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PROBIOTIFICATION OF TROPICAL
FRUIT BLENDS FERMENTED WITH
LACTIC ACID BACTERIA (Lactobacillus
bulgaricus and Lactobacillus plantarum)
Carmencita Duberry
University of Trinidad and Tobago, Caroni North Bank Road, Centeno, Via Mausica,
Trinidad & Tobago, W. I.
E-mail: [email protected]
Tel.: 1 868 642 8888; fax: 1 868 642 1617.
D. Singh-Ackbarali, V. Ganessingh and R. Maharaj
University of Trinidad and Tobago, Caroni North Bank Road, Centeno, Via Mausica,
Trinidad & Tobago, W. I.
E-mail: [email protected]
Tel.: 1 868 642 8888; Fax: 1 868 642 1617.
1
PROBIOTIFICATION OF TROPICAL
FRUIT BLENDS AT UTT
• Probiotics or the “health friendly bacteria” are microorganisms
that when administered in adequate amounts provide health
benefits to the host (Shi et al., 2016).
• Fermented milk products have been usually considered as the
most excellent carriers for probiotics; however, consumption of
milk-based products would be limited to those who are not
lactose-intolerant or vegans (Patel, 2016, Silanikove et al.,
2015).
• Beverages based on fruits, cereals, vegetables and soybeans
have been proposed as new products containing probiotic
strains; essentially, fruit and vegetable juices have been reported
as a novel suitable carrier medium for probiotic.
2
PROBIOTIFICATION OF TROPICAL
FRUIT BLENDS AT UTT
• A major challenge of this type of product is acceptance by
consumers (Ellendersen et al., 2012).
• There is a genuine interest in the development of fruit-juice
based functional beverages, fortified with the probiotic
ingredients (Vasudha and Mishra, 2013).
• In this research the use of fresh pineapple, passion fruit,
orange and carrots were the main ingredients used to create a
product with viable probiotics which is palatable as well as
provide easy to consume nutrients without dairy allergenic
consequences.
3
PRODUCTION OF PROBIOTIC JUICE
Receive, sort, wash, sanitize fruits & vegetables
Use a Juicer to obtain 100% Fruit & Vegetable
Dilute with distilled water add simple syrup
Pasteurize for 80°C for 30 seconds
Cool
Inoculate Probiotic cultures
Pour into sterile bottles
Incubate and ferment for 48hrs
4
PHYSIOCHEMICAL ANALYSES
• All instruments were cleaned and calibrated before used.
• For the titratable acidity the 0.1 M NaOH was previously
standardized, and values were expressed as citric acid
equivalents.
• Ascorbic acid was used for standardization for the vitamin C
test.
Parameter Instrument Name/AOAC Reference Number
pH HANNA desktop pH meter, HI3220
Colour Hunterlab, ColorFlex EZ's 45°/0°
Soluble solids (brix) Reichert, Abbe Mark III 1310499
Titratable acidity
(TA)
AOAC 947.05 (19th Ed. 2012)
Vitamin C AOAC 967.21 (19th Ed. 2012)
5
MICROBIOLOGICAL ANALYSES
• Determine survival of the cultures used to ferment juice blend
as well as to determine possible presence of gram negative
pathogen.
• Serial dilution (10-1 to 10-5) and spread plate method on MRS
and EMB agar plates were used to determine the number of
colony forming units (cfu).
• After inoculation all plates were incubated at 35°C for 48hrs, in
an aerobic atmosphere supplemented with carbon dioxide. The
number of colonies were then counted and cfu/ml was
calculated.
6
SENSORY EVALUATION
• Evaluated and compared to a commercial pineapple orange
kombucha tea fermented drink that was available on the market.
• The sensory evaluation was done with a 10 member panel who
were each given randomized chilled coded samples to evaluate
on attributes of; appearance, fruity flavour, sweet taste, mouth-
feel and overall acceptance.
• A 9 point hedonic scale was used for this comparison, and the
results were plotted on a star diagram.
7
SHELF LIFE TESTING AND STATISTICAL
ANALYSIS • Done under controlled temperatures at 4°C, 23°C and also under
stressed conditions of 35°C for the period of observation ending
at day 21 and 10 respectively. Quality test were also conducted
during this time frame; pH, °Brix and Colour and sensory
attributes.
• The physiochemical and microbiological analyses on the same
sample for each parameter was made in 3 replications and the
results were expressed as mean value, ± standard deviation.
• Student t test was used to compare the results between the
pasteurized and unpasteurized juice blends and ANOVA was
used for sensory evaluation comparison between the three
product samples. Values that were significantly different had
p<0.05. 8
RESULTS
Fruit Juice
Blend
TSS
(Brix)
Titratable
Acidity
Vitamin C
(g of
ascorbic
acid)
pH Colour,
L, a*, b*
CFU/ml
(viable
count in
MRS)
Unpasteurized,
incubated 37°C
for 48hrs
13.70a
(±3.11)
0.67c
(±0.00)
8.07 x 10-4d
g (±0.23)
3.23f
(±0.04)
45.99g, 11.505h,
34.475i (±5.20,
4.48, 1.83)
6.64 ×109 j
(±9.6 ×108)
Pasteurized,
incubated 37°C
for 48hrs
15.9a
(±1.86)
0.73c
(±0.28)
1.27 x 10-3e
g (±0.10)
3.23f
(±0.04)
46.08g, 27.775h,
46.175i (±4.05,
0.06, 0.29)
4.08 × 1010 k
(±9.7 ×109)
T Test (p value) 0.43 0.12 0.054 0.88 0.42, 0.13, 0.95 0.03
* Values are means of three replicates (±standard deviation). The experimental
values within columns that do not have a common superscript are significantly
different (p<0.05).
Table 2: Physicochemical and microbiological properties of the
fermented treated (pasteurized) and untreated juice blends
9
RESULTS
• Decrease in pH and brix, increase in titratable acidity - due to successful carbohydrate fermentation, indicating that the cultures could utilise the sugars present in the fruit juices for cell synthesis.
• The LAB cultures remained viable - proven by the CFU/mL results in the MRS (6.64 ×109 and 4.08 × 1010 cfu/ml) agar.
• The absence of growth on the selective EMB agar medium indicated hygienic and aseptic practices were adhered and that probioticated juices efficiently inhibited the growth of coliforms.
• Significant difference in the CFU between the pasteurized and unpasteurized juice blends, where the pasteurized blends had better survival of the LAB cultures.
• Possibly be because the pasteurization process effectively killed any naturally occurring cultures and so there was less competition for the resources and hence better survival of the LAB.
10
RESULTS
It was also determined that there was no significant differences between the
probioticated pasteurized and unpasteurized juice blends (p>0.05).
Shelf life
Temp
Self life
time
Observations
4°C
21 days
Pasteurized - only insignificant changes (STDEV ± 1.27 and 2.40) in the
“lightness” and “redness” of the pasteurized sample, the brix, pH and sensory
results were insignificant (STDEV < ± 0.5) and still acceptable after the study
period.
Unpasteurized juice also had only insignificant changes (STDEV ± 2.83 and 2.20)
in the brix and “redness”, the sensory results were also still acceptable after the
study period.
23°C
10 days
Least favourable results with respect to the “lightness” (STDEV ± 3.89) and
significant unacceptable difference with the odour for both pasteurised and
unpasteurised.
35°C
10 days
No significant changes in the pH and the colour (STDEV ±2.77 and 0.16),
however there was a noticeable difference in the brix which decreased to 4.4°
(pasteurized) and 9.5° (unpasteurized) from 16.1°, this can be explained by the
higher temperature being a favourable condition for the viable LAB cultures to
break down available sugars in the juice. After the 10 day period the sensory
attributes that were still acceptable was colour and taste however the odour was
not
11
RESULTS
0
1
2
3
4
5
6
7
8
9Appearance
Mouthfeel
Sweet tasteFruity taste
Overall acceptability
Sample 619 Sample 357 Sample 482
Figure 1. Star diagram comparing the five different attributes selected for this
study for the three different probioticated drinks. Sample 619 and 357 were
probioticated fruit blends verses sample 482 commercial fruit kombucha tea. 12
CONCLUSION
• The tropical fruit and vegetable blend, having a pH range of
3.7–3.8 before probiotification, was suitable for addition of
LAB cultures to prepare a non-dairy probioticated drink.
• The results of the present study have demonstrated that Lb.
plantarum and bulgaricus were able to survive in fermented
juices at low pH (3.23) and high acidity (0.67 and 0.73).
• Sensory evaluation showed that the probioticated juice blend
was found to be acceptable.
• This research concluded that fruit juices can be exploited as an
acceptable fermentation medium that is high in nutrients for the
delivery of probiotic LAB to lactose-intolerant people and
vegans.
13
LITERATURE CITED
1. Ellendersen, L.S.N., Granato, D., Guergoletto, B.K. and Wosiacki, G.,
2012. Development and sensory profile of a prebiotic beverage from apple
fermented with Lactobacillus casei. Engineering in Life Science 12: 1–11.
2. Patel, A.R., 2017. Probiotic fruit and vegetable juices- recent advances
and future perspective International Food Research Journal 24(5): 1850-
1857
3. Shi, L. H., Balakrishnan, K., Thiagarajah, K., Mohd Ismail, N. I., and Yin,
O. S., 2016. Beneficial Properties of Probiotics. Tropical life sciences
research 27(2): 73–90.
4. Silanikove, N., Leitner, G., and Merin, U., 2015. The Interrelationships
between Lactose Intolerance and the Modern Dairy Industry: Global
Perspectives in Evolutional and Historical Backgrounds. Nutrients, 7(9):
7312–7331.
5. Vasudha, S. and Mishra, H. N., 2013. Non dairy probiotic beverages.
International Food Research Journal 20(1): 7-15
14
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