**2. Mechanisms of action**

Benefits from the use of dietary acidifiers include positive effects on growth performance and health status (Figure 1). Proposed mechanisms of action include reduction or stabilization of gastric pH, resulting in increased activity of proteolytic enzymes and gastric retention time, and thus led to improvement of protein digestion. Organic acids may influence mucosal morphology or induce alterations in gut microflora through bacteriostatic or bactericidal actions, as well as enhance endogenous enzyme activity, stimulate pancreatic secretions, and they also serve as substrates in intermediary metabolism (Partanen & Mroz, 1999. It is also

The Prophylactic Use of Acidifiers as Antibacterial Agents in Swine 299

efficiency of digestion and scouring problems (Piva et al. 2002a). In addition, the pH activity profile of pepsin seems to be more active at a low pH. Some results indicate that short-chain fatty acids have a stimulatory effect on both endocrine and exocrine pancreatic secretions in pigs; pancreatic exocrine responses are ranked as: formic acid > lactic acid > acetic acid >

There are considerable variations in the results of response to acidification due to possible

• level of intraluminal production of acids in particular segments of the gastrointestinal

• hygiene and welfare standards (density/pen, ventilation intensity and area, cleaning

• quantity of fermentable carbohydrate substrates in the diet for bacterial growth,

• type / composition of diets and their acid-base or buffering capacity,

butyric acid > propionic acid (Harada et al. 1986).

dietary and other factors such as (Mroz, 2005):

• type / pKa / dose of supplemented acids,

• colonization and activity resulting in acids production, • receptors for bacterial colonization on the epithelial villi, • maternal immunity by vaccinations against pathogens,

tract by inhabiting microflora,

Fig. 1. Mode of action of acidifiers in pig

• feed palatability,

• age of pigs,

frequency etc.)

hypothesized that acidifiers could be related to the reduction of gastric emptying rate, the energy source in intestine, the chelation of minerals, the stimulation of digestive enzymes and the provision of an energy source in the distal gastointestinal tract. Organic acid supplementation can reduce dietary buffering capacity, which is expected to slow down the proliferation and/or colonization of undesirable microbes, e.g. *Escherichia coli*, in the gastro-ileal region, resulting in reduction of scouring (Partanen & Mroz, 1999; Partanen, 2001).


Table 3. List of most common salts of acids and their properties

The hypothesis that lowering dietary pH with organic acids reduces gastrointestinal pH has been tested in several studies. The low pH of gastric contents is thought to kill many ingested bacteria, while the gastric pH of newly weaned piglets is notably higher than of older pigs. So in newly weaned pigs this protective action may be enhanced by any low pH which is produced by acids in the feed in comparison to the gastric pH (Ravindran & Kornegay, 1993). Moreover, weaned piglets are physiologically immature and may not produce enough hydrochloric acid (HCl) to keep stomach pH at an optimum of approximately 3.5 (Ravindran & Kornegay, 1993). Weaned piglets are physiologically immature and may not produce enough hydrochloric acid (HCl) in order to keep stomach pH at an optimum of approximately 3.5.

The purpose of adding acidifiers in feed, is to lower the pH in the stomach below pH 5, resulting in an increased activity of proteolytic enzymes, improving protein digestibility and inhibiting the proliferation of pathogenic bacteria in the gastointestinal tract (Partanen & Mroz, 1999). At pH=3.5, digestion of proteins and populations of beneficial bacteria (lactobacilli) are maximized and harmful bacteria are inhibited. Organic acids, in a non dissociated form, are lipophilic and can diffuse across bacterial cell membranes to reach the interior of the cell. There, in the relatively high intracellular pH, organic acids dissociate and disrupt the bacterial cell function and this effect may be stronger in some bacteria than in others (Partanen, 2001). A low pH is required for conversion of pepsinogen to pepsin, which is the active form of the most important gastric proteolytic enzymes. Elevated gastric pH may lead to an ineffective gastric proteolysis as a result of limited pepsin activity, and then a greater proportion of protein may enter the small intestine intact, resulting in lower efficiency of digestion and scouring problems (Piva et al. 2002a). In addition, the pH activity profile of pepsin seems to be more active at a low pH. Some results indicate that short-chain fatty acids have a stimulatory effect on both endocrine and exocrine pancreatic secretions in pigs; pancreatic exocrine responses are ranked as: formic acid > lactic acid > acetic acid > butyric acid > propionic acid (Harada et al. 1986).

There are considerable variations in the results of response to acidification due to possible dietary and other factors such as (Mroz, 2005):

• feed palatability,

298 Antimicrobial Agents

hypothesized that acidifiers could be related to the reduction of gastric emptying rate, the energy source in intestine, the chelation of minerals, the stimulation of digestive enzymes and the provision of an energy source in the distal gastointestinal tract. Organic acid supplementation can reduce dietary buffering capacity, which is expected to slow down the proliferation and/or colonization of undesirable microbes, e.g. *Escherichia coli*, in the gastro-ileal

**Ca/ K / Na salts Antimicrobial activity Improvement of growth** 

Solid Neutral Feed Bosi et al. 2005, 2007

Solid Rancid / Neutral Feed Pallauf & Huter 1993

**(**eg. Amm. formate**)** Liquid Water, feed Eisemann &

The hypothesis that lowering dietary pH with organic acids reduces gastrointestinal pH has been tested in several studies. The low pH of gastric contents is thought to kill many ingested bacteria, while the gastric pH of newly weaned piglets is notably higher than of older pigs. So in newly weaned pigs this protective action may be enhanced by any low pH which is produced by acids in the feed in comparison to the gastric pH (Ravindran & Kornegay, 1993). Moreover, weaned piglets are physiologically immature and may not produce enough hydrochloric acid (HCl) to keep stomach pH at an optimum of approximately 3.5 (Ravindran & Kornegay, 1993). Weaned piglets are physiologically immature and may not produce enough hydrochloric acid (HCl) in order to keep stomach

The purpose of adding acidifiers in feed, is to lower the pH in the stomach below pH 5, resulting in an increased activity of proteolytic enzymes, improving protein digestibility and inhibiting the proliferation of pathogenic bacteria in the gastointestinal tract (Partanen & Mroz, 1999). At pH=3.5, digestion of proteins and populations of beneficial bacteria (lactobacilli) are maximized and harmful bacteria are inhibited. Organic acids, in a non dissociated form, are lipophilic and can diffuse across bacterial cell membranes to reach the interior of the cell. There, in the relatively high intracellular pH, organic acids dissociate and disrupt the bacterial cell function and this effect may be stronger in some bacteria than in others (Partanen, 2001). A low pH is required for conversion of pepsinogen to pepsin, which is the active form of the most important gastric proteolytic enzymes. Elevated gastric pH may lead to an ineffective gastric proteolysis as a result of limited pepsin activity, and then a greater proportion of protein may enter the small intestine intact, resulting in lower

**possible in Beneficial effects** 

Canibe et al., 2001 Øverland et al. 2000 Taube et al. 2009

Kirchgessner & Roth 1990

**performance** 

Roth et al. 1996 Mroz et al. 2002 Øverland et al. 2000 Papenbrock et al. 2005 Partanen et al. 2007 Paulicks et al. 2000 Windisch et al 2001

Piva et al. 2002b Partanen et al. 2007 Mazzoni et al. 2008 Le Gall et al. 2009

Heugte 2007

Eidelsburger et al. 1992b Bosi et al. 2006

region, resulting in reduction of scouring (Partanen & Mroz, 1999; Partanen, 2001).

**Name Physical form Odour Application** 

Solid Neutral Feed

Table 3. List of most common salts of acids and their properties

pH at an optimum of approximately 3.5.

Ca salts (eg Ca-formate, Ca-propionate

K salts (eg K-diformate, K-sorbate )

Na salts (eg Na – butyrate, Na- benzoate, Na - formate)

**Ammonium salts** 


Fig. 1. Mode of action of acidifiers in pig

The Prophylactic Use of Acidifiers as Antibacterial Agents in Swine 301

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

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 feed and also provides acid-binding capacity.

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, whereas certain salts of organic acids can increase it.

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