Alternative to Antibiotic Growth Promoters (AGPs)

Diarrhea can hinder the growth of piglets, being one of the important factors increasing mortality in the breeding process. According to research by relevant scientific teams, nutritional diarrhea is a significant cause of diarrhea. Academician Yin Yulong, Chief Researcher of the Subtropical Agricultural Ecology Research Institute of the Chinese Academy of Sciences, found through research that the imbalance and deficiency of nutrients such as iron, electrolytes, niacin, folic acid, etc., can inhibit the development of pig intestines through signal pathways, reduce the expression of genes related to digestion and absorption, thus causing functional disorders in piglet intestines leading to diarrhea. [1]. Pig feed containing mainly betaine, as well as active substances such as lauric acid, functional peptide EGF, sulfur-containing amino acids, can improve intestinal function and enhance the gastrointestinal absorption capacity of piglets. Sunwin Group from Shandong, China, has adopted this feed additive solution for large-scale production, becoming the largest betaine-based feed additive producer in China.

Pathogens such as Clostridium perfringens, pathogenic Escherichia coli, transmissible gastroenteritis, coccidiosis, proliferative ileitis, swine dysentery spirochete, Salmonella, and other pathogens are the culprits causing bacterial/viral diarrhea in piglets. Treatment is often carried out with antibiotics such as Norfloxacin, Gentamicin, Chloramphenicol, and Sulfonamides. However, the use of these antibiotics not only leads to the development of drug resistance in pathogens but also easily causes imbalance in the microbial community, resulting in secondary infections. If emergency treatment is not administered to piglets, farms are cautious about antibiotic use. Additionally, in accordance with relevant breeding regulations, residue standards for antibiotics such as chloramphenicol are strictly limited to 0.1 μg/kg [2]. The demand for antibiotic-free feed production is therefore growing stronger.

In 2020, according to the announcement No. 194 from the Chinese Ministry of Agriculture and Rural Affairs, Chinese feed producers ceased the production of feed additives containing growth-promoting drugs (excluding traditional Chinese medicine), and sales were fully discontinued in 2021. In 2005, the European Union introduced regulations on setting feed hygiene requirements Regulation (EC) No 183/2005, and FEFANA established the FAMI-QS certification system. According to official information, 137 new feed suppliers complying with this certification system were added throughout 2023. Olmix Group, headquartered in France, is a producer of animal/plant health products that primarily uses algae supplemented with clay in innovative formulations. Their product Ecopiglet™ utilizes marine sulfated polysaccharides extracted from algae to stimulate the secretion of mucins in the pig intestine, enhancing intestinal resistance. Lidervet from Tarragona, Spain, introduced the Liderfeed™ feed additive, containing active ingredients of plant volatile oils that effectively increase the final body weight and average daily weight gain of broiler chickens [3].

Plant Bioactive Compounds (PBCs) for Feed Additives

Eugenol

Clove (Syzygium aromaticum) is a common evergreen tree of the Myrtaceae family in the Indian Ocean region and on islands. Traditionally, it has been used as a cooking spice and traditional medicinal herb. Clove essential oil is primarily extracted commercially from clove flower buds. The secretion of essential oil from clove flower buds is higher than that from its fruit. Eugenol is the main chemical component of clove essential oil, and its relative percentage can range from 50 to 90%, depending on the origin of the cloves [4,5,6,7]. Apart from clove flower buds, eugenol is also found in cinnamon leaves (Cinnamomum zeylanicum), with a relative percentage reaching up to 70% [8]. In sweet basil, the relative percentage is approximately 18-20% [9].

Eugenol is an active substance approved by the European Union for use in feed additives (Regulation (EU) 2023/585, 2022/1452, 2019/898, 2015/1426). Research on the inhibitory effects of eugenol on necrotic enteritis (NE) in different growth stages of broiler chickens [10] has shown that eugenol can improve the intestinal health of broiler chickens. NE is caused by Clostridium perfringens in the chicken’s intestinal tract, and under certain conditions, it can lead to overgrowth of NetB toxin-producing strains, resulting in disease [11]. Due to its extremely high mortality rate, it poses a severe threat to the poultry farming industry. Experimental results [11] indicate that feed additives containing eugenol have beneficial effects on the poultry’s small intestine’s ability to resist pathogens. It increases the number of goblet cells, improves intestinal integrity, and repairs the damage caused by necrotic enteritis to the intestinal mucosa.

In the past, antibiotics were commonly used for treating broiler chickens on farms. With the trend towards antibiotic alternatives, farms are now utilizing plant extracts as substitutes for antibiotics. This not only helps maintain poultry production capacity but also inhibits the spread of avian influenza. According to statistics from the British Egg Industry Council [12], the antibiotic usage in the UK poultry farming industry was 13.66 mg/kg PCU in 2020, which is below the upper limit restriction of 25 mg/kg PCU. CIAs (Fluoroquinolones, Macrolides, Polymixins) class antibiotics are only used as a last resort for treatment.

Eugenol can also be obtained through synthetic preparation, using Guaiacol as the starting material, and undergoing a series of processes [13], followed by distillation to obtain synthetic-grade eugenol.

Carvacrol

Carvacrol is a monocyclic terpene compound and is an isomer of Thymol. It naturally occurs in oregano (Origanum vulgare) and marjoram (Origanum majorana L.), with relative percentages ranging from 13% to 34%. The relative percentages of their isomers fall within the range of 15% to 42%. Both are major chemical components of oregano [14,15]. In the industrial preparation, natural Carvacrol and Thymol are primarily extracted from the volatile oil of oregano leaves, with higher concentrations in flowers and leaves compared to roots and stems [16]. Research on different varieties of oregano and marjoram reveals an inverse relationship between Carvacrol and Thymol content within the plant at different temperatures: Thymol increases as the temperature decreases, while Carvacrol increases as the temperature rises [17].

The synthesis of Carvacrol is primarily achieved by catalyzing o-cresol (ortho-methyl phenol) with a catalyst in a reaction solvent. Alternatively, Carvacrol can be synthesized using carvone as a raw material, undergoing aromatic rearrangement under the influence of a strong acid.

Oregano is a commonly used medicinal herb in traditional medicine, making an in-depth study of carvacrol’s application highly promising. Carvacrol derivatives have made considerable progress in the fields of medicine and agriculture. Carvacrol aldehyde, Schiff base, and copper-Schiff base complex are aldehyde derivatives synthesized from Carvacrol. Relevant studies have demonstrated that the synthesized substance, copper-Schiff base complex, has inhibitory effects on lung cancer cells, suggesting its potential as an antioxidant drug [18]. Another carvacrol benzoyl derivative, Carvacryl benzoate, has been experimentally proven to exhibit extermination efficacy against the genus fire ants (Solenopsis sp.）. This compound can be utilized as an active ingredient in environmentally friendly insecticides [19].

Currently, Carvacrol and Thymol are more directly applied in the formulation of feed additives. After weaning, piglets are susceptible to pathogen infections due to their immature immune and digestive systems. Improving the intestinal barrier can enhance the effectiveness of nutrient absorption in poultry and livestock [20]. Carvacrol and Thymol can play a positive role in nutritional strategies [21]. Their oregano aroma can act as a feed flavor enhancer, enticing poultry and livestock to eat more. The antibacterial, anti-inflammatory, and pharmacological properties of Carvacrol and Thymol can improve the intestinal function of poultry and livestock. After feeding piglets with different control group diets, a comparison of the release of calprotectin (CALP) in their feces revealed that the control groups containing Carvacrol and Thymol significantly reduced the release of CALP [22]. CALP is a marker of gut inflammation, and the experimental results demonstrate the therapeutic effects of plant bioactive compounds such as Carvacrol and Thymol.

Thymol

Thymol is an isomer of Carvacrol, and they share numerous similarities in properties and characteristics. For instance, in terms of their broad-spectrum antibacterial properties, they both exhibit strong inhibitory effects against Staphylococcus aureus [23]. Consequently, they have been widely applied in food packaging applications.

However, they exhibit differences in certain aspects. Researchers have found that Carvacrol and Thymol have independent hydroxylases, which is the reason for the different environmental reactions of the two plant-derived natural compounds [17]. In terms of antibacterial effects, they demonstrate varying abilities against different fungi: Thymol has stronger inhibitory effects on green mold (Penicillium digitatum) and blue mold (Penicillum italicum) [24]; Thymol has a greater inhibitory effect than Carvacrol on the germination of wild-type spores in Trypticase Soy Broth (TSB) [25]. Carvacrol, on the other hand, exhibits the strongest broad-spectrum antibacterial capability against common food-decaying fungi such as Cladosporium spp. [26].

Carvacrol and other plant-derived bioactive compounds were included in the NAFISPACK (Natural Antimicrobials For Innovative and Safe Packaging) support program in 2008. Large companies, SMEs, and institutions such as Danisco A/S, Verdifresh S.L., Nutreco Servicios S.A., among others, participated in this project. The project confirmed the potential application of Carvacrol and other plant-derived bioactive compounds in the innovation of food packaging solutions.

Cinnamaldehyde (Cinnamic aldehyde)

Cinnamaldehyde (as known as Cinnamic aldehyde) naturally occurs in cinnamon essential oil, often referring to trans-Cinnamaldehyde. Cinnamon essential oil is derived from the bark of the evergreen trees, Cinnamomum cassia, commonly found in Guangxi Province, China, or Cinnamomum zeylanicum, known as Ceylon cinnamon, from Sri Lanka. The volatile oil content in the bark is typically higher than in the leaves and branches [27, 28]. The primary compounds vary across different parts of the tree: Cinnamaldehyde is the main compound in the bark of the cinnamon tree, with a relative percentage ranging from 70% to 80%. Eugenol is the main compound in the leaves, while natural camphor is the predominant compound in the roots of the cinnamon tree [28].

Due to the high boiling point and thermosensitive nature of natural Cinnamaldehyde, the primary method for its preparation involves vacuum distillation followed by further purification processes [29]. Synthetic Cinnamaldehyde can be prepared by oxidizing cinnamyl alcohol or by hydroxymethylene condensation reaction using benzaldehyde and acetaldehyde [30]. To further reduce costs, there are instances where products from a petrochemical route are used as substitutes: synthetic benzaldehyde is obtained from toluene and then converted into synthetic Cinnamaldehyde.

Currently, research on replacing monensin with plant bioactive compounds has been ongoing, and related studies [31] have once generated excitement in the market. The market has started introducing feed additive products add to cinnamon and garlic as plant sources. Novus International has introduced NEXT Enhance® 300, with their test results demonstrating increased milk production in cows. Orffa has launched Excential Alliin Plus™, and their test results show the product’s promotion of growth in white leg shrimp (Litopenaeus vannamei), improving feed efficiency. Additionally, when applied in high GC conditions, there is a significant increase in lysozyme activity in white leg shrimp, enhancing the aquaculture’s immune capability.

Simultaneously, Cinnamaldehyde can increase the quantity of probiotics in aquatic products. Relevant study have shown that feeding half-smooth tongue sole (Cynoglossus semilaevis) with fish feed containing Cinnamaldehyde or using it in combination with Bacillus subtilis can enhance the abundance of probiotics in the fish, improving the fish’s gastrointestinal absorption capacity [32]. When used in synergy with Lactobacillus fermentum, it has positive beneficial effects on the absorption function and immune capability of rainbow trout (Oncorhynchus mykiss) [33].

Plant bioactive compounds (PBCs) will be crucial ingredients

Plant bioactive compounds include terpenoids, polyphenols, alkaloids, carotenoids, and other plant-derived substances, possessing the potential to exert physiological effects on poultry, aquaculture, and even the human body. In plants, they play a natural role in defending against pests, pathogens, and inflammation. Compared to plant monomers, the bioactive compounds interact more specifically with the biological systems of animals, enhancing their immune capabilities. Currently, plant-derived natural compounds are often added to feed formulations either as plant essential oils or by incorporating finely ground plant materials. Alternatively, plant monomers can be extracted through distillation of plant essential oils, but their functions may not be fully released. There are several challenges in objectively assessing the current level of application of plant bioactive compounds in the market, and these issues urgently need solutions: stability needs improvement, dosage dependence is relatively high, and the collaborative effectiveness requires validation. Deepening the study of the absolute configuration of plant bioactive compounds contributes to unlocking more application potential in biological processes.

Refference

1. WANG HAOHAO, After 8 years, solves the nutritional ‘diarrhea’ problem in piglets, ScienceNet.cn
2. WANG YUNPENG al., Potential public hazard of using antibiotics in livestock industry, Chinese Journal of Antibiotics 2008
3. efsa Journal 2017
4. ZHANG RONGGUANG al., Analysis of Volatile Oil of Clove Bud from Guangxi and Hainan by GC-MS, Chinese Journal of Ethnomedicine and Ethnopharmacy 2022
5. V. K. Raina al.，Essential oil composition of Syzygium aromaticum leaf from Little Andaman, India，Flavour and Fragrance Journal 2001
6. Hicham Boughendjioua al., Essential Oil Composition of Syzygium aromaticum (L.), International Research Journal of Pharmacy and Medical Sciences 2018
7. Amanda Santos al., Characterization of the raw essential oil eugenol extracted from Syzygium aromaticum L., Journal of Thermal Analysis and Calorimetry, 2009
8. Upali M. Senanayake al., Volatile Constituents of Cinnamon (Cinnamomum zeylanicum) Oils, Journal of Agricultural and Food Chemistry 1978
9. Roberto F. Vieira al., Chemical characterization of basil (Ocimum spp.) based on volatile oils, Flavour and Fragrance Journal 2006
10. Alip Kumar al., Potential of a mixture of eugenol and garlic tincture to improve performance and intestinal health in broilers under necrotic enteritis challenge, Animal Nutrition 2022
11. Alip Kumar al., A Microencapsulated Mixture of Eugenol and Garlic Tincture Supplementation Mitigates the Effect of Necrotic Enteritis on Intestinal Integrity and Increases Goblet Cells in Broilers, Microorganisms 2021
12. Tony Mcdougal al., Antibiotic use indicators met by UK layer and broiler sectors
13. Parent CN111454133A
14. ZHU XIAOFU al., To Study and Compare the Chemical Constituents of Origanum vulgare L. from Different Habitats, Chinese Wild Plant Resources 2022
15. Barbara Teixeira al., Chemical composition and bioactivity of different oregano (Origanum vulgare) extracts and essential oil, Science of Food and Agriculture 2013
16. HAN FEI al., GC-MS analysis on volatile oil extracted from different parts of Origanum vulgare growing in Hubei province, Chinese Traditional and Herbal Drugs 2015
17. J. Novak al., Temperature Influences Thymol and Carvacrol Differentially in Origanum spp. (Lamiaceae), Journal of Essential Oil Research 2010
18. Anu Bansal al., Synthesis of Carvacrol Derivatives as Potential New Anticancer Agent against Lung Cancer, Molecules 2022
19. Jaciele O. Dantas al., Synthetic Carvacrol Derivatives for the Management of Solenopsis Ants: Toxicity, Sublethal Effects, and Horizontal Transfer, Agriculture 2023
20. Amlan Kumar Patra al., Modulation of gastrointestinal barrier and nutrient transport function in farm animals by natural plant bioactive compounds – A comprehensive review, Critical Reviews in Food Science and Nutrition 2019
21. ShuDong Liu al., Effects of oral administration of different dosages of carvacrol essential oils on intestinal barrier function in broilers, Animal Physiology and Animal Nutrition 2018
22. R. Rebucci al., Effects of nature identical essential oils (carvacrol, thymol and cinnamaldehyde) on growth performance of piglets and non-invasive markers of antioxidant status and calprotectin release, Livestock Science 2022
23. Javier Rúa al., Combination of Carvacrol and Thymol: Antimicrobial Activity Against Staphylococcus aureus and Antioxidant Activity, Foodborne Pathogens and Disease 2019
24. C.O. Pérez-Alfonso al., The effects of essential oils carvacrol and thymol on growth of Penicillium digitatum and P. italicum involved in lemon decay, International Journal of Food Microbiology 2012
25. TOSHIO SAKAI al., Different patterns of germination inhibition by carvacrol and thymol in Bacillus subtilis spores, Journal of Microorganism Control, 2023
26. S. Abbaszadeh al., Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungiEfficacité antifongique de thymol, carvacrol, eugenol et menthol comme alternative aux produits actifs sur la croissance des champignons contaminant les aliments， Journal de Mycologie Médicale 2014
27. LIU HONGXING al., Study on chemical component about Cinnamomum cassia oil from the different sections of Cinnamomum cassia by gas chromatography-Mass spectrometry, China Condiment 2011
28. R.O.B. Wijesekera al., The chemistry and technology of cinnamon, Food Science and Nutrition 2009
29. ZHONG CHANGYONG, Process for Purification of Cinnamicaldehyde from Cinnamon Oil, Chemistry and Industry of Forest Products, 2009
30. YU CHUNPING al., Advancements in the Synthesis Research of Cinnamaldehyde, ZHEJIANG CHEMICAL INDUSTRY 2023
31. D. Ferme., Effect of sodium monensin and cinnamaldehyde on the growth and phenotypic characteristics of Prevotella bryantii and Prevotella ruminicola, Folia Microbiologica 2008
32. Yang Wang al., Dietary cinnamaldehyde and Bacillus subtilis improve growth performance, digestive enzyme activity, and antioxidant capability and shape intestinal microbiota in tongue sole, Cynoglossus semilaevis, Aquaculture 2021
33. Saade A. Jasim al., The synergistic effects of the probiotic (Lactobacillus fermentum) and cinnamon, Cinnamomum sp. powder on growth performance, intestinal microbiota, immunity, antioxidant defence and resistance to Yersinia ruckeri infection in the rainbow trout (Oncorhynchus mykiss) under high rearing density, Aquaculture Research 2022