Koozh is a fermented porridge made from millet flour and rice consumed by ethnic communities in Tamilnadu, India. Samples from six street vendors were collected for enumeration of standard plate count (SPC), pathogens, lactic acid bacteria (LAB), yeast and mould count (YMC). Sixty nine bacterial isolates from the samples which were gram positive, catalase negative, cytochrome oxidase negative and non motile were presumed to be LAB. The grouping of isolates was based on their ability to produce ammonia from arginine, sodium chloride tolerance and growth at different temperatures and pH. The pH of the Koozh ranged from 4.33 to 4.91. No Staphaylococcus areus and Listeria sp. were found. Hazard of high risk were in two samples with Salmonella sp. and Shigella, three samples with Clostridia sp., all samples with E. coli. SPC, LAB and YMC were within an average of 8 log cfu/g. Sixty nine percent presumptive LAB could grow at 45 oC, 9.6pH and produce ammonia from arginine. Eighty percent (57/69) were able to grow at 15 oC, eighty four percent in pH3.6 and forty four percent produced gas from glucose. Further test is required to test if the LAB isolates exhibit probiotic properties.
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"Millet" is a generic term for a heterogeneous group of forage grasses known for their small "coarse" grains . It is an important minor cereal in tropical and subtropical regions , India and Africa are considered to be home lands for millets . India is currently the largest producer of millet . Traditional fermented food contains high nutritive value and would develop a diversity of flavour, aroma and texture in food substrate  and provide protection against bacterial pathogens . Fermentation of a food material in a controlled environment leads to a desirable food product but in uncontrolled conditions results in spoilage. Changes that take place during fermentation include increase in amino nitrogen, the breakdown of proteins and destruction of any inhibitors that may be present .
Many traditional fermented products are made from millets both in African and Asian Countries. Koozh a Tamil name for a porridge are ready to eat, popular meal or energy / refreshment drink sold in the streets during the morning to noon in TamilNadu, India. It's either made from finger millet - Eleusine coracana (Kezhvaragu in Tamil) or pearl millet-Pennisetum glaucum (Cumbu) flour and broken rice (noyee) in a mud pot .
Koozh preparation is a two day continuous process shown in flow chart (Fig.1) served from a 12L to 15L plastic or a stainless steel container. The container are moved on a push trolley or a bicycle but in some rural markets it is kept in a clay pot laid on wet sand over a table under thatched roofing. It is served in a 500 to 600 mL wide mouthed stainless steel container (Sembu) along with accompaniments like freshly cut onion, pickle (Oorgai), coriander leaf chutney (Kothamalli Thokku) and dried fried products like- chillies (Moar milagai), turkey berry (Sundakkai Vathal), cluster beans (Kothavarangai Vathal) consumed by daily wage earners, labourers and drivers. The handling during preparation varies depending on regions subsequently altering their organoleptic properties and microbial flora of koozh.
Homemade preparations of millets are fed to growing infants as malted flour porridge which is considered "nourishment for infants" .Finger millet after fermentation contains significant amounts of B-complex vitamins, energy, bioavailable minerals, essential amino acids, iron, calcium, fibre, micro nutrientsÂ and minerals .
The objective of the study is to assess the microbial quality of Koozh that is sold in the street.
Materials and Methods
Finger and pearl millet koozh were collected using a 250 mL autoclaved wide mouthed screw capped plastic containers from market places in Salem, Tamilnadu, India and immediately kept in meal mate (Milton, Mumbai) containing ice packets. It was transported to the Department of Food Technology, SPIC Bioprocess Laboratory, Anna University, Chennai-25 and subjected to enrichment technique for microbial analysis . Finger millet koozh samples sold in road side trolley vendors were bought from four different places located in suburbs of Chennai with autoclaved 500 mL containers and microbial analysis were conducted on the same day.
Media and Supplements
The following media and supplements were used for the microbial analysis: Arginine dihydrolase broth (M619,HiMedia,Mumbai), Bacillus Cereus Agar base (M833, HiMedia, Mumbai) , Baird Parker Agar Base (M043, HiMedia, Mumbai), Clostridial Agar (M497,HiMedia, Mumbai), De Man Rogosa Sharpe broth- MRS (M641,HiMedia, Mumbai), E. coli Direct Agar- ECD (M1357,HiMedia, Mumbai), Egg yolk Emulsion (FD045, HiMedia, Mumbai), Egg yolk Tellurite Emulsion (FD046, HiMedia, Mumbai), Listeria Identification agar (M1064, HiMedia, Mumbai), Listeria selective supplement-PALCAM (FD061, HiMedia, Mumbai), M17 Broth (M1029, HiMedia, Mumbai), Plate count agar (M091, HiMedia, Mumbai), Polymxin B selective supplement (FD003, HiMedia, Mumbai), Potato dextrose agar(M096,HiMedia,Mumbai), SS agar (M108, HiMedia, Mumbai), Standard plate count agar(M096, HiMedia, Mumbai), Violet red bile glucose agar (M581,HiMedia, Mumbai), Yeast Malt Agar (M424, HiMedia, Mumbai), Peptone (RM667,HiMedia, Mumbai) and Agar (RM026,HiMedia, Mumbai)
Chemicals and Reagents
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Benzyl penicillin potassium salt (44197 2W,BDH,UAE), Calcium carbonate precipitated (17630, Merck, Germany), Chloramphenicol (RM218, HiMedia, Mumbai), Gram Stain-Kit (K001-1KT, HiMedia, Mumbai), Sodium chloride (RM031, HiMedia, Mumbai), Streptomycin sulphate (RM220,HiMedia,Mumbai) and Hydrogen peroxide solution 50% Purified (Merck, Germany) were used for the microbial analysis.
Determination of pH
Koozh samples pH were analysed immediately after purchase using AP-1 plus, pH Meter (Susima Technologies, Chennai) containing epoxy body sensor pH probe (Van London pHoenix Company, Texas, USA).
Enumeration of Bacteria, Pathogens and Yeast
All samples were serially diluted prior to microbial analysis. Peptone buffer saline  PBS and CM101 Vortex Mixer (REMI, Mumbai) were used for serial dilution. The following selective media were used for enumeration by spread plate method - Violet red bile glucose agar for Enterobacteria , Clostridial Agar for pathogenic Clostridia, ECD Agar for colifroms especially E.coli, Listeria identification agar supplemented (supp.) with PALCAM for Listeria monocytogenes, Bacillus cereus agar supp. with egg yolk, Baird Parker supplemented supp. with egg yolk tellurite for Staphylococcus, Standard count agar (SPC) for total bacterial count ,SS agar for Salmonella and Shigella. After solidification of the poured selective media and adding the serially diluted PBS,
it was kept over a PlateMasterTM for SpeedPlatingTM (LA622, HiMedia, Mumbai). Yeast and moulds was enumerated on pour plates of Potato dextrose agar (PDA) added with filter sterilized Chloramphenicol .
Bacterial plates were incubated aerobically at 37oC for 24 h but for enumeration of infectious Clostridia sp, inoculated Clostridial agar kept under Anaerobic Gas Pack Systems (LE002, HiMedia, Mumbai). Yeast and mould plates were incubated at 25oC for 72 h. Colonies were enumerated manually using a Lapiz Digital Colony Counter (Medica instruments Mfg. Co., Mumbai).All experiments were carried out in triplicates.
Characterization and Isolation of LAB
MRS and M17 media, were pour plated separately and incubated at 37oC for 24 hours. Colonies on individual culture plates were examined for difference in morphology like shape, size, border, colour and elevation from the surface of the media. Catalase activity, Gram reaction using the HiMedia's Gram's staining kit and cell motility test were conducted . The positive presumptive LAB isolates were named, numbered according to their difference and quadrant streaked on a fresh plates. These isolates were grown in selective broth and cryo-preserved at - 80oC for further tests. MRS, M17 agar media supplemented with 1% CaCO3 was pour plated with presumptive LAB and incubated at 37oC for 24 hours using the Anaerobic Gas Pack Systems  to identify if the isolates could grow in anaerobic condition and produce acid.
Growth at two temperatures -15oC, 45oC and pH - 3.6 and 9.6  were conducted. The turbidity of the inoculated broth, after incubation at 37oC for 72 hours was considered positive. Gas production from glucose  using selective broth as basal medium and ammonia utilization from arginine was conducted using arginine dihydrolase broth .
Result and Discussion:
Koozh is a part of daily diet and during the specific times of religious festivals. Its preparation was learnt from their forefathers and had been practised ever since. As evident from figure one, Koozh is a product of millet and rice, from double fermentation caused by a mixture of flora involving bacteria and yeast. The food uses of finger millet are confined to flour based products because it was not possible to decorticate the millet similar to cereals. This is mainly due to the highly floury endosperm . "Pearl millet malting reduces the anti-nutritient phytic acid consequently increasing the bioavalability of proteins and minerals .Though the mechanism of mousy odour is not known but due to the decrease in pH from the growth of lactic acid bacteria it is almost eliminated" .
The fermentation mediated by the microbial flora would be initiated when the millet is ground to flour, in the pre used milling machine. After adding water to the millet flour, hand is used for mixing were the human skin serves as the starter flora for the first fermentation. This overnight fermented millet slurry kept for 12-16 hours is cooked along with broken rice (Figure 1). Rice sold commercially in Bangladesh, heat resistant spores are a major problem because it survives parboiling and milling stages .Cooking inactivates most contaminating microorganisms; heat resistant bacterial endospores may survive or even be stimulated to germinate  and cooking not only kills vegetative cell of competing bacteria and fungi but also activates spores of bacilli to germinate .
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Allowing this slurry to ferment overnight, the resulting product is called Kali. On the third day small part of the product is transferred into a wide mouthed vessel and diluted with potable water and hand mixed to prepare Koozh. A similar product in Inner Mongolia a traditional food, naturally fermented by residual sour broth, made from millet or rice called Congee (acidic-gruel).Butter milk a product of diluted curd is added directly to the whole lot at the ratio of 1:12 (v/v) or added separately during the time of serving.
The pH of the collected sample was in the acidic, range from 4.3 - 4.9 (Table: 1). Kodo ko jaanr a fermented millet beverage of the Himalayas, the pH ranged from 3.7- 4.5 . Koko sour water of Northern Ghana, made from overnight steeping of pearl millet , the final product pH was 3.9Â± 0.1 .In Uganda's western highlands, from a mixture of millet and sorghum flour the product called bushera with 3.7Â± 0.1pH and sorghum bushera had 4.0-4.5 pH. There was a decrease in number of coliforms, which may be due to high acidity (low pH) as a result of acid production by lactic acid bacteria .
Without considering the first two samples which was subjected to enrichment technique SPC, LAB and yeast all had a similar average of 8 log cfu/g value .A co-metabolism between yeasts and lactic acid bacteria may exist, were the bacteria providing the acid environment, which selects the growth of yeasts that in turn provide vitamins and other growth factors to the bacteria [26, 27]. There was absence of Mould in all the samples.
Gram positive, catalase negative and non-motile isolates were considered as presumptive LAB. Isolates was subjected to various conditions for grouping (Table-3). MRS and M17 supplemented with CaCl2 in both aerobic and anaerobic condition produced clearance zone confirming organic acid production in both Homo and hetero fermentative presumptive LABs.
Out of sixty nine LAB isolates twelve percent (8/69) were identified as bacillus rest were cocci. All isolates were found to be gram positive, catalase negative and non-motile. All grew at 45 oC and produced ammonia from arginine. Forty five percent (31/69) possessed the ability to produce gas from glucose (heterofermentatives), ninety three percent (57/69) were able to grow in 15 oC, and forty three percent (39/69) in pH 3.6.
The Mills are not cleaned prior to grounding of the millet which might serve as the first source of contamination. Adhesion of microorganisms to equipment surfaces has the potential to transmit pathogens to food, and this is apparent in the food processing industry [28, 29]. Exposure of pathogens to surfaces may take place either by direct contact with contaminated objects or indirectly through airborne particles . Recontamination of the cooked porridge may occur through handling or implements / utensils . The surfaces of equipment used for food handling, storage or processing are recognized as a major source of microbial contamination  as bacteria have the ability to attach to surfaces commonly found in the food processing environment, such as polystyrene, hydroxyapatite, glass, rubber, and stainless steel [33,34,35,36]. Earlier studies indicate that cross-contamination from raw products via hands, cleaning cloths or sponges, and utensils used with foods that were not subjected to further cooking contributed to the occurrence of outbreaks of food borne illness [37, 38, 39].
Pathogens presence were confirmed using biochemical test . Microbial contamination has two components: first, the saprophytic flora required in large population responsible for food spoilage and second, the pathogenic flora, a few cells which can cause infections and poisoning to humans and animals .
Listeria and Staphylococcus areus were not detected in any samples. According to http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1259151921557 the pathogenic flora were categorised. E.coli was found in all the samples making it a high risk category. The third sample did not have any other pathogen except E. coli, which is a enteric pathogen , important member of the coliform group, a part of the normal flora of the intestine of human and vertebrates, some strains can cause gastroenteritis, diarrhoea and urinary tract infections . The major reason may be due to the addition of non parboiled potable metro water and handling practices.
The first and the second sample had Clostridium sp., Salmonella sp., Shigella sp at hazardous count levels and Enterobacteria, B.cereus in moderate levels. This is due to the overnight transportation of koozh products.Studies have shown that pathogens such as enterotoxigenicÂ Escherichia coli, Shigella flexneri,Â Salmonella typhimurium,Â Bacillus cereus andÂ Campylobacter jejuniÂ are adversely affected when present in traditionalÂ fermented food [44, 45, 46, 47, 48].
The fourth sample had Clostridum sp. at hazardous and Enterobacteria in moderate risk category. Food poisoning by C. perfringens especially kitchen strains  produces a large amount of heat labile enterotoxins that are cytotoxic and disrupts the membrane of epithelial cells, resulting in diarrhoea . .Its presence in heat treated foods would be due to inadequate cooking or post-processing contamination, but the use of sanitising rinses may reduce but not entirely remove these organisms with accordance to HPA, UK.
Fifth had Closridum at moderate risk and had B.cereus in low level. Bacillus cereus is found more efficient in surviving on processing equipments by implementing itself to germinate a step prior to sanitization [51, 52]. The sixth sample had only B.cereus in low level. B. cereus a food pathogen produces various toxins causes enteric diarrheal disease to consumer" .
During serving of onion, knife and the cutting board were not cleaned prior to use. Hazard analyses carried out in households at Dominican Republic reported that kitchen knives and blenders were contaminated with Salmonella spp . A separate sembu would be dipped with hand and poured in to another cleaned sembu and served. After usage the sembu were washed with prewashed water, without detergent and kept inverted for the next serving. Plastic or metal plates are offered to distribute for self service. But in some places only a spoon would be kept for self service where the consumer needs to keep the accompaniments in hands and consume. The spoon would be again kept back where it was taken without washing.
Accompaniments kept open during the time of serving on busy location like highways or road corner, leads to heavy exposure to smoke and dust. Oil fried products that are kept open increases the possibility of contamination because oil attracts direct dust and air borne pathogens. This street food is prepared by lower income group with little formal education in less hygienic which gives a good opportunity for pathogens to divide, replicate and multiply.
But the Enteric bacterial pathogens inÂ foodÂ and water must survive the acidity of the stomach before they reach the intestinal tractÂ  and cause disease to the host. In conclusion ,koozh contains hazardous count of pathogens but due to the presence of high LAB count, might posses probiotic properties that would suppress the growth of pathogen that is further to be studied.
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