Use of waste acid whey for the cultivation of propionibacteria that produce vitamin B12

Osiromašeno kislo sirotko z manjšim deležem proteinov, ki je nastala med proizvodnjo skute, smo uporabili za gojenje bakterijskega seva Propionibacterium freudenreichii subsp. freudenreichii van Niel 1928, ki proizvaja vitamin B12. Za boljšo rast in proizvodnjo vitamina smo dodali 2,5 % (m/v) kvasnega ekstrakta, mineralne soli ter 5,6-dimetilbenzimidazol (DMBMZ), ki je nujno potreben za sintezo liganda vitamina B12. Izvedli smo procese v manjšem laboratorijskem merilu (v 10 mL centrifugirkah) in v bioreaktorju (1 L in 2,5 L). S preskušanjem v manjšem laboratorijskem merilu smo našli optimalne pogoje za rast propionibakterij, ki smo jih prenesli v večje merilo, v bioreaktor. Ugotovili smo, da sta tako uravnavanje vrednosti pH kot obogatitev gojišča s kislo sirotko izjemno pomembna za uspešno proizvodnjo vitamina B12. Za boljše sproščanje vitamina B12 iz celic smo dodali tudi litične encime. Ugotovili smo, da je za uspešno proizvodnjo vitamina B12 bolje, če pred inokulacijo sev predhodno gojimo v komercialnem gojišču RCM, prva faza bioprocesa pa mora potekati vsaj dva dni. Proizvodnja vitamina B12 doseže maksimum med 5. in 6. dnevom druge stopnje. Med samim procesom propionibakterije izrabljajo laktozo in mlečno kislino, pri tem pa nastaja vitamin B12. Dokazali smo, da je obogatena kisla sirotka ustrezno gojišče za propionibakterije, ki proizvajajo vitamin B12.We used partially deproteinized acid whey, a by-product of cottage cheese production, to culture Propionibacterium freudenreichii subsp. freudenreichii van Niel 1928 which is known to produce vitamin B12. To stimulate the growth of propionibacterium, we added 2,5 % of yeast extract, mineral salts and DMBMZ which is crucial for the synthesis of the lower ligand in vitamin B12. We performed the experiment at the smaller level in a laboratory and after that, we have done the process in 1-L and 2,5-L bioreactors. At the beginning of the experiment, we found optimal conditions for the growth of propionibacteria, which were then used for the bioprocess in bioreactors. We found out that the regulation of pH is crucial for the successful production of vitamin B12, as well as the enrichment of acid whey. Enzymes were also added to break down cell walls and maximize yield of vitamin B12. For a successful production of vitamin B12, we had to pre-cultivate the strain in a commercial medium RCM before the inoculation and the first stage of the biorpocess must last for at least two days. Vitamin B12 production peaks between the 5th and the 6th day of the second stage. During the process, the propionibacteria uses lactose and lactic acid to produce vitamin B12. We have proven that enriched acid whey is a suitable medium for propionibacteria that produce vitamin B12