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: carmencita.duberry146@we.utt.edu.tt

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: rohanie.maharaj@utt.edu.tt

Tel.: 1 868 642 8888; Fax: 1 868 642 1617.

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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.

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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.

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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

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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)

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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.

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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.

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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

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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.

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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

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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.

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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

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