Nghiên cứu điều kiện thu nhận, xác định tính chất và thành phần monosaccharide của exopolysaccharide từ một số chủng thuộc loài lactobacillus plantarum tt tiếng anh

HUE UNIVERSITY HUE UNIVERSITY OF SCIENCES TRAN BAO KHANH STUDY ON PRODUCTION, CHARACTERIZATION AND MONOSACCHARIDE COMPOSITION OF EXOPOLYSACCHARIDE FROM Lactobacillus plantarum Major:

HUE UNIVERSITY HUE UNIVERSITY OF SCIENCES

TRAN BAO KHANH

STUDY ON PRODUCTION, CHARACTERIZATION AND

MONOSACCHARIDE COMPOSITION OF

EXOPOLYSACCHARIDE FROM Lactobacillus plantarum

Major: Organic chemistry Code: 62.44.01.14

HUE - 2019

The work was completed at: University of Sciences – Hue University

Supervisors: Assoc Prof Dr Do Thi Bich Thuy

Reviewer 1: Assoc Prof Dr Truong Thi Minh Hanh

Reviewer 2: Assoc Prof Dr Nguyen Le Doan Duy

Reviewer 3: Assoc Prof Dr Pham Xuan Nui

The dissertation will be presented at………:

……….… At………h……… month………day…….year………

The thesis is stored at

PREAMBLE

Lactic acid bacteria (LAB) are widely used in food industry around the world Beside the lactic acid production, their biosynthesis of enzymes, bacteriocin and exopolysaccharides are used to produce probiotics

Polysaccharides (PS) is used in food and medicine, and has good mechanical properties for applications such as spinning, film, glue, thickener, gel forming agent Supplies for these PSs are now primarily from plants such as starch, agar, galactomannan, pectin, carageenan and aginate Due to the long-chain structure, these PS can satisfy the above requirements However, in order to improve these properties, almost all plant-derived PS compounds have to be treated

by enzymatic methods and chemical methods Therefore, their applicability is still limited

Besides, the production of EPS from microorganisms has many advantages over that from plants such as short production time, inexpensive culture, easy to control production process Microorganisms can synthesize many types of polysaccharide such

as indopolysaccharide, lipopolysacchride, peptidoglycan, exopolysaccharide In addition, if it is synthesized from safe microorganisms, polysaccharide will be a safe and biodegradable material It is even possible to directly use microorganisms capable

of synthesizing exopolysaccharide into some products

Besides contributing to cellular viability, exopolysaccharide as well as other polysaccharide compounds have technological properties that are used as food additives In Europe and America, these compounds are often used to improve the quality of dairy products They play an important role in increasing the sensory value

of the product The technology is based on that new product development

In addition, EPS from lactic acid bacteria has many positive effects on human and animal health such as immune enhancing activity, antiviral, antioxidant, anti-cancer and antihypertensive

Therefore, production, properties, strucrure and application of EPS from lactic acid bacteria have been interests of many scientists

For that reason, we carry out the topic “Study on production, characterization and monosaccharide composition of

exopolysaccharide from Lactobacillus plantarum”

The theme is implemented with the following contents:

1 Determination of exopolysaccharide production conditions

4 Initially investigated the possibility of application L plantarum to ferment soy milk

Chương 1 OVERVIEW 1.1 Overview of lactic acid bacteria

1.1.1 Lactic acid bacteria

1.1.2 Exopolysaccharide from lactic acid bacteria

1.1.3 Structure and classification exopolysaccharide

1.1.4 Biosynthesis of exopolysaccharide from lactic acid bacteria

1.2 Study on exopolysaccharide from lactic acid bacteria

1.2.1 Exopolysaccharide synthesis conditions

1.2.2 Exopolysaccharide extraction conditions

2.3.1 Methods of microorganism experiments

2.3.2 Phenol – sulfuric acid method

2.3.3 Kjeldahl method

2.3.4 Exopolysaccharide extraction method

2.3.5 Determination of solubility of exopolysaccharide method

2.3.6 Determination of water (oil) holding capacity of

exopolysaccharide method

2.3.7 DPPH free radical scavenging activity

2.3.8 Monosaccharide composition and methylation analysis of

Chương 3 RESULT AND DISCUSSION

3.1 Exopolysaccharide production ability of L plantarum

All strains were capable of producing EPS Five strains having highest production ability of EPS (W1, W5, W12, T10 and N5) were selected for further study

3.2 Effect of culture conditions on the biosynthesis of

exopolysaccharide of selected L plantarum strains

Table 3.2 The highest EPS yield in culture medium supplemented

with C source of selected L plantarum strains

L plantarum

The suitable C source

Concentration (%)

EPS increase (%)

Table 3.4 The highest EPS yield in culture medium supplemented

with N source of selected L plantarum strains

L plantarum The suitable

N source

Concentration (%)

EPS increase (%)

3.2.3 Initial cell culture density

Except 107 cfu/mL was suitable for N5 strain, 106 cfu/mL was suitable for the other four strains

306,46 278,90

Figure 3.2 Effect of initial cell culture density on the production

of exopolysaccharide of selected L plantarum strains

L plantarum

Figure 3.3 Effect of initial pH on the production of

exopolysaccharide of selected L plantarum strains

L plantarum

EPS (mg/L)

378.53 402.76

L plantarum

410.44

322.76 335.16

3.3 Effect of the extraction conditions on the yeild of exopolysaccharide obtained

3.3.1 TCA concentration

Figure 3.5 Effect of incubation time on the production of

exopolysaccharide of selected L plantarum strains

EPS (mg/L)

Incubation time (h) EPS (mg/L)

Incubation time (h)

EPS (mg/L)

Incubation time (h) EPS (mg/L)

Incubation time (h) EPS (mg/L)

Incubation time (h)

Figure 3.6 Effect of TCA concentration extraction conditions on the

20% of TCA was suitable for W1 and T10 strain, 20% of TCA

was suitable for the other three strains

3.3.2 Ethanol 99% concentration

Supernatant containing EPS and ethanol 99% in 1:1 ratio was

suitable for all the strains

Table 3.5.Effect of ethanol 99% concentration on the yeild of

3.4 The properties of the exopolysaccharides from the selected

3.4.2 Water (oil) holding capacity

The oil holding capacity of EPS from all the strains is higher than its the water holding capacity The water holding capacity and the oil holding capacity of EPS-W1 are the highest

Solubility (%)

EPS

85.00 a 84.67 a

76.33 ab 74.00 b 72.33 b

Figure 3.8 The water (oil) holding capacity of EPS

from the selected L plantarum strains

Water holding capacity

Oil holding capacity

Hydroxyl radical scavenging activity (%)

3.5.1 Molecular mass of EPS-W1

Figure 3.9 Gel permeation chromatogram of EPS-W1, Molar Mass

Distribution (MMD) pattern

The average molecular weight of EPS from L plantarum W1 was about 1.11x105 Da

3.5.2 Monosaccharide composition of EPS-W1

Table 3.8 Monosaccharide composition of EPS-W1

EPS-W1’s monosaccharide composition included of glucose

and mannose in 1.49:1 ratio

Table 3.9 GC-MS data for the alditol acetates derived from

→2,6)-D-0.30

6 1,3,5,6-tetraacetyl

-2,4-di-O-methyl-mannitol

mannopyranoside-(1→

→3,6)-D-0.14

Methylation analysis showed EPS-W1 had six components

Chemical shifts of sugar components are shown in Table 3.10

Table 3.10 H and C- NMR chemical shifts (, ppm) of EPS-W1 recorded in D 2 O at 353 K

3.6 The gel-forming ability of fermented soybean milk of

selected L plantarum strains

3.6.1 Effect of inocubation time on fermented soybean milk gel

Gel of soybean fermented by L plantarum W1 was the highest

Table 3.11 Gel state of soybean milk fermented by L plantarum

++ : Gel, no water separation

+++ : Gel cracked, separated water

3.6.2 The water holding capacity of fermented soybean milk

The water holding capacity of soybean milk fermented by L

plantarum W1 was the highest

3.6.3 The viscosity of fermented soybean milk

The viscosity of soybean milk fermented by L plantarum W1

was the highest

CONCLUSION

1 The best EPS conditions for Lactobacillus plantarum strains

are as follows:

- Lactobacillus plantarum W1: MRS supplemented with 5%

lactose, 0.3% yeast extract, pH 6, initial cell culture density is 106CFU/mL Fermented temperature and time were 40°C and 36 hours Using 20% TCA to remove protein and precipitate EPS by ethanol

Share rate (s -1 ) Viscosity (Pa.s)

Figure 3.12 The viscosity of soybean milk fermented by L plantarum

L plantarum

with ratio: fermentation solution is 1: 1 for 24 hours With above condition, the yield of EPS-W1 was 446.18 mg/L

- Lactobacillus plantarum W5: MRS supplemented with 4%

saccharose, 0.4% yeast extract, pH 6, initial culture density was 106CFU/mL Fermented temperature and time were 35 oC and 36 hours Using 25% TCA to remove protein and precipitate EPS by ethanol with ratio: fermentation solution is 1: 1 for 24 hours With above condition, the yield of EPS-W5 was 458.25 mg/L

- Lactobacillus plantarum W12: MRS supplemented with 5%

lactose, 0.3% yeast extract, pH 6, initial culture density was 106CFU/mL Fermented temperature and time were 40 °C and 60 hours Using 25% TCA to remove protein and precipitate EPS by ethanol with ratio: fermentation solution is 1: 1 for 24 hours With above condition, the yield of EPS-W12 was 456.22 mg/L

- Lactobacillus plantarum T10: MRS supplemented with 4%

lactose, 0.4% yeast extract, pH 5.5, initial cell culture density was

106 CFU/mL Fermented temperature and time were 35 °C and 48 hours Using 20% TCA to remove protein and precipitate EPS by ethanol with ratio: fermentation solution is 1: 1 for 24 hours With above condition, the yield of EPS-T10 was 454.10 mg/L

- Lactobacillus plantarum N5: MRS supplemented with 5%

lactose, 0.8% meat extract, pH 6, initial culture density was 106 CFU/mL Fermented temperature and time were 40 °C and 36 hours Use 25% TCA to remove protein and precipitate EPS by ethanol

with ratio: fermentation solution is 1: 1 for 24 hours With above condition, the yield of EPS-N5 was 539.71 mg/L

2 Water solubility, water holding, oil holding and antioxidant capacity of EPS-W1 were better than the other EPSs

3 The average molecular weight of EPS-W1 was 1.11x105 Da EPS-W1’s monosaccharide composition included of glucose and mannose in 1.49:1 ratio The repeating unit of this polysaccharide to

be shown D(1 → 6)linked glucosyl,D(1 → 3)linked mannosyl, D-(1 → 3)-linked glucosyl and branch of -D-(1→6)-linked mannosyl, -D-(1 → 2)-linked glucosyl

-4 Initially, survey showed that L plantarum W1 could be

applied to fermented soybean milk

SUGGESTION

- Finding cheaper exopolysaccharide production conditions

- Investigating some other beneficial properties of EPS such

as antibacterial, anti-inflammatory

- Studying on the possibility of applying of the EPSs in other fields such as food and pharmaceuticals

LIST OF RELATED SCIENTIFIC ARTICLE

1 Tran Bao Khanh, Do Thi Bich Thuy, Doan Thi Thanh Thao

(2016), Optimal conditions for high exopolysaccharide production by Lactobacillus plantarum T10, Journal of Science and Technology, 52

(44), 10-47

2 Tran Bao Khanh, Đo Thi Bich Thuy (2016), Optimal

conditions for exopolysaccharide production by Lactobacillus plantarum W5, Hue University Journal of Science, 121 (7) 57-68

3 Tran Thi Ai Luyen, Tran Bao Khanh, Do Thi Bich Thuy, Tran Thi Van Thi (2017), Study on some extraction conditions and

structural characterisation of exopolysaccharides produced by

Lactobacillus fermentum MC3 and Lactobacillus plantarum W12, Vietnam Journal of Chemistry, 55 (4E23), 243-249

4 Tran Bao Khanh, Tran Thi Ai Luyen, Do Thi Bich Thuy (2017), Determination of molecular weights and some physicochemical characterizations of exopolysaccharides produced

by Lactobacillus fermentum MC3 and Lactobacillus plantarum W12, Vietnam Journal of Chemistry, 55 (4E34), 17-21

5 Tran Bao Khanh Do Thi Bich Thuy Nguyen Tran Bao Khuyen (2017), Effect of some conditions on exopolysaccharide

production of Lactobacillus plantarum N5, Journal of Science and Technology (Hue University of Sciences), 1 (10), 131-141