الفهرس 
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Abstract
This study was carried out in order to investigate some factors affecting the growth curve of some lactic acid bacterial starters used in the dairy industry.
These factors are:-
- Effect of water activity ( aw )
- Effect of inoculum size.
- Effect of incubation temperature.
- Effect of fortification of media with proteose peptone.
- Effect of control of culture acidity.
- Combined effect of fortification of media with 1% peptone and control of culture acidity.
- Effect of sodium chloride.
Lactococcus lactis subsp lactis , Lactococcus lactis subsp cremoris and Lactobacillus casei representing LAB were used . While sterilized skim milk was used as acultural media .
In this study MRS media was used for bacterial colony forming units counting.
1- Effect of water activity (aw)
The effect of reduction in water activity on growth and acid production by Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei was studied at water activity values of 0.998, 0.975, 0.967 and 0.959. Reduced water activity resulted in extended lag time of varied magnitude, varied growth rates, different cell population and acid production.
The highest growth rates were observed at an aw of 0.998 (control). Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei reached a maximum cell population of 80x106 CFU/ml, 60x106 CFU/ml and 60x106 CFU/ml after 24th hour of incubation respectively.
At water activity value of 0.975, Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei reached a maximum cell population of 40x105 CFU/ml, 51x105 CFU/ml and 51x105 CFU/ml respectively after 24th hour of incubation , which indicate that Lactococcus lactis subsp lactis reached a maximum cell population which is lower than that observed for the other two strains. Which indicate that for each bacterial strain there is an optimum water activity at which the growth rate is faster comparing with the growth rate at lower or higher water activity. In addition the sensitivity to culture water activity varied between different bacterial strains.
Also the obtained results indicated that the value of culture water activity had approximately simlar effect on rate of increase in developed titratable acidity as that found for the rate of increase in colony forming units.
2- Effect of inoculum size:
The effect of inoculum size being inoculated in sterilized skim milk , incubated at 34°C on bacterial growth was examined at the different levels, i.e., 104, 105 and 108 CFU/ml, 105, 107 and 108 CFU/ml and 104, 105 and 106 CFU/ml, on the growth rates of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei respectively.
The obtained results indicated that the level of inoculum size greatly affected the length of lag time, rate of growth and maximum cell population . The growth rate was increase with the increase of the inoculum size. The length of lag time decreased with increasing the inoculation level, and quantitatively tends to be a linear function of the logarithm representing the number of organisms in the inoculum. It’s noticed that when the size of the inoculum increased, the coagulation occurs more faster than when lower concentration of the inoculum size was used .
When Lactococcus lactis subsp lactis was inoculated with a small inoculum size i.e., 20x104 CFU/ml, the length of lag time as measured by the rate of changes in colony forming units and developed titratable acidity was at intervals of time from zero to 2nd hour of incubation.
However the reduction in the length of the lag time was in accordance with the increase in level of inoculation reaching a minimum at an inoculum level of 10x108 CFU/ml where no lag time was detected as measured by the determination of CFU and DTA.
When Lactococcus lactis subsp cremoris, was inoculated by a small inoculum size (28x105 CFU/ml) the length of logrithimic phase as measured by the rate of increase of colony forming units and developed titratable acidity was at intervals of time zero to 12th hour and from zero to 10th hour of incubation respectively. Reduction in the length logarithmic phase was in accordance with the increase in the level of inoculation reaching a minimum at an inoculum level of 40 x 108 CFU/ml, where logarithmic phase was observed as measured by the determination of CFU and to be at interval of time between zero hour to 8 hour of incubation.
When Lactobacillus casei was inoculated by a small inoculum size 10x104 CFU/ml, the length of lag time as measured by rate of change in CFU and DTA was at intervals of time arranged from zero to 4th hour and from zero to 2nd hour of incubation respectively.
When inoculation was made by higher level of inoculum size, 25x106 CFU/ml, lag time was not detected as measured by the rate of changes in CFU, however, when lag time was measured by the change in DTA, it was at interval of time from zero to2nd hour.
Also , the obtained results indicated that the length of logarithmic phase was decreased in accordance with the increase in the level of inoculation and that was found with Lactococcus lactis subsp lactis , Lactococcus lactis subsp cremoris and Lactobacillus casei .
3- Effect of incubation temperature:
The effect of incubation temperature on the growth rate and the rate of acid production of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei were examined at two different temperature i.e., 30°C and 40°C.
The incubation temperature had affected each of the time needed for milk coagulation, the length of lag time, the growth rate and the maximum cell population.
Milk coagulation occurred after 24 hour of incubation when Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei were incubated at 30°C .
On the other hand , at incubation temperature of 40oC , coagulation was not observed up to 30 hour of incubation .
The obtained results indicated that , rate of increase in developed titratable acidity and colony forming units were faster at incubation temperature of 30°C as compared with incubation temperature of 40oC , and this was found for each of the three bacterial strains used in this study.
At 30°C, Lactococcus lactis subsp lactis grow exponentially between the 4th up to the 12th hour of incubation reached a maximum cell population of 30x106 CFU/ml, after which the growth rate was declined. However at 40°C the growth rate of this strain was lower rate compared with that at 30°C and reached 51x104 CFU/ml after 12 hour of incubation.
Results also showed that the growth rate of Lactococcus lactis subsp cremoris at low temperature i.e., 30°C was characterizedby faster rate of growth compared with Lactococcus lactis subsp lactis and reached a higher colony forming unit after 12th hour of incubation and was reached 46x106 CFU/ml , at incubation temperature of 40oC , colony forming units reached 12 x 105 CFU / ml only .
Lactobacillus casei at 30°C had similar growth rate as Lactococcus lactis subsp lactis and Lactococcus lactis subsp cremoris and reached 32x106 CFU/ml after 12th hour of incubation. On the other hand at incubation temperature of 40°C grow by a faster rate compared with the other two strains and reached 40x105 CFU/ml.
Higher rate of acid production by Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei at lower temperature i.e., 30°C is thought to be due to the higher cell population , which can increase rate of fermentation of lactose to lactic acid .
4- Effect of fortification of media with 1% peptone:
The effect of fortification of media by 1% peptone on the growth rate and rate of increase in developed titratable acidity was studied .
The obtained results indicated that addition of 1% peptone to the medium greatly affected the growth rates and acid production of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei where the developed titratable acidity reached 0.70%, 0.80% and 0.43% respectively after 24th hour of incubation , which is higher than the values of DTA reached in control culture , being o.64 % , 0.72% and 0.35 %respectively.
In the same time, when Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei grow in sterilized skim milk supplemented with 1% peptone the maximum colony forming count reached after 24th hour of incubation incubation were 10x107 CFU/ml, 18x107 CFU/ml and 10x107 CFU/ml, respectively. The corresponding values reached in control samples were considerable lower and reached only 65 x 106 CFU/ ml , 81 x 106 CFU / ml and 56x 106 CFU/ ml respectively.
Regarding coagulation time , the obtained results indicated that fortification of culture medium with 1% peptone leads to a considerable decrease in the time needed for culture coagulation . Which can be explained by the effect of culture fortification by 1% peptone on the rate of increase in colony forming unite and developed titratable acidity .
The obtained results indicated that fortification of sterilized skim milk with 1% peptone yielded an excellent growth of all strains as the maximal level of CFU/ml was obtained.
5- Effect of culture acidity:
This experiment had been carried out in order to investigate and compare the effect of control of control culture acidity on the growth rate of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei in sterilized skim milk incubated at 34°C.
The bacterial growth was estimated on the base of colony forming units, developed titratable acidity and incubation time. Sampling had been carried out at zero time and each 2 hours intervals. After each sampling, a calculated volume of 0.1 N sodium hydroxide solution was added to the culture to readjust the acidity of the remaining samples of the cultures to its initial value at zero time.
The obtained results indicted that:
1- When Lactococcus lactis subsp lactis and Lactobacillus casei under control of culture acidity, lag phase wasn’t detected. While the lag phase was from zero to 2nd hour of incubation only in Lactococcus lactis subsp cremoris.
2- Signification effect of acidity control was noticed on the growth rate of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei. Exponential phase of growth was between 2nd to 12th hour, 4th to 12th hour and 6th to 24th hour of incubation, respectively. However the same cultures being grown with control of culture acidity, the exponential phase of growth was at interval of time from zero to 24th hour, 2nd to 26th hour and zero to 32nd hour of incubation respectively.
3- The maximum cell population in the control sample were 12x106 CFU/ml, 11x106 CFU/ml and 20x106 CFU/ml after 24th hour of incubation. The corresponding values when growth was carried out under control of culture acidity was by a maximum cell population of 30x106 CFU/ml, 20x106 CFU/ml and 70x106 CFU/ml after 30th hour of incubation for Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei, respectively.
The obtained results indicated that, when Lactococcus lactis subsp lactis , Lactococcus lactis subsp cremoris and Lactobacillus casei grown in sterilized skim milk under control of culture acidity grown by a higher specific growth rate as measured by the rate of increase in colony forming units, compared with the same variant being grown without control of culture acidity. In addition, it reached to a higher maximum cell population compared with the control sample.
6- Combined effect of supplementation of media with 1% peptone and control of culture acidity.
This experiment had been carried out in order to investigate and compare the Combined effect of supplementation of media with 1% peptone and control of culture acidity on the growth rate of Lactococcus lactis subsp lactis , Lactococcus lactis subsp cremoris and Lactobacillus casei in sterilized skim milk and incubated at 34°C.
The obtained results indicted that:
- When Lactococcus lactis subsp lactis grow in sterilized skim milk, milk was coagulated after 12th hour of incubation and was reached plate count of 28x106 CFU/ml.
- On the other hand, the same strain being grown in sterilized milk supplemented with 1% peptone and control of culture acidity, the culture stayed liquid up to the end of incubation period of 28th hour at 34°C, and it reached a plate count of 70x106 CFU/ml at the end of the exponential phase after 12th hour of incubation
- After 24th hour of incubation cessation of growth in the culture and started the stationary phase.
The effect of added 1% peptone to sterilized skim milk and control of culture acidity on colony forming units of Lactococcus lactis subsp cremoris, indicated that control sample grow in sterilized skim milk reached a plate count 55x106 CFU/ml after 12th hour of incubation. The corresponding values for Lactococcus lactis subsp cremoris grow with medium supplementation and control of culture acidity reached a plate count of 20x107 CFU/ml after 24th hour of incubation.
When Lactobacillus casei grow in sterilized skim milk incubated at 34°C with medium supplementation with 1% peptone and control of culture acidity the population density was several times higher in culture supplemented with peptone and control of culture acidity reached a population density of 17x107 CFU/ml while only 23x106 CFU/ml were recorded by the same strain being grown as a control after 24th hour of incubation.
Fortification of milk with 1% peptone and control of culture acidity yielded an excellent growth for all studied strains as the maximum level of colony forming units was obtained.
Thus, sufficient cell mass production in a supplemented medium is possible and this may allow exclusive use in the dairy industry.
7- Effect of sodium chloride:
The effect of 0, 1, 3 and 5% sodium chloride on the growth rates and developed titratable acidity of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei when cultivated in sterilized skim milk at 30°C had been studied.
The affect of 0, 1, 3 and 5% sodium chloride on the population level of Lactococcus lactis subsp lactis during 32nd hour of incubation. In the absent of sodium chloride (control) growth was at exponential rate up to the 12th hour reaching a maximum colony forming units of 26x106 CFU/ml. After this period, the growth rate declined and reached 99 x 106 CFU /ml after 32th hour of incubation. In the presence of 1% sodium chloride the growth was by a slower rate reaching a maximum colony forming units 94x105 CFU/ml after 12th hour of incubation. In the presence of 3% and 5% of sodium chloride the growth rate was by a very slow rate reaching 12x105 CFU/ml and 71x104 CFU/ml respectively after 24th hour of incubation.
The results of developed titratable acidity against time for Lactococcus lactis subsp lactis. When growth was in the absence of sodium chloride, exponential phase of acid production between zero and 12th hour of incubation reached 0.29%. After which rate of acid production was decreased. In the presence of 1% NaCl, this strain still gave an exponential phase of increase in DTA but with lower rate reached 0.27% after 12th hour of incubation.
Between 12th hour and 32nd hour of incubation there was increase of the acid production but not reaching the level of the control reached only 0.66% after 30th hour of incubation compared with 0.71% in the control sample.With higher percentages of sodium chloride i.e., 3% NaCl, the population level and acid production by Lactococcus lactis subsp lactis was lower than the concentration 1% salt. The effect of salt was increased at concentration of 5% sodium chloride was added to the culture, where the increase in CFU and DTA was nearly stopped.
Concerning the effect of 0, 1, 3 and 5% sodium chloride on the growth rate of Lactococcus lactis subsp cremoris indicated that , in the absence of sodium chloride growth was exponentially up to the 12th hour of incubation reached a maximum colony forming unit of 64x106 CFU/ml. After that rate of growth was declined. Similar results were obtained when growth was a 1% sodium chloride, reached a maximum cell population of 10x106 CFU/ml after 12th hour of incubation.
In the presence of 3% and 5% NaCl there was a big difference between Lactococcus lactis subsp lactis and Lactococcus lactis subsp cremoris where the effect of sodium chloride either on the rate of increase of CFU and DTA was more dramatic in Lactococcus lactis subsp cremoris as compared with Lactococcus lactis subsp lactis.
The results of developed titratabe acidity against time for Lactococcus lactis subsp cremoris, in the absence of sodium chloride , showed an exponential phase of acid production between 2nd and 12th hour of incubation and was reached 0.30%, the corresponding value reached by Lactococcus lactis subsp lactis was 0.29% only.
In the presence of 1% and 3% salt this Lactococcus lactis subsp cremoris still gave an exponential phase of developed titratable acidity but with a slower rate reached only 0.29 % and 0.28% respectively . On the other hand, in the presence of 5% NaCl, the increase in DTA was considerably affected reached only 0.25% after 12th hour of incubation. The obtained results indicated that rate of increase in DTA was slower than rate of increase in cell population, probably a decoupling effect of salt on growth rate and acid production.
The effect of sodium chloride on the growth rate of Lactobacillus casei in sterilized skim milk at 30°C.
The rate of increase in colony forming units was decreased as the concentration of sodium chloride increased, but there was bigger difference in the effect between percentage of 3% and 5% than between 0% and 1% NaCl. Maximum cell population reached 30x106 CFU/ml, 12x106 CFU/ml, 30x105 CFU/ml and 10x105 CFU/ml when growth was at percentage 0, 1, 3 and 5% sodium chloride respectively. On the other hand differences in developed titratable acidity as a function of salt concentration in milk. It is still noteworthy that differences between percentage of 3% and 5 was more dramatic than between of 0% and 1% NaCl. This may be no more than the consequence of difference in cell population.
These three strains, decreased in growth rate when cultivated in milk containing different concentrations of sodium chloride. However, the effect of sodium chloride varied with these strains, and Lactococcus lactis subsp cremoris seemed to be more sensitive to salt than Lactococcus lactis subsp lactis and Lactobacillus casei.
The obtained results indicated that percentage of 5% NaCl would result in a marked decrease in growth rate and in the rate of acid production.
Which mean that sodium chloride has a potential inhibitory effect on the growth of lactic acid bacteria.