Theoretical exploration of the phenolic compounds' inhibition mechanism of heterocyclic aromatic amines in roasted beef patties by density functional theory.

The ability of spices (bay leaf, star anise, and red pepper) and their characteristic phenolic compounds (quercetin, kaempferol, and capsaicin) to inhibit Heterocyclic aromatic amines (HAAs) in roasted beef patties were compared. Density functional theory (DFT) was used to reveal phenolic compounds interacting with HAAs-related intermediates and free radicals to explore possible inhibitory mechanisms for HAAs. 3 % red chili and 0.03 % capsaicin reduced the total HAAs content by 57.09 % and 68.79 %, respectively. DFT demonstrated that this was due to the stronger interaction between capsaicin and the ß-carboline HAAs intermediate (Ebind = -32.95 kcal/mol). The interaction between quercetin and phenylacetaldehyde was found to be the strongest (Ebind = -17.47 kcal/mol). Additionally, DFT indicated that capsaicin reduced the carbonyl content by transferring hydrogen atoms (HAT) to eliminate HO·, HOO·, and carbon-centered alkyl radicals. This study provided a reference for the development of DFT in the control of HAAs.

An overlooked compound contributing to boar taint and consumer rejection of meat products: 2-Aminoacetophenone.

Mainly skatole and androstenone have so far been considered causative for boar taint. Using a mixed methods approach it is shown herein that 2-aminoacetophenone (AAP) affects human perception of pork, too. We explored the importance of AAP in four trials: (1) chemical analyses of 221 fat samples from boar carcasses revealed that AAP occurs, on average, in similar quantities as skatole while the levels of androstenone being four-fold. (2) ranking tests with mixtures of androstenone and/or skatole with AAP presented on smell strips to trained sensory assessors showed that AAP amplifies boar odour. In order to study AAP's importance in meat products, four experimental variants of Lyon type sausage were then produced: a control, a product with added skatole (0.075 µg/g fat tissue), with added AAP (0.075 µg/g fat tissue), and with addition of both compounds. (3) results of a consumer discrimination test panel (n = 71) showed that, when added to a sausage system, APP causes a sensory difference of similar size as skatole while the methodology chosen affects the effect size: tetrad tests proved to be more sensitive than duo trio difference tests, in the tetrad test a sensory difference expressed as d' (d-prime) of 1.0 was reached. (4) a hedonic consumer test (n = 121) finally revealed that APP decreased consumer liking of the APP-spiked sausage - even to a stronger extent than skatole. APP caused significant drops in smell, taste, mouth-feel, after-taste and overall liking in Lyoner. Overall the findings suggest that, in the context of pork meat, AAP is of similar olfactory importance as skatole.

Development and validation of a classification model for boar taint detection in pork fat samples.

This study aims to characterize a complete volatile organic compound profile of pork neck fat for boar taint prediction. The objectives are to identify specific compounds related to boar taint and to develop a classification model. In addition to the well-known androstenone, skatole and indole, 10 other features were found to be discriminant according to untargeted volatolomic analyses were conducted on 129 samples using HS-SPME-GC×GC-TOFMS. To select the odor-positive samples among the 129 analyzed, the selection was made by combining human nose evaluations with the skatole and androstenone concentrations determined using UHPLC-MS/MS. A comparison of the data of the two populations was performed and a statistical model analysis was built on 70 samples out of the total of 129 samples fully positive or fully negative through these two orthogonal methods for tainted prediction. Then, the model was applied to the 59 remaining samples. Finally, 7 samples were classified as tainted.

Consumer perception of entire male pork coated with spiced edible films as a new product to mask boar taint.

Pork production has undergone a shift towards the rearing of entire male pigs, however, its meat might carry with the presence of boar taint and it would be considered "unfit for human consumption". To offer a new alternative to the pork sector tailored to the needs of consumers, a viable option would be the use of edible spiced gelatin films to help minimize boar taint and improve its marketability. The responses of 120 regular meat consumers to entire pork with high levels of boar taint and castrated pork free of boar taint, both coated with spiced gelatin films were evaluated. They showed a similar response between entire and castrated male pork coated with spiced films, regardless of whether consumers usually detected unpleasant odours (as farm/animal) when consuming pork or not. Therefore, the new spiced films offer a new range of products to consumers as they contribute to the improvement of the sensory quality of entire male pork, especially among consumers who tend to buy new products.

Novel gels and films to mask boar taint in entire male pork.

Boar taint masking strategies were developed using hydrocolloids and spices to produce edible gels and films. Carrageenan (G1) and agar-agar (G2) were used for the gels and gelatin (F1) and alginate+maltodextrin (F2) for the films. The strategies were applied to both castrated (control) and entire male pork with high levels of androstenone and skatole. The samples were evaluated sensorially by a trained tasting panel through quantitative descriptive analysis (QDA). Lower hardness and chewiness of entire male pork, with high levels of boar taint compounds object of study, were found with the carrageenan gel, due to the better carrageenan gel adherence to the loin. The films showed that the gelatin strategy tended to have a certain "sweet" taste, and a higher overall masking than the alginate+maltodextrin film. In conclusion, a trained tasting panel found that gelatin film masked boar taint the most, followed by the alginate+maltodextrin film and the carrageenan-based gel.

Can sensory boar taint levels be explained by fatty acid composition and emitted volatile organic compounds in addition to androstenone and skatole content?

This study aimed at understanding which molecules were responsible for the differences existing in boar taint sensory evaluation. The latter was therefore linked to the results of skatole and androstenone chemical analyses, fatty acid composition and VOC profiles of heated backfat. This study confirmed that some discrepancy exists between chemical analysis and sensory evaluation of tainted backfats. Significant correlations between human nose scores and fatty acid composition were not revealed. Strong correlations between emissions and contents in skatole and androstenone were found. Oxidation products of polyunsaturated fatty acids, with fatty odor descriptors, were found to be more present in the VOC profiles of boar fat considered untainted through the human nose methodology. Weak coefficient of determination for partial least square regression indicates that other factors, yet unknown, are responsible for sensory evaluation outcomes. These findings hence support the idea that high human nose score is mainly due to boar taint compounds rather than general differences in VOC profiles.

Development of an analytical method for the determination of sterol compounds in boars' saliva.

"Boar taint" compounds influence the sexual behavioral responses of sows and stimulate their reproduction. This paper reports a fast, easier, and a non-invasive analytical method for the analysis of three "boar taint" compounds in boar' saliva samples: androstenone, androsten-3α-ol, and androsten-3ß-ol. This method was developed and validated based on solid-phase extraction (SPE) and multidimensional gas chromatography-mass spectrometry (MDGC-MS). All the compounds were detected without derivatization. This method affords good reproducibility (4%-8%), accuracy (80%-105%), precision (5.5%-9.1%), linearity (R2 = 0.98-0.99), and lower limits of quantitation (LLOQ) (0.1-0.2 µg/L). Although the presence of these compounds in saliva has been known for a long time, no simple and easy analytical method has been developed.

The variation of odor characteristics of wastewater sludge treated by advanced anaerobic digestion (AAD) and the contribution pattern of key odorants.

Identification of the odor characteristics of wastewater sludge is important in the evaluation of sludge quality and disposal options considering that sludge odor nuisance may cause major environmental issues. In this study, raw sludge and sludge cake were collected from five WWTPs applied advanced anaerobic digestion (AAD) sludge treatment process to clarify the variation of odor characteristics using sensory analysis and instrumental analysis. The electronic nose, gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS/MS) were used to profile and identify the chemical composition of key odorants. A total of 20 odorants were identified and quantified, including 6 groups of chemicals, among which volatile sulfur compounds (VSCs), indole, 3-methylindole and geosmin were identified as key odorants. The odor of the dewatered digested sludge was improved by means of changing the odor character from fecal/sulfide to earthy odor due to the reduction in VSCs concentration. The AAD and subsequent dewatering process resulted in effective removal of VSCs, which are important constituents that impact the sludge odor characteristics through synergistic effect on fecal odorants and masking effect on earthy odorants. Moreover, due to the variation of sludge quality after AAD treatment, the emission capacity of indole, 3-methylindole, and other volatiles increased, which could not be neglected for the formation of unique sludge odor.

Novel ECL Method for the Determination of Skatole in Porcine Adipose Tissue.

A new method for the determination of skatole present in porcine adipose tissue samples utilizing the electrochemiluminescence of skatole is presented. It has been observed that oxygen radicals produced at a high cathodic voltage can react with oxidized skatole to create an excited intermediate molecule that then relaxes, generating peak photon emission at around 480 nm. A strong electrochemiluminescence or electrogenerated chemiluminescence (ECL) signal using boron-doped diamond (BDD) electrodes was observed optimally when a reduction potential of -1.8 V was applied, held for 40 s, before holding an oxidation potential of 0.8 V for 10 s. Using this principle, a calibration curve using known concentrations of skatole showed good linearity (range 0.025-2 µM) and a very low detection limit (LOD, 0.7 nM). A method that demonstrates for the first time an approach that utilizes this ECL reaction, and has the potential to be developed into an analytical device for use in the slaughterhouse, has been developed. This was achieved by extracting skatole out of the porcine adipose tissues into acetonitrile - giving an extraction efficiency of 67.6%. This method was then validated by analyzing the skatole content of 33 pig fat samples that had been previously tested using a standard technique, high-performance liquid chromatography (HPLC), containing a range of concentrations (0.02-2.58 µg/g). This ECL method exhibited excellent reliability and correlation with HPLC, giving a R2 coefficient of 0.911, thus demonstrating the potential for this method to be developed for an on-line skatole detector.

Genomic background and genetic relationships between boar taint and fertility traits in German Landrace and Large White.

BACKGROUND: Due to ethical reasons, surgical castration of young male piglets in their first week of life without anesthesia will be banned in Germany from 2021. Breeding against boar taint is already implemented in sire breeds of breeding organizations but in recent years a low demand made this trait economically less important. The objective of this study was to estimate heritabilities and genetic relationships between boar taint compounds androstenone and skatole and maternal/paternal reproduction traits in 4'924 Landrace (LR) and 4'299 Large White (LW) animals from nucleus populations. Additionally, genome wide association analysis (GWAS) was performed per trait and breed to detect SNP marker with possible pleiotropic effects that are associated with boar taint and fertility. RESULTS: Estimated heritabilities (h2) were 0.48 (±0.08) for LR (0.39 ± 0.07 for LW) for androstenone and 0.52 (±0.08) for LR (0.32 ± 0.07 for LW) for skatole. Heritabilities for reproduction did not differ between breeds except age at first insemination (LR: h2 = 0.27 (±0.05), LW: h2 = 0.34 (±0.05)). Estimates of genetic correlation (rg) between boar taint and fertility were different in LR and LW breeds. In LR an unfavorable rg of 0.31 (±0.15) was observed between androstenone and number of piglets born alive, whereas this rg in LW (- 0.15 (±0.16)) had an opposite sign. A similar breed-specific difference is observed between skatole and sperm count. Within LR, the rg of 0.08 (±0.13) indicates no relationship between the traits, whereas the rg of - 0.37 (±0.14) in LW points to an unfavorable relationship. In LR GWAS identified QTL regions on SSC5 (21.1-22.3 Mb) for androstenone and on SSC6 (5.5-7.5 Mb) and SSC14 (141.1-141.6 Mb) for skatole. For LW, one marker was found on SSC17 at 48.1 Mb for androstenone and one QTL on SSC14 between 140.5 Mb and 141.6 Mb for skatole. CONCLUSION: Knowledge about such genetic correlations could help to balance conventional breeding programs with boar taint in maternal breeds. QTL regions with unfavorable pleiotropic effects on boar taint and fertility could have deleterious consequences in genomic selection programs. Constraining the weighting of these QTL in the genomic selection formulae may be a useful strategy to avoid physiological imbalances.

Effects of dietary stachyose levels on caecal skatole concentration, hepatic cytochrome P450 mRNA expressions and enzymatic activities in broilers.

Effects of dietary supplemental stachyose on caecal skatole concentration, hepatic cytochrome P450 (CYP450, CYP) mRNA expressions and enzymatic activities in broilers were evaluated. Arbor Acre commercial mixed male and female chicks were assigned randomly into six treatments. The positive control (PC) diet was based on maize-soyabean meal, and the negative control (NC) diet was based on maize-non-soyabean meal. The NC diet was then supplemented with 4, 5, 6 and 7 g/kg stachyose to create experimental diets, named S-4, S-5, S-6 and S-7, respectively. Each diet was fed to six replicates of ten birds from days 1 to 49. On day 49, the caecal skatole concentrations in the PC, S-4, S-5, S-6 and S-7 groups were lower than those in the NC group by 42·28, 23·68, 46·09, 15·31 and 45·14 % (P < 0·01), respectively. The lowest pH value was observed in the S-5 group (P < 0·05). The stachyose-fed groups of broilers had higher caecal acetate and propionate levels compared with control groups, and propionate levels in the S-6 and S-7 groups were higher than those in the S-4 and S-5 groups (P < 0·001). The highest CYP3A4 expression was found in the S-7 group (P < 0·05), but this was not different from PC, S-4, S-5 and S-6 treatments. There was no significant difference in CYP450 (1A2, 2D6 and 3A4) enzymatic activities among the groups (P > 0·05). In conclusion, caecal skatole levels can be influenced by dietary stachyose levels, and 5 g/kg of stachyose in the diet was suggested.

Valorisation of tainted boar meat in patties, frankfurter sausages and cooked ham by means of targeted dilution, cooking and smoking.

Because of the need to abolish the castration of piglets without anaesthesia/analgesia, the pig industry is searching for a mode of action for the valorisation of meat with boar taint, an off-odour in entire male pigs. Carcasses with boar taint were selected by means of sensory and chemical analysis, after which patties with different levels of tainted boar meat were produced, as well as cooked ham and Frankfurter sausages using different smoke condensates and cooking temperatures. For these products orthonasal and retronasal boar taint odour were assessed by a trained expert panel. The results offer guidance regarding dilution of tainted meat (with <400 µg/kg androstenone if skatole is low or <200 µg/kg androstenone in concurrence with ≥37 µg/kg skatole) and the potential application of smoke condensates (e.g., Rudinsmoke C for sausages and Smokez LFBN for ham) as promising boar taint masking strategies.

Sustainable use of tainted boar meat: Blending is a strategy for processed products.

While forming mixtures is a widely used approach for other raw materials in food industry, it has not yet been systematically analyzed for boar tainted meat. That is why we simultaneously studied four factors relevant for the production of emulsion-type sausages: percentage boar meat (skatole concentrations up to 0.3 µg/g, androstenone up to 3.8 µg/g in melted backfat), duration of traditional smoke and concentration levels of two spices. 16 variants of Frankfurters were produced in two independent studies and evaluated by in total 211 consumers. A linear mixed effects model revealed that increased levels of boar tainted meat significantly reduced consumer acceptance which could not be compensated by increased smoke or spice levels. We propose a non-inferiority test to identify the mixture which is similarly accepted as the reference made without boar tainted meat. Up to 33% tainted boar meat is proposed, assuming a liking drop of 0.5 on a 9 point liking scale as benchmark for an inferior product.

Isolation and characterization of two Acinetobacter species able to degrade 3-methylindole.

3-Methylindole (3MI) or Skatole is a volatile lipophilic organic compound produced by anoxic metabolism of L-tryptophan and associated with animal farming and industrial processing wastes. Pure cultures of bacteria capable of utilizing 3MI were isolated from chicken manure using enrichment culture techniques. The bacteria were identified as Acinetobacter toweneri NTA1-2A and Acinetobacter guillouiae TAT1-6A, based on 16S rDNA gene amplicon sequence data. The optimal temperature and pH for degradation of 3MI were established using single factor experiments. Strain tolerance was assessed over a range of initial concentrations of 3MI, and the effects of initial concentration on subsequent microbial 3MI degradation were also measured. During the degradation experiment, concentrations of 3MI were quantified by reverse-phase high-performance liquid chromatography (HPLC). The strains were capable of degrade initial concentrations of 3MI ranging from 65-200 mg/L. The degradation efficiency was >85% in 6 days for both strains when the initial concentration is less than 200 mg/L. The strains were tested for enzymatic activity using 65 mg/L 3MI. The enzyme extracts of NTA1-2A and TAT1-6A from the 3MI medium degraded 71.46% and 60.71% of 3MI respectively, but no appreciable change in 3MI concentration in the control group was witnessed. Our experiment revealed betaine and choline were identified as 3MI degradation metabolites by both strains while nitroso-pyrrolidine and beta-alaninebetaine formed by NTA1-2A and TAT1-6A strains respectively. The NTA1-2A and TAT1-6A strains removed 84.32% and 81.39% 3MI respectively from chicken manure during fermentation in 8 days and showed a statistically significant difference (P < 0.05) compared with the control group. The optimum temperature and pH were 31°C and 6 respectively, for 3MI degradation by A. toweneri NTA1-2A and A. guillouiae TAT1-6A. We concluded that A. toweneri NTA1-2A and A. guillouiae TAT1-6A are potential strains of interest to degrade 3MI and control odorant in poultry and other livestock industries.

Tannin-rich sainfoin pellet supplementation reduces fat volatile indoles content and delays digestive parasitism in lambs grazing alfalfa.

Lambs grazing alfalfa or white clover are prone to flavour taint which can be an impediment to consumer acceptance. Here we investigated whether condensed tannin (CT)-rich sainfoin pellet supplementation of lambs grazing alfalfa influences meat sensory quality. Using three groups of 18 male Romane weaned lambs, we compared three feeding regimes: alfalfa grazing (AF), alfalfa grazing + daily supplementation with CT-rich sainfoin pellets (15 g dry matter (DM)/kg live weight, AS) and stall feeding with concentrate and grass hay indoors (SI). We also investigated the potential interest of sainfoin pellet supplementation for controlling digestive parasitism. The sainfoin pellets contained 42 g of CT/kg of DM and they represented on average 36% of the diet in AS lambs. Skatole and indole were detected in most of the AF and AS lambs, whereas in very few SI lambs. Skatole and indole concentrations in perirenal and dorsal fat were lower in the AS lambs than the AF lambs (P < 0.025 to P < 0.001), but the intensity of 'animal' odour and 'animal' flavour of the chops did not differ between both forage-grazing groups. Longissimus thoracis et lumborum muscle lightness was lower in the AF and AS lambs than the SI lambs (P < 0.001) with the other muscle colour coordinates being unaffected by the treatment and between-treatment group differences in muscle colour coordinates remaining constant throughout the 9-day display period. Subcutaneous fat colour coordinates were not influenced by the treatment. The number of individual anthelmintic drenches necessary to keep nematode faecal egg count below a threshold of 550 eggs/g of faeces was lower in the AS than the AF lambs (0.94 per lamb v. 1.63 per lamb; P < 0.001). Faecal oocyst count was lower in the AS than the AF lambs for the first measurement made 56 days after the beginning of the experiment (P < 0.001) and was not significantly different between both forage-grazing groups thereafter. The use of CT-rich sainfoin pellets to supplement lambs that are concurrently grazing alfalfa reduced fat volatile skatole and indole concentrations and delayed the onset of both helminth and coccidian infections.

Effect of finishing diet and duration on the sensory quality and volatile profile of lamb meat.

Animal production factors can affect the sensory quality of lamb meat. The study investigated the effect of diet composition and duration of consumption on the proximate analysis, volatile profile and sensory quality of lamb meat. Ninety-nine male Texel × Scottish Blackface lambs were raised at pasture for 10 months before being assigned in groups of 11 to one of the following treatments: 100% Silage (S) for 36 (S36), 54 (S54) or 72 (S72) days; 50% Silage - 50% Concentrate (SC) for 36 (SC36), 54 (SC54) or 72 (SC72) days; 100% Concentrate (C) for 36 (C36) or 54 (C54) or 72 (C72) days. A trained sensory panel found Intensity of Lamb Aroma, Dry Aftertaste and Astringent Aftertaste to be higher in meat from lambs on the concentrate diet. Discriminant analysis showed that the volatile profile enabled discrimination of lamb based on dietary treatment but the volatile differences were insufficient to impact highly on sensory quality. Muscle from animals in the S54 group had higher Manure/Faecal Aroma and Woolly Aroma than the SC54 and C54 groups, possibly related to higher levels of indole and skatole. Further research is required to establish if these small differences would influence consumer acceptability.

Characterization of 3-Methylindole as a Source of a "Mud"-like Off-Odor in Strong-Aroma Types of Base Baijiu.

The "mud"-like off-odor, which is an odor reminiscent of musty and strong animal fecal odors, affects the quality classification of the strong-aroma types of baijiu (SAB), but little is known about which compounds are responsible for this aroma, except for certain phenolic compounds. However, not all mud-like off-odor expressions in SAB can be attributed to those phenolic compounds. In this work, volatile compounds in pit mud and SAB samples were isolated by headspace solid-phase microextraction and liquid-liquid extraction. An odoriferous zone was detected by GC-MS/olfactometer and GC-MS and was attributed to 3-methylindole rather than ethyl oleate on the basis of their odor characteristics, MS data, and retention indices. Combined with the detection threshold of 3-methylindole determined by a three-alternative forced-choice (6.09 µg/L) procedure, odor activity values in the tested base SAB were from 1 to 23. Sensory studies and the prepared heat map highlighted the contribution of this compound to the odor characteristics for the first time.

On farm intervention studies on reduction of boar taint prevalence: Feeding strategies, presence of gilts and time in lairage.

One of the challenges in the production of entire male pigs is the occurrence of boar taint. We separately tested the effect of 3 management strategies to reduce boar taint on respectively 2, 3, and 6 Flemish pig farms: 1) adapted feeding strategies, 2) presence of gilts in the compartment, and 3) varying lairage duration at the slaughterhouse. A commercialized feed concept resulted in a significant reduction of olfactory boar taint prevalence when fed for 2 weeks (T2W) compared to control (T-CON) (P = 0.030). For T2W and when fed for 3 weeks (T3W), androstenone (AND) (P = 0.002 for T2W, P = 0.029 for T3W) and skatole (SKA) (P < 0.001 for T2W and T3W) were significantly reduced compared to T-CON. Olfactory boar taint prevalence was significantly reduced when feeding 5% dried chicory roots (FI5%) (P = 0.032), but not for 3% dried chicory roots (FI3%) (P = 0.958). SKA concentration was significantly lower when feeding FI5% (P < 0.001) and when feeding FI3% (P = 0.034). Rearing entire male pigs separately from gilts and increasing lairage duration from <1 h to >3 h did not significantly affect boar taint.

The use of smoke as a strategy for masking boar taint in sausages and bacon.

Smoke has often been recommended as a masking agent for boar taint. However, guidelines on how much smoke is necessary have not been established. We compared different smoking parameters in bacon (smoking times) and sausages (smoking times and use of liquid smoke). In streaky bacon from entire male pigs with skatole concentrations of up to 0.6 µg/g and androstenone concentrations of up to 5.8 µg/g in the neck fat, three smoking times were compared: 10, 30 and 60 min. Boar taint was partially, but not fully, masked. The longer the smoking time, the better the masking effect. In sausages from entire male pigs with an average skatole concentration of up to 0.6 µg/g and androstenone concentration of up to 3.6 µg/g (the meat part) or 2.4 µg/g (the fat part) in the neck fat, smoking for 40 and 80 min fully masked the boar taint, whereas only a minor effect was seen after 10 and 20 min smoking. Liquid smoke (0.1%) did not mask boar taint when added to sausages from entire male pigs with an average skatole concentration of 0.36 µg/g (meat) or 0.38 µg/g (fat) and androstenone concentration of 2.3 µg/g (meat) and 2.9 µg/g (fat). To effectively mask boar taint, an intense smoked flavour is therefore necessary, and the longer the smoking time, the better. In contrast, the use of liquid smoke mixed into the product was not effective in the concentrations used in the current study.

Consumer acceptance of minced meat patties from boars in four European countries.

A consumer study was performed in four EU countries to further clarify the acceptability of meat with boar taint. In Denmark, France, Italy and Poland, a total of 476 female consumers evaluated 8 meat patties from boars with varying levels of skatole (0.10-0.40µg/g fat tissue) and androstenone (0.47-2.00 µg/g fat tissue), in a pair-wise comparison with patties from castrates. Boar meat patties were always less preferred than the castrate meat patties, regardless of the level of androstenone and skatole. Acceptability of the boar meat patties decreased with increasing skatole level. In samples with low skatole levels, higher levels of androstenone also reduced acceptability among androstenone sensitive consumers. No clear threshold levels for androstenone and skatole could be identified. Maps presenting the reduction in preference due to increasing levels of skatole and androstenone, and corrected for the general acceptance of the meat product were developed, taking into account androstenone sensitivity. Further work is needed, covering the whole range of androstenone and skatole levels found in entire male pigs and for a wider set of meat products.