FOOD SPOILAGE & PRESERVATION

PICKLING OF VEGETABLES

T.L.V. Peiris

GS/MSC/FOOD/3630/08

2008/2010

CONTENT PAGE

Introduction 01

Production process 01-02

The microflora of fresh vegetables 02-03

Principles of Pickle Production 03-04

Manufacture of fermented pickles 04-06

Pickle fermentation 06-07

How lactic acid bacteria deal with Acid and Salt in a pickle 07-09

Defects 10-11

Food Additives 11-12

References 13

Introduction

Pickling is a global culinary art. If you were to go on an international food-tasting tour, you’d find pickled foods just about everywhere. You might sample kosher cucumber pickles in New York City, chutneys in India, kimchi in Korea, miso pickles in Japan, salted duck eggs in China, pickled herring in Scandinavia, corned beef in Ireland, salsas in Mexico, pickled pigs feet in the southern United States, and much, much more.

What makes a pickle a pickle? On a most general level, pickles are foods soaked in solutions that help prevent spoilage.

There are two basic categories of pickles. The first type includes pickles preserved in vinegar, a strong acid in which few bacteria can survive. Most of the bottled kosher cucumber pickles available in the supermarket are preserved in vinegar.

The other category includes pickles soaked in a salt brine to encourages fermentation—the growth of "good" bacteria that make a food less vulnerable to "bad" spoilage-causing bacteria. Common examples of fermented pickles include kimchi and many cucumber dill pickles.

Pickling is not only an international food-preservation technique; it’s also an ancient one. For thousands of years, our ancestors have explored ways to pickle foods, following an instinct to secure surplus food supplies for long winters, famine, and other times of need. Historians know, for instance, that over two thousand years ago, workers building the Great Wall of China ate sauerkraut, a kind of fermented cabbage.

But pickling foods does much more than simply preserve them. It can also change their taste and texture in a profusion of interesting—and yummy—ways. It’s no surprise that cultures across the globe enjoy such an assortment of pickled foods, as you would discover on your international food expedition. In fact, food experts say, the evolution of diverse pickled foods in different cultures has contributed to unique cultural food preferences, such as spicy sour tastes in Southeast Asia and acidic flavors in eastern Europe.

Production process

In a general sense, fermented vegetable technology is based on the same principles as other lactic acid fermentations, in that sugars are converted to acids, and the finished product takes on new and different characteristics. In reality, however, the actual production of fermented vegetables occurs quite differently. For example, whereas cheese, cultured dairy products, and fermented meats are usually produced using starter cultures, the fermented vegetable industry still relies on natural lactic microflora to carry out the fermentation. Compared to the relatively few strains used for dairy and meat fermentations, the lactic acid bacteria that are ultimately responsible for vegetable fermentations are quite diverse. Several genera are usually involved, including both heterofermentative and homofermentative species.In addition, although the plant-based substrates (i.e., cabbage,cucumbers, olives) ordinarily contain the relevant lactic acid bacteria necessaryto perform a lactic fermentation, they also harbor a complex microflora consisting of

other less desirable organisms. In fact, the resident lactic acid bacteria population represents only a small faction of the total microflora present in the starting material.And unlike dairy fermentations, where pasteurization can substantially reduce the indigenous microflora present in raw milk, no such heating step can be used to produce fermented vegetables.Although chemical pasteurization procedures have been developed for some products and can effectively reduce the resident flora, these applications, for the most part,are not widely employed. Therefore, the essential requirement for a successful fermentation is to create environmental conditions that are conducive for the lactic acid bacteria, butinhibit or otherwise restrict the non-lactic flora.

The microflora of fresh vegetablesPlant material, including edible vegetables, serves as the natural habitat for a wide variety of microorganisms (Table 7–2). The endogenous or epiphytic floras consist of yeast, fungi, and both Gram positive and Gram negative bacteria. The plant environment is exposed to the air and the surfaces of plant tissue have a high Eh. Thus, aerobic organisms, such as Pseudomonas, Flavobacterium, Bacillus, and various mold species, would be expected to dominate freshly harvested material, as is indeed the case. However, facultative anaerobes, including Enterobacter, Escherichia coli, Klebsiella, and other enteric bacteria, as well as sporeforming clostridia, are also part of the resident flora. Various yeasts, including Candida, accharomyces, Hansenula, Pichia, and Rhodotorula, may also be present. Lactic acid bacteria, mainly species belonging to the genera Lactobacillus, Pediococcus, Streptococcus, and Enterococcus, are ordinarily present, but at surprisingly low numbers. In fact, whereas the total population of Pseudomonas,Flavobacterium,Escherichia, and Bacillus may well reach levels as high as 107 cells per gram, lactic acid bacteria are normally present at only about 103 cells per gram.Thus, the lactic acid bacteria are outnumbered by non-lactic competitors by a thousand timesor more, putting them at a serious disadvantage.Given the diversity of microorganisms initially present in the raw material and the numerical disparity between the lactic and nonlactic bacteria, it would seem that rather severe measures must be adopted to establish the selective environment necessary for a successful lactic acid fermentation. Actually, selection is based on only a few simple factors: salt, temperature, and anaerobiosis. Thus, under appropriate conditions, most non-lactic acid bacteria will grow slowly, if at all. In contrast, lactic acid bacteria will generally be unaffected (but not totally,), and will instead grow and produce acidic end products. The acids, along with CO2 that may also be produced, creates an even more stringent environment for would-be competitors. Within just a few hours, lactic acid bacteria will begin to grow, a lactic acid fermentation will commence, and the number of competing organisms will decline.

The lactic acid fermentation that occurs during most vegetable fermentations depends not on any single organism, but rather on a consortium of bacteria representing several different genera and species (Table 7–3). That is, a given organism (or group of organisms) initiates growth and becomes established for a particular period of time. Then, due to accumulation of toxic end products or to other inhibitory factors, growth of that organism will begin to slow down or cease. Eventually, the initial microbial population gives way to other species that are less sensitive to those inhibitory factors. Microbial ecologists refer to these sorts of processes as a succession. This is one reason why vegetable fermentations are ordinarily conducted without starter cultures, since duplicating a natural succession of organisms likely would not be achieved on a consistent basis.

Principles of Pickle ProductionIn a very general sense,pickles refer to any vegetable that is preserved by salt or acid. Certainly, the vegetable most often associated with pickles is the cucumber. The acid found most often in pickled products is lactic acid, derived from a lactic fermentation. however, not all pickles are fermented. In fact, less than half of the pickles consumed in the United States undergo lactic acid fermentation. Rather, acetic acid can be added directly as the pickling acid, omitting the fermentation step. The pickle slice on the top of a fast-food hamburger is probably not the fermented type. In the United States, pickles are generally divided into three different groups, based on their means of manufacture. Fresh-packed pickles are simply cucumbers that are packed in jars, covered with vinegar and other flavorings, and then pasteurized by heat. They

have a long shelf-life, even at room temperature. Fresh packed pickles are crisp, mildly acidic, and are the most popular.Refrigerated pickles are also made by packing cucumbers jars with vinegar and various flavorings, but they are not heated. Instead these pickles are refrigerated, giving them a crisp, crunchy texture and bright green color. Although a slight fermentation may occur, refrigerated pickle shaves a shorter shelf-life than fresh-packed pickles. Sodium benzoate is usually added as a preservative. The manufacture of both fresh-packed and refrigerated-style pickles is fast and easy and requires few steps (Figure 7–4).

The only pickles that are fully fermented are those referred to as salt-stock or genuine pickles. They may also be referred to as processed, although this may be somewhat confusing since non-fermented pickles can be made into relishes and other processed pickle products. Fermented pickles are nearly as popular as fresh-packed pickles. They have a distinctly different flavor and texture compared to fresh packed or refrigerated pickles, and take much longer to make. Fermented or processed pickles also have a very long shelf-life, about two years. Details regarding their manufacture and the fermentation process are described below. It should be noted that within these three groups, pickles can be further distinguished based on the types of spices, herbs and flavoring agents used, the size or type of cucumber (gherkins and midgets), and the form or shape of the pickle (i.e., whole, spear, slice, etc.). For example, dill pickles, the most

popular, refer to any type of pickle to which dill weed (either the seed or oil) is added. If the dill pickles are labeled as genuine dill, it means the pickles are of the processed type and are dill-flavored. Otherwise, dill pickles are usually made from fresh packed or refrigerated pickles. Other common flavored pickle types include sweet, bread-and butter, kosher (style), and garlic.

Manufacture of fermented picklesThe actual process steps used for the manufacture of fermented pickles are similar to those used for making sauerkraut. Both rely on salt, oxygen exclusion, and anaerobiosis to provide the appropriate environmental conditions necessary to select for growth of naturally-occurringlactic acid bacteria. There are, however, several differences between pickle and sauerkraut fermentations. First, salt concentrations are higher than those used for sauerkraut, resulting in thedevelopment of a less diverse microflora. Inaddition, a brine, rather than dry salt, is used forpickle fermentations.Finally,the pickle fermentation process, unlike sauerkraut, is amenable tothe use of pure starter cultures and a more controlled fermentation. Indeed, such cultures arenow available and some (but not many) pickle manufacturers have adopted controlled fermentation processes.The manufacture of fermented pickles starts with selection and sorting of cucumbers (Figure7–5). Only small or immature cucumbers, harvested when they are green and firm, are used for pickles.They are then washed, sorted,and transferred to tanks, and a brine solution is added. The brine typically contains at least 5% salt (or about 20° salometer, where 100° salometer_ 26% salt). Because the cucumbers-to-brine ratio is nearly 1:1, the actual salt concentration isactually less. For so-called salt stock pickles, which may be held in bulk for long periods, theinitial brine may contain 7% to 8% salt, which is followed by the addition of more salt to raise the total salt concentration to above 12%. For genuine dill-type pickles, the brine concentration isusually between 7.5% and 8.5%.Dill weed is also added, usually in the seed or oil form. Care must be taken when weighing down the pickles, because the buoyancy of the cucumbers may cause those at the top to become damaged. The large tanks used by large pickle manufacturers are usually located outdoors, and temperatures may, therefore, vary between 15°C to 30°C.The lower the temperature, the longer it takes to complete the fermentation. At the end of the fermentation, the pH will be about 3.5, withacidities between 0.6% and 1.2% (as lactic).

Pickle fermentationAs noted above, the high salt concentrations used in pickle manufacturing cause the fermentation to proceed quite differently fromthat in sauerkraut. Only those pickles made using brines at less than 5% salt will allow for growth of L. mesenteroides. Although heterofermentative, fermentations may promote more diverse flavor development, the formation of CO2 is undesirable, because it may lead to bloater or floater defects (see below). Moreover, low salt brines may also permit growth of unwanted members of the natural flora, including coliforms, Bacillus, Pseudomonas, and Flavobacterium. At salt concentrations between 5% and 8%, growth of Leuconostoc is inhibited and instead the fermentation is initiated by Pediococcus sp. and L.

plantarum. Pickle fermentation brines typically contain high concentrations of salt and organic acids and have a pH less than 4.5.These conditions are especially inhibitory to coliforms, pseudomonads, bacilli, clostridia, and other non-lactic acid bacteria that would otherwise cause flavor and texture problems. This environment, in fact, is hard even on lactic acid bacteria. However, the latter have evolved sophisticated physiological systems that enable them to survive under very uncomfortable circumstances.After fermentation, salt stock pickles can be held indefinitely in the brine. However, these pickles cannot be eaten directly, but rather must be de-salted by transfer to water. After several changes (a process called refreshing), the salt concentration is reduced to about 4%. They are then used primarily for relishes and other processed pickle products.

How lactic acid bacteria deal with Acid and Salt in a pickle

The key requirement to ensure the success of vegetable fermentations is to create a restrictive,if not inhospitable, environment, such that most indigenous microorganisms are inhibited orotherwise unable to grow. Typically, this is initially accomplished by adding salt, excluding oxygen,and maintaining a somewhat cool temperature. As lactic acid bacteria grow and produceorganic acids and CO2, the ensuing decreases in pH and Eh provide additional hurdles, especially for salt-sensitive,neutrophilic, aerobic organisms. These conditions, however, may not only affect resident enteric bacteria, pseudomonads, clostridia, fungi, and other undesirable microorganisms, but they can also impose significant problems for the lactic acid bacteria whosegrowth is to be encouraged.In addition, there may be other ionic compounds present in the vegetable juice brine that interferewith growth of lactic acid bacteria. For example, acetate, lactate, and other buffer salts are often added to brines, especially when pure cultures are used to initiate the fermentation.These acids and salts may have significant effects on cell metabolism and growth (Lu et al.,2002). Thus, how plant-associated lactic acid bacteria cope with these challenges may be ofpractical importance. Lactic acid bacteria are prolific producers of lactic acid (no surprise there), and can tolerate high lactic acid concentrations (_0.1 M) and low pH (_3.5), much more so than most of their competitors. At least several physiological strategies have been identified that enable these bacteriato tolerate high acid, low pH conditions. First, lactobacilli and other lactic acid bacteria can generate large pH gradients across the cell membrane, such that even when the medium pH is low (e.g., 4.0), the cytoplasmic pH (the relevant pH for the cell’s metabolic machinery) is always higher (e.g, 5.0). For example, in one study (McDonald et al., 1990), Lactobacillus plantarum and Leuconostoc mesenteroides maintained pH gradients of nearly 1.0 or higher, over a medium pH range of 3.0 to 6.0 (acidified with HCl). In the presence of lactate or acetate, however, somewhat lower pH gradients were maintained. This is because organic acids diffuse across the cytoplasmic membrane at low pH (or when their pKa nears the pH), resulting in acidification of the intracellular medium. For some bacteria, e.g., L. mesenteroides, the pH gradient collapses at low pH (the so-called critical pH). At this point, the cell is in real trouble, as enzymes, nucleic acid replication,ATP generation, and other essential functions are inhibited. In general, these results reflect, and are consistent with the observed lower acid tolerance of L. mesenteroides, as compared to L. plantarum.If maintenance of a pH gradient is important for acid tolerance, then the next question to

ask is how such a gradient can be made.That is, how can the protons that accumulate insidethe cell and cause a decrease in intracellular pH be extruded from the cytoplasm? Althoughthere are actually several mechanisms for the cell to maintain pH homeostasis, one specific system, the proton-translocating F0F1-ATPase (H_-ATPase) is most important. This multi-subunit, integral membrane-associated enzyme pumps protons from the inside to the outside using ATP hydrolysis as the energy source (Figure 1A).This enzyme is widely conserved in bacteria (in fact, throughout nature), but the specific properties of enzymes from different species show considerable variation. Thus, the H_-ATPases from lactobacilli have a low pH optima, accounting, in large part, for the ability of these bacteria to tolerate low pH relative to less tolerant lactic acid bacteria.Although the H_-ATPase system is the primary means by which lactic acid bacteria maintainpH homeostasis, other systems also exist (Figure 1A). For example, deamination of the aminoacid arginine releases ammonia, which raises the pH. Decarboxylation of malic acid, which iscommonly present in fermented vegetables, also increases the pH by conversion of a dicarboxylic acid to a monocarboxylic acid. In fact, when lactobacilli and other lactic acid bacteria are exposed to low pH, a wide array of genes are induced (Van de Guchte et al., 2002). Collectively, this adaptation to low pH is referred to as the acid tolerance response. Some of the induced genes code for proteins involved in the machinery used by the cell to deal with other physical or chemical stresses. Thus, the acid tolerance response may not only protect the cellagainst low pH, but also heat and oxidative stress.In vegetable fermentations, the other important stresses encountered by lactic acid bacteriaare high salt concentrations and high osmotic pressures. Salt concentrations in sauerkrautbrines are around 0.4 M,giving an osmolality of about 0.8 Osm.Pickle and olive brines may contain more than 1.0 M salt, or osmolalities above 2 Osm. Salt is an extremely effective antimicrobial agent due to its ability to draw water from the cytoplasm, thereby causing the cell to become dehydrated, lose turgor pressure,and eventually plasmolyze (and die!).The ability of lactic acid bacteria to tolerate high salt conditions varies (just as it did for acid tolerance), depending on the organism. Given that L. plantarum usually dominates high salt fermentations, it should be no surprise that this organism has also evolved physiological and genetic mechanisms that make it salt- and osmotolerant. Like acid tolerance, the salt tolerance system depends on the activity of membrane pumps.However, in the latter case, the pumps are actually transport systems that take up a special classof molecules called compatible solutes. By accumulating these non-toxic solutes inside the cytoplasm to high concentrations, cell water is retained and osmotic homeostasis is maintained. Compatible solutes are also referred to as osmoprotectants because they not only maintainosmotic balance, they also protect enzymes, proteins, and other macromolecules from dehydration and misfolding.Among the osmoprotectants accumulated by L. plantarum, the quaternary amine, glycine betaine (or simply betaine) is the most effective. Potassium ion, glutamate, and proline are also accumulated, but to lower concentrations, at least in lactobacilli. In L. plantarum, betaine is preferentially transported by the quaternary ammonium compound or QacT transport system (Glaasker et al., 1998).This transporter is a high affinity,ATP-dependent system whose activity is stimulated by high osmotic pressure (Figure 1B). In contrast, at low osmotic pressure, the efflux reaction is activated, and pre-accumulated betaine is released back into the medium. Analysis of the L.plantarum genome sequence indicates QacT may be encoded by the opuABCD operon, which is widely distributed in other Gram positive bacteria (Kleerebezem etal., 2003).The natural source of betaine, it is worth noting, is plant material, so

perhaps it is no coincidence that L. plantarum is unable to synthesize betaine and instead relies on a transport system to acquire it from the environment (Glaasker et al., 1996).

Figure 1. pH and osmotic homeostastis in Lactobacillus plantarum. Panel A shows three main systems whose function is to maintain pH homeostasis in L. plantarum. The F0F1-ATPase (left) is a primary proton pump that extrudes protons from the inside to the outside, using ATP as the energy source. In contrast, the malate and arginine systems rely on product efflux to drive uptake (no energy is required). In the malolactate system (center), proton consumption de-acidifies the medium and raises the pH. In the arginine diiminase system (right), medium pH is raised by virtue of the two molecules of NH3 that are released per mole of arginine. Panel B shows the QacT system responsible for osmotic homeostasis in L. plantarum. The components ofthis putative opuABCD-encoded system (left) include membrane-associated substrate-binding proteins (S-BP), a betaine permease, and ATP-binding proteins (ATP-BP). When the osmotic pressure is high, betaine is bound by the S-BP and taken up by the permease (center). Transport is driven by an ATPase following ATP-binding. If the osmotic pressure is reduced, accumulated betaine is effluxed via membrane channels (right).

DefectsAmong the microbial defects that occur in pickles, the most common are bloaters and floaters. The defect is caused by excessive gas pressure that subsequently results in internal cavity formation within the pickles. The CO2 gas is mainly produced by heterofermentative lactic acid bacteria (some of which may produce CO2 via the malolactic fermentation), although coliforms and yeasts may also be responsible. Floaters and bloaters can still be used for some processed products (i.e., relish), however, they cannot be used for the whole or sliced pickle market. The most common way to control or minimize this defect is to remove dissolved CO2 by flushing or purging the brine with nitrogen gas. Destruction and softening of the pickle surface tissue is another serious defect. When this happens, the pickle loses its crispness and crunch and becomes slippery and soft. These pickles are neither edible nor can anything be done to salvage them. The defect is caused by pectinolytic enzymes produced by microorganisms that are part of the natural cucumber microflora. The organisms responsible include mostly filamentous fungi, especially species of Penicillium, Fusarium, Alternaria, Aschyta, and Cladosporium. Pectins are complex heteropolysaccharides that serve as the main structural component of plant cell walls. Their hydrolysis requires the concerted action of three different enzymes—pectin methylesterase, polygalacturonase, and polygalacturonate lyase. Although Penicillium and other fungi are capable of secreting these enzymes, they may also be produced by various yeasts, as well as by the cucumber flowers. Bacteria, however, do not appear to be a major source of pectin-degrading enzymes.The fungi responsible for the softening defect gain entry into the fermentation tank via their association with cucumber flowers. Thus, excluding these constituents from the fermentationmay reduce or minimize this defect. Other preventative measures include maintaining sufficientsalt concentrations, acidity, anaerobiosis, and temperature. Another method used to controladventitious microorganisms and to ensure a prompt fermentation (especially when the saltconcentration is at the low end) is to partially acidify the cucumbers with acetic acid to aboutpH 4.5 to 5.0.This practice of chemical pasteurization is also used when starter cultures andcontrolled fermentation methods are used to produce pickles.Slimy brine is caused by the development of encapsulated bacteria during fermentation in brines with low salt and acid content. This can be prevented when salt concentration is controlled and maintained at 40o salometer.The spoilage evident condition is caused due to non removal of scum from the top of the brine, pickles were not heated long enough to destroy spoilage microorganism and jars were not sealed air tight while boiling hot. This can be prevented by daily removing of scum during brining process, by heating the pickles long enough or until it boils and each jar should be filled boiling hot and capped immediately.Darkened and discolouration of pickles is caused by minerals present in hard water used for pickle making, usage of iron, Brass, copper or zinc utensils for preparation, usage of ground spices and if whole spices left in pickle jar. This can be prevented by use of soft water, use of enamel ware, glass, aluminum, stainless steel or stone ware for preparation, use of whole spices and spices should not be packed in jars. (Spices are used only to add flavor).Shriveled pickles are caused due to addition of too much salt, sugar or vinegar. This can be prevented by starting with a weaker solution of brine, sugar or vinegar and by gradually adding the full amount in recipe.

Deposition of white sediments at the bottom of pickle jars occur due to growth of harmless yeast on surface and then settled in bottom. There is no preventive measure presence of small white sediments is normal.

Food Additives

The permitted values for food additives for pickle vegetables are given below according to codex standards ACIDITY REGULATORS

ANTIFOAMING AGENTS

ANTIOXIDANTS

COLOURS

FIRMING AGENTS

FLAVOUR ENHANCERS

PRESERVATIVES

SEQUESTRANTS

SWEETENERS

REFERENCE

FOOD PROCESSING TECHNOLOGY, Principles and Practice Second EditionP. Fellows Director, Midway Technology and Visiting Fellow in Food Technology at Oxford Brookes University

Processing Fruits Science and Technology Second Edition Edited by Diane M. BarrettLaszlo Somogyi Hosahalli Ramaswamy CRC PRESS Boca Raton London New York Washington, D.C.