Dietary combination of linseed and hazelnut skin as a sustainable strategy to enrich lamb with health promoting fatty acids.

This study investigated the effect of the inclusion of extruded linseed and hazelnut skin on fatty acid (FA) metabolism in finishing lambs. Forty lambs were divided into 4 groups and fed for 60 d with: a conventional cereal-based diet, or the same diet with 8% of extruded linseed, or 15% of hazelnut skin, or 4% of linseed plus 7.5% of hazelnut skin as partial replacement of maize. Dietary treatments did not affect growth performances, carcass traits, and ruminal fermentation. The combined effect of linseed and hazelnut skin enriched the intramuscular fat with health promoting FA. Particularly, increases in α-linolenic acid (3.75-fold), and very long-chain n-3 poly-unsaturated FA (+ 40%) were attributed to the supplementation with linseed, rich in α-linolenic acid. In addition, increases in rumenic (+ 33%), and vaccenic (+ 59%) acids were attributed to hazelnut skin tannins modulating ruminal biohydrogenation and accumulating intermediate metabolites. The simultaneous inclusion of linseed and hazelnut skin can be a profitable strategy for enriching the intramuscular fat of lambs with health promoting FA, without adverse effects on ruminal fermentation and animal performance.

Role of hazelnut skin supplementation on plasma antioxidant status and cytokine profile in growing lambs.

In this study, the effect of hazelnut skin dietary supplementation on antioxidant status and cytokine profile was evaluated in growing lambs. A total of 22 male lambs at the age of 2 months, balanced for their initial live weight (15.33 ± SD 1.79 kg), were selected and allocated into two experimental groups: the control group (CON) receiving a maize-barley-based concentrated diet, and the hazelnut group (HS) receiving supplementation with hazelnut skin (150 g/kg on the dry matter) as a maize substitute for the concentrate diet. The experiment lasted for 56 days. Peripheral blood was collected at 7, 35, and 56 days of the experiment. The free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, the total antioxidant capacity assay (TAC), the reactive oxygen species (ROS), and reactive nitrogen species (RNS) were determined in plasma. The secretion of IL-1ß, IL-6, and IL-10 cytokines was also determined by ELISA. The DPPH was affected by the interaction between feeding strategy and time of sampling (p = 0.039) with a higher level of DPPH at 7 days in the HS group than the CON group. The time of sampling affected the levels of plasma TAC (p = 0.016), while the ROS/RNS levels showed a higher value in the HS group (p < 0.001), on average. The antioxidant/oxidant index, which combines the TAC and the ROS/RNS levels, was not affected by the inclusion of hazelnut skin in the diet (p = 0.394). The cytokine profile showed a lower IL-6 secretion at both 35 and 56 days than at 7 days, on average. Furthermore, the feeding treatment affected the IL-1ß level, showing a lower level in the HS group than in the CON group on average. Lambs from the HS group had higher IL-10 plasma levels than the CON group at 7 days of the experiment. The present data highlight an antioxidant effect and a modulatory role in the cytokine profile of HS supplementation in growing lambs.

Effects of feeding bergamot pulp and olive leaves on performance and meat quality in Apulo-Calabrese pigs.

To determine the effects of dietary inclusion of bergamot pulp and olive leaves on pork quality, thirty-six barrows (Apulo-Calabrese; 112.5 ± 7.40 kg initial bodyweight) were randomly assigned to four experimental treatments and fed for 100 days a control diet (control group) or a diet similar to the control diet in which part of the cereals were replaced with 20 % (DM on the diet fed) of ensiled bergamot pulp (dry matter on the diet fed, EBP group) or olive leaves (OLL group) or a 1:1 mixture of both by-products (BPOL group). In the present study, the dietary administration of by-products did not influence the accumulation of IMF (P > 0.05) in meat. As for individual fatty acids, no differences were observed among groups. However, the dietary supplementation of bergamot pulp tended to increase (P = 0.073) the level of α-linolenic acid in meat. In raw and cooked meat TBARS values were higher (P < 0.001) in the Control group than in EBP, OLL and BPOL groups. The integration of 20 % of bergamot pulp and/or olive leaves by-products increased the levels of tocopherol and phenolic compounds in the diets, positively influencing the shelf life of meat. No effect of diet was observed on animal performance. The results of present study indicated mainly that partial replacement of cereals with 20 % DM on the diet fed of ensiled bergamot pulp and olive leaves, either alone or in combination, in the pig's diet led to greater oxidative stability of the meat.

In-depth characterization of the sarcoplasmic muscle proteome changes in lambs fed with hazelnut skin by-products: Relationships with meat color.

This study investigated the effect of agro-industrial hazelnut skin by-products supplementation on lamb meat color variation and the changes in the sarcoplasmic muscle proteome during post-mortem storage (0, 4 and 7 days). Gel-based proteomics and bioinformatics approaches were applied to better understand the potential role of feeding strategies in modulating the mechanisms underpinning meat discoloration and post-mortem changes during storage. Therefore, twenty-two Valle del Belice male lambs were randomly assigned to two dietary treatments: control (C), lambs fed with maize-barley diet, and hazelnut skin (H), lambs fed hazelnut skin by-product as maize partial replacer in the concentrate diet. Hazelnut dietary treatment led to better lamb meat color stability as evidenced by the lowest decrease in redness and saturation index values. Proteomics and bioinformatics results revealed changes in the abundance of 41 proteoforms, which were mainly involved in glycolytic processes, responses to oxidative stress, and immune and endocrine system. The proteins allowed revealing interconnected pathways to be behind meat color variation as a consequence of using hazelnut skin by-products to sustainable feed lamb. The proteins can be used as potential predictors of lamb meat color variation. Accordingly, the regression equations developed in this paper revealed triosephosphate isomerase (TPI1) as a reliable candidate biomarker of color stability in lamb meat. SIGNIFICANCE: The use of agro-industrial by-products in animal feeding can be a potential sustainable strategy to reduce the environmental impacts of the food production chain and consequently improve animal welfare and product quality. The inclusion of hazelnut skin by-products in the animal's diet, due to the high concentration of polyphenols, represents an effective strategy to improve the oxidative stability of meat, with significant implications on color. The use of proteomics combined with bioinformatics on the sarcoplasmic proteome is a powerful approach to decipher the underlying mechanism. Accordingly, this approach allowed in this trial a deeper understanding of the molecular mechanisms involved in the post-mortem processes through the discovery of several biological pathways linked with lamb meat color variation. Glycolysis, followed by responses to oxidative stress, and other proteins involved in the immune and endocrine system were found as the major interconnected pathways that could act as potential predictors of lamb meat color stability. Candidate proteins biomarkers were further revealed in this study to be related with multiple meat color traits.

Effect of high levels of almond hulls supplementation on performance and meat oxidative stability in lambs.

The main objective of this study was to evaluate if high dietary levels of almond hulls could influence performance and meat oxidative stability in lambs. Twenty lambs, at an average body weight of 28.8 ± 0.30 (SD) kg, were divided into two experimental groups and fed ad libitum for 40 days either with a control diet (cereal-based concentrate, control group) or with a similar diet in which part of the cereals was replaced with 40% of almond hulls on a dry matter basis (AH40 group). Dietary AH did not affect dry matter intake but reduced final body weight, average daily gain, feed conversion ratio or carcass weight. In meat, TBARS values were lower (P < 0.001) in the AH40 group than in control group. The partial replacement of cereals with 40% almond hulls in the diet negatively affecting the growth parameters of the animals but can improve meat shelf-life by reducing lipid oxidation.

Feeding hazelnut skin to lambs delays lipid oxidation in meat.

This study aimed to investigate the effect of dietary hazelnut skin (HNS), a by-product of confectionery industry, on the oxidative stability of lamb meat. Twenty-two finishing lambs were randomly assigned to 2 groups and fed ad libitum for 56 days on different concentrate-based diets: one control and one experimental, in which 150 g/kg of corn was replaced by HNS. After slaughter, the fat-soluble vitamins content and hydrophilic antioxidant capacity were assessed in fresh meat, as well as color, lipid and protein stability over 7 days of shelf-life trial. Dietary HNS increased (P < 0.001) the tocopherols content of meat and reduced (P < 0.001) lipid oxidation during 7 days of refrigerated storage. Meat from lambs fed HNS showed different (P < 0.05) instrumental color parameters. No diet effect (P > 0.05) was observed on the development of metmyoglobin, hydroperoxides, thiol groups, and carbonyl groups. Feeding HNS to lambs improves the oxidative stability of raw meat by delaying lipid oxidation thanks to the antioxidant molecules (tocopherols and phenolic compounds) contained in this by-product.

Effect of dietary pomegranate by-product on lamb flavour.

This study evaluated the effect of the dietary inclusion of whole pomegranate by-product (WPB) on lamb flavour. Seventeen Comisana male lambs (body weight 14.82 kg ± 2 kg) were assigned to 2 treatments. During 36-day feeding trial, the control group (n = 8) received a conventional concentrate diet; the other group (n = 9) received a concentrate diet containing 200 g/kg (dry matter) of WPB, replacing part of barley and corn. After slaughter, fatty acids were determined in raw meat, while volatile organic compounds, SMart nose, and sensory traits were analysed in cooked meat. The concentrations of total polyunsaturated fatty acids, vaccenic and rumenic acid were higher in WPB meat. Most of volatile compounds that arise from lipid degradation (i.e., aldehydes, alcohols, ketones, and hydrocarbons) were found at higher concentrations in the WPB meat than in the control, except for 2-pentanone that was greater in control meat. Although SMart nose clearly discriminated between dietary treatments, consumer panel did not detect differences in meat flavour.

Multielemental, Nutritional, and Proteomic Characterization of Different Lupinus spp. Genotypes: A Source of Nutrients for Dietary Use.

Among grain pulses, lupins have recently gained considerable interest for a number of attractive nutritional attributes relating to their high protein and dietary fiber and negligible starch contents. The seeds of Lupinus albus (cv. Multitalia and Luxor, and the Modica ecotype); L. luteus (cv. Dukat, Mister, and Taper); and L. angustifolius (cv. Sonet) analyzed in this study were deposited within the germplasm collection of the Research Centre for Cereal and Industrial Crops of Acireale and were sowed in East Sicily in 2013/14. The collected seeds were analyzed for their multielemental micro- and macronutrient profiles, resulting in a wide variability between genotypes. Lupin seed flour samples were subjected to a defatting process using supercritical CO2, with oil yields dependent on the species and genotype. We determined the fatty acid profile and tocopherol content of the lupin oil samples, finding that the total saturated fatty acid quantities of different samples were very close, and the total tocopherol content was about 1500.00 µg/g FW. The proteomic analysis of the defatted lupin seed flours showed substantial equivalence between the cultivars of the same species of Lupinus albus and L. luteus. Moreover, the L. angustifolius proteome map showed the presence of additional spots in comparison to L. albus, corresponding to α-conglutins. Lupin, in addition to being a good source of mineral elements, also contributes vitamin E and, thanks to the very high content of gamma-tocopherols, demonstrates powerful antioxidant activity.

Effect of dietary magnolia bark extract supplementation in finishing pigs on the oxidative stability of meat.

BACKGROUND: Magnolia bark extract (MBE) is a natural supplement with antioxidant, anti-inflammatory, and antimicrobial activities. Its properties suggest that the dietary supplementation in livestock could improve the quality of products. Therefore, the aim of this study was to investigate, for the first time, the effect of dietary MBE supplementation (0.33 mg/kg) in finishing pigs on the oxidative stability of meat. Oxidative stability is of paramount importance for pork, as it affects storage, retail, and consumer acceptance. For the purpose, the fatty acid profile, cholesterol, fat-soluble vitamins, antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase), non-enzymatic antioxidant capacity (TEAC, FRAP, and Folin-Ciocalteu assays), color stability, and lipid stability of pork were assessed. RESULTS: Concerning carcass characteristics, dietary MBE did not affect cold carcass yield, but reduced (P = 0.040) the chilling weight loss. The meat from pigs fed MBE had a lower (P = 0.031) lightness index than the control meat. No effect on intramuscular fat, cholesterol, and fatty acid profile was observed. Dietary MBE did not affect the content of vitamin E (α-tocopherol and γ-tocopherol) in pork, whereas it reduced (P = 0.021) the retinol content. The catalase activity was 18% higher (P = 0.008) in the meat from pigs fed MBE compared with the control group. The MBE supplementation reduced (P = 0.039) by 30% the thiobarbituric acid reactive substances (TBARS) in raw pork over 6 d of aerobic refrigerated storage. Instead, no effect on lipid oxidation was observed in cooked pork. Last, the meat from pigs fed MBE reduced Fe3+-ascorbate catalyzed lipid oxidation in muscle homogenates, with a lower (P = 0.034) TBARS value than the control group after 60 min of incubation. CONCLUSIONS: Dietary MBE supplementation in finishing pigs delayed the lipid oxidation in raw meat. This effect was combined with an increased catalase concentration. These results suggest that dietary MBE could have implications for improving the shelf-life of pork.

Influence of almond hulls in lamb diets on animal performance and meat quality.

Almond hulls (AH) were fed to lambs to study effects on performance and meat quality. Thirty Sarda lambs were allotted to three experimental groups and fed for 40 days either a cereal-based concentrate diet (control) or diets in which cereals were replaced with 15% (AH15) or 30% (AH30) almond hulls on a DM basis. Diets did not affect final body weight, dry matter intake, average daily gain, feed conversion ratio or carcass weight. Replacing part of the cereal mix (i.e., barley and maize) with AH did not affect meat fatty acid profiles. After 5 and 7 days of refrigerated storage respectively for cooked and raw meat, AH15 and AH30 treatments reduced meat lipid oxidation (P < 0.001). Our results suggest feeding almond hulls up to 30% in fattening lamb diets can improve meat oxidative stability without compromising growth performance.

Influence of Dietary Inclusion of Exhausted Bergamot By-Product in Pigs on Animal Performance, Fatty Acid Profile and Oxidative Stability of Meat and Meat Products.

An investigation was carried out on the effect in pig diet of supplementation with exhausted bergamot by-product, stemming from pharmaceutical industry after extraction of phenolic compounds, on growth performance and on the quality of meat and meat products. Twenty pigs were assigned to two dietary treatments and fed a conventional concentrate (control) or a concentrate including exhausted bergamot by-product at the level of 15% on a DM basis (EB). No significant differences between dietary treatments were found for final weight (p = 0.243), carcass weight (p = 0.679), dry matter intake (p = 0.321). In EB pork, the proportion of docosapentaenoic acid was significantly increased (p < 0.05); it tended to have a greater proportion of n-3 PUFA (p = 0.09), and the n-6/n-3 PUFA ratio was lower in EB treatment (p = 0.01). In salami from EB pigs fed, the proportion of α-linolenic acid and the total n-3 PUFA were higher than in the control group (p < 0.001). In salami, the TBARS value was lower after 5 days of storage (p < 0.001) in the EB group. Therefore, the present results suggest that the inclusion of exhausted bergamot by-product in pig diet resulted in a qualitative improvement of meat and meat products.

Effect of different levels of organic zinc supplementation on pork quality.

This study investigated the effect of two supplementation levels of zinc glycinate (ZnGly) on performance, carcass characteristics, and meat quality of growing-finishing pigs. Thirty pigs (bodyweight: 61 ± 4.0 kg) were assigned to three treatments and fed ad libitum for 56 days a diet supplemented with 0 (control), 45 (Zn45), or 100 mg/kg (Zn100) of ZnGly. The highest ZnGly supplementation lowered the average daily gain (P = 0.031); while, cold carcass weight did not differ between treatments. Both ZnGly levels reduced carcass chill loss (P < 0.001). Micromineral content, color stability, and fatty acid profile of meat were not altered by ZnGly. Superoxide dismutase activity was lowered by Zn45 compared to control (P = 0.007); while, catalase activity was enhanced by Zn100 (P = 0.003). Although ZnGly supplementation did not influence lipid oxidation in raw meat and in meat homogenates incubated with pro-oxidant catalysts, Zn45 limited lipid oxidation in cooked meat (P = 0.037). Our results demonstrated that supplementing pigs with 45 mg/kg of ZnGly could improve the oxidative stability of pork subjected to strong pro-oxidant conditions, but this effect needs to be further elucidated.

Effect of feeding pigs with bergamot by-product on fatty acid composition and oxidative stability of meat and salami.

This work investigated the effects of feeding ensiled bergamot pulp to pigs on meat and salami quality. Eighteen pigs were assigned to two experimental treatments and fed a cereal-based concentrate diet (control) or the same diet in which ensiled bergamot pulp replaced 15% dry matter of the diet fed (BP). The BP treatment increased α-linolenic acid (+250%; P < 0.05), docosapentaenoic acid (+62%; P < 0.05), docosahexaenoic acid (+43%; P < 0.05) and consequently n-3 PUFA (+15%; P < 0.01) in meat. In salami, the content of α-linolenic acid, total PUFA and n-3 PUFA increased (+320%, +25% and + 258%, respectively) by feeding the BP diet (P < 0.001). The inclusion of bergamot pulp in the diet did not alter the oxidative stability in raw and cooked meat and colour descriptors. In salami, TBARS values were lower after 5 days of storage (P < 0.001) in BP group (1.54 vs 2.96). Finally, dietary supplementation with ensiled bergamot pulp to pigs improved the nutritional value of meat and meat products.

Fatty acid metabolism in lambs supplemented with different condensed and hydrolysable tannin extracts.

Five groups of lambs (n = 9 each) were used to test the effect of plant extracts rich in hydrolysable (HT) or condensed tannin (CT) on animal performance, fatty acid composition of rumen content, liver and meat. The control group (CO) received a concentrate-based diet without tannins supplementation. The other groups received the same diet as the control lambs plus 4% chestnut (CH) and tara (TA) extracts as a source of HT and mimosa (MI) and gambier (GA) extracts as a source of CT. One-way ANOVA was used to assess the overall effect of dietary treatments, tannins supplementation (CO vs. CH+TA+MI+GA) and the effect of tannin type (HT vs. CT: CH+TA vs. MI+GA) on animal performance, rumen content, liver and intramuscular FA. Dietary CH negatively affected animal performance. The rumen content of the different groups showed similar levels of 18:3 c9c12c15, 18:2 c9c12, 18:2 c9t11, 18:1 t11 and 18:0, whereas 18:1 t10 was greater in CO. Also, 18:1 t10 tended to be lower in the rumen of HT than CT-fed lambs. These data were partially confirmed in liver and meat, where CO showed a greater percentage of individual trans 18:1 fatty acids in comparison with tannins-fed groups. Our findings challenge some accepted generalizations on the use of tannins in ruminant diets as they were ineffective to favour the accumulation of dietary PUFA or healthy fatty acids of biohydrogenation origin in the rumen content and lamb meat, but suggest a generalized influence on BH rather than on specific steps.

Effect of Dietary Hazelnut Peels on the Contents of Fatty Acids, Cholesterol, Tocopherols, and on the Shelf-Life of Ripened Ewe Cheese.

Hazelnut peel (HNP), a by-product from the chocolate industry, is considered to be a suitable ingredient to be included in the diet of ruminants. This study aimed to evaluate the effect of feeding dairy ewes with a diet containing HNP on ripened cheese quality, including fatty acid (FA) profile, cholesterol, and tocopherol content, as well as stability during storage under commercial conditions. In total, 10 experimental cheeses were produced with bulk milk obtained from ewes fed a commercial concentrate (C group; n = 5) or a concentrate containing 36% HNP in dry matter (HNP group; n = 5). After 40 days of aging, each cheese was sub-sampled into three slices: one was analyzed immediately (C0 and HNP0), and the other two were refrigerated and analyzed after seven days (C7 and HNP7) and 14 days (C14 and HNP14), respectively. Compared to C, HNP cheese had more than twice as many tocopherols and mono-unsaturated FA and respectively 38% and 24% less of cholesterol and saturated FA. Tocopherols and cholesterol levels remained rather stable up to 14 days of storage regardless of the experimental group, suggesting no cholesterol oxidation. Therefore, the inclusion of HNP in ewe diets could be a valid resource to produce cheese with a healthier lipid profile and higher tocopherols content.

Influence of dietary inclusion of tannin extracts from mimosa, chestnut and tara on volatile compounds and flavour in lamb meat.

Tannins are compounds able to form complexes with proteins limiting their ruminal degradation and thus the synthesis of some odour-active compounds may be inhibited. Tannins are broadly divided in condensed tannins (CT) and hydrolysable tannins (HT). The study aimed to assess the influence of dietary inclusion of three commercial tannin extracts, namely mimosa (Acacia mearnsii; CT), chestnut (Castanea sativa; HT) or tara (Caesalpinia spinosa; HT) on volatile profile and flavour of meat and kidney fat from lambs. Comisana male lambs were divided into four groups (n = 9 each) and fed for 75 days with a concentrate-based diet (CON) or CON supplemented with 4% of one of the tannin extracts. Tannins reduced "pastoral" odour in perirenal fat of lambs the meat of which was characterized by a very low perception of this attribute. It may be assumed that p-cresol and 8-methylnonanoic acid mostly contributed to "pastoral" odour expression in the diet without condensed or hydrolysable tannins.

Effects of Microencapsulated Blend of Organic Acids and Essential Oils as a Feed Additive on Quality of Chicken Breast Meat.

The present study aims to investigate the effect of dietary supplementation based on a blend of microencapsulated organic acids (sorbic and citric) and essential oils (thymol and vanillin) on chicken meat quality. A total of 420 male Ross 308 chicks were randomly assigned to two dietary treatments: the control group was fed with conventional diet (CON), while the other group received the control diet supplemented with 0.5% of a microencapsulated blend of organic acids and essential oils (AVI). In breast meat samples, intramuscular fat content and saturated/polyunsaturated fatty acids ratio were reduced by AVI supplementation (p < 0.05). Moreover, atherogenic (p < 0.01) and thrombogenic (p < 0.05) indices were lower in AVI than CON treatment. AVI raw meat showed a lower density of psychrotrophic bacteria (p < 0.05) at an initial time, and higher loads of enterococci after 4 days of refrigerated storage (p < 0.05). No contamination of Listeria spp., Campylobacter spp., and Clostridium spp. was found. TBARS values of the cooked meat were lower in the AVI treatment compared to CON (p < 0.01). Among colour parameters, a*, b* and C* values increased between 4 and 7 days of storage in AVI cooked meat (p < 0.05). Overall, organic acids and essential oils could improve the quality and shelf-life of poultry meat.

Effect of Feeding Hazelnut Skin on Animal Performance, Milk Quality, and Rumen Fatty Acids in Lactating Ewes.

The hazelnut skin is waste biomass rich in bioactive compounds that may affect lipid rumen metabolism, ruminant performance, and products' quality. Therefore, we investigated the effect of dietary hazelnut skin on milk production and composition and on rumen fatty acids in ewes. During 28 days, 20 Comisana lactating ewes received alfalfa hay ad libitum plus 800g/head/day of pelleted concentrate containing 36% dried beet pulp (CTRL group; n = 10) or 36% hazelnut skin (HS group; n = 10). The protein percentage was lower in HS milk. Milk fatty acids (FA) partially reflected those of rumen content. Total saturated FA (SFA), odd and branched-chain FA, and n-3 polyunsaturated FA (PUFA) were greater in CTRL milk. Total monounsaturated FA (MUFA) and 18:1 trans were greater in HS milk; moreover, HS milk showed a double percentage of oleic acid than the CTRL group. Individual SFA were greater in CTRL milk except for 18:0. Differing from the rumen content, total PUFA, n-6 PUFA, and 18:2 n-6 were comparable between groups. Vaccenic and rumenic acid were greater in HS milk. To conclude, dietary HS slightly reduced milk protein percentage but improved atherogenic index and healthy FA in milk. The content of the somatic cells suggested a healthier udder in the HS group.

Influence of dietary cardoon meal on volatile compounds and flavour in lamb meat.

Cardoon meal is a by-product retained after oil extraction from the seeds of cultivated Cynara cardunculus var. altilis that has been proposed as a valuable resource for animal feeding. The study aimed to assess the influence of its dietary inclusion on volatile profile and flavour of meat and kidney fat from lambs. Fifteen Sarda × Comisana male lambs were randomly divided in two groups and fed for 75 days with a concentrate-based diet containing 15% cardoon meal (CMD, n = 7) or dehydrated alfalfa (CON, n = 8). Cardoon meal inclusion reduced the "barnyard/animal" odour perceived by a trained panel in kidney fat, which could be associated with the aromatic compound p-cresol (4-methylphenol), detected only in CON diet. Considering the other aroma volatiles regarded as the main contributors for the characteristic lamb flavour, both diets were characterized by moderate to low levels of 4-methyloctanoic acid, skatole and indole while 4-methylnonanoic acid and 4-ethyloctanoic acid were not detected.