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    Application of baby corn husk as a biological sustainable feedstock for the production of cellulase and xylanase by Lentinus squarrosulus Mont.
    (2023-02-01)
    Vichitraka, Asanee
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    Tantratian, Sumate
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    In an effort to use baby corn husk (BCH) as a sustainable feedstock for cellulase and xylanase production by the Lentinus squarrosulus Mont. isolate LS-YA (LSM-LS-YA), a suitable pretreatment method and fermentation strategies were developed. BCH pretreated with 1 M sodium hydroxide for 90 min, an alkaline pretreatment, exemplified an appropriate pretreatment method. In a 10-L external Venturi injector bioreactor, the highest cellulase and xylanase production was 4.12 ± 0.36 unit/mL and 6.15 ± 0.36 unit/mL, respectively, when 1 g/L diammonium hydrogen phosphate was used as the nitrogen source and the aeration rate was controlled at 0.2 vvm. This study provides an informative perspective on the production of cellulase and xylanase from agricultural lignocellulosic materials, which could reduce agricultural waste while supporting a zero-waste circular economy, and this fermentation process would be applicable to larger-scale production.
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    Membrane-based approach for the removal of pigment impurities secreted by Pichia pastoris
    (2023-05-01)
    Kongsinkaew, Chatchol
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    Ajariyakhajorn, Kittisak
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    Boonyaratanakornkit, Viroj
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    Sooksai, Sarintip
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    During recombinant protein expression in Pichia pastoris under the alcohol oxidase promoter, the culture supernatant developed green and yellow pigments, complicating downstream processing. Currently, a chromatography-based purification approach is one of the most efficient strategies to eliminate these pigments, but this method is complicated and often the most expensive step during processing. In this study, we designed a sequential cross-flow filtration by the combination of a microfiltration membrane with a pore size of 0.2 micrometers, followed by an ultrafiltration membrane with a molecular weight cut-off of 10 and 2 kilodaltons (kDa), respectively, and finished with diafiltration of the 10 and 2 kDa retentate fractions. The results show that the microfiltration membrane eliminated host cell impurities and significantly reduced the green pigment, which was measured using the 1976 CIE LAB system, while the 10 and 2 kDa membranes allowed the yellow pigment to pass through. The diafiltration steps also significantly reduced the yellow pigment. The host cell proteins were removed by recirculating in the retentate fraction of the 10 kDa membrane, while the 4.1 kDa target peptide, the candidate cell-penetrating antimicrobial peptide, was recovered by recirculation in the retentate fraction of the 2 kDa membrane.
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    Shelf-life extension of Thai green papaya salad dressing by hurdle technology
    (2024-09-01) ;
    Krusong, Warawut
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    ; ;
    Srisawat, Kraisuwit
    Green papaya salad or Som Tum is the most popular spicy mixed salads in Thailand due to its unique rich flavor. Green papaya salad dressing (GPSD) is made from various ingredients such as fresh chili pepper, fresh garlic, rind tamarind, fish sauce and lime oil, including the limitation in controlling the taste and flavor of salad dressing and its poor shelf-life. In this study, a convenient ready-to-eat GPSD was developed. Hurdle technology was applied to extend shelf-life of the GPSD based on monitoring of microbial contamination and food pathogens throughout the process. Hurdle technology able to decrease total plate count (TPC) from 5.6 ± 0.2 to 1 ± 0.3 log CFU/g and yeast and mold (Y&M) from 4.2 ± 0.3 to <1 log CFU/g. After 12 weeks of storage at 5 ± 2 °C, slightly increase of TPC was detected as 1.5 ± 0.2 log CFU/g and no changes were found for Y&M and other pathogens. At week 12, GPSD stored at 32 ± 2 °C was found to have higher TPC and Y&M (3.7 ± 0.3 and 2.4 ± 0.3 log CFU/g, respectively). Therefore, a combination of hurdles that combines low a<inf>w</inf>, low pH, heat treatment, low temperature after hot filling, and chilled storage could extend the shelf-life of GPSD with satisfy sensorial test result and be suitable for minimally processed salad dressing.
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    Increasing the acetification rate of Acetobacter aceti adsorbed on luffa sponge using recycle of incremental oxygenated medium
    (2020-03-01)
    Krusong, Warawut
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    Vichitraka, Assanee
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    Speeding up the production of vinegar from rice wine by acetification, using a packed-bed bioreactor with a luffa sponge matrix (LSM) as adsorption carrier of acetic acid bacteria (AAB), and the effect of oxygenation of the recycled medium were investigated. The 0.06 L/min recycle of medium resulted in a high oxygen-transfer coefficient, while optimal dissolved oxygen (DO) of the medium maximized planktonic AAB cell growth with no contamination due to high acid in an external reservoir without LSM. The highest acetification rate (ETA) of 2.857 ± 0.1 g/L/day was achieved with DO 3.5–4.5 ppm at 35 ± 1 °C. To increase ETA, the optimized oxygenated medium was externally supplied and recycled at the ratio of 0.1. Therefore, acetification was conducted in both the bioreactor and reservoir resulting in an increased ETA (6 ± 0.2 g/L/day). This also aligned with the highest system AAB biomass (confirmed by scanning electron microscopy). Under the recycled oxygenated medium supply consistently high biotransformation yields (average 77.3%) were observed over nine sequential cycles. Meanwhile, an average ETA of 6.3 ± 0.2 g/L/day was obtained. This method can have practical applications in improving the efficiency and speeding up small-scale vinegar production.
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    Upland rice vinegar vapor inhibits spore germination, hyphal growth and aflatoxin formation in Aspergillus flavus on maize grains
    The efficacy of vapor-phase (VP) upland rice vinegar (URV) was investigated as a bio-fumigant for maize, to reduce consumer health risks associated with spore and toxin formation by Aspergillus flavus. Complete reduction of mycelial growth occurred with in vitro VP exposure to URV (containing 0.0017 mmol/L acetic acid) or with VP exposure to pure acetic acid (PAA) (containing 0.0023 mmol/L acetic acid). No significant differences were observed between the two materials after 90 min exposures. Using gas chromatography-mass spectrometry (GC-MS), URV vapor was shown to contain volatiles having antifungal activities. These are identified as isoamylalcohol, 1-butanol, 3-methyl-, acetate and β-phenylethyl acetate. It is suggested these volatiles increase the antifungal effectiveness of URV. Exposure to VP-URV (containing 0.0043 mmol/L AA) for 5 h completely eliminated viable spores of A. flavus on maize seeds (23% moisture content) previously inoculated with 4.43 ± 0.28 log spores/g). At the same time, aflatoxin production decreased, as VP-URV exposure increased. Hence, VP-URV is shown to be an effective control agent for A. flavus mycelial growth and aflatoxin formation on maize, so effectively reducing the potential for consumer health risks due to this widespread fungus.
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    Enhancing small-scale acetification processes using adsorbed Acetobacter pasteurianus UMCC 2951 on κ-carrageenan-coated luffa sponge
    (2024-01-01) ;
    Krusong, Warawut
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    Samuela, Nialmas
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    Background. This study explored the utilization of luffa sponge (LS) in enhancing acetification processes. LS is known for having high porosity and specific surface area, and can provide a novel means of supporting the growth of acetic acid bacteria (AAB) to improve biomass yield and acetification rate, and thereby promote more efficient and sustainable vinegar production. Moreover, the promising potential of LS and luffa sponge coated with κ-carrageenan (LSK) means they may represent effective alternatives for the co-production of industrially valuable bioproducts, for example bacterial cellulose (BC) and acetic acid. Methods. LS and LSK were employed as adsorbents for Acetobacter pasteurianus UMCC 2951 in a submerged semi-continuous acetification process. Experiments were conducted under reciprocal shaking at 1 Hz and a temperature of 32 <sup>◦</sup>C. The performance of the two systems (LS-AAB and LSK-AAB respectively) was evaluated based on cell dry weight (CDW), acetification rate, and BC biofilm formation. Results. The use of LS significantly increased the biomass yield during acetification, achieving a CDW of 3.34 mg/L versus the 0.91 mg/L obtained with planktonic cells. Coating LS with κ-carrageenan further enhanced yield, with a CDW of 4.45 mg/L. Acetification rates were also higher in the LSK-AAB system, reaching 3.33 ± 0.05 g/L d as opposed to 2.45 ± 0.05 g/L d for LS-AAB and 1.13 ± 0.05 g/L d for planktonic cells. Additionally, BC biofilm formation during the second operational cycle was more pronounced in the LSK-AAB system (37.0 ± 3.0 mg/L, as opposed to 25.0 ± 2.0 mg/L in LS-AAB). Conclusions. This study demonstrates that LS significantly improves the efficiency of the acetification process, particularly when enhanced with κ-carrageenan. The increased biomass yield, accelerated acetification, and enhanced BC biofilm formation highlight the potential of the LS-AAB system, and especially the LSK-AAB variant, in sustainable and effective vinegar production. These systems offer a promising approach for small-scale, semi-continuous acetification processes that aligns with eco-friendly practices and caters to specialized market needs. Finally, this innovative method facilitates the dual production of acetic acid and bacterial cellulose, with potential applications in biotechnological fields.
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    Repeated cultures of Saccharomyces cerevisiae SC90 to tolerate inhibitors generated during cassava processing waste hydrolysis for bioethanol production
    (2019-03-01)
    Palakawong Na Ayutthaya, Pakathamon
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    Charoenrat, Theppanya
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    Krusong, Warawut
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    Large amount of cassava pulp is produced as by-product of industrial tapioca production. The value-added process of this low-cost waste is to use it as a substrate for bioethanol production. However, during the pulp pretreatment by acidification combined with steam explosion, many yeast inhibitors including acetic acid, formic acid, levulinic acid, furfural and 5-hydroxymethylfurfural are generated and these compounds have negative effects on the subsequent fermentation step. Therefore, the objective of this study was to investigate whether the repeated cultures of Saccharomyces cerevisiae SC90 could alleviate this problem. To obtain the inhibitor tolerable cells, the repeated culture was performed by growing yeast cells to a specific growth rate (µ) of 0.22 h <sup>−1</sup> or higher (80% of the µ in control) and then transferring them to progressively higher concentrations of hydrolysate ranging from 20 to 100% (v/v). The results showed a tendency of longer lag phase as well as time to reach maximum cell number (t <inf>maxc</inf> ) with an increase in hydrolysate concentration. However, the repeated culture at the same hydrolysate concentration could shorten both lag period and t <inf>maxc</inf> . Interestingly, the growth and fermentation efficiency of adapted cells in 100% hydrolysate were significantly higher (p ≤ 0.05) than those of non-adapted cells by 38% and 27%, respectively.
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    Enhancing Antimicrobial Peptide Productivity in Pichia pastoris (Muts Strain) by Improving the Fermentation Process Based on Increasing the Volumetric Methanol Consumption Rate
    (2023-03-01)
    Kongsinkaew, Chatchol
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    Chittapun, Supenya
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    Piyapittayanun, Chanitchote
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    Boonyaratanakornkit, Viroj
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    Sooksai, Sarintip
    The instability of the protein expression in Pichia pastoris strains has been an issue for various peptide productions. Some modifications to the traditional fermentation process could potentially solve the problem. Here, we consider a four-stage fermentation process to express the CAP2 (cell-penetrating antimicrobial peptide 2) candidate in P. pastoris KM71H, a slow methanol utilization strain. During the fermentation process, CAP2 productivity is limited (6.15 ± 0.21 mg/L·h) by the low overall methanol consumption (approximately 645 g), which is mainly the result of the slow methanol utilization of the P. pastoris KM71H. To overcome this limitation, we increased the cell concentration two-fold prior to the induction stage. A fed-batch process with exponential and dissolved oxygen tension (DOT) stat feeding strategies was deployed to control the glycerol feed, resulting in an increase in cell concentration and enhancement of the volumetric methanol consumption rate. The improved fermentation process increased the overall methanol consumption (approximately 1070 g) and the CAP2 productivity (13.59 ± 0.24 mg/L·h) by 1.66 and 2.21 times, respectively. In addition, the CAP3 (cell-penetrating antimicrobial peptide 3) candidate could also be produced using this improved fermentation process at a high yield of 3.96 ± 0.02 g/L without any further optimization. Note that there was no oxygen limitation during the improved fermentation process operating at high cell density. This could be due to the controlled substrate addition via the DOT stat system.