Extraction of Heat-Stabilised Defatted Rice Bran Protein by Solid-State Fermentation Using Heterofermentative Microbes from Traditional Asian Starters.

Research background: Heat-stabilised defatted rice bran (HSDRB) is a primary by-product of rice bran oil extraction industry and a nutritious source of protein. However, despite the unique nutritional profile of rice bran protein, the protein-rich by-product, HSDRB is underutilised as a low-value animal feed. Research on protein extraction from HSDRB by enzymatic hydrolysis has attracted the attention of numerous scientists. However, a cost-effective extraction method is required to mitigate the high costs associated with the use of enzymes. Therefore, we have presented an alternative economical and natural approach for protein extraction from HSDRB by solid-state fermentation (SSF) with heterofermentative microbes. Experimental approach: SSF of HSDRB with two types of traditional Asian fermentation starters, namely loog-pang and koji, were evaluated for enzyme production and their efficacy in extracting proteins from HSDRB. For this purpose, HSDRB fermentation was carried out for 0, 12, 24, 48, 72 and 96 h followed by 24-hour hydrolysis to evaluate the extracted rice bran protein. In addition, microbiome diversity in the fermentation starters was also determined by metagenomic sequencing of 16S rRNA and internal transcribed spacer to identify bacteria and fungi, respectively. Results and conclusions: The microbial community in the fermentation starters showed the dominance of lactic acid bacteria (LAB) such as Bacillus subtilis in loog-pang and Streptococcus lutetiensis, Bacillus pumilus, Lactococcus cremoris, Lactococcus garvieae and Pediococcus pentosaceus in koji, while yeast species Saccharomycopsis fibuligera and Saccharomyces cerevisiae dominated the fungal diversity in loog-pang and koji starters, respectively. The results suggest that loog-pang and koji can produce cellulase, neutral and acid proteases during fermentation. Despite the discrepancy in their microbial diversity and the enzyme activity during SSF, both starters could effectively increase protein extraction from HSDRB. A positive relationship between the SSF duration and extracted protein was observed. During SSF with loog-pang and koji after 72 h followed by 24-hour hydrolysis, 65.66 and 66.67 % protein was extracted from HSDRB, respectively. The amino acid analysis of the protein hydrolysate produced by the non-fermented and fermented methods showed no difference and had an abundance of glutamic and aspartic acids, leucine, arginine, alanine and glycine amino acids, which accounted for approx. 58 % of the total amino acids. Novelty and scientific contribution: Loog-pang and koji (traditional Thai and Japanese fermentation starters, respectively) were found to be effective in extracting proteins from HSDRB by SSF although they are inexpensive microbial enzyme sources. Future research aimed at scaling up HSDRB protein extraction for usage in industrial applications can draw on our results.

Nutraceutical Difference between Two Popular Thai Namwa Cultivars Used for Sun Dried Banana Products.

Musa (ABB group) "Kluai Namwa" bananas (Musa sp.) are widely grown throughout Thailand. Mali Ong is the most popular Kluai Namwa variety used as raw material for sun-dried banana production, especially in the Bangkratum District, Phitsanulok, Thailand. The sun-dried banana product made from Nanwa Mali Ong is well recognized as the best dried banana product of the country, with optimal taste compared to one made from other Kluai Namwa varieties. However, the production of Mali Ong has fluctuated substantially in recent years, leading to shortages. Consequently, farmers have turned to using other Kluai Namwa varieties including Nuanchan. This study investigated the nutraceutical contents of two popular Namwa varieties, Mali Ong and Nuanchan, at different ripening stages. Nutraceuticals in the dried banana products made from these two Kluai Namwa varieties and four commercial dried banana products were compared. Results indicated that the content of moisture, total sugar, and total soluble solids (TSS) (°Brix) increased, while total solids and texture values decreased during the ripening stage for both Kluai Namwa varieties. Rutin was the major flavonoid found in both Namwa Mali Ong and Nuanchan varieties ranging 136.00−204.89 mg/kg and 129.15−260.38 mg/kg, respectively. Rutin, naringenin, quercetin and catechin were abundant in both Namwa varieties. All flavonoids increased with ripening except for rutin, gallocatechin and gallocatechin gallate. There were no significant differences (p < 0.05) in flavonoid contents between both varieties. Tannic acid, ellagic acid, gallic acid, chlorogenic acid and ferulic acid were the main phenolic acids found in Mali Ong and Nuanchan varieties, ranging from 274.61−339.56 mg/kg and 293.13−372.66 mg/kg, respectively. Phenolic contents of both varieties decreased, increased and then decreased again during the development stage. Dopamine contents increased from 79.26 to 111.77 mg/kg and 60.38 to 125.07 mg/kg for Mali Ong and Nuanchan, respectively, but the amounts were not significantly different (p < 0.5) between the two Namwa varieties at each ripening stage. Inulin as fructooligosaccharide (FOS) increased with ripening steps. Production stages of sun-dried banana products showed no statistically significant differences (p < 0.05) between the two Namwa varieties. Therefore, when one variety is scarce, the other could be used as a replacement in terms of total flavonoids, phenolic acid, dopamine and FOS. In both Namwa varieties, sugar contents decreased after the drying process. Sugar contents of the dried products were 48.47 and 47.21 g/100 g. The drying process caused a reduction in total flavonoid contents and phenolic acid at 63−66% and 64−70%, respectively. No significant differences (p < 0.05) were found for total flavonoid and phenolic contents between the dried banana products made from the two Namwa varieties (178.21 vs. 182.53 mg/kg and 96.06 vs. 102.19 mg/kg, respectively). Products made from Nuanchan varieties (24.52 mg/kg) contained significantly higher dopamine than that from Mali Ong (38.52 mg/kg). The data also suggest that the banana maturity stage for production of the sun dried products was also optimum in terms of high nutraceutical level.

Preparing spray-dried cholesterol free salad dressing emulsified with enzymatically synthesized mixed mono- and diglycerides from rice bran oil and glycerol.

A spray-dried cholesterol free salad dressing powder was developed using mixed mono- and diglycerides (MG-DG) as emulsifier. The optimum conditions for enzymatic synthesis of the MG-DG from rice bran oil (RBO) and glycerol (Gly) using Candida antarctica lipase was investigated. The synthesis was done by glycerolysis of refined RBO and Gly at molar ratios of 2:1, 2.5:1 and 3:1 (Gly to RBO) and enzyme concentrations of 2% and 5%. Highest MG and DG yield (0.54 ± 0.01 and 0.49.03 ± 0.0 mg/mL) was obtained in sample prepared using 2:1 molar ratio and 5% enzyme concentration and this sample is considered optimum. Salad dressings prepared using 0.5, 1.0, and 1.5% MG-DG concentration (of optimum MG-DG) were spray dried at inlet temperatures of 150, 160 and 170 °C to find the best conditions. Salad dressing of 0.5% MG-DG spray-dried at 170 °C had the highest powder yield (42.70%), solubility (98.04%) and stability (100%). After reconstitution, this optimum sample was compared well next to a control salad dressing prepared using commercial distilled monoglycerides. These findings demonstrate the feasibility of preparing a spray dried salad dressing powder with the synthesized MG-DG as an emulsifier.

Optimization of enzymatic hydrolysis of copra meal: compositions and properties of the hydrolysate.

This study demonstrated that oligosaccharides from copra meal could be prepared by using a commercial enzyme preparation containing mannanase for use as a prebiotic. The conditions giving the highest hydrolysis rate of the copra meal by the enzyme were pH 4 and temperature of 40 °C with an enzyme to substrate ratio (E/S) of 1092.69 ß-mannanase activity unit/g of the dried copra meal. Monosaccharide was the predominant product of the hydrolysis reaction followed by di- and tri-saccharide. Activated carbon treatment of the copra meal hydrolysate reduced the monosaccharide content resulting in 36.13% of monosaccharide, 54.26% of disaccharide and 9.61% of trisaccharide. All monosaccharides were eliminated by incubating the copra meal hydrolysate with Saccharomyces cerevisiae for 48 h, which promoted the growth of B. breve, L. plantrarum, B. bifidum, L. bugaricus, L. acidophilus, L. brevis, L. casei, B. longum, and S. thermophiles while retarding the growth of the pathogenic bacteria, S. aureus and E. coli.

Short-Chain Fructooligosaccharide Synthesis from Sugarcane Syrup with Commercial Enzyme Preparations and Some Physical and Antioxidation Properties of the Syrup and Syrup Powder.

Short-chain fructooligosaccharides (sc-FOS) are prebiotics beneficial to human health, which can be synthesized from raw material containing a high sucrose content. Sugarcane syrup (SS) without molasses removal contains sucrose as a major sugar and is a rich source of several bioactive compounds. The aim of this study is to investigate factors affecting sc-FOS synthesis from SS using commercial enzyme preparations containing fructosyltransferase activity as biocatalysts. sc-FOS content increased significantly as the sucrose concentration of SS in the reaction mixture increased up to 40% (w/v). Changes in carbohydrate compositions during the transfructosylating reaction of a pure sucrose solution and SS prepared from the two sugarcane cultivars Khon Kaen 3 and Suphanburi 50, catalyzed by Pectinex Ultra SP-L and Viscozyme L, showed similar profiles. Both enzymes showed a high ability to transfer fructosyl moieties to produce sc-FOS and a plateau of the total sc-FOS concentration was observed after 4 h of reaction time. For the pure sucrose solution and SS (Suphanburi 50), Viscozyme showed a superior ability to convert sucrose to Pectinex, with a higher sc-FOS yield (g FOS/100 g of initial sucrose), GF2 or 1-kestose yield (g GF2/g of initial sucrose) and GF3 or nystose yield (g GF3/g of initial sucrose). sc-FOS syrup (FOS SS) and the foam-mat-dried syrup powder prepared from SS and FOS SS, respectively, contained a high total phenolic content and possessed higher antioxidant activities than those prepared from pure sucrose, but contained lower calories.

Glutamate Decarboxylase (GAD) Extracted from Germinated Rice: Enzymatic Properties and Its Application in Soymilk.

Glutamate decarboxylase (GAD) is an important enzyme in biological metabolisms acting on catalyzing the irreversible α-decarboxylation of L-glutamic acid to γ-aminobutyric acid (GABA) and CO2, which was focused in this study. Three rice varieties different in color were germinated at different times and used for crude GAD extraction. Crude GADs with an optimal germination time from germinated black (GBR), red (GRR), and white (GWR) rice were evaluated for enzymatic properties, including the effect of pHs, temperatures, and concentrations of both L-glutamic acid and pyridoxal 5'-phosphate (PLP). Crude GAD with optimum enzymatic properties was selected to be partially purified using ammonium sulfate (AMS) precipitation. The obtained GAD was supplemented to soymilk and determined for GABA content. All crude GADs from germinated rice at 10 germination days presented the highest enzyme activity. For enzymatic properties, crude GADs showed the highest activity at pH in a range of 5.6-6.0 at 60ºC. The Km values of crude GADs were in the range of 7.68-8.06 mM for L-glutamic acid and 0.15-0.20 µM for PLP and were the lowest in crude GAD from GBR. GAD from GBR presented the highest enzyme activity in the fraction with 50% saturation (v/v) after AMS precipitation and it was purified for 14.61 folds. The addition of this GAD (1.0%, v/v) resulted in the increasing of GABA content in soymilk to 53.79 mg/100 mL, accounted for 1.23 times compared with control.

Reactivation of broccoli peroxidases: structural changes of partially denatured isoenzymes.

UNLABELLED: Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into an inactive structural species, similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase, as well as molecular rearrangement of the structure around the heme, occurs during incubation at approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed. KEYWORDS: Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.

Purification and partial characterization of broccoli (Brassica oleracea Var. Italica) peroxidases.

Three peroxidase (POD) isoenzymes were purified from a soluble extract of broccoli stems. The acidic and neutral PODs were purified to homogeneity by using ion exchange and hydrophobic chromatography. The basic POD was purified by cation exchange and gel filtration chromatography. The neutral and basic PODs had molecular masses of approximately 43 kDa, and the acidic POD had a molecular mass of 48 kDa by SDS-PAGE. pI was approximately 4, 5, and 8 for acidic, neutral, and basic PODs, respectively. Optimum activity using guaiacol as the H donor was obtained at pH approximately 6 for both neutral and basic PODs and at pH approximately 4 for acidic POD. All three of the purified isoenzymes are glycosylated. Reaction rates with various substrates including guaiacol, guaiacol/MBTH, DMAB/MBTH, and ferulic acid/MBTH were different among the isoenzymes. K(m) and amino acid composition were also determined.

Heat inactivation and reactivation of broccoli peroxidase.

Heat inactivation characteristics differed for acidic (A), neutral (N), and basic (B) broccoli peroxidase. At 65 degrees C, A was the most heat stable followed by N and B. The activation energies for denaturation were 388, 189, and 269 kJ/mol for A, N, and B, respectively. Reactivation of N occurred rapidly, within 10 min after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 50% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperature allowing the maximum reactivation was 90 degrees C for A and horseradish peroxidase (HRP) and 70 and 80 degrees C for B and N, respectively. In all cases, heat treatment at low temperatures for long times prevented reactivation of the heated enzymes. Calcium (5 mM) increased the thermal stability of N and B but had no effect on reactivation. The presence of 0.05% bovine serum albumin decreased thermal stability but increased the extent of reactivation of A..