Microbial encapsulation for enhancing soy sauce aroma development during moromi fermentation

Moromi fermentation is an essential part of soy sauce production. This thesis aimed to characterize and control the growth and interaction of two predominant microorganisms, Tetragenococcus halophilus and Zygosaccharomyces rouxii for enhancing the aroma development during moromi fermentation. Antagonism was observed between T. halophilus and Z. rouxii, regardless of the inoculation sequence. However, sequential inoculation of Z. rouxii resulted in more complex aroma profile than simultaneous inoculation. To eliminate antagonism, chitosan-coated alginate and water-oil-water (W1/O/W2) double emulsions (DEs) were tested for their ability to encapsulate Z. rouxii and stability in high NaCl solutions. Alginate was unstable in high NaCl solutions and chitosan exhibited undesirable antimicrobial activity towards Z. rouxii. DEs minimized the antagonism between T. halophilus and Z. rouxii, by segregation in the external W2 and internal W1 phase, respectively. Physicochemical changes in the fermentation medium indicated that DEs affected microbial growth and cell physiology, contributing to the elimination of antagonism. The destabilization of DEs over 30-day storage depended on glucose concentration in W2, which indicated a possibility of sustained release mechanism of Z. rouxii into the moromi. Furthermore, the application of DEs was tested in a moromi model, formulated with reduced NaCl and/or substitution with KCl. DEs resulted in moromi with similar microbiological and aroma profile to that of high-salt. Overall, this thesis demonstrates the potential of DE for delivering mixed cultures in moromi fermentation, which could be applicable in any fermentation process where multiple species are required to act sequentially

Effects of co-inoculation and sequential inoculation of Tetragenococcus halophilus and Zygosaccharomyces rouxii on soy sauce fermentation

The use of Tetragenococcus halophilus and Zygosaccharomyces rouxii as starter cultures is essential for desirable volatiles production during moromi stage of soy sauce fermentation. In this study, the effect of simultaneous and sequential inoculation of cultures in moromi fermentation models, with respect to viability, physicochemical changes, and volatiles formation (using SPME-GC/MS) was investigated. Interestingly, an antagonism was observed as T. halophilus only proliferated (3 log increase) in the presence of Z. rouxii, while Z. rouxii growth was suppressed by 4 log in concurrence with pH increase to 7.31. Final content of reducing sugars, ethanol, acetic acid, and amino nitrogen did not differ significantly (p < 0.05) between co-inoculation and sequential inoculation. However, Z. rouxii promoted alcohols formation and produced a more complex aroma profile under suppression. According to Principal Component Analysis (PCA), the inoculation sequence (co-inoculation and sequential) has impacts on volatile compound profiles during moromi fermentation

Isolation and Screening of Microbial Isolates from Kombucha Culture for Bacterial Cellulose Production in Sugarcane Molasses Medium

Kombucha tea is a traditional fermented beverage of Manchurian origins which is made of sugar and tea. The fermentation involves the application of a symbiotic consortium of bacteria and yeast (SCOBY) in which their metabolites provide health benefits for the consumer and subsequently allow the product to protect itself from contamination. Additionally, kombucha tea fermentation also produces a byproduct in the form of a pellicle composed of cellulose (Bacterial Cellulose, BC). Compared to plant cellulose, BC properties are more superior, which makes it industrially important. However, BC production at industrial scale has been faced with many challenges, including low yield and high fermentation medium cost. Many researchers have focused their studies on the use of alternative low cost media, such as molasses, which is a by-product&nbsp;&nbsp; of sugar refining process. To&nbsp; maximize the BC production in molasses medium, it&nbsp;&nbsp;&nbsp; is important to select the microbial strains that can grow and produce BC at high yield in molasses. This study aimed to isolate and characterize BC-producing bacteria and a dominant yeast from kombucha culture which had been previously adapted in molasses medium. The isolation of bacteria was performed using Nutrient Agar (NA) and Hestrin and Schramm (HS) supplemented with cycloheximide, while yeast was isolated using Potato Dextrose Agar (PDA) supplemented with chloramphenicol. The most dominant colonies were isolated and then subjected to microscopic observation for morphological analysis. The pure bacteria and yeast isolates were then identified by sequencing the 16S rRNA gene and D1/D2 region of the 26S rRNA, respectively. The bacteria isolates obtained were all from closely related genera: Komagataeibacter sp. DS1MA.62A, Komagataeibacter xylinus, Komagataeibacter saccharivorans, Komagataeibacter xylinus and Gluconacetobacter saccharivorans. The single isolated yeast was identified as Brettanomyces bruxellensis. This study helps to elucidate&nbsp; the BC-producing species which thrive in molasses medium for potential use in the BC production using molasses as alternative cheap carbon source. Also, the study revealed that the co-culture of Komagataeibacter sp. DS1MA.62A and B. bruxellensis could produce BC from molasses supplemented with caffeine and acetate buffer at an average yield of 27.7±1.83 g/L. Keywords: Kombucha, SCOBY, bacterial cellulose, Acetobacter, Komagataeibacter, Brettanomyces, alternative medium, molasses, caffeine, acetate buffer

Primer Design for Isolation of Sucrose:Sucrose 1-Fructosyltransferase (1-SST) Gene from Gembili (Dioscorea esculenta)

Recent studies have reported the presence of inulin, a prebiotic polysaccharide, in gembili (Dioscorea esculenta). Sucrose:sucrose 1-fructosyltransferase (1-SST) is an enzyme that catalyzes the first step of inulin biosynthesis. The identification of this enzyme would be the foundation to improve the yield of inulin in gembili; to modify 1-SST gene for this purpose, its sequence must first be determined. This study aimed to design primers to isolate 1-SST gene from D. esculenta. The primers were designed by using the whole-genome sequence of Dioscorea rotundata due to the lack of genomic information on D. esculenta. Sequences from chromosome 6 and 11 were used as the template of primer design in which four pairs of primers were selected. Amplification products with expected size were gel-purified, then the targets were sequenced and analyzed in-silico. As a result, one of the primer pairs had successfully isolated vacuolar invertase gene, which is closely related to 1-SST gene. On the other hand, the other primer pairs showed either negative or false-positive result. Using the current strategy, 1-SST gene sequence from D. esculenta has not been successfully isolated, thus other approaches in primer design should be considered in further studies. Keywords: 1-SST, Dioscorea esculenta, Gembili, Inulin, Primer desig

Water-in-oil-in-water double emulsion for the delivery of starter cultures in reduced-salt moromi fermentation of soy sauce

This study investigated the application of water-oil-water (W1/O/W2) double emulsions (DE) for yeast encapsulation and sequential inoculation of Zygosaccharomyces rouxii and Tetragenococcus halophilus in moromi stage of soy sauce fermentation with reduced NaCl and/or substitution with KCl. Z. rouxii and T. halophilus were incorporated in the internal W1 and external W2 phase of DE, respectively. NaCl reduction and substitution promoted T. halophilus growth to 8.88 log CFU/mL, accompanied with faster sugar depletion and enhanced lactic acid production. Reducing NaCl without substitution increased the final pH (5.49) and decreased alcohols, acids, esters, furan and phenol content. However, the application of DE resulted in moromi with similar microbiological and physicochemical characteristics to that of high-salt. Principal component analysis of GC–MS data demonstrated that the reduced-salt moromi had identical aroma profile to that obtained in the standard one, indicating the feasibility of producing low-salt soy sauce without compromising its quality

The Effect of Dekkera bruxellensis Concentration and Inoculation Time on Biochemical Changes and Cellulose Biosynthesis by Komagataeibacter intermedius

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (103 and 106 CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 103 CFU/mL compared to K. intermedius alone (0.63 &plusmn; 0.11 g/L). The lowest BC yield was observed when 103 CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 106 CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 106 CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield

The Potential of <i>Pediococcus acidilactici</i> Cell-Free Supernatant as a Preservative in Food Packaging Materials

This study delves into the production and antimicrobial characteristics of cell-free supernatants from Pediococcus acidilactici (CFSs-Pa). Antimicrobial activity was initially observed in CFS-Pa harvested after 12 h of incubation and increased up to the late stationary phase at 48 h. The increase in antimicrobial activity did not align with total protein content, pointing to other factors linked to the accumulation of organic acids, particularly lactic acid. The SDS-PAGE analysis also indicated that the expected proteinaceous compound (pediocin) was not observed in CFS-Pa. Further investigations suggested that the antimicrobial properties of CFS-Pa were exclusively due to organic acids. The MIC values confirmed potent antimicrobial activity, particularly at a 10% dilution of CFS-Pa in MRS broth. The time–kill assays demonstrated bactericidal activity against EHEC, Listeria monocytogenes, and Staphylococcus aureus by 12 h, 18 h, and 24 h using a 10% dilution of CFS-Pa. Additionally, CFS-Pa exhibited dose-dependent antioxidant activity, requiring a 70% (v/v) concentration to inhibit DPPH scavenging activity by 50%. All the experimental results suggested potential applications of CFS-Pa in food preservation. An attempt to incorporate CFS-Pa into bacterial cellulose (BC) for edible food packaging demonstrated promising antimicrobial results, particularly against L. monocytogenes and S. aureus, with room for optimization

Spray Drying Encapsulation of <i>Pediococcus acidilactici</i> at Different Inlet Air Temperatures and Wall Material Ratios

Pediococcus acidilactici has gained research and commercial interest due to its outstanding probiotic properties, yet its survival during storage and consumption requires improvement. This study aims to enhance P. acidilactici survival using spray drying encapsulation. Different inlet air temperatures (120 °C, 150 °C, and 170 °C) and whey protein isolate (WPI):gum arabic (GA) ratios (1:1, 3:1, 1:3) were tested. Cell viability was significantly (p P. acidilactici viability by 1.36 log cycles, from 8.61 log CFU/g to 7.25 log CFU/g. The inlet temperature of 150 °C resulted in a powder yield (63.12%) higher than at 120 °C (58.97%), as well as significantly (p w 0.21). Viable cell counts in all encapsulated P. acidilactici were maintained at 5.24–6.75 log CFU/g after gastrointestinal tract (GIT) simulation, with WPI:GA of 3:1 and inlet temperature 150 °C having the smallest log reduction (0.3 log cycles). All samples containing different WPI:GA ratios maintained sufficient viability (>7 log CFU/g) during the first three weeks of storage at 25 °C. These results could provide insights for further developing P. acidilactici as commercial probiotic products