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Research article
Isolation and Characterization of Lactic Acid Bacteria from Fermented Goat Milk in Tajikistan
1Max Rubner-Insititut, Federal Research Institute for Nutrition and Food, Department of Microbiology and Biotechnology, , 2Department of Agricultural Food Sciences, University of Bologna, Viale Fanin 42, I-40127 Bologna, Italy, 3Department of Safety and Quality of Milk and Fish Products, Hermann-Weigmann-Str. 1, D-24103 Kiel, Germany, 4Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria, 55Physiology and Biochemistry of Nutrition, Haid-und-Neu-Str. 9, D-76131 Karlsruhe, Germany, 6Department of Cultural Heritage, University of Bologna, Via degli Ariani 1, I-48121 Ravenna, Italy, 7A.R. Smith Department of Chemistry and Fermentation Sciences, Appalachian State University, Boone, NC 28608
J. Microbiol. Biotechnol. 2018; 28(11): 1834-1845
Published November 28, 2018 https://doi.org/10.4014/jmb.1807.08011
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Traditional fermentations are in many rural areas of the world still the main method for food processing and preservation. These fermentations are often done empirically, based on cultural knowledge, and they often involve using back-slopping to inoculate a new fermentation with a small portion of a previous successful batch [1]. In traditional fermentations, the biochemical changes of the product during fermentation are brought about by wild bacteria or yeasts, which originate from the raw materials [2, 3]. These are mainly lactic acid fermentations, in which lactic acid bacteria (LAB) such as
While fermentations of milk in Europe predominantly rely on the use of LAB starter cultures and these bacteria are predominantly associated with European milk products, in African and Asian countries milk fermentations appear to be mixed fermentations with LAB and often involving
Goat milk is often fermented in small pastoral communities in northern Tajikistan by traditional methods. These sour milk fermentations mostly rely on spontaneous fermentation and are made from raw, unpasteurized milk without defined starter cultures. An example is fermented goat milk from the Yaghnob Valley in Tajikistan, which is traditionally produced by back-slopping, and has been previously studied with respect to the yeasts occurring in the product [8]. So far however, the LAB, especially the lactobacilli associated with the product, has not been identified. In this study, we report on the identification of the lactobacilli associated with Tajikistan’s traditionally fermented milk products, their technological properties like acidifying and milk coagulating abilities, as well as their antibiotic resistances, in order to assess their suitability for development as starter cultures.
Materials and Methods
Bacterial Strains and Growth Conditions
Twenty three presumptive
Phenotypic Characterization and Determination of Lactic Acid Configuration
The Gram-reaction was determined by the KOH method using 3% (w/v) aqueous KOH and visible amounts of bacterial colonies on glass slides according to Powers [9]. Growth at 10 and 45°C in MRS broth was evaluated after 24 h and 48 h of incubation, and the catalase reaction was tested with 3% (v/v) H2O2 as described by Mathara
16S rRNA Gene Sequencing and Strain Genotyping by RAPD-PCR
The total genomic DNA of the 23 strains was isolated from 4 ml of fresh overnight cultures grown at 37°C in MRS broth using the method of Pitcher
The randomly amplified polymorphic DNA (RAPD) PCR reaction was performed in a 50 µl volume using 100 ng chromosomal DNA, 1 ×
Whole Genome Sequence
The total genomic DNA of selected lactobacilli was isolated using the peqGOLD Bacterial DNA Kit (Peqlab, Erlangen, Germany). For paired-end sequencing, the library of genomic DNA was prepared with an Illumina Nextera XT Library Prep Kit (Illumina, USA) and sequencing was done on the MiSeq sequencer with 2 × 250 cycles. The raw paired-end sequencing data containing adapter sequences were trimmed using the Trimmomatic (v. 0.32) pipeline [15] and then
Fermentation of Goat’s Milk with Lactobacillus Strains
To determine the growth and acidification ability of potential starter strains, commercial, pasteurized goat’s milk was obtained from a local supermarket in Germany and was used in fermentation experiments. The pasteurized goat’s milk was inoculated with the
The milk was fermented in 50 ml volumes at 30°C for 48 h and the pH and numbers of bacteria were determined after 0 h (immediately after inoculation), 24 h and 48 h. For enumeration, 1 ml of milk was removed and diluted in quarter-strength Ringer’s solution in a ten-fold dilution series. Appropriate aliquots from appropriate dilutions were plated out onto MRS agar (Merck, Darmstadt, Germany) plates for determining the
Determination of Rheological Properties
For the measurement of the viscosity, a rheometer MCR 302 (Anton Paar, Ostfildern, Germany) was used. Aliqouts of 0.7 ml of the fermented product were placed into the cone and plate measuring system (diameter 50 mm) using a syringe without needle. A flow curve was recorded at a temperature of 20°C using a logarithmic ramp of the shear rate (10 to 1,000 s-1). These measurements were performed after 24 h and 48 h of fermentation.
Antibiotic Resistance Profile
The susceptibilities of the strains towards antibiotics were determined using the LAB susceptibility test medium (LSM) [21]. The minimum inhibitory concentrations (MICs) of ampicillin, gentamicin, tetracycline, erythromycin, streptomycin, vancomycin and chloramphenicol (Sigma, Germany) were determined. In order to do this, the overnight fresh cultures were inoculated at a concentration of 1 × 106 CFU /ml in 100 µl of LSM using a 96-well plate (Merck, Darmstadt, Germany), which contained a two-fold dilution series of each of the antibiotics. The MIC breakpoint values for each antibiotic were adopted from EFSA [22].
Results
Identification of the Lactic Acid Bacteria Strains and RAPD Strain Typing
Twenty three predominant
The lactobacilli could be identified as belonging to one of five species,
-
Table 1 . Differential characteristics of
Lactobacillus isolates and 16S rRNA sequences.Strain Gas production Growth temperature Lactate enantiomer Related Taxa % Similarity of 16S Sequences [13] 15°C 45°C TJA 1 - - - D L. delbrueckii subspbulgaricus 99.72% TJA 2 - - + D L. delbrueckii subspbulgaricus 99.65% TJA 6A - + + DL L. helveticus 99.65% TJA 7A - + + DL L. plantarum 99.93% TJA 9 - - + D L. plantarum 99.93% TJA 10 - - + DL L. helveticus 100% TJA 11B - + - DL L. helveticus 100% TJA 11S - - + DL L. helveticus 99.79% TJA 12 - - + D L. delbrueckii subspbulgaricus 99.71% TJA 16 - - + DL L. helveticus 100% TJA 17 - - + D L. delbrueckii subspbulgaricus 99.58% TJA 19 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 20 - - + DL L. helveticus 99.93% TJA 21 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 24A - - + DL L. helveticus 100% TJA 26B - + - DL L. plantarum 99.79% TJA 26S - + - DL L. pentosus 99.81% TJA 27 - - + DL L. helveticus 100% TJA 28 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 29 - - + D L. delbrueckii subsplactics 99.71% TJA 31 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 32 - - + DL L. helveticus 99.79% TJB 4 - + - L L. paracasei 99.93% Production of D-, L-, and DL lactic acid as an end product.
+: Positive growth; -: negative growth
-
Table 2 . Carbohydrate fermentation of
Lactobacillus strains.No. of strains L. plantarum (3)L. pentosus (1)L. helveticus (8)L. delbrueckii (10)L. paracasei (1)Carbohydrate fermentation LARA + - - - RIB - - - - GAL + + (75%) - + GLU + + + (80%) + FRU + + (37.5%) + + MNE + + + + RHA + - - - MAN + - - + MDM + - - - MDG - - - - NAG + + (75%) - + AMY + - - - ARB + - - + SAL + + (25%) - + CEL + - - + MAL + - - + LAC + + + + MEL + - - - SAC + - - + TRE + + (50%) - + MLZ + - - + RAF + - - - GEN + - - + TUR + - - + DARL + - - - GNT + - - + LARA L-arabinose, RIB D-Ribose, GAL D-galactose, GLU D-glucose, FRU Dfructose, MNE D-mannose, RHA L-rhamnose, MAN D-mannitol, MDM methyl-α- D-mannopyranoside, MDG methyl-α-D-glucopyranoside, NAG N-acetylglucosamine, AMY amygdalin, ARB arbutin, SAL salicin, CEL D-cellobiose, MAL D-Maltose, LAC D-lactose, MEL D-melibiose, SAC D-saccharose, TRE D-trehalose, MLZ Dmelezitose, RAF D-raffinose, GEN gentiobiose, TUR D-turanose, DARA Darabitol, GNT potassium gluconate
+ : Positive fermentation; percentage was calculated like positive number divided by total number of species, - : negative fermentation.
-
Fig. 1. Dendrogram obtained by UPGMA of correlation value
r of RAPD-PCR fingerprint patterns with primer M13 of strains isolated from fermented milk of Tajikistan.
RAPD fingerprinting furthermore showed that isolates in subgroup II clustered at
Whole Genome Data Analysis of Potential Starter Cultures
The genome sequences of all major technologically important LAB are available, which has given new insight into functional genomics of LAB associated with food fermentations [23]. In our study, the genomes of four strains selected as representative of each species were sequenced and analyzed for typical functions related to fermentation activities and for the absence of transferable antibiotic resistance genes. Briefly, the contigs of the four assembled genomes ranged from 51 to 245 and the genome sizes ranged from 1.75 to 3.24 Mbp (Table 3). The largest N50 value was 131,900 for
-
Table 3 . Genome dataa of selected
Lactobacillus strains from Tajikistani fermented milk.Strain TJA10 TJA26B TJA31 TJB4 GenBank accession no. QNXC00000000 QXEU00000000 QNXB00000000 QXET00000000 No. of contigs 254 122 54 244 Largest contig 61,465 341,241 424,489 180,575 N50 21,570 131,900 107,123 51,965 GC-content (mol%) 36.73 44.5 49.79 46.4 Total length (bp1) 1,889,241 bp 3,243,521 bp 1,742,687 bp 2,945,278 bp Plasmid sequence n.d.2 n.d. n.d. n.d. CDS (coding sequence) 2124 3233 1861 3186 tRNA 52 54 66 56 rRNA 6 4 10 11 ncRNA 3 4 3 3 Bacteriocin Helveticin J Plantaricin EF, N and JK n.d. n.d. Citrate lyase +3 + n.d. + Acetolactate synthase + + n.d. + Acquired antibiotic resistance genes n.d. n.d. n.d. n.d. 1; Base pair, 2; not detected, 3; gene detected.
Characterization of Goat’s Milk Fermentation
One strain of each species,
-
Fig. 2. Determination of total lactic acid bacteria counts in pasteurized goat’s milk after inoculation starter cultures using MRS agar plates. Co-culture 1: four starter lactic acid bacteria with the yeast
Kluyveromyces marxianus MBT-5698, co-culture 2: four starter lactic acid bacteria withS. thermophilus MBT-2 andKluyveromyces marxianus MBT-5698. Counts shown are from triplicate determinations with indicating a standard error.
-
Fig. 3. pH development of goat’s milk inoculated with starter lactic acid bacteria and co-culture with a
S. thermophilus and yeast. Co-culture 1: four starter LAB with the yeastKluyveromyces marxianus MBT-5698, co-culture 2: four starter LAB withS. thermophilus MBT-2 andKluyveromyces marxianus MBT-5698. Goat milk without inoculation of LAB as negative control.
The flow curves of the different cultures are shown in Fig. 4. All samples, except co-cultures 1 and 2, showed similar flow behavior. After 24 h no significant increase in the viscosity was detected. This corresponded well with the results of the pH measurements (Fig. 3). The pH values of samples TJB 4, TJA 10, TJA 26B, and TJA 31 were higher than 5.5 (Fig. 3) and thus no gelation of the milk could occur, as indicated in Fig. 4. The co-cultures on the other hand, show pH values lower than 4.0, and that gelation was nearly completed. Co-culture 2 showed a typical flow-thinning behavior. Viscosity decreased with higher shear rates, which were caused by aggregate destruction. Co-culture 1 shows shear-thickening behavior in the range of 10 to 100/s and shear-thinning behavior in the range above 100/s after 24 as well as after 48 h.
-
Fig. 4. Flow curves of the fermented milk products after 24 h (solid line) and after 48 h (dotted line) of fermentation.
Antibiotic Resistance Profile
In order to characterize the antibiotic resistances of the different lactobacilli, the LSM medium of Klare
-
Table 4 . Antibiotic resistance tests of isolates.
Strain Minimum inhibitory concentration (µg/ml) Amp Ery Tetra Strep Chlor Gent Van L. delbruecki TJA 1 0.25 0.125 1 2 1 8 0.25 TJA 2 Sensitiveb 0.25 16 16 2 2 0.5 TJA 9 0.5 0.125 16 16 4 8 0.5 TJA 12 16 0.5 8 8 4 4 0.5 TJA 17 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 19 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 21 Sensitive 0.5 16 8 2 2 0.5 TJA 28 Sensitive 0.25 16 32 1 2 1 TJA 29 Sensitive Sensitive 16 32 1 16 0.5 TJA 31 0.25 0.5 8 8 2 4 0.5 TJA 32 1 0.25 1 4 4 2 0.25 L. helveticus TJA 6A Sensitive 0.125 >256 1 2 0.25 >256 TJA 10 0.25 0.125 32 4 2 4 0.25 TJA 16 Sensitive 2 16 8 1 4 0.5 TJA 20 Sensitive Sensitive 8 4 1 4 0.5 TJA 24A 0.25 Sensitive 8 2 1 4 0.5 TJA 27 0.25 Sensitive 16 8 2 8 0.5 L. plantarum TJA 7A Sensitive 0.25 >256 2 2 0.5 >256 TJA 11S 0.125 0.25 16 2 2 2 0.5 TJA 11B Sensitive 0.25 >256 4 4 0.25 >256 TJA 26B 0.125 0.25 128 2 4 0.25 >256 L. pentosus TJA 26S Sensitive 0.125 8 4 2 0.5 >256 L. paracasei TJB 4 0.25 0.125 8 4 2 0.5 >256 Breakpointsa for L. plantarum 2 1 32 n.r. 8 16 n.r. Breakpointsa for Lactobacillus obligate homofermentative1 1 4 16 4 16 2 a Breakpoints according to EFSA [22], b Growth inhibition occurs at 0.06 μg/ml As the least concentration, n.r: not required. n.d. : no bacterial growth on ISO medium Amp: ampicillin, Ery: erythromycin, Tetra: tetracycline, Strep: streptomycin, Chlor: chloramphenicol, Gent: gentamicin, Van: Vancomycin.
The value ‘>256’ means no growth inhibition occurred and this was the maximum concentration tested.
Discussion
Most of the
The RAPD strain typing method is a rapid, accurate and sensitive method for monitoring particular strains in a fermentation to which specific starter cultures were added [32]. However, the RAPD fingerprint technique only has limited potential identifying different
Traditionally,
When single strains of each species,
The lower pH brought about by the co-inoculation of the starter strains with the yeast may be explained for the symbiosis of
The flow curves of the different cultures in milk showed no significant increase in the viscosity, while they showed that gelation of the milk after fermentation with the co-cultures was nearly completed. Co-culture 2 showed a typical flow-thinning behavior. Viscosity decreased with higher shear rates, which were caused by aggregate destruction. Co-culture 1 showed shear-thickening behavior in the range of 10 to 100/s and shear-thinning behavior in the range above 100/s after 24 as well as after 48 h. This was probably caused by aggregates which blocked each other in the shear gap at lower shear rates and become destructed at higher shear rates. After 48 hours, cultures TJB 4, TJA 10, TJA 26B, and TJA 31 also showed shear-thinning behavior. The maximum viscosity directly corresponded to the lowest pH value.
There have been some previous reports on aminoglycoside resistances among
In this study, therefore, the lactobacilli from Tajikistani fermented milk could be identified to consist of
The low incidence of antibiotic resistance in the strains isolated from Yaghnob fermented milk could be related to the absence of use of antibiotics in this population: in fact, antibiotic therapy has not been used and traditional medicine with herbs has been mostly utilized to treat diseases. The microorganisms associated with the fermentation of Tajikistani milk have so far not been described. The results of this study clearly show that the microorganisms associated with fermentation are
Nucleotide Sequence Accession Number
The whole-genome shotgun project of Tajikistani starter cultures can be accessed through BioProject number PRJNA479758 and has been deposited at DDB/ENA/GenBank under the accession no. listed in Table 3.
Acknowledgments
The authors acknowledge the team of the Yaghnob Valley Mission directed by Prof. Antonio Panaino (Department of Cultural Heritage, University of Bologna, Italy) for their invaluable support in providing samples and fruitful discussion.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2018; 28(11): 1834-1845
Published online November 28, 2018 https://doi.org/10.4014/jmb.1807.08011
Copyright © The Korean Society for Microbiology and Biotechnology.
Isolation and Characterization of Lactic Acid Bacteria from Fermented Goat Milk in Tajikistan
Gyu-Sung Cho 1, Claudia Cappello 2, Katrin Schrader 3, Olakunle Fagbemigum 4, Folarin A. Oguntoyinbo 4, 7, Claudia Csovcsics 5, Niels Rösch 1, Jan Kabisch 1, Horst Neve 1, Wilhelm Bockelmann 1, Karlis Briviba 5, Monica Modesto 2, Elisabetta Cilli 6, Paola Mattarelli 2 and Charles M.A.P. Franz 1*
1Max Rubner-Insititut, Federal Research Institute for Nutrition and Food, Department of Microbiology and Biotechnology, , 2Department of Agricultural Food Sciences, University of Bologna, Viale Fanin 42, I-40127 Bologna, Italy, 3Department of Safety and Quality of Milk and Fish Products, Hermann-Weigmann-Str. 1, D-24103 Kiel, Germany, 4Department of Microbiology, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria, 55Physiology and Biochemistry of Nutrition, Haid-und-Neu-Str. 9, D-76131 Karlsruhe, Germany, 6Department of Cultural Heritage, University of Bologna, Via degli Ariani 1, I-48121 Ravenna, Italy, 7A.R. Smith Department of Chemistry and Fermentation Sciences, Appalachian State University, Boone, NC 28608
Abstract
The lactobacilli associated with a fermented goat milk product from Tajikistan were isolated to
characterize their technological properties and antibiotic resistances in order to assess their
suitability for development as starter cultures. In this study, twenty three strains were
identified by 16S rRNA sequencing as typical dairy-associated lactic acid bacterial strains, i.e.
L. plantarum, L. pentosus, L. delbrueckii, L. helveticus and L. paracasei. These strains were
generally susceptible to most antibiotics tested in this study and this allowed a selection of
strains as safe starters. The draft genomes of four representative strains were sequenced and
the number of contigs of the four assembled genomes ranged from 51 to 245 and the genome
sizes ranged from 1.75 to 3.24 Mbp. These representative strains showed differences in their
growth behavior and pH-reducing abilities in in vitro studies. The co-inoculation of these
Lactobacillus spp. strains together with a yeast Kluyveromyces marxianus MBT-5698, or together
with the yeast and an additional Streptococcus thermophilus MBT-2, led to a pH reduction to 3.4
after 48 h. Only in the case of fermentation inoculated with the co-culture, the viscosity of the
milk increased noticeably. In contrast, fermentations with single strains did not lead to
gelation of the milk or to a decrease in the pH after 24h. The results of this study provide a
comprehensive understanding of the predominant lactobacilli related to Tajikistani fermented
milk products.
Keywords: Lactic acid bacteria, fermentation, Lactobacillus, whole genome sequencing, milk
Introduction
Traditional fermentations are in many rural areas of the world still the main method for food processing and preservation. These fermentations are often done empirically, based on cultural knowledge, and they often involve using back-slopping to inoculate a new fermentation with a small portion of a previous successful batch [1]. In traditional fermentations, the biochemical changes of the product during fermentation are brought about by wild bacteria or yeasts, which originate from the raw materials [2, 3]. These are mainly lactic acid fermentations, in which lactic acid bacteria (LAB) such as
While fermentations of milk in Europe predominantly rely on the use of LAB starter cultures and these bacteria are predominantly associated with European milk products, in African and Asian countries milk fermentations appear to be mixed fermentations with LAB and often involving
Goat milk is often fermented in small pastoral communities in northern Tajikistan by traditional methods. These sour milk fermentations mostly rely on spontaneous fermentation and are made from raw, unpasteurized milk without defined starter cultures. An example is fermented goat milk from the Yaghnob Valley in Tajikistan, which is traditionally produced by back-slopping, and has been previously studied with respect to the yeasts occurring in the product [8]. So far however, the LAB, especially the lactobacilli associated with the product, has not been identified. In this study, we report on the identification of the lactobacilli associated with Tajikistan’s traditionally fermented milk products, their technological properties like acidifying and milk coagulating abilities, as well as their antibiotic resistances, in order to assess their suitability for development as starter cultures.
Materials and Methods
Bacterial Strains and Growth Conditions
Twenty three presumptive
Phenotypic Characterization and Determination of Lactic Acid Configuration
The Gram-reaction was determined by the KOH method using 3% (w/v) aqueous KOH and visible amounts of bacterial colonies on glass slides according to Powers [9]. Growth at 10 and 45°C in MRS broth was evaluated after 24 h and 48 h of incubation, and the catalase reaction was tested with 3% (v/v) H2O2 as described by Mathara
16S rRNA Gene Sequencing and Strain Genotyping by RAPD-PCR
The total genomic DNA of the 23 strains was isolated from 4 ml of fresh overnight cultures grown at 37°C in MRS broth using the method of Pitcher
The randomly amplified polymorphic DNA (RAPD) PCR reaction was performed in a 50 µl volume using 100 ng chromosomal DNA, 1 ×
Whole Genome Sequence
The total genomic DNA of selected lactobacilli was isolated using the peqGOLD Bacterial DNA Kit (Peqlab, Erlangen, Germany). For paired-end sequencing, the library of genomic DNA was prepared with an Illumina Nextera XT Library Prep Kit (Illumina, USA) and sequencing was done on the MiSeq sequencer with 2 × 250 cycles. The raw paired-end sequencing data containing adapter sequences were trimmed using the Trimmomatic (v. 0.32) pipeline [15] and then
Fermentation of Goat’s Milk with Lactobacillus Strains
To determine the growth and acidification ability of potential starter strains, commercial, pasteurized goat’s milk was obtained from a local supermarket in Germany and was used in fermentation experiments. The pasteurized goat’s milk was inoculated with the
The milk was fermented in 50 ml volumes at 30°C for 48 h and the pH and numbers of bacteria were determined after 0 h (immediately after inoculation), 24 h and 48 h. For enumeration, 1 ml of milk was removed and diluted in quarter-strength Ringer’s solution in a ten-fold dilution series. Appropriate aliquots from appropriate dilutions were plated out onto MRS agar (Merck, Darmstadt, Germany) plates for determining the
Determination of Rheological Properties
For the measurement of the viscosity, a rheometer MCR 302 (Anton Paar, Ostfildern, Germany) was used. Aliqouts of 0.7 ml of the fermented product were placed into the cone and plate measuring system (diameter 50 mm) using a syringe without needle. A flow curve was recorded at a temperature of 20°C using a logarithmic ramp of the shear rate (10 to 1,000 s-1). These measurements were performed after 24 h and 48 h of fermentation.
Antibiotic Resistance Profile
The susceptibilities of the strains towards antibiotics were determined using the LAB susceptibility test medium (LSM) [21]. The minimum inhibitory concentrations (MICs) of ampicillin, gentamicin, tetracycline, erythromycin, streptomycin, vancomycin and chloramphenicol (Sigma, Germany) were determined. In order to do this, the overnight fresh cultures were inoculated at a concentration of 1 × 106 CFU /ml in 100 µl of LSM using a 96-well plate (Merck, Darmstadt, Germany), which contained a two-fold dilution series of each of the antibiotics. The MIC breakpoint values for each antibiotic were adopted from EFSA [22].
Results
Identification of the Lactic Acid Bacteria Strains and RAPD Strain Typing
Twenty three predominant
The lactobacilli could be identified as belonging to one of five species,
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Table 1 . Differential characteristics of
Lactobacillus isolates and 16S rRNA sequences..Strain Gas production Growth temperature Lactate enantiomer Related Taxa % Similarity of 16S Sequences [13] 15°C 45°C TJA 1 - - - D L. delbrueckii subspbulgaricus 99.72% TJA 2 - - + D L. delbrueckii subspbulgaricus 99.65% TJA 6A - + + DL L. helveticus 99.65% TJA 7A - + + DL L. plantarum 99.93% TJA 9 - - + D L. plantarum 99.93% TJA 10 - - + DL L. helveticus 100% TJA 11B - + - DL L. helveticus 100% TJA 11S - - + DL L. helveticus 99.79% TJA 12 - - + D L. delbrueckii subspbulgaricus 99.71% TJA 16 - - + DL L. helveticus 100% TJA 17 - - + D L. delbrueckii subspbulgaricus 99.58% TJA 19 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 20 - - + DL L. helveticus 99.93% TJA 21 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 24A - - + DL L. helveticus 100% TJA 26B - + - DL L. plantarum 99.79% TJA 26S - + - DL L. pentosus 99.81% TJA 27 - - + DL L. helveticus 100% TJA 28 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 29 - - + D L. delbrueckii subsplactics 99.71% TJA 31 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 32 - - + DL L. helveticus 99.79% TJB 4 - + - L L. paracasei 99.93% Production of D-, L-, and DL lactic acid as an end product..
+: Positive growth; -: negative growth.
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Table 2 . Carbohydrate fermentation of
Lactobacillus strains..No. of strains L. plantarum (3)L. pentosus (1)L. helveticus (8)L. delbrueckii (10)L. paracasei (1)Carbohydrate fermentation LARA + - - - RIB - - - - GAL + + (75%) - + GLU + + + (80%) + FRU + + (37.5%) + + MNE + + + + RHA + - - - MAN + - - + MDM + - - - MDG - - - - NAG + + (75%) - + AMY + - - - ARB + - - + SAL + + (25%) - + CEL + - - + MAL + - - + LAC + + + + MEL + - - - SAC + - - + TRE + + (50%) - + MLZ + - - + RAF + - - - GEN + - - + TUR + - - + DARL + - - - GNT + - - + LARA L-arabinose, RIB D-Ribose, GAL D-galactose, GLU D-glucose, FRU Dfructose, MNE D-mannose, RHA L-rhamnose, MAN D-mannitol, MDM methyl-α- D-mannopyranoside, MDG methyl-α-D-glucopyranoside, NAG N-acetylglucosamine, AMY amygdalin, ARB arbutin, SAL salicin, CEL D-cellobiose, MAL D-Maltose, LAC D-lactose, MEL D-melibiose, SAC D-saccharose, TRE D-trehalose, MLZ Dmelezitose, RAF D-raffinose, GEN gentiobiose, TUR D-turanose, DARA Darabitol, GNT potassium gluconate.
+ : Positive fermentation; percentage was calculated like positive number divided by total number of species, - : negative fermentation..
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Figure 1. Dendrogram obtained by UPGMA of correlation value
r of RAPD-PCR fingerprint patterns with primer M13 of strains isolated from fermented milk of Tajikistan.
RAPD fingerprinting furthermore showed that isolates in subgroup II clustered at
Whole Genome Data Analysis of Potential Starter Cultures
The genome sequences of all major technologically important LAB are available, which has given new insight into functional genomics of LAB associated with food fermentations [23]. In our study, the genomes of four strains selected as representative of each species were sequenced and analyzed for typical functions related to fermentation activities and for the absence of transferable antibiotic resistance genes. Briefly, the contigs of the four assembled genomes ranged from 51 to 245 and the genome sizes ranged from 1.75 to 3.24 Mbp (Table 3). The largest N50 value was 131,900 for
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Table 3 . Genome dataa of selected
Lactobacillus strains from Tajikistani fermented milk..Strain TJA10 TJA26B TJA31 TJB4 GenBank accession no. QNXC00000000 QXEU00000000 QNXB00000000 QXET00000000 No. of contigs 254 122 54 244 Largest contig 61,465 341,241 424,489 180,575 N50 21,570 131,900 107,123 51,965 GC-content (mol%) 36.73 44.5 49.79 46.4 Total length (bp1) 1,889,241 bp 3,243,521 bp 1,742,687 bp 2,945,278 bp Plasmid sequence n.d.2 n.d. n.d. n.d. CDS (coding sequence) 2124 3233 1861 3186 tRNA 52 54 66 56 rRNA 6 4 10 11 ncRNA 3 4 3 3 Bacteriocin Helveticin J Plantaricin EF, N and JK n.d. n.d. Citrate lyase +3 + n.d. + Acetolactate synthase + + n.d. + Acquired antibiotic resistance genes n.d. n.d. n.d. n.d. 1; Base pair, 2; not detected, 3; gene detected..
Characterization of Goat’s Milk Fermentation
One strain of each species,
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Figure 2. Determination of total lactic acid bacteria counts in pasteurized goat’s milk after inoculation starter cultures using MRS agar plates. Co-culture 1: four starter lactic acid bacteria with the yeast
Kluyveromyces marxianus MBT-5698, co-culture 2: four starter lactic acid bacteria withS. thermophilus MBT-2 andKluyveromyces marxianus MBT-5698. Counts shown are from triplicate determinations with indicating a standard error.
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Figure 3. pH development of goat’s milk inoculated with starter lactic acid bacteria and co-culture with a
S. thermophilus and yeast. Co-culture 1: four starter LAB with the yeastKluyveromyces marxianus MBT-5698, co-culture 2: four starter LAB withS. thermophilus MBT-2 andKluyveromyces marxianus MBT-5698. Goat milk without inoculation of LAB as negative control.
The flow curves of the different cultures are shown in Fig. 4. All samples, except co-cultures 1 and 2, showed similar flow behavior. After 24 h no significant increase in the viscosity was detected. This corresponded well with the results of the pH measurements (Fig. 3). The pH values of samples TJB 4, TJA 10, TJA 26B, and TJA 31 were higher than 5.5 (Fig. 3) and thus no gelation of the milk could occur, as indicated in Fig. 4. The co-cultures on the other hand, show pH values lower than 4.0, and that gelation was nearly completed. Co-culture 2 showed a typical flow-thinning behavior. Viscosity decreased with higher shear rates, which were caused by aggregate destruction. Co-culture 1 shows shear-thickening behavior in the range of 10 to 100/s and shear-thinning behavior in the range above 100/s after 24 as well as after 48 h.
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Figure 4. Flow curves of the fermented milk products after 24 h (solid line) and after 48 h (dotted line) of fermentation.
Antibiotic Resistance Profile
In order to characterize the antibiotic resistances of the different lactobacilli, the LSM medium of Klare
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Table 4 . Antibiotic resistance tests of isolates..
Strain Minimum inhibitory concentration (µg/ml) Amp Ery Tetra Strep Chlor Gent Van L. delbruecki TJA 1 0.25 0.125 1 2 1 8 0.25 TJA 2 Sensitiveb 0.25 16 16 2 2 0.5 TJA 9 0.5 0.125 16 16 4 8 0.5 TJA 12 16 0.5 8 8 4 4 0.5 TJA 17 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 19 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 21 Sensitive 0.5 16 8 2 2 0.5 TJA 28 Sensitive 0.25 16 32 1 2 1 TJA 29 Sensitive Sensitive 16 32 1 16 0.5 TJA 31 0.25 0.5 8 8 2 4 0.5 TJA 32 1 0.25 1 4 4 2 0.25 L. helveticus TJA 6A Sensitive 0.125 >256 1 2 0.25 >256 TJA 10 0.25 0.125 32 4 2 4 0.25 TJA 16 Sensitive 2 16 8 1 4 0.5 TJA 20 Sensitive Sensitive 8 4 1 4 0.5 TJA 24A 0.25 Sensitive 8 2 1 4 0.5 TJA 27 0.25 Sensitive 16 8 2 8 0.5 L. plantarum TJA 7A Sensitive 0.25 >256 2 2 0.5 >256 TJA 11S 0.125 0.25 16 2 2 2 0.5 TJA 11B Sensitive 0.25 >256 4 4 0.25 >256 TJA 26B 0.125 0.25 128 2 4 0.25 >256 L. pentosus TJA 26S Sensitive 0.125 8 4 2 0.5 >256 L. paracasei TJB 4 0.25 0.125 8 4 2 0.5 >256 Breakpointsa for L. plantarum 2 1 32 n.r. 8 16 n.r. Breakpointsa for Lactobacillus obligate homofermentative1 1 4 16 4 16 2 a Breakpoints according to EFSA [22], b Growth inhibition occurs at 0.06 μg/ml As the least concentration, n.r: not required. n.d. : no bacterial growth on ISO medium Amp: ampicillin, Ery: erythromycin, Tetra: tetracycline, Strep: streptomycin, Chlor: chloramphenicol, Gent: gentamicin, Van: Vancomycin..
The value ‘>256’ means no growth inhibition occurred and this was the maximum concentration tested..
Discussion
Most of the
The RAPD strain typing method is a rapid, accurate and sensitive method for monitoring particular strains in a fermentation to which specific starter cultures were added [32]. However, the RAPD fingerprint technique only has limited potential identifying different
Traditionally,
When single strains of each species,
The lower pH brought about by the co-inoculation of the starter strains with the yeast may be explained for the symbiosis of
The flow curves of the different cultures in milk showed no significant increase in the viscosity, while they showed that gelation of the milk after fermentation with the co-cultures was nearly completed. Co-culture 2 showed a typical flow-thinning behavior. Viscosity decreased with higher shear rates, which were caused by aggregate destruction. Co-culture 1 showed shear-thickening behavior in the range of 10 to 100/s and shear-thinning behavior in the range above 100/s after 24 as well as after 48 h. This was probably caused by aggregates which blocked each other in the shear gap at lower shear rates and become destructed at higher shear rates. After 48 hours, cultures TJB 4, TJA 10, TJA 26B, and TJA 31 also showed shear-thinning behavior. The maximum viscosity directly corresponded to the lowest pH value.
There have been some previous reports on aminoglycoside resistances among
In this study, therefore, the lactobacilli from Tajikistani fermented milk could be identified to consist of
The low incidence of antibiotic resistance in the strains isolated from Yaghnob fermented milk could be related to the absence of use of antibiotics in this population: in fact, antibiotic therapy has not been used and traditional medicine with herbs has been mostly utilized to treat diseases. The microorganisms associated with the fermentation of Tajikistani milk have so far not been described. The results of this study clearly show that the microorganisms associated with fermentation are
Nucleotide Sequence Accession Number
The whole-genome shotgun project of Tajikistani starter cultures can be accessed through BioProject number PRJNA479758 and has been deposited at DDB/ENA/GenBank under the accession no. listed in Table 3.
Acknowledgments
The authors acknowledge the team of the Yaghnob Valley Mission directed by Prof. Antonio Panaino (Department of Cultural Heritage, University of Bologna, Italy) for their invaluable support in providing samples and fruitful discussion.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

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Table 1 . Differential characteristics of
Lactobacillus isolates and 16S rRNA sequences..Strain Gas production Growth temperature Lactate enantiomer Related Taxa % Similarity of 16S Sequences [13] 15°C 45°C TJA 1 - - - D L. delbrueckii subspbulgaricus 99.72% TJA 2 - - + D L. delbrueckii subspbulgaricus 99.65% TJA 6A - + + DL L. helveticus 99.65% TJA 7A - + + DL L. plantarum 99.93% TJA 9 - - + D L. plantarum 99.93% TJA 10 - - + DL L. helveticus 100% TJA 11B - + - DL L. helveticus 100% TJA 11S - - + DL L. helveticus 99.79% TJA 12 - - + D L. delbrueckii subspbulgaricus 99.71% TJA 16 - - + DL L. helveticus 100% TJA 17 - - + D L. delbrueckii subspbulgaricus 99.58% TJA 19 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 20 - - + DL L. helveticus 99.93% TJA 21 - - + D L. delbrueckii subspbulgaricus 99.72% TJA 24A - - + DL L. helveticus 100% TJA 26B - + - DL L. plantarum 99.79% TJA 26S - + - DL L. pentosus 99.81% TJA 27 - - + DL L. helveticus 100% TJA 28 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 29 - - + D L. delbrueckii subsplactics 99.71% TJA 31 - - + D L. delbrueckii subspbulgaricus 99.79% TJA 32 - - + DL L. helveticus 99.79% TJB 4 - + - L L. paracasei 99.93% Production of D-, L-, and DL lactic acid as an end product..
+: Positive growth; -: negative growth.
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Table 2 . Carbohydrate fermentation of
Lactobacillus strains..No. of strains L. plantarum (3)L. pentosus (1)L. helveticus (8)L. delbrueckii (10)L. paracasei (1)Carbohydrate fermentation LARA + - - - RIB - - - - GAL + + (75%) - + GLU + + + (80%) + FRU + + (37.5%) + + MNE + + + + RHA + - - - MAN + - - + MDM + - - - MDG - - - - NAG + + (75%) - + AMY + - - - ARB + - - + SAL + + (25%) - + CEL + - - + MAL + - - + LAC + + + + MEL + - - - SAC + - - + TRE + + (50%) - + MLZ + - - + RAF + - - - GEN + - - + TUR + - - + DARL + - - - GNT + - - + LARA L-arabinose, RIB D-Ribose, GAL D-galactose, GLU D-glucose, FRU Dfructose, MNE D-mannose, RHA L-rhamnose, MAN D-mannitol, MDM methyl-α- D-mannopyranoside, MDG methyl-α-D-glucopyranoside, NAG N-acetylglucosamine, AMY amygdalin, ARB arbutin, SAL salicin, CEL D-cellobiose, MAL D-Maltose, LAC D-lactose, MEL D-melibiose, SAC D-saccharose, TRE D-trehalose, MLZ Dmelezitose, RAF D-raffinose, GEN gentiobiose, TUR D-turanose, DARA Darabitol, GNT potassium gluconate.
+ : Positive fermentation; percentage was calculated like positive number divided by total number of species, - : negative fermentation..
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Table 3 . Genome dataa of selected
Lactobacillus strains from Tajikistani fermented milk..Strain TJA10 TJA26B TJA31 TJB4 GenBank accession no. QNXC00000000 QXEU00000000 QNXB00000000 QXET00000000 No. of contigs 254 122 54 244 Largest contig 61,465 341,241 424,489 180,575 N50 21,570 131,900 107,123 51,965 GC-content (mol%) 36.73 44.5 49.79 46.4 Total length (bp1) 1,889,241 bp 3,243,521 bp 1,742,687 bp 2,945,278 bp Plasmid sequence n.d.2 n.d. n.d. n.d. CDS (coding sequence) 2124 3233 1861 3186 tRNA 52 54 66 56 rRNA 6 4 10 11 ncRNA 3 4 3 3 Bacteriocin Helveticin J Plantaricin EF, N and JK n.d. n.d. Citrate lyase +3 + n.d. + Acetolactate synthase + + n.d. + Acquired antibiotic resistance genes n.d. n.d. n.d. n.d. 1; Base pair, 2; not detected, 3; gene detected..
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Table 4 . Antibiotic resistance tests of isolates..
Strain Minimum inhibitory concentration (µg/ml) Amp Ery Tetra Strep Chlor Gent Van L. delbruecki TJA 1 0.25 0.125 1 2 1 8 0.25 TJA 2 Sensitiveb 0.25 16 16 2 2 0.5 TJA 9 0.5 0.125 16 16 4 8 0.5 TJA 12 16 0.5 8 8 4 4 0.5 TJA 17 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 19 n.d. n.d. n.d. n.d. n.d. n.d. n.d. TJA 21 Sensitive 0.5 16 8 2 2 0.5 TJA 28 Sensitive 0.25 16 32 1 2 1 TJA 29 Sensitive Sensitive 16 32 1 16 0.5 TJA 31 0.25 0.5 8 8 2 4 0.5 TJA 32 1 0.25 1 4 4 2 0.25 L. helveticus TJA 6A Sensitive 0.125 >256 1 2 0.25 >256 TJA 10 0.25 0.125 32 4 2 4 0.25 TJA 16 Sensitive 2 16 8 1 4 0.5 TJA 20 Sensitive Sensitive 8 4 1 4 0.5 TJA 24A 0.25 Sensitive 8 2 1 4 0.5 TJA 27 0.25 Sensitive 16 8 2 8 0.5 L. plantarum TJA 7A Sensitive 0.25 >256 2 2 0.5 >256 TJA 11S 0.125 0.25 16 2 2 2 0.5 TJA 11B Sensitive 0.25 >256 4 4 0.25 >256 TJA 26B 0.125 0.25 128 2 4 0.25 >256 L. pentosus TJA 26S Sensitive 0.125 8 4 2 0.5 >256 L. paracasei TJB 4 0.25 0.125 8 4 2 0.5 >256 Breakpointsa for L. plantarum 2 1 32 n.r. 8 16 n.r. Breakpointsa for Lactobacillus obligate homofermentative1 1 4 16 4 16 2 a Breakpoints according to EFSA [22], b Growth inhibition occurs at 0.06 μg/ml As the least concentration, n.r: not required. n.d. : no bacterial growth on ISO medium Amp: ampicillin, Ery: erythromycin, Tetra: tetracycline, Strep: streptomycin, Chlor: chloramphenicol, Gent: gentamicin, Van: Vancomycin..
The value ‘>256’ means no growth inhibition occurred and this was the maximum concentration tested..
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