CHAPTER 1 INTRODUCTION PREAMBLE Alleviating hunger is a part of the first United Nation's Millennium Development Goal but still malnutrition remains a major fundamental cause of death among children worldwide. Malnutrition is responsible, directly or indirectly, for 54 % of the 10.8 million deaths per year in children under five in developing countries. Traditional household fermented cereal foods are consumed by large number of human beings as they provide important sources of nutrients but have also great potential in maintaining health and preventing diseases. This study deals with traditional fermented cereal-based food “Idli”, in which detailed bacterial flora is analyzed using modern molecular techniques. The work has followed to find out the role of the microflora in souring and leavening action in Idli batter. Selected isolates were also studied for their probiotic role that is essential in promoting health and preventing disease. It is also shown that the enteric pathogens like Salmonella cannot survive during Idli fermentation. This study was essential as the field has not been fully explored even though Idli is a staple diet of large number of individuals in India. Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 1 INTRODUCTION Foods are essential for the vital continued existence of animals and human beings. Since primeval times, different techniques have been used to process and preserve food. Discovery of food fermentation is considered as one of the oldest traditions of food processing and preservation. Fermented food was consumed much before mankind had any knowledge regarding microorganisms and their fermentation capabilities. 1.2.1 Fermented Foods Fermented foods can be defined as foods that have been subjected to the action of microorganisms to bring useful biochemical changes leading to major desirable alterations in the raw material (Campbell-Platt, 1987). During the process of fermentation, microorganisms are not only involved in the preparation and preservation of food products but also give the flavor and aroma to the food (Steinkraus, 2002). Change in texture is one of the important qualities associated with fermented foodstuff. Improvement of nutritional quality after food fermentation occurs due to enrichment of essential amino acids, fatty acids and vitamins. As compared to the raw material, fermented foods are preferred since they are easy to assimilate. Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 2 Fermented food replenishes necessary human gut microflora that is lost during long term antibiotic treatment. Another important aspect is the requirement of less cooking time for fermented foods. This saves heat energy. In almost all over the world, fermented foods and beverages contribute approximately 20- 40 % of the person’s dietary requirements. Fermented foods are geographically different and selective due to the differences in climate, social patterns, consumption practices and availability of raw materials (Nout, 2009). Fermented foods can be classified roughly based on the initial raw material used for preparation. The raw material can be cereals, legumes, milk, fish and meat, fruits, vegetables and starchy tubers. Various fermented foods [Table 1] prepared from different raw materials are consumed all over the world (Scott and Sullivan, 2008). Cereals and pulses are used globally as staple foods. Digestibility of conventional pulses and cereals is considerably lower compared to the foods made from fish and animal sources. In most developing countries belonging to Asia and Africa, pulses and cereals constitute the major dietary constituents. Difficulty in digestion of complex proteins, presence of high levels of insoluble fiber and anti-nutritional factors make plant based diets less nutritive. It is of interest to mention here that these difficult-to- Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 3 digest cereals are converted to nutrition rich food by fermentation, by the people of Africa and Asia (Nout, 2009). RAW FOOD ORGANISM MATERIAL Beer Grain malt Saccharomyces cerevisiae Bread Grain flour S. cerevisiae S. cerevisiae ; Candida rugosa ; Chocolate Cacao bean Kluyveromyces marxianus Coffee Coffee bean Erwinia dissolvens Soy Sauce Soy bean Aspergillus oryzae Miso Soy bean A. oryzae Lactobacillus plantarum ; L. brevis ; Kimchi Streptococcus faecalis ; Leuconostoc Daikon Napa mesenteroides ; Pediococcus pentosaceus Tempeh Soy bean Rhyzopus oligosporus Candida sp. ; Cryptococcus sp. ; Olives Olives Debaryomyces hanseii; Lactobacillus sp.; Saccharomyces sp. Leuconostoc mesenteroides ; Pickles Cucumber Lactobacillus sp. Coliform sp. ; Lactobacillus sp. ; Sauerkraut Cabbage Leuconostoc plantarum S. cerevisiae ; Gluconobacter sp. ; Vinegar Fruit juice Acetobacter sp. Meat + Sausages Pediococcus sp. ; Lactobacillus sp. spices Wine Grapes Saccharomyces cerevisiae Yogurt Milk L. bulgaricus ; Streptococcus sp. Butter Milk Streptococcus sp. ; Leuconostoc sp. Lactobacillus sp. ; Lactococcus sp. ; Pediococcus sp. ; Streptococcus sp. ; Cheese Milk Leuconostoc mesenteroides ; Propionibacter sp. Kefir Milk Saccharomyces kefir ; Torula kefir Table 1: Popular Fermented Foods Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 4 1.2.1.1 Fermented Foods in India India is a vast country which has different climatic conditions and numerous communities. This has resulted in a diverse array of fermented foods. The variety of regional fermented foods supplements the dietary requirements of communities belonging to different cultures. Fermentation is carried out using cereals, pulses, milk, vegetables and unripe fruits as initial raw material. Some of the popular Indian fermented beverages are prepared from rice, sprouted rice, rice husk, cereals, fruits, flowers, jaggery [unrefined sugar]. There is a wide variety of fermented foods consumed in India (Ramakrishnan, 1979). The names of these preparations are mostly colloquial and few very popular foods are listed in Table 2. 1.3 Microorganisms and Food Fermentations Fermentation is an anaerobic cellular process in which organic compounds are converted into simpler compounds, and chemical energy is produced (Frazier and Westhoff, 2008). Fermentation is thought to have been the prime means of energy production in primitive organisms when oxygen was at low concentration in the atmosphere. Food fermentation is the process in which biochemical modification of primary food products are brought about by the action of microorganisms and their enzymes. A variety of bacteria and fungi are involved in the fermentation of Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 5 foods (Nout, 2009) [Table 1-3]. RAW FOOD ORGANISM MATERIAL Rice + Adai Lactic acid bacteria Legume Streptococcus faecalis, Pediococcus Fermented Rice acidilactici, Bacillus sp., ; rice Microbacterium flavum Leuconostoc mesenteroides, Rice + Idli Streptococcus faecalis, pulse Pediococcus cerevisiae Lactobacillus fermentum, Cereal + Dhokla L. mesenteroides, Pichia silvicola, ; pulse Streptococcus faecalis, ; Torulopsis sp. L. mesenteroides, L. fermentum, ; Ambali Cereal Streptococcus faecalis Lactic acid bacteria Nan wheat Saccharomyces cerevisiae Taotjo Cereal Aspergillus oryzae Table 2: Popular Indian [sub-continental] Fermented Foods GRAM GRAM NEGATIVE FILAMENTOU POSITIVE YEAST BACTERIA S FUNGI BACTERIA Acetobacter Arthrobacter Aspergillus Candida Acinetobacter Bacillus Aureobasidium Cryptococcus Alcaligenes Bifidobacterium Fusarium Debaromyces Flavobacterium Lactobacillus Mucor Hansenula Gluconobacter Leuconostoc Neurospora Kluyveromyces Klebsiela Micrococcus Penicillium Pichia Methylococcus Pediococcus Rhizopus Rhodotorula Propionibacterium Streptococci Trichoderma Saccharomyces Xanthomonas Streptomyces Yarrowia Zygosacchar- Zymomonas Weissella omyces Table 3: Various Microorganisms [Genus] involved in Food Fermentation Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 6 Most of these facultative anaerobic microorganisms have catabolic activities to oxidize sugars to yield various acids, alcohols and carbon dioxide. Homo-fermentative microorganisms utilize Embden-Meyerhof-Parnas [EMP] and Entener Doudoroff [ED] pathways to metabolize hexose to obtain ATP. In EMP pathway, one mole of glucose is converted to two moles of pyruvate and it is the most important catabolic pathway in fermentative microorganisms. Conversion of pyruvate to lactic acid or ethanol [Figure 1] balances the redox reaction. Glucose Glycolysis D-Glyceraldehyde-3-phosphate NAD NADH + H+ Pyruvate NAD NADH + H+ Lactic acid Figure 1: Formation of lactate by Homo-fermentative bacteria Two ATP molecules are generated during this reaction, proving EMP pathway as most efficient energy generating pathway in ferme- Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 7 tative microorganisms (Gottschalk, 1986). In dairy fermentations, galactose catabolism from milk sugar “lactose” is essentially done by enzymes involved in ED pathway and during this only one ATP molecule is generated (Montville and Matthews, 2008). Heterofermentative pathway [Figure 2] is characterized by generation of carbon dioxide from hexose sugar. It generates one ATP molecule. Enzymes in this pathway allow the utilization of five and three carbon sugars (Gottschalk, 1986). Glucose Glucose 6-phosphate 6-phospho gluconic acid Carbon dioxide Ribulose 5-phosphate Xylulose 5-phosphate Glyceraldehyde 3-phosphate Acetyl phosphate Acetaldehyde Lactic acid Ethanol Figure 2: Fermentation of glucose by Heterofermentative bacteria Lokesh P. Sharma, Ph. D. Thesis, 2011 Page 8 Cereals are fermented all over the world. Due to