**2.1 Antimicrobial activity**

There are several commercial products with organic acids on the market, all with their own specific chemical and functional properties. As shown in Table 1, the inclusion of organic acids can reduce pH and the feed's buffering capacity, while their antimicrobial effect can prevent the growth of bacteria (especially Gram negative bacterial species, like *Salmonella spp.* and *E. coli*), yeasts and moulds. In the stomach, the pH is decreased, reducing the concentration of all the types of bacteria. In the small intestine, only the organic acids with antibacterial activity are able to inhibit bacteria growth. This is the main reason that the use of these acids has been proposed as a way of preventing or reducing the incidence of diarrhea in young pigs (Jensen et al. 2001; Tsiloyiannis et al. 2001a, 2001b: Piva et al. 2002a; Papatsiros et al. 2011). Thus, the organic acids are divided into two large groups. In the first group are included those with indirect effect on the decrease of the bacterial population by pH reduction and acting mainly on the stomach because the animal organism has the capability of preventing the decline in the acidity in the small intestine by buffering the medium with bicarbonate (fumaric, citric, malic and lactic acids). In the other group, are involved those organic acids (formic, acetic, propionic and sorbic acid), that have the ability to reduce the pH and affect directly Gram- bacteria by interfering in the bacterial cell with complex enzymes. These enzymes destroy the cell membrane and influence the mechanism of DNA duplication which prevents bacterial reproduction (Castro, 2005).

Many studies with dietary acidifiers have shown positive effects in improving growth rate, feed efficiency and acting against bacteria, yeast, fungi, moulds (Table 2), but others have found a negligible and even negative negative response (Radecki et al. 1988; Eidelsburger et al. 1992a; Manzanilla et al. 2004; Štukelj et al. 2010). It is likely that the antimicrobial effects of the organic acid ions, which act by controlling bacterial populations in the upper gastrointestinal tract, are responsible for the beneficial effects of these acids (Roth & Kirchgessner, 1998). Moreover, organic acids can also enhance the effects of antibiotics by improving their absorption (Radecki et al. 1988; Eidelsburger et al. 1992b). In addition, acidifiers can have an initial

Dietary buffering capacity varies substantially between different feedstuffs (Bolduan et al. 1988a, 1988b). The acid-buffering capacity is lowest in cereals and cereal by-products, intermediate or high in protein feedstuffs and very high in mineral sources (Jasaitis et al. 1987). Addition of organic acids reduces dietary pH curvili nearly depending on the acid pKa value and buffering capacity (Bolduan et al. 1988a, 1988b) of the diet. The pH-lowering effect of different organic acids is reduced in the following order: tartaric acid>citricacid>malic acid> fumaric acid>lactic and formic acids>acetic acid> propionic acid. Salts of organic acids have only a small influence on dietary pH, but the addition of protein and mineral sources to the diet weakens the pH-lowering effect of the acid (Roth & Kirchgessner, 1989). It seems reasonable to assume that the buffering capacity of feed can be considerably influenced by the selection of feed ingredients, and it may in part reflect the differences in the effectiveness of acidifiers. In general, organic acids lower dietary buffering capacity,

The greatest acidification benefits have been observed in diets formulated from cereals and plant proteins, while the growth-promoting effect in diets containing milk products is small (Giesting et al. 1991). The latter presumably holds true when lactose in milk products is converted to lactic acid by lactobacilli in the stomach, creating the desired reduction in pH

There are several commercial products with organic acids on the market, all with their own specific chemical and functional properties. As shown in Table 1, the inclusion of organic acids can reduce pH and the feed's buffering capacity, while their antimicrobial effect can prevent the growth of bacteria (especially Gram negative bacterial species, like *Salmonella spp.* and *E. coli*), yeasts and moulds. In the stomach, the pH is decreased, reducing the concentration of all the types of bacteria. In the small intestine, only the organic acids with antibacterial activity are able to inhibit bacteria growth. This is the main reason that the use of these acids has been proposed as a way of preventing or reducing the incidence of diarrhea in young pigs (Jensen et al. 2001; Tsiloyiannis et al. 2001a, 2001b: Piva et al. 2002a; Papatsiros et al. 2011). Thus, the organic acids are divided into two large groups. In the first group are included those with indirect effect on the decrease of the bacterial population by pH reduction and acting mainly on the stomach because the animal organism has the capability of preventing the decline in the acidity in the small intestine by buffering the medium with bicarbonate (fumaric, citric, malic and lactic acids). In the other group, are involved those organic acids (formic, acetic, propionic and sorbic acid), that have the ability to reduce the pH and affect directly Gram- bacteria by interfering in the bacterial cell with complex enzymes. These enzymes destroy the cell membrane and influence the mechanism

whereas certain salts of organic acids can increase it.

**2.1 Antimicrobial activity** 

and thus reducing the need for diet acidification (Easter, 1988).

of DNA duplication which prevents bacterial reproduction (Castro, 2005).

Many studies with dietary acidifiers have shown positive effects in improving growth rate, feed efficiency and acting against bacteria, yeast, fungi, moulds (Table 2), but others have found a negligible and even negative negative response (Radecki et al. 1988; Eidelsburger et al. 1992a; Manzanilla et al. 2004; Štukelj et al. 2010). It is likely that the antimicrobial effects of the organic acid ions, which act by controlling bacterial populations in the upper gastrointestinal tract, are responsible for the beneficial effects of these acids (Roth & Kirchgessner, 1998). Moreover, organic acids can also enhance the effects of antibiotics by improving their absorption (Radecki et al. 1988; Eidelsburger et al. 1992b). In addition, acidifiers can have an initial eradicating effect on bacteria in the feed (Lueck, 1980) and remain there as a first barrier, preventing re-contamination. Even *under good conditions*, all compound feeds have a certain content of germs (bacteria, viruses, fungi and protozoa), which may be proliferate under unfavourable harvest and storage conditions (Schöner, 2001). Preservatives reduce the incidence of germs in the feed and thus the quantity of germs consumed by the animals. The hygienic quality of feed is significantly improved. The addition of organic acid lowers the pH value of the feed and also provides acid-binding capacity.

In fact, organic acids associated with specific antimicrobial activity are short-chain acids (SCFA, C1–C7) and are either simple monocarboxylic acids such as formic, acetic, propionic and butyric acids, or carboxylic acids, bearing a hydroxyl group (usually on the carbon) such as lactic, malic, tartaric, and citric acids. Four organic acids commonly used in feed formic, acetic, propionic and lactic acid - have a specific ability to penetrate the bacterial cell wall and kill bacteria by interfering with their metabolism. These acids only pass the membrane in non dissociated form. Their primary antimicrobial action (strain-selective growth inhibition or delay) is through pH depression of the diet. However, the ability of organic acids to change from undissociated to dissociated form, depending on the environmental pH, makes them effective antimicrobial agents. When acid is in the undissociated form it can freely diffuse through the semi permeable membrane of microorganisms into their cell cytoplasm. Once inside the cell, where the pH is maintained near 7, the acid dissociates and suppress cell enzymes (decarboxylases and catalases) and nutrient transport systems (Lueck, 1980). The efficacy of an acid in inhibiting microbes is dependent on its pKa value which is the pH at which 50% of the acid is dissociated. Organic acids with higher pKa values are more effective preservatives and their antimicrobial efficacy is generally improved with increasing chain length and degree of unsaturation (Foegeding & Busta, 1991). In practice this means that the stomach pH has to be lower than 5 for optimal results. Without these specific antimicrobial acids, the pH needs to be very low to destroy bacteria. Some of the above acids' salts, have also shown to have benefits on growth performance. Other acids, such as sorbic and fumaric acid, have some antifungal activity and are short chain-carboxylic acids, containing double bonds. Organic acids are weak acids and are only partly dissociated; most of them, with antimicrobial activity, have a pKa 3 - 5.

In addition, each acid has its own spectrum of antimicrobial activity. Their antimicrobial effects vary from one acid to another, depending on concentration and pH (Chaveerach et al. 2002). For example, lactic acid is more effective in reducing gastric pH and coliforms (Jensen et al. 2001; Tsiloyiannis et al. 2001a; Øverland et al. 2007), whereas other acids, such as formic, propionic have broader antimicrobial activities and they can be effective against bacteria (e.g. coliforms, clostridia, Salmonella), fungi and yeast (Partanen & Mroz, 1999; Bosi et al. 2005; Creus et al. 2007; Øverland et al. 2007). Several reports have shown that the use of organic acids may reduce the coliform burden along the gastrointestinal tract (Bolduan et al. 1988b) and reduce scouring and piglet mortality or control postweaning diarrhea and edema disease in piglets (Tsiloyiannis et al. 2001a, 2001b; Piva et al. 2002a, Papatsiros et al. 2011). The following order of killing potency of coliform bacteria in the gastric digesta at pH 3, 4, and 5, are: propionic<formic<butyric<lactic<fumaric<benzoic were established (Naughton & Jensen, 2001; Knarreborg et al. 2002). Jensen et al. (2001) demonstrated that the potency of these acids against *Salmonella typhiumurium* in gastric digesta at pH4 was in the following order: acetic <formic < propionic < lactic < sorbic < benzoic. Inconsistent results may be due to the variety of diets with different buffering capacities that were used in these

The Prophylactic Use of Acidifiers as Antibacterial Agents in Swine 303

The use of acidifiers appears to be most beneficial in the early period after weaning. Studies demonstrating the improved feed conversion ratio, weight gain and growth-promoting effects of acidifiers indicated that the effect was greater in young pigs than older pigs (Radcliffe et al. 1998; Øverland et al. 2000; Partanen et al. 2007), but there is some evidence that they may be beneficial for improvement of daily gain and feed efficiency in growing-finishing pigs

The results of trials including the addition of inorganic acids in pig diets has indicated positive responses on growth performance (Walsh et al. 2007; Stein, 2007), especially during the period after weaning (Mahan et al. 1996, 1999). However, the use of other inorganic acids, such as sulfuric acid, has not shown positive effects on growth performance (Ravindran & Kornegay, 1993). In addition, salts of organic acids, such as formates and diformates can be used to significantly improve growth rate and feed conversion in pigs (Table 3). However, there are also studies with no responses (Biagi et al. 2007) or involving risk factors (Pallauf & Huter, 1993; Øverland et al. 2000). For example, calcium formate

a. Acidifiers may have a negative effect on diet palatability, when they are added at excessive levels, resulting in lower feed intake or feed refusal (Partanen & Mroz, 1999). Certain acids, e.g. tartaric and formic acids have a strong odour and flavour, and an increasing dietary acid level, which is generally associated with a dramatic decrease in feed intake, as reflected by lower daily gains (Eckel et al. 1992; Kirchgessner et al. 1993). Addition of excessive amounts of formates to the diet may also disturb the acid-base status of pigs leading to metabolic acidosis, which results in decreased feed intake and slower growth (Giesting et al. 1991; Eckel et al. 1992; Eidelsburger et al. 1992e). Organic acids metabolized via the citric-acid cycle, e.g. fumaric and citric-acids, do not seem to

cause acidosis, irrespective of their dietary inclusion (Eidelsburger et al. 1992c). b. Acids at high levels in feed are corrosive to cement and galvanized steel in pig housing, resulting to pose handling and equipment issues to the feed manufacturer. For example, formic acid is the most corrosive for the equipment and it is dangerous to handle, while fumuric acid is easy to handle (Mateos et al. 1999). Salts of organic acids are generally odorless and less corrosive than their acid forms, making them easier to handle in the

c. The use of organic acids in their free form, at levels that have been proven to be efficacious, can cause palatability problems (Partanen, 2001), damage the stomachal and duodenal mucosas (Argenzio & Eisemann, 1996), as well as cause bone demineralization (Partanen & Mroz, 1999) and an acidic stress, inducing a resistance

In order to minimize these effects, the natural buffering capacity of feeds (related to mineral and protein content) should be evaluated to determine the minimum effective amount of acid to use (Best, 2000). Another strategy to extend the effectiveness of acid supplements and reduce corrosion damage to housing materials is the use of a slow-release form of acids. It consists on the use of organic acids with fatty acids and mono- and diglycerides mixed to form microgranules. A study by Cerchiari (2000) showed that use of these granules, as

mechanism towards organic acids in certain bacteria (Bearson et al. 1997).

compared to use of free acids, results in greater feed intake and growth.

(Øverland et al. 2000; Partanen et al. 2001a; Gauthier 2002; Canibe et al. 2005).

decreased feed intake and daily gain (Pallauf & Huter, 1993; Øverland et al. 2000).

The use of organic acids in feed appears two main problems:

feed manufacturing process (Jacela et al. 2009).

**3. Risk factors of acidifier use** 

experiments. Bacteria are known to develop acid-resistance when exposed to acidic environments for some time (Mroz, 2005).

#### **2.2 Antibacterial activity and growth promoting effects**

The beneficial effects of organic acids and their salts on growth performance have been confirmed in several studies. Acidifiers added to pig diets may potentially help improve growth performance (Table 2 & 3) by improving digestive processes through several mechanisms. It is believed that acidifiers can enhance the growth performance by:


The use of some organic acids has been found to reduce the formation of biogenic amines (such as cadaverine and putrescin) that are produced particularly in high protein feeds and in feeds, containing added synthetic amino acids. Biogenic amines have unfavourable effects on growth and feed conversion. The growth stimulation effects of formic, acetic and propionic acids are partly caused by their inhibitory effect on biogenic amines (Eckel et al. 1992). However, a clear mode of action has not been fully described yet and the magnitude and consistency of the response may vary, depending on inclusion rate and other dietary factors. The use of acidifiers appears to be most beneficial in the early period after weaning. Studies demonstrating the improved feed conversion ratio, weight gain and growth-promoting effects of acidifiers indicated that the effect was greater in young pigs than older pigs (Radcliffe et al. 1998; Øverland et al. 2000; Partanen et al. 2007), but there is some evidence that they may be beneficial for improvement of daily gain and feed efficiency in growing-finishing pigs (Øverland et al. 2000; Partanen et al. 2001a; Gauthier 2002; Canibe et al. 2005).

The results of trials including the addition of inorganic acids in pig diets has indicated positive responses on growth performance (Walsh et al. 2007; Stein, 2007), especially during the period after weaning (Mahan et al. 1996, 1999). However, the use of other inorganic acids, such as sulfuric acid, has not shown positive effects on growth performance (Ravindran & Kornegay, 1993). In addition, salts of organic acids, such as formates and diformates can be used to significantly improve growth rate and feed conversion in pigs (Table 3). However, there are also studies with no responses (Biagi et al. 2007) or involving risk factors (Pallauf & Huter, 1993; Øverland et al. 2000). For example, calcium formate decreased feed intake and daily gain (Pallauf & Huter, 1993; Øverland et al. 2000).
