Kinetic of Carbon Dioxide Production by Leuconostoc mesenteroides Grown in Single and Mixed Culture with Lactococcus lactis in Skimmed Milk
H Kihal, D Prevost, D Henni, Z Benmechernene
acetic acid, citric acid, fermented milk, growth kinetics, lactic acid, lactococcus lactis, leuconostoc, mesenteroides, milk
H Kihal, D Prevost, D Henni, Z Benmechernene. Kinetic of Carbon Dioxide Production by Leuconostoc mesenteroides Grown in Single and Mixed Culture with Lactococcus lactis in Skimmed Milk. The Internet Journal of Microbiology. 2007 Volume 5 Number 2.
The effect of mixed culture of
Lactic acid bacteria are now used extensively as starter cultures in the dairy industry and therefore the optimization of growth conditions appears to be essential for successful industrial applications. Furthermore, studying the effects of some environmental parameters on growth kinetics should provide useful information concerning the physiology of the microorganisms (Carr
The importance of
The shift in metabolic pathways in response to environmental conditions is well documented in the literature in the case of homofermentative species [6,17], but little information is available about the behavior of heterofermentative species such as
The present study was undertaken to determine whether
Materials and Methods
The strains were stored as frozen stock at - 20°C in fortified skimmed milk (10 % skim milk, 0.25 % yeast extract, 0.5 % glucose) containing 30% glycerol as appropriate. Working cultures were prepared from stock cultures by two consecutive transfers in fresh MRS and M17 broth for
Media and conditions cultures
Kinetic growth on milk
The growth rate, lactic acid production and evolved CO2 were studied on reconstituted skim milk. The reconstituted milk was sterilized at 110°C for 10 min, cooled at room temperature (20°C), and inoculated with the strains at 30°C. All the mother culture of
Freshly prepared starter culture of
Determination of pH and total acidity
Measurement of pH was carried out by pH-meter. The total acidity was determined by titrating 10 ml of culture with 0.1
Cultures samples were collected aseptically at 0 hours and every 2 hours post inoculation until 24 hours. Culture sample of 1 ml were submitted to decimal dilutions in sterile tryptone salt solution and agar plate were performed to assess cell count.
The evolved CO2 was measured by a technique based on the pressure which is created by CO2 production by culture in tubes. Evolved CO2 was trapped in burette in which it was measured (Kihal
In order to evaluate the linearity of the method, solution of sodium carbonate 50 mM were used to liberate CO2 in the tube by addition of sulfuric acid (2N). The blank contains 10 ml of sterile milk.
The ability to produce different lactic acid isomers (L-lactate and D-lactate) was tested by an enzymatic method utilizing Boehringer Mannheim GmbH (Mannheim, Germany) Also citric acid and acetic acid were carried out by enzymatic methods (Boehringer) .
Results and discussion
All the used strains were cocci associated in diplococci and chain, Gram+, catalase-, and were can grow under both aerobic and anaerobic conditions. All the strains form on solid medium identical lentil colonies.
In the case of
All this characteristics are in accordance with Carr
Limite of detection
A high correlation was observed when the volume of CO2 measured is lower than 80 mM. The method has a good linearity between 0 to 80 mM of CO2, but the application of the method to the determination of CO2 content in the sample needs a blank which must be prepared and measured before each culture sample. The lower value of coefficient of variation observed was caused by the preparation of sample than by the measurement itself (Tab.1).
By definition, heterofermentative lactic acid bacteria, ferment glucose to produce equimolecular amounts of lactate, carbon dioxide and acetate or ethanol [17,18] However, certain modifications in the conditions of culture may result in the prevalence of one of these products. The kinetics of both evolved CO2 and pH evolution are shown in figure 1 and 2. When
A proportion of 2.5 10 6 cfu/ml of
The lowest titrable acidity and pH evolution were given by the pure culture of
In the first phase, the specific growth rate, lactic acid production rate and evolved CO2 rate exhibited a constant relationship. After the ten first hours an inhibition of growth, lactic acide production and evolved CO2, were observed. Maximum CO2 production rate (Vmax) showed a decrease when the inoculum level of
An interesting way to estimate independently
The estimated results coming from the pure culture relationships applied to mixed culture experiment are presented in (Fig.4). A shift between µmax for
The amount of acetic acid formed from citrate was higher in pure and mixed culture of
In conclusion a greater inhibition of evolved CO2 in