Integration of the Functional Properties of Coconut Water Kefir Into Sourdough Bread
Limbad, Mansi Jayantikumar
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Sourdough technology is an ancient technology that has been widely described and utilized due to its ability to improve the nutritional quality, texture, sensory properties, flavour and volatile compound composition, and shelf life of the bread. Many studies have reported that sourdough fermented using milk kefir has the ability to produce metabolites such as organic acids, amino acids, exopolysaccharides (EPS) and/or enzymes such as phytases, due to the presence of microorganisms such as lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeasts. However, few studies have reported on fermentation of sourdough using a coconut water-based medium and kefir grains. The advantages of using coconut water is its rich nutritional profile with high amounts of glutamic acid, vitamin C, magnesium, vitamin B, arginine, alanine, lysine, calcium, potassium, minerals and antioxidants. These nutrients and sugar content make it an ideal substrate for fermentation. Kefir itself has many claimed health benefits such as the anticarcinogenic and antimutagenic properties, immunomodulating properties, anti-inflammatory and hypocholesterolemic effects, antihypertensive, antidiabetic, antimicrobial properties and enhanced lactose utilisation. These health effects may be attributed to several components such as the microogorganisms such as LAB, AAB and yeast, exopolysaccharides, organic acids, antioxidants, and bioactive peptides. However, few studies have reported on fermentation of sourdough using a coconut water-based medium and kefir grains. This thesis aimed at developing a novel sourdough bread with increased functional properties using fermented coconut water kefir culture and pure culture isolates identified from it. The kefir microorganisms ferment the coconut water by consuming the small quantity of sugar available in the substrate. A study was carried out to understand the fermentation of coconut water kefir supplemented with different sugars (such as glucose and sucrose) at various concentrations. Based on the microbial cell count results, coconut water kefir (CWK) supplemented with 12 g/L of sucrose fermented for 48 h was identified as a suitable material to prepare a sourdough (Chapter 3). Using this CWK culture, a sourdough was prepared and incubated at different temperatures of 4°C, 22°C and 30°C from 0-96h to study the microbial and physico-chemical profile of the sourdough. The sourdough fermentation temperature of 30°C was chosen to incubate the sourdough bread (Chapter 4). The high microbial counts were accompanied by a sharp decline in pH, increase in lactic acid production (D-/L- lactic acid) and increase in TTA, which consequently affected the overall viscosity of the CWK fermented sourdough. The dough volume increased with time when incubated at 30°C temperature. The texture of the CWK fermented sourdough had higher values for hardness, resilience, chewiness, gumminess and springiness after incubation for 48, 72 and 96 h at 30°C. There was significant production of organic acids such as lactic acid (9998.7 ± 18.5 mg/L, 96 h), pyruvic acid (1394.8 ± 2.95 mg/L, 96 h), succinic acid (883.4 ± 2.4 mg/L, 96 h) and malic acid (31.4 ± 8.5 mg/L, 96 h) as well as amino acids such as glutamic acid were produced in notably high quantities. Five (5) lactic acid bacteria species, 3 acetic acid bacteria species and 5 yeast species were identified and confirmed from kefir, fermented CWK and CWK fermented sourdough. Illumina sequencing was carried out to understand the change in the microbial ecology of a CWK fermented sourdough over time (between 0-96h) (Chapter 5). Each confirmed isolate was assessed for possessing any functional properties such as production of glutamic acid (a flavour enhancing amino acid and a precursor of γ-aminobutyric acid (GABA), phytase enzyme (which reacts with phytate) and EPS production (which acts as a hydrocolloid to improve the overall textural properties of the sourdough bread). Each of these properties was quantified for each isolate (Chapter 6). The isolates which produced the highest quantities of phytase enzyme (4052.5 ± 171.1 U/ml and 3151.1 ± 383.0 U/ml, respectively) and glutamic acid (260.3 ± 12.1 µmoles/L and 264.89 ± 8.5 µmoles/L, respectively) were identified as L. fermentum and L. plantarum. These isolates were added in different concentrations (low range of 4.9-5.0 log CFU/ml to the high range of 8.3-9.6 log CFU/ml) to the sourdough to develop a functional sourdough bread. Sensory analyses were carried out for eight experimental sourdough breads but only three sourdough breads were selected based on the outcome of the sensory analysis. Determinations of texture, protein, ash, moisture, carbohydrate, total dietary fibre, total fats, total titratable acidity, and shelf life were carried out on the three finalised sourdough breads. The glycaemic index analysis was also carried out to estimate the impact of the formulated sourdough bread on the blood-glucose level of the consumer. Carboxylic acid analysis and amino acid analysis revealed that significant quantities of carboxylic acids such as lactic acid (828.8 ± 4.8 mg/L) and glutamic acid (187.2 ± 1.5 mg/L) and amino acids such as alanine (654.71 ± 79.24µM/L), proline (310.28 ± 18.93 µM/L) and lysine (156.08 ± 27.11 µM/L) were produced in these sourdough breads which are important flavour components which influence the overall sensory profile of the bread. Production of acids improves the shelf life of the bread due to the lowering of pH and increase in the acidity of the bread, which prevents the growth of spoilage microorganisms (Chapter 7). In conclusion, kefir grains were able to utilise coconut water supplemented with 12 g/L sucrose as a substrate, which could be used to develop a novel and a functional sourdough. Out of the 13 identified species of LAB, AAB and yeast, two LAB species (L. plantarum and L. fermentum) which produced the highest quantities of glutamic acid, EPS, and phytase enzyme were incorporated along with the CWK to prepare a sourdough. The importance of phytase enzyme is attributed to its ability to degrade the phytic acid, which in turn leads to increase in the bioavailability of minerals such as calcium, zinc, and iron in its consumers. Glutamic acid, being the precursor of an important bioactive compound, GABA, is desired in high concentrations in the bread due to the important health properties of GABA to the humans. EPS acts as a hydrocolloid in the bread, which positively influences the textural properties of the final bread product. The isolates which produced significantly highest quantities of glutamic acid and phytase enzyme were selected for addition into a novel CWK-fermented sourdough bread. Eight such sourdough breads were developed and only three were shortlisted based on the sensory analyses carried out on them. The three short listed breads were analysed for textural properties, PROXIMATE properties, metabolite production such as carboxylic acids and amino acid production and finally, the glycaemic index for each of the three formulated sourdough bread was also determined. Overall, the integration of functional properties such as of phytase, glutamic acid, EPS (imparting hydrocolloid-like properties) was successfully achieved due to the identification and incorporation of isolates (Lactobacillus plantarum and Lactobacillus fermentum) into the sourdough. Significant quantity of glutamic acid was detected in the final sourdough bread product and lower GI values for each participant was obtained upon the consumption of the CWK-sourdough bread. Since glutamic acid, phytase, EPS and low GI have been recognised and associated to as health-promoting factors, it could be ascertained that the CWK-sourdough bread is a functional bread product with all of the above-mentioned properties. The textural and sensory profile of the breads were largely liked by the participants. On the whole, the sensory profile of the CWK fermented sourdough bread was pleasant and was associated with many positive attributes such as sour, porous, moist, light, heterogenous, spongy and buttery.