## 3 Results and Discussions
### Biomass Increase  
The increase of kefir grains (growth curve) is essential for optimization, control and monitoring of the process parameters on the kefir grains to achieve the maximum production to obtain the polysaccharide kefiran [50]. [PERSON] and [PERSON] [45] mentioned that some of the factors that affect the chemical, microbiological and sensorial properties of kefir grains are the starting material, process and final product variables. The starting material variables are made up of origin of milk, level of fat and origin of the inoculum, among others. In the present work, after having defined the fermentation conditions in Section 3.1 (2% of kefir grains at 20 \({}^{\circ}\)C), the kinetics of kefir grain biomass growth were investigated using different substrates (FUC, SAC, GAL, GLU and CON), with especial interest in the fructans as a new substrate of carbon source proposal to improve the biomass production of milk kefir grains (Figure 3). During the increase in kefir biomass, it could be observed that the kefir grains present a different behavior depending on the substrate used. It has been mentioned that the reproductive capacity of grains is significantly influenced by growing conditions [50]. The results showed that kefir grains that fermented in the control substrate (CON) presented the least increase in biomass due to the low viability of carbon sources (80% on the seventh day). Conversely, substrates that were enriched with different carbon source such as fructans, sucrose, galactose and glucose showed a greater increase, where the increment from lowest to highest was as follows, SAC \(<\) GLU \(<\) GAL \(<\) FUC, with an increase of 125; 140; 190; 220%, respectively; these results are related to the fermentation process and the role that microorganisms develop in said fermentation. The highest biomass increase was observed with grains that grow in fructans (FUC). This behavior showed a positive effect on the biomass production of the proposal substrate. The ANOVA showed significant differences across samples (\(p<0.05\)), with a \(p=0.001\) and F \(=4.840\). On the other hand, post hoc tests were used to evaluate the comparisons of kefir biomass growth. The post hoc tests showed significant differences across samples where fructans showed significant differences between the control (\(p=0.005\)) and sucrose (\(p=0.006\)). However, they did not show significant differences between glucose (\(p=0.150\)) and galactose (\(p=0.835\)). The modification in the morphology of the kefir grains is related to the type of microorganisms present in the kefir grains. Since this will depend on the fermentation conditions (temperature, pH, concentration) and substrate used, some microorganisms will be favored to remain in the conglomerate and others to form part of the substrate. In an environ  
Figure 3: Biomass increment of kefir grains: (-) nonfat milk powder (control) and nonfat milk powder with: (-) 2% agarose fructans, (-) 2% galactose, (-) 2% glucose and (-x-) 2% sucrose. ment of high cell density with relatively low nutrients and high acid concentrations, the observed morphological change would be a natural response for cells to adapt and survive [51]. What would be directly reflected in the application of kefir grains in the industry is in its application as probiotics. On the other hand, the organization that the organisms have during the fermentation process would allow the sub-products obtained from the conglomerate to have certain characteristics such as exopolysaccharides (EPS), in which their composition and structure are significantly influenced by microorganisms present in the conglomerate [52; 53]. The growth conditions affect the technological (emulsifier, film formation) and the probiotic properties. [PERSON] et al. [54] reported that _Lactobacillus paracasei_ strains showed changes in EPS production and morphology due to the growth conditions used, which were reflected in an increase in EPS production when fermented at low temperatures (20 \({}^{\circ}\)C).