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Communication

Apple Pomace Modulates the Microbiota and Increases the Propionate Ratio in an In Vitro Piglet Gastrointestinal Model

by
Sandrine Dufourny
1,
Sarah Lebrun
2,
Caroline Douny
2,
Benjamin Dubois
1,
Marie-Louise Scippo
2,
José Wavreille
1,
Pierre Rondia
1,
Nadia Everaert
3 and
Véronique Delcenserie
2,*
1
Walloon Agricultural Research Centre, 5030 Gembloux, Belgium
2
Fundamental and Applied Research for Animals & Health (FARAH), University of Liège, 4000 Liège, Belgium
3
Department of Biosystems, KU Leuven, 3000 Leuven, Belgium
*
Author to whom correspondence should be addressed.
Fermentation 2022, 8(8), 408; https://doi.org/10.3390/fermentation8080408
Submission received: 25 June 2022 / Revised: 28 July 2022 / Accepted: 4 August 2022 / Published: 19 August 2022
(This article belongs to the Special Issue In Vitro Fermentation)
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Abstract

:
Apple pomace (AP) contains biomolecules that induce changes in intestinal fermentation of monogastrics with positive expected health effects. The weaning of piglets can induce economic losses due to intestinal disturbances; new weaning strategies are, thus, welcome. The purpose of this study was to test the effect of AP on fermentation products by using baby-SPIME, an in vitro multi-compartment model dedicated to piglet weaning. A comparison was done on short chain fatty acid (SCFA) ratio and the microbiota induced in bioreactors between a control culture medium vs. an AP culture medium. The results of 2 preliminary runs showed that AP medium increased the molar ratio of propionate (p = 0.021) and decreased the molar ratio of butyrate (p = 0.009). Moreover, this medium increased the cumulative relative abundance of Prevotella sp. and Akkermansia sp. in bioreactors. AP could promote an ecosystem enriched with bacteria known as next-generation probiotics (NGP)—likely influencing the energy metabolism of piglets by their fermentation metabolites. AP could be used as a dietary strategy to influence bacterial changes in the intestine by stimulating the growth of bacteria identified as NGP.

1. Introduction

Apple and its by-product apple pomace (AP)—obtained after extraction of juice—have long been studied because of their components of high interest such as dietary fibers, phenolic and terpenic compounds [1,2,3,4,5]. At present, scientists are trying to find the best ways of finding value for AP, given how promising this co-product appears in the context of a circular economy [6,7]. In the food and feed sectors, AP remains poorly used [8]; however, this could change with -omic technological advances [9]. AP is already known to influence the intestinal fermentation of monogastrics. In rats, this ingredient increased intestinal fermentation and resulted in positive antioxidant health effects as well as the reduction of blood glucose levels [10]. In piglets, it increased the number of total colonic bacteria as well as the number of Lactobacilli in feces, and these effects were also observed in the gut morphology, blood parameters and gene expression of immunological markers [11,12]. AP has also been tested in an in vitro batch model inoculated with feces from pre-weaned piglets [13]. Results suggested that the AP matrix offers a certain fermentative potential, but, in a panel of by-products, AP was not classed at the top of the ranking possibilities. However, oligosaccharides derived from apple pectin have also been shown to have strong bacteriostatic effect against Escherichia coli and Staphylococcus aureus [14]. Biomolecules present in the apple skins—triterpenic or polyphenolic compounds such as ursolic acid and phloridzin—are known to be effective against pathogens [5]. This is of particular interest for piglets at weaning, considering that weaning induces multiple stressors [15,16] that lead, among others, to immediate intestinal and immune disorders and may impact long-term performance and health [17,18]. For the swine industry, there is an economic impact associated to post-weaning diarrhea (PWD)—commonly associated with enterotoxigenic Escherichia coli infection [19]. Prebiotics and probiotics are major strategies against PWD [20]. For the above-mentioned reasons and due to the nature of its components, AP appears as an interesting matrix to study in a multi-compartments gastrointestinal model in an attempt to decipher the matrix–microbiota interaction. The baby-SPIME (Simulator of Pig Intestinal Microbial Ecosystem) model was previously developed to study in vitro dietary weaning strategies for piglets [21]. This model was used to study the effects of AP on short chain fatty acid (SCFA) production and on the microbiota of piglets after weaning.

2. Materials and Methods

2.1. Equipment, Inocula, Culture Media and Sample Collection

The baby-SPIME model, requiring the use of SHIME® equipment (ProDigest Bvba, Gent, Belgium), was used for the experiment [21]. One-half cabinet of 3 bioreactors (stomach, inoculated pre-colon and inoculated colon) was used as control and one-half cabinet was dedicated to test AP (Figure 1). The timeline for 1 run included 2 weeks of the lactation phase, a weaning step (modification of the culture medium) and 2 weeks of the post-weaning phase. Two runs were managed with two different donors.
The donors were two [Piétrain × Landrace] suckling piglets (27 days old) free of antibiotics. Feces were used to prepare the inocula for the study as previously described [21]. The two samples were taken at the same farm, at the same time, in two different litters. They were kept on ice under anaerobic conditions during transportation. A single donor was used to prepare the inoculum for a run. A single inoculum was prepared for both pre-colon and proximal colon bioreactors for each run.
Three different culture media were prepared (Table 1): 1° lactation culture medium, 2° post-weaning culture medium and 3° post-weaning culture medium with AP (Extr’Apple SAS, Pleudihen-sur-Rance, France). Culture media and pancreatic juice were prepared as previously described [21].
Samples from pre-colon and proximal colon bioreactors were taken 3 times a week at fixed intervals of days and times for microbial metabolite analysis (2 mL) and/or 16S rRNA sequencing (1 mL) as presented in the timeline of Figure 1. Samples were centrifuged (2 min at 17,000× g). For the microbial metabolite analysis, the supernatants were collected and filtered (0.45 µm). For the sequencing, the pellets were collected. Supernatants and pellets were stored at −20 °C until analyses.

2.2. Microbial Metabolites

Samples were analyzed for their SCFA content as previously described [22] by SPME-GC–MS. The protocol included a step of SCFA extraction with an SPME fiber, a step of separation on a Supelcowax-10 column (30 m × 0.25 mm, 0.2 μm; Supelco, Bellefonte, PA, USA) on a Focus GC gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) and a step of analysis with an ion trap PolarisQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The extraction temperature and time were 60 °C and 20 min. The results (mg/L) were converted into mmol/L. Ratios C2:C3:C4 were then calculated.

2.3. 16S rRNA Gene Sequencing

DNA extraction and sequencing (DNA Vision, Gosselies, Belgium) was done using the DNeasy Blood & Tissue kit (Qiagen Benelux B.V., Venlo, The Netherlands), a NanoDrop 2000 (Thermo Scientific™, Waltham, MA, USA) and PicoGreenVICTOR X3 (PerkinElmer, Waltham, MA, USA) for the quantitative and qualitative assessments of the DNA, using the Quant-it PicoGreen dsDNA Assay kit from Invitrogen. The V3–V4 region was amplified by PCR, purified and tagged. The NEXTERA XT Index kit V2 from Illumina was used to index libraries. The high throughput sequencing was carried out on Illumina Miseq in paired-end sequencing (2 × 250 bp) by targeting an average of 10,000 reads per sample. QIIME2 [23] was used for the bioinformatics analysis. Demultiplexed paired-end sequencing reads were denoised with DADA2 [24] to generate amplicon sequence variants (ASVs). Taxonomy was assigned to ASVs using the q2-feature-classifier plugin [25] together with the Greengenes 13_8 database [26].

2.4. Statistics

A two-way analysis of variance (GLM procedure of Minitab 18, Minitab Inc., State College, Pennsylvania, PA, USA)—including the bioreactor and the culture medium as fixed factors—was also applied to the microbial metabolite results to main relative abundance results of the microbiota. The ANCOM method [27] was used to identify ASVs that were differentially abundant across sample groups at the genus taxonomic rank.
A p-value ≤ 0.05 was significant. A p-value between 0.05 and 0.1 or equal to 0.1 was a trend. A p-value > 0.1 was not significant (ns).

3. Results

3.1. SCFA Results

SCFA and molar ratios C2:C3:C4 for pre-colon and proximal colon effluents are given in Table 2. The SCFA results between PW control and PW AP were not statistically different either in the pre-colon or in the proximal colon. However, statistical differences were observed in molar ratios C2:C3:C4. Apple pomace led to an increase in propionate proportion and a decrease in butyrate.

3.2. 16S rRNA Gene Sequencing

The alpha-diversity parameters—Shannon observed features, phylogenetic diversity (Figure 2) and evenness—were not influenced by the use of AP.
Results of the taxonomic composition analysis are given in Figure 3 at the phylum level. Compared to the control condition (NAP), the use of apple pomace (LAP) tended to decrease the proportions of Firmicutes and Actinobacteria and to increase those of Bacteroidetes (except in the pre-colon of piglet 1) and Verrucomicrobia. The only representative of Verrucomicrobia in bioreactors belonged to the Akkermansia genus.
Relative abundances of the genera observed in the pre-colon and proximal colon in bioreactors are presented in Supplementary Material—Figure S1. The ANCOM analysis showed that the Catenibacterium genus displayed significantly different abundances across NAP and LAP sample groups (respectively 0.0% vs. 3.6%). The ANOVA analysis showed that the cumulative relative abundance of Akkermansia sp. and Prevotella sp. was significantly different across NAP and LAP sample groups (respectively 11.5% vs. 21.2%, p = 0.022). Differences in these microbial genus levels are presented in Figure 4.

4. Discussion

When setting-up a fermentation model, the question arises as to whether or not to pool the feces of individuals. Considering the work of Aguirre and colleagues [28], the preparation of a fecal sample pool is suitable when comparing different experimental conditions such as changes in carbohydrate substrate. The results of their study argue in favor of a pool to reduce the effects of inter-individual variability. However, the aim of the present experiment targeted post-weaning diarrhea, for which some piglets’ microbiota can be more predisposed than others [29]. It is interesting to note the individual particularities, keeping in mind the limitation of the statistics.
AP modified the molar ratios of C2:C3:C4 and the microbiota composition in pre-colon and proximal colon bioreactors. In particular, the proportion of propionate increased when that of butyrate decreased. Although not significant, this molar ratio evolution seemed to originate from a decrease in acetate. Acetate is known to be a common co-substrate for the CoA-transferase route—one of the two metabolic pathways with kinase leading to propionate and butyrate production [30]. As a substrate for the bacterial fermentation and cross-feeding interactions, AP seemed thus to increase this CoA-transferase route in the in vitro piglet model in favor of propionate.
The relative abundance of Actinobacteria and Firmicutes decrease when Bacteroidetes and/or Verrucomicrobia numerically increased in the bioreactors. Observing this dynamic between propionate and the above-mentioned bacteria—independently from AP—is in accordance with the literature. Indeed, in the different microbial metabolic pathways leading to SCFA production in a human study, Bacteroidetes and Verrucomicrobia are known to be users of the succinate pathway leading to propionate production [30]. In pigs, when propionate was directly infused in the caecum, authors showed a decrease of butyrate in the colon, an increase of Bacteroidetes and a decrease of Firmicutes [31].
The effect observed of AP on propionate is also consistent with some observations of studies using rats [32]. Concerning a certain fraction of AP, this fraction is included in the diet supplementation of fiber-rich colloid juices from AP in the study of Sembries and colleagues [33]. The microflora fermentation increased in the caecum—the main site of intestinal fermentation in rats—leading to significantly higher SCFA, acetate and propionate yields. This fraction seems to be also present in the unprocessed AP used in the study of Juskiewicz and colleagues [10] observing the results of acetic, propionic and butyric acid concentrations in digesta. This fraction seemed to be active in the baby-SPIME model. Moreover, in a previous in vivo trial on piglets, with the same AP used, an effect on Bacteroidetes was observed with 4% inclusion of AP in a post-weaning diet [34]. In this trial, Bacteroidetes appeared as the second more abundant phylum in the feces of the 4%AP piglets instead of the third most abundant for 0%AP and 2%AP piglets on the 8th post-weaning day. The results showed that baby-SPIME highlighted an effect of AP on propionate and propionate-producing bacteria, although more runs are required to consolidate the statistics of the study.
Propionate is known to act on the nervous and immune systems [35]—for example, by increasing the gene expression of NF-κB and IL-18 in pigs [31] or by acting on the regulation of Treg cells, which have immunosuppressive activity and participate in the regulation of intestinal inflammation [36]. AP appears in the literature as a matrix with positive immune effect for piglets through the regulation of inflammatory gene expression, e.g., NF-κB, cyclin D1 or caspase 3 [37].
Propionate is also known to act on energy metabolism. Indeed, in humans, propionate exerts beneficial health effects by showing cholesterol-lowering and anti-lipogenic effects and by promoting satiety in the individual [30,38]. Propionate interacts with gluconeogenesis in the intestine and the liver [39,40]. As a particular effect of propionate on the gastrointestinal tract, the fatty acid increases the activity of the glucose-6-phosphatase in the jejunum [39]—an enzyme that ends the glucogenolysis cycle by releasing glucose. AP appears well in the literature as a matrix with positive effect on metabolic disorders for humans [32]. Thus, the results suggest an effect of AP on the regulation of energy metabolism in piglets.
By acting on the immune system and the energy metabolism through SCFA, AP could interact in two important mechanisms of piglets at weaning. The fight against pathogens is also of crucial importance at weaning. In addition, the results suggest that AP promoted the growth of bacteria considered as next-generation probiotics (NGP)—anaerobic gut commensal bacteria suppressing mucosal inflammation [41]—such as Prevotella sp. or Akkermansia sp. [42,43,44,45,46] that appear of interest for the “health” bio-industry. For example, Karasova and colleagues [29] identified Prevotella spp. as an indicator of resistance to post-weaning diarrhea for piglets. A link has also been established in the literature between diet polyphenols (AP contains some), Akkermansia muciniphila and intestinal health in terms of barrier integrity, immune response and resident intestinal microbiota in humans [47,48]. For piglets, Akkermansia muciniphila appears more disturbing. While Karasova and colleagues [29] observed an increased abundance of this bacteria in diarrheic piglets, Luo and colleagues [41] published the promising NGP potential of Akkermansia muciniphila through their in vitro intestinal porcine enterocytes model. NGP are also of interest for swine production. Indeed, adding substrates in the feed that will promote the growth of specific symbiotic bacteria will benefit the gut health of pigs [49]. Introducing dietary components enhancing the growth of specific bacteria is a pathway that can lead to positive changes in the microbiota for better health of the host [50]. Adequate substrate is furnished to the NGPs to promote growth in their niche [48], ensuring an appropriate colonization resistance in the intestine of the host through these bacteria interacting with the host immune system. Considering the concept “nourish the NGPs”, AP appears in this study as an interesting dietary component to achieve this purpose.
To conclude, the preliminary results from the test of dried AP in an in vitro multi-compartment piglet model tended to show that AP could promote an ecosystem enriched in Bacteroidetes and/or Verrucomicrobia with a likely effect on the energy metabolism of the host by their fermentation metabolites. The AP component could be used as a dietary strategy to influence bacterial changes in the intestine by stimulating the growth of bacteria potentially considered as NGP.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8080408/s1, Figure S1: Relative abundances of the genera observed in the pre-colon and proximal colon bioreactors of baby-SPIME when using AP (LAP) or not (NAP) for two donor piglets.

Author Contributions

Conceptualization, S.D. and V.D.; methodology, V.D.; validation, V.D.; formal analysis, S.D., S.L., C.D. and B.D.; investigation, S.D. and S.L.; resources, M.-L.S. and J.W.; writing—original draft preparation, S.D.; writing—review and editing, C.D., P.R., N.E. and V.D.; visualization, S.D.; supervision, V.D.; project administration, P.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was approved by the ethical committee of the University of Liège (ULiège, Liège, Belgium)—file n°1823. The intervention was in compliance with European (Directive 2010/63/EU) and Belgian (Royal Decree of the 29 May 2013) regulations governing the protection of animals used for scientific purposes.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets analyzed for this study can be found in the ENA database (https://www.ebi.ac.uk/ena/browser/home, accessed on 10 March 2021) under the accession number PRJEB43583.

Acknowledgments

The authors thank the colleagues involved in the management of the baby-SPIME.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 08 00408 g001 550
Figure 1. Baby-SPIME model: a SHIME equipment divided into two half cabinets of three double-jacketed bioreactors receiving the culture media three times a day. Bioreactor 1 with medium stomach digestion received pancreatic juice and bile (duodenum/jejunum digestion) following instructions given in the figure. Then the liquid flowed simultaneously toward the pre-colon and proximal colon until a waste container. Bioreactors 2 and 3 were inoculated with feces from one piglet—two different piglets for the two runs. The timeline of the run is described.
Figure 1. Baby-SPIME model: a SHIME equipment divided into two half cabinets of three double-jacketed bioreactors receiving the culture media three times a day. Bioreactor 1 with medium stomach digestion received pancreatic juice and bile (duodenum/jejunum digestion) following instructions given in the figure. Then the liquid flowed simultaneously toward the pre-colon and proximal colon until a waste container. Bioreactors 2 and 3 were inoculated with feces from one piglet—two different piglets for the two runs. The timeline of the run is described.
Fermentation 08 00408 g001
Fermentation 08 00408 g002 550
Figure 2. Faith’s phylogenetic diversity observed in the pre-colon and proximal colon bioreactors of baby-SPIME when using AP (LAP) or not (NAP) for two donor piglets.
Figure 2. Faith’s phylogenetic diversity observed in the pre-colon and proximal colon bioreactors of baby-SPIME when using AP (LAP) or not (NAP) for two donor piglets.
Fermentation 08 00408 g002
Fermentation 08 00408 g003 550
Figure 3. Relative abundances of the phyla observed in the pre-colon and proximal colon bioreactors of baby-SPIME when using AP (LAP) or not (NAP) for two donor piglets.
Figure 3. Relative abundances of the phyla observed in the pre-colon and proximal colon bioreactors of baby-SPIME when using AP (LAP) or not (NAP) for two donor piglets.
Fermentation 08 00408 g003
Fermentation 08 00408 g004 550
Figure 4. Relative abundances [%] of three genera observed in the pre-colon and proximal colon bioreactors of baby-SPIME showing significant differences when using AP (LAP) or not (NAP) for two donor piglets.
Figure 4. Relative abundances [%] of three genera observed in the pre-colon and proximal colon bioreactors of baby-SPIME showing significant differences when using AP (LAP) or not (NAP) for two donor piglets.
Fermentation 08 00408 g004
Table
Table 1. Composition of the culture media.
Table 1. Composition of the culture media.
Ingredients Lactation CMPW Control CMPW AP CM
Mucin
(Sigma-Aldrich, St-Louis, MO, USA) 		6.0 g/L6.0 g/L6.0 g/L
Proteose-Peptone n°3
(BD Bacto Biosciences, Franklin Lakes, NJ, USA)		1.0 g/L1.0 g/L1.0 g/L
Potato starch
(Sigma-Aldrich, St-Louis, MO, USA) 		1.0 g/L1.0 g/L1.0 g/L
L-Cysteine hydrochloride
(Merck, Darmstadt, Germany) 		0.2 g/L0.2 g/L0.2 g/L
Nuklospray Yoghurt 1
(Dumoulin, Andenne, Belgium) 		8.0 g/L0.0 g/L0.0 g/L
Post-weaning diet for piglets 2
(ABZDiervoeding, Nijkerk, The Netherlands)		0.0 g/L8.0 g/L8.0 g/L
Apple pomace
(Extr’Apple SAS, Pleudihen-sur-Rance, France)		0.0 g/L0.0 g/L0.65 g/L
g/L: grams per liter, CM: culture medium, AP: apple pomace, PW: post-weaning. 1 Commercial complementary milk replacer feed for piglets containing, among others, whey powder, vegetable oils and wheat flour. 2 Grinded to particles of 250 µm. Composition: Barley (30.00%), Wheat (14.41%), Maize (5.00%), Oat flakes (5.00%), Toasted soybeans (15.00%), Soya meal (13.87%), Potato protein (2.00%), Bread flour (5.00%), Soya oil (0.36%), Fat-filled whey powder (4.67%), Chalk (1.05%), Monocalciumphosphate (1.01%), Salt (0.54%), Methionine (0.16%), L-lysine HCL (0.47%), L-threonine (0.11%), Lysine/tryptophan mix (0.02%), Flavoring (0.20%), Vitamins (0.40%), Start/BL.15CU (premix containing Cu, Fe, Zn, Mn, Se, I and vitamins A, B2, B3, B5, D3, E, K3; 0.40%), Phytase (0.33%).
Table
Table 2. SCFA results (mg/L) and molar ratio C2, C3 and C4 for pre-colon and proximal colon effluents of baby-SPIME (control vs. AP).
Table 2. SCFA results (mg/L) and molar ratio C2, C3 and C4 for pre-colon and proximal colon effluents of baby-SPIME (control vs. AP).
SCFAPre-Colon Bioreactors Proximal Colon Bioreactorsp-Value
Control
(n = 2)		AP
(n = 2)		Control
(n = 2)		AP
(n = 2)		Bioreactor (B)Medium (M)B * M
C2 2019214621571685nsnsns
C31126135212001165nsnsns
iC438.144.536.144.6nsnsns
C4692579714556nsnsns
iC51039811192nsnsns
C5556531591494nsnsns
C6110137166118nsnsns
Sum4644488749754155nsnsns
Ratio C2 (%)59.358.859.656.1nsnsns
Ratio C3 (%)26.930.427.031.4ns0.021ns
Ratio C4 (%)13.810.813.512.6ns0.009ns
AP: apple pomace, SCFA: short chain fatty acids, C2: acetic acid, C3: propionic acid, iC4: isobutyric acid, C4: butyric acid, iC5: isovaleric acid, C5: valeric acid and C6: hexanoic acid.
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Dufourny, S.; Lebrun, S.; Douny, C.; Dubois, B.; Scippo, M.-L.; Wavreille, J.; Rondia, P.; Everaert, N.; Delcenserie, V. Apple Pomace Modulates the Microbiota and Increases the Propionate Ratio in an In Vitro Piglet Gastrointestinal Model. Fermentation 2022, 8, 408. https://doi.org/10.3390/fermentation8080408

AMA Style

Dufourny S, Lebrun S, Douny C, Dubois B, Scippo M-L, Wavreille J, Rondia P, Everaert N, Delcenserie V. Apple Pomace Modulates the Microbiota and Increases the Propionate Ratio in an In Vitro Piglet Gastrointestinal Model. Fermentation. 2022; 8(8):408. https://doi.org/10.3390/fermentation8080408

Chicago/Turabian Style

Dufourny, Sandrine, Sarah Lebrun, Caroline Douny, Benjamin Dubois, Marie-Louise Scippo, José Wavreille, Pierre Rondia, Nadia Everaert, and Véronique Delcenserie. 2022. "Apple Pomace Modulates the Microbiota and Increases the Propionate Ratio in an In Vitro Piglet Gastrointestinal Model" Fermentation 8, no. 8: 408. https://doi.org/10.3390/fermentation8080408

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