## 4 Discussion  
This study demonstrates that co-culture with the yeast _S. cerevisiae_ was effective in enhancing the exopolysaccharideacharide production of three strains of _L. rhamnus_ R0011, ATCC 9595, and RW-9595M in cultures without pH control. This is a very important aspect because the absence of pH control  
Figure 5: Co-expression gene network of _L. rhamnus_ strains R0011 **(1)**; ATCC 9595 **(2)** and RW-9595M **(3)** obtained using Cytoscape 3.4. The illustrated genes had a significant (\(P<0.05\)) strong correlation (correlation index > 0.8). The largest nodes (circle size) are those with the high level of degree. In the above figure, the blue nodes had higher values of Betweenness Centrality (BC) and the orange nodes had lower BC values. Non-continuous traits show a co-expression pattern found for the three strains of _L. rhamnus_ R0011, ATCC 9595 M, and RW-9595M. could allow the simplification and reduction of cost of the fermentation process coupled to a better EPS production in co-culture. Our results clearly showed that 48 h of co-culture did not show any impact on the growth or viability of strains measured by viable cultivable counts in CFU/mL, total counts by qPCR and by PMA-qPCR. A previous study found that co-culture improved the viability of _L. rhamnus_ HN001 in fermented milk after 1 week of storage at 30 \({}^{\circ}\)C [39]. Further experiments are required in order to evaluate the effect of time of co-culture and storage on viability of the strains in co-culture. Similarities between the three strains were detected during fermentation. The co-culture did not affect lactose consumption or lactic acid production for RW-9595M or R0011. On the contrary, co-culture had an effect on _L. rhamnus_ ATCC 9595 with relation to lactose consumption. These differences showed that the interactions between bacteria and yeast are strain-dependent. Relative gene expression depended on the growth phase for all studied strains. In co-culture, the three strains of _L. rhamnus_ show simultaneous transcription of two pathways to metabolize lactose from whey; through the tagatose-6-phosphate and the Leloir pathway. [PERSON] and [PERSON] (2006) [40] reported that these two pathways depend on the activation of the _lacTEGF_ operon by the inducing gene _lacT_. Lactose can be hydrolyzed by the activation of beta-galactosidase (_bgaC_) to obtain glucose and galactose. Galactose could then be metabolized by the Leloir pathway by the activation of the _galKETRM_ operon. During fermentation, bacterial cells were in an acid medium (pH \(<\) 4), with the presence of lactate issuing from the catabolism of sugars to obtain energy. Linked to lactic acid production, strains showed over-expression of genes coding for pyruvate kinase (_pyK_) and fructose 1,6 biphosphatase (_fbp_) during co-culture. During the stationary phase, the gene coding for acetate kinase (_ackA_) was over-expressed for all three strains. The presence of acetic acid in media was detected and indicated the induction of the mixed acid metabolic pathway for _Lactobacillus_ strains. In co-culture, acetic acid concentration in the culture media was similar (_L. rhamnus_ R0011) or inferior (_L. rhamnus_ RW-9595M and ATCC 9595) to monoculture. In this sense, [PERSON] et al. [41] reported that _S. cerevisiae_ can remove acetic acid and is used to eliminate excessive amounts of it as a practical approach during alcoholic fermentation. For the _L. rhamnus_ RW-9595M strain, producing the highest amount of EPS, gene _ackA_ seems to be an important node for the co-expression network, as gene _wzb_ for the EPS operon (G2). Gene _ackA_ interacts with genes coding for G2 group EPS operon enzymes (_wzd_, _wzb_, _wzm_ and _rmlA_) and one of G1 group sugar metabolism (_bgaC_). For ATCC 9595, gene _ackA_ showed co-expression only with gene _wzm_. For R0011, gene _ackA_ had a positive co-expression with gene _galE_, however it had negative correlation with _groES_. Acetate belongs to the metabolic pathway used for lactate degradation in mixed acid metabolism.