 for 15 min at 45 °C) eliminated an initial inoculum of 1×106 spores/mL of B. subtilis in McIlvaine's buffer (Stewart and others 2000).

Bacteriocins produced by lactic acid bacteria have also shown potential for the control of spores when combined with HPP (Kalchayanand and others 1998a; Black and others 2005a). Nisin is commonly used in both high- and low-acid canned foods to prevent clostridial outgrowth and spoilage by such bacilli as B. stearothermophilus (Cleveland and others 2001). In cheese, nisin is used to prevent “late gas blowing” due to clostridial spore outgrowth (Delves-Broughton and others 1996). Lopez-Pedemonte and others (2003) have combined nisin or lysozyme with pressure to inactivate B. cereus in cheese, and while nisin increased the sensitivity of spores to pressure, lysozyme did not. In a study by Kalchayanand and others (2003), nisin, pediocin, and treatment at 345 MPa for 5 min at 60 °C) were combined to inactivate spores of C. laramie or a mixture of spores from 4 clostridial species, C. sporogenes, C. perfringens, C. tertium, and C. laramie, inoculated into roast beef. Following HPP alone, samples inoculated with a mixture of clostridial spores could be stored for 42 d at 4 °C without spoilage; the use of HPP in combination with either pediocin or nisin extended the shelf-life of the beef to 84 d at 4 °C.

Effect of pH on pressure-induced inactivation of spores

Acidic conditions usually enhance HPP-induced inactivation of vegetative microorganisms (Alpas and others 2000; Smelt and others 2002). Treatment of spores by pressure at low pH has been shown by different authors to either enhance (Roberts and Hoover 1996) or have little or no effect on inactivation (Wuytack and Michiels 2001). Roberts and Hoover (1996) found spores of B. coagulans to be more sensitive to inactivation at 400 MPa suspended in buffer at pH 4.0 than at pH 7.0. Stewart and others (2000) showed C. sporogenes spore numbers were reduced by 2.5 log10 following treatment at 404 MPa for 30 min at 25 °C when the suspending medium was at pH 4.0, whereas a reduction of <0.5 log10 was achieved at neutral pH. In the case of spores of B. subtilis treated at pressures of 100 to 600 MPa at 40 °C, Wuytack and Michiels (2001) found that an acidic environment did not increase inactivation of spores over the pH range 3.0 to 8.0; however, higher levels of inactivation were achieved when spores were HPP-treated at neutral pH and then exposed for 1 h to low-pH conditions. It was concluded that for efficient spore inactivation in acid products, it is better to acidify after pressure treatment, as low pH during HPP treatment inhibits pressure-induced germination; however, the germinated spores are more sensitive post-pressure treatment to the lethal or bacteriostatic effects of acid (Wuytack and Michiels 2001).

If HPP-induced inactivation of spores is to be regarded as a 2-step process, namely, germination followed by inactivation of germinated cells, the pH sensitivity of spore germination can influence this inactivation. Nutrient-induced germination and pressure-induced germination are favored by neutral pH conditions and germinated cells are subsequently sensitized to acidic conditions (Wuytack and Michiels 2001). The pH of the sporulation medium can also have an effect on pressure-induced inactivation; for example, B. cereus spores sporulated at pH 6.0 are more resistant to pressures up to 600 MPa than those sporulated at pH 8.0 (Oh and Moon 2003).

Application of pressure cycling

Pressure-induced germination and subsequent sensitization of the resultant germinated cells has prompted the evaluation of cycling or oscillatory pressure treatments. The use of pressure cycling generally results in enhanced inactivation of microorganisms, compared to a single pressure exposure applied for an equivalent period of time. For example, a single exposure treatment at 600 MPa for 60 min at 70 °C reduced B. stearothermophilus spore counts by 4-log10 CFU/mL, whereas an oscillatory treatment of 6 × 600 MPa cycles of 5 min each at 70 °C gave a 6-log10 reduction (Hayakawa and others 1994). Furukawa and others (2003) compared continuous and cycled pressure treatments for inactivation of spores of B. subtilis where the continuous treatment was comprised of a single 30-min holding time and the cycled treatment consisted of six 5-min cycles of pressurization, holding, and decompression. Both inactivation and injury of spores was higher for the cycled treatment than that from continuous application of pressure. Damage to spore structures, such as spore coats and cortex, was evident from scanning and transmission electron micrographs of the cycled pressure-treated spores. Cycled pressure treatment also enhanced the release of DPA, especially at higher temperatures and pressures.

Other investigators have used an approach of low pressure followed by high pressure. For example, Gola and others (1996) applied a double-pulse treatment of 200 MPa for 1 min followed by an exposure of 900 MPa for 1 min, both at 20 °C, and measured inactivation of 4 × 105 spores/mL of B. stearothermophilus in phosphate buffer. Spores of C. sporogenes were shown to be resistant to treatment at 600 MPa for 30 min at 20 °C by Mills and others (1998), but treatment at 60 MPa at 40 °C or a double-pulse treatment of 60 °C followed by treatment at 400 MPa at 60 °C was more effective, resulting in 3-log10 inactivation. In another study, Lopez-Pedemonte and others (2003) subjected B. cereus in cheese to a germination cycle of 60 MPa at 30 °C for 210 min, followed by an inactivation cycle at 300 or 400 MPa at 30 °C for 15 min, and measured 2.4-log10 of spore inactivation.

Significance of composition of the suspending medium

The suspending medium of spores can have a significant effect on germination, as many foods are rich in germinant nutrients. In addition, some foods, such as milk, offer a protective effect to vegetative cells (Hauben and others 1998; Black and others 2007a); however, the effect of suspending medium on pressure-induced inactivation of spores is not as clear. Moerman and others (2001) showed that neither a fat-rich meat medium nor a carbohydrate-rich potato medium conferred extra baroprotection to Bacillus spores as compared to less organically rich environments. Reddy and others (2003) compared inactivation of C. botulinum by HPP in a buffer to that in crabmeat and found that the meat offered no protection against lethal effects; however, van Opstal and others (2004) found levels of germination to be greater in milk than in buffer, and also that survival of spores of B. cereus was 1 log10 greater at 300 or 600 MPa in milk compared to phosphate buffer, suggesting that although spores germinated readily in milk under pressure, vegetative cells were also somewhat protected from the lethal effects of pressure by the constituents of milk.

A number of studies have considered how water activity (Aw) affected the inactivation of bacterial spores by HPP. In an early study, Timson and Short (1965) found spores of B. subtilis to be protected from pressure effects by glucose and NaCl. Later, Taki and others (1991) found that high concentrations of glucose (10%), sucrose (15%), or glycerol (12%) decreased efficiency of spore inactivation by HPP. Raso and others (1998b) reported that lowering the Aw of the suspending medium to 0.92 by the addition of sucrose increased the resistance of B. cereus spores to inactivation at both 690 and 250 MPa. The resistance of spores to HPP at low Aw can, at least in part, be attributed to incomplete germination in the absence of water.

Conditions of sporulation can also influence the pressure resistance of spores. Raso and others (1998a) and Igura and others (2003) found spores prepared at 20 or 30 °C more resistant to pressure inactivation than those sporulated at > 30 °C. Raso and others (1998a) used 690 MPa at 60 °C for 30 s to inactivate spores of B. cereus, and Igura and others (2003) used a pressure maximum of 300 MPa at 55 °C for 30 min on spores of B. subtilis. The increased pressure resistance with lower sporulation temperature is the opposite as found for wet heat resistance (Nicholson and others 2000; Melly and others 2002).

Spores that are demineralized (such as Ca2+ or Mg2+ replaced by protons) are more resistant to pressure inactivation. One suggested reason for this observation is the involvement of these ions in the activation of cortex-lytic enzymes in germination (Igura and others 2003).

Pressure-assisted thermal sterilization

Whereas food products (mainly refrigerated) produced using high-pressure pasteurization are increasingly available on the international market, the commercial production of low-acid, shelf-stable foods has yet to become a reality. Although attempts have been made to formulate a Tyndallization process, applying pressures of 50 to 400 MPa, to germinate bacterial spores followed by pasteurization by high pressure or by high temperature, it is unlikely that this process will become a commercial reality as typically a small fraction of the spore population does not germinate and survives the pasteurization treatment. A more promising route seems to be the use of pressure-assisted thermal sterilization (PATS; Wilson and Baker 1997; Meyer and others 2000). PATS is a combined process in which both pressure and temperature contribute to the inactivation of spores and enzymes. Pressure alone has a remarkable ability to inactivate vegetative forms of bacteria; however, sole use of high pressure has been found to have little effect on bacterial spores and various enzymes. Of course, spores are a great concern to the food industry, specifically in regards to Bacillus species and Clostridium species, especially C. botulinum. In the production of low-acid, shelf-stable foods, microbiological safety is the single most important prerequisite, with spores of C. botulinum being the critical target for elimination. By combining heat treatment of the product with application of high pressures, inactivation of spores and enzymes can be achieved (Rovere and others 1998). Spore lethality from PATS can therefore be considered a combination of protein denaturation and enzyme inactivation by heat with the aggregation of proteins under pressure (Rodriguez and others 2004).

Of course, this dual process application cannot be considered a nonthermal process, but the heat treatments can occur at significantly lower temperatures, due to the efficiency of heat treatments under pressure and the capacity of pressure to slow down the D values at 95, 105, and 110 °C for the inactivation of spores of B. amyloliquefaciens, but at reaction kinetics. The PATS process can effectively eliminate microbial presence, both vegetative and spore, while producing a product quality that is superior to that of conventional thermal preservation techniques that are used today (Matser and others 2004).

In the compression heating of water, there is an increase of approximately 3°C for every increase of 100 MPa (Balasubramaniam and others 2004). The adiabatic compression characteristics of water were first defined by Bridgman (1912); however, all compressible substances increase in temperature during physical compression (Ting and others 2002). In most foods water is the major component so adiabatic heating is similar to water, except for foods high in fats and alcohol. These foods can demonstrate compression heating approaching 9 °C/100 MPa (Rasanayagam and others 2003). This heat can be integrated into HPP (adiabatic heating or compression heating) and initial product temperature. PATS processes utilize a high initial product temperature (60 to 90 °C) and use thermodynamically induced adiabatic heating to further raise the temperature of the product rapidly and homogeneously. The process temperature for HPP technology is dependent not only on the initial temperature of the product but also the temperature increase/decrease due to adiabatic compression (at the maximum pressure achieved). The temperature increase from adiabatic compression is reversible, so pressure release brings instantaneous cooling. A clear advantage of PATS as compared to traditional heat processing is the theoretical rapid and homogeneous temperature change (heating and cooling) of the product without dependence on product size. In reality, potential heat transfer effects may alter this homogeneous temperature change due to differences between the food product and the processing environment. For example, Balasubramanian and Balasubramaniam (2003) found that target pressure, holding time, product compressibility, initial treatment temperature, and the rate of heat loss to surroundings all influenced inactivation of spores of B. subtilis.de Heij and others (2003) added that heat loss during pressure treatment was a critical factor affecting spore elimination by PATS. In fact, lack of the ability to control temperature during the entire length of treatment may cause the tailing seen in inactivation curves (Ting and others 2002). For reasons such as these, the commercialization of PATS will be dependent on the development of coordinated multidisciplinary programs to assess process efficacy and facilitate the successful development and commercial implementation of this technology.

Inactivation of spores of C. botulinum using PATS

Much of the food industry regulations and sterilizing techniques revolve around the elimination of spores of C. botulinum. Unfortunately, spores of C. botulinum are extremely tolerant to combinations of high pressure and heat. Effective temperature and pressure levels for log10 reductions of these spores can range from 60 to 121 °C and reach approximately 800 MPa (Hendrickx and Knorr 2001). Reddy and others (2003) found that treatment at 827 MPa and 75 °C for 15 min reduced spores of C. botulinum type A by 3.2 log10 CFU/mL. In a more recent study, Reddy and others (2006) treated C. botulinum type B (non-proteolytic) spores with similar pressure and temperature parameters. Four strains were treated in both phosphate buffer and crabmeat, and while 1 strain (C. botulinum KAP8-B) was found to be more sensitive than the others, a 6-log reduction was achieved by treatment at 827 MPa and 75 °C for 30 min for the other 3 strains. Crabmeat conferred no protection to the spores.

A large variation in responses to PATS has been found among C. botulinum spores. Reduction after treatment at 600 MPa and 80 °C ranged from a 5.5-log10 reduction to no reduction at all (Margosch and others 2004a). Heat resistance of spores did not seem to correlate with pressure resistance. It was observed that on combined heat/pressure treatments of C. botulinum where 5-log10 of spores were inactivated, DPA was released; retention of DPA was directly correlated with resistance of C. botulinum spores. Rodriguez and others (2004) also used 80 °C to inactivate spores of C. botulinum; a reduction of 4-log10 spores was realized following a 15-min exposure concurrent with 101.3 MPa.

Ahn and others (2007) demonstrated that a PATS treatment at 700 MPa and 121°C for 1 min inactivated 7 to 8 logs of 2 other clostridial species, C. sporogenes and C. tyrobutyricum, suspended in water. Commercial spore strips of C. sporogenes inserted into egg patties were used as surrogates of C. botulinum by Koutchma and others (2005) to verify PATS treatments of 690 MPa and 100 to 121 °C for 3 to 5 min in a 35-L pilot-scale high pressure unit. A holding time of 4 min at 690 MPa and 110 °C was sufficient to inactivate the spores on the indicator strips.

Overall, inactivation data of C. botulinum spores support the potential of PATS as a process, although variations in resistance among different spore populations seem to skew inactivation results. Survival curves seem to depend highly on which strain and specific organism are targeted. Optimum levels of pressure and temperature need to be established to determine the most efficient and consistent kill rates.

Inactivation of spores of Geobacillus and Bacillus using PATS

Spores of Bacillus amyloliquefaciens have recently been described as the most pressure-resistant bacterial spores of relevance to food processing and have been the target organisms of some recent PATS studies (Margosch and others 2006; Rajan and others 2006a; Ahn and others 2007). Of the 5 sporeforming species studied by Ahn and others (2007), Thermoanaerobacterium thermosaccharolyticum and B. amyloliquefaciens were found to be most resistant to treatments up to 700 MPa and 121 °C, resulting in a 4.5-log10 reduction of spores suspended in water. Scurrah and others (2006) examined the resistance of spores from 40 isolates (8 different species of Bacillus primarily from dairy sources) in skim milk at 600 MPa and 75, 85, and 95 °C for 1 min. In this study, spores of B. sphaericus were the most resistant of those examined, although B. amyloliquefaciens was not studied. Optimum parameters for a 6-log10 reduction of spores of B. subtilis were found by Gao and others (2006a) to be 579 MPa and 87 °C for 13 min, results that correlated well with their predicted values using response surface methodology (r2= 0.9384).

A further target organism of PATS is the highly heat-resistant Geobacillus stearothermophilus, a nonpathogenic species commonly used in establishing thermal sterilization. Gao and others (2006b) reported that a 6-log10 reduction of spores of G. stearothermophilus was achieved at 625 MPa and 86 °C after 14-min treatment in a milk buffer. Patazca and others (2006) compared conventional thermal treatment of G. stearothermophilus spores to PATS in water and found that thermal resistance at 0.1 MPa did not correlate to thermal resistance at high pressure. The DT,P values (decimal reduction time at constant pressure) for 700 MPa were 108.8, 76 and 51.3 s at 92, 100, and 111 °C, respectively, compared to the D value for atmospheric pressure at 121.1 °C which was 5.5 min.

Spore stabilization at high temperature and high pressure

The phenomenon of spore protection or stabilization at high temperature by high pressure has been widely reported by PATS researchers (Rajan and others 2006a; Margosch and others 2006; Patazca and others 2006; Ahn and others 2007). For example, Rajan and others (2006a) reported that an increase in pressure to 700 MPa reduced the D value at 95, 105, and 110 °C, but at 121 °C the contribution of pressure to spore inactivation was less pronounced. Pressures up to 1400 MPa and temperatures up to 120 °C were used by Margosch and others (2006) to study the PATS resistance of spores of C. botulinum and B. amyloliquefaciens. A heat treatment of 100°C alone inactivated mores spores than treatment at 600 or 800MPa and 100 °C or above. Tailing in spore inactivation curves has also been observed in some PATS studies (Margosch and others 2006; Ahn and others 2007), which may be a symptom of pressure stabilization of spores or superdormancy in the spore population.

Other process treatment combinations with HPP

Various combinations of food preservative methods and factors with HPP have been examined in pursuit of an effective nonthermal hurdle approach to render low-acid foods that are shelf-stable. This pursuit continues. Again, bacterial spores are the major issue; HPP alone does not reliably inactivate spores, so elevated temperatures, preservatives, low pH, and refrigerated storage have been required in some part to deal with surviving spores. Used alone, other such nonthermal food processing methods, such as irradiation, pulsed-electric fields (PEF), and ultrasound, have all been equally ineffective against bacterial spores, but have been evaluated in combination with HPP in an effort to find a potential effective synergy (Ross and others 2003).

Sporicidal effects in the combination of HPP and irradiation were found to be additive for inactivation of spores of C. sporogenes. Crawford and others (1996) gave mild treatments of irradiation prior to pressure treatment to demonstrate improved inactivation compared to either process alone. Simultaneous treatments of irradiation and pressure together demonstrated additive spore inactivation of the 2 processes (Gould and Jones 1989). To date, commercial development of a food processing method employing HPP and irradiation does not seem warranted based on current data.

Combination of HPP and PEF also does not appear to hold promise for spore inactivation as a food processing approach. Pagan and others (1998) tried germination of Bacillus spores with HPP followed by inactivation of sensitive germinated cells with PEF in order to inactivate spores. While spore inactivation was demonstrated, the effect was not substantial enough to motivate further investigation.

The combination of ultrasound (20 kHz, 117 μm) and pressure (200 to 600 kPa) to inactivate microorganisms is called manosonication; when heat is added (>50 °C), it is called manothermosonication (Ross and others 2003). Microorganisms can be inactivated using either process. Spores of B. subtilis can be reduced with treatment by manothermosonication, but the degree of inactivation is somewhat limited and the protective effect of foods may be a factor to further reduce effectiveness of the treatment (Raso and others 1998c).

Supercritical CO2 extraction or carbon dioxide gas applied under modest pressures (in the kPa range) has demonstrated microbial inactivation (Kamihara and others 1987; Haas and others 1989). Used with fluid foods, the greatest degree of inactivation is against vegetative forms of bacteria. While bacterial spores have been susceptible, the extent of inactivation is not significant enough to merit commercial development as a sporicidal process.

Inactivation kinetics of bacterial spores by HPP

Modeling of the pressure inactivation kinetics of vegetative forms of bacteria has received far more attention than pressure inactivation kinetics modeling for bacterial spores. One obvious reason is that vegetative forms of bacteria can be pressure-inactivated at ambient and below-ambient treatment temperatures, while spores require the assistance of heat. For potential food processing applications, pressure inactivation of spores normally takes place in the range of 40 to 100 °C, making pressure modeling of spore inactivation a joint exercise with thermal processing as well. Since the 1920s, thermal inactivation of spores has been characterized as a first-order reaction (that is, linear) with standard use of D and z values to explain thermal resistances and predict treatment completion. Application of first-order kinetics often has prevailed even when nonlinear inactivation kinetics were indicated, and this may be with good reason as first-order kinetics has worked remarkably well for decades in predicting thermal inactivation of spores for the safety of low-acid retorted products.

In one of the first kinetic studies involving pressure and spores, Clouston and Wills (1970) identified the initiation of germination and inactivation of spores of Bacillus pumilus E 601 as a first-order process. This work was conducted in phosphate buffer at 25 °C and a rather low 80 MPa; however, the spores were heat-activated (80 °C/15 min) and held 1 wk in buffer at 4 °C prior to pressurization.

More recent studies address the nonlinear inactivation kinetics that is often observed. Ananta and others (2001) examined spores of B. stearothermophilus ATCC 7953 and applied the nonlinear regression method based on nth order kinetics to data obtained for spores in mashed broccoli and cocoa mass which worked well in predicting inactivation when initial spore counts were in the range of 3 × 106 to 7 × 107 CFU/g.

The response of Geobacillus stearothermophilus ATCC 10149 to combination treatments of 500 to 700 MPa and 92 to 111 °C for 0.01 to 360 s in distilled water was evaluated by Patazca and others (2006). The log-linear regression model was found to best-fit data from spore inactivation curves. Resistance of spores to temperature and pressure was characterized with zT and zP values and compared with their resistance to conventional steam heating. It was found that conventional thermal resistance did not correlate to the thermal resistance at high pressure for spores from this strain of G. stearothermophilus.

In their PATS inactivation study of spores of B. stearothermophilus ATCC 7953 in egg patties, Rajan and others (2006b) found the Weibull model for nonlinear inactivation kinetics to best describe spore inactivation at 700 MPa and 105 °C; however, traditional thermal inactivation of spores at 121 °C followed conventional first-order kinetics. Also, D values, applied to nonlinear inactivation from PATS, were not significantly different between spores suspended in distilled water or inoculated into egg patties. The Weibull model also worked well in a similar PATS protocol with egg patty mince in the evaluation of inactivation of spores from B. amyloliquefaciens Fad 82 (Rajan and others 2006a). Rajan and others (2006a) noted that in these applications the Weibull model has additional advantages in mathematical simplicity and accuracy in predicting tailing. It was also added that as the treatment temperature increased to 121 °C (and 700 MPa), pressure contributed less to spore lethality than at lower treatment temperatures of 95 to 110 °C.

In light of temperature fluctuations during pressure treatment (PATS), de Heij and others (2003) suggested a model incorporating process parameters, pressure equipment material and dimensions, characteristics of the product, and the target microorganisms. The 2-step approach involves calculation of temperature distribution by heat conduction in the vessel using an axi-symmetric 1-dimensional finite element model, followed by use of the Eyring–Arrhenius equation to calculate spore inactivation as a function of time, product temperature, and pressure magnitude.

From these examples taken from the literature, it becomes obvious that predictive modeling of spore inactivation kinetics involving pressure has been investigated and developed for commercial application. A variety of models have been applied under different process situations, different spore types, and different foods. Based on this information, the ability to validate and verify PATS processes and pressure/heat pasteurization processes to control spores of spoilage agents or foodborne pathogens such as C. botulinum in commercial products is available.

Fungi—The “Other Spores”

Many studies on the application of HPP treatment have focused on the inactivation of pathogenic microorganisms (Patterson and others 1995; Kalchayanad and others 1998b; Bull and others 2005) and sporeformers as discussed earlier in this review. The effects of HPP on spoilage yeasts have received little attention and those of filamentous fungi even less. In general, yeasts and fungi are considered to be relatively sensitive to HPP; however, the ascospores of heat-resistant molds present a particular challenge, as these ascospores are also resistant to HPP.

Factors affecting pressure inactivation of yeasts and fungal conidia

Pressure-induced inactivation of yeasts and molds in fruit juice was reported by Ogawa and others (1990), who examined the effects of pH, juice concentration, and organic acids on inactivation of 4 yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula anomala, and Rhodotorula glutinis) and 2 mold species (Aspergillus awamori and Mucor plumbeus) in mandarin orange juice. The pressures used were in the range of 100 to 600 MPa (reported as 1000 to 6000 bar). These authors found that the test organisms were effectively inactivated by pressures above 300 MPa for 10 min in single-strength juice, and neither acidic pH (2.5 to 4.5) nor type of organic acid (malic, tartaric, lactic, or acetic) affected inactivation; however, some protective effect was observed in juices concentrated to 20 degrees Brix (°Bx), with the protection increasing with increasing sugar concentration to 40 °Bx, the highest concentration tested.

Ogawa and others (1992), Oxen and Knorr (1993), and Hashizume and others (1995) also reported that pressure resistance of fungi increased as sugar concentration in the media increased. Oxen and Knorr (1993) observed that for Rhodotorula rubra pressure-treated at 400 MPa for 15 min, baroprotection started at Aw values between 0.92 and 0.88, irrespective of solute (sucrose, fructose, glucose, or sodium chloride), and that the effect was pH-independent.

Similar observations were also reported by Palou and others (1997) for the preservative-resistant yeast, Zygosaccharomyces bailii. Using sucrose solutions between Aw 0.998 and 0.90 at pH 5.6, they demonstrated that in sucrose solutions below Aw 0.98 Z. bailii was protected from a treatment of 354 MPa at 21 °C for 5 min. As the Aw of the treatment medium decreased, the number of surviving Z. bailii cells increased, with less than 1-log10 inactivation at Aw 0.92, 0.91, and 0.90. A linear relationship between survival of Z. bailii and the water activity depression factor (1 to Aw) after HPP at 354 MPa for 5 min was reported (Palou and others 1997).

Baroprotection of both yeast cells and mold conidia by sucrose, NaCl, and glycerol was reported by Goh and others (2007), who showed that treatment at 600 MPa for 30 s at Aw 0.90 with NaCl used as the solute provided better protection for conidia of Penicillium expansum and Fusarium oxysporum than did similar treatments in sucrose or glycerol, but at Aw 0.86, all 3 solutes provided a comparable level of protection. For S. cerevisiae, sucrose solutions provided the greatest protection against pressure treatment, with NaCl being the least protective. These results may indicate differing mechanisms of inactivation for the cells of yeasts compared with mold spores.

Hocking and others (2006) investigated the pressure resistances of yeasts and molds responsible for spoilage of fruit and fruit products. Two ascosporic yeasts (S. cerevisiae and Pichia anomala) and 2 conidial fungi (P. expansum and F. oxysporum) were treated up to 400 MPa for periods from 15 to 120 s in an acidified sucrose solution (20°Bx, pH 4.2). Fungal conidia were inactivated more rapidly than the yeast suspensions (containing both ascospores and vegetative cells); there were 5- to 6-log10 reductions in 60 s for the conidia as compared to 3- to 4-log10 reductions for the 2 yeast species. The death curves were nonlinear for both yeasts and molds.

In a more detailed approach, Zook and others (1999) calculated the D and z values after pressure treatment of suspensions of 0.5 to 1.0 × 106 ascospores of S. cerevisiae per milliliter in single-strength orange and apple juices, and a simulated juice [containing (w/v) citric acid (0.8%), sucrose (5%), glucose (2.5%), and fructose (2.5%)] buffered to pH values from 3.5 to 5.0 at pressures ranging from 300 to 500 MPa. The authors ensured that compression heating was not a complicating factor in their results and kept the pressure chamber at a temperature of 5°C. They found that pH had no effect on survival, obtaining similar results for all the juice treatments. D values ranged from D300 MPa of 7.2 to 10.8 min and D500 MPa of 0.14 to 0.18 min; values ranged from 115 MPa for apple juice to 121 MPa for the simulated juice (pH 4.0).

The studies described above were carried out at ambient temperature with the assumption that compression heating would be insufficient to affect inactivation. The combined effects of heat and pressure on inactivation of yeasts were reported by Chen and Tseng (1997) and Donsì and others (2003). As expected, yeasts were inactivated more readily by combinations of pressure with heat than by either treatment alone. Chen and Tseng (1997) reported an additive effect, whereas Donsì and others (2003) proposed more complex inactivation kinetics and attempted to model the combined treatments. Donsì and others (2003) also showed that yeast cells in the exponential phase of growth were more sensitive to pressure treatment than stationary phase cells.

These studies all indicated that yeasts and molds may be protected from inactivation when pressurized in elevated solute concentrations, but that pH and the presence of organic acids had little effect. Other factors that may affect pressure resistance include the physiological state of the cell and the temperature during pressure treatment.

Mechanisms of pressure-induced inactivation of yeasts

The mechanisms of pressure inactivation of yeast cells, particularly S. cerevisiae, have been reported by Abe (2004) and Palhano and others (2004). Pressure interferes with cell membranes and cellular architecture, disrupting microstructures such as microtubules, actin filaments, and nuclear membranes. Proteins may be denatured or polymerized. At nonlethal treatment pressures, yeast cells exhibit a general stress response, producing heat stress proteins similar to those produced in response to temperature stress. The gene expression patterns are those of a general stress response profile, with genes involved in stress defense and carbohydrate metabolism being upregulated.

Ascospores, heat-resistant fungi, and HPP

Heat-resistant molds present significant challenges for the food industry, particularly for acid foods where a mild heat treatment is required. Species such as Byssochlamys fulva, Byssochlamys nivea, Neosartorya fischeri, and Talaromyces macrosporus frequently cause spoilage of heat-processed fruit products, and occasionally dairy products as well (Pitt and Hocking 1997). These fungi produce resistant structures know as ascospores. Ascospores are resistant to heat, pressure, and desiccation, and (like bacterial endospores) are structured to withstand unfavorable conditions in their natural environment, which for these fungi is soil. Fungal ascospores have been studied far less than bacterial spores and, as a consequence, our understanding of the mechanisms of activation and inactivation for fungal spores is relatively poor. The vegetative cells (conidia) of heat-resistant molds are not particularly resistant to pressure, showing sensitivity similar to yeasts (Karatas and Ahi 1992; Butz and others 1996; Voldřich and others 2004). A number of studies have shown that ascospores are resistant to the range of pressures that may be applied during HPP treatment of foods and beverages (300 to 800 MPa).

Factors affecting pressure resistance of ascospores

The efficacy of pressure treatments is affected by a number of factors, including suspension medium (such as buffer versus fruit juice or concentrate), Aw, temperature at which pressure is applied, and the physiological state of the ascospores. pH over the range of 3.0 to 6.0 appears to have little influence on the effects of pressurization (Reyns and others 2003; Chapman and others 2007).

Maggi and others (1994) reported that ascospores of Byssochlamys, Neosartorya, and Talaromyces were more sensitive to pressure treatment (700 MPa) in distilled water than in apricot nectar. At 20 °C, a pressure treatment of 900 MPa for 20 min inactivated Talaromyces flavus ascospores, but only caused a 2-log10 reduction in ascospores of N. fischeri and had no effect on B. fulva or B. nivea ascospores. When the apricot nectar was preheated to 50 or 60 °C, complete inactivation of ascospores was achieved for all species in 1 to 4 min at 800 MPa (50 °C) or 1 to 2 min at 700 MPa (60 °C).

Butz and others (1996) also examined effects of temperature–pressure combinations on ascospores in various matrices from physiological saline solution to concentrated sucrose solution (Aw 0.89) and confirmed the observations of Maggi and others (1994), who reported that reduced Aw was protective. B. nivea was the most resistant of the fungi studied; 700 MPa at 70 °C for 15 min resulted in a 0.4- to 1.0-log10 reduction (depending on strain), and after 60 min at 70 °C and 700 MPa, a 3.2- to 4.0-log10 reduction was observed. At 60 °C and 600 MPa, ascospores of a particularly pressure-resistant strain of B. nivea were reduced by only 1 log10. These workers also reported that different genera and species, Eupenicillium, Neosartorya (reported as Eurotium), and Byssochlamys, reacted differently to temperature-pressure combinations with each exhibiting a different level of survival at 300 MPa. Eupenicillium sp. was most stable from 40 to 60 °C, B. fulva from 25 to 40 °C, and Neosartorya from 10 to 25 °C.

Pressure combined with heat was shown to be more effective than pressure alone in inactivating ascospores of Talaromyces avellaneus.Voldřich and others (2004) obtained a D600 MPa of 32 min at 17 °C, but at 60 °C D600 MPa was reduced to 10 min.

The efficacy of continuous versus oscillatory pressure treatments for inactivation of B. nivea ascospores was investigated by Palou and others (1998). Ascospores harvested at 30 d were suspended in model fruit juices (apple or cranberry) adjusted to Aw 0.98 and 0.94. Application of continuous pressure at 689 MPa at a starting temperature of 21 °C for up to 25 min resulted in ascospore activation, but the same pressure treatment at 60 °C (equipment and spore suspension preheated to 60 °C) resulted in a slight decrease in ascospore viability with juice at Aw 0.94 affording greater protection. Using oscillatory pressurization (689 MPa, 1-s holding time at pressure) at 60 °C, ascospores were inactivated after 3 to 5 cycles in juice at Aw 0.98 but not in Aw 0.94 juice. The same pressure treatment at 21 °C had no effect, irrespective of the Aw of the juice in which the ascospores were suspended. Scanning electron microscopy revealed deep furrows in the surfaces of ascospores that were correlated with the observed inactivation. Palou and others (1998) proposed that the efficacy of oscillatory HPP treatment was due to spore lysis promoted by increased wall permeability at high temperatures and pressures.

Butz and others (1996) found that for B. nivea ascospores the order in which treatments were applied was significant. Pressure treatment (700 MPa at 60 °C) followed by heating (80 °C, 30 min) resulted in 4-log10 reduction, whereas thermal treatment (80 °C, 30 min) followed by the same pressure treatment was much less effective. Pressure treatment appeared to sensitize ascospores to heat treatment, but the reverse was not the case. Similar observations were made by Reyns and others (2003) for ascospores of T. macrosporus pressure-treated at 700 MPa for up to 60 min, followed by heating at 80 °C for 30 min.

Ascospore age can influence pressure resistance. Chapman and others (2007) pressure-treated ascospores of B. fulva, B. nivea, N. fischeri, and Neosartorya spinosa, aged from 3 to 15 wk, at 600 MPa for 10 min in citrate-phosphate buffer, pH 4 and 6. They observed reduction in counts of 2- to 4-log10 in 3-wk-old ascospores, but by 9 wk, most ascospores showed much lower levels of inactivation, for example, 1-log10 or less. Counts of N. spinosa increased by 1-log10 at 9 wk, and in 15-wk-old ascospores, HPP resulted in greater than a 2-log10 increase in counts. Dijksterhuis and Teunissen (2004) noted that both heat and pressure resistances of T. macrosporus ascospores increased 2- to 4-fold between 20 and 40 d, and a further 1.3-fold increase was observed between 40 to 67 d.

Activation or inactivation?

The phenomenon of heat activation of ascospores from heat-resistant molds is well documented; it appears that HPP can have a similar effect on ascospores. Reyns and others (2003) compared heat activation from pressure treatment of ascospores of T. macrosporus harvested after 35-d incubation at 25 °C. Heat activation (30 min at 80 °C) increased the viable count of the ascospore suspension from 3.7-log10 to 6.1-log10. Pressure-induced activation of these ascospores at pressures of ≥200 MPa was measured with maximum activation occurring after ≤ 2 min at 500 to 600 MPa. Under these conditions, the level of ascospore activation was similar to that obtained with the heat treatment. These activated ascospores appeared to be more sensitive to subsequent heat treatment, as described above, but were also sensitive to dehydration. Although no morphological changes were observed in wet microscopic mounts of pressure-treated ascospores, air-dried and pressurized ascospores showed signs of spore collapse that could not be reversed by wetting.

Mechanisms of activation and inactivation of ascospores by pressure

Reyns and others (2003) suggested that HPP affects the permeability and rigidity of the ascospore wall, rendering the spores more permeable to water. The primary event needed for spore germination is hydration of the dehydrated spore core. This hydration triggers metabolic activity, leading to germination and outgrowth if nutrients are available. Pressurized ascospores were also found to be more sensitive than untreated ascospores to the activity of cell wall-lytic enzymes containing β-1–3 glucanases, providing further support for the theory that HPP causes changes to the ascospore wall structure.

Dijksterhuis and others (2002) investigated heat activation of ascospores of T. macrosporus, and observed that ascospores contain large amounts of trehalose which is broken down to glucose during activation and germination within 2 h of heat activation of ascospores. This process is followed about 3 to 4 h after heat activation by rapid ejection of the protoplast through the ascospore wall with hyphal tip formation occurring at about 6 h. Dijksterhuis and Teunissen (2004) examined the effects of HPP on the same strain of T. macrosporus and found that, although pressure was less effective than heat for ascospore activation, treatment at 600 MPa was optimal for ascospore activation with treatment times of 10 s to 3 min being most effective. HPP caused damage to the ascospore wall with extensive deformation after 60 min at 600 MPa. Dijksterhuis and Teunissen (2004) proposed that this damage to the ascospore wall can cause activation when the physical barrier of the outer cell wall is damaged and an influx of water occurs. As with heat activation, pressure activation of ascospores results in ejection of a protoplast, followed by hyphal tip formation.

Summary of HPP and fungal spores

HPP is an effective process for inactivation of yeasts and the conidia and mycelia of fungi. The efficacy of the process is dependent on the pressure-time combination, with pressures greater than 300 MPa generally required for inactivation. The ascospores of heat-resistant molds are highly resistant to HPP and are not inactivated by pressure treatment normally applied to foods. Combined treatments (heat and pressure) may be more effective, as HPP appears to sensitize ascospores to subsequent heat treatment.

Conclusions

While the spores of both bacteria and fungi have been shown to be heat and pressure resistant, spores of bacteria remain the most difficult problem to eliminate for making HPP-treated low-acid foods stable at room temperature. Eliminating all spores in a low-acid commercial food while maintaining nonthermal processing conditions is not possible at the present time. Foods will contain an assortment of spores that are naturally variable in their responses to germination mechanisms. In addition, the enigmatic state of spore superdormancy makes the prediction of success problematic; occasions of tailing seen in survival curves may be due to superdormant spores somehow germinated following exposure to conditions of HPP. Given the unpredictable nature of spores, dependency on the thermal processing of this category of products will continue. While PATS is a thermal process, it remains the only food process incorporating high hydrostatic pressure that can dependably deliver a shelf-stable, low-acid product.

Acknowledgments

Work in the Setlow laboratory was supported by grants from the Natl. Inst. of Health, the Army Research Office, and the US Dept. of Agriculture. P.S. and D.G.H. wish to acknowledge the support of USDA-NRI grant 2003–35201-13553.

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