Comparison of Cultivable Acetic Acid Bacterial Microbiota in Organic and Conventional Apple Cider Vinegar

Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the fi rst stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplifi ed 16S23S rRNA gene ITS regions, we identifi ed four diff erent HaeIII and fi ve diff erent HpaII restriction profi les for bacterial isolates from organic apple cider vinegar. Each type of restriction profi le was further analyzed by sequence analysis of the 16S23S rRNA gene ITS regions, resulting in identifi cation of the following species: Acetobacter pasteurianus (71.90 %), Acetobacter ghanensis (12.50 %), Komagataeibacter oboediens (9.35 %) and Komagataeibacter saccharivorans (6.25 %). Using the same analytical approach in conventional apple cider vinegar, we identifi ed only two diff erent HaeIII and two diff erent HpaII restriction profi les of the 16S‒23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70 %) and Komagataeibacter oboediens (33.30 %). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identifi ed by sequence analysis of the ITS15.8S rDNA‒ ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the fi rst time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a diff erence in microbiota composition infl uences the quality of diff erent types of apple cider vinegar.


Introduction
Apple cider vinegar, alongside wine and alcohol vinegars, is one of the three most common vinegars available on the European market.Given the availability of raw materials, apple cider vinegar is oft en produced by households for their own needs using a traditional spon-ISSN 1330-9862 scientifi c note doi: 10.17113/ft b.54.01.16.4082 taneous process carried out by a native microbiota present on the fruit.Due to market demand, apple cider vinegar is also produced industrially.Alcohol and wine vinegars as well as apple cider vinegar are most oft en produced in submerged bioreactors, which supply the bacteria with a constant infl ow of oxygen and enable an effi cient production process (1).
A key factor in the quality of the product, besides the technology of the bioprocess, is the microbiota converting the main substrate, ethanol, into acetic acid, and also producing by-products that infl uence the chemical composition of the product and, most interestingly from the consumer's point of view, develop the specifi c aroma of the vinegar (2)(3)(4).In recent years some papers have analyzed the bacterial microbiota in the production of diff erent types of vinegars: alcohol, wine, rice, persimmon, strawberry and other types of fruits (5)(6)(7)(8)(9)(10)(11)(12).These data suggest that in a vinegar with high percentage of acetic acid (>6 %) the predominant species are Komagataeibacter europaeus, Komagataeibacter oboediens and/or Komagataeibacter intermedius, whereas in vinegar with low percentage of acetic acid (6 %), the predominant species are Acetobacter aceti, Acetobacter pasteurianus and/or Acetobacter pomorum (5)(6)(7)(8)(9)(10)(11)(12).Komagataeibacter is a novel, recently described genus of acetic acid bacteria to which the species formerly accommodated to the Gluconacetobacter xylinus group have been classifi ed (13,14).
The diffi culties in isolation and identifi cation of acetic acid bacteria have been emphasized many times in recent years (5,10,(15)(16)(17).Molecular biology methods are at present the best choice for quick and accurate identifi cation of acetic acid bacteria (16).There are diff erent methods among molecular biology approaches for identifi cation of the isolated acetic acid bacteria that have been tested in recent years: plasmid profi ling (18,19), random amplifi ed polymorphic DNA-polymerase chain reaction (RAPD--PCR) (5), denaturing gradient gel electrophoresis (DGGE) of the PCR-amplifi ed partial 16S rRNA gene (20), restriction fragment length polymorphism (RFLP) of the PCR--amplifi ed 16S23S rRNA gene ITS region (21), PCR of specifi c region on nifH, nifD (22) or adhA (16), enterobacterial repetitive intergenic consensus (ERIC)-PCR (8), rep--PCR (23), DNA-DNA hybridization (24), (GTG) 5 -PCR (5,23), sequence analysis of housekeeping genes (25) and matrix-assisted laser desorption ionization time-of-fl ight mass spectrometry (MALDI-TOF MS) (26).Some of these methods are very easy to perform and do not need very expensive equipment, such as RFLP of specifi c PCR products, whereas others, for example DNA-DNA hybridization or MALDI-TOF MS, depend on more experienced laboratory stuff and expensive equipment that not all laboratories interested in this subject can aff ord.Taking into account these issues, we think that the method based on the analysis of the 16S23S rRNA gene ITS region is a reasonable choice for identifi cation of acetic acid bacteria, especially among genera Acetobacter and Komagataeibacter (previously Gluconacetobacter), whose 16S rRNA gene sequences are very similar to each other.The method was used quite oft en in recent years for the identifi cation of acetic acid bacteria from diff erent sources, as an alternative genetic marker for novel species description, and it also turned out to be useful for fi shing out novel species of acetic acid bacteria (16,21,27,28).Besides the methods mentioned above, the DGGE analysis of the PCR-amplifi ed partial 16S rRNA gene has been used for direct characterization of vinegar microbiota; it enables microbial identifi cation by culture-independent molecular approach (29)(30)(31).
Nowadays consumers are increasingly aware of the importance of eating healthy food.The number of consumers who regularly buy organic food is therefore growing.The vinegar industry in Slovenia is trying to follow these trends and is producing organic apple cider vinegar in limited amounts in accordance with the availability of organic apple cider.Because of the diff erences in apple tree treatments used in the production of organic and conventional apple cider vinegar, the microbiota brought from the surface of the apples to the cider is expected to be diff erent.The aim of this work is to systematically follow the cultivable bacterial microbiota during a smoothly running oxidation cycle of organic and conventional industrial submerged apple cider vinegar production by restriction and sequence analysis of the PCR-amplifi ed 16S23S rRNA gene ITS regions.

Sampling of vinegars and isolation of microorganisms
Non-fi ltered vinegars were sampled directly from an 8000-litre broth of industrial submerged bioreactors for production of organic and conventional apple cider vinegars from the beginning to the end of one smoothly running oxidation cycle.The oxidation cycles were sampled during diff erent stages of the oxidation, meaning that at the point of sampling the concentration of acetic acid and volume fraction of ethanol were diff erent.Both bioprocesses with sampling points are presented in Fig. 1.At each of the sampling points the unfi ltered vinegar was brought from the bioreactor to the industrial laboratory and immediately spread aseptically onto the reinforced acetic acid and ethanol (RAE) medium (containing in g/L: glucose 40, peptone 10, yeast extract 10, citric acid 1.37, Na 2 HPO 4 •2H 2 O 3.38 and agar 10) containing 1 % (by volume) of acetic acid and 1 % (by volume) of ethanol, and incubated at 30 °C and high humidity (32).Each sampling point was analyzed in triplicates.The conventional apple cider vinegar was sampled between 28 and 31 May 2013 and the organic apple cider vinegar from 15 to 18 January 2014 at the premises of the Šampionka company, located in the sett lement of Volčja Draga in western Slovenia.At each of the sampling points four colonies were selected from each replicate, purifi ed aft er preculturing three times on the same type of medium and preserved in RAE medium with 20 % of glycerol at 80 °C.

Acetic acid and ethanol analysis
The concentration of acetic acid and volume fraction of ethanol were analyzed in each sample of vinegar.The acidity of the samples was determined by titrating the samples with 0.1 M NaOH in the presence of phenolphthalein.The acetic acid in vinegar represents 98 % of acids (33); the total acidity is therefore also a measure for acetic acid concentration.Ethanol volume fraction was determined ebullioscopically (34).

Phenotypic characterization of isolates
Each bacterial isolate was examined microscopically aft er Gram staining.The presence of cytochrome c oxidase and catalase was determined in bacterial smear aft er covering the cells with a few drops of 1 % tetramethyl-p--phenylenediamine and 3 % H 2 O 2 , respectively.
The maximal resistance of yeasts to acetic acid was determined by growing the isolates on a RAE medium with 1 % (by volume) of ethanol and appropriate volume fraction of acetic acid (14 %).

DNA isolation procedure
The bacterial isolates were grown in a liquid RAE medium containing 1 % (by volume) of acetic acid and 1 % (by volume) of ethanol.Aft er reaching the exponential growth phase (A 600 nm =0.51.0), the biomass was harvested and the DNA was isolated using the commercial kit Nu-cleoSpin Tissue (Macherey-Nagel GmbH&Co.KG, Düren, Germany).

Identifi cation of bacteria by 16S-23S rRNA gene ITS restriction and sequence analysis
The PCR amplifi cation of the 16S23S rRNA gene ITS was performed with primers Spacer_Fw (5'-TGCGG(C/T) TGGATCACCTCCT-3') (position 15221540 on 16S rRNA gene, Escherichia coli numbering) and Spacer_Rev (5'-GT-GCC(A/T)AGGCATCCACCG-3') (position 3822 on 23S rRNA gene, E. coli numbering) as described previously (35).The PCR products were digested separately with HaeIII and HpaII restriction enzymes and the restriction profi les obtained were compared to the original database of restriction profi les established with the reference strains published up to 2002 (35) as well as to an extended database established with the recently described reference strains of acetic acid bacteria (kept at the Department of Biology of the Faculty of Natural Sciences and Mathematics at the University of Maribor, Slovenia).The 16S23S rRNA gene ITS regions that gave diff erent restriction profi les were sequenced at Eurofi ns Genomics (Ebersberg, Germany).
The length of restriction fragments of the novel restriction profi les was calculated relative to that of DNA marker by linear regression of the semilogarithmic curve (mobility vs. logarithm of DNA fragment length).
The sequences were compared to the homologous sequences of the reference strains through National Center for Biotechnology Information (NCBI), consisting of Gen-Bank/EMBL/DDBJ databases and assigned to the species with the highest nucleotide identity.The sequences were deposited into EMBL/GenBank/DDBJ databases (International Nucleotide Sequence Database Collaboration) under accession numbers given in Table 1.

Identifi cation of yeasts by ITS1-5.8S rDNA-ITS2 sequence analysis
Morphologically diff erent types of yeast colonies from each type of vinegar were analyzed by sequence analysis of ITS1-5.8SrDNAITS2.PCR amplifi cation of this region was performed with primers 18SF1 (5'-AGGTTTCCGT A-G GTGAACCT-3') and ITS4 (5'-TCCTCCGCTTATTGAT A-TGC-3') as described previously (36).The PCR products were sequenced at Eurofi ns Genomics.The sequences were analyzed as described above for bacteria and deposited into EMBL/GenBank/DDBJ databases under accession numbers given in Table 2.

Typing of bacteria by RAPD-PCR analysis
RAPD-PCR was performed essentially as described previously by Trček et al. (5), with minor modifi cations.PCR was performed in a 20-L reaction mixture containing approx.10 ng of DNA, 2.5 mM of MgCl 2 , 40 pmol of primer, 0.5 U of Taq DNA polymerase (Thermo Scientifi c, Waltham, MA, USA), 0.2 mM of dNTP (Thermo Scientifi c) and 2 L of 10× Taq buff er (Thermo Scientifi c).Based on the number and reproducibility of the amplifi ed bands, two primers were selected: (GTG) 5x and 80 % (G+C).The cycling programme started with initial denaturation of DNA at 95 °C for 3 min and continued with 30 cycles at 95 °C for 30 s, at 56 °C (primer (GTG) 5x ) for 30 s or 41 °C (primer 80 % (G+C)) for 30 s and at 72 °C for 1 min.At the end, a fi nal extension at 72 °C for 7 min was performed, followed by cooling at 4 °C.The PCR products were separated in 1.5 % agarose gel in 1× Tris-acetate running buffer.

Description of the bioprocesses
The oxidation of ethanol to acetic acid during apple cider vinegar production is initiated by microbiota that originates from the surface of apples.These species have to survive the fermentation of sugars to ethanol and the conditions in the submerged bioreactor for the vinegar production.Since the industry strives to conduct the bioprocess economically, part of the previous cycle is typically used as a starter for the next cycle of the bioprocess aft er the process is successfully initiated.The acidity of the oxidation cycle at time zero is therefore above zero, as shown in Fig. 1.The duration of both cycles, for the production of organic and of conventional apple cider vinegar, was approx.the same at 66 and 67 h, respectively.The acidity reached at the end of each cycle was the same, i.e. 57 g/L.This value depends mostly on the sugar content in apples.According to these technological parameters, which are also typically followed by industry technologists to evaluate the performance of the vinegar-producing process, both bioprocesses performed in a similar way.
Although the same vinegar samples were inoculated onto more RAE agar media, with the aim of observing the CFU/mL, the variations were extremely large, up to 100--fold, suggesting that the number of microorganisms as determined by the cultivation approach is not suitable for following the numb er of microorganisms from submerged bioreactors.This observation has already been described for alcohol and wine vinegars (16).
Both processes, for organic and for conventional apple cider vinegar production, were sampled at the beginning of the cycle when the volume fraction of ethanol was maximal, 6.3 % in organic apple cider vinegar and 6 % in conventional apple cider vinegar, then several more times during each oxidation cycle, and at the end of the cycle, when the achieved the maximal concentration of acetic acid (Fig. 1).Because the samples were taken manually and also because the processes started at diff erent times of the day, the samples could not be taken during the process at exactly the same points.However, both processes had the same patt ern from the very initial stage of oxidation, i.e. the lag phase, which lasted approx.30 h during both processes, and later during a typical exponential phase, when the bacteria oxidized the ethanol with the highest effi ciency (Fig. 1).
Acetobacter pasteurianus (3) n.d.=not determined bination of HaeIII and HpaII restriction profi les I 1 J 2 from organic apple cider vinegar.The species identity of the isolates with these restriction profi les was also confi rmed by sequence analysis to be Komagataeibacter oboediens, previously classifi ed as Gluconacetobacter oboedies (Table 1).K. oboediens represented 9.35 % of all isolates from organic cider vinegar (Table 3).In addition, we isolated strains with novel HaeIII and HpaII restriction profi les: Z 1 (480 and 250 bp), W 1 (600 and 100 bp), W 2 (720 bp), SV 1 (320, 240 and 150 bp) and TV 1 (490 and 100 bp) (Table 1).Since we could not allocate them to any restriction profi les available in the database published in 2002 (35), we sequenced the PCR products.Comparison with the sequences in NCBI database identifi ed the isolates with the restriction profi le SV 1 TV 1 as Komagataeibacter saccharivorans (Table 1).K. saccharivorans represented 6.25 % of all isolates from organic cider vinegar but it was detected only at the beginning of oxidation cycle (Table 3, Fig. 1).
For the isolates with restriction profi les Z 1 W 1 and Z 1 W 2 we further sequenced partial 16S rRNA gene and identifi ed them as Acetobacter ghanensis.For this species the 16S-23S rRNA gene ITS sequence has not so far been known, which explains why we did not detect a high pairwise similarity to any sequences deposited in the NCBI database (Table 1).K. ghanensis represented 12.5 % of all isolates from organic cider vinegar and was also detected only at the beginning of oxidation cycle (Table 3, Fig. 1).
In contrast to K. oboediens and K. saccharivorans, which had already previously been isolated from vinegar, the species A. ghanensis has not yet been isolated from vinegar, but has been from cocoa beans in Ghana, tomato and peach in Thailand and palm wine in Burkina Faso (28,37,38).This study thus shows that the species A. ghanensis is not restricted to a highly specifi c ecological niche but that it also colonizes other substrates in nature.
In contrast to organic apple cider vinegar, from conventional apple cider vinegar we isolated only the strains with two diff erent combinations of HaeIII and HpaII restriction profi les.Both combinations P 1 R 1 and I 1 J 2 were previously identifi ed among strains of A. pasteurianus and K. oboediens, respectively (35). A. pasteurianus and K. oboediens represented 66.7 and 33.3 % of all isolates from conventional apple cider vinegar, respectively (Table 3).Both species persisted to the end of ethanol oxidation cycle (Table 3).
The species A. pasteurianus has oft en been isolated in recent years from diff erent fruits and vinegars and therefore its presence in both types of apple cider vinegar is not unexpected (8,(10)(11)(12)39).In both types of vinegar A. pasteurianus was the dominant species, persisting to the end of the bioprocess, when the concentration of acetic acid reached almost 60 g/L (Fig. 1, Table 3).The species K. oboediens is also a common bacterium used for the production of diff erent types of vinegar, and moreover, is a typical species for the production of vinegar with high acetic acid percentage (7,40).All isolates within one type of vinegar identifi ed as A. pasteurianus were genotypically identical to one another as detected by RAPD-PCR but diff erent between the two processes.The same fi ndings were observed for the isolates identifi ed as K. oboediens.According to this data, all isolates of the same species in each type of vinegar are genotypically homogeneous, which is likely to be the result of a strong selection pressure that is forcing the best adapted strain.
Very interestingly, we isolated yeasts from both types of vinegar, four isolates from organic apple cider vinegar and six from conventional apple cider vinegar (Table 2): the species Candida ethanolica from the fi rst sampling point with 6.3 % (by volume) of ethanol and 5.3 g/L of acetic acid, the species Pichia membranifaciens from the vinegar broth with 5.9 % (by volume) of ethanol and 5.5 g/L of acetic acid, and species Saccharomycodes ludwigii from the broth containing 2.7 % (by volume) of ethanol and 30.6 g/L of acetic acid.Similarly, we isolated yeasts from the conventional apple cider vinegar, but only the species C. ethanolica and S. ludwigii.All isolated yeasts were resistant against 30 g/L of acetic acid and it was not lost by any of the strains aft er preservation for half a year at 80 °C.These yeasts probably originate from and have survived through ethanol fermentation to vinegar broth.The presence of so-called non-Saccharomyces yeasts is typical for fermentations yielding low fi nal alcohol content: for example, Hanseniaspora uvarum and Pichia guilliermondii were detected during the fermentation of freshly crushed pineapple juice in Australia and Thailand that yielded 14 % (by volume) of ethanol, and Candida quercitrusa and Issatchenkia terricola were found during gabiroba (Campomanesia spp.) pulp fermentation, which yielded 2.63.8 % (by volume) of ethanol (41,42).The high acetic acid resistance of yeasts isolated and identifi ed in this work is comparable to those of extremely acetic acid--resistant yeast, Zygosaccharomyces bailii (43), and has not been reported before.

Conclusions
The microbiota represents one of the key components of the vinegar-producing bioprocess starting from diff erent sugar-containing materials.In Slovenia, apple cider vinegar is a traditional product produced in households but also at the industrial level.The industry aims to monitor and control the bioprocess and it is therefore important to know the composition of the microbiota responsible for a well-performing process.In this work we compared for the fi rst time the composition of organic and conventional apple cider vinegars.In both types of vinegar the species Acetobacter pasteurianus was dominant and, in addition, the species Komagataeibacter oboediens was detected.In organic apple cider vinegar the species A. ghanensis and K. saccharivorans were also detected.Yeasts were isolated from both types of apple cider vinegar.Very interestingly, these yeasts resisted up to 30 g/L of acetic acid.This study has demonstrated that the microbiota of the organic apple cider vinegar is more heterogeneous than that of conventional apple cider vinegar, which may infl uence the chemical composition and sensorial quality of vinegars.and the European Social Fund under the scheme Practical Knowledge through Creative Pathways is gratefully acknowledged.Mateja Leban from Šam pi onka company is acknowledged for sampling of vinegars.

Fig. 1 .
Fig. 1.Concentration of acetic acid and volume fraction of ethanol during the oxidation cycle of: a) industrial organic apple cider vinegar production, and b) the industrial conventional apple cider vinegar production.The numbers correspond to the sampling points of the microbial cultures

Table 1 .
Typical restriction types of the PCR-amplifi ed 16S23S rDNA ITS regions of bacterial isolates from apple cider vinegars, accession numbers of the corresponding 16S23S rRNA gene ITS sequences and species identity based on these regions

Table 2 .
Yeasts isolated from apple cider vinegars and their species identity based on ITS15.8SrDNAITS2 sequences

Table 3 .
List of species and the number of isolates (in parentheses) from organic and conventional apple cider vinegars isolated at diff erent sampling points (see Fig.1)