Characteristics, antimicrobial capacity, and antioxidant potential of electrospun zein/polyvinyl alcohol nanofibers containing thymoquinone and electrosprayed resveratrol nanoparticles.

The aim of the present study was to fabricate, characterize, and evaluate the in vitro antimicrobial and antioxidant properties of zein/polyvinyl alcohol (ZN/PVA) nanofibers containing 2% and 4% of thymoquinone (TQ), either alone or in combination with electrosprayed ZN nanoparticles containing 1% and 2% of resveratrol (RS). According to scanning electron microscopy analysis, the diameter of nanofibers and nanoparticles increased with increasing TQ and RS concentrations, respectively. The molecular interaction between ZN or PVA polymers and TQ or RS was confirmed by Fourier transform infrared spectroscopy. Thermogravimetric analysis showed that the thermal stability of nanofibers did not change with the addition of TQ and RS. Moreover, incorporation of TQ in nanofibers along with RS nanoparticles increased their antibacterial and free radical scavenging activities based on broth dilution and DPPH methods, respectively (p ≤ .05). Escherichia coli O157:H7 (as a Gram-negative pathogenic bacteria) was more resistant to all treatments than Staphylococcus aureus (as a Gram-positive pathogenic bacteria). In addition, the combined use of TQ in nanofibers and RS nanoparticles had antagonistic antibacterial and synergistic antioxidant effects. The best results were obtained with ZN/PVA nanofiber containing 4% TQ and electrosprayed with 2% RS nanoparticles (p ≤ .05). According to the results of the present study, biodegradable ZN/PVA nanofiber containing TQ and electrosprayed with RS nanoparticles can be used as a novel active packaging material in the food industry.

Characterisation of iron oxide-containing pearlescent pigments used as food colourants: nano-labelling required in the EU?

Pearlescent pigments are used as colourants to increase the attractiveness of food products, especially in the patisserie and confectionery sector. They can be seen as composite materials and consist of thin potassium aluminium silicate (E 555, mica) platelets as carrier material, coated with a thin metal oxide layer of TiO2 (E 171) and/or iron oxides (E 172). The European Food Safety Authority stated in 2020 that mica-based pearlescent pigments as a whole should be evaluated as new food additives. Obtaining dependable data for particle size and layer thickness of these pigments is crucial both for the demanded food additive evaluation itself and also for the nanomaterial labelling assessment of products containing these food colourants according to the 'Food Information to Consumers' regulation. Since it was found in a previous study on TiO2-containing pearlescent pigments (silver and golden coloured) that the coating consisted of nanoscaled constituent titanium oxide particles, in this follow-up study we investigated whether Fe2O3-containing pearlescent pigments exhibit a similar nanostructured morphology. For this purpose, five commercially-available food products containing these pigments were investigated. Static light scattering and flow particle image analysis were used as screening methods to determine the mica platelet size. Scanning electron microscopy combined with energy-dispersive X-ray spectroscopy was used for nanostructure analysis of the metal oxide coating. The carrier mica platelets were 34-96 µm in diameter and 300-800 nm thick. The coating thickness was found to be in the range of 75-105 nm, with the constituent round shaped iron oxide particles contained therein having a minimum Feret diameter of 37-64 nm.

Impact of the fortification of a rice beverage with different calcium and iron sources on calcium and iron bioaccessibility.

Rice beverages are commonly fortified with minerals to improve their nutritional value. However, the effect of fortification on mineral bioaccessibility is poorly understood. Thus, the effects of fortification of a rice beverage on mineral concentration and bioaccessibility using calcium carbonate (CaCO3), tricalcium phosphate (Ca3(PO4)2), sodium iron EDTA (NaFeEDTA) and ferric pyrophosphate (Fe4(P2O7)3) individually and in combination were studied. Recovery of the added minerals in the rice beverage ranged from 71.4 % to 92.0 % and 61.0 % to 93.3 % for Ca and Fe, respectively. Mineral bioacessibility was shown to be higher for CaCO3(≤39.0 %) compared to Ca3(PO4)2 (≤14.4 %) and for NaFeEDTA (≤50.7 %) compared to Fe4(P2O7)3 (≤3.9 %). No interaction of the different Ca sources was identified; the addition of iron sources did not have a significant effect on Ca bioaccessibility. The addition of NaFeEDTA to the rice beverage was found to be better than the addition of iron pyrophosphate and the simultaneous addition of this iron sources did not result in an additive effect on Fe bioaccessibility. These results may be used to develop plant-based beverages with an improved mineral bioaccessibility.

Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities.

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected.

Influence of processing and storage on the iodine content of meat and fish products using iodized salt.

Table salt fortified with KIO3 is commonly used to prevent iodine deficiency disorders. However, there is a lack of reliable data about the stability of KIO3 during food processing. In this study several meat and fish products were prepared with iodized salt and the iodine stability was determined through the whole production process. Applied processes included heating, fermenting, freezing, hot smoking, ripening by enzymes and storing. In all products an increase in iodine content was observed after addition of iodized salt. The iodine content remained constant during most of the applied processes. The only iodine loss was observed in ham after heating and can be explained by loss of iodine containing brine. During subsequent storage no iodine loss was observed in any of the products. The use of KIO3 fortified salt in the investigated products might therefore be beneficial for the iodine supply.

Characterisation of TiO2-containing pearlescent pigments with regard to the European Union labelling obligation of engineered nanomaterials in food.

A wide range of trendy food colourants and ready-to-eat foods containing pearlescent pigments providing glitter effects is currently on the market. These pearlescent pigments consist of mica (potassium aluminium silicate) platelets generally coated with titanium dioxide and/or iron oxides. All single components are approved food additives in the European Union (EU) (E 555, E 171 and E 172). However, the European Food Safety Authority (EFSA) has stated recently, that pearlescent pigments should be evaluated as new food additives. Food grade titanium dioxide was already shown to contain a considerable fraction of nanoparticles. Thus, the question about 'nano'-labelling of TiO2-containing pearlescent pigments according to the 'Novel Food' and 'Food Information to Consumers' regulations arose. In order to provide data for dealing with these issues, in this study four commercially available products of different food categories containing pearlescent pigments were characterised with focus on the structure, size and chemical composition of these pigments. The measurement methods used were flow particle image analysis (FPIA), static light scattering (SLS) and scanning electron microscopy (SEM) combined with energy-dispersive x-ray spectroscopy (EDX). After isolation from various food matrices, the glitter pigments could be easily identified and differentiated by fast FPIA screening from any remaining organic food matrix particles due to their typical platelet-like shape and transparency. The particle size distribution of the platelets was determined by means of SLS and found to be in the range of 8-167 µm. SEM was identified as the most suitable technique for the analysis of the nano-structured coating. For all constituent metal oxide particles (TiO2 and/or Fe2O3) a median minimum Feret diameter (Fmin) of 29.9-46.8 nm was obtained by quantitative SEM image analysis.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies.

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2-3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.

Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases.

BACKGROUND: The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes-Blackburn et al. [J Agric Food Chem 63: 6142-6149 (2015)] on the evaluation of the bioefficacy of phytases. RESULTS: It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+ ) up to a concentration of 10 g kg-1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. CONCLUSION: The in vitro approach was shown to be applicable as a complementary tool in the pre-selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of phytase-supplemented fermentation and household processing on the nutritional quality of Lathyrus sativus L. seeds.

Grass pea (Lathyrus sativus L.) is commonly consumed in cooked, fermented, and roasted forms in Ethiopia. However, the impacts of household processing practices on its nutrients, antinutrients, and toxic compounds have not been adequately studied. Therefore, the effects of household processing and fermentation in the presence and absence of a phytase on the contents of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), myo-inositol phosphates, crude protein, minerals and the in vitro bioaccessibility were investigated. Fermentation exhibited a significant decline in ß-ODAP (13.0-62.0%) and phytate (7.3-90.5%) irrespective of the presence of phytase. Pressure and pan cooking after discarding the soaking water resulted in a 27.0 and 16.2% reduction in ß-ODAP. A 30% reduction in phytate was observed during germination followed by roasting. In addition, germination resulted in a significant (p < 0.05) increase in crude protein. Germination and germination followed by roasting resulted in the highest Fe bioaccessibilities (more than 25 fold higher compared to untreated samples) followed by pressure cooking and soaking. Processing also improved Zn bioaccessibilities by 50.0% (soaked seed without soaking water), 22.5% (soaked seed with soaking water), and 4.3% (germination). Thus, the processing technologies applied were capable of reducing the content of phytate (InsP6) and ß-ODAP with a concomitant increase in mineral bioaccessibilities. Processing of grass peas could therefore contribute to their more widespread utilization.

Influence of salt concentration and iodized table salt on the microbiota of fermented cucumbers.

The fermentation of vegetables is a traditional preservation method, that experiences a renaissance even in domestic households. Table salt is added to the fermentation batches to favor the growth of lactic acid bacteria usually. On an industrial scale, the fermentation brine is typically prepared with non-iodized table salt. In our study, we investigated the microbiota of cucumber fermentations using culture-dependent and -independent methods. We could show that the fermentation process of cucumbers and the involved microbiota is influenced by the concentration of table salt and not by the use of iodized table salt. Therefore, we conclude that the use of iodized table salt does not negatively affect the fermentation process. We could verify that iodine permeates the cucumbers by diffusion, leading to satisfactory iodine concentrations in the final food product. The industrial use of iodized table salt in food fermentations could contribute to maintain a constant iodine supply to the general public.

Effect of phytase treatment of sorghum flour, an alternative for gluten free foods and bioaccessibility of essential minerals.

This study evaluated the effect of phytase treatment on the bioavailability of iron (Fe), calcium (Ca), zinc (Zn), and myo-inositol phosphate fractions in sorghum flour; and characterized its macronutrients and minerals. The proximate composition and mineral content indicated that, sorghum flour has a nutritional potential superior to wheat and maize. The results obtained in the solubility and dialysis assays indicated that, naturally occurring minerals (without phytase treatment) in sorghum flour, presented considerable bioaccessibility; reaching 32, 47 and 67% of dialyzable Fe, Zn, and Ca respectively. The use of phytase had a positive influence on the reduction of myo-inositol phosphates, mainly the IP6 fraction, present in sorghum flour samples, and an increase in the soluble percentage (Fe 52% for one sample, for Zn higher than 266%) and dialyzed minerals (Fe 7.8-150%; Zn 19.7 for one sample; and Ca 5-205%) for most samples. Therefore, the essential minerals naturally occurring in sorghum have an absorption potential; and the use of phytase reduced the IP6 fraction and improved the availability of the minerals evaluated.

In vitro digestion effect on mineral bioaccessibility and antioxidant bioactive compounds of plant-based beverages.

Consumption of plant-based beverages (PBB) is a growing trend; and have been used as viable substitutes for dairy based products. To date, no study has comparatively analyzed mineral composition and effect of in vitro digestion on the bioaccessibility of different PBB. The aim of this research was to investigate the content of essential minerals (calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn)) and to estimate the effect of in vitro digestion in plant-based beverages, and their antioxidant bioactive compounds (phenolic compounds and antioxidant capacity). Moreover, the presence of antinutritional factors, such as myo-inositol phosphates fractions, were evaluated. Samples of PBB (rice, cashew nut, almond, peanut, coconut, oat, soy, blended or not with another ingredients, fortified with minerals or naturally present) and milk for comparison were evaluated. TPC ranged from 0.2 mg GAEq/L for coconut to 12.4 mg GAEq/L for rice and, the antioxidant capacity (DPPH) ranged from 3.1 to 306.5 µmol TE/L for samples containing peanut and oat, respectively. Only a few samples presented myo-inositol phosphates fractions in their composition, mostly IP5 and IP6, especially cashew nut beverages. Mineral content showed a wide range for Ca, ranging from 10 to 1697.33 mg/L for rice and coconut, respectively. The Mg content ranged from 6.29 to 251.23-268.43 mg/L for rice and cashew nut beverages, respectively. Fe content ranged from 0.76 mg/L to 12.89 mg/L for the samples of rice. Zinc content ranged from 0.57 mg/L to 8.13 mg/L for samples of oat and soy, respectively. Significant variation was observed for Ca (8.2-306.6 mg/L) and Mg (1.9-107.4 mg/L) dialyzed between the beverages, with lower concentrations of Fe (1.0 mg/L) and Zn (0.5 mg/L) in dialyzed fractions. This study provides at least 975 analytically determined laboratory results, providing important information for characterization and comparison of different plant-based beverages.

Utilizing Impedance for Quality Assessment of European Squid (Loligo Vulgaris) during Chilled Storage.

This study evaluates the quality of chilled squid Loligo vulgaris by non-destructive measurements of bioelectrical impedance from the first post-mortem day under controlled conditions. Squid samples were stored at 4.5 °C and 55% of relative humidity for 11 days. Impedance magnitude (|Z|) and phase (φ) at 200 frequencies (100Hz to 100MHz) were measured using an Agilent 4294A Precision Impedance Analyzer with needle-type multi-electrode array on days 1, 2, 3, 4, 5, 8, 9, 10, and 11 of storage. The changes in color, sensory properties, total volatile nitrogen, pH, and water holding capacity were also determined. The obtained results indicated that the samples could be classified into five to six distinctive groups by measuring the electrical parameters at frequencies close to 5MHz. In general, φ is less dependent on temperature and measurement setup than |Z|, while records at 5MHz correlate well with the days of storage (R2 = 0.968). The data imply that it is only possible to estimate the length of storage for the samples with measurements of phase angle, which can be useful for the development of new analytical instruments. Biosensors have a practical industrial application, as it is demonstrated that bioelectrical impedance data correlates well with the days of chilled storage.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria as a potential microbial fertilizer.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria (TTPSB) and their use as microbial fertilizers was the main goal of the study. In this study, TTPSB were isolated from soil samples treated for 16 h at 55 °C. Their phosphate solubilizing activity was either evaluated in solid media by forming a clear zone (halo) or in liquid media by quantification of the soluble phosphate in the growth medium. Five colonies (RPS4, RPS6, RPS7, RPS8 and RPS9) were identified to be able to form a halo and two of the isolates (RPS9 and RPS7) tolerated a temperature of 55 °C. With tricalcium phosphate (TCP) as the sole P-source, the phosphate solubilizing capacity of RPS9 and RPS7 was determined to be 563.8 and 324.1 mg P L-1 in liquid Sperber medium, respectively. Both bacterial isolates were identified as Pantoea agglomerans by molecular and biochemical characterization. To be used as a microbial fertilizer a carrier system for the temperature tolerant bacteria consisting of rock phosphate, sulfur and bagasse was used. It could be established that the bacterial cell counts of the microbial fertilizers were acceptable for application after storage for 4 months at 28 °C. In a greenhouse experiment using pot cultures, inoculation of maize (S.C.704) with the microbial fertilizers in an autoclaved soil resulted in a significant effect on total fresh and dry weight of the plant root and shoot as well as on the P content of the root and shoot. The effects observed with RPS9 as a component of the microbial fertilizer on plant growth and P nutrition was comparable with the addition of 50% of recommended triple superphosphate (TSP) dose. Using temperature tolerant bacteria in microbial fertilizers will overcome limitations in production and storage of the microbial fertilizers and contribute to a environmentally-friendly agriculture. The temperature tolerant P. agglomerans strain RPS9 was shown to be effective as part of a microbial fertilizer in supporting the growth and P uptake in maize.

Influence of cooking on the iodine content in potatoes, pasta and rice using iodized salt.

To counteract iodine deficiency in the population in areas of environmental iodine deficiency table salt is often fortified with potassium iodide or iodate. However, most estimations of iodine contribution from the diet rely on calculations based on the added iodized salt and very limited experimental data about the stability of potassium iodate (KIO3) or iodine uptake during food processing is available. Therefore, the influence of cooking on the iodine content of potatoes, pasta, and rice having different size, varieties or composition was investigated. Commonly used cooking procedures were applied, using KIO3-enriched table salt in the cooking water. After iodine extraction with 0.5% NH3 iodine content was measured by ICP-MS. All products showed an increase in iodine content. Waxy potatoes, especially cut in small pieces, and egg pasta showed the highest iodine uptake. Based on the results, the use of KIO3-enriched salt for cooking is recommended to enhance iodine supply.

Effects of Moringa oleifera leaf extract on ruminal methane and carbon dioxide production and fermentation kinetics in a steer model.

Ruminal fermentation produces greenhouse gases involved in global warming. Therefore, the effect of nutrient combinations on methane, carbon dioxide, and biogas production as well as ruminal fermentation kinetics was evaluated in in vitro studies. In total mixed rations, dietary corn grain was partially replaced by two levels of soybean hulls (a highly reusable residue), and a Moringa oleifera extract (a natural extract) at three concentration levels was added. Higher levels of both soybean hulls and M. oleifera extract delayed the initiation of methane production and resulted in a lower methane and carbon dioxide production. Thus, total biogas production was also lower. Replacement of corn grain by soybean hulls tended to lower methane production rates and asymptotic carbon dioxide production, and a delay in biogas and methane formation was observed. Asymptotic biogas and carbon dioxide production, however, were increased. The presence of M. oleifera extract tended to delay methane formation and to decrease methane production rate as well as asymptotic methane production. Higher M. oleifera extract levels decreased asymptotic biogas production with the control and the highest soybean hull levels. In the presence of M. oleifera extract, asymptotic carbon dioxide production was shown to be quadratically increased with the control and lowest soybean hull levels, but quadratically decreased with the highest soybean hull level. With the exception of fermentation pH, the interaction of substrate type and M. oleifera extract level was shown to have an effect on all fermentation parameters. Most fermentation parameters were shown to be higher when replacing corn grain by soybean hulls, including fermentation pH. Thus, the conclusion could be drawn that corn grain replacement by soybean hulls (an agricultural residue) in the presence of M. oleifera extract (a sparing leaf product) could ameliorate greenhouse gas emissions and improve digestion.

Particle size of milled chokeberry pomace did not influence in vitro cellular absorption and transport efficiencies of anthocyanins, phenolic acids and flavonols.

Industrial chokeberry pomace is very rich in polyphenols. The main focus here lies on the possible relationship between the particle size of chokeberry milled pomace and an enhanced absorption and transport of polyphenols by Caco-2 cells. Wet milling was used to produce materials with particle size distributions in the micrometre and in the sub-micrometre to nanometre ranges starting from chokeberry pomace. Milled materials with about 50% of the particles with a mean size (x50,3) of 223 ± 13 µm (coarse milling) and about 90% of the particles with x50,3 of 160 ± 40 nm (fine milling, sonication) were obtained. None of the milled materials exhibited cytotoxic effects within the tested concentration-ranges. The polyphenol absorption and the transport efficiencies from the fine and the coarse milled materials were similar. Thus, no effect of the particle size upon cellular uptake and transport could be established, but agglomeration of particle during incubation cannot be excluded as the cause. Furthermore, based on polyphenol stability we postulate that direct milling may be applied to valorise the processing by-product from commercial fruit juice production.

Reduction of ß-ODAP and IP6 contents in Lathyrus sativus L. seed by high hydrostatic pressure.

Grass pea (Lathyrus sativus L.) seeds contain an endogenous neurotoxic non-proteinogenic amino acid, ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), a major limiting factor-for their human consumption. Furthermore, phytate (IP6), a well-known antinutrient is present in concentration capable of hindering bioavailability of iron (Fe), zinc (Zn), calcium (Ca), phosphorus (P) and other micronutrients from the seeds. Due to the reported capability of high hydrostatic pressure (HHP) to reduce the content of certain antinutritional/toxic agents in seeds and grains, the impact of HHP on the reduction of ß-ODAP and IP6 were investigated. The contents of ß-ODAP of accessions from different regions in Ethiopia were found to be in the range of 51.94 to 806.52 mg/100 g. Accession (GF1- Alemu, AK) exhibiting the highest ß-ODAP content was selected for HHP treatment in soaked and batter forms using Central Composite Face Centered Design of experiments. The best HHP conditions in respect to ß-ODAP reduction were also applied to the accession (GP-240038) with the lowest ß-ODAP-content, a genetically improved variety (Wassie) and a variety from Germany (GR). The HHP treatment at 600 MPa for 25 min of seeds soaked for 6 h and 12 h exhibited the maximum reduction of ß-ODAP (232.11 mg/100 g) and IP6 (21.11 mg/100 g) respectively. The combined incremental effect of pressure and soaking time resulted in a more significant (p ≤ .001) reduction in both compounds than the interaction of pressure with holding time (p ≤ .05). A reduction of ß-ODAP from 36.00 to 71.22% by soaked-HHP treatment was observed. ß-ODAP reductions were always higher for soaked compared to batter grass pea seeds. IP6 contents after HHP treatment ranged from 33.65 mg/100 g to nill. It can be concluded that pressure, soaking and holding time as well as the grass pea seed accession/variety had great impact on molecular structure changes, enhancement of enzyme activity and reduction in ß-ODAP and IP6 content.

Influence of phytase enzyme on ruminal biogas production and fermentative digestion towards reducing environmental contamination.

Environmental impact of livestock production has received a considerable public scrutiny because of the adverse effects of nutrient run-offs, primarily N and P, from agricultural land harboring intensive energy livestock operations. Hence, this study was designed to determine the efficacy of dietary phytase supplementation on fermentation of a sorghum grain-based total mixed ration (TMR) using a ruminal in vitro digestion approach. Phytase was supplemented at three doses: 0 (control), 540 (P540), and 720 (P720) g/t dry matter, equivalent to 0, 2.7 × 106, and 3.6 × 106 CFU/t DM, respectively. Compared to P720 and the control, gas production was higher for P540 after 12 h (P = 0.02) and 24 h (P = 0.03) of fermentation suggesting a higher microbial activity in response to phytase supplementation at lower phytase levels. Correspondingly, dry matter degradability was found to have improved in P540 and P720 compared to the control by 13 and 11% after 24 h of incubation (P = 0.05). For ammonia nitrogen (NH3-N), a tendency towards lower values was only observed for P540 at 24 h of fermentation (P = 0.07), while minimal treatment effects were observed at other fermentation times. The concentrations of total volatile fatty acids (VFA) were higher (P < 0.05) after 48 h of fermentation for P540 and P720 compared to the control (P = 0.03) by 10% and 14%, respectively. Ruminal acetate tended towards higher values in the presence of phytase after 12 h of fermentation (P = 0.10), but towards lower values after 24 h of fermentation (P = 0.02), irrespective of the phytase dose applied. A trend towards lower ruminal propionate levels was observed in the presence of phytase after 6 h (P = 0.10) and 12 h (P = 0.06) of fermentation, while no effects were found at other fermentation times. In conclusion, phytase supplementation has the potential to improve metabolic energy activity of rumen microorganisms and the use of feed constituents. Thus, phytase supplementation could help to reduce environmental contamination in areas of ruminant production.