fermentation of mango juice by lactic acid bacteria

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY FERMENTATI

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY

FERMENTATION OF MANGO JUICE BY

LACTIC ACID BACTERIA

SUPERVISOR STUDENT

MSc HUYNH XUAN PHONG NGUYEN LE VAN

Student code: 3082484 Session: 34

Can Tho, 2013

APPROVAL

MSc HUYNH XUAN PHONG NGUYEN LE VAN

Can Tho, May …, 2013

PRESIDENT OF EXAMINATION COMMITTEE

Abstract

In order to take advantage of available fruit and contribute to the diversification of products from mango, this research was carried out in term of added value of fruit juice fermented by lactic acid bacteria (LAB) Four isolates of lactic acid bacteria from mango were obtained The results of research showed that all 10 strains

of lactic acid bacteria were able to develop in pH from 1.5 to 3.5 after 2 hours of incubation at 37°C with a density from 6.80 to 6.91 log cells/mL After fermentation, the lactic acid content of 10 bacterial strains was in a range from 0.48 to 0.96% w/v, density after 2 hours of incubation at 37°C from 8.85 to 9.3 log cells/mL

Of which, strains isolated Lactobacillus acidophilus from Ybio yeast powder gave the best results with the following levels of lactic acid after fermentation was 0.96% w/v The appropriate ratio for fermentation mango juice was 40% for dilution and 9% for blending sugar The suitable conditions for mango juice fermentation were as follow: at 37°C for 36 hours and at 5 log cells/mL In these conditions the sensory criteria and the density

of bacteria could reach requirements for probiotic products (> 6 log cells/mL) These levels could identically maintain during the storage at temperature 4-6 o C for 2 weeks

Keyword: fermentation, L acidophilus, lactic acid bacteria,

mango, probiotic

CONTENTS

Abstract i

Content ii

1 Introduction 1

2 Materials and methods 3

2.1 Material 3

2.2 Methods 3

2.2.1 Isolation and identification of LAB isolates at genus level 3

2.2.2 Study on the tolerance at low pH of LAB isolates 3

2.2.3 Study on the ability to produce mango juice fermented by LAB 4

2.2.4 Study on the ratio of dilution and blending sugar 4

2.2.5 Study on the effects of LAB density, fermentation temperature and time 5

2.2.6 Study on temperature and time for storage conditions 5

3 Results and discussion 6

3.1 Isolation of lactic acid bacteria 6

3.2 The ability of LAB growing in low pH condition 7

3.3 Applicability of mango juice fermented by LAB 9

3.4 The ratio of dilution and blending sugar 11

3.5 Effect of inoculum density, temperature, fermentation time 16

3.6 The effect of temperature and time for storage conditions 19

4 Conclusions and suggestions 22

4.1 Conclusions 22

4.2 Suggestions 22 References 23

1 INTRODUCTION

Food is an indispensable necessity to humans Growing society, human life is more and more enhanced So, eating is not a main demand The demand of enjoying and exploring the effect

of food on health are top priority Since, the relationship between food and dietary which have health benefits as well as help the body fight the illness has become the trend research of the nutritionist and scientists to create high-value products in terms of biological products, is known as "probiotics" The probiotic concept has been defined by Fuller (1989) to mean “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” Salminen

et al (1999) proposed that probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host There are many probiotic products use for human, livestock, plants It is produced and used of liquid or powder form

Lactic acid are Gram-positive bacteria (Fooks et al., 1999), ferment carbohydrates into energy and lactic acid (Jay, 2000) Depending on the organism, metabolic pathways differ when glucose is the main carbon source: homofermentative bacteria, whereas the heterofermentative transform a glucose molecule into lactate, ethanol and carbon dioxide (Caplice and Fitzgerald, 1999; Jay, 2000; Kuipers et al., 2000) In addition, LAB produce small organic compounds that give the aroma and flavor to the fermented product (Caplice and Fitzgerald, 1999) LAB are widely distributed in the nature They could be isolated from

soils, water, plants, silages, waste products and also from the intestinal tract of animals and humans (Axelsson, 1998) LAB are

widely used in probiotic products such as yogurt, nem chua,

pickle, These products are not only used for eating but also used

to treat intestinal , stomach, due to lactic acid bacteria have the ability to produce antibiotics to prevent and kill bacteria and pathogenic microbes

Juice is a good environment for the growth of bacteria and probiotic products Fruits and vegetables are foods with health benefits because they contain antioxidants, vitamins, fiber and minerals Moreover, they do not contain any allergens for consumers In fruits, mango is a popular fruit and has high nutritional value Ripe mango has attractive yellow color, sweet and sour, savoury aroma is more preferred Mangoes contain vitamin A, C, sugar, organic acids, so mango is widely used both unripe and ripe fruit Mangoes are eaten fresh, making juice, jam, candy

In order to take advantage of available fruit and contribute

to the diversification of products from mango, this research was carried out in term for added value of fruit juice fermented by lactic acid bacteria

Objectives:

Study on the conditions of mango juice fermentation by lactic acid bacteria

2 MATERIALS AND METHODS

2.1 Materials

- Mango: Thanh Ca and Cat Chu

- Six isolates of lactic acid bacteria from Luong Phuoc

Truong (2012)

- Medium: MRS broth, MRS agar

- Chemicals, equipments in Food Biotechnology laboratory

2.2 Methods

2.2.1 Isolation and identification of LAB isolates at genus level

Mango juice was contained in a flask, incubated at 37oC for

Observe the bacteria under the microscope objective lens X100

Identification through the preliminary test: Gram stain, catalase, oxidase test and dissolution of CaCO3

2.2.2 Study on the tolerance at low pH of LAB isolates

Transferring LAB isolates from experiment 1 into tubes containing MRS broth environment with 3 different levels of pH (1.5, 2.5 and 3.5) This was the 2 experimental factors (strain and pH) with 3 replications Count the density of bacteria in individual treatments at incubation time (T0) and after 2 hours of incubation by plate counting method

Monitor the indicators: density of the bacteria strains Analyze the data with statistics program STATGRAPHICS Centurion XV

2.2.3 Study on the ability to produce mango juice fermented

by LAB

Ripe mangoes were pressed (pasteurized with 140 mg/L of

NaHSO3 in 20 minutes) and put in the centrifuge tubes Inoculate each of LAB isolates into each tube This was the 1 experimental factor (strains) with 3 replications

Incubation at 37°C, analyzed after 48 hours

Count density of lactic acid bacteria: plate counting method Monitor the indicators: Brix, pH, level of lactic acid after fermentation and the density of lactic acid bacteria Analyze the

data with statistics program STATGRAPHICS Centurion XV

2.2.4 Study on the ratio of dilution and blending sugar

This was the 2 experimental factors with 3 replications Ratio of dilution (%): 30, 40, 50, 60

Ratio of blending surose (%): 6, 9, 12, 15

After pressing, 480 mL of mango juice was diluted with ratios 30, 40, 50, 60 % and was blended sugar with rate 6, 9, 12,

15 % After dilution and blending, these samples were pasteurized

by NaHSO3 (140 mg/L in 20 minutes) Then the best LAB isolate

in experiment 3 was inoculated in the samples and incubated at

37oC for 48 hours

Monitor the indicators: Brix, pH before and after fermentation, lactic acid content after fermentation and the

density of LAB, the sensory evaluation Analyze the data with statistics program STATGRAPHICS Centurion XV

2.2.5 Study on the effects of LAB density, fermentation temperature and time

This was the 3 experimental factors at 3 levels with 2 replications

Density (log cells/mL): 3, 5, 7

Incubation temperature (ºC): 30, 37, 28-32 Time for fermentation (hours): 24, 36, 48

After pressing, mango juice was diluted and blended sugar

by the appropriate rate in experiment 4, pasteurized by NaHSO3 Then, LAB isolate was inoculated with density were 3, 5, 7 log cells/mL, respectively Incubated these samples at 30oC, 37oC, 28-32oC for 24, 36, 48 hours

Monitor the indicators: Brix, pH and the density of LAB, the sensory evaluation Analyze the data with statistics program STATGRAPHICS Centurion XV

2.2.6 Study on temperature and time for storage conditions

This was the 2 experimental factors with 3 replications Temperature for storage: 4-6oC, 20-25oC, 28-32oC

Time for storage: 1, 2 weeks

Monitor the indicators: Brix, pH and the density of LAB, the sensory evaluation Analyze the data with statistics program STATGRAPHICS Centurion XV

3 RESULTS AND DISCUSSION

3.1 Isolation of lactic acid bacteria

After several transfers in MRS agar medium and visual observations, 4 isolates of lactic acid were isolated from 2 kinds

of mango Colonies of all isolates were round, smooth, grayish white

The cell morphology was shown in Table 2’

Table 2’ The characteristics of isolates

Cell morphology

Note: The strains are marked * which were isolated from Truong (2012)

2 isolates from Cat Chu (C213, C111) were rod in shaped, but the colony of C213 was smaller than C111 2 isolates from Thanh Ca (T223, T133) were rod in pairs-shaped and cocci

pairs-in pairs-shaped.

4 isolates were characterized in Gram positive, lack of catalase and oxydase, produced clear zone around colonies in medium containing CaCO3

From the above characteristics, it could be concluded that

4 isolates belong to lactic acid bacteria group

3.2 The ability of LAB growing in low pH condition

The results in Table 3 showed that all bacterial strains at different pH levels have increased density after 2 hours of incubation at 37°C At time T0, the density of bacteria in the MRS medium at pH 1.5 and 2.5 decreased significantly (from 5 log CFU/mL to 1.46-2.16 log CFU/mL) The reason was that the pH 1.5 and 2.5 are too low So, most bacteria should be shocked But only after 2 hours of incubation at 37°C, they worked to restore and increased the density up to 6.8-6.9 log CFU/mL

Figure 7: CaCO3 test

of T223 isolate

Figure 6: Representative

oxydase test of C213

isolate

At pH 3.5, most of bacteria could tolerate this pH So, the density of LAB at the time T0 decreased slightly from 4.66 to 4.77 log CFU/mL After 2 hours of incubation at 37°C, the density of bacteria increased up to 6.81-6.91 log CFU/mL

Density of 10 isolates at pH 3.5 was higher than at pH 2.5 and density of 10 isolates at pH 1.5 was lowest This showed that the lower the pH affects the ability to survive and activity of LAB Through this experimental study showed that all isolate were able to adapt and develop in a low pH medium, so they could tolerate the environment in the stomach Therefore, these strains were able to apply in probiotic products

Table 3 The density of bacteria

in MRS Broth environment at low pH

(log CFU/mL)

Cv (%)

(log CFU/mL)

Cv (%)

3.3 Applicability of mango juice fermented by LAB

Nowadays, probiotic products are commonly used as fermented milk and yogurt Besides, juice is offered as a good

environment for the fermented probiotic products (Mattila- Sandholm et al., 2002)

Table 4 Some indicators of mango juice after fermentation

ĐĐ 8.0ab 3.07bc 0.69c 4.85ab 9.25a C213 8.0ab 3.42a 0.92ab 4.83abc 9.03a C111 8.0ab 3.39a 0.91ab 4.83abc 9.08a T223 8.12ab 3.21b 0.75c 4.80abc 8.91a T133 8.83a 3.11bc 0.75c 4.77bc 8.85a

Brix of the treatments after fermentation was lower than the original in a range 8.0-8.87, because LAB used sugar in the

environment After 48 hours of fermentation mango juice, pH of the environment of the isolates decreased from 3.83 to 3.02-3.42

As the results presented in the previous experiments, LAB isolates were able to survive and grow at this pH The cause of the decrease in pH was due to the development of lactic acid bacteria that produced lactic acid Content of lactic acid produced from 4 isolates from Ybio powder, Biosubtyl powder and cat chu have no significant differences with each other and higher than the other isolates

Density of lactic acid bacteria strains in time T0 were nearly equal to the initial concentration of bacteria strains (4.76-4.88 log CFU/mL) and after incubation all isolates had a significant enrichment activity (8.85-9.30 log CFU/mL) Results showed statistically processed, the density of lactic acid bacteria strains at time T0 had no significant differences with each other and significantly different from 3 strains P, Bio,T133 After 48 hours of fermentation the density of bacterial strains were > 6 log CFU/mL, consistent with standards of probiotic products Therefore, all strains were capable of application in the production of fermented mango However, lactic acid content of 4 isolates from Ybio powder, Biosubtyl powder and Cat Chu had no significant differences with each other and higher than those of

isolates Among those strains, the L acidophilus isolate from Ybio had the best content of lactic acid So, L acidophilus isolate

was selected for the next experiments

3.4 The ratio of dilution and blending sugar

The results in Table 5’ showed that pH values before and after fermentation was difference After 48 hours of incubation,

pH was reduced from about 4.5-4.61 to 3.63-3.84 It caused by bacterial growth, changed glucose into lactic acid At the same time, content of solution (oBrix) also decreased but not significantly

Table 5’ Some indicators of mango juice fermentation

Blending

Sucrose

(%w/v)

Dilution (%)

pH after fermentation

Brix after fermentation