
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39251803
71961
10.1038/s41598-024-71961-8
Article
Assessment of the antibacterial and antioxidant activities of seaweed-derived extracts
http://orcid.org/0000-0003-3427-9831
Hejna Monika m.hejna@igbzpan.pl

1
http://orcid.org/0000-0002-2171-864X
Dell’Anno Matteo 2
http://orcid.org/0000-0001-7727-4796
Liu Yanhong 3
http://orcid.org/0000-0003-1178-4683
Rossi Luciana 2
http://orcid.org/0000-0003-4766-2434
Aksmann Anna 4
http://orcid.org/0000-0003-3443-4571
Pogorzelski Grzegorz 1
http://orcid.org/0000-0001-5546-9891
Jóźwik Artur 1
1 https://ror.org/0038zp908 grid.460378.e 0000 0001 1210 151X Department of Biotechnology and Nutrigenomics, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Postępu 36A, 05-552 Jastrzębiec, Poland
2 https://ror.org/00wjc7c48 grid.4708.b 0000 0004 1757 2822 Department of Veterinary Medicine and Animal Sciences-DIVAS, Università degli Studi di Milano, Dell’Università 6, 26900 Lodi, Italy
3 grid.27860.3b 0000 0004 1936 9684 Department of Animal Science, University of California, 2251 Meyer Hall, One Shields Ave, Davis, CA 95616 USA
4 https://ror.org/011dv8m48 grid.8585.0 0000 0001 2370 4076 Department of Plant Experimental Biology and Biotechnology, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland
9 9 2024
9 9 2024
2024
14 2104414 6 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
In swine farming, animals develop diseases that require the use of antibiotics. In-feed antibiotics as growth promoters have been banned due to the increasing concern of antimicrobial resistance. Seaweeds offer bioactive molecules with antibacterial and antioxidant properties. The aim was to estimate the in vitro properties of seaweed extracts: Ascophyllum nodosum (AN), Palmaria palmata (PP), Ulva lactuca (UL), and 1:1 mixes (ANPP, ANUL, PPUL). Escherichia coli strains were used to test for growth inhibitory activity, and chemical-based assays were performed for antioxidant properties. The treatments were 2 (with/without Escherichia coli) × 2 (F4 + and F18 +) × 5 doses (0, 1.44, 2.87, 5.75, 11.50, and 23.0 mg/mL). Bacteria were supplemented with seaweed extracts, and growth was monitored. The antioxidant activity was assessed with 6 doses (0, 1, 50, 100, 200, 500, and 600 mg/mL) × 6 compounds using two chemical assays. Data were evaluated through SAS. The results showed that AN and UL significantly inhibited (p < 0.05) the growth of F4 + and F18 +. PP and mixes did not display an inhibition of the bacteria growth. AN, PP, UL extracts, and mixes exhibited antioxidant activities, with AN showing the strongest dose–response. Thus, AN and UL seaweed extracts reveal promising antibacterial and antioxidant effects and may be candidates for in-feed additives.

Keywords

Seaweeds extracts
Bioactive compounds
Antibacterial
Antioxidant
Livestock farming
Subject terms

Microbiology
Plant sciences
European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant and from the Polish Ministry of Education and Science847639 Hejna Monika issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Food production with swine farming ranks among the most profitable agricultural practices, often relying on the use of antibiotic drugs to manage a critical phase of a pig’s development, such as weaning, which is exposed to stressors that cause multifactorial diseases1. Weaning stress is a main factor of diarrhea occurrence linked with Enterotoxigenic and Verotoxigenic Escherichia coli (ETEC and VTEC) strains2. Animals also experience oxidative stress, which can damage proteins, lipids and DNA3. Previously, in-feed antibiotics were commonly applied as preventive treatment for pig diseases. However, the increase in antimicrobial resistance (AMR)4,5 poses a significant challenge to global health, with interconnections among human, animals and the environment. The primary cause of AMR is the overuse of antimicrobials, both in humans and animals, leading to the spread of resistance genes through the food chain or by direct human-animal contact6. Recent reports have highlighted Escherichia coli strains as important vectors for antibiotic-resistance genes with zoonotic spread7. Therefore, in-feed antibiotics as growth promoters have been banned in Europe8 and mass veterinary medication applied to cope with infections has been also recently restricted9,10. In alignment with the One-Health and 3R (reduce, replace and rethink) approaches, today’s challenges in food production require decreasing the use of antibiotics in livestock. This approach aims to improve profitability, increase the sustainability of agriculture, and mitigate the spread of antimicrobial resistance11,12. Novel bioactive sustainable feed-additives serving as substitutes to in-feed antibiotics are essential for improving of sustainability and reducing the antimicrobial resistance in the animal industry4,13. Among the various potential alternatives, seaweeds (macroalgae) emerge as promising natural sources of bioactive molecules for application as functional feed ingredients14,15. Seaweeds are rich in proteins, vitamins, polyphenols and pigments offering antioxidant potential16–18, and in proteins, peptides, phlorotannins, polysaccharides and fatty acids demonstrating broad antibacterial action against pathogenic bacteria19,20. Brown algae, Ascophyllum nodosum (AN), even if is commonly used seaweed species in animal nutrition, though it has not been fully exploited in pig feeding21. AN contains polyphenols and phlorotannins15 with strong antioxidant potential22 and antibacterial activity against enteric pathogens in pigs, respectively23. Fucoidan and laminarin extracted from AN reported to possess the antibacterial property24,25. Another option, red algae such as Palmaria palmata (PP), although rich in bioactive molecules such as lipids, fatty acids, polysaccharides and pigments26–29, has not received much attention as a feed ingredient. Yuan et al.26 proved that flavonoids extracted from PP exerted in vitro antioxidant property stronger than vitamin C and E. Moreover, Lopes et al. also proved that PP is abundant in fatty acids with strong antioxidant properties27. Lastly, green seaweed, such as Ulva lactuca (UL) contains ulvan with strong antioxidant effects30 and phenols with antibacterial properties31 thus has been demonstrated to be a promising additive for monogastric animals32. However, is not yet supplemented in swine feed’ nutrition.

Furthermore, research related to the bio-accessibility and bioavailability of algae compounds after in vitro digestion, as well as the assessment of synergistic and complementary effects of different activities from combined algal extracts is limited33. In vitro digestion models are essential for studying (i) the physiology of specific segments of the digestive tract, and (ii) the digestive properties of algae. Further, the synergistic action of both agents is more effective than the action of a single one achieving the specific activity, while the complementary action of both agents combines two or more different actions34. Thus, testing the seaweed extracts in mixes (1:1) is important to establish their possible synergistic, and complementary interactions reinforcing their effectiveness and reducing the minimal effective dosage against infections using combinatory treatment.

Therefore, the rationale of selecting these seaweed species lies in their broad spectrum of bioactive molecules and diverse chemical compositions, which may enhance the synergistic effects. Additionally, their global geographical distribution facilitates further applications in the field. Thus, several approaches were employed to assess the biological activities of seaweed extracts and their mixes (1:1). The current study targeted algal extracts and their inhibitory activity against ETEC, and VTEC Escherichia coli strains (F4 + and F18 +), which are the most significant pathotypes responsible for post-weaning diarrhea, thereby increasing the utilization of antibiotic treatment in swine35. Besides, two chemical-based assays, including the 2,2-diphenyl-1-picrylhydrazyl (the DPPH) radical scavenging and reducing power assays (the RPA) were used to test the antioxidant property of algal extracts.

Results

Total polyphenol content in seaweed species

The TPC was evaluated in the powder of three seaweed species using tannic acid as a reference standard. The results disclosed that the total polyphenol content was the highest in Ascophyllum nodosum (4951.53 ± 152.47 µg TAE/g of sample). TPC in Palmaria palmata, and Ulva lactuca reached 896.84 ± 27.41 µg TAE/g of sample and 201.48 ± 36.29 µg TAE/g of sample, respectively.

Chemical composition and in vitro digestion of algal powders

The chemical composition results revealed that crude proteins and lipids were under 10% of their value, except for crude proteins for Ulva lactuca (15.86 ± 0.60, Table 1). Moreover, a low content of crude fiber (< 10%) was observed in all species. However, higher content was observed in Ascophyllum nodosum, and Ulva lactuca. Additionally, all tested algae species displayed a high content of ash, and as a consequence, a high content of minerals (Table 1). Moreover, the in vitro digestibility analysis revealed that the digestibility of 100% algal extract ranged from 20.05% in AN and 37.09% in PP (Table 2). Regarding the algae 1:1 mixes the mixture of PPUL exerted the highest digestibility (41.34%; Table 2). The digestibility of the control samples (carbohydrate and protein sources) reached 90.0%. Table 1 Percentage chemical composition on the dry matter basis of Ascophyllum nodosum, Palmaria palmata and Ulva lactuca.

Algae species	DM (%)	CP (%)	EE (%)	CF (%)	Ash (%)	
Ascophyllum nodosum	94.24 ± 0.07	4.37 ± 0.32	2.60 ± 0.02	5.57 ± 0.35	21.16 ± 0.85	
Palmaria palmata	94.93 ± 0.09	9.68 ± 0.15	0.46 ± 0.06	1.14 ± 0.34	24.30 ± 0.20	
Ulva lactuca	89.23 ± 0.08	15.86 ± 0.60	0.28 ± 0.10	6.30 ± 0.34	20.58 ± 0.35	
All values are expressed as mean and standard deviation of the mean ( ±) from triplicated samples.

DM dry matter, CP crude protein, EE ether extract, CF crude fiber.

Table 2 Percentage of in vitro digestibility in all tested samples of algae species.

Digestibility (%)	
AN	20.05 ± 3.94	
UL	36.53 ± 2.97	
PP	37.09 ± 1.84	
ANUL	22.92 ± 0.02	
ANPP	22.88 ± 2.39	
PPUL	41.34 ± 0.34	
All values are expressed as mean and standard error of the mean (±).

Antibacterial activity through the Escherichiacoli growth inhibitory assay

The results of algae species demonstrated that F4 + and F18 + were significantly sensitive to different doses of brown and green seaweed extracts, AN and UL, respectively. A significant (p < 0.05) dose-dependent effect was observed at time points T1 to time points T4 for AN and UL (Figs. 1A,B and 2A,B). The highest dose of AN and UL (23 mg/mL) resulted the significant maximum inhibitory activity against F4 + and F18 + growth at each time point. Red seaweed extract, PP did not exhibit the growth inhibitory effect from T4 to T6 for both strain of Escherichia coli, although the results exhibited significant differences in the highest dose of PP in both T1, T2 and T3 time points (Figs. 1C and 2C). However, algal extract mixes (ANUL, ANPP, PPUL) of seaweed extract show low inhibitory effects on F18 + , and F4 + strains (Figs. 3A,B and 4A,B). A significant dose-dependent effect was observed only at time points T1, and T2 for ANUL, and ANPP, respectively. No dose-dependent effect was resulted for PPUL extract mix in each time point, excluding T2 for F18 + strain (Figs. 3C and 4C).Fig. 1 The impact of different concentrations (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL) of AN (A) UL (B) ant PP (C) on Escherichia coli F4 + growth in 60 min time interval points (T0, T1, T2, T3, T4, T5, T6). Data are expressed as log10 CFU/mL LSMEAN ± SEM (n = 3). Different superscript letters express significant differences at p < 0.05 among different concentrations within the same time point.

Fig. 2 The impact of different concentrations (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL) of AN (A) UL (B) and PP (C) on Escherichia coli F18 + growth in 60 min time interval points (T0, T1, T2, T3, T4, T5, T6). Data are expressed as log10 CFU/mL LSMEAN ± SEM (n = 3). Different superscript letters express significant differences at p < 0.05 among different concentrations within the same time point.

Fig. 3 The impact of different concentrations (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL) of ANUL (A) ANPP (B) and PPUL (C) on Escherichia coli F4 + growth in 60 min time interval points (T0, T1, T2, T3, T4, T5, T6). Data are expressed as log10 CFU/mL LSMEAN ± SEM (n = 3). Different superscript letters express significant differences at p < 0.05 among different concentrations within the same time point.

Fig. 4 The impact of different concentrations (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL) of ANUL (A) ANPP (B) and PPUL (C) on Escherichia coli F18 + growth in 60 min time interval points (T0, T1, T2, T3, T4, T5, T6). Data are expressed as log10 CFU/mL LSMEAN ± SEM (n = 3). Different superscript letters express significant differences at p < 0.05 among different concentrations within the same time point.

Antioxidant properties of algal species through chemical-based assays

The radical scavenging capacity and reducing power were used to define the antioxidant capacity of three different algal species (AN, UL, PP). Moreover, the synergic or combined outcome of antioxidant effects was also assessed by testing 1:1 algae extract mixes (ANUL, ANPP, PPUL) using the same chemical assays. A dose-dependent growth in radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was detected in AN, PP and UL and their 1:1 extract mixes in the range of tested concentrations (Fig. 5A,B). The strongest response was noticed at the highest concentration (600 mg/mL) of each algae extract however, among all tested algae species AN, and its extract mixes (ANUL; ANPP) had the highest dose response starting from 50 mg/mL in comparison to UL and PP confirming the highest antioxidant capacity. Moreover, the maximal plateau was observed in the DPPH assay for Ascophyllum nodosum. All algal species also elicited increased reducing power in a dose-dependent manner, with the highest dose–response for AN and their 1:1 extract mixes (Fig. 6A,B). Moreover, the effective concentrations (EC) calculated on the basis of the DPPH assay, and exerted the highest EC10 for PPUL (504.57 mg/mL), and the lowest for AN (5.10 mg/mL), and ANUL (5.99 mg/mL). EC50 could be calculated only for three experimental variants, namely AN, ANUL and ANPP, with lowest EC50 value for AN (55.86 mg/mL; Table 3, Supplementary Fig. S1, Supplementary Tables S1–S6).Fig. 5 Dose response of (A) Ascophyllum nodosum (AN), Palmaria palmata (PP) and Ulva lactuca (UL) algae species and (B) their 1:1 extract mixes using the 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Data are presented as the mean of 5 observations.

Fig. 6 Dose response of (A) Ascophyllum nodosum (AN), Palmaria palmata (PP) and Ulva lactuca (UL) algae species and (B) their 1:1 extract mixes using reducing power assay. Data are presented as the mean of 5 observations.

Table 3 The values of effective concentrations (EC10 EC20 and EC50; mg/mL) of different algae extracts measured by chemical-based antioxidant activity assays.

Assay	2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity	
EC10 (mg/mL)	EC20 (mg/mL)	EC50 (mg/mL)	
AN	5.10 [3.00–6.61]*	13.48 [12.01–15.02]	55.86 [52.25–60.06]	
UL	313.06 [296.70–329.73]	–	–	
PP	84.69 [59.46–105.88]	307.39 [293.09–321.92]	–	
ANUL	5.99 [1.80–9.61]	22.05 [18.62–25.23]	111.05 [103.30–119.52]	
ANPP	6.12 [3.00–8.41]	16.08 [13.81–18.62]	70.43 [64.26–76.88]	
PPUL	504.57 [478.08–535.14]	–	–	
*95% lower and upper confidence interval.

Discussion

The main purposes of this study were to assess the in vitro biological activities of bioactive molecules from Ascophyllum nodosum (AN), Palmaria  palmata (PP), and Ulva lactuca (UL), and their 1:1 extract mixes. Their inhibitory activity against Escherichia coli F4 + , and F18 + of algae extracts, and their antioxidant effects were evaluated. We also determined the total polyphenol content, the chemical composition, and in vitro digestibility of these compounds and their 1:1 extract mixes. These assessments were crucial to establish the further use of algae as functional additives to counteract antibiotic overuse in food-producing animals.

Seaweeds typically demonstrate a highly diverse chemical composition (protein, polysaccharide, mineral, and lipid contents) influenced by various environmental features, such as season of harvest, water temperature or light and nutrient availability in the water36,37. However, the results regarding the chemical composition of algal powders, including the variation observed, largely align with existing literature, and product labels19,38. The analysis revealed a high percentage of minerals (over 20%) in each algae species to their predisposition to mineral accumulation from seawater, in line with numerous findings in the literature36,39. Thus, these species can serve as rich sources of essential minerals for livestock nutrition19, contributing to a balanced diet, that typically contains less than 5% inclusion of minerals40. However, seaweed can also accumulate non-essential heavy metals from the marine environment, which may pose significant risks to animal health, especially gut health41. Heavy metals such as mercury, lead, cadmium, and arsenic can significantly alter the composition and function of the gut microbiota42. The populations of beneficial gut bacteria such as Lactobacillus and Bifidobacterium are sensitive to heavy metals, thus can decline with their exposure43. Heavy metals can induce oxidative stress and inflammation in the gut, prolonged exposure can cause damage to the epithelial cells lining the gut, resulting in increased intestinal permeability42. Moreover, the presence of heavy metals can trigger an inflammatory response, resulting in chronic gut inflammation, which can further exacerbate gut health issues. Heavy metals absorbed through the gut can also accumulate in various organs, causing damage not only to the gastrointestinal tract but also to the liver, kidneys, and brain44. Consequently, algae commercialized for feed production must have low levels of contaminants, such as heavy metals to ensure both safety and effectiveness45, thus many seaweed species are commercially cultivated in controlled conditions. Additionally, water blanching can be used as one potential method to reduce the total ash content and consequently heavy metals from seaweeds46. Moreover, the bioavailability of minerals may be influenced by the fiber content of macroalgae, which can lead to the formation of insoluble chemical complexes and colloidal structures with minerals, thus reducing their bioavailability47. However, we observed a low content of crude fiber in the analysed samples, especially in Palmaria palmata species, suggesting these species may not influence mineral’ bioavailability. Nonetheless, further studies are essential to explore the diverse interactions of dietary fiber in bio-absorption within the gastrointestinal tract48. Moreover, our results indicated a relative level of protein content in Ulva lactuca (15.86%) and Palmaria palmata (9.68%). The values were in line with literature reports, where Ulva lactuca contains from 7.06 to 23.1% of protein on a dry matter basis19. Instead, the protein content found in Palmaria palmata  was slightly lower than reported range of 15.1–31.4% of dry mass in the literature19. However, the results highlighting these species may be valuable protein sources in farm animals36. Our analysis of the total polyphenol content (TPC) in Ascophyllum nodosum aligned with findings reported in the literature19. However, TPC in Palmaria palmata  and Ulva lactuca was lower than the levels reported in some literature cases. Castejón et al. discovered that the TPC in PP and UL were 1850.5 ± 121.5 (μg GAE/g dry weigh) and 1950.6 ± 109.5 (μg GAE/g dry weigh) in gallic acid equivalent, using hot water extraction, respectively49. The total polyphenol content of detected seaweeds can vary significantly based on the extraction method used, the type of seaweed and the environmental conditions in which seaweeds are grown. Despite these variations, our results disclosed high polyphenol content in three algae Palmaria  species. According to literature, brown, red and green seaweeds are rich in polyphenols. Phlorotannins and phenolic acids are the most abundant types of phenolic compounds found in brown seaweeds, while other phenolic compounds such as flavonoids are commonly found in green and red seaweeds50–52. These phytochemicals contribute to their antioxidant properties and may help protect the body against oxidative stress and damage caused by free radicals53.

Further, a three-step in vitro digestibility test was proceeded to simulate the digestive characteristics of algae, the physiology of certain segments, and their bio-accessibility and bioavailability33. Our results demonstrated that among the tested seaweed extracts, AN had the lowest digestibility (20.05%), and PPUL (41.34%) extract mix and PP (37.09%) showed the highest digestibility. Algae digestibility is influenced by various factors, including the physical characteristics and chemical composition of the algae54. In brown seaweeds, polymers such as alginates and sulphated fucoidans might account for the decrease in digestibility55,56. Alginates form viscous gels that can hinder nutrient breakdown and absorption by reducing the digestive process and decreasing enzyme activity57. Fucoidans instead, can inhibit digestive enzymes and reduce nutrient absorption58. Aside from the well-known polymers such as alginates and fucoidans that affect digestibility, other components in brown seaweeds, such as pigments (fucoxanthin), polysaccharides (laminarin), and polyphenols (tannins), can also reduce the digestibility of brown seaweeds19,25. Fucoxanthin in terms of bioaccessibility is complex due to its non-polar, hydrophobic, and water-insoluble nature. Thus, emulsification and colloid dispersion are necessary to enhance its solubility and adsorption capability. Consequently, the fucoxanthin from Ascophyllum nodosum affects metabolic processes and nutrient absorption59. Moreover, laminarins, a class of polysaccharides found in brown algae, may decrease digestibility due to their resistance to human digestive enzymes60. Additionally, tannins, a type of polyphenol present in brown algae, can bind the proteins, form stable complexes and cause depression of digestive capacity in the small intestine61.

Moreover, the differences in in vitro digestibility could be attributed to the fiber content and the specific dietary fiber characteristics of each algae species. Generally, brown algae are more concentrated in neutral detergent fiber (NDF) and acid detergent fiber (ADF) fractions62. The cell wall of seaweed is rich in various polysaccharides, which can form stable complexes with proteins, making them inaccessible to proteolytic enzymes and thereby reducing the digestibility of seaweed protein37,63. Furthermore, studies have indicated that phlorotannins mostly from brown algae can negatively affect digestibility by binding with other macromolecules such as poorly digestible polysaccharides and proteins, consequently leading to different effects on digestibility37,64. Additionally, phenolic compounds can vary significantly within the same species due to seasonal effects, and the chemical composition of seaweeds. Moreover, to mitigate the decrease in digestibility in the animal feed, polyethylene glycol may be used to counteract the effect on digestibility65.

The literature widely highlights the diversity in in vitro digestibility, nutrient composition, and nutritional values among seaweed species37. Moreover, it has been observed that the nutritional value and digestibility patterns differ among seaweed species and across harvesting seasons39. Hence, the rational use of seaweeds in animal diets and diet formulation will necessitate adequate chemical analysis of each batch of algae biomass to determine its nutrient composition, rather than relying solely on standardized percentage inclusion in the diet36. Likewise, cultivation of the seaweed could be of interest, potentially leading to higher yields with a desired and potentially more consistent or predictable chemical composition36,66. Further study is required to fully recognize the digestibility of different compounds and fully characterize the nutritional value originated in seaweeds to regulate the overall effect of seaweeds on pig feed.

Antibiotics have been widely applied in animal husbandry to treat the bacterial diseases, however, their overuse during the past decades has led to the rise of antibiotic-resistant bacteria in both humans and animals. The overuse has resulted in a diminishing effectiveness of antibiotics, and an increased risk of transmission of antibiotic-resistant pathogens to humans67,68. Hence, there is a pressing need to reduce the reliance on antibiotics and explore novel alternatives such as phytochemicals. In this study, we aimed to estimate the antimicrobial potential of algal extracts against ETEC, and VTEC Escherichia coli, targeting pathogenic strains with two different adhesive fimbriae (F4 + and F18 +), which are responsible for the bacterial adhesive abilities of these strains. Escherichia coli is among the most common pathogens in swine farming, where antibiotics are becoming increasingly ineffective against bacteria. Therefore, it is crucial to avoid the onset of its diseases initiated by this pathogen.

The growth inhibitory activity of Escherichia coli F4 + and F18 + indicated that Ascophyllum nodosum, and Ulva lactuca inhibited the growth of Escherichia coli at varying concentrations (1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL), and time points (1, 2, 3, 4 and 5 h). In contrast, Palmaria palmata  exhibited growth inhibitory effects from T1 to T3 time points. Notably, Ascophyllum nodosum and Ulva lactuca displayed the highest inhibitory activity against VTEC F18 +. Higher concentrations were not evaluated due to potential interference from the color of the extracts, which could affect the absorbance readings and lead to inaccurate results. These findings underscore the importance of employing the highest concentrations to ensure a significant antibacterial effect on the growth of Escherichia coli strains.

Numerous literature studies have confirmed the significant growth inhibition of seaweeds against various bacterial pathogens. Studies have reported their growth inhibition and antibacterial property against Pseudomonas aeruginosa69, Staphylococcus aureus70, and Escherichia coli71. For example, Dell’Anno et al.23 observed that Ascophyllum nodosum (0.12%, 0.06%, 0.03% of inclusion) exhibited antibacterial property against O138 Escherichia coli, and Frazzini et al.40 also revealed inhibitory activity of Ascophyllum nodosum at different doses. Similarly, laminarin from the Irish brown seaweed Ascophyllum nodosum also showed significant inhibition of Escherichia coli growth25 while methanol extracted phlorotannins from the same species displayed bactericidal activity against Escherichia coli72. Besides, ascophyllan extracted from Ascophyllum nodosum resulted in in vitro antibacterial activities against the pathogenic Escherichia coli73. Moreover, Ulva lactuca ethanol extracts had high antibacterial activity against the Escherichia coli strain, decreasing its growth of 69.5% (at 500 μg/mL) attributed to its higher mineral concentration of metals, including copper, zinc, silver and mercury74,75. Tan et al.76 also found that Ulva lactuca consistently formed compounds with activity against various bacteria, indicating that the antibacterial compounds were present seasonably, with the highest production detected in the autumn and winter months. Palmaria palmata displayed moderate antimicrobial activity against pathogenic L. monocytogenes (62.09%), and weak activity against food spoilage E. faecalis77. However, ethanol extraction increased inhibition to 100%. These findings align with our results.

The antibacterial activity of seaweeds arises from various mechanisms, including inhibition of oxidative phosphorylation and the presence of functional groups that act on different levels with the bacterial cell wall. These mechanisms enhance the permeability of the cytoplasmic membrane, resulting in damages of cell membranes, enzyme inhibition, and DNA intercalation, and cell lysis78. Seaweeds represent a promising innovation for animal feed due to their high content of functional molecules. Phenolic compounds, found abundantly in seaweeds, are responsible for their broad spectrum antibacterial activity against various pathogenic microorganisms, such as Escherichia coli19,20,56. Algae are rich sources of phenolic bioactive components such as polyphenols, phlorotannins, bromophenols, alginates, and peptides40,72.

Furthermore, it is crucial to test the algal extracts in combinations mix (1:1) to establish their possible synergistic and complementary interactions, reinforcing their effectiveness and potentially lowering the minimal effective dosage against infections using combinatory treatment. The synergistic outcome of both agents is more effective than the action of a single agent in performing a specific activity34. However, our results indicated that F4 + and F18 + were not sensitive to different doses of algal extract mixes in all-time points (ANUL, ANPP, PPUL). A dose-dependent effect was observed only at points T1, and T2 for ANUL, and ANPP.

Although, our results elicited that AN may have a combined action, increasing the effect of Palmaria palmata or Ulva lactuca in line with literature findings40. The absence of Escherichia coli growth inhibition in the ULPP and ANPP combinations has been observed and these findings may be attributed to several factors, including antagonistic interactions, insufficient effectiveness of active compounds, interference from complex matrices and the extraction efficiency79,80. Our study demonstrated that PP alone did not exhibit inhibitory activity, and combining PP with AN or UL could lead to a lack of interactions. Compounds from PP might antagonistically interact with UL and AN, preventing them from exerting antibacterial effects. As a result, some components may decrease the effectiveness of others, flattening the dose–response curve. These findings may be due to characteristics of seaweed compounds such as proteins, polysaccharides, and lipids. Antimicrobial proteins in PP might be highly potent, achieving maximum activity at low concentrations by inserting into bacterial membranes and causing cell lysis without requiring higher doses81. In fact, our study presented a significant difference in Escherichia coli growth inhibition in ANUL at lower doses (1.4 mg/mL) at time points T1 and T2. Proteins and peptides could also degrade or denature at higher concentrations, leading to a plateau in antibacterial activity82. Furthermore, polysaccharides may affect the dose–response relationship through barrier function and viscosity effect83 by forming protective barriers or disrupting bacterial adhesion, potentially exhibiting a threshold effect84. Once this threshold is reached, further increases in concentration may not improve the barrier properties. Additionally, high polysaccharide concentrations can increase viscosity which may limit their diffusion and interaction with bacterial cells83.

Another factor that could potentially affect inhibition is the extraction efficiency of the compounds. In our study, we used ethanol as the extraction solvent. Ethanol typically dissolves free sugars, amino acids, some phenols, low molecular weight compounds85, and lipids and other lipid-soluble compounds86. Although ethanol is less efficient than water for extracting polysaccharides, proteins and peptides87. Using ethanol as a solvent to extract compounds from Palmaria palmata might thus not recover all bioactive compounds, possibly explaining the lack of a dose–response. Future studies should involve fractionation and targeted isolation of components to identify specific compounds and assess their antibacterial activity. In conclusion, although PP is rich in polysaccharides, its lack of dose–response effect on antibacterial activity may result from interactions among its proteins, polysaccharides, and other bioactive compounds. To our knowledge, no existing literature confirms our findings on the lack of growth inhibition in these seaweed combinations. Further research is needed to identify the specific compounds responsible for antibacterial effects and their interactions. Moreover, although the data from this study require further validation through additional research to evaluate the in vivo effect of tested seaweed extracts in an animal model, our findings are highly promising. They indicate that these seaweed extracts alone should be examined more comprehensively in the pig farming sector due to their potential as phytochemicals and antibacterial agents.

The overproduction of free radicals triggers oxidative stress, resulting in cell damage and cell death88. Antioxidants, capable of slowing or retarding oxidation are essential for human and animal health89–91. The mechanism of action of the antioxidant effects of antioxidants derived from seaweed species include scavenging free radicals and chelating metals. Consequently, these substances can delay the formation of free radicals, and hamper the autoxidation process11.

Macroalgae exhibit antioxidant effects due to the abundance of bioactive molecules such as (i) polysaccharides (fucoidan, alginate, laminarin) in brown algae, ulvan in green algae, and carrageenan in red algae; (ii) phenolic compounds, tannins and phlorotannins and (iii) carotenoid fucoxanthin which influences antioxidant status92. Various literature cases have disclosed the potent antioxidant effects of algal species, thus protecting animals from oxidative stress and cellular damage induced by free radicals19,40,93.

Chemical-based antioxidant assays are cost-efficient and reliable methods for screening for the antioxidant capacity of seaweeds. One of these techniques involves the DPPH radical scavenging analysis. The DPPH exhibits a stable and vibrant violet color, which diminishes upon mixing its solution with a substance capable of donating a hydrogen atom. The formation of hydrazine (DPPH-H) as a result of radical reduction by hydrogen atom transfer from antioxidants causes the change of the solution color from violet to pale yellow. The color change can be easily verified by UV–vis spectroscopy94. The other commonly used method, named reducing power assay (RPA), estimates the capacity of electron donation by assessing the effectiveness of reducing the ferric cyanide complex (Fe3+) to the ferrous cyanide form (Fe2+) which is a detrimental of antioxidant activity of the analyzed material95.

As expected, all seaweed extracts from three species exhibited consistent antioxidative activity, consistent with various literature findings96–99. Moreover, the extract from brown seaweed, Ascophyllum nodosum and its extract mixes with PP and UL displayed the highest increase in DPPH radical scavenging capacity assay as well as the highest capacity of electron donation assessed by the RPA method. The antioxidant capacity of seaweeds relies on their chemical compositions. Brown algae usually exhibit better antioxidant activity compared with green and red algae100–102. Brown algae, Ascophyllum nodosum contains various bioactive compounds such as laminarin, fucoidans and phlorotannin, which have been reported to have strong antioxidant capacity52. The concentrations of laminarin, fucoidans, phenols and phlorotannins in Ascophyllum nodosum reach 5.82% DM, 41.7% (417.6 ± 4.1 mg/g DW) and 12–14% DM, respectively96,103,104. These bioactive compounds serve as electron donors, binding free radicals ions and consequently reducing oxidative damage100. However, even though Palmaria palmata and Ulva lactuca elicited lower antioxidant capacity in our study compared to Ascophyllum nodosum, these algae species also possess antioxidant activity. Many studies have confirmed their bioactive potential to inhibit the oxidation process19,97.

When considering differences in the antioxidant capabilities of individual algae species, it should also considering that the biological activity of seaweeds may be influenced not only from variations in algae origin, cultivation conditions, and environmental conditions, but also from differences in the extraction methods and solvents used to obtain tested compounds105. Therefore, the selection of an appropriate and effective extraction method should be considered when considering species of algae, the target compounds to be extracted, and environmental features40,99.

An additional important issue related to the practical use of algae as antioxidants is the potential combination of two or more species to enhance their effect. Thus, to investigate the synergistic and complementary interactions of algae species, their extract mixes were also tested in this study. The combined effect was estimated on 1:1 extract mixes, and the results from both the DPPH and RPA showed that synergistic or complementary interactions were observed in Ascophyllum nodosum combinations with both Palmaria palmata and Ulva lactuca, enhancing the antioxidant activity of compared with single UL and PP extracts.

The data highlighted that the sum of the antioxidant capacity of the single AN or UL extracts was lower compared to the extracts mix of algae, indicating a potential complementary effect40. Literature studies have shown that the combination of diverse antioxidant sources could improve their effect in scavenging radicals106,107. Therefore, AN species may lower the minimal effective dosage against oxidative stress using combinatory treatment. However, additional studies are needed to fully understand the mechanism of action of the algal combinations in studies of antioxidant properties.

To conclude, this study investigated the in vitro antioxidant activity of three algal species and their extract mixes, with Ascophyllum nodosum exhibiting the strongest antioxidant effect. All tested seaweeds may have promising relevance as feed additives due to their antioxidant activities. However, it is crucial to note that the concentration of antioxidants in algal extracts used in chemical-based assays may not represent their physiological levels when directly administered in the diet in vivo, and chemical-based assays cannot measure indirect antioxidant assets such as alter intracellular antioxidant enzymes in a living organism108. Therefore, further in vitro studies using cell-based tests or in vivo studies would be necessary to fully elucidate the antioxidant properties of tested seaweeds in animal models.

Conclusion

As in-feed antibiotics, and mass veterinary medications have been banned and recently restricted in swine production, novel bioactive feed-additives as plant extracts are of interest to enhance animal disease resistance. Seaweeds may serve as promising sources of bioactive molecules and phytochemicals used as feed additives. Nevertheless, due to the wide variety of algal species and their distinct characteristics, it is essential to assess their individual activities. Thus, we assessed the antioxidant and antibacterial characteristic of three seaweed species extracts and their extract mixes. In this study, we demonstrated the presence of bioactive molecules, such as polyphenols in all tested seaweeds. Further, our study confirmed the antioxidant and some antibacterial activity of the selected seaweed extracts, which may reduce the amount of antibiotics using during the animal’s infection, even if these algae had a lower digestibility level. In the recent study, the brown macroalgae Ascophyllum nodosum extracts were the most effective in terms of antioxidant activity, and antibacterial activity, while AN and their extract mixes were also most efficient in terms of antioxidant activity. The output of this analysis indicated that active molecules derived from Ascophyllum nodosum have a strong inhibitory effect on F4 + and F18 + E. coli strains. Moreover, AN in combinations mix (1:1) may induce a complementary effect with PP and UL. Thus, tested algae may be able to (i) decrease the risk of bacterial infection and (ii) reduce oxidative stress, and may significantly impact the development of new functional nutritional strategies to reduce reliance on antibiotic treatment in swine farming, and provide further guidelines for significantly improving sustainability. Additional research is needed to further explore the therapeutic potential, and perspectives of algae-derived compounds in addressing multifactorial diseases in the pig industry.

Materials and methods

Materials and experimental design

Lyophilized powder (100% pure) of Palmaria palmata (PP; catalog number: 10418) was purchased from Alganex Gmbh (Berlin, Germany), while Ascophyllum nodosum (AN; catalog number: SX 009776) and Ulva lactuca (UL; catalog number: SZ 009874) were purchased from Italfeed Srl (Milan, Italy) in line with European safety requirements. Before further antibacterial and antioxidant experiments, the extraction method of 100% pure lyophilized algal powders of different seaweed and their mixes based on ethanol was proceeded. The extraction method for Total polyphenol content evaluation differs from extraction for subsequent antibacterial and antioxidant experiments. In the antibacterial assays, some of the extracted algal extracts were dissolved in DMSO (˃1%) and then all of them were further resuspended in Luria Bertani broth (LB), and filtered with 0.22 µm syringe filters prior to microbiological assay. In the antioxidant assays, algal extracts were prepared by diluting the stock of the extracted algae solutions with methanol (w/v).

The experimental design for the antibacterial assay comprised a factorial arrangement with 2 (with or without E. coli) × 2 (F4 + and F18 +) × 5 doses (0, 1.44, 2.87, 5.75, 11.50, and 23.00 mg/mL of seaweed extracts) × 6 compounds (AN, PP, UL and their 1:1 extract mixes). The experiment design for antioxidant assays comprised 6 doses (0, 1, 50, 100, 200, 500, and 600 mg/mL of seaweed extracts) × 6 compounds (AN, PP, UL and their 1:1 extract mixes).

Evaluation of total polyphenol content (TPC)

Firstly, Ascophyllum nodosum, Palmaria palmata and Ulva lactuca were extracted according to Attard et al.109. Briefly, 5 g of algae powder were suspended with 30 mL of methanol and left stirred for 48 h at room temperature. The obtained mixtures were centrifuged (5000 rpm for 10 min) and filtered (0.45 µm), and the filtrates were diluted with deionized water in a 1:1 ratio. Subsequently, the TPC of Ascophyllum nodosum, Palmaria palmata and Ulva lactuca was evaluated by the Folin-Ciocalteu microtiter plate method based on Attard109, and measured using a spectrophotometer at 630 nm (BioTek Synergy HTX, Agilent Technologies, Santa Clara, CA, USA). Calibration curves were prepared in five 1:2 dilutions ranging from 960 to 60 μg/mL, with tannic acid as the standard (Sigma Aldrich, St. Louis, MO, USA). Each sample and standard were analyzed in triplicate (n = 3). For Ulva lactuca a proper blank was included according to Attard et al.109 for correcting for the strong background color. The TPC was expressed as µg Tannic Acid Equivalents (TAE) per g of algal powders (µg TAE/ g).

Extraction of algal biomass

Seaweed biomass of the tested species were extracted using ethanol as a solvent, following the literature110,111 with some adaptations. Briefly, algal biomass powder was dissolved in 80% ethanol (1:10 ratio), rubbed in the mortar, and vortexed (3 min). Then, all samples were overnight frozen (− 20 °C) to maximize the efficiency of extraction procedure, centrifuged (5000 rpm × 20 min, 4 °C), supernatant was decanted, and the solid glass beads (3 mm) were added to each sample. The glass beads-solution were then homogenized 30 s × 4.5 RPS (FastPrep-24 classic homogenizer, MP Biomedical, Irvine, CA, USA) and all samples were centrifuged (5 000 rpm × 20 min, 4 °C). Ethanol extraction procedure for the remaining pellet was tripled. The extraction solution was then evaporated by an evaporator (Rotary Evaporator Strike 300, Steroglass srl, Perugia, Italy) at the temperature lower than 50°C, and dried residues were weighed, and the yield was determined considering the weight of the dry algae powder. Each residue was suspended in an appropriate medium for further analysis.

Chemical composition of algal powders

The samples of algae powders were analyzed in triplicate based on the official analysis methods112 for their principal composition, including dry matter, ether extract (EE), crude protein (CP), crude fiber (CF), and total ash contents. Dry matter was determined by forced-air oven at 65 °C for 24 h (AOAC, 930.15). Lipid content (ether extract, EE) was determined using petroleum ether extraction (AOAC, 2003.05). Crude protein content (CP) was measured according to the Kjeldahl method using 6.25 as a nitrogen conversion factor (AOAC, 2001.11), and crude fiber (CF) was assessed using the filtering bags technique (AOCS, Ba 6a-05). Total ash content was measured after incinerating samples at 550 °C for 3 h (AOAC, 942.05).

In vitro digestion of seaweed powders

Escherichia coli growth inhibitory of algal powders was measured by the described procedures113,114 with few adaptations. Briefly, 1 g of each algal powder was mixed with distilled H2O (20 mL) and shaken (150 rpm, 5 min). The control samples including digestion blanks (enzymes) and standard protein and carbohydrate sources, have been included. The process involved three phases. In the oral phase, 150 mg α-amylase (Sigma-Aldrich, Burlington, MA, USA) in 1 mL of 1 mM CaCl2, pH 7 was added, and then the samples were incubated (30 min, 37 °C). In the gastric phase, the pH was decreased to 2 with 6 M HCl and 100 mg of pepsin (Sigma-Aldrich, Burlington, MA, USA) was added in 2 mL of 0.1 M HCl, and incubated (120 min at 37 °C). In the small intestine phase, the pH was increased to 7 with 6 M NaOH, and 200 mg pancreatin (Sigma-Aldrich, Burlington, MA, USA), and 50 g bile extract (Sigma-Aldrich, Burlington, MA, USA), were added with 2 mL of 0.5 M NaHCO3, and incubated (180 min at 37 °C). Samples were then filtered on paper filters for the determination of digestibility (Whatman filters 54). Before further analysis of the antibacterial assay, aliquots were maintained at − 20 °C. Digestibility was calculated based on the formula:Digestibility%=(DMofsample-Undigestedfractiong)DMofsample×100

Measurement of antibacterial activity through Escherichia coli growth inhibitory assay

A liquid culture-based of F4 + and F18 + E. coli growth inhibition assay was completed to estimate the inhibitory activity of previously extracted (from subchapter 5.3.) algal biomass at different concentrations. Two Escherichia coli strains, harboring F4 +, and F18 + adhesive fimbriae, were acquired from a collection of the University of Milan and formerly defined23,115. The bacteria were cultured overnight for 12 h at 37 °C with agitation (150 × rpm) in lysogeny broth (LB) medium under an aerobic conditions, serving as the inoculum for all subsequent experiments. Overnight-grown F4 + and F18 + cultures were inoculated in 96 microplates wells of containing 100 µL of LB medium supplemented with different doses of extracts (0, 1.44, 2.87, 5.75, 11.50, and 23.0 mg/mL) of algal powders, respectively. Before inoculation, bacterial cultures were standardized to initial density (0.05 ± 0.02 OD when read against LB medium) by spectrophotometer (600 nm wavelength). Microplates were incubated aerobically with shaking (150 × rpm) at 37 °C. The bacterial growth was measured via measurement of the optical density of each culture at 620 nm (OD620) at 60 min intervals in a spectrophotometer (ScanReady P-800, Life Real, Zhejiang, China). Bacteria-free wells with equivalent concentrations of algal powders were used as blanks to subtract the background turbidity caused by algal-protein interactions33,116. All data acquired from the optical density measurement were converted to log-transformed based cell count (CFU/mL) using a calibration curve (considering 1 OD = 109 cells/mL). The assay was performed in three biological replicates and four technical replicates. The increase in absorbance determined bacterial growth. The following formula estimated the inhibition rate was calculated based on the formula:Inhibitionrate%=100∗ODCTRL--ODsampleODCTRL--ODblank

Measurement of the antioxidant activity of algal species through chemical-based assays

DPPH radical scavenging capacity and reducing power assays were implemented to estimate the antioxidant activity of previously extracted (from Sect. 5.3) algal biomass. AN, PP, UL, and their 1:1 extract mixes were tested at different doses of extracts: 0, 1, 50, 100, 200, 500, and 600 mg/mL. All assays were repeated with five technical replications.

DPPH radical scavenging capacity assay

The scavenging capacity of algal-based powders against 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma, St. Louis, MO, USA) radical was assessed based on Zhou et al.117 and Wu et al.108. Briefly, samples were mixed with DPPH solution (25 g/mL in methanol) at a ratio of 1:39 (v/v). The optical density (OD) was determined at 540 nm (Synergy 4 Microplate Reader, BioTek, Winooski, VT, USA). The scavenging capacity of each of the algal powders was calculated based on the below equation. A lower EC indicated a higher radical scavenging capacity. Effective concentrations (EC10, EC20 and EC50, mg/mL) of each of the algal powder extracts were defined as the concentrations that cause 10, 20 or 50% reduction of the DPPH radical118,119, and were calculated from the curves fitted to the experimental data (for details see Supplementary graphs S1).DPPH+scavenging capacity%=Ablank-AtestAblank×100

where Ablank was the absorbance of the blank sample, and Atest was the absorbance of the test sample.

Reducing power assay

The ferric iron reducing capacity of algal powders was measured by the procedures of Chung et al.120 and Bhalodia et al.121, with minor modifications according to Wu et al.108. Briefly, equal volumes of test sample, 2 M phosphate-buffered saline solution (PBS, pH 6.6), and 1% potassium ferricyanide (Sigma, St. Louis, MO, USA) were thoroughly mixed. Ascorbic acid prepared at different concentrations (0, 1, 5, 10, 50, 100 and 200 µM) was used as the standard sample. The optical density (OD) was measured at 540 nm (Synergy 4 Microplate Reader, BioTek, Winooski, VT, USA). Higher absorbance indicates higher reducing power. The ferric reducing capacity was calculated as the ascorbic acid equivalent.

Statistical analysis

All data generated from different assays were analyzed by ANOVA using the MIXED procedure (SAS 9.4, SAS Institute Inc., Cary, NC, USA) with different statistical models. Escherichia coli growth data were log10 transformed (normalization) prior to statistical analysis. The model included treatments, time, and time × treatment as fixed effects and block as a random effect. Data from antibacterial assays are presented as least-squares means and the standard error of the means. Data from antioxidant assays are presented as means and standard errors. The data from TPC are presented as means and standard deviation of the mean (n = 3). Probability values of ≤ 0.05 were considered to be significant.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71961-8.

Acknowledgements

The authors would like to thank the Dr Agata Błaszczyk from the Division of Marine Biotechnology, Institute of Oceanography of the University of Gdańsk for setting the procedure of algae extraction method.

Author contributions

M. Hejna: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, roles/writing—original draft, writing—review and editing; M. Dell’Anno: methodology, writing—review and editing; Y. Liu: formal analysis, writing—review and editing; L. Rossi: conceptualization, writing—review and editing; A. Aksmann: formal analysis, writing—review and editing; G. Pogorzelski: writing—review and editing; A. Jóźwik: writing—review and editing.

Funding

This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No 847639 and from the Polish Ministry of Education and Science.

Data availability

All data generated or analysed during this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Moeser AJ Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig Am. J. Physiol. Gastrointest. Liver Physiol. 2007 292 G173 181 10.1152/ajpgi.00197.2006 16901995
Moeser, A. J. et al. Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G173-181 (2007).16901995 10.1152/ajpgi.00197.2006
2. Rossi L Tobacco seed-based oral vaccination against verocytotoxic O138 Escherichia coli as alternative approach to antibiotics in weaned piglets Antibiotics 2023 12 715 10.3390/antibiotics12040715 37107076
Rossi, L. et al. Tobacco seed-based oral vaccination against verocytotoxic O138 Escherichia coli as alternative approach to antibiotics in weaned piglets. Antibiotics 12, 715 (2023).37107076 10.3390/antibiotics12040715
3. Hao Y Xing M Gu X Research progress on oxidative stress and its nutritional regulation strategies in pigs Animals 2021 11 1384 10.3390/ani11051384 34068057
Hao, Y., Xing, M. & Gu, X. Research progress on oxidative stress and its nutritional regulation strategies in pigs. Animals 11, 1384 (2021).34068057 10.3390/ani11051384
4. EMA Committee for Medicinal Products for Veterinary Use (CVMP) and EFSA Panel on Biological Hazards (BIOHAZ) et al. EMA and EFSA Joint Scientific Opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union, and the resulting impacts on food safety (RONAFA). EFSA J. Eur. Food Saf. Auth. 15, e04666 (2017).
5. World Health Organization. WHO Global Principles for the Containment of Antimicrobial Resistance in Animals Intended for Food : Report of a WHO Consultation with the Participation of the Food and Agriculture Organization of the United Nations and the Office International Des Epizooties, Geneva, Switzerland 5–9 June 2000. https://apps.who.int/iris/handle/10665/68931 (2000).
6. ECDC/EFSA/EMA second joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food‐producing animals | EFSA. https://www.efsa.europa.eu/en/efsajournal/pub/4872 (2017).
7. Zalewska, M., Błażejewska, A., Czapko, A. & Popowska, M. Antibiotics and antibiotic resistance genes in animal manure—Consequences of its application in agriculture. Front. Microbiol. 12, (2021).
8. Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on Additives for Use in Animal Nutrition (Text with EEA Relevance). OJ L vol. 268 (2003).
9. Commission Delegated Regulation (EU) 2021/1760 of 26 May 2021 Supplementing Regulation (EU) 2019/6 of the European Parliament and of the Council by Establishing the Criteria for the Designation of Antimicrobials to Be Reserved for the Treatment of Certain Infections in Humans (Text with EEA Relevance). OJ L vol. 353 (2021).
10. Regulation (EU) 2019/6 of the European Parliament and of the Council of 11 December 2018 on Veterinary Medicinal Products and Repealing Directive 2001/82/EC (Text with EEA Relevance). OJ L vol. 004 (2018).
11. Hejna, M., Kovanda, L., Rossi, L. & Liu, Y. Mint oils: In vitro ability to perform anti-inflammatory, antioxidant, and antimicrobial activities and to enhance intestinal barrier integrity. Antioxid. Basel Switz. 10, 1004 (2021).
12. Patel SJ Wellington M Shah RM Ferreira MJ Antibiotic stewardship in food-producing animals: Challenges, progress, and opportunities Clin. Ther. 2020 42 1649 1658 10.1016/j.clinthera.2020.07.004 32819723
Patel, S. J., Wellington, M., Shah, R. M. & Ferreira, M. J. Antibiotic stewardship in food-producing animals: Challenges, progress, and opportunities. Clin. Ther. 42, 1649–1658 (2020).32819723 10.1016/j.clinthera.2020.07.004
13. Wong, B. T. et al. Dietary supplementation of botanical blends enhanced performance and disease resistance of weaned pigs experimentally infected with enterotoxigenic Escherichia coli F18. J. Anim. Sci. 100, skac353 (2022).
14. Madeira MS Microalgae as feed ingredients for livestock production and meat quality: A review Livest. Sci. 2017 205 111 121 10.1016/j.livsci.2017.09.020
Madeira, M. S. et al. Microalgae as feed ingredients for livestock production and meat quality: A review. Livest. Sci. 205, 111–121 (2017).10.1016/j.livsci.2017.09.020
15. Makkar HPS Seaweeds for livestock diets: A review Anim. Feed Sci. Technol. 2016 212 1 17 10.1016/j.anifeedsci.2015.09.018
Makkar, H. P. S. et al. Seaweeds for livestock diets: A review. Anim. Feed Sci. Technol. 212, 1–17 (2016).10.1016/j.anifeedsci.2015.09.018
16. Costa M Cardoso C Afonso C Bandarra NM Prates JAM Current knowledge and future perspectives of the use of seaweeds for livestock production and meat quality: A systematic review J. Anim. Physiol. Anim. Nutr. 2021 105 1075 1102 10.1111/jpn.13509
Costa, M., Cardoso, C., Afonso, C., Bandarra, N. M. & Prates, J. A. M. Current knowledge and future perspectives of the use of seaweeds for livestock production and meat quality: A systematic review. J. Anim. Physiol. Anim. Nutr. 105, 1075–1102 (2021).10.1111/jpn.13509
17. Ganesan AR Tiwari U Rajauria G Seaweed nutraceuticals and their therapeutic role in disease prevention Food Sci. Hum. Wellness 2019 8 252 263 10.1016/j.fshw.2019.08.001
Ganesan, A. R., Tiwari, U. & Rajauria, G. Seaweed nutraceuticals and their therapeutic role in disease prevention. Food Sci. Hum. Wellness 8, 252–263 (2019).10.1016/j.fshw.2019.08.001
18. Michalak, I. et al. Antioxidant effects of seaweeds and their active compounds on animal health and production—a review. Vet. Q. 42, 48–67.
19. Corino C Modina SC Di Giancamillo A Chiapparini S Rossi R Seaweeds in pig nutrition Animals 2019 9 1126 10.3390/ani9121126 31842324
Corino, C., Modina, S. C., Di Giancamillo, A., Chiapparini, S. & Rossi, R. Seaweeds in pig nutrition. Animals 9, 1126 (2019).31842324 10.3390/ani9121126
20. Pina-Pérez MC Rivas A Martínez A Rodrigo D Antimicrobial potential of macro and microalgae against pathogenic and spoilage microorganisms in food Food Chem. 2017 235 34 44 10.1016/j.foodchem.2017.05.033 28554644
Pina-Pérez, M. C., Rivas, A., Martínez, A. & Rodrigo, D. Antimicrobial potential of macro and microalgae against pathogenic and spoilage microorganisms in food. Food Chem. 235, 34–44 (2017).28554644 10.1016/j.foodchem.2017.05.033
21. Pereira L Morrison L Shukla PS Critchley AT A concise review of the brown macroalga Ascophyllum nodosum (Linnaeus) Le Jolis J. Appl. Phycol. 2020 32 3561 3584 10.1007/s10811-020-02246-6
Pereira, L., Morrison, L., Shukla, P. S. & Critchley, A. T. A concise review of the brown macroalga Ascophyllum nodosum (Linnaeus) Le Jolis. J. Appl. Phycol. 32, 3561–3584 (2020).10.1007/s10811-020-02246-6
22. Kim S-K Himaya SWA Medicinal effects of phlorotannins from marine brown algae Adv. Food Nutr. Res. 2011 64 97 109 10.1016/B978-0-12-387669-0.00008-9 22054941
Kim, S.-K. & Himaya, S. W. A. Medicinal effects of phlorotannins from marine brown algae. Adv. Food Nutr. Res. 64, 97–109 (2011).22054941 10.1016/B978-0-12-387669-0.00008-9
23. Dell’Anno M In vitro evaluation of antimicrobial and antioxidant activities of algal extracts Ital. J. Anim. Sci. 2020 19 103 113 10.1080/1828051X.2019.1703563
Dell’Anno, M. et al. In vitro evaluation of antimicrobial and antioxidant activities of algal extracts. Ital. J. Anim. Sci. 19, 103–113 (2020).10.1080/1828051X.2019.1703563
24. Venardou B Evaluation of the antibacterial and prebiotic potential of Ascophyllum nodosum and its extracts using selected bacterial members of the pig gastrointestinal microbiota Mar. Drugs 2021 20 41 10.3390/md20010041 35049896
Venardou, B. et al. Evaluation of the antibacterial and prebiotic potential of Ascophyllum nodosum and its extracts using selected bacterial members of the pig gastrointestinal microbiota. Mar. Drugs 20, 41 (2021).35049896 10.3390/md20010041
25. Kadam SU Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound assisted extraction characterization and bioactivity Mar. Drugs 2015 13 4270 4280 10.3390/md13074270 26184235
Kadam, S. U. et al. Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound assisted extraction characterization and bioactivity. Mar. Drugs 13, 4270–4280 (2015).26184235 10.3390/md13074270
26. Yuan YV Bone DE Carrington MF Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro Food Chem. 2005 91 485 494 10.1016/j.foodchem.2004.04.039
Yuan, Y. V., Bone, D. E. & Carrington, M. F. Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro. Food Chem. 91, 485–494 (2005).10.1016/j.foodchem.2004.04.039
27. Lopes D A new look for the red macroalga Palmaria palmata: A seafood with polar lipids rich in EPA and with antioxidant properties Mar. Drugs 2019 17 533 10.3390/md17090533 31540326
Lopes, D. et al. A new look for the red macroalga Palmaria palmata: A seafood with polar lipids rich in EPA and with antioxidant properties. Mar. Drugs 17, 533 (2019).31540326 10.3390/md17090533
28. Dumay J Clément N Morançais M Fleurence J Optimization of hydrolysis conditions of Palmaria palmata to enhance R-phycoerythrin extraction Bioresour. Technol. 2013 131 21 27 10.1016/j.biortech.2012.12.146 23334315
Dumay, J., Clément, N., Morançais, M. & Fleurence, J. Optimization of hydrolysis conditions of Palmaria palmata to enhance R-phycoerythrin extraction. Bioresour. Technol. 131, 21–27 (2013).23334315 10.1016/j.biortech.2012.12.146
29. Shi Q Overview on the antiviral activities and mechanisms of marine polysaccharides from seaweeds Carbohydr. Res. 2017 453–454 1 9 10.1016/j.carres.2017.10.020 29102716
Shi, Q. et al. Overview on the antiviral activities and mechanisms of marine polysaccharides from seaweeds. Carbohydr. Res. 453–454, 1–9 (2017).29102716 10.1016/j.carres.2017.10.020
30. Guidara M Effect of extraction procedures on the chemical structure, antitumor and anticoagulant properties of ulvan from Ulva lactuca of Tunisia coast Carbohydr. Polym. 2021 253 117283 10.1016/j.carbpol.2020.117283 33278949
Guidara, M. et al. Effect of extraction procedures on the chemical structure, antitumor and anticoagulant properties of ulvan from Ulva lactuca of Tunisia coast. Carbohydr. Polym. 253, 117283 (2021).33278949 10.1016/j.carbpol.2020.117283
31. EL-Sayed, A. I. M., El-Sheekh, M. M. & Makhlof, M. E. M. Synergistic antibacterial effects of Ulva lactuca methanolic extract alone and in combination with different antibiotics on multidrug-resistant Klebsiella pneumoniae isolate. BMC Microbiol. 23, 106 (2023).
32. Bikker P Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels J. Appl. Phycol. 2016 28 3511 3525 10.1007/s10811-016-0842-3 28035175
Bikker, P. et al. Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels. J. Appl. Phycol. 28, 3511–3525 (2016).28035175 10.1007/s10811-016-0842-3
33. Reggi S In vitro digestion of chestnut and quebracho tannin extracts: Antimicrobial effect, antioxidant capacity and cytomodulatory activity in swine intestinal IPEC-J2 cells Animals 2020 10 195 10.3390/ani10020195 31979207
Reggi, S. et al. In vitro digestion of chestnut and quebracho tannin extracts: Antimicrobial effect, antioxidant capacity and cytomodulatory activity in swine intestinal IPEC-J2 cells. Animals 10, 195 (2020).31979207 10.3390/ani10020195
34. Yang S-K Additivity vs synergism: Investigation of the additive interaction of cinnamon bark oil and meropenem in combinatory therapy Mol. Basel Switz. 2017 22 1733
Yang, S.-K. et al. Additivity vs synergism: Investigation of the additive interaction of cinnamon bark oil and meropenem in combinatory therapy. Mol. Basel Switz. 22, 1733 (2017).
35. Reggi S Dell’Anno M Baldi A Rossi L Seed-specific expression of porcine verotoxigenic Escherichia coli antigens in tobacco plants as a potential model of edible vaccines Vet. Res. Commun. 2024 10.1007/s11259-024-10318-y 38319502
Reggi, S., Dell’Anno, M., Baldi, A. & Rossi, L. Seed-specific expression of porcine verotoxigenic Escherichia coli antigens in tobacco plants as a potential model of edible vaccines. Vet. Res. Commun.10.1007/s11259-024-10318-y (2024).38319502 10.1007/s11259-024-10318-y
36. Bikker P Stokvis L van Krimpen MM van Wikselaar PG Cone JW Evaluation of seaweeds from marine waters in Northwestern Europe for application in animal nutrition Anim. Feed Sci. Technol. 2020 263 114460 10.1016/j.anifeedsci.2020.114460
Bikker, P., Stokvis, L., van Krimpen, M. M., van Wikselaar, P. G. & Cone, J. W. Evaluation of seaweeds from marine waters in Northwestern Europe for application in animal nutrition. Anim. Feed Sci. Technol. 263, 114460 (2020).10.1016/j.anifeedsci.2020.114460
37. Ford L Effect of phlorotannins from brown seaweeds on the in vitro digestibility of pig feed Anim. Open Access J. MDPI 2020 10 2193
Ford, L. et al. Effect of phlorotannins from brown seaweeds on the in vitro digestibility of pig feed. Anim. Open Access J. MDPI 10, 2193 (2020).
38. Samarasinghe, M. B. et al. A descriptive chemical analysis of seaweeds, Ulva sp., Saccharina latissima and Ascophyllum nodosum harvested from Danish and Icelandic waters. Anim. Feed Sci. Technol. 278, 115005 (2021).
39. Tayyab U Novoa-Garrido M Roleda MY Lind V Weisbjerg MR Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows Anim. Feed Sci. Technol. 2016 213 44 54 10.1016/j.anifeedsci.2016.01.003
Tayyab, U., Novoa-Garrido, M., Roleda, M. Y., Lind, V. & Weisbjerg, M. R. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows. Anim. Feed Sci. Technol. 213, 44–54 (2016).10.1016/j.anifeedsci.2016.01.003
40. Frazzini S Antioxidant and antimicrobial activity of algal and cyanobacterial extracts: An in vitro study Antioxidants 2022 11 992 10.3390/antiox11050992 35624856
Frazzini, S. et al. Antioxidant and antimicrobial activity of algal and cyanobacterial extracts: An in vitro study. Antioxidants 11, 992 (2022).35624856 10.3390/antiox11050992
41. Cherry P O’Hara C Magee PJ McSorley EM Allsopp PJ Risks and benefits of consuming edible seaweeds Nutr. Rev. 2019 77 307 329 10.1093/nutrit/nuy066 30840077
Cherry, P., O’Hara, C., Magee, P. J., McSorley, E. M. & Allsopp, P. J. Risks and benefits of consuming edible seaweeds. Nutr. Rev. 77, 307–329 (2019).30840077 10.1093/nutrit/nuy066
42. Ghosh, S., Nukavarapu, S. P. & Jala, V. R. Effects of heavy metals on gut barrier integrity and gut microbiota. Microbiota Host 2, (2024).
43. Bist P Choudhary S Impact of heavy metal toxicity on the gut microbiota and its relationship with metabolites and future probiotics strategy: A review Biol. Trace Elem. Res. 2022 200 5328 5350 10.1007/s12011-021-03092-4 34994948
Bist, P. & Choudhary, S. Impact of heavy metal toxicity on the gut microbiota and its relationship with metabolites and future probiotics strategy: A review. Biol. Trace Elem. Res. 200, 5328–5350 (2022).34994948 10.1007/s12011-021-03092-4
44. Tchounwou PB Yedjou CG Patlolla AK Sutton DJ Heavy metals toxicity and the environment EXS 2012 101 133 164 22945569
Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K. & Sutton, D. J. Heavy metals toxicity and the environment. EXS 101, 133–164 (2012).22945569
45. Commission Regulation (EU) No 1275/2013 of 6 December 2013 amending Annex I to Directive 2002/32/EC of the European Parliament and of the Council as regards maximum levels for arsenic, cadmium, lead, nitrites, volatile mustard oil and harmful botanical impuritiesText with EEA relevance.
46. Pandey D Differential impacts of post-harvest hydrothermal treatments on chemical composition and in vitro digestibility of two brown macroalgae (Fucales, Phaeophyceae), Ascophyllum nodosum and Fucus vesiculosus, for animal feed applications J. Appl. Phycol. 2023 35 2511 2529 10.1007/s10811-023-03044-6
Pandey, D. et al. Differential impacts of post-harvest hydrothermal treatments on chemical composition and in vitro digestibility of two brown macroalgae (Fucales, Phaeophyceae), Ascophyllum nodosum and Fucus vesiculosus, for animal feed applications. J. Appl. Phycol. 35, 2511–2529 (2023).10.1007/s10811-023-03044-6
47. Circuncisão AR Catarino MD Cardoso SM Silva AMS Minerals from macroalgae origin: Health benefits and risks for consumers Mar. Drugs 2018 16 400 10.3390/md16110400 30360515
Circuncisão, A. R., Catarino, M. D., Cardoso, S. M. & Silva, A. M. S. Minerals from macroalgae origin: Health benefits and risks for consumers. Mar. Drugs 16, 400 (2018).30360515 10.3390/md16110400
48. Palafox-Carlos H Ayala-Zavala JF González-Aguilar GA The role of dietary fiber in the bioaccessibility and bioavailability of fruit and vegetable antioxidants J. Food Sci. 2011 76 R6 R15 10.1111/j.1750-3841.2010.01957.x 21535705
Palafox-Carlos, H., Ayala-Zavala, J. F. & González-Aguilar, G. A. The role of dietary fiber in the bioaccessibility and bioavailability of fruit and vegetable antioxidants. J. Food Sci. 76, R6–R15 (2011).21535705 10.1111/j.1750-3841.2010.01957.x
49. Castejón N Thorarinsdottir KA Einarsdóttir R Kristbergsson K Marteinsdóttir G Exploring the potential of icelandic seaweeds extracts produced by aqueous pulsed electric fields-assisted extraction for cosmetic applications Mar. Drugs 2021 19 662 10.3390/md19120662 34940661
Castejón, N., Thorarinsdottir, K. A., Einarsdóttir, R., Kristbergsson, K. & Marteinsdóttir, G. Exploring the potential of icelandic seaweeds extracts produced by aqueous pulsed electric fields-assisted extraction for cosmetic applications. Mar. Drugs 19, 662 (2021).34940661 10.3390/md19120662
50. Cotas J Seaweed phenolics: From extraction to applications Mar. Drugs 2020 18 384 10.3390/md18080384 32722220
Cotas, J. et al. Seaweed phenolics: From extraction to applications. Mar. Drugs 18, 384 (2020).32722220 10.3390/md18080384
51. Matos J Cardoso C Serralheiro ML Bandarra NM Afonso C Seaweed bioactives potential as nutraceuticals and functional ingredients: A review J. Food Compos. Anal. 2024 133 106453 10.1016/j.jfca.2024.106453
Matos, J., Cardoso, C., Serralheiro, M. L., Bandarra, N. M. & Afonso, C. Seaweed bioactives potential as nutraceuticals and functional ingredients: A review. J. Food Compos. Anal. 133, 106453 (2024).10.1016/j.jfca.2024.106453
52. Sardari RRR Prothmann J Gregersen O Turner C Nordberg Karlsson E Identification of phlorotannins in the brown algae, Saccharina latissima and Ascophyllum nodosum by ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry Mol. Basel Switz. 2020 26 43
Sardari, R. R. R., Prothmann, J., Gregersen, O., Turner, C. & Nordberg Karlsson, E. Identification of phlorotannins in the brown algae, Saccharina latissima and Ascophyllum nodosum by ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Mol. Basel Switz. 26, 43 (2020).
53. Gupta S Abu-Ghannam N Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality attributes of foods Innov. Food Sci. Emerg. Technol. 2011 12 600 609 10.1016/j.ifset.2011.07.004
Gupta, S. & Abu-Ghannam, N. Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality attributes of foods. Innov. Food Sci. Emerg. Technol. 12, 600–609 (2011).10.1016/j.ifset.2011.07.004
54. Pootthachaya P Investigation of nutritional profile, protein solubility and in vitro digestibility of various algae species as an alternative protein source for poultry feed Algal Res. 2023 72 103147 10.1016/j.algal.2023.103147
Pootthachaya, P. et al. Investigation of nutritional profile, protein solubility and in vitro digestibility of various algae species as an alternative protein source for poultry feed. Algal Res. 72, 103147 (2023).10.1016/j.algal.2023.103147
55. Øverland M Mydland LT Skrede A Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals J. Sci. Food Agric. 2019 99 13 24 10.1002/jsfa.9143 29797494
Øverland, M., Mydland, L. T. & Skrede, A. Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals. J. Sci. Food Agric. 99, 13–24 (2019).29797494 10.1002/jsfa.9143
56. Shannon E Conlon M Hayes M Seaweed components as potential modulators of the gut microbiota Mar. Drugs 2021 19 358 10.3390/md19070358 34201794
Shannon, E., Conlon, M. & Hayes, M. Seaweed components as potential modulators of the gut microbiota. Mar. Drugs 19, 358 (2021).34201794 10.3390/md19070358
57. Lu LW Chen J-H Seaweeds as ingredients to lower glycemic potency of cereal foods synergistically—A perspective Foods 2022 11 714 10.3390/foods11050714 35267347
Lu, L. W. & Chen, J.-H. Seaweeds as ingredients to lower glycemic potency of cereal foods synergistically—A perspective. Foods 11, 714 (2022).35267347 10.3390/foods11050714
58. Mensah EO Kanwugu ON Panda PK Adadi P Marine fucoidans: Structural, extraction, biological activities and their applications in the food industry Food Hydrocoll. 2023 142 108784 10.1016/j.foodhyd.2023.108784
Mensah, E. O., Kanwugu, O. N., Panda, P. K. & Adadi, P. Marine fucoidans: Structural, extraction, biological activities and their applications in the food industry. Food Hydrocoll. 142, 108784 (2023).10.1016/j.foodhyd.2023.108784
59. Din NAS Brown algae as functional food source of fucoxanthin: A review Foods 2022 11 2235 10.3390/foods11152235 35954003
Din, N. A. S. et al. Brown algae as functional food source of fucoxanthin: A review. Foods 11, 2235 (2022).35954003 10.3390/foods11152235
60. Devillé C Damas J Forget P Dandrifosse G Peulen O Laminarin in the dietary fibre concept J. Sci. Food Agric. 2004 84 1030 1038 10.1002/jsfa.1754
Devillé, C., Damas, J., Forget, P., Dandrifosse, G. & Peulen, O. Laminarin in the dietary fibre concept. J. Sci. Food Agric. 84, 1030–1038 (2004).10.1002/jsfa.1754
61. Caprarulo V Evaluation of dietary administration of chestnut and quebracho tannins on growth, serum metabolites and fecal parameters of weaned piglets Anim. Open Access J. MDPI 2020 10 1945
Caprarulo, V. et al. Evaluation of dietary administration of chestnut and quebracho tannins on growth, serum metabolites and fecal parameters of weaned piglets. Anim. Open Access J. MDPI 10, 1945 (2020).
62. Mišurcová L Kráčmar S Klejdus B Vacek J Nitrogen content, dietary fiber, and digestibility in algal food products Czech J. Food Sci. 2010 28 27 35 10.17221/111/2009-CJFS
Mišurcová, L., Kráčmar, S., Klejdus, B. & Vacek, J. Nitrogen content, dietary fiber, and digestibility in algal food products. Czech J. Food Sci. 28, 27–35 (2010).10.17221/111/2009-CJFS
63. Azizi MN Chemical compositions of brown and green seaweed, and effects on nutrient digestibility in broiler chickens Animals 2021 11 2147 10.3390/ani11072147 34359273
Azizi, M. N. et al. Chemical compositions of brown and green seaweed, and effects on nutrient digestibility in broiler chickens. Animals 11, 2147 (2021).34359273 10.3390/ani11072147
64. Catarino MD Silva AMS Cardoso SM Fucaceae: A source of bioactive phlorotannins Int. J. Mol. Sci. 2017 18 1327 10.3390/ijms18061327 28635652
Catarino, M. D., Silva, A. M. S. & Cardoso, S. M. Fucaceae: A source of bioactive phlorotannins. Int. J. Mol. Sci. 18, 1327 (2017).28635652 10.3390/ijms18061327
65. Makkar HPS Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds Small Rumin. Res. 2003 49 241 256 10.1016/S0921-4488(03)00142-1
Makkar, H. P. S. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Rumin. Res. 49, 241–256 (2003).10.1016/S0921-4488(03)00142-1
66. Westermeier R Patiño DJ Müller H Müller DG Towards domestication of giant kelp (Macrocystis pyrifera) in Chile: selection of haploid parent genotypes, outbreeding, and heterosis J. Appl. Phycol. 2010 22 357 361 10.1007/s10811-009-9466-1
Westermeier, R., Patiño, D. J., Müller, H. & Müller, D. G. Towards domestication of giant kelp (Macrocystis pyrifera) in Chile: selection of haploid parent genotypes, outbreeding, and heterosis. J. Appl. Phycol. 22, 357–361 (2010).10.1007/s10811-009-9466-1
67. Scott AM Is antimicrobial administration to food animals a direct threat to human health? A rapid systematic review Int. J. Antimicrob. Agents 2018 52 316 323 10.1016/j.ijantimicag.2018.04.005 29660406
Scott, A. M. et al. Is antimicrobial administration to food animals a direct threat to human health? A rapid systematic review. Int. J. Antimicrob. Agents 52, 316–323 (2018).29660406 10.1016/j.ijantimicag.2018.04.005
68. Sharma, C. et al. Antimicrobial resistance: Its surveillance, impact, and alternative management strategies in dairy animals. Front. Vet. Sci. 4, (2018).
69. Brameyer S Heermann R Specificity of signal-binding via Non-AHL LuxR-type receptors PLOS ONE 2015 10 e0124093 10.1371/journal.pone.0124093 25923884
Brameyer, S. & Heermann, R. Specificity of signal-binding via Non-AHL LuxR-type receptors. PLOS ONE 10, e0124093 (2015).25923884 10.1371/journal.pone.0124093
70. Eom S-H The mechanism of antibacterial activity of phlorofucofuroeckol-A against methicillin-resistant Staphylococcus aureus Appl. Microbiol. Biotechnol. 2014 98 9795 9804 10.1007/s00253-014-6041-8 25267155
Eom, S.-H. et al. The mechanism of antibacterial activity of phlorofucofuroeckol-A against methicillin-resistant Staphylococcus aureus. Appl. Microbiol. Biotechnol. 98, 9795–9804 (2014).25267155 10.1007/s00253-014-6041-8
71. Rodrigues D Antitumor and antimicrobial potential of bromoditerpenes isolated from the Red Alga Sphaerococcus coronopifolius Mar. Drugs 2015 13 713 726 10.3390/md13020713 25629386
Rodrigues, D. et al. Antitumor and antimicrobial potential of bromoditerpenes isolated from the Red Alga Sphaerococcus coronopifolius. Mar. Drugs 13, 713–726 (2015).25629386 10.3390/md13020713
72. Wang Y Xu Z Bach SJ McAllister TA Sensitivity of Escherichia coli to Seaweed (Ascophyllum nodosum) Phlorotannins and terrestrial tannins Asian-Australas. J. Anim. Sci. 2009 22 238 245 10.5713/ajas.2009.80213
Wang, Y., Xu, Z., Bach, S. J. & McAllister, T. A. Sensitivity of Escherichia coli to Seaweed (Ascophyllum nodosum) Phlorotannins and terrestrial tannins. Asian-Australas. J. Anim. Sci. 22, 238–245 (2009).10.5713/ajas.2009.80213
73. Yu G A low-molecular-weight ascophyllan prepared from Ascophyllum nodosum: Optimization, analysis and biological activities Int. J. Biol. Macromol. 2020 153 107 117 10.1016/j.ijbiomac.2020.02.334 32135255
Yu, G. et al. A low-molecular-weight ascophyllan prepared from Ascophyllum nodosum: Optimization, analysis and biological activities. Int. J. Biol. Macromol. 153, 107–117 (2020).32135255 10.1016/j.ijbiomac.2020.02.334
74. Boisvert C Beaulieu L Bonnet C Pelletier É Assessment of the antioxidant and antibacterial activities of three species of edible seaweeds J. Food Biochem. 2015 39 377 387 10.1111/jfbc.12146
Boisvert, C., Beaulieu, L., Bonnet, C. & Pelletier, É. Assessment of the antioxidant and antibacterial activities of three species of edible seaweeds. J. Food Biochem. 39, 377–387 (2015).10.1111/jfbc.12146
75. Shannon E Abu-Ghannam N Antibacterial derivatives of Marine Algae: An overview of pharmacological mechanisms and applications Mar. Drugs 2016 14 81 10.3390/md14040081 27110798
Shannon, E. & Abu-Ghannam, N. Antibacterial derivatives of Marine Algae: An overview of pharmacological mechanisms and applications. Mar. Drugs 14, 81 (2016).27110798 10.3390/md14040081
76. Tan SP Extraction and bioautographic-guided separation of antibacterial compounds from Ulva lactuca J. Appl. Phycol. 2012 24 513 523 10.1007/s10811-011-9747-3
Tan, S. P. et al. Extraction and bioautographic-guided separation of antibacterial compounds from Ulva lactuca. J. Appl. Phycol. 24, 513–523 (2012).10.1007/s10811-011-9747-3
77. Cox, S., Abu-Ghannam, N. & Gupta, S. An assessment of the antioxidant and antimicrobial activity of six species of edible irish seaweeds. Articles.10.21427/D7HC92 (2010).
78. Lobiuc A Future antimicrobials: Natural and functionalized phenolics Molecules 2023 28 1114 10.3390/molecules28031114 36770780
Lobiuc, A. et al. Future antimicrobials: Natural and functionalized phenolics. Molecules 28, 1114 (2023).36770780 10.3390/molecules28031114
79. Caesar LK Cech NB Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2 Nat. Prod. Rep. 2019 36 869 888 10.1039/C9NP00011A 31187844
Caesar, L. K. & Cech, N. B. Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat. Prod. Rep. 36, 869–888 (2019).31187844 10.1039/C9NP00011A
80. Emu SA Effects of extracting solvents on phytochemical, antioxidant, and antibacterial activity of some seaweeds from the Bay of Bengal offshore Island Food Hum. 2023 1 1157 1166 10.1016/j.foohum.2023.09.005
Emu, S. A. et al. Effects of extracting solvents on phytochemical, antioxidant, and antibacterial activity of some seaweeds from the Bay of Bengal offshore Island. Food Hum. 1, 1157–1166 (2023).10.1016/j.foohum.2023.09.005
81. Echave J Seaweed-derived proteins and peptides: Promising marine bioactives Antioxidants 2022 11 176 10.3390/antiox11010176 35052680
Echave, J. et al. Seaweed-derived proteins and peptides: Promising marine bioactives. Antioxidants 11, 176 (2022).35052680 10.3390/antiox11010176
82. Akbarian M Chen S-H Instability challenges and stabilization strategies of pharmaceutical proteins Pharmaceutics 2022 14 2533 10.3390/pharmaceutics14112533 36432723
Akbarian, M. & Chen, S.-H. Instability challenges and stabilization strategies of pharmaceutical proteins. Pharmaceutics 14, 2533 (2022).36432723 10.3390/pharmaceutics14112533
83. Lovegrove A Role of polysaccharides in food, digestion, and health Crit. Rev. Food Sci. Nutr. 2017 57 237 253 10.1080/10408398.2014.939263 25921546
Lovegrove, A. et al. Role of polysaccharides in food, digestion, and health. Crit. Rev. Food Sci. Nutr. 57, 237–253 (2017).25921546 10.1080/10408398.2014.939263
84. Wang X Liu M Yu C Li J Zhou X Biofilm formation: Mechanistic insights and therapeutic targets Mol. Biomed. 2023 4 49 10.1186/s43556-023-00164-w 38097907
Wang, X., Liu, M., Yu, C., Li, J. & Zhou, X. Biofilm formation: Mechanistic insights and therapeutic targets. Mol. Biomed. 4, 49 (2023).38097907 10.1186/s43556-023-00164-w
85. Lohvina, H., Sándor, M. & Wink, M. Effect of ethanol solvents on total phenolic content and antioxidant properties of seed extracts of fenugreek (Trigonella foenum-graecum L.) varieties and determination of phenolic composition by HPLC-ESI-MS. Diversity 14, 7 (2022).
86. Saini RK Prasad P Shang X Keum Y-S Advances in lipid extraction methods—A review Int. J. Mol. Sci. 2021 22 13643 10.3390/ijms222413643 34948437
Saini, R. K., Prasad, P., Shang, X. & Keum, Y.-S. Advances in lipid extraction methods—A review. Int. J. Mol. Sci. 22, 13643 (2021).34948437 10.3390/ijms222413643
87. Plaskova A Mlcek J New insights of the application of water or ethanol-water plant extract rich in active compounds in food Front. Nutr. 2023 10 1118761 10.3389/fnut.2023.1118761 37057062
Plaskova, A. & Mlcek, J. New insights of the application of water or ethanol-water plant extract rich in active compounds in food. Front. Nutr. 10, 1118761 (2023).37057062 10.3389/fnut.2023.1118761
88. Poljsak B Šuput D Milisav I Achieving the balance between ROS and antioxidants: When to use the synthetic antioxidants Oxid. Med. Cell. Longev. 2013 2013 e956792 10.1155/2013/956792
Poljsak, B., Šuput, D. & Milisav, I. Achieving the balance between ROS and antioxidants: When to use the synthetic antioxidants. Oxid. Med. Cell. Longev. 2013, e956792 (2013).10.1155/2013/956792
89. Amorati R Foti MC Valgimigli L Antioxidant activity of essential oils J. Agric. Food Chem. 2013 61 10835 10847 10.1021/jf403496k 24156356
Amorati, R., Foti, M. C. & Valgimigli, L. Antioxidant activity of essential oils. J. Agric. Food Chem. 61, 10835–10847 (2013).24156356 10.1021/jf403496k
90. Bartel I Effect of dried apple pomace (DAP) as a feed additive on antioxidant system in the rumen fluid Int. J. Mol. Sci. 2022 23 10475 10.3390/ijms231810475 36142387
Bartel, I. et al. Effect of dried apple pomace (DAP) as a feed additive on antioxidant system in the rumen fluid. Int. J. Mol. Sci. 23, 10475 (2022).36142387 10.3390/ijms231810475
91. Khan, I. & Ahmad, S. The Impact of Natural Antioxidants on Human Health. In Functional Food Products and Sustainable Health (eds. Ahmad, S. & Al-Shabib, N. A.), pp. 11–24 (Springer, Singapore, 2020). 10.1007/978-981-15-4716-4_2.
92. Jacobsen, J., Am, S., Sl, H., Cc, A. & Db, H. Source, extraction, characterization, and applications of novel antioxidants from seaweed. Annu. Rev. Food Sci. Technol. 10, 1 (2019).
93. Liu Y Non-antibiotic feed additives in diets for pigs: A review Anim. Nutr. 2018 4 113 125 10.1016/j.aninu.2018.01.007 30140751
Liu, Y. et al. Non-antibiotic feed additives in diets for pigs: A review. Anim. Nutr. 4, 113–125 (2018).30140751 10.1016/j.aninu.2018.01.007
94. Gulcin İ Alwasel SH DPPH radical scavenging assay Processes 2023 11 2248 10.3390/pr11082248
Gulcin, İ & Alwasel, S. H. DPPH radical scavenging assay. Processes 11, 2248 (2023).10.3390/pr11082248
95. Ferreira ICFR Baptista P Vilas-Boas M Barros L Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity Food Chem. 2007 100 1511 1516 10.1016/j.foodchem.2005.11.043
Ferreira, I. C. F. R., Baptista, P., Vilas-Boas, M. & Barros, L. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food Chem. 100, 1511–1516 (2007).10.1016/j.foodchem.2005.11.043
96. Kadam SU Tiwari BK O’Donnell CP Extraction, structure and biofunctional activities of laminarin from brown algae Int. J. Food Sci. Technol. 2015 50 24 31 10.1111/ijfs.12692
Kadam, S. U., Tiwari, B. K. & O’Donnell, C. P. Extraction, structure and biofunctional activities of laminarin from brown algae. Int. J. Food Sci. Technol. 50, 24–31 (2015).10.1111/ijfs.12692
97. Nishida, Y., Kumagai, Y., Michiba, S., Yasui, H. & Kishimura, H. Efficient extraction and antioxidant capacity of Mycosporine-like amino acids from red alga dulse palmaria palmata in Japan. Mar. Drugs 18, (2020).
98. Pappou S Extraction of bioactive compounds from Ulva lactuca Appl. Sci. 2022 12 2117 10.3390/app12042117
Pappou, S. et al. Extraction of bioactive compounds from Ulva lactuca. Appl. Sci. 12, 2117 (2022).10.3390/app12042117
99. Prasedya, E. S. et al. Antioxidant activity of Ulva lactuca L. from different coastal locations of Lombok Island, Indonesia. in 020003 (Lombok, Indonesia, 2019). 10.1063/1.5141281.
100. Bai L Antioxidant activities of natural polysaccharides and their derivatives for biomedical and medicinal applications Antioxidants 2022 11 2491 10.3390/antiox11122491 36552700
Bai, L. et al. Antioxidant activities of natural polysaccharides and their derivatives for biomedical and medicinal applications. Antioxidants 11, 2491 (2022).36552700 10.3390/antiox11122491
101. Chen L Physicochemical characterization, antioxidant and immunostimulatory activities of sulfated polysaccharides extracted from Ascophyllum nodosum Molecules 2018 23 1912 10.3390/molecules23081912 30065217
Chen, L. et al. Physicochemical characterization, antioxidant and immunostimulatory activities of sulfated polysaccharides extracted from Ascophyllum nodosum. Molecules 23, 1912 (2018).30065217 10.3390/molecules23081912
102. Yuan Y Macquarrie D Microwave assisted extraction of sulfated polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant activity Carbohydr. Polym. 2015 129 101 107 10.1016/j.carbpol.2015.04.057 26050894
Yuan, Y. & Macquarrie, D. Microwave assisted extraction of sulfated polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant activity. Carbohydr. Polym. 129, 101–107 (2015).26050894 10.1016/j.carbpol.2015.04.057
103. Holdt SL Kraan S Bioactive compounds in seaweed: functional food applications and legislation J. Appl. Phycol. 2011 23 543 597 10.1007/s10811-010-9632-5
Holdt, S. L. & Kraan, S. Bioactive compounds in seaweed: functional food applications and legislation. J. Appl. Phycol. 23, 543–597 (2011).10.1007/s10811-010-9632-5
104. Rajauria G Purification and molecular characterization of Fucoidan isolated from Ascophyllum nodosum brown seaweed grown in Ireland Mar. Drugs 2023 21 315 10.3390/md21050315 37233509
Rajauria, G. et al. Purification and molecular characterization of Fucoidan isolated from Ascophyllum nodosum brown seaweed grown in Ireland. Mar. Drugs 21, 315 (2023).37233509 10.3390/md21050315
105. Lee J-C Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties Cancer Cell Int. 2013 13 55 10.1186/1475-2867-13-55 23724847
Lee, J.-C. et al. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 13, 55 (2013).23724847 10.1186/1475-2867-13-55
106. Assadi I Nutritional quality and antioxidant capacity of a combination of pomegranate and date juices Int. J. Fruit Sci. 2019 19 300 314 10.1080/15538362.2018.1512438
Assadi, I. et al. Nutritional quality and antioxidant capacity of a combination of pomegranate and date juices. Int. J. Fruit Sci. 19, 300–314 (2019).10.1080/15538362.2018.1512438
107. Bag A Chattopadhyay RR Evaluation of synergistic antibacterial and antioxidant efficacy of essential oils of spices and herbs in combination PLOS ONE 2015 10 e0131321 10.1371/journal.pone.0131321 26132146
Bag, A. & Chattopadhyay, R. R. Evaluation of synergistic antibacterial and antioxidant efficacy of essential oils of spices and herbs in combination. PLOS ONE 10, e0131321 (2015).26132146 10.1371/journal.pone.0131321
108. Wu Z Chemical composition and antioxidant properties of essential oils from peppermint native spearmint and scotch spearmint Molecules 2019 24 2825 10.3390/molecules24152825 31382468
Wu, Z. et al. Chemical composition and antioxidant properties of essential oils from peppermint native spearmint and scotch spearmint. Molecules 24, 2825 (2019).31382468 10.3390/molecules24152825
109. Attard E A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols Cent. Eur. J. Biol. 2013 8 48 53
Attard, E. A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols. Cent. Eur. J. Biol. 8, 48–53 (2013).
110. López, A., Rico, M., Rivero, A. & Suárez de Tangil, M. The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts. Food Chem. 125, 1104–1109 (2011).
111. Mazur-Marzec H Occurrence of cyanobacteria and cyanotoxin in the Southern Baltic Proper Filamentous cyanobacteria versus single-celled picocyanobacteria Hydrobiologia 2013 701 235 252 10.1007/s10750-012-1278-7
Mazur-Marzec, H. et al. Occurrence of cyanobacteria and cyanotoxin in the Southern Baltic Proper Filamentous cyanobacteria versus single-celled picocyanobacteria. Hydrobiologia 701, 235–252 (2013).10.1007/s10750-012-1278-7
112. Official Methods of Analysis of AOAC International (AOAC International, Gaithersburg, Maryland, 2019).
113. Minekus M A standardised static in vitro digestion method suitable for food—an international consensus Food Funct. 2014 5 1113 1124 10.1039/C3FO60702J 24803111
Minekus, M. et al. A standardised static in vitro digestion method suitable for food—an international consensus. Food Funct. 5, 1113–1124 (2014).24803111 10.1039/C3FO60702J
114. Giromini C Fekete ÁA Givens DI Baldi A Lovegrove JA Short-communication: A comparison of the in vitro angiotensin-1-converting enzyme inhibitory capacity of dairy and plant protein supplements Nutrients 2017 9 1352 10.3390/nu9121352 29236035
Giromini, C., Fekete, Á. A., Givens, D. I., Baldi, A. & Lovegrove, J. A. Short-communication: A comparison of the in vitro angiotensin-1-converting enzyme inhibitory capacity of dairy and plant protein supplements. Nutrients 9, 1352 (2017).29236035 10.3390/nu9121352
115. Rossi L Protective effect of oral administration of transgenic tobacco seeds against verocytotoxic Escherichia coli strain in piglets Vet. Res. Commun. 2014 38 39 49 10.1007/s11259-013-9583-9 24249478
Rossi, L. et al. Protective effect of oral administration of transgenic tobacco seeds against verocytotoxic Escherichia coli strain in piglets. Vet. Res. Commun. 38, 39–49 (2014).24249478 10.1007/s11259-013-9583-9
116. Min BR Attwood GT McNabb WC Molan AL Barry TN The effect of condensed tannins from Lotus corniculatus on the proteolytic activities and growth of rumen bacteria Anim. Feed Sci. Technol. 2005 121 45 58 10.1016/j.anifeedsci.2005.02.007
Min, B. R., Attwood, G. T., McNabb, W. C., Molan, A. L. & Barry, T. N. The effect of condensed tannins from Lotus corniculatus on the proteolytic activities and growth of rumen bacteria. Anim. Feed Sci. Technol. 121, 45–58 (2005).10.1016/j.anifeedsci.2005.02.007
117. Zhou H-C Lin Y-M Wei S-D Tam NF Structural diversity and antioxidant activity of condensed tannins fractionated from mangosteen pericarp Food Chem. 2011 129 1710 1720 10.1016/j.foodchem.2011.06.036
Zhou, H.-C., Lin, Y.-M., Wei, S.-D. & Tam, N. F. Structural diversity and antioxidant activity of condensed tannins fractionated from mangosteen pericarp. Food Chem. 129, 1710–1720 (2011).10.1016/j.foodchem.2011.06.036
118. Chen F Enzymatic and non-enzymatic bioactive compounds, and antioxidant and antimicrobial activities of the extract from one selected wild berry (Rubus coreanus) as novel natural agent for food preservation LWT 2022 171 114133 10.1016/j.lwt.2022.114133
Chen, F. et al. Enzymatic and non-enzymatic bioactive compounds, and antioxidant and antimicrobial activities of the extract from one selected wild berry (Rubus coreanus) as novel natural agent for food preservation. LWT 171, 114133 (2022).10.1016/j.lwt.2022.114133
119. Mahmoudi M Boughalleb F Maaloul S Mabrouk M Abdellaoui R Phytochemical screening, antioxidant potential, and LC–ESI–MS profiling of ephedra alata and ephedra altissima seeds naturally growing in Tunisia Appl. Biochem. Biotechnol. 2023 195 5903 5915 10.1007/s12010-023-04370-8 36719522
Mahmoudi, M., Boughalleb, F., Maaloul, S., Mabrouk, M. & Abdellaoui, R. Phytochemical screening, antioxidant potential, and LC–ESI–MS profiling of ephedra alata and ephedra altissima seeds naturally growing in Tunisia. Appl. Biochem. Biotechnol. 195, 5903–5915 (2023).36719522 10.1007/s12010-023-04370-8
120. Chung SI Kang MY Lee SC In vitro and in vivo antioxidant activity of aged ginseng (Panax ginseng) Prev. Nutr. Food Sci. 2016 21 24 30 10.3746/pnf.2016.21.1.24 27069902
Chung, S. I., Kang, M. Y. & Lee, S. C. In vitro and in vivo antioxidant activity of aged ginseng (Panax ginseng). Prev. Nutr. Food Sci. 21, 24–30 (2016).27069902 10.3746/pnf.2016.21.1.24
121. Bhalodia NR Nariya PB Acharya RN Shukla VJ In vitro antioxidant activity of hydro alcoholic extract from the fruit pulp of Cassia fistula Linn Ayu 2013 34 209 214 10.4103/0974-8520.119684 24250133
Bhalodia, N. R., Nariya, P. B., Acharya, R. N. & Shukla, V. J. In vitro antioxidant activity of hydro alcoholic extract from the fruit pulp of Cassia fistula Linn. Ayu 34, 209–214 (2013).24250133 10.4103/0974-8520.119684
