1. UOIT
Glucose Supplementation
is Effective for Growth of
Lactobacillus acidophilus
in Fermented Milk
Vita Koren (100481835)
Keywords: Fermented Milk; Glucose supplementation; Lactobacillus acidophilus; 2% Milk

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Abstract
Lactobacillus acidophilus culture was suspended in 2% milk with 5% glucose
supplemented milk and non-supplemented milk to see the effect on fermentation. Results showed
that overall bacterial growth had a 2 fold increase in growth under glucose conditions, this was
significantly different compared to non-glucose. Lactobacillus culture also had a 2-fold increase
in glucose conditions. A statistical analysis concluded that results were significantly different
and glucose had an impact on Lactobacillus production. However, Lactobacillus bacteria initially
comprised 2.7% of the population and after fermentation only 0.01%. Other bacteria grew at
higher rates in fermented milk under the given conditions. Factors other than glucose
supplementation (pH and time) need to be studied in order to further L. acidophilus growth.
Introduction
The production of yogurt occurs due to bacterial cultures growing and fermenting in the
milk. Lactobacillus bacteria are one of the most popular bacteria in yogurt. They contain
exopolysaccharides (EPS) as a biothickener, help absorb Vitamin B12 and K and lower lactose
intolerance in individuals (Beausoleil, et al. 2007).
It has been determined that Lactobacillus growth is highest at temperatures of 42°C at a
slightly acidic pH of 6.2 (Patel, 2013). Lactobacillus bacteria is able to metabolize glucose sugar
as an additional energy source to its primary lactose source found in 2% milk (Patel, 2013). In
other words, milk supplementation with glucose sugar has been found to increase growth of
Lactobacillus bacteria (Patel, 2013). Li, et al. (2011) found that strain 111 of Lactobacillus
acidophilus had an enhanced growth rate when 2% and 3% glucose supplementation was added
to milk already containing the bacteria – meaning glucose had a positive effect on growth.

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In Li, et al. (2011) research, 5% glucose supplementation yielded more S. thermophilus
growth, hence, the current experiment was conducted to study the effect of 5% glucose
supplemented milk on the bacterial count of Lactobacillus bacteria after fermentation.
Methods and Materials
Preparation of Fermentation Flasks
Initially, 12 Lactobacillus acidophilus RO052 colonies from Dr. J. Green-Johnson’s
laboratory were suspended into 5 mL of Sealtest pasteurized 2% milk (Shoppers Drug Mart) to
create an inoculum. The control group was prepared by adding 0.25 mL of this inoculum into 25
mL of milk containing 5% water supplementation. The experimental group was prepared by
adding 0.25 mL of inoculate into 25 mL of 5% glucose supplemented milk. There was 3
replicates for both the experimental and control group. The experimental group and control
group were fermented for 24 hours at 42°C.
Bacterial Counts
Serial dilutions in a ratio of 1/10 of milk to saline solution were performed using the
initial inoculum. These dilutions were plated on MRS (De Man, 1960) and PCA (Baris, et al.
1953) plates for counts of Lactobacillus bacteria (MRS) and overall bacteria (PCA). After
fermentation, diluted samples from the glucose supplemented milk and the non-supplemented
milk were plated onto PCA media for total bacterial count and onto MRS agar media for
Lactobacillus count to compare the impact on growth and fermentation.
Statistical Analysis

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Average bacterial colony growth in fermented milk were calculated in CFU/ mL -
specifically, Lactobacillus bacteria and overall bacteria were counted. Standard deviations were
calculated to compare lactobacillus growth with or without glucose as well as for the assessment
of total bacterial growth in glucose versus non glucose milk. Two follow-up two-tailed t-test
were conducted with a degree of freedom of 1 and a P-value of 0.05 to observe statistical
difference between glucose and non-glucose milk (Dawson, 1997).
Results
Bacteria found naturally in 2% Milk
Lactobacillus bacteria found in 2% milk before experimentation was 1.0x104 CFU/ mL
compared to total number of bacteria was 3.7x105 CFU/ mL, data not shown. Thus, Lactobacillus
bacteria was a small contribution – 2.7% to the overall bacteria that are found in milk.
Effects of Glucose on Lactobacillus Growth and Overall Bacterial Growth
The effect of 5% glucose supplemented milk versus non-supplemented milk on the
growth of Lactobacillus in 2% milk at 42°C for 24 hours is illustrated in Table. 1. Glucose
yielded 2x the growth than non-glucose for Lactobacillus bacteria. This was significantly
different – glucose had an impact. Comparatively, significant difference was determined for
overall bacteria also due to glucose yielded 2x the growth related to non-glucose. However,
Lactobacillus bacteria accounted for only 0.01% of the total population of bacteria found in
glucose supplemented milk and in non-glucose milk as oppose to the initial 2.7% contribution.
So, other bacteria found in the fermented milk (99.91%) overgrew the Lactobacillus bacteria.
Discussion

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Lactobacillus acidophilus is a popular strain used to ferment milk. L. acidophilus grew
double the amount in glucose milk. According to Gard et al. (1990) glucose increased L.
acidophilus bacterial growth 4-fold using 3% glucose supplemented milk. The current
experiment used 5% glucose which may have been a factor to slow down the growth rate.
Additionally, the current L. acidophilus population went from 2.7% to 0.01% in the
overall bacterial growth after fermentation in glucose and non-glucose milk. This was
determined after 24 hrs of fermentation. Gard et al. (1990) found that L. acidophilus needed 6
days to yield larger plate counts of approximately 5% from its initial 2%. This occurred because
L. acidophilus strives in the process of the fermentation after other bacteria begun the process by
lowering the pH to <5 (Beausoleil, et al. 2007).
Bacteria other than Lactobacillus bacteria consisted of 99.91% percent of the bacteria in
fermented milk. This meant these other bacteria were modifying the properties of the milk so that
L. acidophilus growth could occur later on in the process. S. thermophiles, B. bifidum, and S.
lactis are some other popular bacteria able to grow well under 42°C (Czarnecki, et al. 2008) and
further identification could help account for their percentages in the fermented milk.
In summary, Lactobacillus bacteria and overall bacteria both had a 2-fold increase in
growth in glucose milk compared to non-glucose milk; this was significant. However, the
decrease in Lactobacillus bacteria from 2.7% to 0.01% in both glucose and non-glucose milk
after fermentation indicated that other conditions were not optimal for L. acidophilus growth
during fermentation. Other bacterial strains must initiate the fermentation process and after a
significant pH drop (<5), L. acidophilus growth may strive to help with milk fermentation.
Testing should be done during a 5-7 day period for a better evaluation of glucoses (5%) impact.

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References
Baris Y., Buchbinder L., and Goldstein, L., (1953),”Further studies on new milk-free media for
the standard plate count of dairy products.” American Journal of Public Health 43.1, 869-872
Beausoleil, M.., Fortier, N., and Weiss, K., (2007)”Effect of fermented milk combining
Lactobacillus acidophilus CL1285 and Lactobacillus casei in the prevention of antibiotic-
associated diarrhea: A randomized, double-blind, placebo-controlled trial.” Canadian Journal of
Gastroenterology 21.11, 732-736
Czarnecki, Z., Goderska, K., and Nowak, J. (2008) "Comparison of the growth of Lactobacillus
acidophilus and Bifidobacterium bifidum species in media supplemented with selected
saccharides including prebiotics" Scientiarium Polonorium 07.02, 5-20.
De Man, J.C., Rogosa, M., and Sharpe, M.E. (1960). “A medium for the cultivation of
lactobacilli.” J. Appendix. Bacteriology, 23.1, 130-135.
Dawson, R. (1997). “A t-Table for Today” Journal of Statistics Education 05.02, 55
Garg, S., Mital, B., Srinivas, D. (1990). Utilization of sugars by Lactobacillus acidophilus
strains, International Journal of Food Microbiology 10.01, 51-57
Li, S., Yang, Z., Zhang, C., Zhang, T. and Zhang, Y. (2011)"Growth and Exopolysaccharide
Production by Streptococcus Thermophilus ST1 in Skim Milk." Brazilian Journal of
Microbiology 42.4, 1470-478
Patel, A. (2013) "Food and Health Applications of Exopolysaccharides Produced by Lactic Acid
Bacteria." Advances in Dairy Research 01.107, 384-392.

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Table 1. Effect of glucose supplementation on growth of Lactobacillus bacteria and on overall
bacterial culture
Growth of Bacteria (CFU/ mL) b
Type of Bacteria a Non-glucose Supplemented
Milk
5% Glucose Supplemented
Milk
Lactobacillus
Bacteria
1.01x105 + 1.0x103 c 2.1x105 2 + 1.2x103 c
Overall
Bacteria
1.11x108 + 0.2x104 c 2.01x108 + 0.2x104 c
a. Bacteria were quantified as Lactobacillus bacteria (grown on MRS agar) and overall bacteria
(grown on PCA agar) in order to compare the impact of glucose on Lactobacillus bacteria versus
other strains of bacteria.
b. Growth of bacteria after 24 hrs of fermentation in milk with glucose supplementation and
without glucose supplementation.
c. Values are the means of triplicate determinations of bacterial growth + standard errors.