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Listeria is a psychrophile and ice-cream is highly nutritious complete dairy based food stored in freezing or refrigerated conditions unless consumed. Detection for the Listeria spp. from the ice-cream sample was performed from twenty different Ice-cream samples. From these, two samples showed presence of Listeria sps. based on cultural, morphological, biochemical and physiological tests. Of these two isolates, one was taken and D50°C Value and effect of preservatives were determined. D50°C Value was determined to be 41 minutes with decrease rate - 0.07011 log10 CFU per minute being 4.42 times quicker than the regular increase rate of 0.016525 log10 CFU per minute at favorable incubation conditions at 25°C. Both of the preservatives were found out to be bactiostatic in action, and of the two preservatives, benzoic acid was found out to be the better preservatives than potassium nitrate. The growth rate was found to decrease extensively 5.3 times less using potassium nitrate and 6.3 times using sodium benzoate than without using any preservatives.
KEYWORDS: Listeria, D-Value, Preservatives, Ice-cream, Prakash
According to Subarna M. Pradhan, 2005, ice cream is an emerging dairy product in Nepal. Ice cream parlours are becoming common in major cities. Estimated annual ice cream production from DDC and private sector is about 1,000 tons and the main market is Kathmandu valley and Pokhara. Eighty three percent of ice cream production is in the private sector followed by imported brands from India.
Ice-cream represents a congealed dairy product produced by freezing a pasteurized mixture of milk, cream, milk solids other than fat, sugars, emulsifier and stabilizers (FEHD, HKSAR, 2001). Ice cream as defined by The Food And Drug Administration (FDA, 2003), “is a food produced by freezing, while stirring, a pasteurized mix consisting of one or more of the optional dairy ingredients specified in paragraph (b) of this section, and may contain one or more of the optional caseinates specified in paragraph (c) of this section subject to the conditions here in after set forth, one or more of the optional hydrolyzed milk proteins as provided for in paragraph (d) of this section subject to the conditions hereinafter set forth, and other safe and suitable non milk-derived ingredients; and excluding other food fats, except such as are natural components of flavoring ingredients used or are added in incidental amounts to accomplish specific functions. With all ingredients, ice- cream is a complete food, thus can act as a complete medium for the proper growth and development of the microorganisms. Added to that if stored in cold conditions and microorganisms are present, they can remain viable but only metabolically dormant.
Listeria monocytogenes is a Gram-positive bacterium that occurs widely in both agricultural (soil, vegetation, silage, faecal material, sewage, water), aquacultural, and food processing environments. L. monocytogenes is a transitory resident of the intestinal tract in humans, with 2 to 10% of the general population being carriers of the microorganism without any apparent health consequences. Although frequently present in raw foods of both plant and animal origin, sporadic cases or outbreaks of listeriosis are generally associated with ready-to-eat, refrigerated foods, and often involves the post processing recontamination of cooked foods (CAC, 2007). Similar to L. monocytogenes, L.ivanovii is also a potential threat to people with impaired immunity, and mostly pregnant women are the closest categorized into threat (CDC, 2010). Cell wall compositions of Listeria spp. show peptidoglycan, teichoic acids, lipoteichoic acids and more-or-less comparable to other Gram positive bacteria. But, one of the most confusing characters while observing under microscope is that they appear differently. Though Gram positive rods, they show variable Gram reaction, as when observed under microscope not clearly seen as rods. Sometimes they are confused with coccus in arrangements like diplococci, and some other time they appear to be rods arranged in angles, and some other times as single rods too. Thus, they don’t have that clear arrangements and they don’t show that clear Gram’s reaction (Goldfine et al., 2007; Garrity et al., 1984). They show umbrella like motility in Listeria motility agar, show actin-based motility, are oxidase negative, most are catalase positive. These are esculin positive, non-pigmented, but show bluish sheen at Henry’s angle at 10 X glass stage Microscope. Listeria monocytogenes was first isolated in 1918 from cerebrospinal fluid (meningitis) of a soldier. 1926: Sudden death of young rabbits; mononuclear leucocytosis; documented isolation (Murray et al.) of Bacterium monocytogenes. In 1940: named after Joseph Lister; “Listeria monocytogenes”. In 1979, these were identified to be the emerging food borne pathogens. In 1980s, numerous outbreaks were faced due to Listeria monocytogenes (USDA-FSIS, 2006). All together there are only six species of Listeria; L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L.ivanovii and L. grayi. Of these all, the only pathogenic strain to human is Listeria monocytogenes. They are mainly of two types, hemolytic and non-hemolytic. Even within Listeria monocytogenes there are different strains which appear to be hemolytic and some others non hemolytic. Hemolysis in Blood agar or any chromogenic agar is designed to detect the production of the Listeriolysin-O. This Listeriolysin-O is responsible for the disruption of the phagolysosome membrane and so it is the major virulence factor of L. monocytogenes (Collee et al., 2006). L. monocytogenes has 13 known serovars, but four of them (1/2a, 1/2b, 1/2c, and 4b) are responsible for over 95% of reported human listeriosis cases, and major outbreaks of listeriosis have been caused by L. monocytogenes 4b (USDA, 2007).
No detailed study on Listeria monocytogenes have been performed in any food products of Nepal. This study focuses the current situation and existence of Listeria species in ice-cream and also towards the control of the isolated Listeria species by temperature or with the use of preservatives.
MATERIALS AND METHODOLOGY
Sample collection was done by using the sealable-pouches. The sealable pouches had first been treated with alcohol and hot-air-oven dried at very low temperature of around 30°C, wrapped in the aluminum foil. All samples were collected in early morning time, and were reached to lab as soon as possible. At one time five different samples were collected in separate disinfected pouches, and labeled as soon as collected. Sample collections were done in the general manner in which the sample were sold. The sample was directly received into the pouches, the condition they are given or served to the local customers. All of the samples in pouches were kept in Ice-box, so that it won’t gain heat. As soon as reaching the lab the samples were again kept at deep-freezing temperature (-18°C) till sample is processed.
All together twenty different samples were collected and duplicate sampling was done. Based on colony morphology one hundred and eleven different colonies were taken from plate count agar after pour plating. The colonies so observed were then further enriched in Listeria enrichment broth and plated each by each on Listeria selective agar. By this, seventy-nine different isolates were determined to be more proximately Listeria in appearance. Of these, further test with esculin hydrolysis showed only sixty-two isolates more close to be Listeria species. With late gram positive, catalase positive and oxidase negative reactions; only thirty-six different isolates were more close to be Listeria species. With biochemical and cultural characters as methyl red positive/negative, voges proskauer positive, triple sugar iron agar acid/ acid, and typical characters on Listeria chromogenic agar, (rhamnose positive and glucosidase positive) and Listeria motility agar nineteen (umbrella-like motility) different colonies were then undergone the carbohydrate fermentation tests for confirmation with final five different proximate isolates. These isolates were then further undergone urease test for negative results and then slant agar of tryptone soy agar for blue sheen colonies. And finally two of the total isolates were determined to be the Listeria species.
Of all five proximate isolates, Listeria species i3 was processed for the determination of D-value and effect of the preservatives. D-value was determined at 50 ËšC using waterbath with shaker (sterile conditions maintained) and then enumeration at zeroth and regular time intervals.
For the determination of the effect of preservatives with potassium nitrate and sodium benzoate, the Log10 CFU change with respect to time and preservative being used in fresh milk with reference standards of potassium nitrate and sodium benzoate were determined, incubated at optimum 25 ËšC in water bath with shaker (sterile conditions maintained).
RESULT AND DISCUSSIONS
D-value was determined to be fourty-one minutes at 50 ËšC. Effect of potassium nitrate was found to be more distinct than that of sodium benzoate, with growth rate in milk with potassium nitrate (30mg/lit), with sodium benzoate (450 mg/lit), and without any preservatives respectively being 0.001988889 log10 CFU per minute, 0.001677778 log10 CFU per minutes and 0.010592222 log10 CFU per minute.
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