physiological relevance of food grade microcapsules: impact of milk protein based microcapsules on...

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Received: 03-Dec-2014; Revised: 27-Mar-2015; Accepted: 13-Apr-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201400885. This article is protected by copyright. All rights reserved.

Physiological relevance of food grade microcapsules: Impact of milk protein based microcapsules on inflammation in mouse models for inflammatory bowel diseases

Rebecca Würtha, Ilias Lagkouvardosb, Thomas Clavelc, Julia Wilkea, Petra Foerstd, Ulrich Kulozika, Dirk Hallerb, Gabriele Hörmannspergerb

a Chair for Food Process Engineering and Dairy Technology, ZIEL Research Center for Nutrition and Food Science, Technology Unit, Technische Universität München, Weihenstephaner Berg 1, D-85354 Freising-Weihenstephan

b Chair of Nutrition and Immunology, ZIEL Research Center for Nutrition and Food Science, Biofunctionality Unit, Technische Universität München, Gregor-Mendel-Str. 2, D-85354 Freising-Weihenstephan

c Junior Research Group Intestinal Microbiome, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Gregor-Mendel-Str. 2, D-85354 Freising-Weihenstephan

d Chair of Process Engineering of Disperse Systems, Technische Universität Mün-chen, Maximus-von-Imhof-Forum 2, D-85354 Freising-Weihenstephan

Keywords: functional food, microencapsulation, immunity, IBD, probiotics, intestinal microbiota Correspondence: Gabriele Hörmannsperger, PhD, Chair of Nutrition and Immunology, ZIEL Research Center for Nutrition and Food Science, Biofunctionality Unit, Technische Universität München, Gregor-Mendel-Str. 2, D-85354 Freising-Weihenstephan Tel +49.8161.71.2374 Fax +49.8161.71.2824 [email protected] Abbreviations: IBD; inflammatory bowel diseases, SoCas; sodium caseinate, ReGel; Rennet gel; TNF; tumor necrosis factor, IL-10; interleukin 10

mailto:[email protected]


This article is protected by copyright. All rights reserved.

Abstract

In order to increase beneficial effects of bioactive compounds in functional food and

dietary supplements, enormous efforts are put in the technological development of

microcapsules. Although these products are often tailor-made for disease suscepti-

ble consumer, the physiological impact of microcapsule uptake on the respective

target consumer has never been addressed. The present study aimed to assess the

relevance of this aspect by analyzing the impact of milk-protein based microcapsules

on experimental inflammatory bowel disease (IBD).

Long-term feeding of sodium caseinate (SoCas) or rennet gel (ReGel) microcapsules

resulted in significant alterations in the intestinal microbiota of healthy mice. In

TNFΔARE/wt mice, a model for chronic ileal inflammation, ReGel microcapsules re-

sulted in further increased splenomegaly whereas ileal inflammation was unchanged.

In IL10-/- mice, a model for chronic colitis, both types of microcapsules induced a

local increase of the intestinal inflammation.

The present study is the first to demonstrate that, independent of their cargo, micro-

capsules have the potential to affect the intestinal microbiota and to exert unprece-

dented detrimental effects on disease-susceptible individuals. In conclusion, the im-

pact of microcapsule uptake on the respective target consumer groups should be

thoroughly investigated in advance to their commercial use in functional food or die-

tary supplements.



Introduction

Microencapsulation of bioactive food additives is thought to be a promising techno-

logical approach to enhance the beneficial health effect of the respective bioactive

food supplement, like probiotics, polyphenols or enzymes. [1, 2] Technological ef-

forts have been put into the development of food-grade microcapsules that reduce

the loss of bioactivity during production and storage or that enable the supplementa-

tion of a broader range of food matrices in addition to dairy products, like e.g. fruit

juices. Apart from protection in the product, microencapsulation often aims to protect

the bioactive structure from the harsh conditions during the gastrointestinal passage

after consumption. Highly sophisticated encapsulation techniques are currently de-

veloped to enable targeted release of the bioactive structure at the respective intes-

tinal target site. Abundant encapsulation techniques using different food grade en-

capsulation matrices, ranging from algae- or citrus derived polysaccharides to milk

proteins, have been used to generate tailor-made microcapsules. The hypothesized

protective effect of various microcapsules, e.g. on the survival of bioactive microor-

ganisms under different production, food matrix, storage or simulated gastric and

intestinal conditions, has already been proven in various studies. [3-6] However, tak-

ing into account that functional food and dietary supplements are specifically de-

signed to be frequently taken up by disease susceptible or diseased consumers, it is

obvious that the impact of the encapsulation material on the health of the respective

targeted consumer needs to be taken into consideration. Nevertheless, nothing is

known with regard to the effects microcapsules might have on disease development

in gastrointestinal disturbances, gastrointestinal infections, allergic diseases, irritable

bowel syndrome (IBS) or inflammatory bowel diseases (IBD), all of which are dis-

cussed as target diseases for orally applied active ingredients like probiotics.

The present proof of concept study aimed at assessing the physiological impact of

two newly developed, milk-protein based, food-grade microcapsules on IBD as mod-

el diseases. IBD, comprising Crohn´s Disease and Ulcerative Colitis, are immune

mediated chronic diseases of the gastrointestinal system that affect up to 0.3% of

western populations and that cannot be cured yet. [7] IBD are characterized by a

deregulated inflammatory immune response towards the intestinal microbiota and

alterations in the composition and functionality of the intestinal microbiota are strong-

ly associated with IBD. [8] In consequence, prebiotic and probiotic supplementations

are of high therapeutic interest. [9, 10] The impact of specific food constituents on



IBD development and severity is mostly unclear, but IBD patients frequently develop

food intolerances mainly towards dairy products (e.g. lactose), cereals and yeast.

[11] Therefore, IBD are relevant model diseases for studying the impact of long-term

feeding of microcapsules on the disease outcome.

Material and Methods

The effect of long-term feeding of SoCas and ReGel microcapsules (experimental

schedule in Fig. 1A) on the intestinal microbiota was analyzed in healthy wildtype

mice. The effects of the respective microcapsule feeding on body weight, spleen

weight (a well-recognized and broadly used marker of inflammation in experimental

models for IBD and arthritis [12-14]), plasma TNF (a pro-inflammatory cytokine) and

parameters of intestinal inflammation were analyzed in healthy BL/6 wildtype mice,

TNFΔARE/wt mice (a model for chronic ileitis (and arthritis)) and IL10-/- mice (a

model for chronic colitis). ELISA, Histopathological analysis, Immunofluorescence

staining, Alcian Blue staining and 16sRNA gene sequencing were performed as indi-

cated in Supporting Information Material and methods.

Results

Long-term feeding of SoCas as well as ReGel microcapsules (Fig. 1A/B) was suffi-

cient to induce significant shifts in dominant gut bacterial communities of healthy

mice (Fig.1 C-E, Supporting Information S1 A, B). ReGel- but not SoCas feeding re-

sulted in a significant drop of bacterial richness compared to control mice (Fig. 1D).

At the family level, both types of microcapsules significantly reduced the relative

abundance of Helicobacteriaceae, whereas that of Verrucomicrobiacea increased

(Fig. 1E). In addition, the presence/absence of 22 OTUs was found to be dependent

on the respective feeding regimen (Supporting Information Fig. S1 B). In contrast to

the observed shifts in gut bacterial diversity and composition, none of the investigat-

ed host parameters (body weight, spleen weight, plasma TNF, intestinal histology)

was affected by the microcapsule feedings in healthy mice (Fig. 2A/C, Supporting

Information S2).

As expected, 18 week old TNFΔARE/wt mice showed significantly reduced body

weight (Fig. 2A), increased plasma TNF levels (Supporting Information Fig. S2) and

increased spleen to body weight ratios (splenomegaly) (Fig. 2B/C) compared to

healthy BL/6 wildtype mice. Long-term feeding of SoCas or ReGel capsules did not



result in altered body weight of TNFΔARE/wt mice compared to control mice (Fig.

2A). Plasma levels of TNF were also not affected by the microcapsules (Supporting

Information Fig. S2). However, ReGel fed TNFΔARE/wt mice showed a marked fur-

ther increased splenomegaly compared to ctr and SoCas fed TNFΔARE/wt mice

(Fig. 2B/C, Supporting Information S3). Histopathological analysis of the terminal

ileum revealed severe inflammation in TNFΔARE/wt mice from all three feeding

groups. TNFΔARE/wt mice in all groups showed similar levels of infiltrated immune

cells in the mucosa, submucosa and muscularis as well as architectural distortions in

the distal ileum. (Fig. 2D,E)

Analogous to wt and TNFΔARE/wt mice, body weight and plasma TNF levels (Fig.

3A, Supporting Information S3) were unchanged between IL10-/- mice fed different

diets. In contrast to TNFΔARE/wt mice, spleen to body weight ratios (Fig. 3B) in

IL10-/- mice were unaffected by ReGel feeding. However, histopathological analysis

of the cecum revealed that long-term feeding of SoCas as well as ReGel microcap-

sules resulted in significantly increased, mild-to moderate cecal inflammation in IL10-

/- mice (Fig. 3C,D). The increased cecal inflammation was characterized by a promi-

nent loss of Goblet cells, [15-17] and significantly increased T cell infiltration (Fig. 3

E-H). Colonic inflammation in IL10-/- mice was unaffected by microcapsule feeding

(Supporting Information Fig. S4).

Discussion and Conclusion

In summary, the results of the present study demonstrate that long-term feeding of

protein-based microcapsules can affect the diversity and composition of the intestinal

microbiota and can aggravate disease-specific alterations of secondary immune

compartments (splenomegaly) as well as intestinal inflammation in murine models

for IBD. The physiological impact of microcapsule feeding was found to be depend-

ent on the type of microcapsule and the IBD mouse model used. The relevant micro-

capsule ingredients as well as the underlying mechanisms for the observed micro-

capsule-mediated increase in splenomegaly (TNFΔARE/wt) or cecal inflammation

(IL10-/-) are unclear and remain to be investigated in further studies. Of note, the

high amount of lactose in ReGel microcapsules is a major difference between the

two types of microcapsules (~50 % w/v in ReGel vs. ~0.2 % w/v in SoCas dry mass).

Hence, one might speculate that lactose could be causally linked to the observed

ReGel-mediated increase in spleen weight in TNFΔARE/wt mice. This hypothesis is



supported by the fact that IBD patients show a high incidence of lactose malabsorp-

tion/intolerance [18, 19]. In line with this, ReGel feeding results in significantly re-

duced intestinal pH in the ileitis model (TNFΔARE/wt), which is indicative of in-

creased microbial fermentation of lactose to SCFA due to impaired small intestinal

capacity to digest and absorb lactose [20, 21], whereas ReGel feeding does not af-

fect intestinal pH nor spleen weight in the colitis model (IL-10-/-) (Table S1). Howev-

er, the potential pathophysiological relevance of lactose for arthritis, ileitis and spleen

weight development in TNFΔARE/wt is unclear and needs to be investigated in sep-

arate studies using pure lactose. As both types of microcapsules mediated increased

cecal, but not colonic, inflammation in IL10-/- mice, it may be speculated that specific

milk proteins, that are present in SoCas and ReGel microcapsules, might have me-

diated the observed local, pro-inflammatory effect. As the intestinal microbiota is

known to play a major role in IBD, [8] the observed detrimental effects of SoCas and

ReGel microcapsules in TNFΔARE/wt and IL10-/- mice might also be due to micro-

capsule induced alterations in the intestinal microbiota. Although the pathophysiolog-

ical mechanisms are unknown, the finding that microcapsules per se can have the

potential to modulate the intestinal microbiota as well as consumer immunity clearly

underlines the importance of thorough investigation of microcapsules with regard to

potential pathophysiological effects in diseased target consumer groups.

Encapsulation of bioactive substances aims at protecting these bioactive compounds

and in consequence, at increasing their potential to mediate beneficial effects on the

healthy, disease susceptible or diseased consumer. However, the results of the pre-

sent study show that the encapsulation materials per se can affect disease develop-

ment in disease susceptible organisms. In conclusion, one should not neglect the

fact that frequent consumption of these products by disease susceptible individuals,

which most often constitute an important target consumer group, may harbor disease

specific risks, although the encapsulation materials used are food-grade and show a

history of safe use. To date, abundant studies report successful development of mi-

croencapsulation technologies for specific food applications [3-6] but no study ad-

dressed the impact of these microcapsules on the health of the target consumer.

However, it can be assumed, and has partly already been shown, that the commonly

used encapsulation matrices like alginate, pectin, chitosan, guar gum, carrageenan,

lipids, whey and caseins, may affect the composition of the intestinal microbiota or

may even modulate the metabolism and/or the immune function of specific consumer



populations. [22-24] In consequence, the encapsulation material itself may impose

beneficial or detrimental effects on specific disease susceptible or diseased con-

sumers. Therefore, risk assessment of newly developed microcapsules in adequate

in vivo model systems should become a standard procedure before microcapsules

are used in functional food products or dietary supplements.

Author contributions

G.H. designed the experiments and wrote the manuscript. R.W., J.W. and G.H. per-

formed the experiments. I.L and T.C. performed the 16S rRNA gene sequencing and

data analysis, D.H., U.K. and P.F. critically revised the manuscript.

Acknowledgement

This research project was supported by the German Ministry of Economics and

Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V.,

Bonn) (Project AiF 16537 N). The authors thank George Kollias (Institute of Immu-

nology, Biomedical Sciences Research Center (BSRC), “Alexander Fleming”,

Greece) for providing TNFΔARE/wt mice. The authors are also very thankful to Simone

Daxauer, Melanie Klein, Julia Riker, Yangfang Sun, Alexandra Buse, David Himbert

and Danika Grätz for technical support.

Conflict of Interest

The authors declare that there is no commercial conflict of interest



References

1. Mitropoulou, G., et al., Immobilization technologies in probiotic food production. J Nutr

Metab, 2013. 2013: p. 716861. 2. Shah, N.P., Probiotic bacteria: selective enumeration and survival in dairy foods. J Dairy Sci,

2000. 83(4): p. 894-907. 3. Abbaszadeh, S., et al., The effect of alginate and chitosan concentrations on some properties

of chitosan-coated alginate bead and survivability of encapsulated Lactobacillus rhamnosus in simulated gastrointestinal conditions and during heat processing. J Sci Food Agric, 2013.

4. Zhang, F., et al., Effect of microencapsulation methods on the survival of freeze-dried Bifidobacterium bifidum. J Microencapsul, 2013. 30(6): p. 511-8.

5. Ortakci, F. and S. Sert, Stability of free and encapsulated Lactobacillus acidophilus ATCC 4356 in yogurt and in an artificial human gastric digestion system. J Dairy Sci, 2012. 95(12): p. 6918-25.

6. Guerin, D., J.C. Vuillemard, and M. Subirade, Protection of bifidobacteria encapsulated in polysaccharide-protein gel beads against gastric juice and bile. J Food Prot, 2003. 66(11): p. 2076-84.

7. Ananthakrishnan, A.N., Epidemiology and risk factors for IBD. Nat Rev Gastroenterol Hepatol, 2015.

8. Kostic, A.D., R.J. Xavier, and D. Gevers, The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology, 2014. 146(6): p. 1489-99.

9. Shen, J., Z.X. Zuo, and A.P. Mao, Effect of probiotics on inducing remission and maintaining therapy in ulcerative colitis, Crohn's disease, and pouchitis: meta-analysis of randomized controlled trials. Inflamm Bowel Dis, 2014. 20(1): p. 21-35.

10. Jonkers, D., et al., Probiotics in the management of inflammatory bowel disease: a systematic review of intervention studies in adult patients. Drugs, 2012. 72(6): p. 803-23.

11. Rajendran, N. and D. Kumar, Role of diet in the management of inflammatory bowel disease. World J Gastroenterol, 2010. 16(12): p. 1442-8.

12. Wang, Y., et al., Protective role of tumor necrosis factor (TNF) receptors in chronic intestinal inflammation: TNFR1 ablation boosts systemic inflammatory response. Lab Invest, 2013. 93(9): p. 1024-35.

13. Snider, A.J., et al., A role for sphingosine kinase 1 in dextran sulfate sodium-induced colitis. FASEB J, 2009. 23(1): p. 143-52.

14. Xu, Y., N.H. Hunt, and S. Bao, The role of granulocyte macrophage-colony-stimulating factor in acute intestinal inflammation. Cell Res, 2008. 18(12): p. 1220-9.

15. Zhang, H., et al., Mast cell deficiency exacerbates inflammatory bowel symptoms in interleukin-10-deficient mice. World J Gastroenterol, 2014. 20(27): p. 9106-15.

16. Strugala, V., P.W. Dettmar, and J.P. Pearson, Thickness and continuity of the adherent colonic mucus barrier in active and quiescent ulcerative colitis and Crohn's disease. Int J Clin Pract, 2008. 62(5): p. 762-9.

17. Vereecke, L., et al., A20 controls intestinal homeostasis through cell-specific activities. Nat Commun, 2014. 5: p. 5103.

18. Eadala, P., et al., Association of lactose sensitivity with inflammatory bowel disease--demonstrated by analysis of genetic polymorphism, breath gases and symptoms. Aliment Pharmacol Ther, 2011. 34(7): p. 735-46.

19. Mishkin, S., Dairy sensitivity, lactose malabsorption, and elimination diets in inflammatory bowel disease. Am J Clin Nutr, 1997. 65(2): p. 564-7.

20. Holtug, K., et al., The colon in carbohydrate malabsorption: short-chain fatty acids, pH, and osmotic diarrhoea. Scand J Gastroenterol, 1992. 27(7): p. 545-52.

21. Alexandre, V., et al., Lactose malabsorption and colonic fermentations alter host metabolism in rats. Br J Nutr, 2013. 110(4): p. 625-31.



22. Arnberg, K., et al., Skim milk, whey, and casein increase body weight and whey and casein increase the plasma C-peptide concentration in overweight adolescents. Journal of Nutrition, 2012. 142(12): p. 2083-90.

23. Razavi, A., et al., Therapeutic effect of sodium alginate in experimental chronic ulcerative colitis. Iran J Allergy Asthma Immunol, 2008. 7(1): p. 13-8.

24. Tobacman, J.K., Review of harmful gastrointestinal effects of carrageenan in animal experiments. Environ Health Perspect, 2001. 109(10): p. 983-94.



Figure 1: Experimental design and analysis of the intestinal microbiota. Figure

A gives an overview on the study design including the IBD models used and the

feeding regimen. Figure B shows microscopic pictures (100x) of SoCas and ReGel

capsules (left picture) and the capsule mash (food matrix) (right picture) as it was fed

to the mice. The size of the microcapsules was 73.6±23.9 µm (SoCas) to 54.1±18.5

µm (ReGel). C; Multidimensional scaling plot based on phylogenetic distances be-

tween colonic bacterial profiles of all individual healthy mice (wt) fed the three differ-

ent diets. (round circle: female, triangle: male). Figure D shows the molecular spe-

cies counts in the colon content from mice in the different groups. Figure E shows all

bacterial families for which relative sequence abundances were significantly affected

by microcapsule feeding in healthy mice.



Figure 2: Impact of microcapsule feeding on healthy and TNFΔARE/wt mice.

Body weight (A) was unaffected by microcapsule feeding in wildtype (wt) and

TNFΔARE/wt (ARE) mice. Figure B shows representative pictures of spleens isolated

from wt mice and TNFΔARE/wt mice fed different microcapsule diets. ReGel feeding

significantly increased spleen to body weight ratio in TNFΔARE/wt mice compared to

ctr and SoCas feeding (C). Ileal morphology (D) and histopathological inflammation

of distal ileum (dI) at 18 weeks of age (E) were unchanged by microcapsule feeding.



Figure 3: Impact of microcapsule feeding on IL10-/- mice. Body weight (A) and

spleen to body weight ratio (B) were unchanged by microcapsule feeding in IL10-/-

mice. Figure C and D show that ReGel and SoCas microcapsules significantly in-

crease immune cell infiltration, architectural distortion and histopathological inflam-

mation of the cecal tip (CT) in IL10-/- mice. Both microcapsule feedings resulted in a

prominent loss of Goblet cells (blue) (E/F) and significantly increased numbers of

infiltrated CD3+ T cells (red) (G/H) in the cecum of IL10-/- mice.

physiological relevance of food grade microcapsules: impact of milk protein based microcapsules on...

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