Evaluation of oil-in-water (O/W) emulsifying properties of galactan exopolysaccharide from Weissella confusa KR780676 (2024)

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J Food Sci Technol
v.57(4); 2020 Apr
PMC7054585

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J Food Sci Technol. 2020 Apr; 57(4): 1579–1585.

Published online 2020 Jan 24. doi:10.1007/s13197-020-04262-3

PMCID: PMC7054585

PMID: 32180655

Digambar Kavitake, Sangeeta Balyan, Palanisamy Bruntha Devi, and Prathapkumar Halady Shetty

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Abstract

Galactan exopolysaccharide (EPS) was extracted from Weissella confusa KR780676 isolated from idli batter. The present study reports the effect of galactan EPS concentration, pH, ionic strength, temperature, salinity, monovalent salts on the emulsion formed with vegetable oils (virgincoconut oil, groundnut oil, olive oil and mustard oil). Emulsion determination test revealed the oil-in-water type of emulsion. Microscopic observation showed the phase of oil droplet distribution of emulsions. Groundnut and olive oil showed significant emulsifying activity (50–60%) and stability (90%) irrespective of the EPS concentration. One percent of EPS also showed good emulsifying activity (50–70%) and stability (100%) with groundnut and olive oil in wide range of temperature (− 20, 4, 30, 40, 50 and 60°C), pH (3–8), salinity (1, 5, 10, 20 and 30% of NaCl) and monovalent salt (1, 5, 10, 20 and 30% of KCl). Galactan could be used as a prominent emulsifier for applications in the food industry as it possesses significant activity with most of the influential factors in wide range and also obtained from lactic acid bacteria that are regarded as Generally Regarded as Safe (GRAS).

Keywords: Galactan EPS, Weissella confusaKR780676, Oil emulsion, Emulsion stability

Introduction

Emulsion are heterogeneous system formed as a mixture of two or more liquids (immiscible) usually contains very small droplets of oil dispersed in a fluid typically water. Oil-in-water emulsion finds its immense application in preparation of bakery, candy, dairy, meat products and beverages. Various physicochemical factors such as gravitational and phase separation, flocculation, coalescence, etc., collapses the emulsion and makes unstable (McClements and Jafari 2018). Emulsifier, an amphiphilic molecule possessing both hydrophilic and hydrophobic groups on same molecule is used as an important emulsion stabilizing agent (Kralova and Sjöblom 2009). Currently natural food-grade emulsifiers such as proteins, polysaccharides, phospholipids and saponins are widely used in food industries.

Bacterial exopolysaccharides (EPS) is gaining wide importance nowadays in food, pharma and cosmetic industries due to its potential pseudo-plastic rheological behaviour, viscosity and emulsifying properties. They are used as thickeners, gelling agent, adhesive, emulsifier, coating agents, stabilizers in these industries (Satpute et al. 2010; Kavitake et al. 2019a). Many novel EPS has been characterized from various bacterial strains possessing significant emulsifying and stabilization properties at low concentration with less toxicity (Song et al. 2009). EPS has the capacity to form and stabilize water-in-oil and oil-in-water emulsions and improves the texture of food thereby enhancing the organoleptic characteristics.

EPS from lactic acid bacteria (LAB) is widely attracted due to its potential technological and functional properties as well as Generally Regarded as Safe (GRAS) status. Earlier, we have reported a novel linear galactan EPS containing α-(1→6)-linked galactose units produced by Weissella confusa KR780676 isolated from Idli batter (an Indian traditionalcereal-legume based fermented food) (Kavitake et al. 2016). Our previous study on the physico-chemical properties of galactan EPS from Weissella confusa showed the remarkable emulsifying activity (69.4%) and emulsion stability up to 15days with sunflower oil (Devi et al. 2016). The present study reports the oil-in-water (O/W) emulsifying properties of galactan EPS with other vegetable oils (virgin coconut, groundnut, olive and mustard oil). Various external factors like pH, ionic strength, temperature, salinity, monovalent salts are also reported to influences the performance of emulsifiers. This study enlarges the effects of these parameters on galactan emulsion activity and stability to find its application in food industries.

Materials and methods

Materials

Lyophilized powder form of galactan EPS from Weissella confusa KR780676 was used for emulsion studies. Vegetable oils were purchased from local super market. All the reagents and chemicals used were of analytical grade.

Emulsion preparation

Emulsions were prepared with different oils (virgin coconut oil, groundnut oil, olive oil and mustard oil) and EPS suspension in a screw cap glass tube (100 × 13mm) and vortexed for 20min at 40Hz (Top mix FB 15024, Fisher Scientific, UK) as per the method explained by Freitas et al. (2009). Emulsification activity (EA) was determined after 1, 24 and 48h (EA₁, EA₂₄ and EA₄₈).

Effect of EPS concentration on emulsifying activity and stability

The emulsions prepared with virgin coconut oil, groundnut oil, olive oil and mustard oil were selected for further studies. Effect of different EPS concentrations (1, 0.75, 0.50, 0.25% (w/v)) on emulsion activity and stability were studied as described by Prasanna et al. (2012) with slight modifications.

$Emulsifying activity (EA) = \frac{Height of the emulsion layer}{Overall height of the mixture} \times 100$

$Emulsifying stability (ES) = \frac{Final EA}{Initial EA} \times 100$

Emulsion determination test

Emulsion type was determined by wetting filter paper test as described by Yadav et al. (2014). Hundred microlitre emulsions of virgin coconut oil, groundnut oil, olive oil and mustard oil were added as drop on Whatman filter paper 1. Droplet dispersesin oil-in-water (o/w) emulsion, whereas droplet remains on filter paper in water-in-oil (w/o) emulsion.

Microscopic observation of emulsions

As mentioned in Kavitake et al. (2019b), emulsion formed after 24h storage was observed microscopically (10 and 40X objective lens) using inverted microscope (Nikon eclipse E100 microscope) by placing 100μL of emulsion in cavity microscope slide.

Stability of emulsions

Effect of temperature

Thermal stability of the emulsions was checked by incubating emulsions at various temperatures (− 20, 4, 30, 40, 50 and 60°C) and emulsion index EA₁, EA₂₄ and EA₄₈ were calculated.

Effect of pH

Emulsion activity and stability were determined at different pH ranges from 3 to 8 as described by Davidov-Pardo et al. (2016). The pH was monitored using a digital pH meter (Hydrus 400, Fisher Brand). Calibration was carried out using phosphate buffer solution (0.005M) of pH 7 and the pH was adjusted with small volumes of 0.01M NaOH or 0.01M HCl. The emulsifying activity and stability were determined at emulsion index EA₁, EA₂₄ and EA₄₈.

Effect of salinity and monovalent salt

Various salts are commonly used in food stuffs, therefore effect of salts on emulsifying property of galactan EPS was investigated. Salinity influence was explored with sodium chloride (NaCl), monovalent salts consequences on emulsions were studied with potassium chloride (KCl) (Cui et al. 2016). Effect of NaCl and KCl on the EPS-oil emulsification was evaluated at different concentrations (1, 5, 10, 20 and 30%) by using salt aqueous solutions instead of water during microemulsion preparation.

Statistical analysis

All the experiments were carried out in triplicates. Results were expressed in mean ± SD. Data were processed by using Microsoft Excel 2016 and SPSS software.

Result and discussion

Effect of EPS concentration on emulsifying activity and stability

The emulsification index (EA₁, EA₂₄ and EA₄₈) and stability for all the concentrations (0.25, 0.50, 0.75, and 1%) of EPS revealed different emulsification activity and stability for virgin coconut, groundnut, olive and mustard oil (Fig.1A). Groundnut and olive oil showed activity and stability about 60 and 90% respectively. Emulsifying activity for coconut and mustard oil were between 50 and 70% at 1h and started to decline on further incubation. Bifidobacterium infantis NCIMB 702205 and Bifidobacterium longum CCUG 52486 EPS showed emulsion activity with coconut (71.4 and 61.9%) and olive oil (68.5 and 58.1%) respectively at 1mg/mL (Prasanna et al. 2012). However, there is no significant difference between the activity of 0.75 and 1% EPS. Therefore, 1% concentration of EPS was carried for further studies.

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Fig.1

A Effect of different EPS concentration on emulsifying activity and stability by vegetable oils. a Virgin coconut oil, b groundnut oil, c olive oil, d mustard oil. B Emulsion determination test by filter paper wetting method. a Virgin coconut oil, b groundnut oil, c olive oil, d mustard oil. C Microscopic observation of galactan emulsion formed with different oils

Emulsification determination test

Filter paper wetting method was used to determine the emulsification type. All the oil emulsions drops were dispersed on the paper which revealed oil-in-water type emulsions. In Fig.1B, clearly noticeable that outer layer of water dispersed first and then oil at the centre. This kind of emulsions plays a significant role in food industries which may find diverse food applications (Nitschke and Costa 2007).

Microscopic observation of emulsions

Photomicrograph of galactan emulsion formed with different oils was shown in the Fig.1C. Emulsion with virgin coconut oil showed small and large droplets whereas mustard showing small evenly distributed droplets. Emulsion with olive oil showed enlargement of large droplets consisting small densely packed evenly distributed droplets inside whereas emulsion with groundnut oil showed evenly distributed small droplets. Similar pattern of emulsion droplets was reported by Prasanna et al. (2012) for the EPS of Bifidobacterium infantis NCIMB 702205 and Han et al. (2015) for EPS of Bacillus amyloliquefaciens LPL061 with sunflower oil emulsion.

Stability of emulsions

Effect of temperature

Thermal stability and activity of the EPS emulsion with different vegetable oil were studied at different temperatures (− 20, 4, 30, 40 and 50, 60°C) (Fig.2A). Emulsion stability and activity started to decline drastically with increase in temperature for virgin coconut and mustard oil. Emulsion activity ranged between 50 and 70% with stability 90–100% at − 20, 40, 50, and 60°C for groundnut oil emulsion. Emulsion activity for olive oil was between 40 and 65% and stability 100% at EPS20°C; 95% at 50 and 60°C. Highest stability (> 90%) for groundnut and olive oil emulsions were observed at 40–60°C; this might be due to the decrease in viscosity of emulsions at the cause of temperature.

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Fig.2

A Effect of temperature on emulsifying activity and stability. a Virgincoconut oil, b groundnut oil, c olive oil, d mustard oil. B Effect of pH on emulsifying activity and stability. a Virgin coconut oil, b groundnut oil, c olive oil, d mustard oil

Effect of pH

Effect of pH (3–8) on EPS emulsion activity and stability with different vegetable oil were shown in the Fig.2B. Emulsion activity ranged between 70 and 80% and stability ranged between 90 and 95% for both groundnut and olive oil irrespective of increase in pH, whereas, there was no significant activity and stability for virgin coconut and mustard oil. Same trend of emulsifying activity 70–80% were observed for Enterobacter cloaceae EPS (1mg/mL) at pH range 2–10 with groundnut oil (Iyer et al. 2006).

Effect of salinity and monovalent salt

Influence of salinity (NaCl) (Fig.3A) and monovalent cation (KCl) (Fig.3B) on the EPS-oil emulsification and stability was evaluated at different concentrations (1, 5, 10, 20 and 30%). Emulsifying activity was maintained at 60% and 100% stability for olive oil irrespective of increase in NaCl concentration. For groundnut oil EA was 60% and ES ranged from 90 to 100% with increase in concentration. Emulsion activity was 40% at 1h; however, activity started to decline drastically with increase in concentration and incubation time for both mustard and virgin coconut oil. Similar trend was observed by Sahana et al. (2018) for coconut oil emulsion with EPS of Cronobacter sp. YU-R3. Also, there was no significant stability for these two oils. Emulsifying activity maintained between 60 and 70% for virgin coconut, groundnut and olive oil irrespective of increase in KCl concentration and incubation time. Stability was 100% for virgin coconut and groundnut oil, and 85–90% for olive oil. For mustard oil, emulsion activity ranged between 10 and 40%, however stability was 100% at 8 and 10% concentration. Better emulsifying activity and stability was observed with KCl compared to the NaCl.

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Fig.3

A Effect of NaCl salt on emulsifying activity and stability by a virgin coconut oil b groundnut oil c olive oil d mustard oil. B Effect of monovalent salt (KCl) on emulsifying activity and stability by a virgin coconut oil, b groundnut oil, c olive oil, d mustard oil

Conclusion

Emulsifying properties of the galactan EPS with different vegetable oils (Virgin coconut, groundnut, olive and mustard oil) has been studied with its effect to different pH, temperature, salinity, monovalent salts. Microscopic observation and particle size of EPS-oil emulsion revealed the size and phase of oil-in-water emulsion distribution. Groundnut and olive oil showed significant emulsifying activity (50–60%) and stability (90%). One percent of EPS also showed good emulsifying activity and stability with groundnut and olive oil in wide range of temperature, pH, salinity and monovalent salt. This galactan EPS could be used as a potential emulsifier in food industries as a green additives/ingredient.

Acknowledgement

Authors DK and PBD acknowledged University Grants Commission (UGC), New Delhi for providing financial assistance in the form of Senior Research Fellowship and Post-Doctoral Fellowship respectively. Authors are grateful to Central Instrumentation Facility (CIF), Pondicherry University for availing the instrumentation facility.

Compliance with ethical standards

Conflicts of interest

Authors declare that they do not have any conflict of interest.

Footnotes

Publisher's Note

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