Dairy food staff such as soft cheese,cream cheese,raw milk,sour cream,yoghurt and probiotic yoghurt products can be a rich source of diverse lactic acid bacteria.The objective of this lab practical was to isolate lactic acid bacteria(LAB) form raw milk,establishment of pure cultures of LAB,identify LAB and phage recovery and enumeration of recoverd phage.Raw milk was chosen as a sample so as to have a more positive result.To identify bacteria Lab isolated from raw milk,biochemical,morphological ,physiological and cultural characteristics were employed. The purification of isolates was done by moving Gram +ve ro ds and cocci 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 cocci and lactic acid bacilli,respectively.
Also, our biochemical tests showed the occurrence 11 and 13 of 11 and 13 Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris among lactic acid cocci.In the case of lactic acid bacilli, Lactobacillus helveticus 18; Lactobacillus plantarum 37; Lactobacillus brevis 8; Lactobacillus casei subsp. casei 18 and Lactobacillus delactobacillirueckii subsp. bulgaricus 47 was found. In the lactic acid cocci and bacilli, Leuconostoc mesenteroides subsp. cremoris and Lactobacillus delactobacillirueckii subsp. bulgaricus 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.
To produce flavor and acidity at desired levels,fermented milk products are prepared in controlled fermentation of milk. (Thapa, 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 Lactobacillus, Lactococcus and Leuconostoc are added to this group. 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 biological ‘revolution’ of foodstuffs has LAB an ecomonic boost because they are important in safety aspects of fermented products. Lactic acid is used by food industry as an acidulent and preservative for the production of sour curd cheese and yoghurt (Linkater and Griffin, 1971). Lactococci are the major mesophilic 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 bottles 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. lactic acid bacteria isolation from raw milk: The samples were weighed and homogenized aseptically.Each sample, a 1.10 dilution was made by using peptone water then by making a 10 pack of continued dilution. The 0.1 ml taken from each dilution was then sub cultured duplicately into the M 17 and MRS agars used for isolating LAB (Badis et al., 2004a; Guessas and Kihal, 2004).In order to counter yeast growth the media were then added with 100 mg of cyclo-heximide prior to being incubated in optimum temperatures ( 30°C) for 3 days (Beukes et al., 2001; Kalavrouzioti et al., 2005).
The agar plates of MSR were incubated in anaerobic conditions using the Gas-Pack system at 30°C for 3 days to provide an optimum temperature for growing the different genus of bacteria. M17 agar plates were also incubated in anaerobic conditions at 30°C for 2 days to set up an optimal temperature for growing lactococci.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 conditions including at 4°C (for MRS and M17 plates ) and at -20°C (for M17 and MRS broth) withh by 20% glycerol.
Identification of the bacterial strains:The strains were subjected to gram staining,catalase and spore formation tests. (Harrigan and McCance, 1976).All Colonies were characterized in MRS and M 17 agars.The strains that gave gram positive and catalse negative results were set aside for further identification.(Sharpe, 1979).The growth of the bacteria at different temperatures of between Growth 10-45°C for 3-6 days , resistance to 60°C for 30 min (Sherman test), growth in the presence of 2 to 6 % NaCl and different pHs (4.5 and 6.5) were used to identify the strains of LAB. Arginine and asculin hydrolysis,citrate utilistaion, acetone productionformation of gas from glucose and production of dextran from sucrose were also determined. The starins were then tested for fermentation of L-arabinose, D-xylose, galactose, D-fructose, sorbitol, lactose, melibiose, saccharose, D-raffinose, melezitose, mannose and glucose.
Bacterial growth in the different temperatures were confirmed by turbidity change in MRS or M17 after incubation(after 24,48 and 72 hrs).Microbial tolerance to the diverse levels of salt, pH and heat was evaluated. Arginine dihydrolase agar and asculin acid agar were used to perform the hydrolysis tests. For determination of citrate utilization and acetone production, citrate and MR-VP agars were used. MRS or M17 broths with Durham tubes were used for determination of gas production and the detrin production from sucrose was done in MSR.To assess the sugars fermentation in a medium a solution with the following composition was used (gL-1): bovine extract, 10.0; neopepton, 10.0; yeast extract, 5.0; K2HPO4, 2.0; CH3COONa+3H2O, 5.0; diamonium citrate, 2.0; MgSO4, 0.2; MnSO4, 0.05; brom-cresol-purple, 0.17; tween 80, 1 mL. Carbon utilization was also tested.
MRSA broth liquid culutures were equally divided into two sterile tubes.Each tube was labeled as ‘mitomycin C’ and the other as ‘control’. 500µl of micomycin was added to the tube labelld as ‘mitomycin’ and ascpetic techniques of flaming the neck before and after adding the mycomycin. A starch agar plate marked STA containing nutrient agr 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 MRSA Lactobacillus lattis 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 37oC. 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 pfu (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 0.3ml 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 suface 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 celcious for 8 hours.
After incubation, hydrogen peroxide was added to the one colony on the nutrient agar plate. Small bubbles of oxygen wereformed which indicated a positive result for catalase.
Figure 2 – The catalase test
Starch hydrolysis test
When iodine solution was poured to the starch agar plate and allowed to rest for close to 2 minutes,the plate turned blue which indicated the presence of starch that has not been hydrolysed.
Fig 1 -Growth of MRSA Lactobacillus lattis on starch agar plate (A) before the addition of iodine solution and (B) after the addition of iodine solution.
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 aerotolerant.
Figure 3- The bacteria stabbed in both the tubes containing NA and MRS.
Carbohydrate fermentation substrates
API test strips were used to identify the bacteria and the results showed it was Lactococcus lactis sspcremoris 1.
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.
Saturated ammonium sulpahate was added onto the gelatine 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 mitomycin C was added to the MRS liquid broth, it was observed for the induction of phages. It showed there was a clear lysis of the turbid culture.
Figure 6 – Comparison between a control and bacteria culture containing mitomycin C
All 150 Gram +non-sporeforming ans catalase negative were charcterised as follows:
Mesophilic homo-fermentative cocci, 11 isolated:It was characterized by arginine dihydrolase negative, arginine hydrolysis negative, citrate negative and acetoin negative This gropu was identifies as Lactococcus lactis subsp. Cremoris .The microorganisms were spherically shape.They occurred in pairs with non motile, facultative anaerobic fermentative metabolism. Mesophilic heterofermentative cocci, 13 isolated:Microorganisms in this group had a close relation with Leuconostoc mesenteroides subsp. cremoris .
They were arginine negative,glucose positive,acetonoine positive and dextrane positive. Lactobacilli bacteria, 128 isolated: The group was divided into 3: (1) Mesophilic facultative heterofermentative Lactobacilli (55 isolates)Included Lactobacilli plantarum (37 isolates)and Lactobacilli. casei subsp. casei (36 isolates, (2) Thermophilic obligate homo-fermentative Lactobacilli (64 isolates) Included Lactobacilli. helveticus (17 isolates) and Lactobacilli. delactobacillirueckii subsp. bulgaricus (47 isolates). They were lactose positive andfructose positive.
(3) mesophilic obligate hetero-fermentative Lactobacilli (8 isolates) Included Lactobacilli. brevis (18 isolates)
It was discovered that mesophilic facultative hetero-fermantative lactobacilli group was divided into two;37 isolates were identified to be lactobacilli plantarum ans 18 isolates as lactobacilli casei subsp.casei.This results are also consistent with other research works such as the isolation of lactic acid bacteria from Maasai traditional fermented milk(Mathara et al.,2004). For the second group,17 isolates were identified as Lactobacilli plantarum and 47 isolates identified as lactobacilli delactobacillirueskii subs.bulgaricus. Furthermore,lactobacilli brevis isolates(8) were identified using mannose and melezitose fermentation. In the cocci group,12 and 22 isolates were identified as Leuconostoc mesenteroides subsp. cremoris and Lactococcus lactis subsp. cremoris. Respectively.This number is low an its attributed to the fact lactic acid cocci are not able to compete with lactic acid bacilli in mixed cultures(Teuber and Geis, 1981; Togo et al., 2002).
LAB are presenta in dairy manufacturing as starter cultures.There are specific fermentation processes that have been developed to maximize the growth of desired LAB species.Some of the species are fastidious organisms like Lactobacilli delactobacillirueckii subsp. bulgaricus and Lactobacilli helveticus (Bottazzi, 1988). Isolates that belong to lactobacilli plantarum group are shown to be dominant members of LAB flora of acid –fermented stuff(tempoyuk).Morever, Lactobacilli. brevis group and Ln. mesenteroides isolates were also found (Leisner et al., 2001).
This isolates have alos been found in South African Traditional fermeneted products.There are also other isolates that have been found in raw gaot’s milk of Algerian origin.This species include Lactobacilli. helveticus, Lactobacilli. plantarum, Lactobacilli. delactobacillirueckii subsp. bulgaricus, Lactobacilli. brevis and Lc. lactis subsp. lactis (Badis et al., 2004b). In chili bo, Lactobacilli. plantarum isolates were found to be the most dominant organism(Leisner et al. 1999).
Accolas, J.P. and J. Auclair, 1977. Determination of the acid producing activity of concentrated frozen suspensions of lactic acid bacteria. Lait, 50: 609-626.
Ammor, S., C. Rachman, S. Chaillou, H. Prevost and X. Dousset et al., 2005. Phenotypic and genotypic identification of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages. Food Microbiol., 22: 373-382. CrossRef |
Badis, A., D. Guetarni, B. Moussa-Boudjema, D.E. Henni and M. Kihal, 2004. Identification and technological properties of lactic acid bacteria isolated from raw goat’s milk of four Algerian races. Food Microbiol., 21: 579-588. CrossRef |
Badis, A., D. Guetarni, B. Moussa-Boudjema, D.E. Henni, M.E. Tornadijo and M. Kihal, 2004. Identification of cultivable lactic acid bacteria isolated from Algerian raw goat’s milk and evaluation of their technological properties. Food Microbiol., 21: 343-349. CrossRef |
Beukes, E.M., B.H. Bester and J.F. Mostert, 2001. The microbiology of South
African traditional fermented milks. Int. J. Food Microbiol., 63: 189-197. CrossRef | PubMed | Direct Link |
Bottazzi, V., 1988. An introduction to rod shaped lactic-acid bacteria. Biochemie, 70: 303-315. PubMed |
Collins, M.D., B.A. Phillips and P. Zanoni, 1989. Deoxyribonucleic acid homology studies of Lactobacillus casei, Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. Tolerans and Lactobacillus rhamnosus sp. nov., comb. nov. Int. J. Syst. Bacteriol., 39: 105-108. CrossRef |
Guessas, B. and M. Kihal, 2004. Characterization of lactic acid bacteria isolated from Algerian arid zone raw goats milk. Afr. J. Biotechnol., 3: 339-342. Direct Link |
Harrigan, W.F. and M.E. MaCance, 1976. Laboratory Methods in Food and Dairy Microbiology. Revised Edn., Academic Press, New York, pp: 33-200.
Hemme, D. and C. Foucaud-Scheunemann, 2004. Leuconostoc, characteristics, use in dairy technology and prospects in functional foods. Int. Dairy J., 14: 467-494. CrossRef |
Herrero, M., B. Mayo, B. Gonzalez and J.E. Suarez, 1996. Evaluation of technologically important traits in lactic acid bacteria isolated from spontaneous fermentation. J. Applied Bacteriol., 82: 565-570. Direct Link |
Holt, J.G., 1994. Bergeys Manual of Determinative Bacteriology. 9th Edn., Williams and Wilkins, Baltimore, Pages: 787.
Kalavrouzioti, I., M. Hatzikamari, E. Litopoulou-Tzanetaki and N. Tzanetakis, 2005. Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts. Int. J. Dairy Technol., 58: 30-38. CrossRef |
Lee, B., 1996. Bacteria- Based Processes and Products. In: Fundamentals of Food Biotechnology, Lee, B. (Ed.), Wiley-Inter Science, New York, pp: 219-290.
Leisner, J.J., B. Pot, H. Christensen, G. Rusul and J.O. Olsen et al., 1999. Identification of lactic acid bacteria from Chilli Bo, a Malaysian food ingredient. Applied Environ. Microbiol., 65: 599-605. PubMed |
Leisner, J.J., M. Vancanneyt, G. Rusul, B. Pot, K. Lefebvre, A. Fresi and L.K. Tee, 2001. Identification of lactic acid bacteria constituting the predominating microflora in an acid-fermented condiment (Tempoyak) popular in Malaysia. Int. J. Food Microbiol., 63: 149-157.
Linkater, P.M. and C.J. Griffin, 1971. Immobilized living cell fermentation. J. Dairy Res., 38: 127-136.
Lipinsky, E.S., 1981. Growth and activity of Lactococcus lactis ssp. cremoris in skim milk. J. Sci., 212: 1465-1471.
Mathara, J.M., U. Schillinger, P.M. Kutima, S.K. Mbugua and W.H. Holzapfel, 2004. Isolation, identification and characterization of the dominant microorganisms of kule naoto: The Maasai traditional fermented milk in Kenya. Int. J. Food Microbiol., 94: 267-278. PubMed |
Mayeux, J.V., W.W.E. Sandine and P.R. Elliker, 1962. A selective medium for detecting Leuconostoc organisms in mixed strain starter cultures. J. Dairy Sci., 45: 655-656.
Muyanja, C.M.B.K., J.A. Narvhus, J. Treimo and T. Langsrud, 2003. Isolation, characterization and identification of lactic acid bacteria from bushera: A Ugandan traditional fermented beverage. Int. J. Food Microbiol., 80: 201-210. CrossRef |
Olarte, C., S. Sanz, E. Gonzalez- Fandos and P. Torre, 2000. The effect of a commercial starter culture addition on the ripening of an artisanal goats
cheese (Cameros Cheese). J. Applied Microbiol., 88: 421-429. PubMed |
Samelis, J., F. Maurogenakis and J. Metaxopoulos, 1994. Characterization of lactic acid bacteria isolated from naturally fermented Greek dry salami. Int. J. Food Microbiol., 23: 179-196. PubMed |
Server-Busson, C., C. Foucaud and J.Y. Leveau, 1999. Selection of dairy Leuconostoc isolates for important technological properties. J. Dairy Res., 66: 245-256. CrossRef |
Sharpe, M.E., 1979. Identification of the Lactic Acid Bacteria. In: Identification Methods for Microbiologists, Skinner, F.A. and D.W. Lovelock (Eds.). Academic Press, London, pp: 233-259.
Stiles, M.E. and W.H. Holzapfel, 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol., 36: 1-29. Direct Link |
Terzic-Vidojevic, A., M. Vukasinovic, K. Veljovic, M. Ostojic and L. Topisirovic, 2007. Characterization of microflora in homemade Int. J. Food Microbiol., 114: 36-42. PubMed |
Teuber, M. and A. Geis, 1981. The Family Streptococaceae (Non-Medical Aspect). In: The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria, Starr, M.P., H. Stolp, H.G. Trueper, A. Balows and H.G. Schlegel (Eds.). Springer-Verlag, Berlin, pp: 1614-1630.
Thapa, T., 2000. Small-Scale Milk Processing Technologies: Other Milk Products. FAO, Rome, Italy.
Togo, C.A., S.B. Feresu and A.N. Mutukumira, 2002. Identification of lactic acid bacteria isolated from Opaque beer (Chibuku) for potential use as a starter culture. J. Food Technol. Afr., 7: 93-97. Direct Link |
Tserovska, L., S. Stefanova and T. Yordanova, 2002. Identification of lactic acid bacteria isolated from Katyk, goats milk and Cheese. J. Cult. Collect.,
3: 48-52. Direct Link |
Ward, L.J.H., G.P. Davey, H.A. Heap and W.J. Kelly, 2002. Lactococcus Lactis. In: Encyclopedia of Dairy Science, Roginski, H., J.W. Fuquay and P.F. Fox (Eds.). Elsevier Sci. Ltd., London, pp: 1511-1516.