fermentation of dragon fruit juice by lactic acid bacteria

Abstract This study was carried out to isolate and select the isolates of lactic acid bacteria applied for the fermentation of dragon fruit juice.. Six strains of lactic acid bacteria i

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENT THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY

FERMENTATION OF DRAGON FRUIT JUICE

BY LACTIC ACID BACTERIA

Student code: 3082567 Session: 34

Can Tho, 2013

Abstract

This study was carried out to isolate and select the isolates

of lactic acid bacteria applied for the fermentation of dragon fruit juice Experiments were done including dilution rates, sugar content, inoculum level, incubation temperature, fermentation time, and storage conditions Six strains of lactic acid bacteria isolated from dragon fruit juice and three strains of probiotic powder were tested All 6 tested bacterial strains could grow in low pH conditions (1.5 – 3.5) and could be used for fermentation

of dragon fruit juice, with the bacterial level was at 6 log CFU/mL A strain Lactobacillus acidophilus isolated from Antibio powder gave the best capable of dragon fruit juice fermentation (acid content was 1.38% w/v and bacteria concentration was 8.44 log CFU/mL) The favourable conditions

of fermentation were determined as follow: pure dragon fruit juice, 9% of supplemented sugar, 37ºC of fermentation temperature, 48 hours of fermentation, and 6 log CFU/mL of inoculum level The appropriate temperature and time storage for fermented juice were found at 4 – 6ºC for 3 weeks

Keywords: dragon fruit juice, fermentation, lactic acid bacteria,

Lactobacillus acidophilus, probiotic

Contents

Abstract i

Contents ii

1 INTRODUCTION 1

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Methods 3

2.2.1 Isolation and identification of LAB isolates at genus level 3

2.2.2 Study on the acidity tolerant ability of LAB isolates 4

2.2.3 Study on application in producing of fermented dragon fruit juice by LAB 4

2.2.4 Study on dilution rates of dragon fruit juice and sucrose rates 5

2.2.5 Study on inoculum levels, incubation temperature and fermentation time 5

2.2.6 Study on storage temperature and storage time 5

3 Results and discussion 6

3.1 Isolation and identification of LAB isolates at genus level 6 3.2 Acidity tolerant ability of LAB isolates 7

3.3 Application in producing of fermented dragon fruit juice by LAB 9

3.4 Dilution rates of dragon fruit juice and sucrose rates 10

3.5 Inoculum levels, incubation temperature and fermentation time 15

3.6 Storage temperature and storage time 18

4 Conclusions and suggestions 21

4.1 Conclusions 21

4.2 Suggestions 21

References 22

1 INTRODUCTION

Probiotics are defined as the non-pathogenic microorganisms capable of survive in the digestive process, having positive impact on the health and physiology of the hosts There are many probiotic products for human and livestock Lactic acid bacteria (LAB) are able to produce probiotics (Temmerman et al., 2002) Thus, LAB are used in probiotic products such as yogurt, fermented meat, fermented vegetables… These products are not only used as food but also used for treating intestinal and stomach diseases because LAB can produce antibiotics to prevent and skill pathogenic bacteria and germs Also, LAB are widely used in food fermentation because of their ability to improve flavour, texture and safety of perishable raw materials (Caplice and Fitzgerald, 1999)

Fruit juice is a good environment for the growth of bacteria and probiotic products Fruits and vegetables are good for health because of antioxidants, vitamins, fibers and minerals in components

“Dragon fruit” are commonly known as “thanh long” as in Vietnamese meaning “green dragon” Dragon fruit contains vitamins A, C, sugar, organic acids,… Dragon fruit juice is a suitable medium for lactic acid bacteria to produce new probiotic products Therefore, the study “Fermentation of dragon fruit juice

by lactic acid bacteria” was carried out

Objectives:

To isolate LAB strains from dragon fruit juice, and to study fermented dragon fruit juice producing process by lactic acid bacteria

Contents made

- Isolation and identification of LAB isolates at genus level

- Study on the acidity tolerant ability of LAB isolates

- Study on application in producing of fermented dragon fruit juice by LAB

- Study on dilution rates of dragon fruit juice and sucrose rates

- Study on inoculum levels, incubation temperature and fermentation time

- Study on storage temperature and storage time

2 MATERIALS AND METHODS

2.1 Materials

- Dragon fruit bought from Xuan Khanh market (Can Tho City)

- Probiotics powders:

+ Antibio powder: Product of Han Wha Pharma Co., Ltd

(Number 472, Namgog-Ri, Yangji - Myon, Yongin - Si, Kyonggi - Do, Korea)

+ PROBIO powder: Made in the Corporation Imexpharm

(No 4, 30/4, Cao Lanh Town, Dong Thap)

+ Lactomin powder plus: RexGene Biotech Co products.,

Ltd (Number 641-2, Ochang - Myun, Cheongwon - Kun, Chungbuk, Korea)

- Medium:

+ MRS broth (Merck, Germany): Peptone from casein: 10 g/L, meat extract: 8 g/l, yeast extract: 4 g/L, D-glucose: 20 g/L, dipotassium hydrogen phosphate: 2 g/L, tween 80: 1 mL, diammonium hydrogen citrate: 2 g/L, sodium acetate: 5 g/L, magnesium sulfate: 0.2 g/L, manganese sulfate: 0.04 g/L

+ MRS agar: MRS broth + agar 14 g/L

- Chemicals: NaOH 0.1N, HCl 0.1N, H2O2 3%, NaHSO3, the Gram stain (Crystal violet, Iodine, Acetone, Ethanol, Fuchsin)

- Equipments in Food Biotechnology Laboratory, Biotechnology Research and Development Institute, Can Tho University

2.2 Methods

2.2.1 Isolation and identification of LAB isolates at genus level

- Sterilized dragon fruit juice was incubated at 37ºC for 24 – 48 hours Fermented juice and probiotic powders were incubated in MRS broth medium for 24 hours LAB were isolated

by culturing the incubated media on MRS broth agar medium until getting the pure bacteria

- LAB isolates were identified at genus level through the preliminary tests: Gram stain, catalase, oxidase test and inspection capabilities resolution of CaCO3

2.2.2 Study on the acidity tolerant ability of LAB isolates

LAB isolates were inoculated in MRS broth medium adjusted to different levels of pH (1.5, 2.5 and 3.5) Bacterial density levels of LAB were determined at incubation time (T0) and after 2 hours of incubation by the counting living method Acidity tolerant LAB isolates were chosen

2.2.3 Study on application in producing of fermented dragon fruit juice by LAB

4 mL of LAB with 5 log cells/mL density were inoculated

to 36 mL of pasteurized dragon fruit juice The inoculated juices were incubated at 37ºC for 48 hours After 48 hours of incubation, the juices were analyzed the assessment criteria including:

2.2.4 Study on dilution rates of dragon fruit juice and sucrose rates

0.4 mL of LAB was inoculated to 39.6 mL of pasteurized dragon fruit juice adjusted to different dilution rates (0, 20, 30 and 40% w/v) and sucrose rates (6, 9, 12 and 15%) The inoculated juices were incubated at 37ºC for 48 hours After 48 hours of incubation, the juices were analyzed the assessment criteria including: pH, soluble matter content, content of generated lactic acid, density of LAB and sensory evaluation

2.2.5 Study on inoculum levels, incubation temperature and fermentation time

0.4 mL of LAB was inoculated to 39.6 mL of pasteurized dragon fruit juice adjusted to different levels of inoculum (4, 5, and 6 log cells/mL) The inoculated juices were incubated at different temperature levels (25, 30, and 37ºC) at 12, 24, and 36 hours After incubation, the juices were analyzed the assessment criteria including: pH, soluble matter content, content of generated lactic acid, density of LAB and sensory evaluation

2.2.6 Study on storage temperature and storage time

The fermented dragon juice products were stored at different temperature levels including: 4 – 6ºC (refrigerator), 20 – 25ºC (cooler) and 28 – 32ºC (room temperature) in 1, 2, 3, and 4 weeks After storage time, the juices were analyzed the assessment criteria including: pH, soluble matter content, content

of generated lactic acid, density of LAB and sensory evaluation

3 RESULTS AND DISCUSSION

3.1 Isolation and identification of LAB isolates at genus level

Isolation results

The features of isolated bacteria colonies: round, glossy, smooth, creamy white The colonies of each bacteria strain are identical Colonies of bacteria isolated from Antibio and Probio powder were larger than dragon fruit ones

There were six isolated bacteria strains, two from dragon fruit juice and four from probiotic powder Cell morphology of isolated bacteria was various as shown in Table 3’

Table 3’ Cell morphology of isolated bacteria

Characteristics of isolated bacteria strains:

- Bacteria strains isolated from Antibio and Probio powder were rod-shaped bacteria They had the same characteristics of

Lactobacillus acidophilus, the bacteria in probiotic powder

components

- Bacteria strains isolated from Lactomin were streptococci

- Bacteria strains isolated from dragon fruit juice were shaped bacteria However, the length of these bacteria strains was shorter than the length of rod-shaped bactera strains isolated from Antibio and Probio powder

rod-Genus identification

Some simple biochemistry experiments were carried out to determine the isolated bacteria strains at genus level The results showed that:

- Gram staining: Bacteria strains remained purple color after staining crystal violet, thus they were Gram-positive bacteria

- Catalase test: All six bacteria strains could not produce bubbles when adding H2O2 3%, indicated negative results

It can be concluded that these isolated bacteria strains belonged to lactic acid bacteria because they:

- Are Gram-positive bacteria

- Has no enzyme catalase

- Are rod-shaped or streptococcus

- Can grow on MRS medium

3.2 Acidity tolerant ability of LAB isolates

According Guarner and Schaafsma (1998), one of the criteria of probiotic products is survival ability of microorganisms after consuming process In human digestive system, the stomach

pH is very low, about 1 – 2, so majority of microorganisms are difficult to survive in this pH However, microorganisms existed only 1 – 2 hours in the stomach and then move to the large intestine with a neutral pH

After 2 hours of incubation at low pH, the bacteria increased concentration Initial inoculum level of LAB was 4 log cells/mL But when inoculating to MRS medium with pH in the range of 1.5 to 2.5, the bacterial density reduced significantly (only 1.08 to 1.40 log CFU/mL), because of shock of bacteria

under low pH condition But only after 2 hours incubation at 37ºC, they recovered and increased concentration (up to 6.31 to 6.62 log CFU/mL)

Almost bacteria could resist to pH 3.5 The decrease of bacterial density was not significantly at T0 (3.34 – 3.58 log CFU/mL) After 2 hours of incubation, the concentration reached

to 6.45 – 6.66 log CFU/mL

At T0, the concentration of 6 bacteria strains in pH 3.5 medium were higher than theirs in pH 2.5 medium And the lowest concentration of bacteria was in pH 1.5 medium It was showed that the lower pH affect to the survival ability of LAB

Table 4 Density of bacteria in MRS broth with low pH

Note: Value in the table was average value of triplication; the average values with the same letter were not significantly different at the 95% confidence level

(Log CFU/mL)

T2 (Log CFU/mL)

All six LAB strains increased the density after 2 hours of incubation at 3 different pH levels (ranging from 6.31 to 6.66 log CFU/mL) The bacterial densities after fermentation were significantly different at the 95% confidence level (Table 4’) The Pro strain reached the highest value (6.66 log CFU/mL) at pH 3.5, next to A strain at pH 1.5 (6.62 log CFU/mL) and TL1 strain at

pH 2.5 (6.55 log CFU/mL)

All isolated lactic acid bacteria strains were able to adapt and develop in a low pH environment, so they could survive in the environment of the stomach Therefore, these strains were able to apply in probiotic products

3.3 Application in producing of fermented dragon fruit juice by LAB

This experiment was carried out to determine whether the lactic acid bacteria strains could be applied in the production of fermented dragon fruit juice to create probiotic products, and to compare individual application abilities of LAB strains together After 48 hours, all lactic acid bacteria strains increased their concentration; solvent content (ºBrix) decreased; lactic acid was produced to reduce the pH of medium (Table 5)

Table 5 Following indicators fermented dragon fruit juice

Bacteria

Acid (% w/v)

Log T0 (log CFU/mL)

Log T48 (log CFU/mL)

Brix concentration of all treatments after fermentation decreased insignificantly (from 8.9ºBrix to about 7.1 – 8.0ºBrix) After 48 hours of fermentation, pH decreased from 4.52 to about 3.23 – 4.31 At this pH, lactic acid bacteria could remain The pH decreased because due to the production of lactic acid during development of bacteria Concentration of lactic acid produced from A strain (1.38%) and Pro strain (1.36%) were higher than other strain’s and they were significantly different to others at the 95% confidence level

After incubation, densities of LAB increased significantly (from 3.82 – 3.87 to 8.27 – 8.44 log CFU/mL) As statistical data analysis results, at T0, densities of Bio and A strains were highest (3.87 and 3.86 log CFU/mL), they were significantly different to others at the 95% confidence level

After 48 hours, densities of LAB were higher than 6 log CFU/mL The highest concentration (8.44 CFU/mL) belonged to

A strain (Lactobacillus acidophillus) and it was significantly

different to others at the 95% confidence level

Therefore, the Lactobacillus strain isolated from Antibio powder (notated as A) was used in further experiments

3.4 Dilution rates of dragon fruit juice and sucrose rates

The pH and Brix values in different dilution rates of dragon fruit juice and sucrose rates before and after fermentation were presented in Table 5