www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 59
Formulation of Guava Fruit Beverage with Isolated Probiotic Strains
Sai Bramari.G1, Anitha.L
4* Kalpana.P
2 and Pooja Mandlik
3
1, 2, 3Research Scholars,
4Associate Professor
Department of Microbiology and Food Science & Technology, Institute of Science, GITAM
University, Visakhapatnam, A.P, India -530 045.
Email: [email protected]
ABSTRACT
The suitability of Guava fruit beverage for the production of probiotic juice with Lactic acid bacteria
isolated from milk, curd, whey and Lactobacillus plantarum has been studied. The probiotic strains
isolated from dairy products are characterized morphologically and biochemically. The formulated
Guava fruit beverage was inoculated with these isolates and incubated at 300C. Changes in pH,
acidity, sugar content, and viable cell counts during fermentation under controlled conditions were
measured. The pH of the Guava fruit beverage was initially 5.5 at 300 C and decreased gradually to a
suitable range. The titrable acidity at 300 C found to be 0.28% – 0.32% and was increased on further
fermentation. The sugar at 300 C was initially 15-22g/ 100ml. The viable cell counts at 30
0C were
estimated and they ranged between 5.6 x 10 6- 8.9x10
6. The fruit beverage was assessed for
acceptability through sensory evaluation. Molecular confirmation of isolate no.8 through 16s rDNA
sequence has been done and was found to be L. coryneformis. The final product resulted in suitable
pH, acidity, sugar content and ideal number of viable cell counts. The Probiotic Guava fruit beverage
could serve as a healthy beverage for consumers with dairy allergy, beneficial to gut health, prevention
of diarrhea and best nutrient source for under nourished vulnerable population.
Key words: Probiotics, Fermentation, Guava fruit beverage, Lactic acid bacteria, Diarrhea.
1. INTRODUCTION:
‘Let food be thy medicine and medicine be thy food’, the age-old quote by Hippocrates, is certainly the
tenet of today. With the growing interest in self-care and integrative medicine coupled with our health-
embracing baby boomer population, recognition of the link between diet and health has never been
stronger. Empirically, high levels of population, health go hand in hand with high levels of national
income. Higher incomes promote better health through improved nutrition, better access to safe water
and sanitation (Bloom and Canning, 2000).
Ever since the population is growing, the problems are also increasing very rapidly. The high
prevalence of malnutrition contributes to over 50% of child deaths. The second leading cause of death
among children under age five globally is Diarrhoea (Progress for Children Report, December 2007).
National surveys estimate that almost 10% of the population suffers from diarrheal disease in India.
(NFHS-3, 2005-06).
Diarrhea is mainly due to lack of adequate sanitation facilities for disposal of excreta, poor hygiene
practices which results in spread of pathogens in to the human environment (Progress for Children
Report ,December 2007; WHO, 2008). The current treatment for diarrhea includes the oral rehydration
therapy to replace the fluid and electrolyte loss. ORT (Oral Rehydration Therapy) was spread
worldwide to decrease the mortality rate due to diarrhea (Lochery, 2005). A search of alternative
therapy in treating various forms of diarrhea finally led to the conclusion that “Probiotics” can be the
treatment of choice (FAO/WHO, 2002). The studies demonstrated that the use of Probiotics reduced
the duration and the chances of symptoms lasting longer than four days by 60% (Sunil Kumar Mishra
et al., 2012). So, Probiotics are fast emerging as an alternative to conventional antimicrobial therapy
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 60
and are less likely to induce resistance due to their multifaceted mechanisms of action (Sujatha
Narayan et al., 2010).
Probiotics are defined as live microbial feed supplement that beneficially affects the host by improving
its intestinal balance (Gorbach, 2000). The term “Probiotics” meaning “for life” was first coined by
Lilly and Stillwell in 1960’s (Lilly and Stillwell, 1965). In the early 20th
century, Elie Metchnikoff
(1907) first proposed the concept of Probiotics. He suggested that beneficial bacteria could be
administered to replace harmful microbes with useful ones. FAO/ WHO (2002) defined Probiotics as
live microorganisms that when administered in adequate amounts confer a health benefit on the host
(FAO/WHO, 2002; Fuller, 1989). It has been estimated that 100 different species (with a total bacterial
population between 1010
and 1012
) are present in the human intestinal tract. Maintenance of an optimal
gut flora balance requires that ‘friendly’ bacteria, such as the gram-positive Lactobacilli and
Bifidobacterium which dominate and form a barrier to pathogenic bacteria. Probiotics are the most
natural and safe means of maintaining this balance (Anuradha and Rajeshwari, 2005).
Pribiotic dairy products and non dairy products like vegetable and fruits juices are the currently used
Probiotic foods (Vasudha and Mishra, 2011). The fermented milks and dairy products are the first
Probiotic products (Metchnikoff, 1907). Lactose intolerance and allergy towards dairy products are
problems with Probiotic dairy products (Amal Hassan et al., 2013). Hence it was thought that fruit
juices could be a better medium for fermentation with Probiotics (Panesar, 2011). The objective of this
research is to develop a Probiotic fruit beverage that contains Probiotic characters and healthy
properties of fruits.
2.0 Materials and Methods
2.1 Isolation of Lactic acid bacteria from dairy products
The Lactobacilli were isolated from different sources like raw milk, Curd, pasteurized milk, goat’s
milk and from whey (Renuka Goyal et al., 2012). MRS medium was used for the isolation of
Lactobacilli (Rogosa, et al, 1951). The raw milk of cow, curd of cow milk and whey were used as
inoculums and incubated at 370 C for 24-48 hours. (Talat Mehamood et al, 2009).
2.2 Morphological and Biochemical identification:
The phenotypic characterization of isolated strains was carried out by Grams staining (Talat
Mehamood et al, 2009) and Catalase test was also performed (James Cuppucino & Natalie Sherman,
2004). Along with isolated species of Lactobacilli, L. plantarum (NCIM NO. 2592) obtained from
NCIM, NCL; Pune was also tested to check its potentiality as a Probiotic strain. The isolated species
were found to be Gram positive and Catalase negative which gives a preliminary idea about the
isolated strains as Lactobacilli. These isolated strains were further subjected to the biochemical tests
like sugar fermentation tests, Arginine hydrolysis, Growth in different concentrations of NaCl and
growth at different temperatures.
After the confirmation of isolates through morphological and biochemical tests as Lactic acid bacteria
species, fermentation of Guava fruit beverage was carried out. (James Cuppucino & Natalie Sherman,
2004).
2.3 Formulation of Guava fruit beverage:
Fresh guavas were purchased from the market and were thoroughly washed. The skin was
removed and the fruits were made into pulp using kitchen mixer and juicer. Guava fruit beverage was
prepared as per composition (Table1) (Rakesh Gehlot et al. 2011).
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 61
TABLE: 1 Formulation of Guava Fruit Beverage
The concentrated Guava fruit beverage so prepared was diluted in 1:3 proportions and distributed into
clean, dry and sterilized glass bottles.
The isolated Lactobacilli were introduced into the diluted Guava fruit beverage and subjected
to fermentation by incubating the bottles at 300C (room temperature) and 4
0C (refrigeration
temperature). Shelf life of fermented Guava fruit beverage was checked at 300C and 4
0C. Fermentation
analysis of the Guava fruit beverage was carried out by checking its pH, titrable acidity, sugar
estimation and viable cell counts (Kyung Young Yoon et al., 2004).
2.4Fermentation analysis
2.4.1 pH:
The pH was measured with a pH meter (ELICO, sl.no. - 3456/2008 type 101).The pH was
measured at 300 C and 4
0 C.
2.4.2 Titrable acidity:
It was measured by titrimetric method. Initial and final readings of burette were noted. Titrable acidity
was calculated.
2.4.3. Sugar estimation:
The sugar content of fermented guava fruit beverage was estimated at frequent intervals by
phenol sulphuric acid method. It was estimated as grams of glucose per 100ml of guava fruit beverage.
O.D values were taken using spectrophotometer (Hitachi, u-2900) model.
2.4.4 Viable cell counts:
The viable cell counts were measured by the standard plate technique on MRS medium. The
inoculated and incubated Guava fruit beverage sample was taken and serially diluted. From 10 5
dilution, 0.1 ml of inoculum was taken and spread on to MRS agar plates and incubated for 24-48
hours. The colonies were counted using a colony counter. (Digital Colony Counter Sr. No- 1275)
(Kyung Young Yoon et al., 2005).
2.5 Antibacterial activity of isolates on Enterobacter aerogens, E.coli and S. aureus
The antibacterial activity by isolated samples on Enterobacter aerogens, Escherichia coli and
Staphylococcus aureus has been carried out by agar well diffusion method (Verma, et al., 2013).
2.6 Antibiotic sensitivity tests of isolates
The Antibiotic sensitivity of the isolated strains was carried out by disc diffusion method. Five oral
antibiotics like Ciprofloxacin, Ceftriaxone. Gentamycin, Lomefloxacin and Sublactum were used for
testing the isolated strains (Maharoshi Nayeem et al., 2012).
2.7 Molecular analysis:
One of the isolated strain (sample 8) was analyzed by 16s r DNA sequencing at Bioserve Biological,
India Lab at Hyderabad.
2.8 Statistical analysis:
All experiments were carried out in triplicates and each sample analyzed as duplicates. The
results were expressed as Mean ± S.D (Standard Deviation). Statistical analysis was done through
multiple comparison method (Scheffe’s post hoc test) of every isolate with each other. The P value
was analyzed by ANOVA (Kyung Young Yoon et al., 2004).
2.9 Organoleptic evaluation:
The sensory evaluation of fermented Guava fruit beverage was done by a selected panel
consisted of students from department of Food science who regularly participate in sensory analysis
studies and had experience in profiling food products. Sensory descriptors were developed during
GUAVA PULP 1LITRE
SUGAR 1.5 Kg
CITRIC ACID 22 Grams
WATER(FILTERED) 750ml.
SODIUM BENZOATE 2.0 Grams (Preservative)
RASPBERRY RED COLOUR 1 DROP
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 62
initial session by panelists. The score card consists of sensory attributes like appearance, color, flavor,
taste and overall acceptability. The scores given by panelists measured with five point hedonic rating
scale were recorded as mean± S.D (standard deviation) (Mohammad Daneshi et al., 2013).
3. Results
3.1 Morphology of isolates:
The present study was undertaken to study the suitability of Guava fruit beverage as substrate for
Lactic acid fermentation by Probiotic Lactobacillus strains. The experiment was started with the
isolation of Lactobacillus strains from dairy products like milk, curd and whey. As many as 50
colonies were isolated from each product but only 5 individual colonies were picked up after
screening. These isolated colonies were maintained as pure cultures. Out of these 5 isolates two were
isolated from raw milk of Cow, two strains from Curd of Cow milk and one isolate from whey. L.
plantarum (NCIM no. 2592) was obtained from NCIM, NCL, Pune. The isolated strains were
characterized morphologically. The isolated species were found to be Gram positive and Catalase
negative were shown in fig. 1 (James Cuppucino and Natalie Sherman, 2004). One isolate was
confirmed with molecular analysis of 16s rDNA sequencing (Isolate no. 8 shown in figure 4).
Fig 1: Morphology of Isolates of Lactobacillus from Diary Products (samples 2, 3, 8, 9 and 10
respectively)
3.2 Fermentation analysis
3.2.1 pH of the fermented Guava fruit beverage at 300C and 4
0C:
The pH of the Guava fruit beverage was initially 5.5 at 300 C and decreased gradually. (Table: 5) The
increase in the titrable acidity leads to decrease of the pH. The change in pH at 40C was shown in table
6. The same was represented graphically (fig. 2) which reveals that there was no significant difference
among pH values of each and every pair of sample at 300C. The decrease in the pH at 4
0 C was much
nearer to the changes in the pH at 300C. The similar results can be observed from the study of Kyung
Young Yoon et.al (2004).
TABLE 5: Changes in pH at 300 C during fermentation of Guava fruit beverage
Samples
0
days
3
days
5
days
7
days
9
days
12
days
Mean± S.D
F-
Value
P-
value
Decision
2 5.5 5.316 5.19 4.89 3.772 3.732 4.7333±0.7874 0.03 0.999 N.S
3 5.5 5.291 5.097 4.796 3.805 3.76 4.7100±0.7528
8 5.5 5.25 5.082 4.742 3.74 3.359 4.6117±0.8671
9 5.5 5.286 5.143 4.836 3.765 3.297 4.64±0.8949
10 5.5 5.321 5.168 4.739 3.83 3.402 4.66±0.8584
L.p 5.5 5.257 5.088 4.726 3.561 3.231 4.56±0.9436
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 63
TABLE 6: Changes in pH at 40C in fermented Guava fruit beverage
3.2.2 Titrable acidity (TA) at 300 C and at 4
0C in the fermented Guava fruit beverage:
The TA at 300 C in the Guava fruit beverage found initially to be 0.28% which was increased to 0.34%
for sample 2, 0.35% for sample 3, 0.37% for sample 8, 0.34% for sample 9, 0.36% for sample 10 and
0.38% for L. plantarum respectively. It was further increased and the same was shown in table 7 and in
the graph (fig. 2). TA at 40C was increased after fermentation (table 8) and was shown in the graph
(fig3) (Kyung Young Yoon et al. 2004).
TABLE 7: Variations in Titrable Acidity of Guava fruit beverage incubated at 300C
TABLE 8: Variations in Titrable acidity in fermented Guava fruit beverage at 40C
Samples
0 weeks
1stweek
2nd
week
3rd
week
4th
week
Mean± S.D
F -
Value
P-
value
Decision
2 5.5 5.384 5.182 4.983 4.741 5.156±0.3054 0.04 0.999 N.S
3 5.5 5.373 5.16 4.976 4.789 5.160±0.2868
8 5.5 5.356 5.107 4.945 4.654 5.114±0.3361
9 5.5 5.388 5.193 4.98 4.772 5.166±0.2974
10 5.5 5.379 5.204 4.963 4.734 5.154±0.3124
L. p 5.5 5.353 5.098 4.921 4.643 5.102±0.3415
Samples 0 days 3 days 5 days 7 days 9 days
12
days
Mean± S.D
F-
Value
P-
value
Decision
2 0.288 0.342 0.3708 0.3816 0.3888 0.3816 0.35833±0.03764 0.68 0.640 N.S
3 0.3024 0.3564 0.3924 0.3888 0.414 0.3888 0.37333±0.03933
8 0.3168 0.3708 0.396 0.4068 0.4188 0.414 0.38833±0.03764
9 0.288 0.3456 0.378 0.396 0.4032 0.4104 0.37167±0.04535
10 0.2952 0.3672 0.3924 0.396 0.3996 0.3996 0.37667±0.03933
L. p 0.3204 0.3852 0.4068 0.4188 0.4248 0.4161 0.39667±0.03933
Samples
0weeks
1stweek 2
ndweek 3
rdweek 4
thweek
Mean± S.D
F-
Value
P-
value
Decision
2 0.288 0.3924 0.432 0.522 0.5292 0.432±0.099 0.24 0.943 N.S
3 0.3024 0.3672 0.4476 0.5292 0.5328 0.436±0.100
8 0.3168 0.432 0.4896 0.5472 0.5508 0.468±0.0965
9 0.288 0.4032 0.4608 0.5328 0.5256 0.442±0.1008
10 0.2952 0.4176 0.4692 0.5292 0.5292 0.450±0.0956
L. p 0.3204 0.4896 0.4989 0.558 0.5652 0.488±0.1003
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 64
TABLE 9: Changes in Sugar estimation of Guava fruit beverage incubated at 300C
Table10: Changes in Sugar estimation of fermented Guava fruit beverage at 40C
3.2.3 Sugar estimation (SE) of fermented Guava fruit beverage at 300 C and at 4
0C:
The SE value at 300 C was initially 15-22g/ 100ml for sample 2 to L.p respectively and has been
reduced during fermentation. The results of SE at 300C were tabulated in table 9 and at 4
0C in table 10
and were shown in the graph (fig. 2). The findings were on par with the study of Kyung Young Yoon
et al. (2005).
3.2.4 Viable cell counts of fermented Guava fruit beverage at 300C and at 4
0C in 10
5 dilutions:
The viable cell counts at 300C were estimated in 10
5 dilutions. The cell counts increased rapidly after
incubation of 24hours. They ranged between 5.6 x 10 6- 8.9x10
6 in 10
5 dilution. The cell counts were
found to be increased up to 7days after that these was a fall in the cell counts at 300C. This was shown
in the table 11 and fig.2. Viable cell counts at 40C found to be increased and were observed till
4weeks. The cell counts were shown in table 12 and fig.3. The increase in the cell counts and the
survival of culture in the fruit beverage makes it suitable as a probiotic fruit beverage. (Kyung Young
Yoon et al. (2005).
TABLE 11: Viable cell counts of isolates in Guava fruit beverage at 300C:
Samples 0
weeks
1st
week
2nd
week
3rd
week
4th
week
Mean± S.D F-
Value
P-
value
Decision
2 15.2 14.8 13.6 12.4 11.2 13.44±1.664 0.81 0.552 N.S
3 16.4 16.4 15.2 14 13.2 15.04±1.431
8 21.2 20.8 19.2 17.6 16 15.1±2.184
9 16.8 16.4 14.8 13.6 12.4 14.80±1.855
10 17.2 16.8 15.6 13.6 11.2 14.88±2.488
L. p 22 21.2 20 17.6 18 15.2±1.931
Samples 0
days
3
days
5
days
7
days
9
days
12
days
Mean± S.D F-
Value
P-
value
Decision
2 15.2 14.4 13.6 12.4 11.2 9.2 12.667±2.215 2.39 0.062 N.S
3 16.4 15.2 14 14 12.4 10.8 13.80±1.988
8 21.2 20 20.4 20.4 19.2 16.8 15.4±1.906
9 16.8 15.6 14 14.8 13.6 11.2 14.333±1.917
10 17.2 16.4 16 16.4 14.4 12 14.967±0.689
L.p 22 20.8 20.4 21.2 20 18 15.433±0.841
Samples 0 days 3 days 5 days 7 days
9 days
12 days
2 5.6x106 6.3x10
6 7.4x10
6 6.8x10
6 3.8x10
6 1.6x10
6
3 6.1x106 7.2x10
6 7.8x10
6 7.3x10
6 5x10
6 3.2x10
6
8 8.5x106 9.2x10
6 10.3x10
6 9.2x10
6 5.8x10
6 3.4x10
6
9 6.8x106 7.3x10
6 7.4x10
6 7.8x10
6 4.4x10
6 1.9x10
6
10 6.3x106 7x10
6 7.2x10
6 7x10
6 4.9x10
6 1.8x10
6
L. p 8.9x106 10x10
6 11.1x10
6 10x10
6 6x10
6 3.9x10
6
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 65
TABLE 12: Viable cell counts of isolates in Guava fruit beverage at 40C:
Fig. 2: Fermentation analysis at 30˚C; TA ---- Titrable acidity; SE ---- Sugar estimation
0
2
4
6
8
10
12
14
16
18
Ph at 30°C TA at 30°C SE at 30°C Viable Cell Count at
30°C
Parameters
Aver
age
Val
ue
Sample 2
Sample 3
Sample8
Sample 9
Sample10
Sample Lp
Samples 0 weeks 1stweek 2
nd week 3
rd week 4
th week
2 5.6x106 5.9x10
6 6x10
6 6.4x10
6 7x10
6
3 6.1x106 6.5x10
6 6.8x10
6 7x10
6 7.1x10
6
8 8.5x106 8.9x10
6 9x10
6 9.3x10
6 9.6x10
6
9 6.8x106 7x10
6 7.2x10
6 7.5x10
6 7.7x10
6
10 6.3x106 6.6x10
6 7x10
6 7.7x10
6 8x10
6
L. p 8.9x106 9.2x10
6 9.2x10
6 9.5x10
6 9.6x10
6
mple 2
ple 3
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 66
Fig 3: Fermentation analysis at 4˚C; TA ---- Titrable acidity; SE ---- Sugar estimation
3.2.5 Antibacterial activity:
The diameter of zone of inhibition was measured and recorded and shown in the table 13. The highest
zone of diameter was observed for L. plantarum followed by sample 8. All other strains have shown
specific antibacterial activity. Similar results can be observed from the work of Verma, et al. (2013).
3.2.6 Antibiotic sensitivity:
All the samples have shown resistance against the antibiotics like Ciprofloxacin, Ceftriaxone,
Lomefloxacine, Sublactum and Gentamycin. The results were shown in the table 14. The results of the
antibiotic sensitivity can be compared with the results of the study carried out by Maharoshi nayeem et
al. (2012).
3.2.7 Molecular analysis of isolate 8 through 16s r DNA sequencing:
The molecular analysis of sample 8 was carried out at Bioserve Biological India Labs,
Hyderabad. By the 16s r DNA sequence report the isolate was confirmed as L. coryniformis species.
The phylogeny of sequence was constructed using MEGA software (Fig 4). The sample 8 was chosen
for molecular analysis because it has shown highest antibacterial activity next to L. plantarum and the
sample 8 was shown resistance to all antibiotics. The molecular analysis was carried out similar to the
study of Mirzaei Hamid and Barzgari Abulfazl, (2012). Phylogenetic and molecular evolutionary
analyses were conducted using MEGA version 4 (Tamura et al, 2007).
Table 13: Anti Bacterial activity of isolates on Enterobacter aerogens , Escherichia coli and
Staphylococcus aureus:
Bacterial sp. Diameter of zone of
inhibition for E.
aerogens(mm)
Diameter of zone of
inhibition for E.
coli(mm)
Diameter of zone of
inhibition for S.
aureus(mm)
Sample-2 83 66 61
Sample-3 64 52 70
Sample-8 120 108 119
Sample-9 60 48 54
Sample-10 92 76 96
Sample-L. p 180 172 182
0
2
4
6
8
10
12
14
16
Ph at 4°C TA at 4°C SE at 4°C Viable cell Count at
4°C
Parameters
Av
erag
e V
alu
e
sample2
sample 3
sample8
sample9
sample 10
sample Lp
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 67
Table 14: Antibiotic sensitivity test of isolates on various antibiotics:
R- resistant S-sensitive
Fig 4: Evolutionary relationships of 8 taxa- Phylogenetic tree
4. Discussion:
The isolated colonies were identified as Lactobacillus species. L. plantarum was proved as best
probiotic strain in the earlier works (Kyung Young Yoon et al., 2004 & 2005). Sample 8 was analyzed
and identified through 16s rDNA sequencing as L. coryneformis. (Mirzaei Hamid and Barzgari
Abulfazl, 2012).
The Guava fruit beverage formulated, optimized and evaluated in the laboratory (Rakesh
Gehlot et al. 2011). The isolated five Lactobacillus species were inoculated into Guava fruit beverage
and fermentation was carried out at 300 C and 4
0 C.
All isolates used for fermentation of Guava fruit beverage were capable of utilizing the nutrients for
their growth, synthesis and metabolism. From the earlier works it can be known that the Lactobacillus
species were able to ferment vegetables such as Tomato, Cabbage (Kyung Young Yoon et al., 2004 &
2005) and fruits like Raspberries, Blue berries and Rock melon etc (Lin Kiat Saw et al., 2011). The
appearance, color, flavor, taste and overall acceptability of fermented Guava fruit beverage was found
be acceptable. The mean scores of the sensory attributes were given in the table 15. In fermented
Guava fruit beverage, the color was deep pink with creamy tinge and was accepted by 100%. The taste
was accepted by 99% and flavor was accepted by 95% of panelists. The sensory evaluation reports
were similar with the findings of Mohammad Daneshi et al. (2013). The fermented final product
resulted with desirable properties like suitable pH, acidity, the amount of reducing sugar and adequate
number of viable cell counts as in case probiotic Tomato, Cabbage and other fruit juices (Kyung
Young Yoon et al., 2004 & 2005). The isolated strains have shown significant antibacterial activity
against bacterial pathogens (Verma, et al., 2013). The isolated strains have shown good resistance
against antibiotics (Maharoshi Nayeem et al., 2012).
Sample
Ciprofloxacin
Ceftriaxone
Lomefloxacine
Sublactum
Gentamycin
2 R R R R S
3 R R S R R
8 R R R R R
9 S R R R R
10 R S R R R
L.P R R R R R
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 68
The richness of Guava fruit qualities and goodness of Probiotic strains blend together to make
Guava fruit beverage as an ideal Probiotic fruit beverage and acceptable to all vegetarians who are
allergic to dairy products.
TABLE 15: Mean scores of sensory attributes of Guava fruit beverage
CONCLUSION AND SIGNIFICANCE OF THE WORK:
.The Guava fruit is rich in vitamin C and is a good source of nutrients. The alkaline astringent
compound present in Guava fruit binds the bowels during diarrhea. So, the current research has been
undertaken to formulate and develop a Guava fruit beverage with isolated probiotic strains that
controls diarrhea and also provides the rich nutrients to consumers. The potentiality of the formulated
fruit beverage needs to be tested in vitro and in vivo to confirm and declare it as an anti diarrheal
probiotic fruit beverage.
REFERENCES
1. Amal A. Hassan, Mona M, A. Aly and Soher T. El-Hadidie, (2013) Production of Cereal-
Based Probiotic Beverages, Int Food Res Journal 20(1): 7-15.
2. Anuradha, S, Rajeshwari, K. (2005). ‘‘Probiotics in health and disease’’. J Indian Acad Clin
Med. 6, 67-72.
3. Atkins, J. P.; Mazzi, S.; Easter, C.D. (2000). A Commonwealth Vulnerability Index for
Developing Countries: The Position of Small States. Commonwealth Economic Paper Series
40, Commonwealth Secretariat, London, www.thecommonwealth.org/.../...
4. Bloom and Canning, (2000). ‘‘Population and Economic growth available at’’
www.growthcommission.org/storage/cgdev/documents/gcwp024web.pdf
5. FAO/WHO (2002) ‘‘Guidelines for the evaluation of probiotics in food’’. FAO website:
ftp://ftp.fao.org/es/esn/food/ wgreport2.pdf. Accessed 8 Apr 2010.
6. Fuller, R. (1989) Probiotics in man and animals, J. of Appl. Bacteriol 66, 365-367.
7. Gorbach, S.L. (2000). Probiotics and gastrointestinal health. American J.of Gastroent, Vol 95,
Iss 1, Suppl. S, pp S2-S4.
8. James Cuppucino and Natalie Sherman, (2004). MICROBIOLOGY A LABORATORY MANUAL
Pearson Education (US) Benjamin-Cummings Publishing Company, Subs of Addison Wesley
Longman, Inc 05 Apr 2004.
9. Kyung Young Yoon; Edward, E.; Woodams, L.; Yong D Hang, L. (2004). December
Probiotication of Tomato Juice by Lactic Acid Bacteria, The J.of Microbio, p.315-318.
10. Kyung Young Yoon; Edward, E.; Woodams, L.; Yong D Hang, L. (2005). June, Production of
probiotic cabbage juice by lactic acid bacteria. Biores tech, Vol. 97, No. 12. (August 2006), pp.
1427-1430.
11. Lilly, D.M.; Stillwell, R.H. (1965). Probiotics: growth-promoting factors produced by
microorganisms. Science; 147,747-8.
12. Lin Kiat Saw, Suijing Chen, Siew Hwa Wong, Soon Ann Tan, Kelvin K.T Goh, 2011. ‘‘The
12th ASEAN FOOD CONFERENCE 2011’’ 16 -18 June, 2011 BITEC Bangna, Bangkok,
Thailand.
ITEM CODE APPEARANCE COLOUR FLAVOR TASTE OVER ALL
ACCEPTABILITY
Guava fruit
beverage at 30˚C
4.6±0.514
4.31±0.61
4.54±0.54
4.54±0.54
4.54±0.50
Guava fruit
beverage at 4˚C
4.4±0.66
4.6±0.43
4.5±0.54
4.65±0.39
4.35±0.39
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 69
13. Lochery, P. (2005). ‘‘The impact of water and sanitation on the health of children under five:
Global Health Council’’ available at 217.218.200.220/documents/ 10129/20978/02-
Feb.pdf?version=1...
14. Mahrosh naeem, M. Ilyas, Saleem haider, Shahjahan baig and m. Saleem, (2012). “Isolation
characterization and identification of lactic acid bacteria from fruit juices and their efficacy
against antibiotics”. Pak. J. Bot., 44: 323-328, Special Issue March 2012.
15. Metchnikoff, E. (1907). In the Prolongation of Life. In: Optimistic Studies, Chalmers M. (Ed.).
William Heinemann, London
16. Mirzaei Hamid and Barzgari Abulfazl, (2012). “Isolation and Molecular Study of Potentially
Probiotic Lactobacilli in Traditional White Cheese of Tabriz in Iran”. Annals of Biological
Research, 2012, 3 (4):2019-2022
17. Mohammad Daneshi, Mohammad Reza Ehsani, Seyed Hadi Razavi, Mohsen Labbaf (2013).
“Effect of refrigerated storage on the probiotic survival and sensory properties of milk/carrot
juice mix drink”. Electronic Journal of Biotechnology; ISSN: 0717-3458;
http://www.ejbiotechnology.info; DOI: 10.2225/vol16-issue5.
18. Mortality and disease burden rate (2004), www.who.int/healthinfo/...burden disease/.
19. NFHS-3 (2005-06), www.measuredhs.com/pubs/pdf/FRIND3/FRIND3-VOL2.pdf
20. PS Panesar (2011), Fermented Dairy Products: Starter Cultures and Potential Nutritional
Benefits PP. 47-51 DOI: 10.4236/fns.2011.2100
www.scirp.org/Journal/PaperInformation.aspx?paperID=3643
21. Pochapin, M. (2000). The effect of Probiotics on Clostridium difficile diarrhea. American J.of
Gastroent, Vol 95, Iss 1, Suppl. S, pp S11-S13.
22. Progress for Children Report (December 2007)-A Statistical Review,
www.unicef.org/.../files/Progress_for_Children_No_6_revised.pdf
23. Rakesh Gehlot et al. 2011 January, Nutritional and Therapeutic Beverages from Guava,
Beverage and Food world, hau.ernet.in/cobs/cfst.pdf
24. Renuka Goyal, Harish Dhingra, Pratima Bajpai and Navneet Joshi, (2 October, 2012)
Characterization of the Lactobacillus isolated from different curd samples, African Journal of
Biotechnology,Vol.11(79), pp.14448-14452, http://www.academicjournals.org/AJB,
AJB11.310 ISSN 1684–5315.
25. Rogosa, M, Mitchell, J.A. Wiserman, R.F (1951). A selective medium for isolation and
enumeration of oral and fecal lactobacilli. J. of. Dental. Re. 30, 682.
26. Saarela, M.; Mogensen, G.; Fonden, R.; Matto, J.; Matilla-Sandholm,T. (2002). “Probiotic
bacteria: safety, functional and technological properties’’. J. of Biotech.
27. Sujatha S Narayan, Sharmila Jalgaonkar, S Shahani, Vijaya N Kulkarni, SS
Narayan (2010) Probiotics: Current trends in the treatment of diarrhea’’
www.hkmj.org/article_pdfs /hkm1006p213.pdf
28. Sunil Kumar Mishra, Pratibha Mishra, Mohit Saxena (1 April – June, 2012) Probiotics: An
Approach for Better Treatment, Volume 3 Issue 2 Page No. 1042, RJPBCS.
www.rjpbcs.com/pdf/2012_3(2)/[122].pdf
29. Talat mehamood et.al, (2009), ‘‘Isolation and identification of wild strains of Lactic acid
bacteria for yoghurt preparation from indigenous dahi, Pak jou of Nutr 8 (6): 866-871, ISSN
1680 – 5194, Asian network of scientific information.
30. Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.
31. Vasudha, S. and Mishra, H. N. (2011) Non dairy probiotic beverages. Food and Nutr Sci, 2,
47-51,doi:10.4236/fns.2011.21006.
32. Verma, S., Paliya, B.S., Prasad, S., Chaudhary, H.S (2013) Antimicrobial Activity of Probiotic
Microorganisms from Probioticated Carrot Juice against Selective Pathogenic Strains.
International Journal of Scientific & Engineering Research, Volume 4, Issue 8, August-2013
2130 ISSN 2229-5518.
www.theinternationaljournal.org > RJSITM: Volume: 03, Number: 10, August-2014 Page 70
33. World Health Organization. Global burden of disease report: (2004 update; 2008).
www.who.int/healthinfo/global_burden_disease/2004_report_update/.
34. WHO. (2002) Improving child health in the community. WHO/FCH/CAH/02.12.Geneva:
WHO, www.coregroup.org/storage/ .../who_key family practices evidence.pdf.