Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis.

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

INTRODUCTION

Culture-based methods are not designed to detect unculturable cells and often fail to characterize less abundant microbial populations or those microorganisms that require selective enrichment (17). These limitations have led to the development of culture-independent techniques. The most commonly used techniques are PCR-based and nucleic acid-based detection methods (28). Compared to conventional methods, culture-independent methods are generally less time consuming and well suited for analyzing microbial communities over time. These methods may also help in the study of microbial populations and their diversity in food ecosystems (16).

Sour cassava starch (“polvilho azedo”) and Minas cheese are traditional fermented foods produced in Brazil. These products are mostly manufactured on a small scale using traditional recipes (5, 18). During sour cassava starch production, the starch from cassava root is fermented in a tank for a period of time that ranges between 20 and 70 days (7, 18). Using culture-based methods, Lactobacillus plantarum and other lactic acid bacteria have been reported to be the most prevalent microorganisms associated with spontaneous fermentation of cassava starch in Brazil (18). These microorganisms are responsible for generating organic acids and aromatic compounds in this food product (3, 4, 9, 10, 18). Cheese production in Brazil involves milk fermentation by lactic acid bacteria, which are either deliberately added as starter cultures or are adventitious microbial populations, the growth of which is the result of the selective pressures encountered under the conditions used in the fermentation process (5, 20). Lactic acid bacteria occur naturally as part of the indigenous microbiota of raw milk, and the microbial diversity contributes to the large differences in organoleptic characteristics found in raw-milk cheeses (1).

Cachaça is the most important Brazilian distilled beverage (13, 26). During traditional cachaça production, lactic acid bacteria are considered contaminants in the fermentation process. These bacteria compete with yeasts for the sucrose found in the sugar cane juice, which then reduces the ethanol yield of fermentation (12). However, a precise correlation between bacterial contamination and reduced alcohol content during cachaça fermentation has not yet been shown (25).

The bacterial communities associated with sour cassava starch, cachaça and Minas cheese have been determined using cultivation techniques in combination with the molecular identification of isolates (14, 18, 20, 14). The aim of the present study was to examine the structure of the bacterial communities found in these traditional fermented products using direct DNA extraction, 16S rRNA gene amplification, cloning and sequencing.

MATERIALS AND METHODS

Sample collection

Sour cassava starch samples were collected from a factory in the city of Conceição dos Ouros, Minas Gerais, Brazil. The samples were collected 5, 27 and 45 days after the fermentation process started, according to Lacerda et al. (18). Cachaça fermentation samples were collected from a traditional distillery in the city of Esmeraldas, Minas Gerais at 7, 21 and 35 days after fermentation process started. Preparation procedures and sample collection for sour cassava starch and cachaça are described in Lacerda et al. (18) and Gomes et al. (13), respectively. After 7 and 60 days of maturation, Minas cheese samples were collected from a farm in the city of São Roque de Minas, Minas Gerais. This semi-hard Minas cheese, also known as Canastra cheese, is produced with raw milk and has been made at the farmhouse production level for the last 200 years using traditional procedures. Milk coagulation is a result of employing natural whey cultures as starter cultures (indigenous lactic acid bacteria) and commercial rennet. The natural microbiota present in the milk and in the environment is responsible for ripening (6). All samples were aseptically Fermentation using culture-independent 16S rRNA gene sequence collected using sterile flasks and were stored at -20°C until the DNA was extracted.

DNA extraction, amplification and clone libraries

DNA was extracted from 1 g of each sour cassava starch and cachaça fermentation sample, which was diluted in 9 mL sterile distilled water. Each 1-gram cheese sample was homogenized in 9 mL of 0.1% buffered peptone water. These suspensions were centrifuged at 15,000 rpm for 10 min, and DNA was extracted from the pellet using the procedure described by Lacerda et al. (18).

16S rRNA gene sequences were PCR amplified using the bacterial domain specific primers 27f (5 ´AGAGTTTGATCCT GGCTCAG3’) and 1492r (5’GGTTACCTTGTTACGACTT3’) (19). These primers amplify approximately 1,600 base pairs of the 16S rRNA gene. PCR reactions were performed in a final reaction volume of 50 µL, containing 0.1–0.5 µg DNA, dNTPs (200µM each), primers (0.8µM each), MgCl2 (1.5 mM), 1 U Taq DNA polymerase (Invitrogen) and the supplied buffer. The amplification conditions were as described in Morlon-Guyot et al. (22). The amplified fragments were purified using the Wizard system, ligated into the pGEM®-T Vector System II (Promega, Madison, USA) and transformed into competent E. coli (XL1-Blue) cells according to the manufacturer’s instructions (Invitrogen). Blue/white screening on Luria-Bertani (LB) agar containing ampicillin (Sigma), X-gal (Invitrogen) and isopropyl β-D-1-thiogalactopyranoside (IPTG, Promega) was used to randomly select approximately 30 white colonies for each sample. The clones were stored in freezing medium at -80°C.

Sequencing of cloned DNA and identification

Both strands of the cloned inserts were sequenced using the universal sequencing primers that correspond to the regions flanking the cloning site. Sequencing was performed using a DYEnamic Terminator Cycle Sequencing Kit in a MegaBACETM 1000 automated sequencing system (Amersham Biosciences, USA). These partial sequences were searched against sequenced deposited in GenBank using the advanced BLAST similarity search option (2). Partial sequences were approximately 600 base pairs long (range 350–940 bp). The nucleotide sequences and other related sequences were aligned using MEGA version 4. The 16S rRNA gene clone sequences that had a greater than 97% similarity to sequences deposited in GenBank were designated as belonging to the corresponding species (see accession numbers in Table 1).

Table 1

Most closely related bacterial species to the sour starch cassava, cachaça and cheese species based on BLASTn analysis.

UFMGCB	Most closely related species/GenBank accession number	Timesa			Percent similarity	bp analyzed	Identification	GenBank accession number
		I	II	III
Sour cassava starch
UFMG-PB10	Enterobacter aerogenes JCM1235 [NR_024643]	-	2	-				GQ918333
					97.4%	735	Enterobacter aerogenes
UFMG-PB11	Enterobacter cloacae ATCC13047T [AJ251469.1]	-	1	-				GQ999778
					98.4%	680	Enterobacter cloacae
UFMG-PB6	Leuconostoc mesenteroides LM2 [AY675249]	3	-	-				GQ999779
					98.8%	827	Leuconostoc mesenteroides
UFMG-PB20	Lactobacillus manihotivorans LSAF000162 [AF000162.1]	-	1	-				GQ999780
					97%	694	Lactobacillus manihotivorans
UFMG-PB8	Leuconostoc pseudomesenteroides NRIC 1777 [AB023237.1]	1	-	-				GQ999781
					97%	837	Leuconostoc pseudomesenteroides
UFMG-PB9	Lactobacillus plantarum DSM 20205 [M58827]	1	3	1				GQ999782
					99.6%	815	Lactobacillus plantarum
UFMG-PB03	Lactobacillus plantarum DSM 20205 [M58827]	-	1	-				GQ999783
					96%	556	Lactobacillus sp.
UFMG-PB12	Lactococcus lactis subsp.lactis NCDO 604T [AB100803]	5	1	-				GQ999784
					99.6%	538	Lactococcus lactis subsp. lactis
UFMG-PB07	Leuconostoc citreum ATCC49370 [AF111948]	10	-	-				GQ999785
					99%	770	Leuconostoc citreum
UFMG-PB02	Enterobacter sp. [GQ478379]	-	2	6				GQ999786
					90%	933	Uncultured bacterium clone
UFMG-PBOl	Enterobacter sp TGO3600721	1	-	-				GQ999787
					99%	765	Enterobacter sp.
UFMG PB30	Enterobacteriaceae bacterium [AB461659]	-	5	-				GQ999788
					99%	589	Uncultured bacterium clone
UFMG-PB44	Enterobacter sp. [GQ360072]	-	-	1				GQ999789
					99%	721	Enterobacter sp.
Cachaça fermentation
UFMG-CAB11	Lactobacillus nagelii ATCC 700692 [AB162131]	1	-	-				GQ999790
					90%	541	Lactobacillus sp.
UFMG-CAB16	Lactobacillus paracasei subsp. paracasei ATCC 25302 [D79212]	3	1	1				GQ999791
					100	812	Lactobacillus paracasei subsp. paracasei
UFMG-CAB5	Lactobacillus nagelii ATCC 700692 [AB162131]	9	1	-				GQ999792
					99%	800	Lactobacillus nagelii
UFMG-CAB6	Lactobacillus satsumensis NRIC 0604 [AB154519]	-	1	-				GQ999793
					96%	533	Lactobacillus sp.
UFMG-CAB12	Lactobacillus harbinensis SBT10908 [AB196123.1]	-	9	4				GQ999794
					100%	868	Lactobacillus harbinensis
UFMG-CAB9	Streptococcus salivarius ATCC 25975(FJ154797.1)	-	-	1				GQ999795
					100%	449	Streptococcus salivarius
UFMG-CAB10	Lactobacillus buchneri ATCC 4005 [AB205055]	-	-	11				GQ999796
					99%	781	Lactobacillus buchneri
UFMG-CAB14	Lactobacillus acetolerans JCM 9904 [AB289008]	-	-	1				GQ999797
					99%	763	Lactobacillus acetolerans
UFMG-CAB17	Lactobacillus plantarum DSM 20205 [M58827]	-	-	1				GQ999798
					98.8%	421	Lactobacillus plantarum
Minas cheese
UFMG-QB5	Lactobacillus plantarum DSM 20205 [M58827]	1	4					GQ999799
					99.5%	390	Lactobacillus plantarum
UFMG-QB6	Lactococcus lactis NCDO 604T [AB100803]	9	4					GQ999800
					100%	499	Lactococcus lactis
UFMG-QB3	Lactobacillus brevis ATCC 14869 [M58810.1]	-	1					GQ999801
					99%	700	Lactobacillus brevis
UFMG-QB16	Sphingomonas sp. Clone FI012 [AY349411]	-	2					GQ999802
					99%	614	Sphingomonas sp.
UFMG-QB55	Lactobacillus arizonensis NRRLB14768 [AJ965482]	-	7					GQ999803
					99.8%	496	Lactobacillus arizonensis
UFMG-QB26	Streptococcus salivarius ATCC 7073 (AY188352.1)	1	10					GQ999804
					99.6%	720	Streptococcus salivarius
UFMG-QB20	Uncultured Streptococcus sp. clone SC002B48 [AY807774.1]	-	1					GQ999805
					99.6%	721	Uncultured clone Streptococcus sp. clone

Sour starch cassava: I-5 days, II-27 days, III-45 days. Cachaça fermentation: I-7 days, II-21 days, III-35 days; Minas cheese: I-7 days, II-60 days.

RESULTS AND DISCUSSION

We sequenced 129 clones to identify the specific phylotypes of bacteria associated with the traditional processes used in sour cassava starch and Minas cheese fermentation and cachaça production. The overall structure of the bacterial communities found associated with these food products are summarized in Table 1.

Out of 45 clones examined from sour cassava starch fermentation, the most common species found after five days of fermentation were Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Leuconostoc citreum and Enterobacter sp. Leuconostoc citreum was the most prevalent species at the start of the fermentation process, representing 47.6% of the total clones analyzed. After 27 days of fermentation, two and five clones (GenBank accession number GQ999786 and GQ999788, respectively) were similar to uncultured bacterium clones, representing 43.8% of the bacterial community analyzed at this stage of the fermentation process. Enterobacter cloacae, Lactobacillus manihotivorans, Lc. lactis subsp. lactis, Lb. plantarum and Lactobacillus sp. were found at lower frequencies. After 45 days of fermentation, an uncultured bacterium clone (GenBank accession number GQ999786) was the most prevalent. This uncultured bacterium clone was distantly related (90% identity) to some sequences deposited in GenBank, such as Enterobacter sp. (GenBank accession numbers GQ478379 and FJ646658). At this fermentation time, Lb. plantarum and Enterobacter sp. were found at a low frequency.

Ampe et al. (3) used denaturing gradient gel electrophoresis (DGGE) to show that the dominant bacteria involved in the spontaneous fermentation of sour cassava starch in Colombia were all lactic acid bacteria, mainly those closely related to Bifidobacterium minimum, Lc. lactis, Streptococcus sp., Enterococcus saccharolyticus and Lb. plantarum.

The presence of Lb. manihotivorans was only detected when 16S rRNA genes were hybridized with phylogenetic probes. Using culture-dependent methods, Lacerda et al. (18) showed that Lb. plantarum and Lactobacillus fermentum were the species most frequently associated with sour cassava starch fermentation at two factories in the state of Minas Gerais. However, the results obtained using clone library sequence analysis showed that the prevalence of lactic acid bacteria was limited to the first five days of fermentation, and a lower prevalence of these bacteria was detected at later time points. Lb. plantarum was detected in the three fermentation samples assayed but at low frequencies. Uncultured bacterium clones were prevalent after 27 and 45 days of fermentation. Figueroa et al. (10) suggested that the traditional sour cassava starch fermentation process is characterized by a succession of microbial populations. Ampe et al. (3) suggested that this succession could be determined by the varying sensitivities of microorganisms to the acidic conditions that develop during the fermentation process. Our results show that unculturable bacteria were found at a high frequency during the middle and at the end of the sour cassava fermentation process. These microorganisms may potentially play an important role in the fermentation process that should be examined further.

Most of the clones obtained during the fermentation of cachaça corresponded to lactic acid bacteria belonging to the genus Lactobacillus. The bacterial communities present in this fermentation process are very similar regardless of the raw material used and are characterized by the presence of lactic acid species, especially those from the genera Lactobacillus, Leuconostoc, Weissella and Lactococcus (14). Many species from these genera are characteristically resistant to low pH and high alcohol concentrations, which explains their presence and wide distribution in different fermentation processes, including both alcoholic and lactic fermentation processes (15). After 7 days of fermentation, the most prevalent species was Lb. nagelli, representing 76.9% of the total clones analyzed. The other clones were Lactobacillus sp. and Lactobacillus paracasei subsp. paracasei. After 21 days, Lb. harbinensis was the most prevalent species, representing 75% of the total clones. Lb. paracasei subsp. paracasei, Lb. nagelli and Lb. satsumensis were found at lower frequencies. After 35 days of fermentation, Lb. buchneri was the most prevalent species, representing 57.9% of the clones analyzed, followed by Lb. harbinensis (21%). Lb. paracasei subsp. paracasei, Streptococcus salivarius, Lb. acetolerans and Lb. plantarum were also found at lower frequencies. Leuconostoc mesenteroides, Lb. plantarum, and Lb. brevis were described as contaminants of the cachaça fermentation vats (23, 27). Carvalho-Netto et al. (6) also characterized the bacterial community during cachaça production in the state of São Paulo using partial 16S rRNA gene sequencing. Their analysis included 587 sequences and revealed the presence of 170 operational taxonomic units. The genus Lactobacillus was the most predominant, accounting for approximately 66% of the sequences, and Lb. hilgardii and Lb. plantarum were the species most frequently found. Other species were found at lower frequencies, such as Curtobacterium flaccumfaciens, Lactobacillus casei, Leuconostoc mesenteroides and Ln. citreum. Gomes et al. (14) identified populations of culturable lactic acid bacteria involved in cachaça fermentation using both physiological and molecular methods. A high frequency of lactic acid bacteria was found during the entire fermentation process, and the species Lb. plantarum and Lb. casei were the most prevalent in the vats. These differences may be related to the different approaches used and the regional differences in the composition of the lactic acid bacterial community; Carvalho-Netto et al. (6) worked with fermentations from the state of São Paulo, whereas our work was performed with distilleries from the state of Minas Gerais.

The characterization of the bacterial communities involved in the production of cachaça using culture-dependent and culture-independent techniques shows the prevalence of a variety of Lactobacillus species during the fermentation process. Lactobacillus plantarum was the only species found during cachaça fermentation using both techniques. Although lactic acid bacteria are considered to be contaminants in cachaça fermentation, it is important to understand the influence these microorganisms have on the cachaça fermentation process (14). Lactic acid bacteria ferment glucose and produce lactic acid as their main metabolic byproduct, thus reducing the yield of alcohol. The microbial community present during fermentation is also responsible for producing secondary compounds that determine the flavor of the beverage. The presence of certain concentrations of bacteria during fermentation is not considered a contaminating factor but rather an essential component of the final quality of the beverage (6). However, there is little information available regarding the composition and concentration of these microorganisms during cachaça production. Therefore, a better understanding of the relationship between the microorganisms involved in the fermentation process and the final chemical quality of the beverage is needed.

We obtained 40 clones from cheese samples after 7 and 60 days of ripening. After 7 days of ripening, 9 clones of Lc. lactis and 1 clone of both Lb. plantarum and S. salivarius were found. After 60 days of ripening, S. salivarius was the most prevalent, representing 34.5% of the clones obtained. Lactobacillus arizonensis was the second most prevalent species, representing 24.1% of the total clones. Lactobacillus plantarum and Lc. lactis were also isolated at this ripening time with other bacteria, such as Lactobacillus brevis, Sphingomonas sp. and an uncultured Streptococcus sp. Lactic acid bacteria found during cheese ripening are collectively referred to as non-starter lactic acid bacteria, and they are considered to be the species involved mainly in flavor development. The composition of these bacterial species varies with the method used in milk processing and the age of the cheese (21). The presence of lactic acid bacteria during cheese maturation has been described in several reports (5, 11, 21, 24). Duthoit et al. (8) used clone libraries and 16S rRNA gene sequencing to show that the lactic acid bacteria Lc. lactis, Lb. plantarum, S. termophilus, Enterococcus faecium, Ln. mesenteroides, Ln. pseudomesenteoides and Lb. pentosus were the most predominant during the manufacturing and ripening of Salers, an artisanal cheese produced in France. The first two species were also found during the ripening of Canastra Minas cheese in our study.

The succession of the bacterial populations observed using an analysis of a 16S rRNA gene clone library showed that the fermentation of sour cassava starch, cachaça and Minas cheese is mediated by complex microbial ecosystems, which include both lactic acid and uncultured bacteria. The acidic conditions found during these fermentation processes may be the most important factor in determining the prevalence of the different bacterial species during traditional fermentation. Lactic acid bacteria were the most prevalent species during the fermentation processes for cachaça and Canastra Minas cheese production. Unculturable bacteria predominated during the middle and at the end of sour starch cassava fermentation. These unculturable bacteria may play an important role in the fermentation process, and more studies are necessary to understand the contribution these microorganisms make to the quality of this product.