Fruits are composed of polysaccharides, sugars, organic acids, vitamins, and minerals, which function as reservoirs or substrates for microbial growth. Fresh fruits exhibit the presence of a mixed microbial population, and the growth rate of each microbial type would depend upon an array of factors listed below.
Intrinsic Factors
These factors are an inherent part of the plant tissues (Mos- sel and Ingram 1955) and are characteristics of the growth substrates. They include the following.
Hydrogen Ion Concentration (pH)
Microbial cells lack the ability to adjust their internal pH, and most microorganisms grow best around neutral pH. Bacte- ria exhibit a narrow pH range, pathogenic bacteria being the most fastidious; yeasts and molds are more acid-tolerant than bacteria. Typically, fruits possess acidic pH (⬍4.0), favoring growth of yeasts and molds. Microbes, in general, experi- ence increased lag and generation times at either extremes of the optimum pH range. However, small fluctuations in pH impact microbial growth rates, and the pH changes become more profound if the substrate has low buffering capabilities, leading to rapid changes in response to metabolites produced by microorganisms (Table 4.1).
The intracellular pH of microbial cytoplasm remains rea- sonably constant due to relative impermeability of cell mem-
Table 4.1. Approximate pH Values of Some Fresh Fruits
Fruits pH Values
Apples 2.9–3.3
Bananas 4.5–4.7
Grape fruit 3.4–4.5
Watermelons 5.2–5.6
Oranges 3.6–4.3
Limes 1.8–2.0
Melons 6.3–6.7
Figs 4.6
Plums 2.8–4.6
brane to hydrogen (H+) and hydroxyl (OH−) ions as key cellular compounds such as ATP and DNA require neutrality (Brown 1964). The pH changes also affect the morphology of some microbes such asPenicillum chrysogenumthat show decreased length of hyphae at pH above 6.0. Corlett and Brown (1980) observed varying ability of organic acids as microbial growth inhibitors in relation to pH changes.
Water Activity (Moisture Requirement)
Water is a universal constituent required by all living cells, and microbes are no exceptions, but the exact amount of water required for growth of microorganisms varies. Hence, several preservation methods involve drying or desiccation of the produce (Worbo and Padilla-Zakour 1999). The water requirement of microbes is defined as water activity (aw) or ratio of water vapor pressure of food substrate to that of vapor pressure of pure water at the same temperature,
aw= p po,
wherep is the vapor pressure of the solution andpo is the vapor pressure of the solvent.
Christian (1963) related water activity (Table 4.2) to rela- tive humidity (RH) as
RH=100×aw.
Table 4.2. Lower LimitawValues of Certain Microorganisms
Bacteria
Minimum
awValues Fungi
Minimum awValues
Pseudomonas 0.97 Mucor 0.62 (0.94)
E. coli 0.96 Rhizopus 0.62
Staphylococcus aureus 0.86 Botyritis 0.62 Bacillus subtilis 0.95 Aspergillus 0.85 Clostridium botulinum 0.93 Penicillum 0.95 Enterbacter aerogenes 0.94
In general, most fresh produce hasawvalue above 0.99, which is sufficient for the growth of both bacteria and molds;
however, bacteria, particularly gram-negative, are more strin- gent regardingawchanges, while molds could grow atawas low as 0.80. The lowest range ofawfor halophilic bacteria, xerophilic fungi, and osmophilic yeasts is 0.75–0.61. Morris (1962) elaborated the interaction ofawvalues with tempera- ture and nutrition and observed that at optimum temperature, range ofawvalues remain wide, while lowering/narrowing aw values reduces growth and multiplication of microbes, and nutritive properties of substrate increase the range ofaw
over which microorganisms can survive.
Each microbe has a characteristicaw range for optimum growth and multiplication. Theawis affected by temperature, pH, oxygen, nutritive properties of substrate, and organic acids or other secondary metabolites performing inhibitory action. Thus, narrowing theaw range decreases microbial growth (Wodzinsky and Frazier 1961). Lowering of water activity builds up stress and exerts adverse influence on all vital metabolic activities that require aqueous environment.
Charlang and Horowitz (1974) observed the appearance of nonlethal alterations in cell membrane permeability ofNeu- rospora crassa cells, resulting in loss of various essential molecules, as the dynamic cell membrane should remain in fluid state.
The exception to normal awrequirements are halophilic (salt tolerant) bacteria that grow under lowawvalues by ac- cumulating potassium ions in the cell (Csonka 1989), while osmophilic yeasts concentrate polyols as osmoregulators and enzyme protectors (Sperber 1983). Brown (1976) reported proline accumulation in response to lowawin halotolerant Staphylococcus aureusstrains. Xerotolerant fungi accumu- late polyhydric alcohols (Troller 1986). Microbes thus at- tempt to compensate for increased stress by accumulating compatible solutes.
Redox (Oxidation–Reduction) Potential
Microbial growth depends upon oxidation–reduction (O/R) potential of a substrate or its ability to lose or gain electrons.
Aerobic microbes require oxidized (positive Eh values) sub- strate for growth, and it is opposite for the anaerobes (Walden and Hentges 1975). Fruits contain sugars and ascorbic acid for maintaining reduced conditions, though plant foods tend to have positive Eh values (300–400 mV). Hence, aerobic bacteria and molds most commonly spoil fruits and fruit products. The O/R potential of food can be determined by
r pH of food, r Poising capacity,
r Oxygen tension of the atmosphere, r Atmospheric access of food.
Poising capacity alters the ability of the living tissues to metabolize oxygen at specifically low Eh values that ex- ist in the vacuum-packed foods. Aerobic microbes include
bacilli, micrococci, pseudomonas, and actinobacters and require positive Eh values, while anaerobes such as clostridia and bacteriodes require negative Eh values. However, most yeast and molds are aerobic and few tend to be facultative anaerobes. In the presence of limited amount of oxygen, aerobic or facultative microbes may produce incompletely oxidized organic acids. Pasteurization of fruit juices would render microbes lose reducing substances, but this would not limit yeast growth.
Available Nutrients
Each microbe has a definite range of food requirements, with some species having wide range and ability to grow on a va- riety of substrates, while others having narrow range and fas- tidious requirement, allowing growth on limited substrates.
Fruits are a reservoir of sugars (source of energy), water, min- erals, vitamins, and other growth-promoting factors, but the protein content or nitrogen source is not as high. Microorgan- isms have varied nutrient requirements, which are influenced by temperature, pH, and Eh values. The microbes become more demanding at reduced temperatures, while at optimum temperature, nutrients control microbial growth. Thus, pecti- nolytic bacteria such as Erwinia cartovora, Pseudomonas sp., or pectinolytic molds grow best on fruits and vegetables.
Nitrogen requirement is usually met by proteolysis of pro- tein present in substrates, use of amino acids, nucleotides, cer- tain polysaccharides, and fats under usual microbe-specific conditions. The accessory food substances or vitamins are to be furnished by substrate since microorganisms are unable to synthesize essential vitamins. In general, gram-positive bac- teria are least synthetic and require supply of certain vitamins before growth, while gram-negative bacteria and molds are relatively independent and can synthesize most of the vita- mins. Thus, these two groups of microbes grow profusely on foods relatively low in B-complex vitamins, such as fruits under the influence of usual low pH and positive Eh values.
Antimicrobial Factors
Certain naturally occurring substances in food work against the microbes, thus maintaining stability of food; however, these are directed toward a specific group of microorgan- ism and have weak activity. Song et al. (1996) reported that the presence of aroma precursor hexal readily gets converted to aroma volatilesin vivo by fresh-cut apple slices. Hexal acts as antibrowning agent as well as inhibits growth of molds, yeasts, mesophilic, and psychrotropic bacteria (Lan- ciotti et al. 1999). Hexanal and (E)-hexenal in modified at- mosphere packaging (MAP) of sliced apples reduce spoilage microbe populations (Corbo et al. 2000).
Spices contain essential oils such as eugenol (clove), al- licin (garlic), cinnamic aldehyde and eugenol (cinnamon), allyl isothiocynate (mustard), eugenol and thymol (sage),
thymol, and isothymol (oregano) that have antimicrobial ac- tivity (Shelef 1983). Buta and Molin (1998) observed re- duction in mold growth on fresh-cut peppers by exogenous application of methyl jasmonate.
The antimicrobial compounds may originally be present in food, added purposely or developed by associated micro- bial growth, or by processing methods. Certain antifungal compounds applied to fruits include benomyl, biphenyl, and other phenylic compounds that exist in small quantities as by-product of phenol synthesis pathways. Beuchat (1976) observed that essential oils of oregano, thyme, and sassafras have bacteriocidal activity, at 100 ppm, toV. parahaemolyti- cusin broth, while cinnamon and clove oils at 200–300 ppm inhibit growth and aflatoxin production byAspergillus para- siticus(Bullerman et al. 1977). The hydroxy-cinnamic acid derivatives such asp-coumaric, ferulic, caffeic, and chloro- genic acids and benzoic acid in cranberries have antibacterial and antifungal activities and are present in most plant prod- ucts including fruits.
Extrinsic Factors
Extrinsic factors imposed from the external environment dur- ing storage can affect food and the microbes that tend to develop on it. These factors include the following.
Temperature
Microbes grow over a wide range of temperature, and change in temperature at both extremes lengthens the generation time and lag periods. The temperature required for microbial growth varies from –34◦C to 90◦C. The following is the list of microbes based on temperature requirements.
1. Psychrotrophs: These microorganisms can grow well at 7◦C or below, with temperature optima ranging from 20◦C to 30◦C. For example,Lactobacillus,Micro- coccus,Pseudomonas,Enterococcus,Psychrobacter, Rhodotorula,CandidaandSaccharomyces(yeasts), Mucor,Penicillum,Rhizopus(molds) andClostridium botulinum,L. monocytogenes,Y. enterocolitica, and Bacillus cereus(pathogenic psychrotrophs). The groups of microbes, which grow from –10◦C to 20◦C with the optima of 10–20◦C, are included asPsychrophilesand include certain genera mentioned above.
2. Mesophiles:These include microbes growing best be- tween 20◦C and 45◦C, with optimum range of 30–40◦C.
For example,Enterococcus faecalis,Streptococcus, Staphylococcus, andLeuconostoc.
3. Thermophiles:Microbes, which grow well above 45◦C with the optima ranging between 55◦C and 65◦C and with maximum of above 60–85◦C, are known as ther- motolerant thermophiles. For example,Thermussp.
(extreme thermophile),Bacillus sternothermophilus, Bacillus coagulans, andClostridium thermosaccha-
rolyticumare endospore-forming thermotolerants and grow between 40◦C and 60◦C and create major prob- lems in the canning industry.
4. Thermotrophs:This group includes microbes similar to mesophiles but with slightly higher temperature optima and includes pathogenic bacteria in foods. For example, Salmonella,Shigella, enterovirulentE. coli,Campy- lobacter, toxigenicBacillus cereus,Staphylococcus aureus, andClostridium perfringens.The relationship between temperature and growth rate of microorgan- isms between minimum and maximum temperature is given by the following equation:
√r =b(T−T0),
whereris the growth rate,bis the slope of regression line,Tis temperature, andT0is the conceptual tem- perature of no metabolic significance (Ratowsky et al.
1982).
Relative Humidity of Environment
Success of a storage temperature depends on the RH of the en- vironment surrounding the food. Thus, RH affectsawwithin a processed food and microbial growth at surfaces. A lowaw
food kept at high RH value tends to pick up moisture until equilibrium is reached, and foods with highawlose moisture in a low-humidity environment. Fruits and vegetables un- dergo a variety of surface growth by yeasts and molds as well as bacteria, and thus are liable to spoilage during storage at low RH conditions. However, this practice may cause certain undesirable attributes, such as firmness and texture loss.
Modified Atmosphere Storage
Altering the gaseous composition of the environment, which retards the surface spoilage without reducing humidity in- cludes the general practice of increasing CO2 (to 10%) is referred as “controlled or modified atmosphere packaging”
(CAP or MAP). CAP/MAP retard senescence, lower respi- ration rates, and slow the rate of tissue softening or texture loss (Wright and Kader 1997a, Qi et al. 1999, Rattanapanone and Watada 2000). CAP/MAP storage has been employed for fruits (apples and pears) with CO2applied mechanically or as dry ice, and this retards fungal rotting of fruits probably by acting as competitive inhibitor of ethylene action (Wright and Kader 1997b, Gil et al. 1998).
The inhibitory effect increases with decrease in temper- ature due to increase in solubility of CO2 at lower temper- atures (Bett et al. 2001). Elevated CO2levels are generally more microbiostatic than microbiocidal, probably due to the phenomena of catabolite repression. However, an alterna- tive to CO2 application includes the use of ozone gas at low ppm concentrations, which acts as ethylene antagonist as well as a strong oxidizer that retards microbial growth.
Sarig et al. (1996) and Palou et al. (2002) reported control of
postharvest decay of table grapes caused by Rhizopus stolonifera. A similar report on effect of ozone and stor- age temperature on postharvest diseases of carrots was ob- served by Liew and Prange (1994). In general, gaseous ozone introduction to postharvest storage facilities or refrigerated shipping and temporary storage containers is reported to be optimal at cooler temperatures and high RH (85–95%; Gra- ham 1997). The most reproducible benefits of such storage are substantial reduction of spore production on the surface of infected produce and the exclusion of secondary spread from infected to adjacent produce (Kim et al. 1999, Khadre and Yousef 2001).
MAP is the most commonly used for fresh-cut fruits (Jayas and Jeyamkondan 2002, Soliva-Fortuny and Martin-Belloso 2003, Ayhan and Esturk 2009). Timon (2005) reported better product quality and enhanced shelf life of the fresh-cut fruits by using approximately 3–5% O2 and 5–10% CO2 within the package. MAP technique has been used in combination with physical, chemical, or radiation techniques. The texture and quality of fresh fruits packed by MAP technique can be enhanced by treating with essential oils having antimicro- bial properties. A study of fresh sweet cherry fruits revealed that treatment with antifungal essential oils like eugenol, thy- mol, or menthol imparts certain positive benefits on several quality parameters. The treated fruits had less weight loss, color degradation, and texture softness than the control fruits (Serranoa et al. 2005). Aromatic compounds (e.g., hexanal, 2- (E)-hexenal and hexyl acetate) have been reported to have an- timicrobial effects on gram-negative bacteria (Lanciotti et al.
2004).
Implicit Factors
While growing in a food substrate, microorganisms may pro- duce one or more inhibitory substances such as acids, alco- hols, peroxides, and antibiotics that affect the growth of other microorganisms.
General Interference
Normal microflora of fresh produce helps prevent the colo- nization of pathogens and succeeds in overcoming the con- taminant number by overgrowth and efficient utilization of available nutrients.
Production of Inhibitory Substances
Some microbes can produce inhibitory substances, includ- ing “bacteriocins,” for example, “nisin” produced by cer- tain strains ofLactobacillus lactis. As an inhibitor of spore- formingClostridiumspp., which causes cheese blowing due to undesirable gas production, nisin was the first bacteriocin produced by lactic acid bacteria to be isolated and approved for use in cheese spreads. Other bacteriocins produced by lactic acid bacteria include lactococcins, lacticins, lactacins,
diplococcin, sakacins, acidophilocins, pediocins, and leuconosins. Although mostly active against gram-positive bacteria, bacteriocins can be microbiocidal under certain con- ditions, even toward gram-negative bacteria and yeasts. The antimicrobial action of nisin and of similar bacteriocins is believed to involve cell membrane depolarization, leading to leakage of cellular components and to loss of electrical po- tential across the membrane.Propioniobacterium produces propionic acid that has inhibitory effect on other bacteria.
The bacteriocins may be of broad or narrow spectrum depending on the kind of microbe. Narrow-spectrum bacte- riocins selectively inhibit high-risk bacteria in foods likeL.
monocytogeneswithout affecting harmless microorganisms;
this is of importance for extending shelf life of high sugar and high moisture fruits like honeydew melons, berries, ap- ple, etc. (Leverentz et al. 2003). Thus, these peptides have a future as preservatives, shelf-life extenders, additives, or ingredients that could be produced in situ by bacteriocino- genic starters, adjunct, or protective cultures (Galvez et al.
2007). Certain bacteriocins, which are available for com- mercial applications, are nisin, pediocin PA-1/AcH, lacticin 3147, enterocin AS-48 or variacin, and many more. Penney et al. (2004), however, reported that nisin did not prevent growth of spoilage-causing microbes in fruit yogurt made with minimally processed wild blueberries. They advocated application of phytopreservatives such as vanillin as “natural”
antimicrobial agents in minimally processed fruit yogurt.
Certain microorganisms can produce a wide-spectrum an- timicrobial substances or secondary metabolites called “an- tibiotics,” capable of killing or inhibiting a wide range of microbes. Further, growth of one kind of microbe can lower pH and make the environment unsuitable for other microbes to grow. Organic acid production or hydrogen peroxide for- mation can also interfere with the growth of microbial popu- lation (Jay 1992).
Biofilm Formation
Most of the gram-negative bacteria exhibit quorum sensing or a cell-to-cell communication phenomena leading to for- mation of a multicellular structure that provides protection to bacterial species from deleterious environment by precip- itation. Adoption of biofilm formation involves release of autoinducers, particularly called theN-acyl homoserine lac- tones that either activate or repress the target genes involved in biofilm formation (Surette et al. 1999). Quorum sensing can regulate prime events such as spore germination, viru- lence factor production, and biofilm formation on surfaces (Frank 2000b).
Bacterial cells, which have slow growth on biofilm, are more resistant to heat, chemicals, and sanitizers due to the dif- fusional barrier created (Costerton 1995). Morris et al. (1997) reported certain methods for observing microbial biofilms directly on leaf surfaces and also to recover the constituent microbes for isolation of cultivable microorganisms. Biofilm
formation has emerged as a challenge for routine decontam- ination techniques used in the food and beverage industries (Frank 2000a).