**6. Probiotics**

Metchnikoff recommended, since the early twentieth century, the intake of beneficial microbes for health, particularly in the treatment of pathologies of the gastrointestinal tract. In 2001, the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) officially defined probiotics as those live microorganisms that can confer a health benefit to the host, when consumed in adequate amounts [48]. Probiotics have had a considerable increase as an alternative over antibiotics used as growth promoters and pathogen control. This phenomenon has motivated the development of effective probiotic products for use in animal production [49, 50]. Prado et al. [50, 51] conducted an experiment where they evaluated an aerosolized probiotic formulation as a bactericidal method during incubation, compared against formaldehyde fumigation, where the results showed that the number of recovered non-selective aerobic bacteria and lactic acid bacteria increased in the incubation environment, thus suggesting the application of lactic acid bacteria in setters and hatchers. Likewise, lactic acid bacteria (*Lactobacillus acidophilus* and *Bacillus animalis*) administered *in ovo*, with the use of a commercial automated multiple egg injection system, have been used without affecting the hatch of fertile eggs. Although it recommends against administering *Bacillus animalis* at high concentrations of 105 and 106 CFU/mL, because they increase the number of chicks that bite and die, as well as contaminated eggs, the *Bacillus subtilis* strain is not recommended because it affects all stages of embryonic development, due to competition for nutrients or secretion of byproducts, such as bacteriocins, enzymes, and 2,3 butanediol, which is toxic to biological systems and damages the defense system and central nervous system [32, 52].

## **7. Chitosan**

Chitosan is a modified natural carbohydrate polymer derived from the deacetylation of chitin; it is insoluble in water but soluble in weak, organic acid solutions [53]; it is part of the exoskeleton of crustaceans, cuticle of insects, algae, and fungal cell walls [54]; it has physical and chemical properties, including antibacterial activity; and it has a high degree of biocompatibility [55]. Chitosan is primarily used as a reinforcement in vegetation development, due to its anti-fungal properties. Chitosan is a biomaterial that can be used as a biofilm with a selective permeability effect for O2 and CO2 with good properties to effectively control pathogenic microbial growth. Its antimicrobial activity is dose-dependent, and it exhibits simultaneous cell membrane permeability to small components [56–58].

Prado et al. [51] developed a chitosan biofilm to preserve table and fertile eggs; chitosan concentrations were 0.1, 5, and 10%; table and fertile eggs were impregnated with chitosan and subsequently challenged with *Salmonella enteritidis*, then stored for 1, 24, 96, and 168 h at 4°C. The lowest concentration of *Salmonella enteritidis* was for the 5 and 10% concentrations in the table egg. For the fertile egg, incubation variables showed no differences for the different concentrations of chitosan [51].

From the most recent studies, chitosan has been used in combination with essential oils across a wide application in the food industry, although for applications in table and fertile eggs, there are no reports of its effectiveness [59]. Another combination has been with slightly acidic, electrolyzed water, as a protective alternative against bacteria present in the eggshell. However, this process damages the cuticle, so after disinfection with slightly acidic, electrolyzed water, a chitosan-based coating was used to form a new, artificial cuticle to prevent loss of humidity and CO2 from the damaged cuticle, which had positive effects on eggs stored at 25°C for 42d, without loss of internal egg quality [14, 26].

## **8. Organic acids**

Organic acids, being natural products, have emerged as viable alternatives to formaldehyde for disinfection of fertile eggs in commercial hatcheries. These acids exist in a non-dissociated form and exhibit a measure of their dissociation through the Ka (acid dissociation constant) value. Organic acids are commonly found in nature and can be derived from various sources such as fruits, vegetables, and fermentation processes. Examples of organic acids include acetic acid (found in vinegar), citric acid (found in citrus fruits), lactic acid (found in dairy products), and formic acid (found in ants).

In their non-dissociated form, organic acids remain intact, allowing them to effectively penetrate the eggshell and target potential pathogens without harming the developing embryo inside. This characteristic makes them suitable for disinfecting fertile eggs in commercial hatcheries, where maintaining a sterile environment is crucial for successful incubation. The Ka value, also known as the acid dissociation constant, measures the extent to which an organic acid dissociates into its constituent ions in an aqueous solution. It provides an indication of the acid's strength and its ability to release hydrogen ions (H+ ) when in contact with water. The higher the Ka value, the greater the extent of dissociation and the stronger the acid.

By considering the Ka value of organic acids, hatchery operators can select appropriate disinfectants that effectively combat pathogens while minimizing any potential adverse effects on the developing embryos. The choice of organic acid for disinfection can be based on factors such as its antimicrobial efficacy, safety, and compatibility with the hatchery environment. Overall, organic acids offer a natural and sustainable alternative to formaldehyde for disinfection of fertile eggs in commercial hatcheries. Their non-dissociated form allows for effective penetration of the eggshell, while the Ka value helps determine the acid's dissociation extent and strength, aiding in the selection of appropriate disinfectants for optimal hatchery operations.

Acetic, ascorbic, citric, formic, lactic, propionic, and peracetic organic acids are regularly used in food disinfection processes at concentrations of 0.05–2.5%, with no toxic residues [60]. Some organic acids, such as lactic, acetic, citric, and peracetic acids, are weak acids in solution, since one part of their molecule is dissociated [H<sup>+</sup> ] [A<sup>−</sup> ] and the other is not [A]. The ratio between the dissociated and non-dissociated part is expressed by the dissociation constant pKa. By determining the acid concentration, pH and pKa, the concentration of the non-dissociated acid present in the solution is established [61].

Lactic acid or its ionized form, lactate, known by the official nomenclature 2-hydroxypropanoic acid, is a carboxylic acid, with a hydroxyl group on the carbon adjacent to the carboxyl group. There are two optical isomers: D (−) lactic and L (+) lactic, as well as a racemic form consisting of equimolar fractions of the L (+) and D (−) forms. Unlike the D (−) isomer, the L (+) configuration is metabolized by the human organism [62]. It is a slightly brown liquid; it is the natural component of meat produced by post-mortem glycolysis; and it is used in carcass washing with doses of 2.5–5.0% at temperatures not exceeding 55°C with application before or after the carcass cooling stage [63].

*Natural Products as an Alternative to Formaldehyde for Disinfection of Fertile Eggs… DOI: http://dx.doi.org/10.5772/intechopen.112568*

Acetic or ethanoic acid of natural origin is present in most fruits. It is produced by bacterial fermentation, is present in all fermented products, and its commercial form (vinegar) has been used as a disinfectant since the beginning of civilization. Doses used range from 1.5 to 14.4% or 52°C in spray for 10s. Negative Gram bacteria are more susceptible to acids than Positive Gram bacteria [64].

Citric acid is the main organic acid in fruits, such as lemons, which contain between 7 and 9% citric acid on a dry weight basis. The three carboxylate groups of citric acid mono-hydrate have different pKa values ranging from 3.15, 4.78, and 6. At doses of 2–5%, it reduces the count of pathogenic bacteria [65]. The antimicrobial action is due to the dissociated form; being an anion, it is highly polar, so it does not cross the plasma membrane of microorganisms easily, but its non-dissociated form does cross the membrane [66]. The references found on the use of organic acids as antimicrobials only refer to their use in carcasses and parts of raw poultry, where they measured the effectiveness in reducing the native flora or inoculated bacteria that were mostly *Salmonella* or *Campylobacter*; in the case of the use of organic acids in the disinfection of the eggshell, they can demineralize the eggshell and eliminate the cuticle [67], which is why it is important to conduct experiments that consider the form of preparation, concentration, and measurement of cuticle integrity and calcium carbonate levels.
