Effect of Fungal and Fungal-Bacterial Tempe-Type Fermentation on the Bioactive Potential of Grass Pea Seeds and Flaxseed Oil Cake Mix.

Tempe is an Indonesian food product traditionally obtained from soybeans through solid-state fermentation with Rhizopus. A variety of substrates can be processed into tempe in the presence of other microorganisms. In this study, grass pea seeds with the addition of flaxseed oil cake (20% w/w) were either fermented using individual mould strains-Rhizopus oryzae, R. oligosporus, and Mucor indicus-or cofermented with the moulds and Lactiplantibacillus plantarum. In the obtained products, the content of dietary fibre, B group vitamins, and the level of peptides and antioxidant potential in aqueous extracts were measured. Moreover, peptides, angiotensin I convertase inhibitor, and antioxidant activity were determined after in vitro digestion. The effect of digestates on the differentiation of enterocytes was also investigated. Fermentation generally resulted in a decrease in the dietary fibre, especially the insoluble fraction (30-50%). The product obtained with R. oryzae was the best source of riboflavin and thiamine among all tested. The fermentation process promoted the accumulation of water-soluble peptides and antioxidant compounds. After in vitro digestion, the largest amount of antioxidant and antiradical compounds was released from tempe obtained with R. oryzae. However, the enrichment of the products with antioxidants resulting from solid-state fermentation did not simply translate into an improvement in antioxidant potential after digestion. Generally, fermentation carried out in the presence of L. plantarum brought positive effects only in the case of R. oligosporus DSM 1964. Digestion products obtained from R. oryzae tempe had a positive effect on the viability of Caco-2 cells differentiated into enterocytes. Interestingly, a higher activity of differentiation markers (alkaline phosphatase and sucrase-isomaltase) was observed under the influence of digestate of R. oryzae and L. plantarum tempe.

Phenolic Profile, Antioxidant Activity and Amino Acid Composition of Moringa Leaves Fermented with Edible Fungal Strains.

Solid-state fermentation (SSF) is widely recognised as a technique to increase the bioactive potential and nutritional value of plant materials. However, the effect of this biotreatment differs for individual substrates. This study aimed to evaluate the impact of SSF with filamentous fungi (Rhizopus, Aspergillus, and Neurospora) on a moringa leaf phenolic profile, antioxidant activity, and amino acid composition. A total of 43 phenolic compounds were determined in the dried leaves analysed by HPLC-ESI-TOF-MS. The leaves contained 11.79 mg/g of free phenolics: flavonols (80.6%, mainly quercetin and kaempferol glycosides), hydroxycinnamic acid derivatives (12.3%), vitexin and vicenin (6.9%), and a small amount of lignan (isolariciresinol isomers). The result of the 1-day fermentation was a slight enhancement in the concentration of individual free phenolics (flavones) and the antioxidant activity of the leaves. However, extending the incubation period caused a significant decrease in those parameters and cannot be recommended for obtaining a food fortificant from moringa leaves. In contrast, the 3-day fermentation with N. intermedia led to a 26% average accumulation of individual amino acids. Therefore, the SSF with Neurospora can be a promising method for improving the nutritional composition of moringa leaves and needs further investigation.

Solid State Fermentation of Olive Leaves as a Promising Technology to Obtain Hydroxytyrosol and Elenolic Acid Derivatives Enriched Extracts.

Extraction of valuable bioactive compounds from olive leaves is a hot topic and the use of sustainable and green technologies is mandatory in terms of circular economy. In this way, the use of fermentation technologies showed very interesting results in terms of phenolic compound recovery. Because of that in this work the use of solid state fermentations, as valuable tool to improve the phenolic extraction has been checked. Aspergillus oryzae (in mycelium and spore form), Aspergillus awamori and Aspergillus niger were used as fermentation microrganisms. Phenolic compounds were determined by HPLC-ESI-TOF-MS and, to our knowledge, new compounds have been tentatively identified in olive leaves. Fermentation using mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae were effective to increase both hydroxytyrosol and elenolic acid derivatives whereas the use of spores of Aspergillus oryzae caused a loss of hydroxytyrosoyl derivatives, contrary the content of elenolic derivatives are comparable with the other fermentation treatments and higher than control. The proposed fermentation processes using the mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae lead to an increase the hydroxytyrosyl and elenolic acid derivatives and could be used at industrial scale to obtain enriched extracts.

Fermentation with Edible Rhizopus Strains to Enhance the Bioactive Potential of Hull-Less Pumpkin Oil Cake.

Solid-state fermentation with food-grade fungal strains can be applied to enhance the bioactive parameters of agro-industrial by-products. Tempe-type fermentation can be adapted to various substrates, but the key factor is the appropriate strain selection. The aim of this study was to compare the potential of Rhizopus strains for obtaining products of improved antioxidant activity from pumpkin oil cake. For this purpose, substances reacting with the Folin-Ciocalteu reagent, with free radical scavenging potential, as well as reducing power were assessed. The effect of the fermentation on the phytate level and inositol phosphate profile in the material was also monitored. The fermentation resulted in the significant enhancement of the antioxidant potential of pumpkin oil cake in the case of all the strains tested, but the most efficient one was R. oligosporus ATCC 64063. During the course of fermentation, the level of phytate in the material decreased (the highest reduction rate was observed in the oil cake fermented with R. oryzae CBS 372.63), while peptides and fungal glucosamine were accumulated. Tempe-type fermentation can be considered as an alternative way of improving the bioactive parameters of pumpkin oil cake and, thanks to the various activities of different Rhizopus strains, it is possible to obtain products of desired parameters.

Rhizopus oligosporus and Lactobacillus plantarum Co-Fermentation as a Tool for Increasing the Antioxidant Potential of Grass Pea and Flaxseed Oil-Cake Tempe.

Tempe-type fermentation originating from Indonesia can enhance the antioxidant activity of plant material. However, this biological potential depends on substrates and applied microorganisms. This study aimed to determine whether co-fermentation with Rhizopus oligosporus and Lactobacillus plantarum improved antioxidant activity of tempe obtained from grass pea seeds with flaxseed oil-cake addition (up to 30%). For this purpose, substances reacting with Folin-Ciocalteu reagent and free radicals scavenging potential were measured in water-soluble fractions and dialysates from simulated in vitro digestion. Additionally, the water-soluble phenolic profile was estimated. The higher level of water-extractable compounds with antioxidant activity was determined in co-fermentation products than in fungal fermentation products. Moreover, the fermentation process with the use of L. plantarum contributed to a greater accumulation of some phenolic acids (gallic acid, protocatechuic acid) in tempe without having a negative effect on the levels of other phenolic compounds determined in fungal fermented tempe. During in vitro digestion simulating the human digestive tract, more antioxidant compounds were released from products obtained after co-fermentation than fungal fermentation. An addition of 20% flaxseed oil-cake and the application of bacterial-fungal co-fermentation, can be considered as an alternative tool to enhance the antioxidant parameters of grass pea tempe.

Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake.

Flaxseed oil cake was subjected to fermentation with Rhizopus oligosporus (DSM 1964 and ATCC 64063), and the phytate (InsP6) content, myo-inositol phosphate profile and in vitro bioavailability of essential minerals were studied. Flaxseed oil cake had a phytate mass fraction of 13.9 mg/g. A 96-hour fermentation of flaxseed oil cake by R. oligosporus DSM 1964 and R. oligosporus ATCC 64063 decreased the InsP6 content by 48 and 33%, respectively. The strains had different phytate-degrading activities: fermentation of flaxseed oil cake with R. oligosporus DSM 1964 was more advantageous, yielding InsP3-5 as a predominating myo-inositol compound, while fermentation with R. oligosporus ATCC 64603 produced predominantly InsP5-6. Solid-state fermentation of flaxseed oil cake enhanced in vitro bioavailability of calcium by 14, magnesium by 3.3 and phosphorus by 2-4%.

Endo-xylanase and endo-cellulase-assisted extraction of pectin from apple pomace.

Pectins were extracted from apple pomace with monoactive preparation of endo-xylanase and endo-cellulase. The process was conducted for 10 h in conditions of pH 5.0 at 40 °C, with constant shaking. Endo-xylanase application resulted in the highest extraction efficiency of pectins (19.8%). The obtained polymer was characterised by a very high molecular mass, high level of neutral sugars - mainly arabinose, galactose and glucose, and very high DM (73.4). It also contained the highest level of protein and phenols. Pectin extracted with endo-cellulase had 1.5 fold lower molecular mass but contained significantly more GalA (70.5%) of a high degree of methylation (66.3%). The simultaneous application of both enzymatic preparations resulted in their cooperation, leading to a decrease of both the extraction efficiency and the molecular mass of pectin. However, this pectin was distinguished by the highest GalA (74.7%) and rhamnose contents.

Application of Celluclast 1.5L in apple pectin extraction.

Pectins were extracted from apple pomace with Celluclast 1.5L at a dose of 25, 50 and 75 µl per 1g of material. In obtained pectin, the galacturonic acid (GalA) content, the neutral sugars (NS) profile, the degree of methylation (DM) and acetylation (DAc), the molecular mass, protein, ash and polyphenol levels as well as antioxidant and antitumor activity were determined. The lowest dose of enzymatic preparation resulted in the yield of pectin isolation comparable with acidic treatment (15.3%). Application of higher dose caused further, almost 4% increase in polymer recovery. Enzymatically isolated pectin was characterised by larger molecular mass and contained more GalA of higher DM and DAc than polymer extracted with acid. It was also richer in protein and polyphenols, and had different NS profile, which resulted in higher antiradical activity as well as the ability to inhibit the proliferation and invasion of Caco-2 adenocarcinoma cells.

Proteolysis in tempeh-type products obtained with Rhizopus and Aspergillus strains from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Tempeh is a food product obtained from legumes by means of solid-state fermentation with Rhizopus sp. Our previous research proved that mixed-culture inoculum may also be successfully applied. The objective of present research was to study the proteolytic activity of R. microsporus var. chinensis and A. oryzae during tempeh-type fermentation of grass pea seeds, and the effect of inoculum composition on the protein level and in vitro protein bioavailability in products. METHODS: Fermentation substrate were soaked and cooked grass pea seeds. Material was mixed with single- or mixed-culture inoculum, and incubated in perforated plastic bags at 30°C for 32 hrs. In the products, the proteolytic activity (pH 3, 5 and 7), glucosamine, total protein and free amino acids levels, as well as protein in vitro bioavailability and degree of protein hydrolysis were obtained. RESULTS: The significant correlation was found between glucosamine content and proteolytic activity in grass pea seeds fermented with Rhizopus or Aspergillus. The activities of Rhizopus proteases were higher than Aspergillus ones, which corresponded with the degree of seed protein hydrolysis. Both strains showed the highest activity of protease at pH 3. Tempeh made with pure culture of Rhizopus had 37% protein of 69% in-vitro bioavailability. Mixed-culture fermentation improved nutritional parameters of products only when the dose of Aspergillus spores in the inoculum was equal and lower than that of Rhizopus. This process resulted in higher in-vitro bioavailability of protein, slightly more efficient protein hydrolysis and higher level of free amino acids, as compared to standard tempeh. CONCLUSIONS: The activity of A. oryzae in tempeh-type fermentation is beneficial as long as it does not dominate the activity and/or growth of Rhizopus strain.

Development of complete hydrolysis of pectins from apple pomace.

Enzymatically extracted pectins have a more complex structure than those obtained by conventional methods. As a result, they are less susceptible to hydrolysis, which makes the precise determination of their composition difficult. The aim of the study was to develop a method of complete hydrolysis of enzymatically extracted apple pectins. Substrates were pectins isolated from apple pomace by the use of xylanase and multicatalytic preparation Celluclast and apple pomace. Hydrolysis was performed by a chemical method with 2M TFA at 100 °C and 120 °C and a combined acidic/enzymatic method. After hydrolysis, the contents of galacturonic acid and neutral sugars were measured by HPLC. Complete hydrolysis of polygalacturonic acid occurred after 2.5h incubation with 2M TFA at 120 °C. The efficient hydrolysis of neutral sugars in pectins was performed with 2M TFA at 100 °C for 2.5h. Monomers most susceptible to concentrated acid were rhamnose, mannose and arabinose.

Effect of controlled lactic acid fermentation on selected bioactive and nutritional parameters of tempeh obtained from unhulled common bean (Phaseolus vulgaris) seeds.

BACKGROUND: Tempeh is a traditional Indonesian food of high nutritional quality obtained by fungal fermentation of dehulled, soaked and cooked legumes. The aim of this research was to study the effect of Lactobacillus plantarum DSM 20174 activity on selected parameters of tempeh made from unhulled seeds of common bean (Phaseolus vulgaris). Lactobacillus plantarum cells were applied during soaking of seeds (submerged fermentation) or during solid state fermentation with Rhizopus microsporus var. chinensis (co-cultivation). RESULTS: Tempeh obtained from common beans contained 200 g kg⁻¹ protein of 34% in vitro bioavailability. Fungal fermentation caused decomposition of raffinose, stachyose and verbascose levels in seeds, on average by 93, 84 and 73% respectively. Enhanced antiradical (DPPH•, ABTS•+) capacity was accompanied by increased soluble phenol content. Application of Lactobacillus in the fermentation procedure increased tempeh protein and in vitro protein bioavailability by 18 and 17% respectively. Mixed culture tempeh contained lower levels of stachyose (25%), verbascose (64%) and condensed tannins (20%). Co-cultivation enhanced both DPPH•-scavenging activity and antioxidant capacity. CONCLUSION: The application of Lactobacillus in most cases improved the nutritional parameters of tempeh from unhulled common beans. It may also be recommended to obtain products with diverse antioxidant properties as compared with fungal fermentation alone.

Antioxidant properties of extracts from fermented and cooked seeds of Polish cultivars of Lathyrus sativus.

Antiradical and total antioxidant activities of extracts from raw, prepared for inoculation, fermented (tempeh) and cooked seeds of grass pea (Lathyrus sativus Krab and Derek cultivars) were measured. Tempeh fermentation with Rhizopus oligosporus resulted in higher scavenging activity towards DPPH and ABTS(+) radicals which correlated well with the content of total phenols. In Derek cultivar, fermentation caused a significant inhibition of linoleic acid oxidation by methanol extracts. In buffer extracts the highest TAA values were observed in raw seeds. Cooking of seeds lowered RSA values as compared to fermentation, especially for the DPPH assay. Methanol and buffer extracts from cooked seeds showed prooxidant activity towards linoleic acid.