Ammonia Assay Kit (Rapid)

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

The aim of this study was to determine the effect of milk thistle (Silybum marianum L., SM) oil supplementation on proinflammatory cytokines, acute phase proteins, rumen metagenomic profile, rumen fluid variables and performance during the milk feeding period of Holstein calves. In the present study, 24 calves that consumed quality and sufficient amount of colostrum (≥50 mg/ml IgG) after birth were divided into three groups, with 8 animals in each group (4 males + 4 females). Individually fed calves were given added SM oil as 0 µL/day/calf (Control group, SM0), 100 µL/day/calf (SM100) and 200 µL/day/calf (SM200). The ration containing concentrated feed mix (90%) + wheat straw (10%) was offered to the calves. SM oil did not change the ammonia nitrogen and pH values of the rumen fluid of calf (P>0.05). The molarities of PA, IBA, IVA and BSCFA in the rumen fluid increased linearly with SM oil dose (P<0.05). The relative abundance of Firmicutes linearly increased and the relative abundance of Actinobacteriota decreased with the addition of SM oil (P<0.05). Relative abundances of Erysipelotrichaceae_UCG_002, Eubacterium_coprostanoligenes_group, Clostridia_UCG_014, Lachnospiraceae_Unknown_1, Lachnospiraceae_NK3A20_group, Shuttleworthia, Selenomonadaceae_Uncultured_1, Rikenellaceae_RC9_gut_group and Succinivibrionaceae_UCG_001 linearly increased with SM oil (P<0.05). Relative abundances of Methanobrevibacter, Acetitomaculum, Olsenella and Megasphaera in calf rumen fluid linearly decreased with SM oil (P<0.05). Concentrations of TNF-α, IFN-ɣ, and SAA of calf’ serum at weaning stage linearly decreased with SM oil doses (P<0.05). Serum IgA concentration increased with 100 µL SM oil /day (P<0.05). As a result, the addition of SM oil to calves has the potential to reduce the immune suppression of calves during the milk feeding period and at weaning time, has a positive effect on the microbiome involved in starch and protein catabolism in the rumen fluid, and increases the fermentation end products (PA, IVA and BA). Milk thistle oil has an inhibitory effect on methanogenic archaea and can be used as an anti-methanogenic feed additive and will contribute to the effective use of feed energy.

The probiotic Bacillus subtilis 29784 (Bs29784) sustains chicken's intestinal health, enhancing animal resilience and performance through the production of the bioactive metabolites hypoxanthine (HPX), niacin (NIA), and pantothenate (PTH). Here, using enterocyte in vitro models, we determine the functional link between these metabolites and the three pillars of intestinal resilience: immune response, intestinal barrier, and microbiota. We evaluated in vitro the capacity of Bs29784 vegetative cells, spores, and metabolites to modulate global immune regulators (using HT-29-NF-κB and HT-29-AP-1 reporter cells), intestinal integrity (HT-29-MUC2 reporter cells and Caco-2 cells), and cytokine production (Caco-2 cells). Finally, we simulated intestinal fermentations using chicken's intestinal contents as inocula to determine the effect of Bs29784 metabolites on the microbiota and their fermentation profile. Bs29784 vegetative cells reduced the inflammatory response more effectively than spores, indicating that their benefit is linked to metabolic activity. To assess this hypothesis, we studied Bs29784 metabolites individually. The results showed that each metabolite had different beneficial effects. PTH and NIA reduced the activation of the pro-inflammatory pathways AP-1 and NF-κB. HPX upregulated mucin production by enhancing MUC2 expression. HPX, NIA, and PTH increased cell proliferation. PTH and HPX increased epithelial resilience to an inflammatory challenge by limiting permeability increase. In cecal fermentations, NIA increased acetate, HPX increased butyrate, whereas PTH increased acetate, butyrate, and propionate. In ileal fermentations, PTH increased butyrate. All molecules modulated microbiota, explaining the different fermentation patterns. Altogether, we show that Bs29784 influences intestinal health by acting on the three lines of resilience via its secreted metabolites.

Three commercial Saccharomyces cerevisiae strains with low, medium, and high H₂S-producing capacity were chosen to investigate the effect of yeast assimilable nitrogen (YAN) levels and composition on volatile compounds in a chemically defined medium, specifically high, medium, and low initial YAN levels with varying proportions of DAP or sulfur-containing amino acids (cysteine and methionine). The results revealed that the initial YAN containing a larger proportion of diammonium phosphate resulted in a higher YAN consumption rate during the early stages of fermentation. The yeast strain had a greater effect on the volatiles than the YAN level and composition. Keeping the total YAN constant, a higher proportion of sulfur-containing amino acids resulted in a considerably higher production of 3-methylthiopropanol. The sensory impact of three key volatile sulfur compounds was investigated in a Chardonnay wine matrix, indicating that 3-methylthiopropanol at subthreshold or greater concentrations was effective in enhancing the cantaloupe aroma.

Most reduced organic matter entering activated sludge systems is particulate (1-100-µm diameter) or colloidal (0.001-1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge.

This study evaluated the role of increasing salinity in brackish-water aquaponics, also defined as haloponics, during an 8-month cycle characterized by two fish monoculture phases in autumn and spring and a polyculture phase in winter. The effects of three water salinity levels (low: 0.5‰; medium: 3.0‰; high: 6.0‰) were assessed on the health, growth performance and carcass traits of black bullhead catfish (Ameiurus melas) and rainbow trout (Oncorhynchus mykiss), and the concurrent production of Swiss chard (Beta vulgaris, ssp. vulgaris) and cherry tomato (Solanum lycopersicum).

Energy-consuming processes should be avoided while separating biomass components. This study investigated the dewatering and extraction of components from Sorghum bicolor silage using liquefied NH₃ (NH₃(l)). Using a plug-flow-type reactor, NH₃(l) was passed through the specimen at 20°C and 0.85 MPa. When the NH₃/wet sample weight was 4.7, a 31.0 wt% dry ammonia-treated sample (AT) was obtained as an extract, achieving a 96.5 wt% dewatering ratio. Glucose, xylose, and an acid-insoluble fraction were retained in the AT, whereas lactic and acetic acids were separated as an extract. Crystalline cellulose was transformed, and the increase in glucose and xylose yields from the enzymatic hydrolysis of the AT was similar levels to those from sulfuric acid hydrolysis. Therefore, NH₃(l) treatment can dewater S. bicolor silage without severe pretreatment. In addition, it can be used in biofuel and chemical production using enzymatically degradable ATs from which organic acids are separated as extracts.

The increasing popularity of Greek yogurt generates large amounts of acid whey worldwide. The use of yogurt acid whey in animal nutrition is limited. The aim of this study was to determine the effect of a yogurt acid whey powder (YAWP) addition to maize forage prior to ensiling on the nutritional, microbial and fermentation quality of maize silage. Depending on the addition level of the YAWP to maize forage, there were the following four experimental treatments: YAWP 0, 2.5, 5 and 10% w/w. An increasing YAWP inclusion level linearly increased the maize silage dry matter, crude protein and ash concentrations, whereas it reduced the crude fiber, neutral-detergent fiber and acid-detergent fiber concentrations. The silage pH decreased quadratically with the increasing YAWP level, with the lower plateau noted for the YAWP 5% addition. Concentrations of total bacteria in the silage and Lactobacillus spp. decreased linearly with the YAWP increase. The silage acetic acid content decreased linearly, whereas propionic acid, lactic acid and the ratio of lactic to acetic acid increased linearly with the increasing YAWP level. The ammonia-N content decreased linearly with the increasing YAWP level. In conclusion, the incorporation of the 5 and 10% YAWP addition in silage preparation improved the nutritional and fermentative quality of the produced silage.

Chitosan is a fining agent used in winemaking, although its use in juice and wine beyond fining has been limited until now. Therefore, this study's first aim was to determine if chitosan derived from Agaricus bisporus (button mushrooms) could reduce caffeic and caftaric acid concentrations in Pinot noir grape juice (Study A). The second aim was to determine if chitosan, when added to base wine, could influence the synthesis of furan-derived compounds during storage (Study B). In Study A, Pinot noir grape juice was stored at 10°C for 18 hours after the following treatments: control (no addition), bentonite/activated charcoal (BAC), low molecular weight (< 3 kDa; LMW) chitosan, med. MW (250 kDa; MMW) chitosan, and high MW (422 kDa; HMW) chitosan (all 1 g/L additions). Caftaric acid was decreased, and total amino acid concentration was increased in the LMW chitosan-treated juice, while the estimated total hydroxycinnamic acid content, turbidity, and browning were decreased in the MMW chitosan-treated juice compared to the control. In Study B, Pinot noir base wine destined for sparkling wine was stored at 15 and 30°C for 90 days with the following treatments: control (no addition), LMW chitosan, MMW chitosan, and HMW chitosan (all 1 g/L additions). The three chitosan treatments stored at 30°C had increased furfural, homofuraneol, and 5-methylfurfural formation in the base wine compared to the control. At 15°C, furfural and homofuraneol had greater concentrations in all chitosan-treated wines after 90 days of storage. Our results demonstrate the potential of mushroom-derived chitosan to remove caftaric acid from grape juice and suggest that chitosan can influence the synthesis of furan-derived compounds in wine after short-term storage.

Yeast assimilable nitrogen (YAN), besides the oenological parameters (sugar content, titratable acidity, and pH) in grape musts of sixteen native and international varieties of Vitis vinifera cultivated in six regions of Northern Greece, was assessed in the frame of the present study. Low levels of YAN are frequently thought to be the cause of problematic fermentations and originate significant changes in the organoleptic aspects of the finished product. The objective of this multi-variety study was to assess factors affecting the YAN amount and composition in technologically mature grapes and, therefore, to evaluate the necessity of YAN supplementation with ammonium salts in musts across different native and international grape varieties. Free amino nitrogen was measured colorimetrically, ammoniacal nitrogen was measured enzymatically, and their values for each must sample were summed to obtain the total amount of YAN. Statistical analysis was carried out including principal component analysis (PCA) to discover relationships among must samples and the parameters studied. PCA analysis classified samples depending on grape varieties and region of origin, bringing knowledge about native and international cultivars of great commercial interest. Moreover, these findings could help to understand how commercial varieties can behave in different climates in the climate change context.

Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.

The aim of this study comprises the effect of fermented grape pomace (FGP) in experimental total mixed rations (TMR) at different rates (0, 7.5%, 15%, and 22.5%) on the in vitro cumulative gas production (6th, 12th, 18th and 24th hours), methane production, ruminal fermentation values, pH and ammonia-nitrogen and straight and branched short-chain fatty acids (SCFA and BCFA) concentration. The method of in vitro total gas production was carried out in glass syringes. Ruminal in vitro methane production linearly decreased by adding up to 22.5% FGP in experimental TMR (p < 0.05). The molarities of acetic, propionic, butyric, and valeric acids in the in vitro fermentation fluid linearly decreased with the addition of FGP to TMR (p < 0.05). FGP up to 22.5% in experimental TMRs decreased the molarity of iso-valeric acid and iso-butyric acid from BSCFAs (p < 0.05). As a result, it was concluded that the use of FGP, containing a low level of total condensed tannins (TCTs), up to 22.5% in the experimental TMR based on dry matter (DM) did not adversely affect the in vitro ruminal fermentation value and had an anti-methanogenic effect. In addition, some SCFA (acetic, propionic, butyric, and valeric acids) molarities and iso-acid BSCFA (iso-butyric and iso-valeric acid) did not change up to 15% rate of FGP in the ration. Still, these values decreased by using a 22% rate of FGP. The dose-dependent effect of FGP on ruminal iso-acids has been associated with the ability of TCTs to inhibit ruminal protein degradation partially.

Maize silage suffers from several factors that affect the final quality and, to some extent, pre-ensiled conditions that can be potentially tuned during harvesting. After assessing new indices for silage quality under lab-scale conditions, several trials have been conducted to find associations between fresh maize characteristics and silage features. Among the first, we included field input levels, FAO class, maturity stage, use of bacterial inoculants, sealing delay and chemical traits, whereas, among the latter, we assessed density and porosity, pH, fermentative profile, dry matter loss and aerobic stability. The trials were conducted using vacuum bags or mini silo buckets. More than 1500 maize samples harvested in Northeast Italy were analysed during the 2016-2022 period. Moreover, to evaluate silage aerobic stability, the fermentative profile and temperature were measured 14 days after the opening of the silo. The association between silage quality and aerobic stability was assessed, and a prognostic risk score was used to calculate the probability of aerobic instability. The dataset could provide baseline information to promote the continuous improvement of maize silage management from different botanical and crop fields, thus improving agronomic and animal farm resource allocation from a precision agriculture perspective.

Gas fermentation is an upcoming technology to convert gaseous substrates into value-added products using autotrophic microorganisms. The hydrogen-oxidizing bacteria Cupriavidus necator efficiently uses CO₂ as its sole carbon source, H₂ as electron donor and O₂ as electron acceptor. Surplus CO₂ is stored in microbial storage material poly-(R)-3-hydroxybutyrate. O₂ supply is the most critical parameter for growth and poly-(R)-3-hydroxybutyrate formation. A narrow O₂ optimum between ~0.2 and ~4 mg/L was previously reported. Here, a standard benchtop bioreactor was redesigned for autotrophic growth of C. necator on explosive mixtures of CO₂, H₂ and O₂. The bioreactor was equipped with mass flow control units and O₂ and CO₂ sensors. A controller for automated gas dosage based on a mathematical model including gas mass transfer, gas consumption and sensor response time was developed. Dissolved O₂ concentrations were adjusted with high precision to 1, 2 and 4% O₂ saturation (0.4, 0.8 and 1.5 mg/L dissolved O₂, respectively). In total, up to 15 g/L cell dry weight were produced. Residual biomass formation was 3.6 ± 0.2 g/L under all three O₂ concentrations. However, poly-(R)-3-hydroxybutyrate content was 71, 77 and 58% of the cell dry weight with 1, 2 and 4% dissolved O₂, respectively.