Vital Role In The Fermentation Process Biology Essay

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Fermented foods are important components of the diet in a number of developing countries, importantly South East Asian countries. They are either consumed as main course or as condiments. Fermentation is also one of the oldest methods of preservation, dating back at least 6000 years . The process involves microorganisms that play a vital role in the physical, nutritional and organoleptic modification of the starting material. It may lead to destruction of undesirable factors present in raw foods such as cyanide, phytates, tannins and polyphenols and also removal of lactose. Its importance in today's life is highlighted by the wide range of fermented foods marketed both in developing and industrialized countries for their highly appreciated sensory attributes other than their safety and preservation benefits. Traditional skills are being improved for controlling technical parameters during fermentation processes through trial and error experiments. Experience has also underlined that the inoculation of raw substrates with a residue from a previous batch accelerates the initial phase of fermentation and promotes the desirable changes during the fermentation process .

Lactic acid bacteria (LAB)

This group of bacteria plays a vital role in the fermentation process. Principal genera include Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissell. Their growth is encouraged in a variety of fermented foods including meat, fish, vegetables and cereals . The metabolism of these bacteria can be of two types: homofermentative, where 95% of the total end product is lactic acid or heterofermentative, in which ethanol, acetic acid, CO2 and lactic acid are, produced (BOOK).

Lactic acid bacteria also produce bacteriocins, antimicrobial proteins or peptides, that insert into the membranes of closely related bacteria, causing formation of pores, thereby leakage and hence the inability to sustain metabolism which causes death The bacteriocins have been generally classified into 4 major groups according to their molecular weight: Class I (<5kDa), Class II (<10kDa), Class III (>10kDa) and Class IV (large complex molecules). However, only the first 2 groups qualify themselves to be used in the food industries as preservatives. . Nisin, a bacteriocin, is a very good example of the same BOOK). Nisin, produced by a lactococcal culture, is widely used in the manufacture of cheese. The pathogen of primary concern for cheese manufacturers is mainly Listeria monocytogenes, which is able of growing at refrigeration temperatures and can survive in low pH range. Moreover, L. monocytogenes can resume their growth in cheeses exhibiting a rise in pH during the ripening stage, as seen on the surface of mould-ripened cheese. Extensive research has been done to check the effectiveness of nisin for inhibition of L. monocytogenes in cheese and undoubtedly it has shown promising results . Some nonstarter LAB cultures like Lactobacillus. Paracasei NdP78 produce bacteriocin like substances that are effective against L .monocytogenes and increase the shelf life of Tosela cheese . Another bacteriocin, Sakacin C2, isolated from a fermented vegetable source has shown antimicrobial activity against not only a wide range of LAB but also against several food borne spoilage and harmful pathogenic strains like S. aureus and E.coli .

Fermented foods

Various traditional methods are still being followed in households and small scale industries to produce fermented food. In most of these methods, there is no commercial starter culture added. The indigenous bacteria present in the hands of workers, the vessels in which they are fermented and the bacteria in the surrounding environment aid to the fermentation process. However the end product is safe for consumption. In some cases, the residues from the previous batch are added to help in the fermentation process. Let us look at some examples where fermented foods are prepared traditionally.

Milk based fermented products

In Burindi, Urubu, a fermented milk product, is produced by leaving the raw bovine milk in an earthen pot for 3 days to ferment. The microbes present in the milk, container and the environment ferment the milk within 1-3 days depending upon the temperature. The absence of starter cultures and unhygienic conditions are responsible for shorter shelf life due to large variety of microorganisms present in it. However it is an important part of the diet in Burindi. The end product may be consumed directly or churned to butter. More research to develop starter cultures for urubu needs to be done in order to develop a safe product with a longer shelf life .

In Africa, many people enjoy sour milk products. Fermentation takes place in calabashes or clay pots. A calabash is generally inoculated with microbial culture and placed outside the hut for fermentation. This was a relatively faster method where fermentation occurs within 2-3 hours . LAB dominates the micro flora, especially the genera Leuconostoc, Lactococcus and Lactobacillus. However, today industries have replaced calabashes and clay pots with modern containers using commercial mesophilic starter cultures including some predominant species like Lactococcus .lactis subsp. cremoris, Leuconostoc mesenteroides and a few more .

In Zimbabwe, most of the rural populations ferment the milk to amasi. But, in urban areas this is substituted by an industrially processed product called 'lacto'. Lacto is prepared using standardized milk, followed by pasteurization at 90°C for 20 min. In the former method, the microorganisms present in the container, the surrounding environment and the milk itself ferment the milk in 1-3 days . Whereas, lacto is prepared in a very controlled environment by adding a starter culture in just 18h, followed by refrigeration until sold . There was no major difference in the total count, LAB number and the amount of lactic acid produced at the end of fermentation in both products. 200 strains of LAB were identified from amasi, some at sub species level including genera like lactococcus, enterococcus and lactobacillus. However, upon sensory evaluation amasi was more acceptable than lacto. But from the safety point of view, lacto was much safer to drink and had a longer shelf life than amasi. Lacto inhibited E.coli more than amasi at all temperatures i.e. at the ambient temperature and the refrigeration temperature probably because pasteurized milk was used for production. L.monocytogens count was more in number at refrigeration temperatures while E.coli count was higher at ambient temperatures . Conversely, it was reported that E.coli O157:H7 was detected in commercial grades of amasi but not in traditional amasi. This difference was also attributed to the possibility of higher LAB count in traditional amasi than the commercial one. But it was proved that, E. coli O157:H7 could survive during fermentation and storage of this fermented product. Moreover, it was known that E.coli O157:H7 can survive better under cold storage conditions .Extensive research needs to be done in overcoming the current problems including inconsistency, safety and longer shelf life in producing amasi at a larger scale.

Warankasi is a soft ripened cheese produced from goat or cow milk in Nigeria. The raw milk is mixed with a few drops of juice from plant sources and allowed to ferment for 12h at ambient temperature. The fermented mixture is then heated for 45 minutes to form a junket. This is allowed to 'break' and is carefully removed with a scoop, exposing white curd. It is then heated at higher temperatures to stop the enzymatic activity, to develop flavor and better texture in the final product. The resulting product called 'wara' is moulded and the whey is allowed to drain off for some time. The carrot-shaped cheese is thrown back into its whey from where it is consumed. Scientific evaluations were done and the methods to develop a safer product were discussed. The nutritional composition was also found out . However, Iwuoha et al failed to report on the predominant micro flora and the optimal conditions to produce warankasi.

Where most of the above milk based fermented products discussed above, very less or no alcohol is produced, Koumiss, a Chinese fermented milk product, contains 2% alcohol with a pH of 4.0. Traditional koumiss is prepared from camel or mare milk by mixing it with previous batch of prepared fermented koumiss. The mixtures are fermented in a suitable bag made from animal skin. During the fermentation, a regular beating and storage temperature of 20-30 °C is required in order to control the process and develop aroma. When strong foam and particular sour flavor are sensed, koumiss is ready for consumption . Predominantly, the LAB micro flora includes Lactobacilli, such as Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus casei, and Lactobacillus kefiri because of their high acid tolerance property. .The lactobacillus acidifies the milk whereas yeasts change the raw material into a carbonated, mildly alcoholic drink. Despite the fact that fermented products are potentially safe and serve better nutrition, the industrialization and large levels of production of Koumiss and several other Chinese fermented milk products still remain a challenge due to high variability in conditions that influence the final quality of the end product .

Fermented seafood products

The role of lactic acid bacteria (LAB) in seafood products is very complex, depending on the fish type, storage conditions, treatments, the flora used, and interaction between them . While salting and smoking are very frequently used to preserve fish in European countries, fermentation is used in a number of developing countries such as Scavendia because of its well known benefits. L. monocytogenes can grow on sea foods like cold- smoked salmon, cooked crab meat, smoked mussels and are frequently isolated from such sources even when stored at refrigeration temperatures . Table 1 shows the occurrence of L. monocytogenes and Listeria sp. in lightly preserved sea food products from different countries. Implementation of cleaning and sanitizing programs particularly designed at reducing the presence of L. monocytogenes in the factory, the safe eradication of L. monocytogenes from heat treated products and prevention of growth in ready to eat products are necessary to prevent outbreaks .

Huss pointed out that Clostridia sp can be traced even in vacuum and MAP packed fish fillets. Hazard Analysis Critical Control Point (HACCP) system is the most preferred approach for assuring safety and quality of sea food . Traditionally, sea food fermentation involves curing fish fillets with a sugar and salt mixture and normally herbs or spices. However, a very complex microbial flora develops. For instance, more than 50 strains of lactobacilli and carnobacteria were isolated from fermented salmon product. Hence the use of starter cultures was introduced to produce fermented fish products of better organoleptic qualities, safe and edible. L. sake LAD and L. alimentarius BJ33, because of their fast acidifying properties are thought to be starters best suited for fermentation of salmon fillets .

However, in som-fak, a Thai fermented fish product, pathogens such as salmonella and vibrio sp could survive probably due to low salt (< 4%), garlic (0.5-1%) and lactic acid (<1%) concentrations. Lactic acid was found to be inhibitory at more than 1.5% in such products. Lactobacillus sp. alone was inefficient to eliminate Salmonella sp, hence was to be used with high concentrations of garlic (6%).The use of garlic fermenting starter culture was then suggested to ensure the safety of the product . Pseudomonas and Shewanella can easily reach levels of 106-108 CFU per gram of flesh in few days when kept on ice for storage. This was attributed to low carbohydrate and high protein content in fishes which help metabolize these bacteria rapidly.

Marinated seafood salad is typically an Italian processed product. The typical ingredients of seafood salad are mussels, shrimps, squid, octopus, surimi and cuttlefish. Ingredients are boiled and then marinated in vinegar, salt, a few additives and water solution. When the pH of the product drops to 5, the marinade is dripped off and the product is packed after adding sunflower seed oil. Though, vinegar used is this formulation drops the pH to the desired value and makes the product relatively safe for consumption, bacteria like L.monocytogenes may still survive in such acidic conditions . Hence a study was conducted to study the biodiversity of LAB bacteria in such products. As pointed out earlier, LAB produces bacteriocins that inhibits the growth of such harmful pathogens and increase the shelf life of the product. Lactobacillus curates, Enterococcus sp. and Lactobacillus sanfranciscensis were some among the LAB flora found present in all the salad batches studied with a total LAB count reaching up to 8.0 log CFU/g . L. fuchuensis and L. gelidum also seem to be promising inhibitors of most of the pathogenic and spoiling strains . Contrary to this, Gram (1996) reported that LAB does not retard the growth of some marine bacteria. Many of them, predominantly Shewanella putrefaciens, Photobacterium phosphoreum and the Vibrionaceae use TMA-O as a terminal electron acceptor for anaerobic respiration. They mainly require sodium for their growth. These bacteria reduce TMA-O to trimethylamine, producing rotten fish odors and also sulphurous odors resulting from the breakdown of amino acids like cysteine and methionine even in vacuum packed containers .

Jeotgal is a salted and fermented food, used as an important additive for enhancing the taste of foods or as main course in Korean cuisine. It is prepared by adding 20-30% (w/w) salt to a variety of seafood such as, oyster, shellfish, shrimp etc and becomes edible through preservation and fermentation . More than 160 kinds of jeotgal exist in Korea, of which about 30 are of commercial importance. Principal genera include LAB and non-LAB flora such as Acromobacter, Bacillus, Clostridium, Flavobacterium, Halobacterium, Leuconostoc, Micrococcus, Pediococcus, Pseudomonas, Sarcina, and Serratia, and also yeasts such as Saccharomyces and Torulopsis. Strains of Staphylococcus and virgibacillus, also involved in proteolytic activities were thought to be playing an important role in such fermentations where high salt concentrations were used. These could be used to prepare starter cultures for the same .

Sikhae is another Korean cuisine where fermentation is acid based. The fish is stripped, cured with salt brine and kept overnight. It is then mixed with cooked millet, garlic and red pepper and left to ferment in an earthen jar for a week. Followed by fermentation it is left in a cool place for 3-4 weeks before consumption, where softening takes place. Leuconostoc mesenteroides and Lactohacillus plantarum are the dominant micro organisms in sikhae fermentation. Similar flora is found in Nazerushi, a fermented Japanese cuisine. The use of garlic makes sikhae and nazerushi a relatively safe product to consume by inhibiting a a majority of microbes in the raw material. However, sikhae is on the verge of suitable growth conditions of micro organisms like Bacillus cereus and Staphylococcus aureus, and hence consumed only in colder regions of Korea . Recently, Zeng et al found that Enterobacteria and Pseudomonads were under the detection limits in a variety of Chinese, low salt fermented fish products .

Other than salted fish and fermented fish sauces, fermented fish sausages are gaining commercial importance in Asian and European countries in a past few years. In China, silver carp is harvested in abundance and fermented at a larger scale. The use of mixed starter cultures restrains the growth of the micro flora present in raw materials, hydrolyze the muscle proteins and lower TBARS value indicating decreased lipid oxidation during fermentation . However, Lyhs et al reported the detection of some LAB groups such as Lactobacillus sakei, Lactobacillus curvatus and L. alimentarius and Lactobacillus fuchuensis in spoiled maatjes herring .

Meat based fermented foods.

Dry fermented sausages are a significant part of traditional meat products using lean pork/ beef meat. 'Feldkieker' sausages and other meat based fermented sausages are increasingly liked and consumed by the Germans. Most of the ones prepared in Central Germany are air dried i.e. unsmoked; and are made from fresh meat immediately after slaughter. The rate and degree of acid formation during fermentation is limited by low concentrations of sugar and low temperatures maintained i.e. below 15°C. About 2% salt is added, and nitrate is used as curing agent. In traditional practices, mould growth on the surface of non smoked sausages was obvious, but this had its own advantages. It helps to avoid fluctuations in relative humidity and also protect from detrimental effects of oxygen. However, the typical flavor of mould-ripened sausages is not accepted. Therefore, mould growth is limited by removing excess mould by brushing. Whereas, in Central Germany, air dried sausages are prepared by using starter cultures, post rigor pork and adjusting the ripening conditions to reduce the growth of moulds. These traditionally made sausages have a very good record of safety, attributed to factors like relatively cold fermentations and ageing. No cases of listeriosis have been verified in dry sausages. The bacterial counts of shigatoxin-forming E. coli and L. monocytogenes decreased by about 2 log cycles during ripening .

India is the world's largest producer of buffalo meat. Sausages from buffalo meat are prepared using meat, fat, spices, salt, sucrose, dextrose, MSG and a mixed LAB culture. The mixture of the above ingredients is kept in a pan at 7C for two days to help stabilize the microbial flora, followed by fermentation at 15C for two days. These fermented sausages are then smoked and dried. These sausages have enhanced texture and are edible. They have a shelf life of up to 60 days under refrigerated conditions .

During manufacturing of sausages, a wide range of contaminating micro organisms including salmonella, listeria and S. aureus are present on knives, mincing machines, cold rooms and other equipments. Hence, efficient cleaning and disinfection procedures should be followed on a regular basis . Although, Adams et al reviews that no growth of a five strain mix of E.coli O157:H7 is observed during the manufacture of fermented meat sausages, many studies prove that acid tolerant species of the same can survive during fermentation process irrespective of the acid conditions (pH> 4.5) even when stored at refrigerated conditions. A few studies point out that E.coli O157:H7 grow very well under salty and low temperature storage conditions . It is partially known that vacuum packaging also aids to the survival of such bacteria .Contradicting to this, Holley et al reported that fermented ham when sliced and vacuum packed with films of low oxygen permeability increases the shelf life and is safe for consumption for up to 21 days. However nothing was mentioned about E.coli O157:H7 in particular . Added MSG could have a synergistic effect in inhibiting these strains via fermentation .