Comparative Study on the In Vitro Antibacterial Activity of Different Types of Acid Preparations

Abstract: To evaluate the in vitro antibacterial efficacy of different types of acid preparations, six common types of compound acid formulations available on the market were selected, and the plate culture method was employed. The study evaluated the in vitro antibacterial effects of six different types of acid preparations—formic acid type I, phosphoric acid, lactic acid, fumaric acid, citric acid, and formic acid type II—against four harmful bacteria (Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens) and four beneficial bacteria (Bifidobacterium, Butyricum, Lactobacillus, and Lactobacillus acidophilus), with the aim of identifying a composite acidifier formulation that exhibits superior in vitro antimicrobial activity. The results showed that formic acid type I, citric acid, and fumaric acid exerted strong inhibitory effects on both harmful and beneficial bacteria; meanwhile, lactic acid–based and phosphoric acid–based acid preparations performed relatively well, effectively suppressing harmful bacteria while still allowing a substantial population of beneficial bacteria to survive.

Keywords: Acidifier; organic acid; antibacterial effect; harmful bacteria; beneficial bacteria

There is a wide variety of acid preparations, most of which promote digestion; they consist of phosphoric acid, lactic acid, citric acid, fumaric acid, formic acid, and their salts. In practical production, citric acid and fumaric acid are the most commonly used. Both play important roles in the animal tricarboxylic acid cycle, participate in the metabolism of the three major nutrients, and exert multiple physiological functions. ^[1] In animal production, citric acid and fumaric acid, as weak acids, exhibit good palatability. Their primary functions in feed include modulating the gastrointestinal environment, enhancing pepsin activity, lowering intestinal pH to inhibit the proliferation of harmful bacteria, and improving feed utilization, thereby increasing average daily gain. These effects contribute to improved animal health and growth performance. ^[2-3] Lactic acid is a metabolic product of lactic acid bacteria; it has good palatability, can lower the gastrointestinal pH in piglets, and helps regulate gut microbiota, reduce feed-to-meat ratio and diarrhea incidence, thereby improving piglet performance. ^[4] Formic acid is an organic acid with strong antibacterial properties. Adding formic acid to weanling pig diets can effectively inhibit the growth of harmful bacteria, thereby reducing diarrhea rates, while also enhancing feed intake, promoting the digestion and absorption of nutrients, and improving production performance. Phosphoric acid is an important inorganic salt that is commonly used as a key component in compound acidifiers added to weanling pig diets. It lowers the pH of the feed, inhibiting the growth of harmful bacteria and reducing diarrhea incidence. Additionally, phosphoric acid promotes the absorption of minerals such as calcium and phosphorus, improves feed utilization, supports skeletal development and production performance in weanlings, and increases economic returns. ^[5] 。

The intestinal lining of animals harbors numerous diverse bacterial species. Within this microbial community, beneficial bacteria and harmful pathogens coordinate and regulate one another, forming a complex yet stable microbial ecosystem. Among the beneficial microbes, lactic acid bacteria and bifidobacteria predominate. Most species and strains of these beneficial bacteria can adhere to the surface of intestinal epithelial cells. They play crucial roles in providing nutrients, detoxifying harmful substances, and activating immune responses. ^[6] Among harmful microbial groups, common intestinal pathogens include Bacteroides, Clostridium, and Bacillus. Among these, Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens are widespread and highly prevalent; they are four common pathogenic bacteria that can cause various intestinal and extraintestinal diseases in livestock and poultry. A balanced gut microbiota plays a crucial role in maintaining animal health. However, during animal production, factors such as feed ingredient composition, functional additives like acidifiers and probiotics, antimicrobial agents, and infection with extraintestinal pathogens can all disrupt the composition of the gut microbiota, thereby affecting production performance through their impact on intestinal health.

It is worth noting that while bacteriostatic growth-promoting antibiotics kill pathogenic bacteria, they can also cause the massive death of beneficial bacteria, leading to disruptions in the gut microbiota and the emergence of antibiotic-resistant strains, ultimately compromising health. ^[7] The European Union and China have successively banned the use of growth-promoting antibiotics in animal feed. Acidifiers, by enhancing nutrient digestion and inhibiting the proliferation of intestinal bacteria, have emerged as strong alternatives to antibiotics. Consequently, numerous studies have investigated the effects of various compound acids on gastrointestinal microorganisms, particularly pathogenic bacteria; however, comparative research examining their impacts on both beneficial and harmful microbes remains relatively limited. In this experiment, four common beneficial bacterial strains and four pathogenic strains were selected to compare the antibacterial effects of different types of acidifiers.

1.1 Experimental Materials

Different types of acid preparations are all supplied by Guangdong Suandongli Biotechnology Co., Ltd. The main parameters are shown in Table 1.

1.2 Strain

Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538), Salmonella (ATCC 9120), Clostridium perfringens (ATCC 13124), Bifidobacterium longum subsp. infantis (GDMCC 1.2817), Clostridium butyricum (B80602), lactic acid bacteria (CCTCC NO:M2011381), and Lactobacillus acidophilus (ATCC 4356) were purchased from Guangdong HuanKai Microbial Technology Co., Ltd.

1.3 Main Instruments and Culture Media

Autoclave (model BXM-30R), constant-temperature incubator (model SPX-250B-Z), laminar flow hood (model SWCJ-2FD), and anaerobic incubator (model HYQX-) were used; nutrient broth, nutrient agar, and liquid thioglycolate medium were all purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

1.4 Preparation of Bacterial Suspension

A sample was taken from the cryopreserved strain and inoculated into a flask containing nutrient broth liquid medium, which was then shaken at 37°C for 18–24 hours. Subsequently, another aliquot of the cryopreserved strain was inoculated into a flask containing liquid thioglycolate medium and similarly incubated with shaking at 37°C for 18–24 hours. Bacterial counts were determined using the plate count method, and a suspension was prepared at a concentration of 1×10 ⁷ ~1×10 ⁸ cfu·mL ^-1 The bacterial suspension.

1.5 Determination of the Minimum Inhibitory Concentration (MIC)

(1) Once the culture medium has melted and cooled to approximately 45°C, add the required amount of sample to 100 mL of medium to achieve the final concentration intended for the sample. Shake the medium thoroughly to ensure uniform mixing. Do not add any sample to the blank control medium. Immediately pour the medium into Petri dishes, allow it to cool and solidify, thereby preparing culture media containing the sample at the appropriate final concentration. Perform two replicates for each sample at each concentration.

(2) Activate the test strain for two passages. For the second passage, use the streak plate method on agar plates to obtain linear colonies. To use, pick 2–4 loops of cells with a 1-mm diameter inoculation loop and transfer them into a flask containing 100 mL of 0.9% sterile sodium chloride solution (with an appropriate number of glass beads). Mix well and set aside for later use.

(3) On the outer bottom of the Petri dish, place a pre-drawn sheet of paper marked with 4 × 5 points, and transfer a loopful of bacterial suspension onto the corresponding points.

(4) Place the MRS agar plates, RCM agar plates, and Columbia blood agar plates in an anaerobic jar, along with the other plates, and incubate them in a constant-temperature incubator at 37°C, inverted, for 24 hours; record the results.

1.6 Data Analysis

The experimental data were processed using Excel 2010.

2.1 Minimum inhibitory concentrations of different types of acidifiers against harmful bacteria

As shown in Table 2, formic acid type I exhibits the strongest inhibitory effect on Escherichia coli at a concentration of 1 g·L ^-1 It can achieve a bacteriostatic effect immediately; secondly, the fumaric acid type at 1.2 g·L ^-1 , citric acid 1.5 g·L ^-1 , lactic acid type 2.5 g·L ^-1 , phosphate type 2.5 g·L ^-1 , whereas the minimum inhibitory concentration of formic acid type II is greater than 10 g·L ^-1 , indicating no significant inhibitory effect on Escherichia coli; formic acid type I and fumaric acid type showed better inhibition against Salmonella, with a minimum inhibitory concentration of 1.2 g·L for both. ^-1 , followed by citric acid and formic acid; both types II were 1.5 g·L ^-1 , again for the lactic acid type 2 g·L ^-1 Finally, the phosphate type exhibited the weakest inhibitory effect on Salmonella; citric acid showed the best inhibitory effect on Staphylococcus aureus, with a minimum inhibitory concentration of 0.8 g·L. ^-1 , followed by formic acid type I at 1 g·L ^-1 , fumaric acid type 1.5 g·L ^-1 , phosphoric acid type and lactic acid type, with a minimum inhibitory concentration of 1.8 g·L for all. ^-1 , finally, formic acid type II has a minimum inhibitory concentration greater than 10 g·L ^-1 , with no significant inhibitory effect on Staphylococcus aureus; citric acid exhibited the strongest inhibitory activity against Clostridium perfringens, with a minimum inhibitory concentration of 2.2 g·L ^-1 , followed by formic acid type I and formic acid type II, both at 3.2 g·L ^-1 , fumaric acid type 3.8 g·L ^-1 , and finally for the lactic acid type and the phosphoric acid type, both at 4.8 g·L ^-1 , the inhibitory effect on Clostridium perfringens is relatively weak.

2.2 Minimum Inhibitory Concentrations of Different Types of Acid Preparations Against Beneficial Bacteria

As shown in Table 3, the six different types of acid preparations exhibited stronger inhibitory effects on Clostridium butyricum than on other strains, indicating that Clostridium butyricum has poorer tolerance compared to other strains. Among them, the lactic acid type showed the weakest inhibitory effect, at 4.8 g·L. ^-1 , citric acid is strongest at 1.8 g·L ^-1 ; Among them, fumaric acid exhibited the strongest inhibitory effect on Bifidobacterium longum subsp. infantis, with a minimum inhibitory concentration of 6 g·L ^-1 , the weakest inhibitory effects were observed for the lactic acid and phosphate types, with minimum inhibitory concentrations both exceeding 10 g·L. ^-1 ; The strongest inhibitory effects against Bacillus coagulans were observed with formic acid type I and fumaric acid, both having a minimum inhibitory concentration of 3 g·L. ^-1 The weakest inhibitory effects were observed with the phosphate and formic acid type II formulations, both at concentrations of 10 g·L⁻¹ or higher. All six different types of acid preparations exhibited relatively weak inhibition against Lactobacillus acidophilus, with no significant differences among them, indicating that L. acidophilus possesses good acid tolerance.

Acidifiers are highly effective, green, safe, non-polluting, and residue-free feed additives available in both liquid and solid forms. The market offers a wide variety of products with complex formulations, and their applications are becoming increasingly mature. ^[8] . Compound acid preparations are feed acidifiers formulated from a blend of organic acids and their salts, used to enhance digestion. Their primary function is to regulate and lower the pH in the digestive tract, compensating for insufficient gastric acid secretion caused by the underdeveloped gastrointestinal tract in young animals, thereby promoting food digestion and preventing indigestion and digestive disorders. ^[9] . Compound organic acid preparations can prevent alkaline substances in feed from neutralizing gastric acid, maintaining a lower pH environment in the gastrointestinal tract, thereby facilitating the digestion and absorption of nutrients by animals, while also inhibiting the growth of harmful microorganisms in the gastrointestinal tract and improving feed palatability. ^[10] 。

3.1 Inhibitory Effects of Six Acidifiers on Major Harmful Bacteria in the Gastrointestinal Tract of Livestock and Poultry

During livestock and poultry farming, inadequate sanitation and environmental cleaning can easily lead to infection with various intestinal pathogens, causing diarrheal and other gastrointestinal diseases and resulting in economic losses for farmers. Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens are four common pathogenic bacteria that can cause a range of intestinal and extraintestinal diseases in livestock and poultry, posing a serious threat to the animal husbandry industry. ^[11] Acidifiers can inhibit the growth of common pathogenic bacteria, and their antibacterial mechanisms primarily include macromolecular synthesis, energy competition, increasing intracellular osmotic pressure, and inducing the host to mount nutritional immunity. ^[12] In an in vitro study comparing six single-acid formulations with compound acid formulations, Lei Yan et al. demonstrated that fumaric acid and citric acid exhibited strong antibacterial activity against Escherichia coli and Staphylococcus aureus. ^[13] Similarly, in a study comparing the inhibitory effects of eight single-acid formulations against standard strains of Escherichia coli, Staphylococcus aureus, and Salmonella, formic acid, fumaric acid, and tartaric acid exhibited the strongest antibacterial activity. ^[14] In this experiment, the overall inhibitory effect of six acid formulations against four harmful bacteria was as follows: citric acid type > formic acid type I > fumaric acid type > phosphoric acid type > lactic acid type > formic acid type II, which is consistent with the findings of the aforementioned studies.

It is worth noting that formic acid type I exhibits superior antibacterial activity compared to type II, likely because it contains a higher proportion of formic acid. This finding is consistent with the results reported by Guo Hua et al., who demonstrated in a study comparing different compound organic acids against pathogenic bacteria that compound organic acids with a higher formic acid content are more effective at inhibiting Escherichia coli, Staphylococcus aureus, and Salmonella than those with lower formic acid levels. ^[15] Formic acid and citric acid exhibit slightly superior antibacterial activity compared to other acid-type acidifiers, likely because formic acid has a lower pKa value and thus dissociates more readily. Research by R. C. Beier et al. demonstrated that inhibition of Enterococcus faecium, Campylobacter jejuni, and Campylobacter coli correlates with the amount of dissociated formic acid, rather than with pH or undissociated formic acid. ^[16] . Citric acid, owing to its relatively low pKa value within the tricarboxylic acid family and its weak chelating ability toward metal cations, serves as a highly effective antimicrobial agent. ^[7] 。

Studies by Ma Xin et al. have shown that formic acid and potassium diformate inhibit Salmonella typhimurium, Clostridium perfringens, and Campylobacter jejuni, a finding consistent with previous experimental results. ^[17] Similarly, the study by Zhang Yan et al. also demonstrated that citric acid and fumaric acid exhibit inhibitory effects against both Escherichia coli and Staphylococcus aureus. ^[18] Furthermore, studies by Dong Airong et al. have shown that fumaric acid exhibits the strongest inhibitory effect against Escherichia coli, followed by Staphylococcus aureus, with Salmonella showing the weakest inhibition. This is consistent with the antibacterial results obtained in this study using a fumaric-acid-based acidifying agent, which primarily contains fumaric acid. ^[19] In this experiment, the lactic‑acid‑based and phosphoric‑acid‑based acidifiers exhibited comparable antibacterial activity against four harmful bacterial strains. However, their inhibitory effects on Escherichia coli and Staphylococcus aureus differed from those reported by Wang Zhu et al. in their screening of compound acidifiers: their results indicated that a phosphoric‑acid‑based acidifier containing 30% phosphoric acid was more effective against E. coli, whereas a lactic‑acid‑dominant lactic‑acid‑based acidifier showed superior inhibition of S. aureus. ^[20] 。

In this experiment, the antibacterial efficacy of the six acid preparations was as follows: Salmonella > Escherichia coli > Clostridium perfringens > Staphylococcus aureus, which is consistent with the findings of Yan Xinru et al. regarding the antibacterial effects of organic acids containing varying concentrations of phosphoric acid against harmful bacteria. ^[21] Different types of acidifiers exhibit varying inhibitory effects on four harmful bacteria—Salmonella, Escherichia coli, Clostridium perfringens, and Staphylococcus aureus. This may be attributed to differences in the acid tolerance of the bacterial strains, as well as variations in the content and ratio of active ingredients within the acidifiers; it may also relate to the structural properties of the acidifiers themselves, including their inherent charge.

3.2 Inhibitory Effects of Six Acid Preparations on Major Beneficial Bacteria

Clostridium butyricum, Lactobacillus acidophilus, Bacillus coagulans, and Bifidobacterium are the major beneficial bacterial species in the gastrointestinal tracts of livestock and poultry. They play a crucial role in maintaining gut microbiota balance, promoting intestinal health, and supporting optimal growth performance. ^[22] . There are few reports on the in vitro inhibitory effects of organic acids on beneficial bacteria; research has mainly focused on their probiotic effects on the intestinal microbiota of livestock and poultry. Adding acidified feed additives can improve the gut microbiota and promote the growth of beneficial bacteria. ^[23] . H. Diao et al. conducted a study in which 5000 mg·kg was added to the feed of 24-day-old piglets. ^-1 A trial of benzoic acid capsules was conducted, and changes in the composition of the gut microbiota were observed. ^[24] After two weeks of benzoic acid supplementation, colonic pH decreased; after six weeks, ileal and cecal pH also decreased. Concurrently, the populations of lactic acid bacteria such as Bifidobacterium and Bacillus increased. R. Lan et al. found that adding a 0.2% composite acid preparation (fumaric acid, citric acid, and capric acid) to sow diets resulted in a 1.2% increase in lactic acid bacteria during the farrowing period, a 2.0% reduction in Escherichia coli during the lactation period, and a 2.2% increase in lactic acid bacteria. ^[25] S. T. Ahmed et al. added a 0.4% compound organic acid blend (containing 4.1% propionic acid, 9.5% phosphoric acid, 10.2% lactic acid, and 17.2% formic acid) to the diets of weaned piglets and found that the concentrations of Lactobacillus and lactic acid bacteria in the intestinal microbiota increased significantly, while the numbers of Salmonella and Escherichia coli decreased. In this experiment, the six acid preparations exhibited the strongest inhibitory effect against Clostridium butyricum, with MIC values significantly lower than those for the other three beneficial bacteria. Among them, citric acid showed the strongest inhibition of Clostridium butyricum, followed by Formic Acid Type II and Formic Acid Type I preparations. These results indicate that Clostridium butyricum has poorer tolerance compared to the other three beneficial bacteria.

Analysis of the MIC values of different acidifiers against four beneficial bacterial strains revealed that formic acid type I and fumaric acid exhibited the lowest MIC values compared with other acidifiers, indicating their strongest inhibitory effect on beneficial bacteria. Consequently, while suppressing harmful bacteria, they also inhibit the growth of beneficial bacteria. In contrast, lactic acid and phosphoric acid showed the highest MIC values, suggesting minimal impact on beneficial bacteria; thus, they can suppress harmful bacteria without harming beneficial ones, thereby helping to maintain intestinal microbiota balance and support gut health.

This study used a nutrient medium method to evaluate the inhibitory effects of six different types of acidifiers on eight bacterial groups in the gastrointestinal tracts of livestock and poultry, yielding the following conclusions: Formic acid type I, citric acid, and fumaric acid—these digestible organic acids—although more effective than other types of acidifiers at inhibiting harmful bacteria, also suppress the growth of beneficial bacteria, which is detrimental to the balance of the gut microbiota in livestock and poultry. Among the various acidifier types, lactic acid–based and phosphoric acid–based acidifiers performed relatively better: they significantly inhibit harmful bacteria while still allowing a large population of beneficial bacteria to survive, thereby helping to maintain gut microbial balance.