Potential Antimicrobial Activity of Weak Acids in Combination with pH and Temperature on Alicyclobacillus acidoterrestris †

: Alicyclobacillus acidoterrestris causes spoilage in fruit juices. This research investigated the effects of organic acids on the growth of two strains of A. acidoterrestris at different pH values and temperatures over 2, 7 and 14 days. The results show different impacts of weak acids on A. acidoterrestris ’s growth and survival. Ascorbic and malic acids have the highest antimicrobial activity; also, pH played a crucial role. Moreover, the results suggest a possible activity of acids on outgrowing spores rather than on spores. Understanding the organic acids interactions with A. acidoterrestris is crucial for implementing strategies to ensure quality and reduce spoilage incidents


Introduction
Alicyclobacillus is a group of Gram-positive, thermophilic, acidophilic and nonpathogenic bacilli species, with optimal temperatures and pH values at 42-53 • C and 3.5-5.0,respectively [1].This spoiler is frequently associated with fruit juices and acidic drinks and its presence is often attributed to inefficient pasteurization processes.Among the various species in this genus, A. acidoterrestris is the major species responsible for causing spoilage in juices [2].The spores of A. acidoterrestris can survive pasteurization and subsequently germinate, leading to the production of compounds such as guaiacol and halophenols, resulting in undesirable sensory characteristics such as sediment, cloudiness, or discoloration in the affected products [3].For juices, the threshold level of A. acidoterrestris cells for spoilage to be evident is typically 4-5 log CFU/mL [4].
The acid resistance of A. acidoterrestris is well-documented, and it has been extensively studied for its ability to survive and grow in acidic conditions.However, less is known about how different weak acids, commonly present in fruits [5] and used as acidulants and preservatives in the food industry [6], impact the behavior of A. acidoterrestris.Additionally, the response of A. acidoterrestris to different acids typically found in foods has not been thoroughly explored.
Organic acids, especially weak acids, are naturally present in fruits and contribute to their taste and suitability for juice processing.Furthermore, they exhibit antimicrobial activities and have various mechanisms, such as pH reduction, disturbance of membrane transport and permeability, anion accumulation, enzyme inhibition, cytoplasm acidification and specific antimicrobial effects of anionic species [7].
While some research has been conducted on the effects of different acids on A. acidoterrestris, the focus has mainly been on inactivation or resistance in combination with other factors.There is a need for a comprehensive study to investigate the impact of weak acids, which are commonly found in juices and used as preservatives, on soil strains of A. acidoterrestris.Thus, the aim of this research was to study the effects of specific weak acids present in juices (ascorbic, malic, citric, tartaric) in comparison to a weak acid used as an acidulant in food processing (lactic acid) and a strong acid (hydrochloric acid) on A. acidoterrestris soil strains, in combination with pH, contact time and storage temperature.

Materials and Methods
Two isolates of A. acidoterrestris (C1 and C13), which were genotypically and phenotypically characterized [8], from the Culture Collection of the Department of Agriculture, Food, Natural Resources and Engineering (DAFNE), University of Foggia, were used in this study.Spores were produced and quantified through a standardized protocol.
For sample preparation, a full randomized design was used to combine the isolates, pH levels (2, 3 and 4), acids (ascorbic, malic, citric, tartaric, lactic and hydrochloric acids) (J.T. Baker, Milan, Italy) and temperatures (5 • C and 45 • C) in 72 combinations.Each combination was prepared in duplicate, and three technical replicates were performed for each batch.All experiments were performed in Malt Extract broth (Oxoid, Milan, Italy), acidified to a pH from 2 to 4 through a 10% solution of the different acids; then, samples were separately inoculated to 4-5 log CFU/mL of the spores of the two strains.After incubation, either at 5 or 45 • C, the viable count was assessed immediately after inoculation and after 2, 7 and 14 days.
For the statistical analysis, the data were preliminary standardized as increase/decrease with reference to inoculum and then modelled using a multifactorial analysis of variance (MANOVA) by using the software Statistica for Windows (Version 7, Tulsa, OK, USA).Type of acid and strain, pH, temperature and sampling time were used as categorical predictors.

Results and Discussion
The first output of MANOVA is the table of standardized effects, which gives statistical weight of the predictors through an F-test.All single predictors have different statistical weights; the most significant predictor was pH (F-value, 1099.75),followed by temperature (F-value, 728.65) and time (F-value, 72.09).The viable count of alicyclobacilli was also affected by some interactive terms, e.g., temperature*pH (F-value, 521.18), temperature*isolate (F-value, 223.17) and acid*pH (F-value, 34.33).
The second output of MANOVA is the decomposition of the statistical hypothesis.However, many predictors that are used as well as the decomposition of the statistical hypothesis could provide outputs difficult to understand; thus, the predictors were reduced, and Figure 1 shows the effects of pH and type of acid on the C1 strain after 2 days.Concerning the weight of the type of acids, independently from the pH or time of sampling, the statistical analysis showed a strong quantitative effect of ascorbic acid, which caused a mean decrease in the viable count of 1.6-1.7 log CFU/mL.There are two mechanisms related to the antimicrobial activity of weak acids according to the bibliog- A significant antimicrobial action in lactic and citric acid occurred only at pH 2, probably due to depending on their higher concentrations and the amount of undissociated acid (98.7% for lactic acid and 90.45% for citric acid) compared to pH 3 or 4, with a mean reduction in the viable count of 4 log CFU/mL.Malic, tartaric and ascorbic acid also perform an antimicrobial action at pH 3 as well as pH 4 (only for ascorbic acid).
The C13 Strain showed a similar trend, although it appeared more sensitive in some combinations.
Concerning the weight of the type of acids, independently from the pH or time of sampling, the statistical analysis showed a strong quantitative effect of ascorbic acid, which caused a mean decrease in the viable count of 1.6-1.7 log CFU/mL.There are two mechanisms related to the antimicrobial activity of weak acids according to the bibliography.The first mechanism depends on the ability of entering the cell in an undissociated form with subsequent cytoplasm acidification, while the second mechanism is related to the ability of reducing the external pH of an acid [9].In addition, the stronger effects at 45 • C suggest that weak acids probably act on outgrowing spores, as alicyclobacilli spores germinate at this temperature, and do not at 5 • C.
In conclusion, the findings of this study demonstrate the potential of weak acids, especially ascorbic and malic acids, in effectively reducing the viable count of A. acidoterrestris.These acids can play a crucial role in controlling the spoilage of juices and concentrates when employed in proper combinations with low pH levels and appropriate temperatures.
However, further validation in real food products is necessary to develop predictive models for effective control measures against this spoiling bacterium.Understanding weak acid interactions with A. acidoterrestris in food systems is crucial for implementing preventive or corrective measures in the juice and concentrate industries to ensure product quality and reduce spoilage incidents.
This research provides valuable insights into the behavior of A. acidoterrestris in the presence of weak acids and contributes to the body of knowledge on controlling microbial spoilage in the food industry.Future studies should explore additional factors that might influence the antimicrobial efficacy of weak acids and focus on real juice products to assess their practical applicability in industrial settings.The combination of weak acids with other preservation methods could also be investigated to enhance their antimicrobial potential and provide an integrated approach to ensure the microbiological stability of fruit juices and concentrates.

of 4 Figure 1 .
Figure 1.Change in the viable count of A. acidoterrestris C1 strain after 2 days, depending on pH and type of acids used for acidification.Bars denote 95% confidence intervals.

Figure 1 .
Figure 1.Change in the viable count of A. acidoterrestris C1 strain after 2 days, depending on pH and type of acids used for acidification.Bars denote 95% confidence intervals.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/Foods2023-15092/s1,Conference Presentation Video: Potential Antimicrobial Activity of Weak Acids in Combination with pH and Temperature on Alicyclobacillus acidoterrestris.Author Contributions: Conceptualization, M.R.C.; methodology, B.S.; investigation, A.D.S. and B.S.; writing-original draft preparation, A.D.S.; writing-review and editing, all authors.All authors have read and agreed to the published version of the manuscript.