Growth Pattern of Lactic Acid Bacteria in Probiotic Rice Washed Water

Nelfa C. Gil1, Jocelyn G. Daclag2, Journal Science, Engineering and Technology

and Allen Glen C. Gil3 Vol. 3:126-138(2015)

Abstract

Readily available rice washed water posed as potential medium for lactic
acid bacteria, thus its utilization as probiotic drink for animals was
explored. This study investigated the LAB growth pattern in rice washed
water using factorial design following Completely Randomized Design.
Highest bacterial count was from 1:3 rice-water ratio 1st washing (T5) at
12h (1.5 x109cfu/ml) and 1:2 rice-ratio 1st washing (T3) at 30h (1.5x109cfu/
ml). The 1:3 rice-water ratio 1st washing (T5) and 1:1 rice-water ratio 2nd
washing (T2) provided an early rapid growth environment. Logarithmic
phase started at 18h with T5 having the highest microbial count. Decline
phase ranged from 18h to 36h. Morphological and culture characteristics
were identical to the activated LAB from the source. Results indicate that
rice washed water can be used as probiotic drink within 12h to 42h after
fermentation.

Keywords: microbial count, growth curve, logarithmic growth phase, LAB

culture, morphological characteristics

1.0 Introduction undated) and intestinal balance

(Fuller, 1991; FAO/WHO, 2001).
By and large, probiotics had The beneficial effects of probiotics
already gained distinction in both include: modification of the
human and animal health. The health intestinal microbiota, increase
benefits it offered had been proven production of VFA (volatile fatty
effective, thus it obtained popularity acid), stimulation of immune system,
in the market today. It is generally increased biomass and stool bulking,
agreed that a probiotic is a reduced inflammatory reactions,
preparation of live microorganisms increased B vitamin synthesis,
which, when applied in adequate prevention of pathogen colonization,
amounts to humans or animals, improved mineral absorption,
beneficially affects the host by enhanced animal performance,
improving the properties of the prevention of cancer, decreased
indigenous microbiota (Rush, carcass contamination, lower serum
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Southern Leyte State University, Sogod, Southern Leyte
2,3
Visayas State University, Visca, Baybay City
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cholesterol, decreased ammonia and treatment of day-of-hatch broiler

urea excretion, and lower skatol, chicks (Higgins et al., 2007).
indol and phenol (Patterson & Commercial producers of lactic acid
Burkholder, 2003). The inhibition of products have promoted lactic acid
pathogens by the intestinal feeds for poultry for the following
microbiota has been called bacterial reasons: improved performance of
antagonism, bacterial interference, broilers, improved digestion,
barrier effect, colonization protection against microbial
resistance, and competitive contamination, and increased food
exclusion. Mechanisms by which the safety. These only show that
indigenous intestinal bacteria inhibit production of lactic acid and use of
pathogens include competition for lactic acid bacteria posed high
colonization sites, competition for potential for use as probiotic
nutrients, and production of toxic product.
compounds or stimulation of the LAB needs a good medium
immune system (Patterson & for growth and survival in order to
Burkholder, 2003). maintain its integrity as probiotic.
Lactic acid bacteria (LAB) One potential medium is rice
are among the probiotics used in the washed water which contain starch
health industry. It has been used on as source of LAB food. Being a rice
foods for human consumption for -eating country, rice washed water
the numerous benefits that it offers as a waste product in cooking, is
to consumers. LAB and lactic acid always available in the households.
has also been used on plants and The prospects of LAB in rice-
animals. Organic farming used LAB washed water necessitates thorough
for the improvement of the quality evaluation prior to its usage as
of produce. Scientific studies probiotic drink, specifically for
revealed positive results on the use poultry, hence this study.
of lactic acid on broilers. Lactic acid Generally, this study
bacteria have been used on the feed determined the growth pattern of
and drinking water. It had caused lactic acid bacteria (LAB) in
significant reduction of Salmonella probiotic rice washed water.
and Campylobacter incidence in Specifically, the
poultry product (Bryd et al., 2001). investigation focused to: determine
Administration of LAB likewise the microbial count of the viable
caused a significant reduction of LAB at the different phases of its
salmonella recovered 24 hours after growth; determine the typical
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Southern Leyte State University, Sogod, Southern Leyte
2,3
Visayas State University, Visca, Baybay City
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growth curve of LAB in probiotic different treatments used in the

rice-washed water; identify the study.
logarithmic growth phase of LAB on
probiotic rice-washed water Preliminary Activities
medium; and examine the cultural
and morphological characteristics of Working area was cleaned
LAB growing on probiotic rice and completely sanitized with
washed water. chlorinated water and 70% ethyl
alcohol. Tools and equipment were
all washed, dipped in a 150 – 200
2.0 Methodology ppm chlorine solution while all
glassware were sterilized
Research Design and Experimental (autoclave).
Treatment
Preparation of microbial starter
The study utilized factorial culture
design employing Completely
Randomized Design (CRD) in three Starter culture for LAB was
replications. Independent variables obtained from a commercial yogurt.
included the rice-water ratio (1:1, LAB culture from yogurt was
1:2, 1:3) used for washing and the activated using fresh carabao’s
number of washings (1st and 2nd milk. Activated starter culture was
washing). Table 1 shows the inoculated into the probiotic rice
washed water at constant

Table 1. Experimental treatments of the probiotic rice washed water.

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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City
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and Allen Glen C. Gil3 Vol. 3:126-138(2015)

concentration, using 10% starter of The starter culture was

the total volume of the water. inoculated into the different
treatments of probiotic rice washed
Washing of Rice water after pasteurization. The
amount of starter culture was
To obtain the probiotic rice equivalent to 10% of the measured
washed water, the rice (red rice volume of the probiotic rice washed
variety) was washed with distilled water. The probiotic rice washed
water following the rice-water ratios water and starter culture were
(1:1, 1:2, 1:3) and the number of thoroughly mixed and incubated at
washings (1st washing and 2nd 370C to allow optimum growth of
washing). The rice was washed LAB. Standard microbial procedure
thoroughly and strained to separate was followed for the inoculation
the water from the rice. Final with the application of aseptic
volume of the water was determined techniques.
using the graduated cylinder. This
determined the volume of the starter Preparation of Dilution Water
culture that was inoculated into the
probiotic rice washed water. Peptone water was used for
the dilutions of the sample. The
Pasteurization of Rice Washed amount of peptone powder used was
Water in proportion to the recommended
amount of 20g of peptone powder to
The probiotic rice washed 1 liter of distilled water. Weighing
water was heated until it reaches the of peptone powder was done using
temperature of 720C. This was an analytical balance to obtain its
maintained for 15 minutes to kill any exact weight. Diluted peptone water
microorganisms that might cause was then sterilized in an autoclave at
spoilage and unnecessary 121oC for 15 minutes.
fermentation that may affect the
growth of LAB. After heating, the Preparation of MRS Agar
probiotic rice washed water was
cooled down to 37 - 400C ready for The amount of MRS agar
inoculation of starter culture. powder used was in proportion to
the recommended amount of 66g of
Inoculation of Starter Culture MRS agar powder to 1 liter of
distilled water. MRS mixture was
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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City
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heated to completely dissolve the distilled water was poured.

MRS agar powder. It was sterilized Afterwards, a loopful of colony
in an autoclave at 121oC for 15 taken from the petri plate where
minutes. LAB is growing was spread in the
drop of water. To detain the bacteria
Microbial Evaluation for examination, the glass slide was
heat-fixed until the liquid
Samples from the different evaporated. The slide is now ready
treatments of the probiotic rice for gram staining in order to be
washed water was subjected to observed under an electronic
microbial evaluation at 6 hours microscope.
interval to determine the growth
pattern of LAB. Serial dilution of
the sample was prepared using the Statistical Analysis
peptone water as diluents. The 10-7
dilution of the sample was pour Results of the study were
plated on MRS agar using standard subjected to Analyses of Variance
pour plating method. Standard plate (ANOVA) using SPSS to determine
count (SPC) of the microorganisms the effects between treatments
was evaluated and the growth following the Completely
pattern was analyzed by plotting the Randomized Design (CRD) with
data on a graph. The cultural and three replications. Treatments with
morphological characteristics of the significant difference were subjected
LAB growing on the probiotic rice to post hoc analysis using Duncan’s
washed water were also evaluated. Multiple Range Test (DMRT).

Examination of the Cultural and

Morphological Characteristics of 3.0 Results and Discussion
LAB
Microbial Count
In order to examine the
cultural and morphological At twenty-four (24) hours
characteristics of LAB, a glass slide after incubation, the microbial
was prepared and sterilized by counting revealed less than
applying alcohol and heating it until 1x107cfu/ml of all experimental
the alcohol evaporates. At the treatments. The condition remained
center of the glass slide, a drop of the same 6 hours thereafter except
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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City
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for treatment with 1:3 rice-water provided better media for early
ratio second washing (T6) where proliferation of viable LAB. On the
microbial count reached 2.8x108cfu/ other hand, highest count of viable
ml (Fig. 6). It is interesting to note LAB in T3 and T4 (1.5x109 and
that the said medium offered the 1.4x109 cfu/ml, respectively)
earliest high growth of LAB occurred 30 hours after incubation
although the population dropped at (Figs. 3 & 4). This suggests that 1:2
18 hours, regained maximum rice-water ratio, whether 1st or 2nd
strength six hours after (at 24 hours), washing, offered better probiotic
and gradually dropped in the medium but took longer lag
succeeding hours. This growth duration. Of all the experimental
behavior points out that the medium treatments, T1 had the lowest
provided conducive environment for (5.4x108 cfu/ml) recorded microbial
the multiplication of LAB in a count throughout the duration (Fig.
longer duration compared to others. 1). It is basically due to its high
In one hand, T5 and T2 registered the starch content (1:1 rice-water ratio
first two highest microbial counts 1st washing) which is not readily
(1.5x109 and 1.3x109 cfu/ml, consumable/fermentable by the
respectively) at 12 hours after LAB. At 42 hours, most of the LAB
incubation (Figs. 5 & 2), while T4 counts from various treatments
registered the lowest (2x107 cfu/ml) dropped (<1x107) until finally at 48
(Fig. 4). This indicates that both 1:3 hours, scanty LAB were left due to
rice-water ratio 1st washing and 1:1 its death basically caused by acidic
rice-water ratio 2nd washing environment.

Fig.1. Growth trend of lactic acid Fig.2. Growth trend of lactic acid
bacteria from 0-48 hours bacteria from 0-48 hours after
incubation of T2.
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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City
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Fig.3 Growth trend of lactic acid Fig.4 Growth trend of lactic acid
bacteria from 0-48 hours after bacteria from 0-48 hours after
incubation of T3. incubation of T4.

Fig.5 Growth trend of lactic acid Fig.6 Growth trend of lactic acid
bacteria from 0-48 hours after bacteria from 0-48 hours after
incubation of T5. incubation of T6.

Typical Growth Curve of LAB growth of LAB already started. At

12 hours, all other experimental
During the first six hours treatments entered logarithmic phase
after the incubation period, all with T5 having the highest and T4
experimental treatments exhibited a having the lowest microbial count
lag phase or adjustment period in (1.5x109 and 1.3x109 cfu/ml,
their growth except for T6. At 6 respectively) (Fig. 7). It was
hours after the incubation period, T6 observed that there was a variation
showed 2.8x108 cfu/ml bacterial in the peak growth of LAB from 12
count which indicates that rapid hours (T5 and T2) to 30 hours (T3
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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City
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and Allen Glen C. Gil3 Vol. 3:126-138(2015)

and T4) and the rest in between (T1 Logarithmic Growth Phase of LAB
and T6). Likewise, decline or death
phase vary from 18 hours (T5 and In general, the logarithmic
T2) to 36 hours (T3 and T4). Beyond growth phase of LAB in various
this, majority of the experimental experimental treatments started at 12
treatments exhibited an estimated hours after the incubation period.
plate count (ESPC) of<1x107. The The stationary phase differs among
results specify that 1:3 rice-water treatments as well as the decline
ratio 1st washing (T5) and 1:1 rice- phase. Overall, the LAB in all
water ratio 2nd washing (T2) treatments greatly dropped after 42
provided a growth environment hours (Table 2). The rice-water ratio
which enhances early rapid growth of 1:1 1st washing (T1) attain
while 1:2 rice-water ratio, whether logarithmic phase at 12 hours
1st or 2nd washing, provided longer (3x107); at its highest (5.4x108) at 18
adjustment period prior to the hours; and gradually declined
attainment of rapid growth. thereafter. The highest bacterial

Figure 7. Graphical representation of the growth pattern of LAB in probiotic

rice washed water.

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Visayas State University, Visca, Baybay City
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count of T1 is the lowest among the Despite the similarity in the

peak bacterial counts of other microbial count at 12 hours, T5 and
treatments. The highest bacterial T2 exhibited significantly different
count attained among all treatments decline in microbial count
was that of T5 at 12 hours (1.5 x109) 8 7
(7.7x10 and 4x10 , respectively) at
and T3 at 30 hours (1.5x109) 18 hours. Treatment 5 generally
followed by T4 at 30 hours showed higher viable bacterial count
(1.4x109 ) and T2 at 12 hours in longer duration (12 hours to 30
(1.3x109) (Table 2). hours) (Table 2).
The comparison of treatment At 18 hours, T5 remained as
means using one-way analysis of the treatment with highest microbial
variance (ANOVA) unveiled highly count (7.7x108) followed by T1
significant difference (0.01 level of (5.4x108). At 24 hours, T6 posted the
significance) from 12 to 36 hours; highest microbial count (1.1x109).
significant difference at 42 hours At 30 hours, T3 (1.5x109) and T4
(0.05 level of significance) and no (1.4x109) marked the significantly
significant difference from 0, 6 and highest microbial count and were in
48 hours (Table 2). The post hoc their peak growth. The LAB
test (DMRT) result further revealed population gradually declined at 36
that at 12 hours after incubation to 42 hours, but still the two highest
period, T5 and T2 (first 2 highest treatments excelled from the rest.
microbial count) were not This result manifests the longer lag
significantly different from each period of T3 and T4 to attain the peak
other (1.5x109 and 1.3x109, of LAB growth. Longer lag period
respectively) but significantly means longer time of waiting prior
different from the rest of the to utilization thus, higher cost is
treatments. This suggests that, needed. However, depending on the
utilization-wise, either of the 1:3 rice urgency of the need, appropriate rice
-water ratio 1st washing or 1:1 rice- -water ratio and number of washing
water ratio 2ndwashing, can be can be employed to obtain the
employed as probiotic medium to optimum viable LAB and maximize
grow and harvest viable probiotic the use of this resource at lowest
earlier. Viable LAB can then be possible cost. At 48 hours, most of
harvested and stored in the the LAB in all treatments were
refrigerator to temporarily deactivate already dead (Table 2).
it while not yet used or directly use
it as probiotic drink for animals.
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Table 2. Comparison of microbial count means at 6-hour evaluation interval.

Morphological and Culture shape, color and stain reaction were

Characteristics of LAB in Probiotic identical to the activated LAB from
Rice Washed Water the source (Fig. 8), Nestle plain
yogurt. This proved that the LAB
Gram staining process of the LAB in from the source were the same LAB
various experimental treatments of that grew in the different
probiotic rice washed water experimental treatments regardless
disclosed that their morphological of rice-water ratio and number of
and cultural characteristics such as washing (Figs. 9 to 14).

Fig. 8. Lactic acid bacteria growing in Fig. 9. Lactic acid bacteria growing in
activated yoghurt and fresh 1:1 rice-water ratio 1st
milk mixture (oil immersion). washing (oil immersion).
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Fig. 10. Lactic acid bacteria growing in Fig.11.Lactic acid bacteria growing in
1:1 rice-water ratio 2nd 1:2 rice-water ratio 1st
washing (oil immersion). washing (oil immersion).

better media for highest and

early proliferation (12 hours) of
viable LAB.

2. It takes at least 12 hours, after

incubation, for the LAB to attain
logarithmic growth phase and 42
hours and beyond for LAB to
greatly drop in microbial count.
Fig.12.Lactic acid bacteria growing in
1:2 rice-water ratio 2nd 3. The rice washed water, as a
washing (oil immersion). probiotic medium, maintained
the integrity of LAB within 12 to
42 hours after incubation.

4.0 Conclusion
5.0 References Cited
Based on the foregoing
results, it can be deduced that: Byrd JA, Hargis BM, Caldwell DJ,
Bailey RH, Herron KL,
1. Both 1:3 rice-water ratio 1st McReynolds JL, Brewer R L,
washing (T5) and 1:1 rice-water Anderson RC, Bischoff KM,
ratio 2nd washing (T2) provide Callaway TR, Kubena LF.
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Visayas State University, Visca, Baybay City
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and Allen Glen C. Gil3 Vol. 3:126-138(2015)

2001. Effect of lactic acid Fuller R. 1992. Probiotics: The

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Visayas State University, Visca, Baybay City
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and Allen Glen C. Gil3 Vol. 3:126-138(2015)

Overview. Institute for

Integrative Biology, Herborn
-Dill, Germany.

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Southern Leyte State University, Sogod, Southern Leyte
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Visayas State University, Visca, Baybay City