Alicyclobacillus Strains Research Articles

Exploration of Alicyclobacillus spp. Genome in Search of Antibiotic Resistance.

The study investigates the antibiotic resistance (AR) profiles and genetic determinants in three strains of guaiacol-producing Alicyclobacillus spp. isolated from orchard soil and pears. Their phenotypic characteristics, such as spore formation; resistance to different factors, including drugs or disinfectants; or production of off-flavor compounds, can affect the taste and aroma of spoiled products. Food and beverages are potential vectors for the transfer of antibiotic resistance genes, which is a growing health concern; thus, microorganisms in food and beverages should not be a potential source of drug resistance to consumers. Whole-genome sequencing (WGS) was utilized to identify antibiotic resistance genes, metabolic pathways, and elements associated with guaiacol and halophenol production. Minimum inhibitory concentration (MIC) testing revealed that all strains were susceptible to eight out of nine tested antibiotics (ampicillin, gentamycin, kanamycin, streptomycin, clindamycin, tetracycline, chloramphenicol, and vancomycin) but exhibited high resistance to erythromycin. Analysis indicated that the erythromycin resistance gene, ribosomal RNA small subunit methyltransferase A (RsmA), was intrinsic and likely acquired through horizontal gene transfer (HGT). The comprehensive genomic analysis provides insights into the molecular mechanisms of antibiotic resistance in Alicyclobacillus spp., highlighting the potential risk of these bacteria as vectors for antibiotic resistance genes in the food chain. This study expands the understanding of the genetic makeup of these spoilage bacteria and their role in antimicrobial resistance dissemination.

Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC–MS) of spoilage compounds and spore's resistance to thermal shocks

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC–MS) were used to identify and quantify the spoilage compounds. All GC–MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Genomic insight and physiological characterization of thermoacidophilic Alicyclobacillus isolated from Yellowstone National Park.

Alicyclobacillus has been isolated from extreme environments such as hot springs, volcanoes, as well as pasteurized acidic beverages, because it can tolerate extreme temperatures and acidity. In our previous study, Alicyclobacillus was isolated during the enrichment of methane oxidizing bacteria from Yellowstone Hot Spring samples. Physiological characterization and genomic exploration of two new Alicyclobacillus isolates, AL01A and AL05G, are the main focus of this study to identify their potential relationships with a thermoacidophilic methanotroph (Methylacidiphilum) isolated from the same hot spring sediments. In the present study, both Alicyclobacillus isolates showed optimal growth at pH 3.5 and 55°C, and contain ω-alicyclic fatty acids as a major lipid (ca. 60%) in the bacterial membrane. Genomic analysis of these strains revealed specific genes and pathways that the methanotroph genome does not have in the intermediary carbon metabolism pathway such as serC (phosphoserine aminotransferase), comA (phosphosulfolactate synthase), and DAK (glycerone kinase). Both Alicyclobacillus strains were also found to contain transporter systems for extracellular sulfate (ABC transporter), suggesting that they could play an important role in sulfur metabolism in this extreme environment. Genomic analysis of vitamin metabolism revealed Alicyclobacillus and Methylacidiphilum are able to complement each other's nutritional deficiencies, resulting in a mutually beneficial relationship, especially in vitamin B1(thiamin), B3 (niacin), and B7 (biotin) metabolism. These findings provide insights into the role of Alicyclobacillus isolates in geothermal environments and their unique metabolic adaptations to these environments.

Kefir fermented fruit by-products: anti-Alicyclobacillus spp. activity, and antioxidant activity

This study aimed to investigate the antimicrobial activity of kefir fermented fruit by-products against strains of Alicyclobacillus spp., and determine their chemical characterization and antioxidant activity. According to the results, extracts fermented for a longer period (72 h) showed greater inhibition, and the extract of acerola by-product fermented for 72 h achieved the best results. For all strains, the Minimal Inhibitory Concentration (MIC) was 0.78%, except for A. acidocaldarius subsp. rittmannii, (1.56%). The same applies to the Minimal Bactericidal Concentration (MBC), in which 1.56% of the extract was capable of inactivating A. cycloheptanicus and A. acidocaldarius. In addition, damages to the structure of the microorganisms caused by the extracts were detected by Scanning Electron Microscopy. Metabolite identification through liquid chromatography (UHPLC-Qtof-MS) showed that the fermented extracts had a greater number of compounds compared to the non-fermented ones, such as glucuronic, succinic and glutaric acids. In conclusion, the extracts of fruit by-products showed to have bioactive properties, such as antibacterial potential and antioxidant activity against the Alicyclobacillus strains tested in this study, not to mention the value added by the use of a food by-product.

Mobile genetic elements mediate the mixotrophic evolution of novel Alicyclobacillus species for acid mine drainage adaptation.

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions.

Thermoacidophilic Alicyclobacillus Superoxide Dismutase: Good Candidate as Additives in Food and Medicine.

Thermoacidophilic Alicyclobacillus strains attract great interests as the resource of thermostable or acidic enzymes. In this study, a putative gene encoding superoxide dismutase (AaSOD) was identified in a thermoacidophilic Alicyclobacillus strain. With a 16-fold activity observed, the AaSOD activity expressing in the medium of manganese enrichment was much higher than that in the iron medium. In addition, the purified AaSOD can be reconstituted exclusively with either Fe2+ or Mn2+, with its Mn-bound protein showing 25-fold activity than that of Fe-bound form. The optimal temperature for AaSOD reaction was 35°C, and was highly stable at any certain temperature up to 80°C. Of particular interest, the enzyme is found to be very stable across a wide pH range spanning from 2.0 to 10.0, which confers its robust stability in the acidic stomach environment and implies striking potentials as food additive and for medical use.

A Deeper Look into the Biodiversity of the Extremely Acidic Copahue volcano-Río Agrio System in Neuquén, Argentina.

The Copahue volcano-Río Agrio system, on Patagonia Argentina, comprises the naturally acidic river Río Agrio, that runs from a few meters down the Copahue volcano crater to more than 40 km maintaining low pH waters, and the acidic lagoon that sporadically forms on the crater of the volcano, which is studied for the first time in this work. We used next-generation sequencing of the 16S rRNA gene of the entire prokaryotic community to study the biodiversity of this poorly explored extreme environment. The correlation of the operational taxonomic units (OTUs)s presence with physicochemical variables showed that the system contains three distinct environments: the crater lagoon, the Upper Río Agrio, and the Salto del Agrio waterfall, a point located approximately 12 km down the origin of the river, after it emerges from the Caviahue lake. The prokaryotic community of the Copahue Volcano-Río Agrio system is mainly formed by acidic bacteria and archaea, such as Acidithiobacillus, Ferroplasma, and Leptospirillum, which have been isolated from similar environments around the world. These results support the idea of a ubiquitous acidic biodiversity; however, this highly interesting extreme environment also has apparently autochthonous species such as Sulfuriferula, Acidianus copahuensis, and strains of Acidibacillus and Alicyclobacillus.

High alkaline activity of a thermostable α-amylase (cyclomaltodextrinase) from thermoacidophilic Alicyclobacillus isolate

It is well demonstrated that glycosyl hydrolases from Alicyclobacillus strains are general thermoacidophilic enzymes and are ideal proteins for industrial applications. In this study, a thermophilic Alicyclobacillus α-amylase of glycoside hydrolases 13_20 subfamily, AMY1, was identified from an Alicyclobacillus strain and efficiently expressed in the host Escherichia coli BL21 CodonPlus. In agreement with other reported Alicyclobacillus hydrolases, the purified AmyY1 had an optimal pH of 6.0–6.5 in phosphate or citrate/Na2HPO4 buffers, and a remarkably decreased activity at pH 8.0. Differently, much higher activity was detected in the alkaline glycine/NaOH reaction mixtures. Compared to the highest amylolytic activity at pH 6.0, AmyY1 exhibited 230 and 116% activities at pH 8.0 and 9.0, respectively. This glycine-activation was further confirmed by a supplementation of glycine into the assay mixtures. During the digestions of various raw starches, AmyY1 also exhibited high hydrolysis efficiency under acidic or alkaline conditions. Findings in this study not only endow AMY1 with much broad applications, but also may provide a novel field for the application potentials of some other Alicyclobacillus hydrolases.

Effective Adsorption of Patulin from Apple Juice by Using Non-Cytotoxic Heat-Inactivated Cells and Spores of Alicyclobacillus Strains.

Patulin (PAT) is a major threat to many food products, especially apple and apple products, causing human health risks and economic losses. The aim of this study was to remove PAT from apple juice by using the heat-inactivated (HI) cells and spores of seven Alicyclobacillus strains under controlled conditions. The HI cells and spores of seven strains adsorbed PAT effectively, and the HI cells and spores of Alicyclobacillus acidocaldarius DSM 451 (A51) showed maximum PAT adsorption capacity of up to 12.6 μg/g by HI cells and 11.8 μg/g by HI spores at 30 °C and pH 4.0 for 24 h. Moreover, the PAT adsorption process followed the pseudo-first order kinetic model and the Freundlich isotherm model; thermodynamic parameters revealed that PAT adsorption is a spontaneous exothermic physisorption process. The results also indicated that PAT adsorption is strain-specific. The HI cells and spores of Alicyclobacillus strains are non-cytotoxic, and the bioadsorption of PAT did not affect the quality of the juice. Furthermore, the cell wall surface plays an important role in the adsorption process.

The Effect of RF Treatment Combined with Nisin Against Alicyclobacillus Spores in Kiwi Fruit Juice

In the present study, the efficiency of the treatment of radio frequency (RF) combined with nisin against the spores of three strains of Alicyclobacillus from two species was examined with the response surface methods (RSM). The results showed that the combination of RF and nisin was effective on killing Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans spores in kiwi fruit juice. The killing effect increased with the increase of temperature, holding time, and nisin concentration, but the increment was not significant at high levels of temperature and nisin concentration. The killing effect between A. acidoterrestris and A. contaminans strains was different in kiwi fruit juice. The strain with the highest killing effect was DSM 17975T, followed by DSM 3923T and C-ZJB-12-33. The reduction of spore number of DSM 17975T at the central design points of RSM was about 2.4. Under experimental conditions, nisin showed hardly any effect on killing DSM 17975T spores when combined with RF.

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A.acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A.acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A.acidocaldarius isolates analysed in this study. Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species.

Discrimination of Alicyclobacillus Strains by Lipase and Esterase Fingerprints

Alicyclobacillus spp. have recently become a major issue in the fruit juice industry due to its implication in the spoilage of pasteurized juices/beverages. In this study, lipase and esterase fingerprints of 37 Alicyclobacillus strains and 14 Bacillus strains in apple juice were collected using a five-substrate cocktail including four C8-esters and one C4-diester. Characterization of each strain could be finished in a single assay followed by high-performance liquid chromatography (HPLC) analysis for product separation and quantification. The obtained lipase and esterase fingerprints were highly species-dependent. Differences between the relative peak areas of each product for different species were significant. Coupled to chemometric techniques including principal component analysis (PCA) and stepwise linear discriminant analysis (SLDA), the lipase and esterase profiles led to 100 % of correct species identification for Alicyclobacillus and Bacillus strains. This approach is promising, reliable, and with good repeatability and could be used as an alternative tool to discriminate Alicyclobacillus spp. in fruit juices.

Control of Alicyclobacillus spp. vegetative cells and spores in apple juice with rosemary extracts

In this study, we initially investigated the antimicrobial activities of two commercial rosemary extracts (V20, V40) against Alicyclobacillus strains, with the ultimate purpose to determine whether these can be used in apple juice for the control of growth of alicyclobacilli. Minimum inhibitory concentrations (MICs) were first determined, and put through sensory analysis. Addition of the rosemary extracts to apple juice at their MICs did not change the colour, odour, taste or opacity of the apple juice. Growth kinetics studies with these rosemary extracts indicated a reduction in vegetative cells for Alicyclobacillus acidoterrestris, Alicyclobacillus hesperidum and Alicyclobacillus cycloheptanicus in Bacillus acidoterrestris broth and in apple juice. Further studies with A. acidoterrestris spores showed that the MICs of these rosemary extracts had relatively low effects on spore numbers in B. acidoterrestris broth, but had a spore number inhibition index >15% in apple juice. A four-fold increase in the rosemary extract concentrations showed the opposite effects: greater reduction in spores in B. acidoterrestris broth (inhibition index, >60%) than in apple juice (inhibition index, <10%). These data indicate that at their MICs, the V20 and V40 rosemary extracts allow outgrowth of spores but reduce vegetative cells, acting together with the low pH or other particular constituents of the apple juice. Rosemary extracts applied at their MICs thus represent an alternative method for the control of A. acidoterrestris in apple juice.

Discrimination between Bacillus and Alicyclobacillus Isolates in Apple Juice by Fourier Transform Infrared Spectroscopy and Multivariate Analysis

Alicyclobacillus is a causative agent of spoilage in pasteurized and heat-treated apple juice products. Differentiating between this genus and the closely related Bacillus is crucially important. In this study, Fourier transform infrared spectroscopy (FT-IR) was used to identify and discriminate between 4 Alicyclobacillus strains and 4 Bacillus isolates inoculated individually into apple juice. Loading plots over the range of 1350 and 1700 cm(-1) reflected the most distinctive biochemical features of Bacillus and Alicyclobacillus. Multivariate statistical methods (for example, principal component analysis and soft independent modeling of class analogy) were used to analyze the spectral data. Distinctive separation of spectral samples was observed. This study demonstrates that FT-IR spectroscopy in combination with multivariate analysis could serve as a rapid and effective tool for fruit juice industry to differentiate between Bacillus and Alicyclobacillus and to distinguish between species belonging to these 2 genera.

Alicyclobacillus dauci sp. nov., a slightly thermophilic, acidophilic bacterium isolated from a spoiled mixed vegetable and fruit juice product.

A novel, moderately thermophilic, acidophilic, Gram-variable, rod-shaped, endospore-forming bacterium was isolated from a spoiled mixed vegetable and fruit juice product that had the off-flavour of guaiacol. The bacterium, strain 4F(T), grew aerobically at 20-50 °C (optimum 40 °C) and pH 3.0-6.0 (optimum pH 4.0) and produced acid from glycerol, d-galactose and d-glucose. It contained menaquinone-7 (MK-7) as the major isoprenoid quinone and the DNA G+C content was 49.6 mol%. The predominant cellular fatty acids of strain 4F(T) were ω-alicyclic (ω-cyclohexane fatty acids), which are characteristic of the genus Alicyclobacillus. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belongs to the Alicyclobacillus cluster, and is related most closely to the type strains of Alicyclobacillus acidoterrestris (97.4 % similarity) and Alicyclobacillus fastidiosus (97.3 %). Strain 4F(T) produced guaiacol from vanillic acid. It can be distinguished from related species by its acid production type and guaiacol production. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, it can be concluded that the strain represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus dauci sp. nov. is proposed; the type strain is 4F(T) ( = DSM 28700(T) = NBRC 108949(T) = NRIC 0938(T)).

Patulin reduction in apple juice by inactivated Alicyclobacillus spp.

This study aimed to investigate the reduction of patulin (PAT) in apple juice by 12 inactivated Alicyclobacillus strains. The reduction rate of PAT by each strain was determined by high-performance liquid chromatography (HPLC). The results indicated that the removal of PAT was strain specific. Alicyclobacillus acidoterrestris 92 and A.acidoterrestris 96 were the most effective ones among the 12 tested strains in the removal of PAT. Therefore, these two strains were selected to study the effects of incubation time, initial PAT concentration and bacteria powder amount on PAT removal abilities of Alicyclobacillus. The highest PAT reduction rates of 88·8 and 81·6% were achieved after 24-h incubation with initial PAT concentration of 100μgl(-1) and bacteria powder amount of 40gl(-1) , respectively. Moreover, it was found that the treatment by these 12 inactivated Alicyclobacillus strains had no negative effect on the quality parameters of apple juice. Similar assays were performed in supermarket apple juice, where inactivated Alicyclobacillus cells could efficiently reduce PAT content. Taken together, these data suggest the possible application of this strategy as a means to detoxify PAT-contaminated juices. Inactivated Alicyclobacillus cells can efficiently reduce patulin concentration in apple juice. It provides a theoretical foundation for recycling of Alicyclobacillus cells from spoiled apple juice to reduce the source of pollution and the cost of juice industry. This is the first report on the use of Alicyclobacillus to remove patulin from apple juice.

Development and evaluation of an immunomagnetic separation–ELISA for the detection of Alicyclobacillus spp. in apple juice

The immunomagnetic separation (IMS) technique was used in combination with an enzyme-linked immunosorbent assay (ELISA) procedure to shorten the total analysis time and improve the sensitivity for the detection of Alicyclobacillus spp. in apple juice samples. The specificity of IMS–ELISA for twenty strains of Alicyclobacillus spp. and eighteen strains of non-Alicyclobacillus spp. was determined and there was little cross-reaction with non-Alicyclobacillus strains. Artificially contaminated apple juice with different concentrations of Alicyclobacillus acidoterrestris was detected by IMS–ELISA, and the detection limit of the assay in apple juice was 103CFU/mL. Furthermore, the sample inoculated with 1CFU/mL of A. acidoterrestris could be detected as positive after incubation for 24h. The IMS–ELISA described, allows for the identification of suspect positive samples within 3h of testing versus 3–5days required by standard culture methods while significantly reducing the materials and labor required for the detection of Alicyclobacillus spp. in apple juice samples. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS–ELISA for 102 naturally contaminated apple juice samples were 91.3%, 96.02% and 95.09%, respectively. These results demonstrated that the newly proposed IMS–ELISA procedure can be a potentially useful analytical method for the detection of Alicyclobacillus spp. in apple juice.

Micronized benzoic acid decreases the concentration necessary to preserve acidic beverages against Alicyclobacillus

The aim of this study was a challenge testing the effect of lower concentrations of micronized benzoic acid against two strains of Alicyclobacillus. The effect of micronized benzoic acid was compared with the usual levels of untreated commercial sodium benzoate and benzoic acid, at the challenge temperature of 45°C. The size of the benzoic acid particles was determined by scanning electron microscopy. The diameter of the micronized particles was around 10 μm with a maximum length of 200 μm, while the untreated preservative structures were irregular with lengths up to 500 μm. A continuous bactericidal effect against two Alicyclobacillus strains, throughout the 28-day period, was observed with 50 mg l(-1) of micronized benzoic acid, but when the untreated preservative was used, the same lethal effect was not achieved even after doubling its concentration. The antimicrobial activity of benzoic acid was improved by micronization. The process proved to be an effective alternative to reduce the benzoic acid concentration necessary to ensure stability of an orange juice matrix. The results proved that the micronization process represents an alternative to reduce the required food preservative concentration; this method increased the stability of the compound, which maintains its bioavailability.

Evaluation of a New Chromogenic Agar Medium for Alicyclobacillus acidoterrestris

Spoilage of fruit juices by a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris, is a big problem for fruit juice industries worldwide. We have developed a novel chromogenic selective agar medium (EAATSM) for the isolation and enumeration of A. acidoterrestris. A. acidoterrestris strains appeared as blue colonies on the EAATSM. Other Alicyclobacillus strains appeared as white colonies or were inhibited. A study comparing EAATSM and YSG agar was carried out using artificially contaminated samples of 50 fruit juice products. The correlation coefficient between EAATSM and YSG was 0.991.

Discrimination of Alicyclobacillus Strains Using Nitrocellulose Membrane Filter and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Alicyclobacillus spp. are thermoacidophilic, spore-forming bacteria, some of which cause spoilage in pasteurized and heat-treated apple juice products through the production of guaiacol. It would be helpful if a rapid method to detect and discriminate Alicyclobacillus strains was available. A simple and rapid sample preparation method using nitrocellulose membrane filter (NMF) and a single reflection horizontal attenuated total reflection (HATR) accessory with Fourier transform infrared (FT-IR) was developed here. Fourier transform infrared (FT-IR) spectroscopy was used and tested on 8 Alicyclobacillus strains (KF, WAC, NWN-13501, NWN-12697, NWN-12654, NWN-10682, 1016, 1101). A linear discriminant analysis (LDA) was established to discriminate Alicyclobacillus strains. The sample preparation method could successfully separated strains into different groups by principal component analysis (PCA). High identification accuracy (95%) was achieved with the LDA model. The method developed in the paper can be used to discriminate different Alicyclobacillus strains from each other making it possible to easily determine whether the strain of Alicyclobacillus present is associated with juice spoilage.