In vitro assessment of the effect of lactose‐free milk on colon microbiota of lactose‐intolerant adults

Abstract

Milk is an essential food, but a large part of adult's population is uncapable to digest lactose. Lactose intolerance can seriously affect the intestinal ecology and compromise the host's well-being. In this scenario, the role of human gut microbiota is crucial, but little is known about that because few research studies were conducted, via either clinical trials or in vitro models. Also, due to the call to reduce animal testing in science, an in vitro model with the gut microbiota of lactose-intolerant adults is necessary. This paper proposes an in vitro model coupling oro-gastro-duodenal digestion to colonic fermentation to evaluate lactose impact on colon microbiota of lactose-intolerant adults. Microbiomics and metabolomics with respect to a baseline of fermentation were compared. Generally, when the insult was given, taxa specialised for dairy sugars were unaffected, but Bacteroidaceae and Lachnospiraceae were underrepresented. Lactose triggered raise of opportunistic Proteobacteria dominated by harmful Klebsiella. In addition, an important reduction of essential short-chain fatty acids was observed, and in particular, that of butyrate. Although more observations need to be conducted, as well as a comparison with the healthy condition, the present work gives results for pre-clinical application in the sight to reduce animal testing.

Experimental protocols for in vitro gut models in food research: Editorial

“…The protocol presented in this paper is able to discriminate among variables of the microbiota up to the species level (Xie et al, 2023). Casciano et al (2022) investigated the impact of lactose containing milk on colon ecology of certified lactose intolerant adults (25-50 y), focusing on the modulation of bacterial populations of colon microbiota and changes in its metabolites production. This paper, in addition to known microbial SCFAs and medium chain fatty acids, proposed also the study of the volatilome derived from the entire set of volatile organic compounds of the colon microbiota ecology, and evidenced the changes in single VOCs production by multivariate analysis.…”

Impact of cooking methods of red-skinned onion on metabolic transformation of phenolic compounds and gut microbiota changes

“…Colonic fermentations were conducted for 24 hours in independent vessels using an in vitro gut model, MICODE [8][9][10]26,27 , obtained by the assembly of Minibio Reactors (Applikon Biotechnology BV, Delft, NL) and controlled by Lucullus PIMS software (Applikon Biotechnology BV, NL). The preparation of the experiments was made according to published procedures [8][9][10]23,27,28 and recently described in details 9 .…”

“…Bacterial DNA was extracted just after sampling (Purelink Microbiome DNA Purification Kit by Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and quantified (Biodrop, Biochrom Ltd., Cambridge, UK). Enumeration of bacterial groups was made by qPCR to evidence changes in the microbiota after fermentation [31][32][33][34] and expressed as cells/mL (obtained from values in gene copy numbers/mL multiplied by three for multicopy genes, as V3-V4 16S, and by one for monocopy genes, as RecA), following previous protocols [8][9][10]26,27,35 S1) were assessed by qPCR on a QuantStudio 5 System (Applied Biosystem, Thermo Fisher, USA) and SYBR Green I chemistry. The qPCR Prebiotic Index (qPI) was calculated as previously reported 9,10 .…”

“…Colonic fermentations were conducted for 24 h in independent vessels using an in vitro gut model, MICODE, [8][9][10]26,27 obtained by the assembly of Minibio Reactors (Applikon Biotechnology BV, Delft, NL) and controlled by the Lucullus PIMS software (Applikon Biotechnology BV, NL). The formulation for the experiments was made according to published procedures, [8][9][10]23,27,28 and recently described in detail. 9 Briefly, four fermentation bioreactors were filled with 90 mL of basal medium, 23 and then each one loaded as follows: (i) with 10 mL of a mixture of FS and 1 g of the solid portion of in vitro digested raw onion (RRSO); (ii) with 10 mL of FS including 1 g of the solid portion of in vitro digested grilled onion (GRSO); (iii) with 10 mL of FS including 1 g of the solid portion of in vitro digested fried onion (FRSO), and (iv) with 10 mL of FS including 1 mL of digestive enzymes, as the blank control (BC).…”

“…Enumeration of the bacterial groups was done by qPCR to evidence the changes in the microbiota after fermentation [31][32][33][34] and expressed as cells per mL (obtained from values in gene copy numbers per mL multiplied by three for multicopy genes, as V3-V4 16S, and by one for monocopy genes, as RecA), following previous protocols. [8][9][10]26,27,35 Specifically, 14 different bacterial taxa (Eubacteria, Firmicutes, Bacteroidetes, Lactobacillales, Bifidobacteriaceae, Enterobacteriaceae, Clostridium group I, Clostridium group IV, Bacteroides-Prevotella-Porphyromonas (BPP) group, Atopobium-Collinsella-Eggerthella (ATOP) group, Bifidobacterium longum, Escherichia coli, Faecalibacterium prausnitzii, and Akkermansia muciniphila) (Table S1 †) were assessed by qPCR on a QuantStudio 5 System (Applied Biosystem, Thermo Fisher, USA) and SYBR Green I chemistry. The qPCR prebiotic index (qPI) was calculated as previously reported.…”