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.

A new horseradish peroxidase (HRP)-based assay for the determination of aqueous ammonia was developed. This new assay utilizes the concentration-dependent, linearly increasing activation effect of aqueous ammonia on the enzyme HRP at alkaline pH values. The special feature of this assay is that the analyte, ammonia, is not directly involved in the reaction catalyzed by the enzyme HRP. Instead, the concentration of the analyte is determined by measuring the increase of the activity of the HRP in the absence and presence of the sample. Accordingly, all relevant assay parameters and concentrations of the components were first carefully evaluated to enable the determination of low aqueous ammonia concentrations while simultaneously achieving a broad linear range. The optimized conditions in the newly developed method for the determination of ammonia were pH 10.0, 4.8 nkat mL⁻¹ HRP, 2.3 mmol L⁻¹ o-dianisidine, and 400 µM hydrogen peroxide. The limit of detection and the limit of quantification of the new method were 0.24 mmol L⁻¹ and 0.36 mmol L⁻¹, respectively. One possible application for the newly developed ammonia determination assay was the determination of protein-glutamine glutaminase (PG) activities. This assay was employed as a two-step assay, starting with the PG reaction conducted at the optimal pH value for the PG used. After this step, the pH was increased to 10, and the ammonia released was measured in a second reaction. The results obtained with this method showed less than 10% variation compared to two established methods.

Purpose: Chronic Obstructive Pulmonary Disease (COPD) is associated with poor dietary quality that may lead to gut microbiota imbalances. A healthy gut microbiome contributes to metabolic health and immune function through production of metabolites like short-chain fatty acids. Prebiotic fibers positively influence microbiota by promoting the production of beneficial metabolites. This study aimed to assess altered gut microbiota composition in patients with COPD and to explore the effects of targeted multi-nutrient supplementation including prebiotic fibers on these outcomes. Methods: An exploratory analysis was performed within the double-blinded placebo-controlled NUTRECOVER-trial to gain preliminary insights into the effects of the nutritional intervention. The cross-sectional baseline comparison included 32 patients with COPD and 32 age-matched healthy references. Subsequently, patients were randomly assigned to a multi-nutrient supplement including prebiotic fibers, vitamin D, tryptophan, and N-3 long-chain poly unsaturated fatty acids (n = 16) or placebo (n = 16) for three months. Stool samples, blood samples and food diaries were obtained before and after the intervention. Results: Higher relative abundance of Bacteroidota (0.50 ± 0.13 vs. 0.41 ± 0.14, p = 0.010), and lower Firmicutes (0.40 ± 0.14 vs. 0.49 ± 0.12, p = 0.007) were found in patients compared with healthy controls. Patients also showed lower alpha diversity (5.80 ± 0.32 vs. 5.99 ± 0.30, p = 0.017) and higher inter-individual variability (0.51 ± 0.16 vs. 0.48 ± 0.10, p < 0.001). No effects of the nutritional intervention on gut microbiome and systemic inflammation were shown at 3 months. Conclusion: Patients with COPD exhibit differences in gut microbiota composition compared with healthy controls. Three months of multi-nutrient supplementation is insufficient to show changes in microbiome composition. The ongoing NUTRECOVER-trial will show the potential of long-term prebiotic fiber supplementation in this susceptible patient population.

The poor technological functionality of oat protein presents a challenge in food applications. This study investigated the effect of extraction method, gelling conditions (pH and NaCl concentration), and enzymatic modification on heat-induced gelation of oat protein isolate (OPI). Fairly strong gels (G′ = 5000 Pa, tan δ = 0.26) were formed from OPI, but gelation was highly sensitive to pH and NaCl concentration. Extraction at Ph ~10 negatively affected the gelation properties compared to extraction at pH 8. Partial hydrolysis with alcalase, bromelain, or papain increased the solubility of OPI but was detrimental to gelation, leading to liquid expulsion from the gel. After enzymatic deamidation with protein glutaminase, up to 87% solubility of OPI at pH 7 was achieved, and it formed soft, elastic, and slightly translucent gels (G′ = 1100 Pa, tan δ = 0.21). The extraction process and modifications of oat proteins have a great impact on their techno-functionality and need optimization for food applications.

The impact of six alpine herbs (Alchemilla vulgaris L., Sanguisorba officinalis L., Tanacetum vulgare L., Cicerbita alpina (L.) Wallr., Galium odoratum (L.) Scop., and Sisymbrium officinale (L.) Scop.), was compared with grass hay on rumen degradability and fermentation parameters. The seven plants were fermented using an automatic in vitro system to evaluate the kinetics of gas production (GP), degraded dry matter (dDM) and fermentation end products [volatile fatty acids (VFA), carbon dioxide, methane, and hydrogen]. Gas and methane productions were also computed from VFA using specific stoichiometric relationships. The partitioning factor (PF: ratio between dDM and GP) was calculated as an index of microbial growth. Compared to grass hay, the alpine herbs exhibited lower degradability (on average −12.8%) due to their high lignified fibre content. The alpine herbs also increase the PF (+9.1%), suggesting a reduction in microbial growth efficiency, and altered the VFA profile by increasing the proportion of acetic acid (+9.9%) at the expense of propionic (−11.9%) and n-butyric acids (−19.4%). Stoichiometric relationships typically associate these variations with an increase in methane proportion. However, this was not observed; in fact, Sanguisorba officinalis L. (−15.0%) and Galium odoratum L. Scop. (-13.9%) reduced methane production. The discrepancy between the measured and expected methane production indicates that part of the hydrogen, not used for methane synthesis, was redirected to alternative sinks such as reductive acetogenesis. This change in the fermentation profile appears to be modulated by bioactive compounds present in the medicinal herbs, which are potentially found in grazing pastures.

Drama represents one of the distinct winegrape regions in the continuous growth of northern Greece. Yet, little is known about grape, must, and wine quality in various parts of the region. Determining the physicochemical characteristics of the Merlot, Cabernet Sauvignon, and Agiorgitiko grape varieties grown in different locations in the area across two vintages, their chromatic characteristics, phenolic profile, and organoleptic characteristics were the goal of this study. The results reveal a high variation in the physicochemical characteristics of must and wine among the locations. The location affects the profile of phenolic compounds with procyanidin B2, catechin, procyanidin B3, procyanidin B1, epicatechin, and gallic acid being present in high amounts. Merlot wines from the location with the highest altitude showed a higher amount of quantified phenolics. Altitude positively affected the yeast assimilable nitrogen (YAN) levels and negatively the wine’s purple hue. Moreover, the grape weight negatively affects the levels of some of the quantified phenolic compounds. On the other hand, both altitude and YAN positively impact the taste of wines. The altitude represents a significant parameter in the location studied that influences the quality parameters and taste of the wines. As part of climate adaptation strategies for viticulture in the Drama region and other comparable regions, high-altitude vineyard development should be studied to preserve the grape’s acidity and counteract warming trends.

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.