(177u) Volumetric Oxygen Transfer Coefficient As a Parameter for Scale-up Probiotic Bacteria Production (Lactobacillus fermentum K73)

Global sales of ultra-processed food have increased, at the same time the prevalence of some chronic diseases, like, obesity, diabetes, hypertension, and cancer¹. It has been evidencing that consumption of ultra-processed food influences chronic diseases development ². Functional food emerges from the necessity of products consumption that has benefits effects on organism health and prevents the development of diseases; one of these products are probiotics ³.

Probiotics production is growing worldwide. In 2015 global probiotics production and commercialization were 36.6000 million dollars. Besides between 2016-2023, the average annual of probiotic’s production is expected to increase by 7.3% ⁴. To satisfy population nutritional necessities, it is important to produce probiotics at the industrial level. Scale-up of the microbiological industrial process has an objective to develop a product that then is commercialized for beneficiary the population ⁵. Scale-up bioprocess must guarantee a similar concentration of probiotic bacteria to replicate the fermentation behavior at a laboratory and large scale (industrial or pilot plant) ^6,7.

During industrial probiotic production is pivotal to keep probiotic viability, due to a least a concentration of 10⁶ -10⁷ UFC/ g must reach the colon to guarantee minimal therapeutic dose. Also, it is important to keep the microorganism functionality to tolerate the storage and gastrointestinal conditions as acidic pH, enzymes activity and the presence of bile salts, with the objective of exerting the benefits effects on the host ^8,9.

Nevertheless, the problem in scale-up bioprocess is the changes in bioreactor geometries, because affect the mixing quality. As the bioreactor sizes increase the mixing quality decreases, generating substrate and, oxygen depletion zones, therefore, in the process will exist zones with excess or depletion of the substrate and, oxygen ¹⁰. It has been evidence that the scale-up process affects the yield, productivity and, microorganism functionality ^11,12.

A critical factor in aerated and, micro-aerated cultures is the oxygen supply due to is necessary for respiration and microorganism growth. This parameter is described by the volumetric oxygen transfer coefficient (K_La) during the process ¹³. This coefficient is related to oxygen transfer from the gas to the liquid phase and to the cell. In stirred tank bioreactors describes the intensity of mass transfer and, is the main factor for scale-up a bioprocess ¹¹.For scale-up bioprocess must keep constant this coefficient (K_La) in the different sizes bioreactors, through adjusting operational parameter as the agitation and, oxygen supply ¹⁴.

The oxygen is a crucial factor in Lactic acid bacteria production even though the LAB considered as an Oxygen-tolerant anaerobe. There has been evidence that in some cases aerobic cultures for Lactobacillus species improves the cells tolerance of oxidative stress and, heat, also increases biomass yield by the activation of a minimal electron transport chain and, the oxygen can act as a different electron acceptor for the generation of NAD+H⁺, this process rises ATP yield ^15,16. For those reasons is considered that the volumetric oxygen transfer coefficient (K_La) is an adequate parameter for scale-up the biomass production of Lactobacillus fermentum K73.

According to Salazar-Magallon. et al (2016), when the production of protein by Bacillus thurungiensis, with pesticide effect, was scaled-up from shake flask to bioreactor based on volumetric oxygen transfer coefficient, it obtained the same concentration of protein ¹⁷. In another study, at micro-aerated conditions was scaled-up the process of ethanol production by E. coli, using the volumetric oxygen transfer coefficient as a criterion of scale-up the bioprocess, it obtained that the productivity and, yield of ethanol kept at similar levels from laboratory scale to 0,75 L bioreactor.

The present study investigated the scale-up process of Lactobacillus fermentum K73 biomass production. Lactobacillus fermentum K73 has relevance on the food industry, due to it is a probiotic with a high hypocholesterolemic potential due to produces the hydrolase enzyme BSH and absorb cholesterol In-vitro ¹⁸. That is why, the aim of this work was to scale-up biomass production of a probiotic bacteria, Lactobacillus fermentum K73, from bioreactor of 1 L (Bioflo 110, New Brunswick Scientific) to a 10 L (Bioflo 3000, New Brunswick Scientific).

The scaled-up process performed by a geometric similarity of the bioreactors and keeping constant the volumetric oxygen transfer coefficient (KLa). The KLa measured experimentally at the two bioreactor scales, by the dynamic gassing-out method. Also, it used a dimensionless correlation for the prediction of KLa in a stirred tank reactor. The prediction of operational conditions (agitation), to keep constant this parameter. It was analyzed the growth kinetics of cultures by plate counting (UFC/mL), the changes of pH and oxygen concentration during ten hours at the two bioreactors of different scales. Also, it was studied the functionality of the microorganism, related to the ability to tolerate conditions of the in-vitro gastrointestinal digestion model of the Infogest COST Action. It was measured, the viability by plate counting of the probiotic at different phases of the digestion, oral, gastric and intestinal phases.

The viability of probiotic bacteria was successfully scaled-up from 1 L to 10 L bioreactor, achieving a similar concentration of Lactobacillus fermentum K73 ranging from 8.48 to 8.65 log UFC/mL, no significant differences obtained. Growth curves analyzed with the web version DMFit (https://browser.combase.cc/DMFit.aspx). Growth fitted well to the Baranyi and Roberts model for the culture on all the bioreactors (R^2= 0.98). The functionality of probiotic bacteria was scaled-up too, obtaining similar survival percentage when was subjected to digestion phases: oral, gastric and intestinal, ranging from 60% to 88% of survival.

Volumetric oxygen transfer coefficient (Kla), is an adequate criterion for scale-up production of Lactobacillus fermentum K73. At the scaled-up process keeping constant Kla, was obtained similar biomass concentration and survival percentage. These results are relevant to the food industry because Lactobacillus fermentum K73 can keep the viability and tolerate the gastrointestinal conditions at the two scales. These properties could allow the probiotic growth on gut for exerts a beneficial effect on the host and to satisfy the nutritional necessities of the population. Also, in a future study, these results are a basis for scale-up the process at pilot/industrial level.

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