Isolation and enumeration
We selected seventeen samples of bacteria from six traditional Kazakh homemade dairy products. Their colonies (2 ‒ 6 mm diameter) had an oval or round shape, smooth surface, plat profile, shiny and white color appearance on MRS agar. Identification of Lactobacilli isolates was carried out by examination of colony characteristics and cell morphology.
The isolated LAB strains was identify with 16S RNA. The 16S RNA sequence data were aligned to conduct a phylogenetic tree and the phylogenetic position of studied strains. The sequence of the 16S rRNA gene (around 1,400 bp) was determined and searched with the NCBI BLAST program (http://www.ncbi.nlm.nih.gov) for their closest relatives/reference strains with a homology of over or equal to 99%. In the process of genetic identification, it was found that the studied cultures belong to taxonomic group such as Lactobacillus (fig. 1).
The phylogenetic trees showing were Lactobacilli (Lb.) containing (Lb. fermentum, Lb. gorillae, Lb. durianis, Lb. agilis, Lb. equi, Lb. alimentarius, Lb. zymae, Lb. saniviri, Lb.rhamnosus Lb.paracasei, Lb. zeae, Lb. algidus, Lb.dextrinicus, Lb. acidophilus, Lb. equicursoris).
Previously we have studied probiotic properties of all the Lactobacilli isolates: antagonistic activity against pathogenic microorganisms, biocompatibility, viability and lactose utilization activity (Raikhan et al. 2020). Further research will focus on Lb. acidophilus and Lb.rhamnosus. The ability of Lb. acidophilus and Lb.rhamnosus to ferment lactose had been investigated using Cole’s ferricyanide methods (Sydney 1933). The lactose content of the PM with cultivated Lb. acidophilus and Lb.rhamnosus was 3.1 % (data not shown). In the traditional Kazakh homemade dairy products Lb. acidophilus and Lb.rhamnosus was identified as dominant species and present in the most collected samples.
Antagonistic activity
The antagonistic activity of the sixteen tested probiotic strains was studied by agar spot test. The antagonistic activity of LAB against three indicator pathogens widely differ, the results are showed in table 1. The Lb. acidophilus and Lb. rhamnosus possessed the high and middle zone of inhibition effect against all 3 indicator pathogens S. typhimurium, S.marcescens and E.coli (table 2).
Table 1 Antagonistic activity of 16 strain of LAB isolated from traditional Kazakh homemade dairy products
Strains
|
Identifications
|
I
|
II
|
III
|
Lb 1
|
Lb. fermentum
|
++
|
+
|
++
|
Lb 2
|
Lb. gorillae
|
+
|
++
|
++
|
Lb 3
|
Lb. durianis
|
++
|
+
|
++
|
Lb 4
|
Lb. agilis
|
+
|
-
|
-
|
Lb 5
|
Lb. equi
|
-
|
++
|
+
|
Lb 6
|
Lb. alimentarius
|
++
|
+
|
++
|
Lb 7
|
Lb. zymae
|
-
|
-
|
+
|
Lb 8
|
Lb. saniviri
|
+
|
+
|
-
|
Lb 9
|
Lb.rhamnosus
|
+++
|
++
|
+++
|
Lb 10
|
Lb.paracasei paracasei
|
++
|
+
|
+
|
Lb 11
|
Lb.paracasei tolerans
|
-
|
+
|
+
|
Lb 12
|
Lb. zeae
|
+
|
-
|
++
|
Lb 13
|
Lb. algidus
|
-
|
+
|
-
|
Lb 14
|
Lb.dextrinicus
|
++
|
+
|
+
|
Lb 15
|
Lb. acidophilus
|
+++
|
++
|
+++
|
Lb 16
|
Lb. equicursoris
|
++
|
-
|
+
|
+: low zone of inhibition (1.0-4.9 mm), ++: middle zone of inhibition (5.0-8.9 mm), +++: high zone of inhibition (10 – 5.5 mm), -: no inhibition. Indicators, I: E. coli, II: S. typhimurium, III: S. marcescens
|
Table 2 Antagonistic activity of potential probiotic cultures againstGram-negative pathogen
The inhibition high zone range was exhibited by Lb. acidophilus against E. coli and S. marcescens from 10-12 mm, respectively. While, obtained middle inhibition zone against S. typhimurium was 6.0 mm. Lb. rhamnosus of also was effective against all indicator bacteria. The Lb. rhamnosus showed high zone inhibition against E.coli and S. marcescens were 10 mm. A meddle zone of inhibition was showed against S. typhimurium 5.0 mm, respectively.
pH treatment and viability
The pH effect on the growth properties of potential probiotic culture Lb. acidophilus and Lb. rhamnosus was carried out as described in the material and methods section. The ability of potential probiotic strains to survive in acid conditions is a required criterion for the production of probiotic dairy products (Abosereh et al. 2016).
It is suggested that a good probiotic must have the ability to withstand a pH of range 3.5 – 4.5 because it is used to evaluate the acid tolerance of a probiotic culture (Jung et al., 2016).
As pH is an important criterion for the functioning and viability of bacteria, we research the effect of various pH values (3.0, 4.0, 5.0, 6.4, 8.5). The evolution of cell populations during cultivation of Lb. acidophilus and Lb. rhamnosus are shown in fig. 2, fig. 3.
Lb. acidophilus and Lb. rhamnosus shows a good growth at pH 3.0. The cell populations remained ≥ 106 CFU ml during 72 h incubation. When Lb. acidophilus was treated of pH values 3.0, 4.0, 5.0 the cells population was significantly higher than that of the pH 6.4, 8.5 treatment group (p ≤0.05).
As, shown in fig. 2, fig. 3 the Lb. acidophilus had been reached the population from 6, 38 to 6, 52 log CFU. Lb. rhamnosus from 6, 35 to 6, 38 log CFU, during fermentation from 24 to 72 h of pH 4.0, it increasing was statistically significant (p ≤ 0, 05). A rapid cells raised in the pH 3.0 was observed by Lb. acidophilus, reaching from 6, 67 to 6, 72 at 72 h of fermentation (p ≤ 0, 05). However, at pH 4.0 from 48 to 72 h and pH 5.0 from 48 to 72 h the cell population decreased slightly, but it was not statically significant (p ≥ 0,05).
Lb. acidophilus and Lb. rhamnosus doesn’t grow well above 5.0. The viable cell counts decline rapidly in the range of pH 6.4 – 8.5. At the end of the study, Lb. acidophilus decreased by ~ 1.4 log CFU, Lb. rhamnosus ~ 1.6 log CFU, respectively from the initial counts.
NaCL treatment and viability
To perform a selection of possible probiotic candidates we investigate their ability to grow in various salt concentrations. Salt tolerance of probiotic strains is one of the important technological properties for the production of probiotic dairy products. The osmotic stress may cause pronounced inhibition for bacterial growth. Therefore, that a high concentration of NaCL caused a decline in the adhesion ability of functional groups of lactobacilli and provides increases in the cell membrane damage (Jiage et al. 2020).
Thus, Lb. acidophilus and Lb. rhamnosus were tested for their ability grow under different salt concentration (2%, 5%, 7% NaCL). Results of the cells growth are presented in fig. 4, fig. 5. The Lb. acidophilus and Lb. rhamnosus showed not significantly increases of the cell population during 24, 48 and 72 hours under 2 % NaCL treatment, which do not differ statistically (p ≥ 0.05). The number of the cells in medium containing 5% NaCL was significantly higher than that in 2 % NaCL (p ≤0.05). The tested strains show the best survival rates in 5% and 7% tolerance to NaCl. Lb. acidophilus and Lb. rhamnosus were found to grow at ~ 7 log CFU at 5 – 7 % NaCl during 72 h.
Co-fermentation of Lb. acidophilus and Lb. rhamnosus
The counts of Lb. acidophilus and Lb. rhamnosus strains during co-fermentation are shown in Fig. 6. At the beginning of co-fermentation ~ 7 log CFU/ml of Lb. acidophilus and Lb. rhamnosus were inoculate into 500 ml PM. Co-culture fermentation process was carried out 24 h. The viable cell populations of tested strains increased slowly over 24 h and reached ~8 log CFU/ml, which showed an increase of 1 log CFU/ml. Within 8 hours of co-fermentation, their growth was above 7 log CFU/ml. Lb. rhamnosus population ranged from ~ 7.29 to 7.57 log CFU/ml at 20 h. Lb. acidophilus showed a population was 8 log CFU/ml at the end of co-cultured fermentation. In the co-culture of Lb. acidophilus and Lb. rhamnosus both presented increasing their population from ~ 7 log CFU/ml to ~ 8 log CFU/ml in 12, 16, 20 and 24 h of fermentation than when cultivated in 4 h of fermentation, this differences was significant ( p ≤ 0.05). Thus, the LAB reached significantly viable counts of cell population in 24 h of fermentation, than when cultivated in 12, 16, and 20 h ( p ≤ 0.05).
The acidification rate during co-fermentation of mixed culture in PM is showed in fig.7. At the 4 hours, the pH values fall in the PM substrate with inoculum. Co-cultured fermentation with Lb. acidophilus and Lb. rhamnosus followed decrease pH from 6 to 3 at 24 hours.