Organic Acid Redux

Organic acids have long been used to ensure the safety and improve the quality of food products. In addition to their potential antimicrobial activity, organic acids are applied in food products as antioxidants to delay lipid and protein oxidation, stabilize color, inhibit off odor, and enhance nutrition and quality attributes such as flavor, taste, tenderness, and juiciness of foods. Many organic acids–acetic, citric, formic, lactic, propionic, sorbic, and benzoic acids and their salts–are generally recognized as safe (GRAS). As product developers look for effective ways to formulate healthy, tasty, and safer clean label foods, revisiting the fundamentals of these acidic compounds and their applications can help spark new formulation strategies.

Modes of Action

Organic acids occur naturally as normal constituents of animal tissues and plants, or can be added as preservatives, acidity regulators or stabilizers to food products. Examples abound: citric acid in fruits and vegetables, malic acid in berries, cherries, apricots, and apples, tartaric acids in grapes and citrus, and benzoic acid in cranberries, to name a few. Friendly bacteria, fungi, and yeasts produce organic lactic and acetic acids during fermentation.

Organic acids are weak acids and do not dissociate completely in water. The undissociated form of organic acids crosses the cell membranes of microorganisms. The release of weak acid protons from intracellular dissociation causes two problems for the cell: acidification of the cytoplasm, and accumulation of acid anion in the cytoplasm. If the internal pH of the cell drops, cellular metabolism is impaired, and the cumulative effect of acid anion accumulation and reduced internal pH results in cell death. Microbial cells respond to the critical drop in pH by employing enzyme-catalyzed reactions to consume protons, producing basic compounds to help raise the low pH and eliminating protons at the expense of ATP consumption.

The mode of action of organic acids and their salts is generally attributed to the “classical weak-acid theory,” in which weak acids inhibit organisms by diffusion of undissociated acids through the membrane, dissociation within the cell to protons and anions, and consequent acidification of the cytoplasm. However, additional modes of action also have been suggested for the microbiological inhibition by weak organic acids. For example, lactic acid destabilizes the membrane of Gram-negative bacteria, which results in the formation of pores, denatures acid-sensitive proteins and DNA, and interferes with both metabolic and anabolic processes. Citric acid destabilizes the cellular outer membrane by chelation of essential ions. Sorbic acid inhibits intracellular enzymes and interferes with the microbial respiration chain crucial for cellular energy generation. The inhibitory activity of organic acid on the survival of pathogens and spoilage microorganisms could be due to one or more of these effects.

Factoring in Variables

The bacteriostatic or bactericidal properties of organic acids depend on the physicochemical properties of the surrounding environment and the extent of dissociation of the acid (i.e., pKa value). This means that the effectiveness of an organic acid in inactivating foodborne spoilage and pathogenic bacteria depends on the percentage of undissociated acid at a given pH. For example, in cold-fill hold acidified food products at pH 3.3 to 3.8, researchers have found that Escherichia coli O157:H7 is significantly more acid resistant than Salmonella enterica or Listeria monocytogenes at 10°C (50°F). At pH 4.1, E. coli O157:H7 and L. monocytogenes had similar heat and acid resistance in acidified vegetable brine (Breidt et al., 2005). In another study investigating cucumber juice with acetic acid at pH 4.6, researchers found that L. monocytogenes was the most heat- and acid-resistant organism tested, followed by S. enterica or E. coli O157:H7, depending on temperature between 56°C and 66°C. At a reference temperature of 71°C (160°F), the average 5-log reduction time was 4.9 min for L. monocytogenes, 4.4 min for E. coli O157:H7, and 3.3 min for S. enterica (Breidt et al. 2014).

Microorganisms also respond to changes in the external and internal concentrations of protons (i.e., hydrogen ions) to sustain life. The concentration of protons is measured as pH. At low pH where the proton concentration is high, acidic environments adversely affect cell structure and function. For coping with acid stress, some bacteria produce fatty acids at higher levels to modify the lipid composition of the membrane and reduce membrane permeability to protons. Acid-adapted cells have increased resistance to inactivation by organic acids and can survive better in acidified food compared to non-adapted cells. In acidified foods, acid-resistant E. coli O157:H7, Salmonella and L. monocytogenes may survive long enough to cause disease.

Processing Pointers

Acidic foods are high-acid foods that have a natural pH of 4.6 or below. Acidified foods are low-acid foods to which acid(s) or acid food(s) are added and that have a finished equilibrium pH of 4.6 or below and a water activity greater than 0.85. In the manufacture of acidified foods, organic acids such as acetic, lactic, or citric acid are added singly or in combination to control pathogenic and spoilage organisms, improve sensory attributes, and extend shelf life. Processors of acidified foods are required to validate the microbiological stability of their products based on process (such as thermal inactivation) or formula based preventive controls (such as acidification) according to the U.S. Food and Drug Administration’s Food Safety and Modernization Act.

Regulations governing acidified foods in the United States were primarily established to prevent botulinum toxin production by Clostridium botulinum, which will be inhibited if the pH is maintained at or below 4.6. While pathogenic microorganisms such as E. coli O157:H7 and Salmonella have not been found to grow in products at pH 4.0 or lower, they may adapt to acid conditions and survive for an extended period.

Traditionally, direct acidification of vegetables by high concentrations of acids without thermal processing was the primary method of preservation. Spoilage usually occurs due to improper acidification and pasteurization. Most acids play a major role in creating the overall taste and aroma profile and maintaining the color and texture of foods. High acetic acid products (pH <4.0) are the most microbiologically safe food products. However, strong aroma and flavor of acetic acid has led food manufacturers to consider milder organic acids such as citric, malic, and lactic acids.

Today, preservation is usually achieved by the addition of the low concentration of organic acids and mild pasteurization. In heat processed acidified foods, the processing conditions are determined to assure a 5-log reduction of vegetative bacterial pathogens such as E. coli O157:H7, Salmonella, and L. monocytogenes.

Thermal tolerance of pathogens in heat-treated products may vary at different pH levels and depending on the acidulant used. For example, E. coli O157:H7 was found to be significantly more heat- and acid-resistant than Salmonella and L. monocytogenes in tomato puree acidified to pH 4.2 using citric or acetic acid, while L. monocytogenes was the most heat- and acid-resistant in puree acidified to pH 3.8 using acetic acid but not citric acid (Dufort 2017).

Minimum growth requirements for pathogens are well established. However, the survival of pathogens varies significantly by acid type and the pH level associated with specific microbes. In one study, minimum pH values for growth of enterohemorrhagic E. coli in broth were 4.25 and 5.5 for hydrochloric acid and acetic acid, respectively. Salmonella was later found to be less heat resistant than E. coli O157:H7 or L. monocytogenes in brine solution adjusted to pH 4.1 with acetic acid. In commercial fermented vegetables, there has been a correlation between pH level and a 5-log reduction in E. coli O157:H7.

Similarly, a recent study found that E. coli O157:H7, Salmonella and L. monocytogenes were inactivated 0.5 h after inoculation in sauces acidified to pH 3.2 with acetic acid while at least 48 h was required to achieve the same inactivation when citric acid was used (Lobo et al. 2019). Researchers also determined the specific effects of acetic acid concentration and pH (independently and combined) on the survival of E. coli O157:H7. At an equivalent pH, acetic acid was more effective compared with the use of non-inhibitory acids such as hydrochloric acid to control pH.

Future Forward

Microbiological responses to acidic stress and acid resistance can be determined by using well known approaches from microbiology fields, or through many rapidly advancing novel technologies and approaches. For example, laboratory evolution experiments combined with whole genome sequencing (WGS) have given new insights into acid resistance and tolerance. WGS can provide higher resolving power than subtyping methods, as well as provide insights into acid resistance and tolerance. A combined approach using WGS and metagenomics may generate synergistic information in determining adaptive responses to acidic environment.

In addition, predictive microbiology, which is used to predict the effects of intrinsic, extrinsic, and processing factors on the growth of microorganisms, will likely advance understanding of acid applications for food safety and quality. These mathematical models are used to determine food shelf life or to evaluate the potential growth of pathogens in food. Such models have been developed to determine the effect of pH, concentration of the undissociated form of the acid used at a given pH, and other parameters such as temperature, water activity, salt, and preservatives (e.g., nitrite).