The purification of isolates was done by moving Gram +eve rods ND Cisco shaped bacteria to selective media MRS.. And M-17 plates. The isolates were sub cultured till pure isolates were got. From 20 raw milk samples a total of 150 LAB positives were got, in which 22 and 128 were identified as lactic acid Cisco and lactic acid bacilli,respectively. Also, our biochemical tests showed the occurrence 11 and 13 of 11 and 13 Lactose’s lactic subs. Careerism and Leucosis misidentifies subs. Careerism among lactic acid Cisco. N the case of lactic acid bacilli, Lasciviously Helvetica 18; Lasciviously plantar 37; Lasciviously braves 8; Lasciviously cases subs. Ease 18 and Lasciviously telecommunications subs. Bulgarians 47 was found. In the lactic acid Cisco and bacilli, Leucosis misidentifies subs. Careerism and Lasciviously telecommunications subs. Bulgarians were found to be the more dominant species, respectively. Bacterial phages were inducted from the Lactic acid bacteria and enumerated by using several biochemical techniques.
INTRODUCTION To produce flavor and acidity at desired levels,fermented milk products are prepared in controlled fermentation of milk. (Tap, 2000). Starter culture organisms in this fermentations belongs to bacteria family known as the Lactic Acid Bacteria (LAB). These LABS are identified by of morphological,and physiological characteristics. LAB are widely found in nature and almost in all micro flora. LAB are gram positive bacteria and are important in food fermentation. Other species of the genus Lasciviously, Lactose’s and Leucosis are added to this group.
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The lactic acid fermentation process has been known by human for long time and even applied in some activities. LAB has also been an efficient method of natural preservation. Furthermore lab determine the nutritional value, flavor and texture of food and feeds). Industrialization of the illogical ‘revolution’ of foodstuffs has LAB an economic boost because they are important in safety aspects of fermented products. Lactic acid is used by food industry as an acidulate and preservative for the production of sour curd cheese and yoghurt (Linkable and Griffin, 1971).
Lactose are the major mesospheric bacteria used for acid production in dairy fermentations and used as starter cultures in the manufacture of a vast range of dairy foods including fermented milks, lactic butter, cheese and lactic casein (Ward et al. , 2002). MATERIALS AND METHODS Raw milk samples: Raw milk samples were collected in sterilized specimen tootles from the local dairy shops around the university,including the raw milk from the university’s dairy department. The raw milk were kept at 4 for more use. Acetic acid bacteria isolation from raw milk: The samples were weighed and homogenates aseptically-Each sample, a 1. 10 dilution was made by using potent water then by making a 10 pack of continued dilution. The 0. 1 ml taken from each dilution was then sub cultured duplicate into the M 17 and MRS.. Gars used for isolating LAB (Baddish et al. , AAA; Guesses and Kill, 2004). Len order to counter yeast growth the media were then added with 100 MGM of yuck-hedgier prior to being incubated in optimum temperatures ( 3000 for 3 days (Buses et al. , 2001; Collaboration et al. , 2005).
The agar plates of MS were incubated in anaerobic conditions using the Gas-Pack system at ICC for 3 days to provide an optimum temperature for growing the different genus of bacteria. MI 7 agar plates were also incubated in anaerobic conditions at ICC for 2 days to set up an optimal temperature for growing lactose. Higher dilutions were used to perform total counts. Colonies were then selected randomly and the streak plating method employed to purify the stains. The strains were kept in 2 notations including at ICC (for MRS.. And MI 7 plates ) and at -ICC (for MI 7 and MRS.. Broth) with by 20% glycerol.
Identification of the bacterial strains:The strains were subjected to gram staining,catalane and spore formation tests. (Harridan and Menace, 1976). All Colonies were characterized in MRS.. And M 17 gars. The strains that gave gram positive and catalyst negative results were set aside for further identification. (Sharpe, 1979). The growth of the bacteria at different temperatures of between Growth 10-ICC for 3-6 days , resistance to ICC for 30 min (Sherman test), growth in the presence of 20th% NCAA and different pHs (4. And 6. 5) were used to identify the strains of LAB.
Argentine and calculi hydrolysis,citrate utilization, acetone procrastinations of gas from glucose and production of Textron from sucrose were also determined. The strains were then tested for fermentation of L-rabbinate, D-close, calaboose, D-fructose, servitor, lactose, melodies, saccharine, D-refines, milestone, manses and glucose. Bacterial growth in the different temperatures were confirmed by turbidity change in MRS.. Or MI 7 after incubation(after 24,48 and 72 hrs). Microbial tolerance to the diverse levels of salt, pH and heat was evaluated.
Argentine dehydrates agar and calculi acid agar were used to perform the hydrolysis tests. For determination of citrate utilization and acetone production, citrate and MR..-UP gars were used. MRS.. Or MOM broths with Durham tubes were used for determination of gas production and the dirtier production from sucrose was done in MS. TO assess the sugars fermentation in a medium a solution with the following composition was used (gel-1): bovine extract, 10. 0; neophyte, 10. 0; yeast extract, 5. 0; KAPPA, 2. 0; Cinchona+AH, 5. 0; diamond citrate, 2. 0; Mages, 0. 2; Mains, 0. 5; broom- cresol-purple, 0. 17; teen 80, 1 ml. Carbon utilization was also tested. Phage induction MRS.. Broth liquid cultures were equally divided into two sterile tubes. Each tube was labeled as ‘mitotic C’ and the other as ‘control’. Pill of neomycin was added to the tube labeled as ‘mitotic’ and aseptic techniques of flaming the neck before and after adding the neomycin. A starch agar plate marked SAT containing nutrient agar with soluble starch was already provided A casein agar plate that contained nutrient agar mixture added skim milk was given and marked CA.
All the three plates were inoculated by streaking of the MRS.. Lasciviously lattes culture. This was done with the help of the loop. The loop was flamed and a colony of the culture was collected. The plates were then streaked with the culture. The plates were then incubated for 12-18 hours at ICC. The bacteria were also transferred into the nutrient agar plate to set up for biochemical tests.. Enumeration of bacterial phages Phage stock was diluted to achieve a plaque count on plates of 100-250 pouf (plaque forming units). All the dilutions were mixed thoroughly in a sterile saline.
The phage was then plated by removing one soft agar at a time,then adding 0. Ml of bacterial suspension to it. This was also followed by 0. 1 of diluted phage The agar tube was rolled between palms to mix and quickly pour to suffice of warm base agar plate. Quick gentle figure patterns were done on the surface of the base plate agar The agar was allowed to harden and incubated for 35 degree Celsius for 8 hours. Results Catalane test After incubation, hydrogen peroxide was added to the one colony on the nutrient agar plate. Small bubbles of oxygen Hereford which indicated a positive result for catalane.
Figure 2 – The catalane test Starch hydrolysis test When iodine solution was poured to the starch agar plate and allowed to rest for lose to 2 minutes,the plate turned blue which indicated the presence of starch that has not been hydrolysis. Fig 1 -Growth of MRS.. Lasciviously lattes on starch agar plate (A) before the addition of iodine solution and (B) after the addition of iodine solution. Agar test The position of the growth in the tube was observed. The growth was throughout the tube, but near the surface, the growth was highest which indicated being rattlebrain. Figure 3- The bacteria stabbed in both the tubes containing AN and MRS…
Carbohydrate fermentation substrates API test strips were used to identify the bacteria and the results showed it was Lactose’s lactic coprocessors 1. Casein Hydrolysis The casein agar plates were examined to see any clearing around the colonies after being incubated for 48 hours. There was no clearing of the agar around the bacterial growth. Therefore, the results showed negative casein hydrolysis. Gelatin hydrolysis Saturated ammonium sultanate was added onto the gelatin agar plate there was no precipitation indicating negative hydrolysis Figure 4 – The results obtained after the data was entered on the computer database.
Figure 5 – The difference between a control and the samples of bacteria. Test for phage induction Once mitotic C was added to the MRS.. Liquid broth, it was observed for the induction of phages. It showed there was a clear lysine of the turbid culture. Figure 6 – Comparison between a control and bacteria culture containing mitotic C All 150 Gram *non-cosponsoring mans catalane negative were characterized as follows: Mesospheric homo-fermentation Cisco, 1 1 isolated:alt was characterized by argentine dehydrates negative, argentine hydrolysis negative, citrate negative and action negative This group was identifies as Lactose’s lactic subs.
Careerism . The microorganisms were spherically shape. They occurred in pairs tit non motile, facultative anaerobic fermentation metabolism. Mesospheric heterogeneity’s Cisco, 13 isolated:Microorganisms in this group had a close relation with Leucosis misidentifies subs. Careerism . They were argentine negative,glucose positive,acetone positive and dextrose positive. Laconically bacteria, 128 isolated: The group was divided into 3: (1) Mesospheric facultative heterogeneity’s Laconically (55 isolates)launched Laconically plantar (37 isolates)and Laconically. Cases subs. Ease (36 isolates, (2) Thermometric obligate homo-fermentation Laconically (64 isolates) Included Laconically. Leviticus (17 isolates) and Laconically. Telecommunications subs. Bulgarians (47 isolates). They were lactose positive understood positive. (3) mesospheric obligate hetero-fermentation Laconically (8 isolates) Included Laconically. Braves (18 isolates) Discussion It was discovered that mesospheric facultative hetero-fermentation laconically group was divided into two;37 isolates were identified to be laconically plantar mans 18 isolates as laconically cases subs. Ease. This results are also consistent with other research works such as the isolation of lactic acid acetate from Mammas traditional fermented milk(Mahatma et al. ,2004). For the second group, 1 7 isolates were identified as Laconically plantar and 47 isolates identified as laconically delactobacillirueskii subs. Bulgarians. Furthermore,laconically braves isolates(8) were identified using manses and milestone fermentation. In the Cisco group, 12 and 22 isolates were identified as Leucosis misidentifies subs. Careerism and Lactose’s lactic subs. Careerism. Respectively.