Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali
Creators: name: Zanoni, Simona <1979>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: CHIM/11 Chimica e biotecnologia delle fermentazioni

description: The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting
GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model
for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic
parameters were calculated with a Matlab algorithm. Galactose was the best carbon source;
lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest
carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes
toward fermentation products. Similar distribution between 3- and 2-carbon products was
observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was
higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the
stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the
products, ATP yield was calculated on the different carbohydrates. ATP yield was the
highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose,
respectively. Therefore, a correspondance among ethanol production, low ATP yields, and
low biomass production was established demonstrating that carbohydrate preferences may
result from different sorting of carbon fluxes through the fermentative pathway. During
GOS fermentation, stringent selectivity based on the degree of polymerization was
exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was
observed until longer oligosaccharides were utilized. Throughout the growth on both lactose
and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides
can be hydrolysed before they are taken up.
The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides
(XOS) was also studied and our attention was focused on an extracellular
glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS
as sole carbon source. The extracellular enzyme was purified from the the supernatant,
which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol
was developed: the sample was loaded on a Mono-Q anion exchange chromatography and
then, the active fractions were pooled and β-Xylosidase was purified by gel filtration
chromatography on a Superdex-75. The enzyme was characterized in many aspects. β-
Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a
termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0.
The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate
specificity with different di-, oligo- and polysaccharides was tested. The reactions were
carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates
hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase
activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose
and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher
than the Xylanase one.
These studies on the carbohydrate preference of a strain of Bifidobacterium
underlined the importance of the affinity between probiotics and prebiotics. On the basis of
this concept, together with Barilla G&amp;R f.lli SpA, we studied the possibility to develop a
functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum
BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were
studied to assess their suitability for utilization in synbiotic products on the basis of
antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates
fermentation and viability in food matrices. Bile and human gastric juice resistance was
tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis
and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to
bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides
and fructo-oligosaccharides were successfully utilized by all the strains
and could be considered the most appropriate prebiotics to be used in effective synbiotic
formulations. The vitality of the three strains inoculated in different food matrices and
maintained at room temperature was studied. The best survival of Lactobacillus plantarum
BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was
found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20
healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the
efficacy of the pre-prototype was promising for the future develop of potential commercial
products.