Journal of Functional Foods 116 (2024) 106196

Contents lists available at ScienceDirect

Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Effects of Aronia melanocarpa juice-powder on hindgut function and

performance in post-weaned pigs
Sarah C. Pearce a, *, Christopher L. Anderson b, Brian J. Kerr a
a
Agroecosystems Management Research Unit, National Laboratory for Agriculture and the Environment, USDA-ARS, Ames IA, USA
b
Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, USDA-ARS, Ames, IA, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Twenty-four gilts and barrows (n = 8/trt) were divided into three treatment groups: control (CON), 0.5 % Aronia
Aronia melanocarpa melanocarpa juice powder (AM; LoBerry), and 1.0 % AM juice powder (HiBerry). Pigs were fed for 14 d and
Inflammation tissues were collected. No differences in performance were observed. Ileum IL-18 tended to be lower in LoBerry
Intestinal
pigs, while colonic IFN-γ was increased in pigs fed either AM diet. There were several changes in ileal gene
Polyphenols
Microbiota
expression in pigs fed the LoBerry diet compared to pigs fed the CON diet, including BMI1, CLDN2, JAM2, and
Pigs MYLK, which are largely related to barrier and stem cell function. In the colon, CLDN2, REG3G, SI, and SLC6A19
were increased in pigs fed the LoBerry diet. There were no differences found in the colonic microbiome due to
diets. In conclusion, feeding AM juice powder may have a positive impact in young pigs, but may require longer
feeding time to observe performance differences.

1. Introduction
Aronia melanocarpa (AM), more commonly known as aronia berries
or black chokeberries, are a native shrub species to Iowa and have been
used for centuries by Native Americans for medicinal purposes. In the
Midwest United States, AM are largely converted into juice or wine for
human consumption (Watrelot & Bouska, 2022). These berries have one
of the highest concentrations of antioxidants of all fruits but are un­
common in North American human and animal diets (Bushmeleva et al.,
2022; Tolić et al., 2015). Berries are good sources of phytochemicals
including flavonoids, tannins, and phenolic acids, among others (Agui­
lera, 2023). Aronia berries are known to be high in polyphenols; spe­
cifically, proanthocyanidins, anthocyanins, flavonols, and flavanones,
all of which are known antioxidants and anti-inflammatory compounds
(Sidor & Gramza-Michałowska, 2019). Due to intestinal disturbances
and diet change, the period following weaning represents a difficult
challenge period that can have drastic negative impacts on intestinal
health and performance in young pigs (Lynegaard et al., 2022).
Typically, weaned pigs may experience low feed intakes and
increased incidence of post-weaning diarrhea (PWD) which is often
associated with intestinal inflammation and oxidative stress (Lallès
et al., 2007; Li et al., 2023). Although zinc and copper are often used in
pig diets within in the United States at the time of weaning, other non-
antibiotic feed additives may represent a potential alternative inter­
vention strategy during this transition period (Canibe et al., 2022).
Because polyphenolic compounds require the gut microbiota for meta­
bolism, the distal small intestine and large intestine are of greatest in­
terest (Mithul Aravind et al., 2021).
There is limited research examining AM in pigs, however, a few
studies have shown promising effects on intestinal development (Ren
et al., 2022) and improved growth performance while reducing inci­
dence of diarrhea (Liu et al., 2021). In addition, AM polyphenols have
been shown to modulate the gut microbiota including increasing short-
chain fatty acid concentrations while inhibiting colonic inflammation in
a rat model of high fat diet (Zhu et al., 2023; Zhu et al., 2022). AM also
improved symptoms of colitis in rats (Valcheva-Kuzmanova et al.,
2018). In inflammatory bowel disease models, AM was shown to reverse
intestinal dysbiosis and improve inflammatory markers in mice (Li et al.,
2022) as well as prevent artificial sweetener induced dysbiosis in vitro
(Vamanu et al., 2022).
To our knowledge the current study is the first study to examine AM
juice powder in pigs. Therefore, objectives of the current experiment

Abbreviations: AM, Aronia melanocarpa; CLDN, Claudin; CON, Control; IL, Interleukin; JAM, Junctional adhesional molecule; LYZ, Lysozyme; PWD, Post weaning
diarrhea; SI, Sucrase isomaltase.
* Corresponding author.
E-mail address: sarah.pearce@usda.gov (S.C. Pearce).

https://doi.org/10.1016/j.jff.2024.106196
Received 6 February 2024; Received in revised form 27 March 2024; Accepted 18 April 2024
Available online 20 April 2024
1756-4646/Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S.C. Pearce et al.
were to examine two concentrations of AM juice powder fed for 14 d to
assess markers of pig performance, intestinal inflammation, gut micro­
biota changes, and host intestinal gene expression in newly weaned pigs.
2. Materials and methods
2.1. Animal care
All animal procedures were approved by the Iowa State University
Institutional Animal Care and Use Committee (IACUC #22-200) and
adhered to the guidelines for ethical and humane use of animals for
research.
2.2. Experimental design and diets
Twenty-four mixed sex pigs (n = 8/trt) were blocked by body weight
and divided into three treatment groups; control (CON), the CON with
0.5 % AM Juice Powder (LoBerry), and the CON with 1 % AM Juice
Powder (HiBerry). Pig body weights (BW) were recorded on d 0, 7, and
14. Individual feed intake and growth rates were measured and calcu­
lated for the overall 0–14 d test period. Feces was collected to determine
fecal scoring as a measure of diarrhea. At the end of the feeding
experiment (d 14 post-weaning), pigs were euthanized using captive
bolt followed by exsanguination for sample collection.
2.3. Sample collection
Following euthanasia, ileum (36 cm proximal from the cecum) and
colon (apex of the spiral colon) contents and sections were collected
along with colon contents. Contents was snap frozen and tissue were
flushed with Krebs-Henseleit buffer (25 mM NaHCO3, 120 mM NaCl, 1
mM MgSO4, 6.3 mM KCl, 2 mM CaCl2, and 0.32 mM NaH2PO4, pH 7.4)
and, snap frozen and stored at − 80 ◦ C until further analysis.
2.4. Aronia berry powder sample analysis
Commercially available AM Juice Powder (Aronia Advantage, Fair­
bank, IA) was sent for analysis of flavonoids and phenolic acids to a
commercial laboratory (Creative Proteomics, Shirley, NY). Briefly, 50
mg of each sample was weighed to a tube for extraction. All samples
were extracted for flavonoids and phenolic acids according to a standard
protocol using 100 % methanol. An internal standard was spiked at the
beginning of the extraction. The samples were reconstituted in 100 μL of
30 % methanol and diluted further 50 times and injected at 1 μL. The
following phenolic compounds were included in the targeted assay:
apigenindin, apigenin, caffeic acid, catechin, chalconaringenin,
chlorogenic acid, cinnamic acid, daidzein, delphinidin, epicatechin,
ferulic acid, gallic acid, genistein, hesperetin, kaempferol, luteolin,
naringenin, p-coumaric acid, phloretin, proanthocyanindin A2, pro­
cyanidin B2, protocatechuic acid, quercetin, quercetin-3-galactoside,
quercetin-3-glucoside, resveratrol, rutin, syringic acid, and vanillic acid.
2.5. Tissue inflammation
Protein was extracted from one frozen ileum and colon sample per
pig using Tissue Protein Extraction Reagent (T-PER) with protease and
phosphatase inhibitors (Thermo Fisher Scientific, Waltham MA). Protein
concentration was then analyzed using the Bicinchoninic Acid Assay
(Thermo Fisher Scientific, Waltham MA). Cytokine concentrations in
protein extracts were analyzed in duplicate using a commercially
available kit (Porcine Cytokine and Chemokine MILLIPLEX, Millipore
Sigma, St. Louis, MO) and analyzed on a quantitative fluorescent flow
cytometer (MAGPIX Multiplexing System, Luminex, Austin, TX). Cyto­
kines analyzed included Granulocyte-macrophage colony-stimulating
factor (GM-CSF), Interferon-γ (IFN-γ), as well as interleukins IL-1β, IL-
1ra, IL-8, IL-12, and IL-18.
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Journal of Functional Foods 116 (2024) 106196
2.6. Multiplex gene assay
The QuantiGene™ Plex Sample Processing Kit (Thermofisher Sci­
entific, Waltham, MA) was utilized to process whole ileum tissue sam­
ples ileum. Briefly, 50 mg of powdered ileum tissue and 1 mL of working
homogenization solution were added to a 2-mL microcentrifuge tube
containing silica beads. Samples were loaded into a bead beater for 3
min at maximum speed. Homogenized samples were then incubated at
65 ◦ C on a heat block for 30 min. Each sample was vortexed at maximum
speed for 1 min every 10 min during incubation. Samples were centri­
fuged at 16,000g for 15 min to pellet remaining cellular debris and su­
pernatant was transferred to a 1.5 mL microcentrifuge tube for storage.
Supernatants were then analyzed on a QuantiGene™ Plex Gene
Expression Array (ThermoFisher Scientific, Waltham, MA). Samples
were lysed at 37 ◦ C for 30 min and then diluted 1:5 with homogenization
solution. A working bead mix containing, in order, nuclease-free water,
lysis mixture, blocking reagent, proteinase K, capture beads, and probe
set was then prepared. The working bead mix was vortexed and added
into a hybridization plate with diluted tissue homogenate, sealed, and
placed overnight at 54 ◦ C at 600 rpm. The following day samples were
transferred from a hybridization plate to the magnetic separation plate
and wells were washed 3X. Pre-amplifier solution was then added and
placed at 50 ◦ C at 600 rpm for 1 h. Next, the plate was washed 3X and
amplifier solution was added and placed at 50 ◦ C at 600 rpm for 1 h. The
plate was then washed, and label probe solution was added for 1 h at
50 ◦ C at 600 rpm and then washed again. Proprietary probes were design
for Sus scrofa by Thermosteric Scientific (Waltham, MA) and include the
genes: atonal BHLH transcription factor 1 (ATOH1), BMI1 proto-
oncogene, polycomb ring finger (BMI1), cyclin dependent kinase in­
hibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1C (CDKN1C),
chromogranin A (CGA), claudin-2 (CLDN2), claudin-3 (CLDN3),
claudin-4 (CLDN4), catenin beta 1 (CTNNB1), defensin beta 1 (DEFB1),
fatty acid binding protein 2 (FABP2), free fatty acid receptor 3 (FFAR3),
ghrelin (GHRL), beta-glucuronidase (GUSB), Hes family BHLH tran­
scription factor 1 (HES1), hypoxanthine phosphoribosyltransferase 1
(HPRT1), interferon-γ (IFNG), interleukin-1β (IL1B), interleukin 1 re­
ceptor agonist (IL1RN), interleukin 10 (IL10), interleukin 17A (IL17A),
junctional adhesion molecule 2 (JAM2), lactase (LCT), leucine rich
repeat containing G protein coupled receptor 5 (LGR5), lysozyme (LYZ),
microtubule actin cross-linking factor 1 (MACF1), mucin 2 (MUC2),
mucin 5AC (MUC5AC), myosin light chain kinase (MYLK), notch re­
ceptor 1 (NOTCH1), occludin (OCLN), phosphoglycerate kinase (PGK1),
regenerating family member 3 gamma (REG3G), ribosomal protein L32
(RPL32), sucrase isomaltase (SI), solute carrier family 1 member 2
(SLC1A2), solute carrier family 2 member 5 aka glucose transporter 5
(SLC2A5/GLUT5), solute carrier family 5 member 1 aka sodium/glucose
transporter 1 (SLC5A1/SGLT1), solute carrier family 6 member 19 also
known as sodium-dependent neutral amino acid transporter B(0)AT1
(SLC6A19/B0AT1), solute carrier family 27 member 4 aka long-chain
fatty acid transporter protein 4 (SLC27A4), solute carrier family 38
member 2 also known as sodium-coupled neutral amino acid transporter
2 (SLC38A2/SNAT2), SRY-box transcription factor 9 (SOX9), trefoil
factor 2 (TFF2), trefoil factor 3 (TFF3), tight junction protein 1 (TJP1),
tumor necrosis factor (TNF), toll-like receptor 2 (TLR2), toll-like re­
ceptor 3 (TLR3), toll-like receptor 4 (TLR4), and wingless-type MMTV
integration site family, member 3A (WNT3A). Finally, Streptavidin R-
Phycoerythrin Conjugate (SAPE) working reagent was added and the
plate was covered with foil for 30 min at room temperature. Afterwards,
SAPE wash buffer was added and the plate was run on a Luminex
MAGPIX Instrument (Luminex, Northbrook, IL). Data were analyzed on
the QuantiGene™ Plex Analysis Software using the GEOMEAN of mul­
tiple housekeeping genes to generate normalized expression of values of
target genes (ThermoFisher Scientific, Waltham, MA) where house­
keeping genes included HPRT1, RPL32, and GUSB. Heatmaps were
generated using free online software through Displayr.
S.C. Pearce et al. Journal of Functional Foods 116 (2024) 106196

2.7. Microbial 16S rRNA gene sequencing Table 1

Diets.
DNA was extracted from colon contents with the Zymo ZymoBIO­ Ingredient, % CON LoBerry HiBerry
MICS 96 MagBead DNA Kit (Irvine, CA). Mechanical lysis was conducted
Corn, yellow dent 29.39 28.89 28.39
in individual Zymo BashingBead Lysis Tubes (Irvine, CA) using a Vortex Soybean meal, 46.5 % CP 40.00 40.00 40.00
Genie with a microtube adaptor. Amplicons of the V4 region of the 16S Oats, groat 5.00 5.00 5.00
rRNA gene were generated from the resulting DNA using the dual index Whey, dried 20.83 20.83 20.83
sequencing strategy outlined in Kozich et al. (Kozich et al., 2013) with Soybean oil 1.77 1.77 1.77
Limestone 0.82 0.82 0.82
30 PCR cycles. Negative DNA extractions and no template PCR reactions Monocalcium phosphate 21 % 0.34 0.34 0.34
were conducted and included for sequencing. Barcoded amplicons were Salt 0.50 0.50 0.50
pooled and sequenced on an Illumina MiSeq (La Jolla,CA) using the L-lysine HCl 0.19 0.19 0.19
MiSeq Reagent Kit V2 (2 × 250 bp). DL-methionine 0.16 0.16 0.16
L-threonine 0.06 0.06 0.06
L-tryptophan 0.10 0.10 0.10
2.8. Statistical analysis Optiphos 2500 0.03 0.03 0.03
Vitamin Premixa 0.25 0.25 0.25
Experiments were analyzed in JMP SAS (Version 9.2, SAS institute). Trace Mineral Premixb 0.15 0.15 0.15
Zinc oxide, 72 % Zn 0.4 0.4 0.4
Individual wells were used as technical replicates. Data were analyzed Aronia berry powderc − 0.5 1.0
with a one-way ANOVA with the Tukey–Kramer adjustment for pairwise
comparisons. Figures are presented as least square means ± standard
Calculated composition
error of the mean while tables are presented with the pooled standard Crude protein, % 24 24 24
error. For tables, means not sharing any letter are significantly different MEd, kcal/kg 3388 3388 3388
by the Tukey–Kramer test P < 0.05. For Fig. 1, *P < 0.05 and #P < 0.10. Lys, SIDe % 1.45 1.45 1.45
From the 16S rRNA gene sequencing data, amplicon sequence variants Phosphorus, available, % 0.45 0.45 0.45
Calcium, total % 0.65 0.65 0.65
(ASVs) were delineated with DADA2 (version 1.18.0) (Callahan et al., ADF, % 3 3 3
2016) and classified with the IDTAXA algorithm (Murali et al., 2018) NDF, % 4.79 4.79 4.79
using the SILVA training set (r138) (Quast et al., 2012). ASVs classified Crude fiber, % 2.58 2.58 2.58
as domain bacteria were retained for downstream analyses. The DEI­ a
Diet was provided with 6,125 IU vitamin A, 700 IU vitamin D3, 50 IU
CODE QIIME 2 plugin was used to normalize ASV counts across samples vitamin E, 30 mg vitamin K, 0.05 mg vitamin B12, 11 mg riboflavin, 56 mg
based on the robust centered log ratio, calculate the Aitchison Distance niacin, and 27 mg pantothenic acid per kilogram of diet.
between samples, and visualize the relationship between samples b
Diet was provided with 22 mg Cu (as CuSO4), 220 mg Fe (as FeSO4), 0.40
(Bolyen et al., 2018; Martino et al., 2019). A PERMANOVA test (Oksa­ mg I (as Ca(IO3)2), 52 mg Mn (as MnSO4), 220 mg Zn (as ZnSO4), and 0.40 mg
nen et al., 2007) was used to assess differences in the microbial com­ Se (as Na2SeO3) per kilogram of diet.
c
munity structure between treatment groups. Species richness and Aronia Advantage, Fairbank, IA.
d
diversity metrics were calculated using breakaway (Willis & Bunge, Metabolizable energy.
e
2015; Willis et al., 2017) and DivNet (Willis & Martin, 2022) and the Standardized ileal digestible.
differences in alpha diversity metrics were assessed with a Krus­
kal–Wallis test.
Table 2
Flavonoid and phenolic acids, DM basis.
3. Results
Compound Concentration, ng/g

Diet formulations are presented in Table 1 and were formulated to Apigenin 178.3
Caffeic acid 13862.1
meet energy and nutrient requirements for pigs of this age (National
Catechin 1893.1
Research Council). All 23 compounds for flavonoids and phenolic acids Chlorogenic acid 1217061.2
were found in the AM Juice Powder with the highest concentration of Cinnamic acid 5348.8
compounds being chlorogenic acid, protocatechuic acid, and rutin p-Coumaric acid 7622.1
(Table 2). Epicatechin 22922.6
Ferulic acid 7348.4
Gallic acid 12724.3
Genistein 58.9
Kaempferol 1809.2
Luteolin 859.8
Naringenin 325.7
Procyanidin B2 27371.3
Phloretin 26.4
Protocatechuic acid 375482.3
Quercetin-3-glucoside 140433.5
Quercetin-3-galactoside 142205.8
Quercetin 82622.3
Resveratrol 73.0
Rutin 278985.6
Syringic acid 2241.9
Vanillic acid 3973.5
Total content 2,345,430

Regardless of week of evaluation (wk 1 or 2) or over the entire 14

Fig. 1. Fecal scoring. Average fecal scoring over the 14-day test period in
control (CON) pigs compared to pigs fed 0.5% aronia melanocarpa (AM;
LoBerry) juice powder or 1% AM juice powder (HiBerry).
d period, there were no differences in body weight, average daily gain,
average daily feed intake, or gain:feed ratio observed between pigs fed
the CON, HiBerry, or LoBerry (P > 0.05; Table 3). While not statistically

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S.C. Pearce et al. Journal of Functional Foods 116 (2024) 106196

Table 3 Table 5
Performance. Colon cytokines, pg/mL.
Parameter Treatment Parameter Treatment

CON LoBerry HiBerry SEM P-Value CON LoBerry HiBerry SEM P-Value

BW, D0 5.43 5.39 5.48 0.128 0.889 IFN-γ 1.490a 2.266b 2.248b 0.221 0.044
BW, D14 6.67 7.16 6.84 0.347 0.594 IL-1β 1.358 1.638 1.665 0.512 0.897
ΔBW, D 0–14 1.47 2.01 1.40 0.216 0.118 IL1RA 0.338 0.295 0.286 0.040 0.626
ADG 0–14 0.09 0.13 0.10 0.021 0.433 IL-8 0.721 0.585 0.667 0.098 0.618
ADFI 0–14 0.14 0.16 0.16 0.016 0.643 IL-12 0.030 0.0242 0.031 0.006 0.720
G:F 0–14 0.61 0.74 0.64 0.075 0.665 IL-18 7.835 6.860 7.780 1.278 0.833

Pa,b,c,< 0.05.
significant when compared across all three treatments, there was a trend Px,y,z < 0.10.
for a difference in d-14 BW gain where pigs fed the LoBerry diets gained
0.54 kg more BW than pigs fed the CON (P = 0.10) and 0.61 kg more BW either the HiBerry or CON diets (P < 0.05; Fig. 2b). Epithelial structural
than pigs fed the HiBerry diet (P = 0.10). Similarly, there were no dif­ molecule, MYLK was decreased in pigs fed both AM diets compared to
ferences in fecal scores averaged between d 0–14 among pigs fed the pigs fed the CON diet (P < 0.05; Fig. 2b). Lastly, long chain fatty acid
three diets (P > 0.05; Fig. 1). transporter, SLC27A4, was increased in pigs fed the HiBerry diet
There was a tendency for a difference in ileum cytokine IL-18 con­ compared to pigs fed the CON diet (P < 0.05; Fig. 2a).
centration between treatments (P = 0.075; Table 4) where ileal IL-18 in Bacterial 16S rRNA gene sequencing of colonic contents yielded
pigs fed the LoBerry diet was 11 % lower compared to pigs fed the CON 2,927 ASVs with an average of 187,154 sequences per sample to assess
diet (Table 4). No significant differences between the other ileum cy­ shifts in the microbial community structure due to dietary treatments.
tokines including IFN-γ, IL-1β, IL1RA, IL-8, and IL-12 were observed due There were no significant differences observed in the microbial com­
to dietary treatment (P > 0.05; Table 4). However, when pigs fed the munity structure due to dietary treatment (Fig. 3a, PERMANOVA P =
CON diet were only compared to pigs fed the LoBerry diets, IFN-γ was 0.47, R2 = 0.39). Further, no significant differences in species richness
decreased by more than 100 % (P < 0.05). Interestingly, IFN-γ in the (Fig. 3b, Kruskal-Wallis P = 0.97) or Shannon diversity index (Fig. 3c,
colon was increased 50 % in pigs fed both the HiBerry and LoBerry diets Kruskal-Wallis P = 0.52) were detected between pigs fed the different
compared to pigs fed the CON diet (P < 0.05; Table 5). IL-1β, IL1RA, IL- dietary treatments.
8, IL-12, and IL-18 were not different between treatments (P > 0.05;
Table 5). 4. Discussion
In the ileum, the tight junction gene CLDN2 tended to increase in the
pigs fed the LoBerry diet compared to pigs fed the CON diet, with pigs Few studies have evaluated the effects of AM on pig health and
fed the HiBerry diet being intermediate (P < 0.10; Fig. 2a). Interestingly, performance and to our knowledge this is the first study to evaluate AM
gene expression of IL10 was significantly decreased in pigs fed the juice powder in pigs or any other livestock species. Native to North
HiBerry diet compared to pigs fed the CON diet (P < 0.05; Fig. 2a). America and grown in the Midwest, AM fruit is often not consumed
Antimicrobial peptide gene REG3G tended to increase in pigs fed the directly due to its bitter taste. However, AM byproducts such as pomace
LoBerry diet compared to pigs fed the CON diet, with pigs fed the and juice are more palatable and may be useful additives to animal
HiBerry diet being intermediate (P < 0.10; Fig. 2a). Digestive enzyme feedstuffs to improve performance (Bushmeleva et al., 2022). AM is rich
sucrase isomaltase (SI) and amino acid transporter SLC6A19 also tended in polyphenols including anthocyanins, procyanidins, flavonols, and
to increase in pigs fed the LoBerry diet compared to pigs fed the CON phenolic acids (Jurendić & Ščetar, 2021). Although lower concentra­
diet (P < 0.10; Fig. 2a). Stem cell marker, SOX9 was upregulated in both tions are observed in the AM skin and seeds, AM juice still contains high
berry treatment groups compared to pigs fed the CON diet (P < 0.05; levels of polyphenols (Oszmiański & Wojdylo, 2005). Interestingly, the
Fig. 2a). juice contains higher concentrations chlorogenic acid compared to
In the colon, there were several gene expression changes observed. pomace or the whole fruit, while still containing high levels of antho­
Stem cell marker BMI1 was decreased in pigs fed the LoBerry diet cyanins (Oszmiański & Wojdylo, 2005). Analysis of the juice powder
compared to pigs fed the CON diet (P < 0.05; Fig. 2b). There was a utilized in the current study showed presence of several different fla­
tendency for tight junction gene CLDN2 to be increased in pigs fed the vonoids and phenolic acids which have been reported in prior studies,
LoBerry diet compared to pigs fed the CON diet (P < 0.10; Fig. 2b). Fatty including high amounts of chlorogenic acid.
acid transporter FABP2 tended to be increased in pigs fed the HiBerry There was little to no effect of feeding AM juice powder on pig
diet compared to pigs fed the CON diet (P < 0.10; Fig. 2b). In contrast, performance in the current study. There was, however, a tendency for an
another junctional related gene, JAM2, was decreased in the pigs fed the improvement in body weight in pigs fed the LoBerry diet compared to
LoBerry diet compared to pigs fed the CON diet. Lysozyme, a Paneth cell pigs fed the CON diet when evaluated over the 14-d trial, but no other
marker, was increased in pigs fed the LoBerry diet compared to pigs fed trends were noted. AM pomace is a residue left over from juice pro­
cessing, which still contains a high number of phenolic compounds.
When AM pomace was fed to weaned piglets for 28 d at 0.5, 1, and 2 %
Table 4 inclusion levels, the 2 % inclusion rate increased average daily gain,
Ileum cytokines, pg/mL. average daily feed intake, and reduced incidence of diarrhea (Liu et al.,
Parameter Treatment 2021). Given this information, the minimal performance results
CON LoBerry HiBerry SEM P-Value observed in the current study could be due to timing of feeding or in­
clusion level differences.
IFN-γ 0.588 0.254 0.589 0.072 0.137
IL-1β 0.987 2.139 0.855 0.528 0.184 Although this study was conducted in non-immune challenged pigs,
IL1RA 0.237 0.271 0.245 0.027 0.615 fecal scoring was utilized to determine whether dietary treatment would
IL-8 6.556 4.683 6.559 0.941 0.245 cause diarrhea. Excess AM has been shown to cause digestive issues due
IL-12 0.101 0.088 0.089 0.017 0.773 to presence of sorbitol (Mayer-miebach et al., 2012), which due to poor
IL-18 15.775x 14.018y 15.045xy 0.581 0.075
absorption in the small intestine has been shown to induce laxative ef­
Pa,b,c,< 0.05. fects (Hattori et al., 2021). In the current study, we did not observe any
Px,y,z < 0.10.

4
S.C. Pearce et al.
Fig. 2a. Ileal gene expression. Heatmap of ileal gene expression in control (CON) pigs compared to pigs fed 0.5 % aronia melanocarpa (AM; LoBerry) juice powder or
1 % AM juice powder (HiBerry) for 14 days. Pa,b,c < 0.05.
differences in fecal scoring or the microbial community structure across
treatments.
There have been only a few studies using AM products in pigs in vivo
and in vitro. It has been shown that feeding 4–8 % AM pomace for 7 wks
increased expression of tight junction genes, positively affected intesti­
nal morphology, and increased beneficial microbes, while decreasing
inflammatory markers (Ren et al., 2022). Likewise, feeding dried pom­
aces of chokeberry has been shown to increase pig feed intake (Pieszka
et al., 2017). Furthermore, in porcine intestinal epithelial cells, AM
extract showed antioxidant and anticytotoxic activity while increasing
cellular proliferation (Kšonžeková et al., 2016). Although not many
studies have been conducted in pigs, there are several related animal
models that have shown positive effects of AM consumption. In
5
Journal of Functional Foods 116 (2024) 106196
humanized mice which were inoculated with human gut microbes, AM
fruit juice was fed for 2 wk and improved microbial metabolism
including producing metabolites associated with improved intestinal
barrier function (Wilson et al., 2023). In addition, AM fruit juice
ameliorated inflammatory bowel symptoms in a rodent colitis model
(Valcheva-Kuzmanova et al., 2018).
Polyphenols are generally considered to be of low bioavailability
with only 5–10 % being absorbed in the small intestine (Gowd et al.,
2019). Instead, a majority require biotransformation by the gut micro­
biota found in the hindgut (Mithul Aravind et al., 2021). Thus, we were
mainly interested in examining tissue effects in the hindgut for this
study. As polyphenolic compounds are thought to be anti-inflammatory,
we examined several cytokines in ileum and colon tissue. Although pigs
S.C. Pearce et al.
Fig. 2b. Colonic gene expression. Heatmap of colonic gene expression in control (CON) pigs compared to pigs fed 0.5 % aronia melanocarpa (AM; LoBerry) juice
powder or 1 % AM juice powder (HiBerry) for 14 days. Pa,b,c < 0.05.
used in the current study did not have an additional immune challenge,
they were obtained from a commercial farm immediately after weaning.
In the current study, ileal IL-18 was decreased in pigs fed the LoBerry
diet. Interleukin-18 can be produced by intestinal epithelial cells (Somm
& Jornayvaz, 2022) and is known as a potent pro-inflammatory mole­
cule (Ihim et al., 2022) and is thought to be involved in host defense as
well as intestinal barrier function and intestinal epithelial cell turnover
because it is primarily found in intestinal crypts (Chiang et al., 2022).
Increased levels of IL-18 are often indicative of inflammatory disorders
(Landy et al., 2024). IL-18 is also involved (along with IL-22) in coor­
dinating anti-microbial responses of Paneth cells with a link to IFN-γ to
6
Journal of Functional Foods 116 (2024) 106196
signal a T-cell response (Chiang et al., 2022). Interferon-γ, when only
compared to pigs fed the CON diet was significantly decreased in pigs
fed the LoBerry treatment as well. Conversely, in the colonic IFN-γ was
increased in pigs fed both the HiBerry and LoBerry diets which may
represent regional differences in dietary treatment effects.
Although in the current study we did not observe any changes in gut
microbial composition, prior research has shown a positive effect of
feeding AM. AM polyphenols have been shown to increase short-chain
fatty acid concentrations and alter microbial composition in a rat
model of high fat diet including increases in relative abundance of
Bacteroides, Prevotella, Romboutsia and Akkermansia (Zhu et al., 2023;
S.C. Pearce et al.
Fig. 3. 16S sequencing. 16S sequencing data (a) microbial community struc­
ture, (b) species richness and (c) Shannon diversity in control (CON) pigs
compared to pigs fed 0.5 % aronia melanocarpa (AM; LoBerry) juice powder or
1 % AM juice powder (HiBerry) for 14 days.
Zhu et al., 2022). In an inflammatory bowel disease model, AM was
shown to reverse intestinal dysbiosis in mice by improving the compo­
sition and diversity of microbes (Li et al., 2022). Administration of AM
juice into a digestor simulator for two weeks showed increased abun­
dance of firmicutes (Wu et al., 2018) while another in vitro study
demonstrated that AM could prevent artificial sweetener induced dys­
biosis (Vamanu et al., 2022). Based on prior research, this area warrants
further investigation in future pig studies.
There were several effects of feeding AM on gene expression of
several intestinal epithelial genes related to cell turnover, metabolism,
immune function, and barrier function. Regenerating family member
gamma (REG3G) which is anti-microbial peptide produced mainly by
Paneth cells in the small intestine, tended to increase in the ileum of pigs
fed the LoBerry diet. REG3G is thought to target gram-positive bacteria
(Shin et al., 2023) and stimulation of REG3G production affects mucus
distribution which impacts the intestinal epithelial layer and helps
maintain the physical barrier between the epithelium and gut microbes
(Vaishnava et al., 2011).
Digestive enzyme SI tended to increase in pigs fed the LoBerry diet.
7
Journal of Functional Foods 116 (2024) 106196
Sucrase isomaltase is mainly located on the brush border of the small
intestine and is responsible for breaking down disaccharides (Gericke
et al., 2016). Increased abundance may indicate better carbohydrate
metabolism. Neutral amino acid transporter SLC6A19 also tended to be
increased in LoBerry fed pigs. SLC6A19 is responsible for Na+-coupled
animo acid absorption from dietary protein on the epithelial brush
border of the small intestine (Yang et al., 2013). Increased SLC6A19
could indicate more efficient absorption of amino acids or may be
related to the small percentage of available free polyphenols.
Stem cell renewal is an important epithelial process which depends
on cell turnover and a homeostatic balance of cells proliferating and
differentiating (Liu et al., 2024). SOX9 is a transcription factor present in
intestinal crypts which is regulated by the Wnt pathway (Blache et al.,
2004). It was significantly increased in the ileum of pigs fed both berry
treatment groups. High expression levels of SOX9 are associated with
reserve stem cells (Roche et al., 2015) and is important for differentia­
tion of the Paneth cells (Bastide et al., 2007).
Changes in stem cell markers were also observed in the colon. BMI1
is involved in stem cell renewal and was used as a marker to help
identify stem cell subsets (Sangiorgi & Capecchi, 2008). Specifically,
BMI1 marks +4 quiescent stem cells which only activate during times of
epithelial injury (Yan et al., 2012). Colon levels of BMI1 were decreased
in pigs fed the LoBerry diet. Elevated levels of BMI1 have been associ­
ated with inflammation or infection (Yu et al., 2021). Tight junction
marker CLDN2 also tended to be increased in the ileum and colon in pigs
fed the LoBerry diet. Caudin-2 is a pore-forming claudin that helps
regulate paracellular transport of ions and water into the intestine and is
largely found in intestinal crypts (Garcia-Hernandez et al., 2017) and
has been shown to protect against intestinal inflammation and promote
mucosal healing (Ahmad et al., 2023). Lysozyme gene expression was
increased in pigs fed the LoBerry diet in the colon as well. Lysozyme is an
anti-microbial peptide produced in Paneth cells. Interestingly, IL-18 has
been shown to be a regulator of Paneth and stem cells (Chiang et al.,
2022). JAM2 was significantly decreased in LoBerry fed-pigs. Junctional
adhesion molecules (JAMs) belong to the immunoglobulin family and
JAM2 or JAM-B is involved in epithelial barrier permeability (Luissint
et al., 2014). While no changes in the colonic microbiome were noted
between treatments, metagenomic and metatranscriptomic sequencing
of the colonic mucosa could be used in the future to determine the
impact of feeding AM on the abundance and expression of microbial
genes at the intestinal epithelium.
5. Conclusions
In the current study, AM fruit juice powder was utilized as it is more
easily commercially available and easy to add into the feed and to our
knowledge, this is the first study to publish data on feeding AM juice
powder to pigs. The feeding level of 0.5–1 % was chosen based on prior
livestock phenolic studies and, to potentially be scale-able, and to avoid
potential issues with high concentrations of sorbitol. At these concen­
trations and a single time point some potential benefits were observed at
a tissue level, but no changes in performance were noted. Specifically,
some positive effects in both inflammatory status (decreased IL-18 in the
ileum) as well as gene expression related to metabolism (SI, SLCA19,
FABP2), barrier function (CLDN2), and cell differentiation (SOX9),
mainly at 0.5 % AM juice powder were also observed. In future studies,
testing additional concentrations as well as varying timepoints to assess
the potential benefits in post-weaned and growing pigs would be of in­
terest. Overall, non-antibiotic alternatives such as plant-based antioxi­
dants may represent a sustainable way to improve pig performance post-
weaning (Patience & Ramirez, 2022). Native AM represent a unique
opportunity to explore as they are in an area where pork production is
highest.
S.C. Pearce et al.
Funding
This project was funded by the USDA CRIS Projects #5030-31000-
007-00D and #5030-32000-225-000D.
CRediT authorship contribution statement
Sarah C. Pearce: Writing – original draft, Visualization, Validation,
Methodology, Formal analysis, Data curation, Conceptualization.
Christopher L. Anderson: Writing – review & editing, Visualization,
Methodology, Formal analysis, Data curation. Brian J. Kerr: Writing –
review & editing, Supervision, Project administration, Methodology,
Funding acquisition, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
Authors would like to thank Shari R. Stedham of the USDA-ARS
National Laboratory for Agriculture and the Environment for help
with lab analyses. Authors would also like to thank Dr. Nicholas Gabler
and Mitchell Nisley at Iowa State University for help with the animal
portion of the study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2024.106196.
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