Comparative influence of medium composition on biomass growth, lactic acid and Exopolysaccharides Production by some Strains of Lactic Acid Bacteria
B Adebayo-Tayo, A Onilude
B Adebayo-Tayo, A Onilude. Comparative influence of medium composition on biomass growth, lactic acid and Exopolysaccharides Production by some Strains of Lactic Acid Bacteria. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
To promote the overall quality of fermented dairy products (FDP) which depends on the types and characteristics of the starter organisms used in the production, the effect of medium composition on biomass growth, viscosity, exopolysaccharide and lactic acid production by EPS- producing lactic acid bacteria isolates was investigated. Medium composition had profound effect on the studied parameters. Among the five medium used for the cultivation of the isolates, maximum biomass growth and viscosity production was achieved in partially de-protenised whey medium (PDW) in which
The overall quality of fermented dairy products (FDP) depends on the types and characteristics of the starter organisms used in the production. The essential criteria for starter selection include acidification, aroma, flavour, stability and texture. Manufacturers can improve the quality of their FDP by substituting stabilizers with EPS-producing lactic acid bacteria (LAB) as starter. Lactic acid bacteria (LAB) are a group of gram-positive bacteria which produce lactic acid as the major end products of fermentation of carbohydrates (Axelsson, 1998). The growth of LAB is often accompanied by the production of polysaccharides, which are found outside the cell wall. These exopolysaccharides (EPS) may be found as capsule attached to the bacteria or they may be released to the environment as slime or both (Sutherland, 1977). Polysaccharides even though important to bacteria for adhesion, infection and protection may as well have commercial value. Some polysaccharides are known to have gelling properties that is; agar and gel rite (Davis
This study was therefore aimed at investigating the effect of medium composition on some strains of lactic acid bacteria for selecting LAB strains with good biomass growth, high production of viscosity, lactic acid and exopolysaccharide.
Materials and methods
The pure cultures of the EPS -producing LAB isolates made up of 16
The working cultures were prepared by transfering 0.5ml of the stock frozen culture to 10ml of MRS broth and incubated for 16h at 30 oC. The resulting culture was transferred (2% v/v) to modified exopolysaccharide selection medium (mESM) (van den Berg
The growth media used were; (a) whey (whey only), (b) Semi - defined medium (SDM) with the following composition(grams/liter); dextrose, 20g; Tween-80, lml: Ammonium citrate; 2g; sodium acetate, 5g Mg504.7H20, 0.1mg; MnS04, 0.05mg; K2HP04, 2mg; yeast extract, 5g and bactocasitone, 10g; pH 6.5; (c) modified MRS medium (mMRS); (d) Partially deprotenized whey (PDW) containing 1% peptone prepared by adjusting to 4.6 using 1N HCI, the pH of the Skim milk (from raw milk by centrifugation) and removing the precipitate by centrifugation. The whey recovered was adjusted to pH 6; (e) Sweet whey made up of Lactose, 50g; MgSO4.7H2O, 40.7mg; MnSO4.7H20, 4.0mg; FeSO4.7H2O, 1.4mg; CaCl2, 0.9mg, pH 6.2, H2O 1 liter. 100ml of each medium was dispensed into 250ml Erlenmeyer flask and autoclave at 121oC for 15mins by autoclaving.the flask were inoculated with 10ml inocula of the 16h old culture containing 2.5x106 cfu/ml and incubated at 30oC for 48h. Samples were taken from each flask and analyzed for pH, lactic acid concentration, cell growth, viscosity and EPS production.
Isolation and partial purification of EPS
Partial purification of exopolysaccharide was carried out according to the method described by Gancel and Novel (1994). Ten grams of culture medium were accurately weighed into a 50ml centrifuge tube and the content was heated in a boiling water bath for 10 min to inactivate enzymes potentially capable of polymer degradation (Cerning
Total sugar determination
The total sugar concentration was determined by the phenol-sulfuric acid method using glucose as a standard (Chaplin, 1986). The results are expressed in milligrams of glucose per liter.
This was measured as described by Schellhass and Morris (1985), using a Haake Rotovisco Rv20 coaxial cylinder viscometer with No. 1 sensor system (Haaker, Inc, Saddle Brook; NJ) at incubation temperatures of 25, 30 and 40oC. The shear rate increased from O to 1000/sec over a 3 min period.
Measurement of growth
Growth of the test organisms was determined spectrophotometrically by taking the Optical Density reading at 650mm after appropriate dilution of fermented samples earlier incubated at 300C for 48h (A.O.A.C. 1990).
Quantitative estimation of lactic acid
The production of lactic acid was determined by titrating 10ml of the homogenized sample against 0.25mol l-1 NaOH using 1 ml of phenolphthalein indicator (0.5 % in 50% alcohol). The titratable acidity was calculated as percentagelactic acid (v/v). Each milliliter of 1N NaOH is equivalent to 9.008mg of lactic acid. (A.O.A.C. 1990).
Result and Discussion
The effect of different medium on cell growth of twenty EPS-producing LAB strains is shown in Table 1.
The result of effect of medium composition on biomass growth by the 20 LAB isolates were in order SW>PDW>SDM>mMRS>WHEY. The biomass growth ranges from 0.0025cfu/ml - 1.561cfu/ml in which
Among the 20 EPS producing LAB tested.
Comparative effect of medium composition on EPS production from various LAB isolates was shown in Table 2. Medium composition had profound and significant effects on EPS production by the EPS producing LAB isolates. Comparatively, the result of the effect of medium composition on EPS - production by the 20 LAB isolates were in order Whey> SDM> mMRS>SW>PDW. The highest EPS was obtained from
There was variation in EPS production from cultures shown to be the same isolated species. For example the two
The higher EPS yields in mMRS during fermentation may be due to small amounts of nitrogen sources present in the media. This result agrees with the finding of Garicia-Garibay (1991) who reported the production of exopolysaccharides when the organism was grown in mMRS broth. mMRS, the usual medium for laboratory fermentation by LAB contains compounds (e.g. beef extract, peptone, yeast extract) that enhanced EPS synthesis.
The comparative effect of medium composition on lactic acid production by the isolates was shown in Table 3.
Medium composition had significant effect on lactic acid production by the isolates. In comparism with SDM, SW and mMRS, Whey and PDW medium was not favourable for lactic acid production by the isolates. In SDM, the highest (5.57g/l) lactic acid production was recorded from
The comparative effect of medium composition on viscosity development by the isolates was showed in Table 4; it was observed that medium composition had a significant effect on viscosity development by the isolates. The entire medium used enhanced viscosity development by the isolates. PDW, Whey, Sweet whey and SDM had a profound effect on development of viscous medium.
Lower viscosity development in Sweet whey, deproteinised whey and whey than Semi-defined medium, may probably be as a result of the removal of the peptide and peptones in the deproteised whey medium and non-supplementation of such in the medium. Despite this result, however whey has been reportedly used for production of ethanol, lactic acid and citric acids, biomass proteins, and food yeas (Moulin and Galzy, 1984; Kennedy, 1985; Mann, 1987) Production of EPS by LAB in milk is an important factor in assuring the proper consistency and texture of fermented food(Ricciard and Clementi, 2000). Ability of these EPS producing LAB to produced viscous medium is an added advantage over stabilizer which can adversely affect the true taste, aroma and mouthfeel of fermented dairy products. An alternative way to improve texture and stabulity of fermented dairy is the use of lactic acid bacteria which are food–grade organisms and are generally recognised as safe status (GRAS). Their exopolysaccharides function as thickeners, stabilizers, emulsifiers, gelling and water- binding agents. It contributes to the specific rheology and texture of fermented milk products. These exopolysaccharides represent safe additives for novel food formulations and may have applications in non-food products [(Crescenzi, 1995, Giraffa, 1994).
Comparatively, medium composition had a significant influence on the final pH of the fermentation broth as showed in Table 5. The initial pH of the fermentation broth of
Conclusively, from the aforementioned results, growth, viscosity, pH, lactic acid development and exopolysaccharide production of LAB vary widely with respect to the LAB species and their nutritional status and most of the isolates can serve as good starter for the production of quality fermented foods. This result may provide a sustainable and means of adding value to fermentation of which will result in production of this promising industrial biomolecule and high quality fermented products.