The Effects of Different Roasting Methods on the Phenolic Contents, Antioxidant Potential, and In Vitro Inhibitory Activities of Sacha Inchi Seeds.

Roasted sacha inchi seeds are now commercialized as a health food product, but the influence of roasting methods on their proclaimed health effects has yet to be explored. This study investigated the total phenolic contents (TPCs), antioxidant potential, and inhibitory activities of raw and roasted sacha inchi seeds in vitro. Individual phenolics in raw seeds were also identified in an attempt to explain the bioactivities of the seeds. The results suggested that roasting in a cooking pan, vacuum oven, and tray dryer had distinct impact on TPC in sacha inchi seeds, and thus differentially altered their antioxidant and inhibitory properties. Seeds that underwent roasting exhibited 1.5-2.7-fold higher antioxidant potentials than raw seeds. Certain roasting methods provided the products with anti-α-amylase and anti-cholinesterase activities, while inhibitions of these enzymes were not detected in raw seeds. Roasted seeds also possessed superior anti-lipase and anti-glycation activities when compared with raw seeds (up to 1.7- and 4.8-fold, respectively). The inhibitory properties observed in the seed samples might be attributed to their p-coumaric acid, ferulic acid, and quercetin, as these potential enzyme inhibitors were predominant in raw seeds. The overall results showed that pan-roasting could be used to obtain relatively high health benefits from the antioxidant and inhibitory activities of sacha inchi seeds. The information obtained from this study may serve as the basis for the proper processing of sacha inchi seeds to optimize their functional food and nutraceutical applications.

Effects of Maturity and Thermal Treatment on Phenolic Profiles and In Vitro Health-Related Properties of Sacha Inchi Leaves.

Sacha inchi (Plukenetia volubilis L.) has been adopted as a novel economic crop with well-studied nutritional and bioactive benefits for human health. Sacha inchi seeds and oil have high commercial value but scant research has focused on its leaves. This study investigated and compared phenolic compositions, antioxidant potentials and in vitro health-related properties of both young and mature sacha inchi leaves after freeze-drying and oven-drying processes. Results showed that p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid and gallic acid were predominantly detected in both young and mature leaves that also exhibited similar total phenolic contents (TPCs), while higher TPCs were detected in freeze-dried than in oven-dried leaves. Mature leaves exhibited higher antioxidant potential than young leaves after freeze-drying, while the opposite results were observed for oven-drying. Overall in vitro health-related activities were higher in mature leaves compared to young leaves regardless of the drying process. Knowledge gained from this study can be used to encourage prospective utilization of sacha inchi leaves as a source of health-promoting compounds. This, in turn, will increase the commercial value of the leaves and provide a wider market variety of sacha inchi products.

Mangifera indica 'Namdokmai' Prevents Neuronal Cells from Amyloid Peptide Toxicity and Inhibits BACE-1 Activities in a Drosophila Model of Alzheimer's Amyloidosis.

Alzheimer's disease (AD) is a progressive neurological illness with few effective treatments. Thus, ameliorating the effects of AD using natural products has attracted global attention with promising efficacy and safety. In this study, ten tropical fruits including Ananas comosus 'Phulae', Ananas comosus 'Pattavia', Carica papaya 'Khaekdum', Carica papaya 'Khaeknuan', Durio zibethinus 'Monthong', Durio zibethinus 'Chanee', Psidium guajava 'Kimju', Psidium guajava 'Keenok', Mangifera indica 'Kaew' and Mangifera indica 'Namdokmai' were screened for their inhibitory activities against the key enzymes, cholinesterases and ß-secretase (BACE-1), involved in AD pathogenesis. The top three fruit extracts with promising in vitro anti-AD activities were further investigated using rat pheochromocytoma PC-12 neuronal cell line and Drosophila AD model. Data showed that M. indica 'Kaew', M. indica 'Namdokmai' and P. guajava 'Kimju' reduced Aß1-42-mediated neurotoxicity by promoting glutathione-dependent enzymes, while M. indica 'Namdokmai' limited Aß1-42 peptide formation via BACE-1 inhibition and amended locomotory behavior of the Drosophila AD model. Results indicated the potential anti-AD properties of tropical fruits, particularly M. indica 'Namdokmai' in the prevention of Aß1-42-mediated neurotoxicity and as a BACE-1 blocker.

Impact of Drying Processes on Phenolics and In Vitro Health-Related Activities of Indigenous Plants in Thailand.

Thailand has vast areas of tropical forests with many indigenous plants, but limited information is available on their phytochemical profile and in vitro inhibitions of enzymatic and nonenzymatic reactions. This study investigated phenolic profiles using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), antioxidant activities, and in vitro inhibitory activities of 10 indigenous plants on key enzymes related to obesity (lipase), diabetes (α-amylase and α-glucosidase), and Alzheimer's disease (cholinesterases and ß-secretase). The nonenzymatic anti-glycation reaction was also investigated. The 10 indigenous plants were Albizia lebbeck (L.) Benth, Alpinia malaccensis (Burm.) Roscoe, Careya arborea Roxb., Diplazium esculentum (Retz.) Swartz, Kaempferia roscoeana Wall., Millettia brandisiana Kurz., Momordica charantia, Phyllanthusemblica L., Zingiber cassumunar Roxb, and Zingiber citriodorum J. Mood & T. Theleide. Preparations were made by either freeze-drying or oven-drying processes. Results suggested that the drying processes had a minor impact on in vitro inhibitions of enzymatic and nonenzymatic reactions (<4-fold difference). P. emblica was the most potent antioxidant provider with high anti-glycation activity (>80% inhibition using the extract concentration of ≤6 mg/mL), while D. esculentum effectively inhibited ß-secretase activity (>80% inhibition using the extract concentration of 10 mg/mL). C. arborea exhibited the highest inhibitory activities against lipase (47-51% inhibition using the extract concentration of 1 mg/mL) and cholinesterases (>60% inhibition using the extract concentration of 2 mg/mL), while Mi. brandisiana dominantly provided α-amylase and α-glucosidase inhibitors (>80% inhibition using the extract concentration of ≤2 mg/mL). Information obtained from this research may support usage of the oven-drying method due to its lower cost and easier preparation step for these studied plant species and plant parts. Furthermore, the information on in vitro inhibitions of enzymatic and nonenzymatic reactions could be used as fundamental knowledge for further investigations into other biological activities such as cell culture or in vivo experiments of these health-beneficial plants.

Analysis of Phytonutrients, Anti-Mutagenic and Chemopreventive Effects of Tropical Fruit Extracts.

Thailand is located in the tropics and a wide variety of fruits are grown commercially. However, studies regarding the phytonutrients, anti-mutagenic and chemopreventive effects of these fruits are limited. Thus, phytochemical profiles and inhibition of key enzymes involved in obesity and diabetes, together with anti-mutagenic and chemopreventive properties of eight tropical fruit extracts cultivated in Thailand, including Psidium guajava 'Kimju', Psidium guajava 'Keenok', Ananas comosus 'Pattavia', Ananas comosus 'Phulae', Durio zibethinus 'Chanee', Durio zibethinus 'Monthong', Carica papaya 'Khaekdum' and Mangifera indica 'Namdokmai' were investigated. Different cultivars were also compared. Results showed that M. indica 'Namdokmai' was the most antioxidant-rich extract containing abundant 4-hydroxybenzoic acid and its derivative, gallic acid, as the main phenolics. M. indica 'Namdokmai' also exhibited high inhibitory capacities (>60% inhibition under studied conditions) against lipase, α-amylase and α-glucosidase, key enzymes as drug targets for controlling obesity and type 2 diabetes. Interestingly, all fruit extracts suppressed food mutagen-induced DNA mutations assayed by the Ames test, especially M. indica 'Namdokmai' and C. papaya 'Khaekdum' (>50% inhibition at 200 µg/plate). The M. indica 'Namdokmai' was also the most potent extract for suppression of cancer promotion (>90% inhibition at 200 µg/mL) followed by P. guajava 'Kimju', P. guajava 'Keenok' and C. papaya 'Khaekdum'. Results potentially indicated that fruit intake after overcooked meat consumption might supplement nutrients and fiber and also reduce DNA mutation sources.

Development of Optimal Digesting Conditions for Microplastic Analysis in Dried Seaweed Gracilaria fisheri.

Currently, research on the accumulation of microplastics (MPs) in the marine food web is being highlighted. An accurate and reliable digestion method to extract and isolate MPs from complex food matrices has seldom been validated. This study aimed to compare the efficacy of MP isolation among enzymatic-, oxidative-, and the combination of two digestion methods on red seaweed, Gracilaria fisheri. The dried seaweed sample was digested using three different methods under various conditions using enzymes (cellulase and protease), 30% H2O2, and a combination of enzymes and 30% H2O2. The method possessing the best digestion efficiency and polymer recovery rate of MPs was selected, and its effect on spiked plastic polymer integrity was analyzed by Raman spectroscopy. As a result, the enzymatic method rendered moderate digestion efficiency (59.3-63.7%) and high polymer recovery rate (94.7-98.9%). The oxidative method using 30% H2O2 showed high digestion efficiency (93.0-96.3%) and high polymer recovery rate (>98%). The combination method was the most effective method in terms of digestion efficiency, polymer recovery rate, and expenditure of digestion time. The method also showed no chemical changes in the spiked plastic polymers (PE, PP, PS, PVC, and PET) after the digestion process. All the spiked plastic polymers were identifiable using Raman spectroscopy.

Characterization of Sporidiobolus ruineniae A45.2 Cultivated in Tannin Substrate for Use as a Potential Multifunctional Probiotic Yeast in Aquaculture.

At present, few yeast species have been evaluated for their beneficial capabilities as probiotics. Sporidiobolus ruineniae A45.2, a carotenoid-producing yeast, was able to co-produce cell-associated tannase (CAT), gallic acid and viable cells with antioxidant activity when grown in a tannic acid substrate. The aim of this research study was to identify the potential uses of S. ruineniae A45.2 obtained from a co-production system as a potential feed additive for aquaculture. S. ruineniae A45.2 and its CAT displayed high tolerance in pH 2.0, pepsin, bile salts and pancreatin. Furthermore, its viable cells were characterized by moderate hydrophobicity, high auto-aggregation and moderate co-aggregation with Staphylococcus aureus, Salmonella ser. Thyphimurium and Streptococcus agalactiae. These attributes promoted S. ruineniae A45.2 as a multifunctional probiotic yeast. In addition, the intact cells possessed antioxidant activities in a 100-150 µg gallic acid equivalent (GAE)/mL culture. Remarkably, the fermentation broth demonstrated higher antioxidant activity of 9.2 ± 1.8, 9.0 ± 0.9, and 9.8 ± 0.7 mg GAE/mL culture after FRAP, DPPH and ABTS assays, respectively. Furthermore, higher antimicrobial activity was observed against Bacillus cereus, Staphylococcus aureus and Strep. agalactiae. Therefore, cultivation of S. ruineniae A45.2 with a tannic acid substrate displayed significant potential as an effective multifunctional feed additive.

Co-production of gallic acid and a novel cell-associated tannase by a pigment-producing yeast, Sporidiobolus ruineniae A45.2.

BACKGROUND: Gallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive. RESULTS: Tannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs. CONCLUSION: Co-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.

Structural Comparison of Different Galacto-oligosaccharide Mixtures Formed by ß-Galactosidases from Lactic Acid Bacteria and Bifidobacteria.

The LacLM-type ß-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhß-gal) and Lactobacillus plantarum (Lp609Lhß-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by ß-galactosidase from L. helveticus, together with three GOS mixtures produced using ß-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using ß-galactosidases (ß-gal I and ß-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1H NMR spectroscopy. ß-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form ß-(1→6)- and ß-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ∼90% of all GOS components. GOS mixtures obtained with ß-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).

Immobilization of ß-Galactosidases on the Lactobacillus Cell Surface Using the Peptidoglycan-Binding Motif LysM.

Lysin motif (LysM) domains are found in many bacterial peptidoglycan hydrolases. They can bind non-covalently to peptidoglycan and have been employed to display heterologous proteins on the bacterial cell surface. In this study, we aimed to use a single LysM domain derived from a putative extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WCFS1 to display two different lactobacillal ß-galactosidases, the heterodimeric LacLM-type from Lactobacillus reuteri and the homodimeric LacZ-type from Lactobacillus delbrueckii subsp. bulgaricus, on the cell surface of different Lactobacillus spp. The ß-galactosidases were fused with the LysM domain and the fusion proteins, LysM-LacLMLreu and LysM-LacZLbul, were successfully expressed in Escherichia coli and subsequently displayed on the cell surface of L. plantarum WCFS1. ß-Galactosidase activities obtained for L. plantarum displaying cells were 179 and 1153 U per g dry cell weight, or the amounts of active surface-anchored ß-galactosidase were 0.99 and 4.61 mg per g dry cell weight for LysM-LacLMLreu and LysM-LacZLbul, respectively. LysM-LacZLbul was also displayed on the cell surface of other Lactobacillus spp. including L. delbrueckii subsp. bulgaricus, L. casei and L. helveticus, however L. plantarum is shown to be the best among Lactobacillus spp. tested for surface display of fusion LysM-LacZLbul, both with respect to the immobilization yield as well as the amount of active surface-anchored enzyme. The immobilized fusion LysM-ß-galactosidases are catalytically efficient and can be reused for several repeated rounds of lactose conversion. This approach, with the ß-galactosidases being displayed on the cell surface of non-genetically modified food-grade organisms, shows potential for applications of these immobilized enzymes in the synthesis of prebiotic galacto-oligosaccharides.

ß-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry.

ß-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant ß-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native ß-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant ß-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-ß-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of ß-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.

Fermentability of a Novel Galacto-Oligosaccharide Mixture by Lactobacillus spp. and Bifidobacterium spp.

This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a ß-galactosidase from a potential probiotic Lactobacillus isolate that contained mainly oligosaccharides of ß-1,3 and ß-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight Lactobacillus spp. strains and three Bifidobacterium spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4'GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when L. reuteri Lb46 and the two Bifidobacterium strains, B. animalis subsp. lactis Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4'GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.