Minimum Inhibitory Concentration (MIC) of Propionic Acid, Sorbic Acid, and Benzoic Acid against Food Spoilage Microorganisms in Animal Products to Use MIC as Threshold for Natural Preservative Production

Unprocessed animal products and processed animal products were selected based on following criteria; i) cases of research on natural preservatives, ii) food items and raw materials with high consumption (MFDS, 2020), iii) fat content. For unprocessed animal products, eggs, chicken breast, chicken legs, pork ribs, pork sirloin, beef ribs, beef chuck, and milk samples were used. For processed animal products, processed butter, fermented milk, ground meat product, natural cheese, and smoked egg samples were used. These samples were purchased from local supermarkets and butcher shops.

Considering the strain variation of microorganisms, a strain mixture for each microorganism was prepared as inoculum. Bacteria strains were cultured in 10 mL of culture media at optimal incubation temperature for 24 h. Aliquots (0.1 mL) of the cultures were inoculated in 10 mL fresh culture media and subcultured at optimal temperature for 24 h. Yeast and mold strains were cultured in 10 mL of culture media at optimal incubation temperature for 24–48 h. Aliquots (0.1 mL) of the cultures were inoculated in 10 mL fresh culture media and subcultured at optimal temperature for 24–48 h. The cultures of the strains for each microorganism species were mixed. Each mixture was then centrifuged at 1,912×g and 15 min for 4°C, and the cell pellets were washed twice with phosphate-buffered saline [PBS; KH₂PO₄ 0.2 g, Na₂HPO₄ 1.5 g, NaCl 8.0 g, KCl 0.2 g, 1 L of distilled water (DW), pH 7.4]. For the bacteria and yeast inocula, cell pellets were diluted with PBS to have 6 Log CFU/mL. For the mold inocula, the resulting suspensions of conidia were vigorously vortexed, and sterile DW was added to the suspension to have 5 Log CFU/mL. Mold cell counts were measured by a hemacytometer, which was confirmed by a serial dilution plate count. The microorganism strains and culture media used in this study were presented in Table 1.

Bacteria that were the most sensitive to propionic acid, benzoic acid, and sorbic acid were used to determine MIC of preservatives in unprocessed animal products (eggs, chicken breast, chicken legs, pork ribs, pork sirloin, beef ribs, beef chunk, and milk) and processed animal products (processed butter, ground meat product, natural cheese, and smoked eggs). The selected bacteria were Campylobacter coli ATCC33559, Campylobacter jejuni ATCC33560, Erwinia carotovora KCCM11319, Micrococcus luteus KCCM11211, and Moraxella catarrhalis KCCM42707. A mixture of the bacteria was prepared according to the procedure described in the section of ‘Inoculum preparation’. Inoculum 0.1 mL was inoculated to 25 g of food sample in a sample bag to obtain a concentration of 4 Log CFU/g. A hundred microliters of the preservatives were then spiked in samples to have 0, 100, 500, 1,000, and 1,500 (1,200 ppm for sorbic acid) ppm. Pork ribs, pork loin, beef ribs, beef chunks, milk, processed butter, fermented milk, and natural cheese were stored at 10°C. Poultry and processed meat products were stored at 5°C, and smoked eggs were stored at 25°C. The sample (25 g) was aseptically transferred to a sample bag containing 225 mL of buffered peptone water (BPW; Becton Dickinson, Sparks, MD, USA), and the sample was pummeled for 60 s in a pummeler (BagMixer^® 400, Interscience, Saint Nom la Bretehe, France). One milliliter of the homogenate was serially diluted with BPW, and the homogenates were dispensed on an aerobic bacteria count plate (AC Petrifilm; 3M^TM Petrifilm aerobic count plate, 3M, St. Paul, MN, USA) to quantify the total bacteria. The AC Petrifilms were incubated at 35°C for 48 h, and the colonies were then manually counted. The end time of the storage was determined as the time when the bacterial cell counts in the 0-ppm sample increased to 6 Log CFU/g. This experiment was repeated three times. The bacterial cell counts for each concentration of preservatives at the end of the storage were compared to the cell counts on day 0. This comparison was conducted by pairwise t-test at α=0.05 with the general linear model procedure (proc glm) of SAS^® (ver.9.4, SAS Institute, Cary, NC, USA). If the difference was not significant, the concentration was determined as MIC per each replication. Among the MIC of 3 replications, the lowest MIC was determined as a final MIC.

To measure pH of the samples, 18 mL of DW was added to 2 g of the sample, and it was homogenized for 60 s in a pummeler. The pH of homogenate was measured using a pH meter (Thermo Fisher Scientific).

Control of microorganism growth in raw food materials and products is important in ensuring product safety, shelf life, and consumers’ health. In meat, Pseudomonas, Acinetobacter, and Brochothrix mainly affect the quality and may cause spoilage (Liang et al., 2021; Wei et al., 2021). Also, pathogenic bacteria such as Escherichia coli, Salmonella, Campylobacter, Listeria monocytogenes, and Staphylococcus aureus are frequently detected in meat (Kim et al., 2020; Lee and Yoon, 2021; Park et al., 2021; Yang et al., 2022). Spoilage yeasts mainly include Zygosaccharomyces, Saccharomyces, Candida and Brettanomyces, and spoilage molds include Zygomycetes, Penicillium, Aspergillus, etc. (Blackburn, 2006). Especially, spoiled meats and cheeses often have high cell counts of Debaryomyces, Yarrowia, and Rhodotorula (Blackburn, 2006). The MIC of propionic acid, sorbic acid, and benzoic acid to these microorganisms in broth media were determined at pH 7.0 (Table 2). To increase the solubility of preservatives, salts were combined with the preservatives. Calcium propionate, sodium propionate, sodium benzoate, and potassium sorbate were also examined, and they had higher MIC than acid-type preservatives (Table 2). C. coli, C. jejuni, M. catarrhalis, E. carotovora, and M. luteus had lower MIC for the preservatives (propionic acid, benzoic acid, and sorbic acid), compared to other microorganisms. The preservative used in this study is a weak-acid type, which increases the number of non-dissociated molecules, when the pH is lowered. Thus, the molecules easily penetrate the microbial cell membrane or protoplasm, which prevents microbial growth (Theron and Lues, 2007). Unlike the acidic-preservatives, salt preservatives are considered to have a high MIC, because their pH were close to neutral. To investigate the antibacterial activity of preservatives according to pH, MIC of the preservatives were investigated by adjusting the pH of the medium to 4.5, 5.5, and 6.0. The five bacterial strains showed lower MIC of the preservative at lower pH (Table 3). The MIC of the preservative for E. carotovora were 50 ppm for propionic acid, 25 ppm for sorbic acid, and 50 ppm for benzoic acid at pH 5.5, which were lower MIC than those at pH 6.0. These results confirmed that the microbial growth prevention efficacy of the weak-acid type preservatives increased at low pH as presented in other research.

Unprocessed animal products were inoculated with a mixture of the most sensitive foodborne bacteria selected by MIC to the preservatives, and the samples were stored at 10°C until the bacterial cell counts of the control increased to >10⁶ CFU/g, which is considered to be the level that the spoilage started. At this time the total bacteria in other samples were counted.

The MIC of preservatives in animal products are presented in Table 4. The MIC of propionic acid were 100 ppm in chicken legs, pork ribs, pork sirloin and beef ribs, 500 ppm in chicken breast, beef chunk and milk, and 1,500 ppm in eggs. The MIC of benzoic acid were 100 ppm in chicken legs, pork ribs, and pork sirloin, 500 ppm in chicken breast, beef ribs, beef chunk, and milk, and 1,500 ppm in eggs. The MIC of sorbic acid were 100 ppm in chicken breast, chicken legs, pork ribs, pork sirloin, beef ribs, and beef chunk, and 500 ppm in milk, and 1,200 ppm in eggs. The MIC of propionic acid, benzoic acid, and sorbic acid in processed butter and natural cheese were 100 ppm. In smoked eggs, MIC of propionic acid were 1,000 ppm, and MIC of benzoic acid and sorbic acid were 500 ppm. In our study, the MIC investigated in food were higher than pH in broth media. Specifically, the pH of ground meat was close to 6.0 and the MIC of propionic acid, benzoic acid, and sorbic acid were 1,500, >1,500, and >1,500 ppm, respectively. However, the MIC in the broth of the five strains of microorganisms used as inoculum were below 500 ppm at pH 6.0.

Preservatives are food additives that inhibit microbial growth in food, but most studies have identified MIC in microbiological media rather than food. Although few studies have evaluated the MIC of preservatives in food, it is known that the MIC of preservatives in food were higher than those in microbiological media (Brocklehurst et al., 1995; Weiss et al., 2015). While the media have homogeneous structure and consist of simple composition, the food consists of various components (fat, protein, fiber, and antibacterial substances) and structures (Weiss et al., 2015). Lipid content and preservative activity are correlated (Glass and Johnson, 2004; Weiss et al., 2015). Organic acids such as propionic acid bind to phospholipids in the bacterial cell membrane. However, the fat component in food also competitively binds to lipophilic molecules, making it difficult for preservatives to bind to bacteria. Electrostatic and hydrophobic interactions also significantly affect the activity of acid-type preservatives that are dissociated (Weiss et al., 2015). These reasons may also have caused the differences in MIC between the broth media and animal products in our study.