fermentation of carrot juice by lactic acid bacteria

In the second stage, the fermented carrot juice processing was tested with the selected lactic acid bacteria.. The favorable conditions for fermented carrot juice were determined based o

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

FERMENTATION OF CARROT JUICE

BY LACTIC ACID BACTERIA

SUPERVISOR STUDENT

MSc HUYNH XUAN PHONG NGUYEN THI AI XUAN Student code: 3082569 Session: 34 (2008- 2013)

Can Tho, 2013

APPROVAL

SUPPERVISOR STUDENT

MSc HUYNH XUAN PHONG NGUYEN THI AI XUAN

Can Tho, May , 2013

PRESIDENT OF EXAMINATION COMMITTEE

Abstract

This study was carried out in term to contribute for added value of the available, inexpensive and high nutrition raw materials as well as to diversify the production with good characteristics The study was divided into 2 stages In the first stage, isolation of the strain for fermentation was done from the nature fermented carrot juice Four isolates of lactic acid bacteria were compared with other 6 strains from products of digested powder (Luong Phuoc Truong, 2012) A strain of Lactobacillus helveticus gave the best results (1.46% w/v) In the second stage, the fermented carrot juice processing was tested with the selected lactic acid bacteria Of four testing media, carrot juice with 15% (w/v) of saccharose and 30% dilution ratio was found as the best medium The favorable conditions for fermented carrot juice were determined based on the results of sensory evaluation and bacterial density determination and they were found as follow: at

5 log cell/mL of the bacterial inoculum, for 24 hours of fermentation time and at 37ºC The result of storage testing showed that suitable temperature for product storage was 4-6ºC, sensory evaluation (4.21) and bacterial density (7.13 log CFU/mL) of final product maintained after 4 weeks

Key words: carrot juice, lactic acid bacteria, Lactobacillus

helveticus, probiotic

ii

CONTENTS

1 INTRODUCTION 1

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Methods 3

3 RESULTS AND DISCUSSION 7

3.1 Isolation of lactic acid bacteria 7

3.2 Study on low pH tolerance of isolates 8

3.3 Study on lactic acid fermentation ability of isolates in carrot juice 10

3.4 Identification of the selected lactic acid bacteria by molecular technique 11

3.5 Study on the effect of dilution ratio and sugar content 12

3.6 Studying the effect of inoculums density, temperature and time of lactic acid fermentation 14

3.7 Study on temperature and time of storage 16

4 CONCLUSIONS AND SUGGESTIONS 18

REFERENCES 19

1 INTRODUCTION

Probiotication is one of the methods used to produce fermented functional foods Addition of probiotics to food provides several health benefits including reduction in the level of serum cholesterol, improvement of gastrointestinal function, enhancement of immune system and reduction in risk of colon cancer (Yoon et al., 2004)

Probiotic bacteria are increasingly used in food and pharmaceutical applications to balance disturbed intestinal microflora and related dysfunction of the human gastrointestinal tract Presently, the majority of products containing probiotics are dairy-based, which include yogurt and fermented milk beverage However, due to some drawbacks related to dairy products, there are emerging interests in using non-dairy ingredients as substrates for delivering the physiological benefits of probiotics to wider group of consumers (Mattila-Sandholm et al., 2002)

Non-dairy substrates that have been used for lactic acid bacteria (LAB) fermentation include soy protein and cereals In recent years, several studies have reported the use of fruits and vegetables juices as base medium for LAB fermentation Juices from these sources are deemed to be advantageous because of their low allergenicity and perceived health benefits (Luckow and Delahunty, 2004)

Carrots are good source of carbohydrate, calcium, phosphorous, iron, potassium, magnesium, copper, manganese and sulphur It is an excellent source of vitamin A, B1, B2, C, E, thiamin, folic acid and riboflavin The intake of carrot as potent antioxidants, appear to be associated with better health It is not

The content of this study includes:

- Isolation of lactic acid bacteria

- Study on low pH tolerance of isolates

- Study on lactic acid fermentation ability of isolates in carrot juice

- Identification of the selected lactic acid bacteria by molecular technique

- Study on the effect of dilution ratio and sugar content

- Studying the effect of inoculums density, temperature and time of lactic acid fermentation

- Study temperature and time of storage

2 MATERIALS AND METHODS

- Culture medium: MRS (De Man Rogosa and Shape) agar and broth

- Chemicals: Yeast extract, peptone, glucose, manitol, CaCO3, NaCl, NaOH 1N, H2SO4 1N

- Devices:

+ Petri dish, pippetteman, eppendorf 1.5 mL

+ Incubator shaker Innova 4200, New Brunskick Scientific, USA

+ Optical microscope Olympus DP12 BX41

+ Autoclave pbi international 22793

+ RefrigeratorSanaky Tokyo, Japan

+ Eppendorf centrifuge 5417R

2.2 Methods

2.2.1 Isolation of lactic acid bacteria

Carrot juice solution was pasteurized Then, it was incubated for naturally fermented at 37ºC for 24-48 hours After that, it was cultured in MRS broth at 37ºC for 24 hours

The bacterial suspension was streaked into MRS agar medium, anaerobically incubated at 37ºC for 48 hours Then, typical colonies were subcultured several times until the purity of culture was obtained

2.2.2 Study on low pH tolerance of isolates

Low pH tolerant ability of isolates was determined by isolate density in growing media containing different level of pH The cultures were grown at 30ºC for 24 hours in MRS broth and were used as an inoculum (5 log cell/ mL) Then, isolates were inoculated in MRS broth medium with different pH levels (1,5; 2,5 and 3,5) in 2 hours (sterilized already at 121ºC for 15 minutes) Isolates were measured on plant count method at T0 and

T2 (afer 2 hours inoculation) The colonies were counted as viable numbers of isolates in low pH

2.2.3 Study on lactic acid fermentation ability of isolates in carrot juice

Carrot juice (pasteurized already with 140 mg/L of NaHSO3

in 20 minutes) was distributed in sterile tubes The juice was supplied with a inoculum with 5 log cell/mL isolates mixture (each tube contained 36 mL carrot juice + 4 mL of inoculum) Each tube represented a single sample and the experiments were performed in triplication

The lactic acid fermentation was performed in 37ºC for 48 hours The microorganisms were counted on MRS plates Samples were taken at 48 hours intervals for chemical and microbiological analysis pH was measured with a pH meter Sugar content was measured by Brix handheld meter Lactic acid content expressed as percent lactic acid was determined by

titrating with 0.1N NaOH to pH 8.2 Viable cell counts (CFU/mL) were determined by the standard plate method with MRS medium after 48 hours incubation at 30ºC

2.2.4 Identification of the selected lactic acid bacteria by molecular technique

The bacterial isolate was identified by sequence analysis of partial 16S rRNA gene A couple of primer including forward primer 8F: (5’-AGA GTT TGA TCC TGG CTC AG-3’) and reverse primer 1492R: (5’-GGC TAC CTT GTT ACG ACT T-3’) were employed to amplify the target gene through PCR technique DNA sequence of bacterial isolate was determined by DNA machine Nucleotide sequence was aligned and compared with the data obtained from Gene Bank

2.2.5 Study on the effect of dilution ratio and sugar content

The following variants differing with sugar levels: juice with the addition of 6, 9, 12 and 15% sugar and variants differing with dilution ratio 0, 10, 20 and 30% v/v

Fermentation experiments were conducted in test tubes, each containing 40 mL of pasteurized carrot juice All samples were inoculated with a 24 hours culture (5 log cell/mL) and incubated

at 37ºC for 48 hours

The fermentation dynamics was analyzed on basis of the most important physico-chemical (reducing sugar content, pH, lactic acid content) and microbiological parameters (numerical evolution of the microbial inoculum) at 48 hours In addition, sensory evaluation of the fermented carrot juice show a statistically significant effect of dilution ratio and sugar content

on its total quality

6

2.2.6 Studying the effect of initial inoculum density, temperature and time of fermentation

The activity was designed with three factors: LAB density at

103, 105 and 107 cells/mL; fermentation temperatures at: room temperatures, 30ºC and 37ºC; fermentation time at: 24, 36 and 48 hours Total number of treatments was 3 x 3 x 3 = 27 treatments The total number of experimental units was 27 treatments x 2 replicates = 54 units Carrot juice was pasteurized by NaHSO3

140mg/L in 20 minutes and then transferred into tube Inoculate 0.4 mL of bacterial suspension into tube containing 40 mL of pasteurized carrot juice and incubated room temperatures, 30ºC and 37ºC, respectively Measure Brix level, pH, acid lactic content and evaluated sensory after fermentation 24, 36, and 48 hours

2.2.7 Study temperature and time of storage

From previous activities, selected the favorable conditions including: isolate strains, inoculum density, sugar content (Brix), dilution ratio, temperature and time of fermentation to produce carrot juice at high quality After fermentation, fermented carrot juice tubes were stored at the different temperature conditions: at room temperature (between 28ºC and 34ºC), in the cabinet cooler (about 20ºC), in the refrigerator (4-6ºC) about 4 weeks of storage Found the better storage conditions

2.2.8 Statistical data analysis

The statistical data were analyzed by Microsoft Office Excel

2003 and Statgraphics centurion XV (USA) software

3 RESULTS AND DISCUSSION

3.1 Isolation of lactic acid bacteria

All pure isolates were classically defined by observing macro and micro morphology As the result, 4 isolates of lactic acid bacteria were isolated from 2 kinds of carrot (Dalat and Chinese) Colonies of all isolates were round, smooth, grayish white Because of the differentiation of collected samples, the cell morphology was various as shown in Table 6’

Moreover, 4 isolates were characterized in Gram positive, lack of calatase and oxydase, produced clear zone around colonies in medium containing CaCO3 The result of Gram staining of 2 representative isolates is described in Figure 14

Figure 14 Gram staining of isolates

Two lactic acid bacteria were isolated from Dalat carrot DL52 were defined as V rod-shaped whereas DLII3 looked like rod-shaped The isolates TQ13 and TQ25 from Chinese carrot were rod-shaped

Four isolates were characterized of basically characteristics of lactic acid bacteria, they had varieties of both morphology and isolated samples

8

Table 6’ Cell morphology of isolates

Note: The isolates were marked * from Phuoc Luong Truong (2012)

3.2 Study on low pH tolerance of isolates

Guarner and Schaafsma (1998), Schrezenmier and de Vrese (2001) reported that a probiotic properties is the ability to survive gastrointestinal transit

Low pH tolerance of isolates was determined by monitoring the density of LAB cells after 2 hours inoculated to MRS broth medium at different pH: 1.5; 2.5; 3.5 The results of counting plate density of isolates cells were showed in Table 7

Table 7 Density of isolates in MRS broth at low pH

The density of isolates was used to evaluate the development and survival of isolate cells Initial cell density was about 4.21-

10

4.46 log CFU/mL When supplied into the pH 1.5 and 2.5 medium, these 10 isolates could grow and survive after 2 hours incubation at 37ºC temperature In pH 3.5 medium, isolates grew well, reaching density of about 6.55 to 6.99 log CFU/mL In low

pH supplied media, density of isolates decreased correlatively with the decrease of pH level

3.3 Study on lactic acid fermentation ability of isolates in carrot juice

Table 8 Following indicators fermented carrot juice The

The results in Table 8 showed that pH after fermentation was lower than initial, roughly 3.21 to 4.18 During fermentation process, isolates converted glucose into lactic acid, reduced the

pH of the fermenting liquid Similarly, Brix levels also decreased after fermentation

One of the basic requirements demanded from probiotic products is a proper number of probiotic bacteria at the level of at least 6 log CFU/mL The survival rate of all isolates at the level

of about 8.02-8.69 log CFU/mL of fermented carrot juice Addition, the result of lactic acid concentration measured of different level, ranging 0.46-1.46% w/v The highest lactic acid concentration was Pro with 1.46% w/v Therefore, this isolates were chosen for next activities

3.4 Identification of the selected lactic acid bacteria by

molecular technique

Isolate Pro was selected to sequence 16S rRNA gene and

BLAST on NCBI website (http://www.ncbi.nlm.nih.gov) The

result showed that the gene sequences of isolate Pro shared 99%

similarity with Lactobacillus helveticus

Result of BLAST on NCBI of isolate Pro

Lactobacillus helveticus is an important industrial thermophilic starter that is predominantly employed in the fermentation of milk for the manufacture of several cheeses In addition to its technological importance, a growing body of

scientific evidence shows that strains belonging to the Lb

helveticus species have health-promoting properties Several in

vitro studies showed that Lb helveticus possesses many common

probiotic properties, such as the ability to survive gastrointestinal transit, adhere to epithelial cells, and antagonize pathogens (Valentina and Simone , 2012)

12

3.5 Study on the effect of dilution ratio and sugar content

The results of Lb helveticus density and lactic acid content of

carrot juice fermentation were presented in Furgre 17’

Furgre 17’ Changes of lactic acid content

and Lb helveticus density

In the same dilution ratio carrot juice Figure 17’ compared

the lactic acid generated by Lb helveticus at 6, 9, 12, and 15% sugar Lb helveticus was able to ferment well at 15% glucose

(1.17% w/v) However, they did not have the same results at ratio