Ken-Yuon Li
Tung-Hai University, Taichung, Taiwan
I. INTRODUCTION
The use of microorganisms to process or preserve foods is an ancient technique. Yeast was the first microorganism used in the production of wine and beer and the leavening of dough. These techniques have been known for at least 4000 – 5000 years. When these processes are underway, bubbles form as in gentle boiling. This bubbling is due to the liberation of carbon dioxide from the degradation of sugar. The word fermentation signifies the gentle bubbling or boiling condition in these processes.
The nature of the fermentation reaction did not become clearly understood until the late part of the nineteenth century when Louis Pasteur discovered the relationship between living cells and fermentation. In 1854, Pasteur demonstrated the relationship between yeast and this reaction. The word fermentation became associated with microorganisms. Pasteur also showed that true fermentation occurs only in the absence of free oxygen. He called life without air anaerobiosis. Actually, the definition of fermentation in biochemistry is the extraction of energy from
carbohydrates and other organic substrates without using O2 as an electron acceptor. Hence
fermentation is an energy-yielding catabolic pathway that proceeds with no net change in the oxidation state of the products compared to that of the substrate. The common usage of the word fermentation frequently overlooks the strict biochemistry definition. A broad sense was adopted, that is, a process in which microorganisms produce chemical changes in organic substrates through the action of enzymes produced by these microorganisms. According to the common usage, the term fermented foods is used to describe a special class of foods that contain a complex mixture of carbohydrates, proteins, fats, etc., undergoing simultaneous modification under the action of a variety of microorganisms and enzymes. Reactions involving carbohydrates and carbohydratelike materials are referred to as fermentative. Changes in proteinaceous materials are designated proteolytic, and the breakdown of fatty substances are described as lipolytic. When complex foods are fermented under natural conditions, they invariably undergo different degrees of each type of change. Whether fermentative, proteolytic, or lipolytic end products dominate will depend upon the nature of the food, the types of microorganisms present, and the environmental conditions affecting their growth and metabolic patterns.
The basic concept of fermentation is to facilitate the proliferation and predomination of desirable microorganisms in raw plant materials. The desirable microorganisms will metabolize sugars into chemicals such as lactate, ethanol, and acetate that infuse the plant materials with various characteristics. The addition of salt and the inoculation of a defined microbial culture are
the two basic methods for controlling the growth of microorganisms during fermentation. In this chapter, we will describe the predominant bacterial strains occurring in some popular fermented vegetables, illustrate their sugar metabolic reactions, and discuss how fermentation is manipulated with these organisms.
II. THE FERMENTATION OF VEGETABLES
At present only cabbage (sauerkraut and Korean kimchi), cucumbers (pickles), and olives are of real economic importance. In this chapter, the discussion is focused on these vegetable products. In addition to these vegetables, fermented carrots, the potential new products, and fermented bamboo shoots will be described.
A. Cabbage Fermentation
Sauerkraut is a fermented product made from fresh cabbage. In the cabbage fermentation process lactic acid bacteria are favored. The addition of 2.25 – 2.5% salt restricts the activities of undesirable gram-negative bacteria. The fermentation is started by Leuconostoc mensenteroids. This bacterium converts sugar to lactic acid, acetic acid, alcohol, CO2, and other products that
contribute to the flavor of sauerkraut. CO2helps maintain the anaerobic conditions necessary in
fermenting cabbage. As the acids accumulate, Leu. mensenteroids is inhibited, but the fermentation continues with Lactobacillus brevis, Pediococcus cerevisiae, and finally, Lactobacillus plantarum. Lb. plantarum and Lb. brevis effect the final stages of sauerkraut production. P. cerevisiae and Enterococcus faecalis may also contribute to product development (1).
Kimchi is a traditional Korean fermented vegetable product. Kimchi fermentation is the Korean method for preserving a fresh and crispy vegetable texture for consumption during the winter, when fresh vegetables are not available. Although the history of kimchi fermentation in Korea can be traced to the 3rd and 4th centuries, the earliest description of the processing methods is found in 17th century works of literature (2). A fresh cabbage is cut in half or shredded, soaked in brine with an approximately 10% salt concentration overnight, and then washed and drained. The minor ingredients (garlic, red pepper, green onion, ginger) are chopped and mixed with shredded radish and stuffed between the salted cabbage leaves. The kimchi is packed in an earthen jar, buried in the ground, and pressed with a stone placed inside in order to keep the ingredients immersed in the juice. Before ripening, Lu. mensenteroides is the dominant microorganism, while Lactobacillus spp. are the major organisms in over-ripened kimchi. Lactobacillus species may be dominant in the later stages of kimchi fermentation depending on the temperature (2).
The difference between sauerkraut and kimchi is that of the of fermentation end-point. The best-tasting kimchi is attained before Lb. brevis and Lb. plantarum overgrowth occurs with an optimal pH of 4.5. The Lb. brevis and Lb. plantarum overgrowth diminishes the product quality, but sauerkraut production depends on these organisms.
B. Cucumber Fermentation
In the natural fermentation of pickles, selected cucumbers are placed in brine with about 5% NaCl. The brine strength is gradually increased during fermentation until it reaches around 16% NaCl. The sugars that diffuse from the cucumbers are fermented sequentially by Leu. mensenteroides, P. cerevisiae, Lb. brevis, and Lb. plantarum. Depending on the fermentation condition, about 0.6 to 1.2% lactic acid is formed in about 7 to 14 days. When the pH is lowered to
3.2, the metabolism of Lb. plantarum is inhibited and the fermentation is completed. In this process, the high salt level is used to protect against spoilage. The fermented cucumber must be desalted before being used in products. However, the NaCl level in the desalting solution creates a serious dumping problem. Procedures have been developed for brining cucumbers in closed anaerobic tanks at substantially lower salt concentrations (3). This approach to fermentation may allow cucumber fermentation and storage at sufficiently low salt concentrations that require no desalting.
In natural fermentation, bloating in defective pickles often occurs. Bloating is due to the accumulation of CO2gas inside the cucumber during fermentation. The respiration of cucumber
tissue and fermentation by P. cerevisiae and Lb. plantarum produces sufficient CO2 to cause
bloating (4). The degradation of malic acid to lactic acid is a major source of CO2 when Lb.
plantarum ferments brined cucumbers. Research has demonstrated that using a mixed culture with a malolactic-deficient mutant and normal malolactic strain of Lb. plantarum in brined cucumber fermentation could reduce the level of released CO2(5).
In cucumber fermentation, yeasts have conventionally been viewed as undesirable because it produces CO2. However, when N2is used in purging cucumber fermentation tanks to prevent
bloater damage, using yeast (Saccharomyces cerevisiae or S. rosei) in the mixed culture can facilitate complete sugar metabolization (6).
Softening in defective pickles is another problem. Softening is attributed to pectinolytic enzymes that degrade the cucumber tissue. The source of these enzymes may be the microorganisms growing in or on the cucumbers. To reduce fermentation defects, a controlled fermentation process is used. The controlled fermentation method employs a chlorinated brine
with a 258 salinometer, acidification with acetic acid, the addition of sodium acetate, and
inoculation with P. cerevisiae and Lb. plantarum (7).
C. Olive Fermentation
Olive fermentation is similar to that in sauerkraut except that the olives are soaked in a 1.6 to 2.0% lye solution before brining. The lye treatment is necessary to remove oleuropein, a bitter factor in olives. The olives are brined in containers following the complete removal of lye by rinsing the olives in fresh water. The brine concentration varies from 5 to 15%, depending on the variety and size of the olives (8). Lactic acid bacteria become prominent during the intermediate stage of fermentation. Leu. mensenteroides and P. cerevisiae are the first lactic acid bacteria to become prominent. These bacteria are followed by lactobacilli, with Lb. plantarum and Lb. brevis being the most important (9). The lye treatment may affect the microbial flora. Inoculation with Lb. plantarum may be required. A study has showed that using a strain of Lb. plantarum with the capability to produce bacteriocin as a starter controls lactic acid fermentation much better (10). The entire fermentation process may take 2 weeks to several months. The acid content of the final product varies from 0.18 to 1.27% (11).
D. Carrot Fermentation
Carrots are not a traditional vegetable for fermentation. Until 1969, carrots were fermented using a home-based process (12). Fermentation provides a simple method of preserving raw carrots. The raw carrot slices contain a high level of reducing sugar that might cause Maillard reactions and produces dark compounds with a burnt smell during thermal processes. Using lactic acid fermentation, the reducing sugar content in the raw carrot can be decreased to a level that allows
the carrot slices to be processed using hightemperature deep frying to yield chips. The deep-fried carrot chips have a light red – yellow color and pleasant taste that makes them a potential new product (13).
A mixed culture of Lb. plantarum, Lb. brevis, P. cerevisiae, and Leu. mesenteriodes is used to ferment carrots (14). Use of carrot-adapted inocula significantly reduced the lag period for early acid production despite the salt concentration. The repressive effects of increased salt concentrations on the rate of fermentation means that carrots treated with the lowest level of salt, 1.5%, require only 10 days incubation to produce a 1.0% acid level, whereas a 3.0% salt concentration requires 18 days incubation to reach a similar acid value. The acidic properties of fermented brines resemble the fermentation properties of the cabbage head brining solution (15). A new process for carrot fermentation using an alkaline treatment with lye before inoculating a pure culture of Lb. plantarum was developed (16). The alkaline treatment helps inoculum establishment over the natural flora in the fermentation. However, most of the sucrose remains unmetabolized after 7 days of fermentation. Thus long-term stability in the fermented carrots is not ensured. A high risk of secondary fermentation may present in the package product. This process was further modified using a mixed culture of Lb. plantarum and S. cerevisiae to replace the single culture of Lb. plantarum. The result indicated that the mixed culture was able to completely use up all of the sugars and, at same time, improve the flavor of the fermented carrots (17).
E. Bamboo Shoot Fermentation
People in the bamboo-growing regions of Asia have traditionally consumed fermented bamboo shoots. The dried Ma bamboo (Dendrocalamus latiflorus) shoot is a special product of Taiwan (18). Mesu is a similar product from India (19). Both are produced by using nonsalted fermentation with natural cultures.
Using mesu as a pickle and as the base for curry is a tradition in the Darjeeling hills and Sikkim area of India. A study has shown that a total of 327 strains of lactic acid bacteria, representing Lb plantarum and Lb. pentosaceus were isolated from 30 samples of mesu. These species were present in all of the raw bamboo shoot samples tested. Mesu is dominated by Lb. plantarum followed by L. brevis. P. pentosaceus was isolated less frequently and recovered from only 40 – 50% of the mesu samples. Fermentation is initiated by P. pentosaceus, followed by L. brevis, and finally succeeded by L. plantarum species. During the fermentation, the titratable acidity increased from 0.04 to 0.95%, resulting in a decline in pH from 6.4 to 3.8 (20). Ma- bamboo shoots are fermented using a traditional natural culture. After 10 days of fermentation, the fermented bamboo shoots contain about 109cfu=g of lactic acid bacteria, and 104– 106cells=g of yeast and mold. The final pH was 3.3 to 4.1, and the titratable acid was 1.05 – 1.20% (19).
III. FERMENTATION TECHNIQUES
The procedures for vegetable fermentation are varied and complicated. Basically, vegetable fermentation can be considered as a three-staged process.
A. Stage 1: The Pretreatment Steps
In this stage, the common operations include sorting and grading raw vegetables, cleaning the selected vegetables, specific pretreatment, such as peeling carrots, blanching green beans, shredding cabbage, or lye-treating olives.
B. Stage 2: The Fermentation Environment Adjustment Operation
Adding salt and inoculating the defined starter culture are two methods to set up a suitable environment around the vegetables to allow the desirable microflora to proliferate and predominate. Salt addition is necessary in most kinds of vegetable fermentation. The major contributions of salt are to inhibit the growth of pathogens and destructive spoilage microorganisms, to exert a selective effect on the microorganisms present on vegetables, to enhance the release of tissue fluids from the fermenting vegetables, and to impose a special flavor on the fermented vegetables. The amount of salt used depends on the particular vegetables. In the fermentation of cucumbers and olives, the salt concentration is 5 – 8% at equilibrium. For cabbages, the salt concentration is less than 2.5% at equilibrium. The difference in salt concentration between that used in sauerkraut fermentation and that used in pickle fermentation probably accounts for the difference in the types of lactic acid bacteria that grow in each fermentation environment (21).
The application of a defined starter culture is an another method of facilitating the predomination of desirable microflora in the fermenting vegetables. The lactic acid bacteria used for this purpose include Lactobacillus species (Lb. plantarum and Lb. casei are the most often used.), Lactococcus lactis, and Leu. mensenteroides. The defined starter cultures are capable of growing rapidly and are highly competitive under the environmental conditions used to ferment products.
C. Stage 3: The Vegetable Fermentation Process
Temperature, pH value, and anaerobiosis maintenance are major factors that influence the course of fermentation. The temperature range for vegetable fermentation is 16 to 358C. Vegetables fermenting at 108C lead to good quality products. Usually, the optimal temperature is between 15 and 208C. Various microorganisms may dominate a mixed fermentation depending on the temperature. For sauerkraut fermentation, the preferred temperature is 188C or lower. The predominant strain Leu. mensenteroides grows optimally at a lower temperature than the homofermentative Lb. plantarum, presumably resulting in a higher ratio of volatile to nonvolatile acids than at higher temperatures. For cucumber fermentation, the predominant cultures of P. pentosaceus and Lb. plantarum are capable of rapid growth at 188C (22). The optimal temperature for vegetable fermentation depends on the predominant cultures during the fermentation.
The buffering capacity of the vegetable affects the extent of proliferation of the predominant culture used to ferment the natural sugars. Several methods have been adopted to maintain the pH during fermentation. Sodium acetate (23) and calcium acetate (24) have been used as buffering agents to assure complete sugar utilization during the primary fermentation of cucumbers. Acid neutralization during fermentation with a pH controller has also been used to assure complete sugar utilization (25). In the fermentation of carrots, sodium hydroxide treatment of peeled and trimmed carrots is a useful alternative to pasteurization to achieve controlled fermentation. Subsequent neutralization of the NaOH by adding acetic acid to the brine could lead to the formation of a buffer system in the brine. The buffer system benefits greater utilization of the fermentable sugars by the starter culture (26). For preserving fermented vegetables for long periods of time, the pH should be controlled below 4.0 (27).
During fermentation, to maintain anaerobic conditions the plant materials must be totally covered by the brine in the vessels. Open filled vessels are normally covered with plastic sheets or wooden plates weighted down with stones or heavy matter to exclude oxygen from the air. For
cucumber fermentation, anaerobic tanks provide more suitable anaerobic conditions (23). Anaerobic tanks replaced open tanks in the olive fermentation industry of the USA and Spain many years ago (28).
IV. VEGETABLE FERMENTATION MICROORGANISMS
Fresh plant material harbors numerous and varied types of microorganisms. The microflora in vegetables and fruits is largely made up of Pseudomonas spp., Erwinia herbicola, Flaeobacterium, Xanthomonas, and Enterobacter agglomerans as well as various molds. Lactic acid bacteria such as Leu. mesenteroids and Lactobacillus spp. are also commonly found, as are several species of yeasts (29). Between 40 and 75% of the bacterial flora in peas, snap beans, and corn was shown to consist of leuconostocs and streptococci, whereas many of the gram-positive, catalase-positive rods resembled corynebacteria (30,31). An analysis of 30 different samples of white cabbage from four growing seasons has shown that the microflora normally is dominated by aerobic bacteria (e.g., pseudomonads, enterobacteria, and coryneforms) and yeasts, while lactic acid bacteria represent 0.15 to 1.5% of the total bacterial population (32). Vegetable fermentation involves controlling specific microorganisms or a succession of microorganisms that dominate the microflora in vegetables. Although lactic acid bacteria are present as a small population, the metabolic activities of this microorganism are indispensable in the vegetable fermentation process. Lactic acid fermentation is the most important contribution to the fermentation of vegetables.
A. The Major Lactic Acid Bacteria in Vegetable Fermentation
The major lactic acid bacteria involved in vegetable fermentation are located in three genera, Lactobacillus, Leuconostoc, and Pediococcus. Among the lactobacilli, several species and strains have been isolated from fresh vegetables. These include the homofermentative species Lb. plantarum, Lb. casei, Lb. arabinosus, and Lb. homohiochii, and the heterofermenters Lb. brevis, Lb. fermentum, and Lb. buchneri. The genus Pediococcus comprises two species, P. pentosaceus and P. acidilactici. Currently, Leuconostoc comprises a single species, Leu. mensenteroides (33). The lactic acid bacteria share some common features: they are Gram-positive; mesophilic, but some can grow at temperatures as low as 58C or as high as 458C; growing at pH 4.0–4.5, (some are active at pH 9.6 and others at pH 3.2); generally weakly proteolytic and lipolytic and require preformed amino acids, purine and pyrimidine bases, and B vitamins for growth; do not contain a citric acid cycle or a cytochrome system so no energy is derived from oxidative phosphorylation, but energy is obtained via substrate level phosphorylation during the fer- mentation of sugars into lactic acid, ethanol or acetate, and CO2.
There are four important species of lactic acid bacteria associated with vegetable fermentation: Leu. mensenteroides, Lb brevis, P. pentosaceus, and Lb plantarum. These species are successively predominant during sauerkraut fermentation in the approximate order listed (7). Lb brevis, P. pentosaceus, and Lb plantarum have also been reported to ferment cucumbers (34) and olives (35). The properties of these four species are described as follows.
1. Leu. mensenteroides
The colorless bacterial cell is spherical or egg-shaped and appears usually in pairs. The size of the bacterium is 0.5 – 0.7 mm. Leuconostoc is distinguished among the lactic acid bacteria in being heterofermentative and also in lacking aldolase, a key enzyme in glycolysis. Under anaerobic
conditions, this bacterium metabolizes glucose via the phosphoketolase pathway and producesD-
lactate. At the temperature range of 20 to 258C, this bacterium produces dextrans from sucrose. This bacterium is capable of metabolizing citrate into CO2and diacetyl, which is an important
flavor component in many dairy products.
2. Lb. plantarum
Lb. plantarum is the final and predominant lactic acid bacterium species at the completion of fermentation in many vegetables. This is attributed to its metabolic diversity and its tolerance for