Industrial Fermentation nondairy products
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Ministry of Education, Science, Culture and Sport of Ukraine
National Aviation University
Institute of Ecological Safety
Department of Chemistry and Chemical Technology
From discipline: “ Biotechnology of enzyme product and probiotics”
Theme:“ Industrial Fermentation nondairy products”
done by student of IES 305a
1. Meat products……………………………………………………3
4. Plants products………………………………………………….11
Fermented sausages are produced generally as dry or semidry products, although some are intermediate. Dry or Italian-type sausages contain 30–40% moisture, are generally not smoked or heat processed, and are eaten usually without cooking. In their preparation, curing and seasonings are added to ground meat, followed by its stuffing into casings and incubation for varying periods of time at 80–95◦F. Incubation times are shorter when starter cultures are employed. The curing mixtures include glucose as substrate for the fermenters and nitrates and/or nitrites as color stabilizers. When only nitrates are used, it is necessary for the sausage to contain bacteria that reduce nitrates to nitrites, usually micrococci present in the sausage biota or added to the mix. Following incubation, during which fermentation occurs, the products are placed in drying rooms with a relative humidity of 55– 65% for periods ranging from 10 to 100 days, or, in the case of Hungarian salami, up to 6 months. Genoa and Milano salamis are other examples of dry sausages.
In one study of dry sausages, the pH was found to decrease from 5.8 to 4.8 during the first 15 days of ripening and remained constant thereafter. Nine different brands of commercially produced dry sausages were found by these investigators to have pH values ranging from 4.5 to 5.2, with a mean of 4.87. With respect to the changes that occur in the biota of fermenting dry sausage when starters are not used, Urbaniak and Pezacki found the homofermenters to predominate overall, with Lactobacillus. plantarum being the most commonly isolated species. Heterofermenters such as L. brevis and L. buchneri increased during the six-day incubation period as a result of changes in pH and Eh brought about by the homofermenters.
Semidry sausages are prepared in essentially the same way as dry sausages but are subjected to less drying. They contain about 50% moisture and are finished by heating to an internal temperature of 140–154◦F (60–68◦C) during smoking. Cervelat, summer sausage, and Lebanon bologna are some examples of semidry sausages. “Summer sausage” refers to those traditionally of northern European origin, made during colder months, stored, aged, and then eaten during summer months. They may be dry or semidry.
Lebanon bologna is typical of a semidry sausage. This product, originally produced in the Lebanon, Pennsylvania area, is an all-beef, heavily smoked, spiced product that may be prepared by the use of a Pediococcus cerevisiae starter. The product is made by the addition of approximately 3% NaCl along with sugar, seasoning, and either nitrate, nitrite, or both, to raw cubed beef. The salted beef is allowed to age at refrigerator temperature for about 10 days during which time the growth of naturally occurring lactic acid bacteria or the starter organisms are encouraged and Gram negatives are inhibited. A higher level of microbial activity occurs along with some drying during the smoking step at higher temperatures. A controlled production process for this product has been studied,52 and it consists of aging salted beef at 5◦C for 10 days and smoking at 35◦C with high relative humidity (RH) for 4 days. Fermentation may be carried out either by the natural biota of the meat or by the use of a commercial starter of P. cerevisiae or P. acidilactici. The amount of acidity produced in Lebanon bologna may reach 0.8–1.2%.
The hazard of eating improperly prepared, homemade, fermented sausage was indicated by an outbreak of trichinosis: of the 50 persons who actually consumed the raw summer sausage, 23 fell ill with trichinosis. The sausage was made on two different days in three batches according to a family recipe that called for smoking at cooler smoking temperatures, believed to produce a better-flavored product. All three batches of sausages contained home-raised beef. In addition, two batches eaten by victims contained pork inspected by the U.S. Department of Agriculture (USDA) in one case and home-raised pork in the other, but Trichinella spiralis larvae were found only in the USDA-inspected pork. This organism can be destroyed by a heat treatment that results in internal temperatures of at least 60◦C or 140◦F.
In the production of dry sausages, lactobacilli produce aminopeptidases that aid in the generation of amino acids from sausage proteins. The amino acids contribute to the overall flavor of dry sausages. In the case of Lactobacillus sakei, it produces decarboxylases that give rise to biogenic amines, and these compounds can inhibit aminopeptidases and thus reduce flavor enhancement in dry-fermented sausages.
Fermented sausages produced without the use of starters have been found to contain large numbers of lactobacilli such as L. plantarum. The use of a P. cerevisiae starter leads to the production of a more desirable product.19,36 In their study of commercially produced fermented sausages, Smith and Palumbo77 found total aerobic plate counts to be in the 107–108/g range, with a predominance of lactic acid types. When starter cultures were used, the final pH of the products ranged from 4.0 to 4.5, whereas those produced without starters ranged between 4.6 and 5.0. For summer-type sausages, pH values of 4.5–4.7 have been reported for a 72-hour fermentation.2 These investigators found that fermentation at 30◦C and 37◦C led to a lower final pH than at 22◦C and that the final pH was directly related to the amount of lactic acid produced. The pH of fermented sausage may actually increase by 0.1 or 0.2 unit during long periods of drying due to uneven buffering produced by increases in the amounts of basic compounds. The ultimate pH attained following fermentation depends on the type of sugar added. Although glucose is most widely used, sucrose has been found to be an equally effective fermentable sugar for low pH production. The effect of a commercial frozen concentrate starter (P. acidilactici) in fermenting various sugars added to a sausage preparation is illustrated in Figure 8–1. Lactobacillus gasseri, when employed in a meat fermentation was shown to prevent enterotoxin formation by Staphylococcus aureus in a model sausage preparation. This species was the most effective of five other Lactobacillus species.
Prior to the late 1950s, the production of fermented sausages was facilitated by either back inoculations, or a producer took the chance of the desired organisms being present in the raw materials. Until recently, the manufacture of these, as well as of many other fermented foods, has been more of an art than a science. With the advent of pure culture starters, not only has production time been shortened, but more uniform and safer products can be produced. Although the use of starter cultures has been in effect for many years in the dairy industry, their use in many nondairy products worldwide is a recent development with great promise. “Micrococcus aurantiacus” has been employed along with starters in the production of some European sausages. The addition of a Micrococcus or a Staphylococcus, especially S. carnosus, to a lactic culture is a common practice in Europe. The nonlactic member reduces nitrates to nitrites and produces catalase that benefits the lactic culture.
Figure 8–1 Rate of pH reduction in fermenting sausage containing 0% or 1% of various carbohydrates.
Molds are known to contribute to the quality of dry European-type sausages such as Italian salami. In an extensive study of the fungi of ripened meat products, Ayres et al. found nine species of penicillia and seven of aspergilli on fermented sausages and concluded that the organisms play a role in the preservation of products of this type. Fewer species of other mold genera were found. A study of thefungal biota of naturally fermented sausages in northern Italy revealed that penicillia made up 96% and aspergilli 4%. The initial biota of the sausage was made up of>95% yeasts. After 2 weeks, yeasts and molds were about 50:50, but after 4–8 weeks, molds constituted >95% of the biota. Fifty percent of the mold biota was P. nalgiovensis. The addition of Penicillium camemberti and P. nalgiovensis during the curing of raw, dry, sausages was used in an effort to prevent the growth of mycotoxigenic house molds, and it was more successful than potassium sorbate.
Country-cured hams are dry-cured hams produced in the southern United States. During the curing and ripening period of 6 months to 2 years, heavy mold growth occurs on the surfaces. Although Ayres et al. noted that the presence of molds is incidental and that a satisfactory cure does not depend on their presence, it seems likely that some aspects of flavor development of these products derive from the heavy growth of such organisms, and to a lesser extent from yeasts. Heavy mold growth obviates the activities of food-poisoning and food-spoilage bacteria, and in this sense the mold biota aids in preservation. Ayres et al. found aspergilli and penicillia to be the predominant types of molds on country-cured hams.
The processing of country-cured hams takes place during the early winter and consists of rubbing sugar cure into the flesh side and onto the hock end. This is followed some time later by rubbing NaCl into all parts of the ham not covered by skin. The hams are then wrapped in paper and individually placed in cotton fabric bags and left lying flat for several days between 32◦C and 40◦C. The hams are hung shank end down in ham houses for 6 weeks or longer and may be given a hickory smoke during this time, although smoking is not essential for a desirable product.
Italian-type country-cured hams are produced with NaCl as the only cure. Curing is carried out for about a month, followed by washing, drying, and ripening for 6–12 months or longer.33 Although halophilic and halotolerant bacteria increase as Italian hams ripen, the biota, in general, is thought to play only a minor role. In Europe, molds are critical in the production of safe and high quality products such as salami and hams (Parma from Italy and Solano from Spain). For more detailed information on meat starter cultures and formulations for fermented sausages, along with cure ingredients for country-style hams.
Fermented fishery products are rather widespread in parts of Asia where marine sources contribute more protein to the human diet than is the case in the Western world. Only two classes of fermented seafood products are noted below–sauces and pastes.
Fish sauces are popular products in Southeast Asia, where they are known by various names such as ngapi (Burma), nuoc-mam (Cambodia and Vietnam), nam-pla (Laos and Thailand), ketjap-ikan (Indonesia), and so on. The production of some of these sauces begins with the addition of salt to uneviscerated fish at a ratio of approximately 1:3, salt to fish. The salted fish are then transferred to fermentation tanks generally constructed of concrete and built into the ground or placed in earthenware pots and buried in the ground. The tanks or pots are filled and sealed off for at least 6 months to allow the fish to liquefy. The liquid is collected, filtered, and transferred to earthenware containers and ripened in the sun for 1–3 months. The finished product is described as being clear, dark-brown in color with a distinct aroma and flavor. In a study of fermenting Thai fish sauce by the latter investigators, the H from start to finish ranged from 6.2 to 6.6 with the NaCl content around 30% over the 12-month ermentation period.70 These parameters, along with the relatively high fermentation temperature, result in the growth of halophilic aerobic spore formers as the predominant microorganisms of these products. Lower numbers of streptococci, micrococci, and staphylococci were found, and they, along with the Bacillus spp., were apparently involved in the development of flavor and aroma. Some part of the liquefaction that occurs is undoubtedly due to the activities of fish proteases. Although the temperature and pH of the fermentation are well within the growth range of a large number of undesirable organisms, the safety of products of this type is due to the 30–33% NaCl.
Fish pastes are also common in Southern Asia, but the role of fermenting microorganisms in these products appears to be minimal. Among the many other fermented fish, fish-paste, and fish-sauce products, are the following: mam-tom of China; mam-ruoc of Cambodia; bladchan of Indonesia; shiokara of Japan; belachan of Malaya; bagoong of the Philippines; kapi, hoi-dong, and pla-mam of Thailand; fessik of Africa; nam-pla , pla-ra, pla-chom, and pla-com of Thailand. A fermented shrimp product of Thailand is kung-chom.
Soy sauces are fermented condiments of various plant materials. Typically, the plant material first undergoes a fungal fermentation followed by a brine fermentation in which Tetragenococcus spp. Are active. In Chinese soy sauce only soy beans are used, whereas in Japanese both wheat and soy beans are used. T. halophilus, which can tolerate 18% NaCl, is active in the brine of the soy sauces noted. Another Tetragenococcus sp., T. muriaticus, has been isolated from fermented squid liver sauce. This species can grow in 1–25% NaCl, and it produces histamine. Some soy sauces are made by acid hydrolysis of soy beans.
San Francisco sourdough bread is similar to sourdough breads produced in various countries. Historically, the starter for sourdough breads consists of the natural biota of baker’s barm (sour ferment or mother sponge, with a portion of each inoculated dough saved as starter for the next batch). The barm generally contains a mixture of yeasts and lactic acid bacteria. In the case of San Francisco sourdough bread, the yeast has been identified as Saccharomyces exiguus (Candida holmii and the responsible bacteria are Lactobacillus sanfranciscensis, L. fermentum, L. fructivorans, some L. brevis strains, and L. pontis. The key bacterium is L. sanfranciscensis, and it preferentially ferments maltose rather than glucose and it requires fresh yeast extractives and unsaturated fatty acids. The souring is caused by acids produced by these bacteria, and the yeast is responsible for the leavening action, although some CO2 is produced by the bacterial biota. The pH of these sourdoughs ranges from 3.8 to 4.5. Both acetic and lactic acids are produced, with the former accounting for 20–30% of the total acidity. Lactobacillus paralimentarius is another of the sourdough bacteria. Sourdoughs are placed into three groups and each has its unique fermentation consortium. Type I sourdoughs are fermented at 20–30◦C and the two primary organisms are L. sanfranciscensis and L. pontis. Type II doughs employ baker’s yeast as a leavening agent, and the dominant lactics are L. pontis, L. panis, and from one to nine other lactobacilli. Type III doughs are dried products of traditional fermentations. Greek wheat sourdoughs belong to Type I, and the fermentation consortium in the traditional wheat product consists of L. sanfranciscensis, L. brevis, L. paralimentarius, andWeissella cibaria. Among other organisms found in some sourdough fermentations are Candida humilis, Dekkera bruxellensis, Saccharomyces cerevisiae, and Saccharomyces uvarum. Idli is a fermented bread-type product common in southern India. It is made from rice and black gram mung (urd beans). These two ingredients are soaked in water separately for 3–10 hours and then ground in varying proportions, mixed, and allowed to ferment overnight. The fermented and raised product is cooked by steaming and served hot. It is said to resemble a steamed, sourdough bread. During the fermentation, the initial pH of around 6.0 falls to values of 4.3–5.3. In a particular study, a batter pH of 4.70 after a 20-hour fermentation was associated with 2.5% lactic acid, based on dry grain weight.46 In their studies of idli, Steinkraus et al.78 found total bacterial counts of 108–109/g after 20–22 hours of fermentation. Most of the organisms consisted of Gram-positive cocci or short rods, with L. mesenteroides being the single most abundant species, followed by E. faecalis. The leavening action of idli is produced by L. mesenteroides. This is the only known instance of a lactic acid bacterium having this role in a naturally fermented bread.46 The latter authors confirmed the work of others in finding the urd beans to be a more important source of lactic acid bacteria than rice. L. mesenteroides reaches its peak at around 24 hours, with E. faecalis becoming active only after about 20 hours. Other probable fermenters include L. delbrueckii subsp. delbrueckii, L. fermentum, and Bacillus spp. Only after idli has fermented for more than 30 hours does P. cerevisiae become active. The product is not fermented generally beyond 24 hours because maximum leavening action occurs at this time and decreases with longer incubations. When idli is allowed to ferment longer, more acidity is produced. It has been found that total acidity (expressed as grams of lactic acid per gram of dry grains) increased from 2.71% after 24 hours to 3.70% after 71 hours, whereas the pH decreased from 4.55 to 4.10 over the same period. (A review of idli fermentation has been made by Reddy et al.65)
4. Plants products
Sauerkraut is a fermentation product of fresh cabbage. The starter for sauerkraut production is usually the normal mixed biota of cabbage. The addition of 2.25–2.5% salt restricts the activities of Gram-negative bacteria, while the lactic acid rods and cocci are favored. Leuconostoc mesenteroides, Lactobacillus plantarum, and Leuconostoc fallax are the three most dominant lactics in sauerkraut production, with the two Leuconostoc spp. having the shorter generation time and the shorter life span. The activities of the cocci usually cease when acid content increases to 0.7–1.0%. The final stages of kraut production are effected by L. plantarum and L. brevis. P. cerevisiae and E. faecalis may also contribute to product development. The final total acidity is generally 1.6–1.8%, with lactic acid at 1.0–1.3% and pH in the range of 3.1 to 3.7.
The microbial spoilage of sauerkraut generally falls into the following categories: soft kraut, slimy kraut, rotted kraut, and pink kraut. Soft kraut results when bacteria that normally do not initiate growth until the late stages of kraut production actually grow earlier. Slimy kraut is caused by the rapid growth of Lactobacillus cucumeris and L. plantarum, especially at elevated temperatures. Rotted sauerkraut may be caused by bacteria, molds, and/or yeasts, whereas pink kraut is caused by the surface growth of Torula spp., especially T. glutinis. Due to the high acidity, finished kraut is generally spoiled by molds growing on the surface. The growth of these organisms effects an increase in pH to levels where a large number of bacteria can grow which were previously inhibited by conditions of high acidity.
Olives to be fermented (Spanish, Greek, or Sicilian) are done so by the natural biota of green olives, which consists of a variety of bacteria, yeasts, and molds. The olive fermentation is quite similar to that of sauerkraut except that it is slower, involves a lye treatment, and may require the addition of starters. The lactic acid bacteria become prominent during the intermediate stage of fermentation. L. mesenteroides and P. cerevisiae are the first lactics to become prominent, and these are followed by lactobacilli, with L. plantarum and L. brevis being the most important.
The olive fermentation is preceded by a treatment of green olives with 1.6 to 2.0% lye, depending on the type of olive, at 21–25◦C for 4–7 hours for the purpose of removing some of the bitter principal. Following the complete removal of lye by soaking and washing, the green olives are placed in oak barrels and brined so as to maintain a constant 28◦–30◦ salinometer level. Inoculation with L. Plantarum may be necessary because of destruction of organisms during the lye treatment. The fermentation may take as long as 6–10 months, and the final product has a pH of 3.8–4.0 following up to 1% lactic acid production.
Among the types of microbial spoilage that olives undergo, one of the most characteristic is zapatera spoilage. This condition, which sometimes occurs in brined olives, is characterized by a malodorous fermentation. The odor is due apparently to propionic acid, which is produced by certain species of Propionibacterium.61 In addition to propionic acid, formic, butyric, succinic, isobutyric, n-valeric, and cyclohexacarbolic acids, as well as methanol, ethanol, 2-butanol, and n-butanol may be produced.
A softening condition of Spanish-type green olives has been found to be caused by the yeasts Rhodotorula glutinis var. glutinis, R. minuta var. minuta, and R. rubra. All of these organisms produce polygalacturonases, which effect olive tissue softening. Under appropriate cultural contions, the organisms were shown to produce pectin methyl esterase as well as polygalacturonase. A sloughing type of spoilage of California ripe olives was shown by Patel and Vaughn to be caused by Cellulomonas flavigena. This organism showed high cellulolytic activity, which was enhanced by the growth of other organisms such as Xanthomonas, Enterobacter, and Escherichia spp. The production of some biogenic amines has been shown to occur primarily in Spanish-style green olives during the brining process. The amines found were cadaverine, histamine, tyramine, tryptamine, and putrescine with the latter being in highest concentration after 3 months of brining. The others were found in samples taken after 12 months.
4. Plants products………………………………………………….11