Characterization of yeast mutant strains for starter culture in Arabica coffee fermentation.

Arabica coffee is the most popular and best-selling type of coffee. During coffee fermentation, microorganisms are essential for the production of metabolites and volatile compounds that affect coffee flavor quality. This work aimed to study the mutation, selection, and characterization of the Wickerhamomyces anomalus strain YWP1-3 as a starter culture to enhance the flavor quality of Arabica coffee. The results revealed that six mutants could produce relatively high levels of the pectinase enzyme on pectin agar media and exhibited high activity levels, ranging from 332.35 to 415.88 U/ml in mucilage broth. Strains UV22-2, UV22-3, UV41-1 and UV32-1 displayed higher levels of amylase activity than did the wild type. The UV22-2 and UV22-3 mutants exhibited the highest pectin degradation indices of 49.22% and 45.97%, respectively, and displayed significantly enhanced growth rates in nitrogen yeast base media supplemented with various sugars; thus, these mutants were evaluated for their ability to serve as a starter for fermentation of Arabica coffee. The cupping scores of coffees derived from UV22-2 and UV22-3 were 83.5 ± 1.5 and 82.0 ± 2.14, respectively. The volatile compounds in the roasted coffee fermented by UV22-2 were analyzed by GC‒MS, which revealed higher levels of furfuryl alcohol and furfuryl acetate than did the other samples. These findings suggested that UV22-2 could be an influential starter culture for Arabica coffee fermentation.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Influence of Different Extraction Methods on the Changes in Bioactive Compound Composition and Antioxidant Properties of Solid-State Fermented Coffee Husk Extracts.

In brewing coffee, a huge amount of food waste is generated; that waste, coffee husks in particular, should be comprehensively exploited. They offer a rich source of bioactive compounds such as caffeine, chlorogenic acid, and trigonelline. The aim of this study was to investigate the effects of extraction methods on the bioactive compounds and antioxidant activity of such waste. Coffee husks in this study were fermented with S. cerevisiae based on a solid-state fermentation technique. The study method included ethanolic or water extraction with varied controllable factors, i.e., temperature (60, 100°C) and extraction technique. Bioactive contents were investigated with the Folin-Ciocalteu assay and 1H-NMR spectroscopy. The antioxidant activity was investigated with DPPH and FRAP assays. Results show that yields were the highest in the extract of fermented coffee husks at 100°C. The highest levels of bioactive contents (total trigonelline content at 3.59% and antioxidant activity at 23.35% (DPPH) and 25.9% (FRAP)) were found in the ethanolic extract of fermented coffee husks at 60°C. The bioactive content and bioactivity, including antioxidant activity, depended on different raw materials, preparation methods, and extraction conditions. This study illustrates the potential for using food waste such as coffee husks as a sustainable source of bioactive compounds or bioactive extracts.

New approach for screening of microorganisms from Arabica coffee processing for their ability to improve Arabica coffee flavor.

The purpose of this research was to isolate microorganisms from coffee fermentation processes and screen them for their potential to improve the flavor of Arabica coffee using a new approach that included pectin degradation ability and growth in mucilage broth. All of the studied microorganisms were isolated from 38 different samples of fresh coffee cherries, coffee mucilage and coffee pulp. A total of 262 microbial isolates were obtained and subjected to screening using pectinase screening agar medium for pectinolytic organisms. The results of the pectinase production test showed that 18 yeast isolates were found to produce pectinase that could degrade the pectin present in solid media. The sugar assimilation profiles and growth of selected strains in mucilage broth were studied. Therefore, 18 isolates from the selected yeasts were subjected to molecular identification by the use of 18S rRNA gene sequencing. The diversity of the yeast isolates was studied, and they were identified as Wickerhamomyces anomalus, Naganishia liquefaciens, Pichia kudriavzevii, Kazachstania naganishii and Kazachstania sp. Moreover, isolates SWU3YWP1-3, SWU3YSK9 and INFCY1-4 were used as a seed culture for Arabica coffee fermentation. The cupping sensory scores of the control (without yeast inoculation) and those inoculated with three isolated yeast strains that were determined by Q-Arabica Graders were 73.75, 84.75, 80.25 and 75.00, respectively. Unique flavors and aromas were detected. This is the first report of screening microorganisms from the Arabica coffee fermentation process by the combination of various properties with success in improving the quality of coffee beverage.

Biodegradation of polystyrene by three bacterial strains isolated from the gut of Superworms (Zophobas atratus larvae).

AIMS: To isolate polystyrene-degrading bacteria from the gut of superworms and investigate their ability to degrade polystyrene (PS). METHODS AND RESULTS: Three PS-degrading bacteria identified as Pseudomonas sp. EDB1, Bacillus sp. EDA4 and Brevibacterium sp. EDX were successfully isolated from the gut of superworms (Zophobas atratus Larvae) that ingest PS. Incubating PS with each strain for 30-day led to the formation of biofilm on the PS film. Scanning electron microscopy (SEM) revealed considerable damage (in terms of pits formation) on the surface of the PS films. FTIR analysis suggested the incorporation of carbonyl group into the carbon backbone of PS. Decreasing of WCA of microbial-treated PS film confirmed a chemical change from hydrophobicity to hydrophilicity on the PS surface. Based on these results, we conclude that all isolates had the ability to degrade PS. CONCLUSIONS: Brevibacterium sp. EDX (GenBank MZ32399) was isolated as the most efficient PS-degrading strain based on the most changing in both PS surface morphology (SEM and WCA analyses) and chemical modification (FTIR analysis) in its PS degradation process. SIGNIFICANCE AND IMPACT OF THE STUDY: This was the first study to describe the PS degradation by Brevibacterium sp. EDX, and thus provided for its development in the plastic remediation process.

Production of poly (l-lactide)-degrading enzyme by Actinomadura keratinilytica strain T16-1 under solid state fermentation using agricultural wastes as substrate.

Poly (l-lactide) (PLLA) is an aliphatic polyester that can be obtained from renewable resources and degraded by various microorganisms. In previous reports, Actinomadura keratinilytica strain T16-1 demonstrated high ability to degrade PLLA under various conditions. PLLA-degrading enzyme production under solid state fermentation has been sparsely studied. PLLA-degrading enzyme production by A. keratinilytica strain T16-1 was investigated using agricultural wastes as substrate under solid state fermentation (SSF). Three agricultural wastes as soybean meal, cassava chips and duckweed were tested as substrates for PLLA-degrading enzyme production by statistical methods using mixture design. Results revealed that using duckweed as the substrate gave the highest enzyme production (138.66 ± 13.57 U/g dry substrate). Maximum enzyme activity of 391.24 ± 15.57 U/g dry substrate was obtained under 10 g duckweed, 10% inoculum size, 7 days of cultivation time, pH 7.0, 2.8% PLLA powder, and 60% moisture content at 45 °C. It can be concluded that duckweed is an inexpensive substrate, which reduces the costs of PLLA-degrading enzyme production, as an alternative to effective water weed management.

Utilization of oil palm decanter cake for valuable laccase and manganese peroxidase enzyme production from a novel white-rot fungus, Pseudolagarobasidium sp. PP17-33.

Oil palm decanter cake (OPDC) in the current study was converted to valuable products as laccase and manganese peroxidase (MnP) by an undescribed strain of the white-rot fungus, Pseudolagarobasidium sp. PP17-33. The optimization to enhance the production of enzymes through solid-state fermentation was performed using Plackett-Burman design and response surface methodology. The highest observed laccase was 5.841 U/gds and observed MnP was 5.156 U/gds, which enhanced yield by 2.59-fold and 1.94-fold from the non-optimization. The optimized medium (mg/g of OPDC) consisted of 0.852 mg CuSO4·5H2O, 13.512 mg glucose, 2 mg yeast extract, 0.2 mg KH2PO4, 1.5 mg MgSO4·7H2O, 0.01 mg FeSO4·7H2O, 0.15 mg MnSO4·H2O, 0.01 mg ZnSO4·7H2O and 0.3 mg Tween 80 (pH 5.0) when incubated at 30 °C for 7 days. The most significant variables of laccase and MnP productions were CuSO4·5H2O and glucose concentrations. This study is the first to report on the production of ligninolytic enzymes from OPDC waste using white-rot fungi. In addition, five different white-rot fungi, Coriolopsis aspera, C. retropicta, Dentipellis parmastoi, Nigroporus vinosus and Tyromyces xuchilensis, are newly observed producers of ligninolytic enzymes in Thailand. The results obtained from this study are significant not only for agro-industrial waste management but also for value-added enzyme production.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Poly(DL-lactide)-degrading enzyme production by immobilized Actinomadura keratinilytica strain T16-1 in a 5-L fermenter under various fermentation processes

Background: Poly(DL-lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results: Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions: This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).