Development of novel in vitro human digestion systems for screening the bioavailability and digestibility of foods

Journal of Functional Foods 22 (2016) 113–121

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Development of novel in vitro human digestion systems for screening the bioavailability and digestibility of foods Seung-Jae Lee a,b, Seung Yuan Lee a, Myung-Sub Chung c, Sun Jin Hur a,* a

Department of Animal Science and Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi-do, 456-756, Republic of Korea b Eco-friendly Biomaterial Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 580-185, Republic of Korea c Department of Food Science and Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi-do, 456-756, Republic of Korea

A R T I C L E

I N F O

A B S T R A C T

Article history:

The in vitro models employed have included all steps involved in digestion upon passage through

Received 17 June 2015

the mouth, stomach, small intestine, and the colon. Importantly, enterobacter bacteria (Es-

Received in revised form 16

cherichia coli and Lactobacillus casei) are included to simulate the colon step. After in vitro human

December 2015

digestion in the small and large intestines, the antioxidant activity of rutin increased dra-

Accepted 5 January 2016

matically, whereas the antioxidant activity was not influenced by digestion in the mouth or

Available online

the stomach. Before in vitro human digestion, the antioxidant activity of quercetin and chlo-

Keywords:

intestines (with enterobacteria), the antioxidant activity of quercetin increased although en-

In vitro human digestion

terobacteria did not influence the antioxidant activity. However, the antioxidant activity of

rogenic acid was increased, and after in vitro human digestion in the small intestine and large

Enzymes

chlorogenic acid was not influenced by in vitro human digestion in the small intestine and

Gastrointestinal tract

large intestines (with enterobacteria). This study provides data supporting an alternative to

Enterobacter bacteria

the use of animals and humans for rapid screening of food digestibility and bioavailability.

Bioavailability

1.

Introduction

In vivo studies are time consuming and costly. As a result, much effort has been devoted to the development of in vitro procedures (Boisen & Eggum, 1991). In vitro digestion methods are ethically superior, faster, and less expensive than in vivo techniques, and provide a useful alternative to animal and human models for rapidly screening food ingredients (Coles, Moughan, & Darragh, 2005). An ideal in vitro digestion model would provide highly accurate results in a short time (Coles et al., 2005) and could serve as a tool to study the digestibility or bioavailability

© 2016 Elsevier Ltd. All rights reserved.

of various foods (Hur, Lim, Decker, & McClements, 2011). However, any in vitro method is inevitably going to fail to match that achieved by studying food digestion in vivo (Coles et al., 2005). This is because the results of human digestion are dependent on many factors associated with food composition, structure, amount, and enzyme characteristics (Hur, Lim et al., 2011). In the past ten years, we have worked to develop a more realistic in vitro human digestion system for food applications. The purpose of the current study was to develop an in vitro model that included all steps of the human digestion system and to use this model to rapidly screen the digestibility and bioavailability of food materials.

* Corresponding author. Department of Animal Science and Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi-do, 456-756, Republic of Korea. Tel.: +82 31 670 4673; fax: +82 31 675 3108. E-mail address: [email protected] (S.J. Hur). http://dx.doi.org/10.1016/j.jff.2016.01.005 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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2.

Materials and methods

2.1.

Materials

1996; Hur, Lim et al., 2011) although the transit times depend on the condition of each sample. In general, transit times were 5 min for the mouth step, 2 h for the stomach step, 2 h for the small intestine step, and 4 h for the large intestine step.

Analytical grade potassium chloride, potassium hydroxide, potassium persulphate, sodium sulphate, sodium bicarbonate, hydrogen chloride, potassium phosphate monobasic, magnesium chloride, hexane, methanol, acetate, phosphoric acid, ferric chloride, hydrochloric acid, sulphuric acid, chloroform, ether, and ethanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). Analytical grade bicarbonate, potassium thiocyanate, sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride, calcium chloride, ammonium chloride, urea, glucose, glucuronic acid, glucosamine, α-amylase, uric acid, mucin, bovine serum albumin, pepsin, pancreatin, lipase, bile salt extract, phenolphthalein, L-ascorbic acid, 2,2′-azino-bis(3ethylbenzothiazoline-6-sulphonic acid (ABTS), nile red, and rutin were purchased from Sigma-Aldrich (St Louis, MO, USA). Wheat flour, starch, and palm oil were purchased from general commercial sources. LB broth and MRS broth were purchased from Difco (Sparks, MD, USA).

2.2.

Compartments of in vitro human digestion

A human gastrointestinal digestion model (for adults) that simulates the mouth, stomach, small intestine, and large intestine was used in this study. This was a modified version of that described previously (Hur, Lim et al., 2011; Oomen et al., 2003; Versantvoort, Oomen, Van de Kamp, Rompelberg, & Sips, 2005). For simulation of digestion by the large intestine, enterobacter bacteria such as Escherichia coli and Lactobacillus casei were applied to the sample after digestion by the small intestine.

2.3.

Temperature of in vitro human digestion

Normal human body temperature is 37 °C and most metabolic processes and digestive enzymes are optimized to this temperature. Therefore, in vitro human digestion was carried out at 37 ± 0.3 °C.

2.4.

Transit times of in vitro human digestion

Transit times were chosen on the basis of physiology as reported previously (Christensen et al., 1985; Degen & Phillips,

2.5.

pH of in vitro human digestion

The pH values for the digestive juices and gastrointestinal tract were selected based on existing human anatomy and medical physiology literature (Evans et al., 1988; Guyton & Hall, 2006; Marieb & Hoehn, 2010) and a previous study (Hur, Lim et al., 2011). In general, pH values were 6.8 ± 0.2 for the mouth step, 1.5 ± 0.2 for the stomach step, 8.0 ± 0.2 for the small intestine step, and 7.0 ± 0.2 for the large intestine step.

2.6. Digestive enzymes, inorganic and organic solutions of in vitro human digestion Digestive enzymes, and inorganic and organic solutions used in this study were from those described previously (Hur, Lim et al., 2011; Oomen et al., 2003; Versantvoort et al., 2005). The compositions of the simulated saliva, gastric, duodenal, and bile juices are listed in Table 1.

2.7. Enterobacter bacterial preparations used for large intestine digestion During in vitro human digestion, enterobacter bacteria were applied to samples during the large intestine digestion step. This is the first in vitro method to be developed that includes this step of human digestion. E. coli (American Type Culture Collection (ATCC) 25922, Manassas, VA, USA) liquid agar was prepared using 2.5 g Luria– Bertani (LB) Broth (Sparks, MD, USA) with 100 mL deionized– distilled water (DDW). L. casei (ATCC 393) liquid agar was prepared using 5.5 g Lactobacilli MRS Broth (Sparks, MD, USA) mixed with 100 mL DDW. Each agar preparation was sterilized by autoclave at 121 °C for 15 min and cooled in tap water. Frozen (−80 °C) stock E. coli and L. casei were melted at room temperature then warmed to 37 °C. One per cent of E. coli and L. casei stocks were added to 100 mL of the appropriate sterilized liquid agar. E. coli and L. casei agar solutions were incubated at 37 °C for 12 h for activation. The activated E. coli and L. casei

Table 1 – Constituents and concentrations of the various synthetic juices used in the in vitro human digestion model representing fed conditions. Saliva (mouth step)

Gastric juice (stomach step)

Duodenal juice (small intestine step)

Bile juice (small intestine step)

Organic and inorganic components

1.7 mL NaCla (175.3 g/L)b 8 mL urea (25 g/L) 15 mg uric acid

6.5 mL HCl (37 g/L) 18 mL CaCl2 2H2O (22.2 g/L) 1 g bovine serum albumin

6.3 mL KCl (89.6 g/L) 9 mL CaCl2 2H2O (22.2 g/L) 1 g bovine serum albumin

68.3 mL NaHCO3 (84.7 g/L) 10 mL CaCl2 2H2O (22.2 g/L) 1.8 g bovine serum albumin 30 g bile

Enzymes

290 mg α-amylase 25 mg mucin 6.8 ± 0.2

2.5 g pepsin 3 g mucin 1.50 ± 0.02

9 g pancreatin 1.5 g lipase 8.0 ± 0.2

pH a

7.0 ± 0.2

The numbers are the concentration of chemicals used to make digestive juices. b The numbers in parentheses are the concentrations of inorganic or organic components per 1 L distilled water. After mixing all ingredients (inorganic components, organic components, and enzymes), the volume was increased to 500 mL with distilled water. If necessary, the pH of the juices was adjusted to the appropriate value.

Journal of Functional Foods 22 (2016) 113–121

were applied again to 100 mL of sterilized liquid agar for an additional 12 h at 37 °C. After incubation, the final number of E. coli and L. casei colonies was log 108~1010. For the large intestine digestion system, 38 mL of the liquid agar E. coli and L. casei solutions were applied to samples (after small intestine digestion) and incubated for 4 h at 37 °C. The large intestine digestion procedure with enterobacter bacteria is illustrated in Fig. 1.

2.8. In vitro digestion procedure for the analysis of sample bioavailability or structural changes 1. Initial system: 5 g samples. 2. Mouth: 5 g samples were mixed with 6 mL of simulated saliva solution (pH 6.8) and then stirred for 5 min at 37 °C. 3. Stomach: 12 mL of simulated gastric juice (pH 1.5) were added and the mixture was stirred for 2 h at 37 °C. 4. Small intestine: 12 mL of duodenal juice, 6 mL of bile juice, and 2 mL of 70% bicarbonate solution (pH 8.0) were added and the mixture was stirred for 2 h at 37 °C. 5. Large intestine: after small intestine digestion, 38 mL of liquid agar containing E. coli and L. casei were applied to a sample previously digested in the small intestine step and incubated for 4 h at 37 °C (see above Enterobacter method). The in vitro human digestion procedure for analysis of bioavailability is illustrated in Fig. 1.

2.9. In vitro procedure for the analysis of sample digestibility or absorption ratio 1. Initial system: 5 g samples. 2. Mouth: 5 g samples were mixed with 6 mL of simulated saliva solution (pH 6.8) and then stirred for 5 min at 37 °C. 3. Stomach: 12 mL of simulated gastric juice (pH 1.5) were added and the mixture was stirred for 2 h at 37 °C. 4. Small intestine: 12 mL of duodenal juice, 6 mL of bile juice, and 2 mL of sodium bicarbonate solution (pH 8.0) were added. The total solution was placed in a 250 mL flask. Dialysis tubing (molecular weight cutoff of 50 kDa, flat width 34 mm, thickness 18 µm (Membrane Filtration Products, Inc., Seguin, TX, USA)) containing 10 mL of phosphate buffer (pH 7) was then placed in the flask and the mix was stirred for 2 h at 37 °C. During the simulated human gastrointestinal digestion, the samples were swirled (60 rpm) in a shaking water bath (Model 3582, Labline Instruments, Inc., Melrose Park, IL, USA) to simulate the motility of the gastrointestinal tract. The digestibility of samples was determined by the extent of infiltration through the dialysis tubing. Briefly, the dialysis tubing was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA) with a molecular weight cutoff of 12– 14 kDa. The dialysis tubing was cut into pieces of 10 cm and soaked in distilled water at 4 °C before use. After stomach digestion, the buffer was transferred to the dialysis bags. The bags were then placed into a 250 mL flask (containing digestive juice and sample) and incubated at 37 °C for 2 h in a stirred water bath. The digestibility percentage was calculated as: (sample concentration inside the dialysis tube − sample concentration

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outside the dialysis tube)/(sample concentration inside the dialysis tube) × 100. The in vitro human digestion procedure for analysis of digestibility is illustrated in Fig. 2.

2.10. Antioxidant activity assessed by ABTS radical scavenging activity ABTS was dissolved in water (7 mmol/L). The ABTS radical cation (blue in colour) was produced by reacting the ABTS stock solution with 2.45 mmol/L potassium persulphate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For testing samples, the ABTS radical cation stock solution (5 mmol/L) was diluted with phosphate-buffered saline, pH 7.4, to an absorbance of 0.70 at 734 nm. After the addition of 980 µL of diluted ABTS to 20 µL of sample, the absorbance reading was taken 5 min after the initial mixing. The percentage ABTS scavenging activity was calculated as:

[(control absorbance 2.11.

sample absorbance) (control absorbance)] × 100

Confocal laser scanning microscopy

Images of lipid digestibility were analysed through confocal laser scanning microscopy (Carl Zeiss, LSM 5 Live, GmbH, Jena, Germany) with a 20× objective lens. Five grams of each sample were weighed into 50 mL test tubes and mixed with 15 mL of DDW. Nile red (a lipid fluorescent dye) was excited at 488 nm with an argon laser. The fluorescence emitted from each sample was monitored at an emission wavelength of 543 nm with a pinhole size of 150 µm. The resulting images consisted of 512 × 512 pixels, with a pixel size of 414 nm and a pixel dwell time of 5 s.

3.

Result and discussion

We previously reported the effects of biopolymer encapsulation on the digestibility of lipids and cholesterol oxidation products in beef patties during in vitro human digestion (Hur, Lee, & Lee, 2015). Marounek, Volek, Synytsya, and Copikova (2007) reported that gel-forming polysaccharides increased the viscosity and affected the processes of digestion and absorption in the small intestine. Several studies have reported the production of oxidized lipids and cholesterol in cooked meats. Lipid digestibility during in vitro human digestion in samples with different amounts of beef tallow is presented in Fig. 3. After in vitro digestion in the small intestine, lipid digestibility was increased to approximately 80 to 90% regardless of the tallow content. Fig. 4 shows that the size of the lipid droplets decreased after in vitro digestion in the small intestine. Armand et al. (1999) reported that initial fat droplets of smaller sizes were effectively digested by gastric lipase both in the stomach and in the duodenum. Our previous studies (Hur, Joo, Lim, Decker, & McClements, 2011; Hur, Kim, Chun, Lee, & Keum, 2013; Hur, Kim, Choi, & Lee, 2013; Hur, Lim et al., 2011; Hur, Park, & Jeong, 2011; Hur, Decker, & McClements, 2009) also showed that the droplet size of lipids decreased with in vitro human digestion, although the composition of digestive juices

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Fig. 1 – Schematic view of in vitro human digestion for analysis of bioavailability.

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Fig. 2 – Schematic view of in vitro human digestion for analysis of digestibility.

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Fig. 3 – Lipid digestibility in samples with different contents of beef tallow during in vitro human digestion. Error bars represent means ± SD.

was similar. Thus, a full step in vitro human digestion system, as described in this study, can be used to analyse lipid digestibility. The antioxidant activity of a rutin standard during in vitro human digestion, using the ABTS method, is presented in Fig. 5. After in vitro human digestion in the small and large intestines, the antioxidant activity values of rutin increased dramatically, whereas the antioxidant activity was not influenced by digestion in the mouth or stomach. The antioxidant activity of quercetin and chlorogenic acid standard during in vitro human digestion with enterobacteria, using ABTS method, are presented in Tables 2 and 3. Before in vitro human digestion, the antioxidant activity values of quercetin and chlorogenic acid were increased linely, and after in vitro human digestion in the small intestine and large intestines (with enterobacteria), the antioxidant activity of quercetin increased although enterobacteria did not influence the antioxidant activity, whereas the antioxidant activity of chlorogenic acid was not influenced by in vitro human digestion in the small intestine and large intestines (with enterobacteria). In this study, the increased antioxidant activity measured after in vitro human digestion by the small intestine and large

intestines (with enterobacteria) may have been due to changes in the structure resulting from different pH conditions, digestive juices, or the intervention of enterobacter bacteria. Bermúdez-Soto, Tomás-Barberán, and García-Conesa (2007) reported that most dietary polyphenols are quite stable during gastric digestion. Conversely, dietary polyphenols are highly sensitive to the mild alkaline conditions found in the small intestine. Therefore, during digestion in the duodenum, a portion of these compounds may be transformed into different structural forms with different chemical properties (Bermúdez-Soto et al., 2007). Moreover, digestive enzymes, ion strength, and temperature will also affect the phytochemicals’ structure during in vitro human digestion. In our previous studies (Hur, Joo et al., 2011; Hur, Lim et al., 2011; Hur, Park et al., 2011), we found that the aglycone quercetin was released from the glycosidic form in rutin during in vitro human digestion. In general, quercetin exhibits strong antioxidant activity by scavenging free radicals and chelating transition metal ions (Murota & Terao, 2001). Thus, the increase the conversion of rutin to quercetin by in vitro human digestion was likely one of the primary reasons for the increase in the antioxidant activity in Fig. 5. During in vitro human digestion in this study, the pH shifts dramatically

Table 2 – Effect of in vitro human digestion and enterobacteria on the ABTS scavenging activity of quercetin. Quercetin concentration (%) 0.5

1.0

2.0

55.31Bb ± 4.02 67.23Aa ± 3.68 72.31Aa ± 4.97 71.84Aa ± 2.47 74.47Aa ± 6.02

60.52Ba ± 3.52 71.23Aa ± 2.17 70.98Aa ± 4.67 74.66Aa ± 5.23 75.64Aa ± 2.97

ABTS scavenging activity (%) Before digestion After small intestine digestion In vitro digestion + E. coli 14In vitro digestion + L. casei In vitro digestion + E. coli + L. casei 1),A,B a,b,c

41.23Bc1) ± 3.26 60.78Ab ± 4.31 61.17Ab ± 4.69 62.25Ab ± 2.47 64.38Ab ± 3.97

Means ± SD with different superscripts in the same column differ significantly at p < 0.05. Means ± SD with different superscripts in the same row differ significantly at p < 0.05.

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Fig. 4 – Representative confocal images of samples with different contents of beef tallow during in vitro human digestion.

between the stomach and the small intestine, from pH 1.5 to 7.5. This change of pH is the main factor involved in the irreversible breakdown of rutin. Our previous survey found that enterobacteria such as E. coli and lactobacillus possess various activity enzymes such as amylase, dehydrogenases, glucosidase, phenolic acid decarboxylases, and phenol reductase (Hur, Lee, Kim, Choi, & Kim, 2014). Rutin contains hydrogen bond that could be cleaved by the hydrolysis enzymes possessed by the enterobacteria used in this study. However, quercetin and chlorogenic acid were not influenced by enterobacteria during in vitro human digestion. These results indicate that the enterobacteria e.g. E. coli and lactobacillus could selectively influence the structural changes of phytochemicals. Chlorogenic acid is a combination of caffeic acid and quinic acid molecules, and all three molecules have a bioavailability after chlorogenic acid ingestion (Dos Santos, Almeida, Lopes, & de Souza, 2006).

Couteau et al. (2001) reported that when the chlorogenic acid reaches the colon, there is a chance that the gut microflora can break the quinic bond and release caffeic acid. However, antioxidant activity of chlorogenic acid may be the same after break down into quinic acid and caffeic acid because quinic acid and caffeic acid also have antioxidant activity. In this study, the ability of antioxidant activity of chlorogenic acid was not different before and after in vitro human digestion. This result may be because quinic acid and caffeic acid released from chlorogenic acid by in vitro human digestion also have the antioxidant activity, whereas the antioxidant activity of quercetin was increased after in vitro human digestion in the small intestine although enterobacteria seem not to influence the changes of antioxidant activity in this study. The antioxidant activity of quercetin could be influenced by the changes of pH during in vitro human digestion in this study. Jurasekova,

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Fig. 5 – Antioxidative activity of rutin during in vitro human digestion. Error bars represent means ± SD. Bars with different letters are significantly different (P < 0.05).

Table 3 – Effect of in vitro human digestion and enterobacteria on the ABTS scavenging activity of chlorogenic acid. Chlorogenic acid concentration (%) 0.5

1.0

2.0

52.58b ± 6.20 55.23b ± 3.12 56.74b ± 2.14 51.34b ± 3.65 50.19b ± 4.25

72.36a ± 3.87 76.25a ± 4.87 70.68a ± 6.07 69.78a ± 2.65 68.07a ± 3.92

ABTS scavenging activity (%) Before digestion After small intestine digestion In vitro digestion + E. coli In vitro digestion + L. casei In vitro digestion + E. coli + L. casei 1),a,b,c

43.12c1) ± 1.56 42.56c ± 2.15 40.35c ± 3.54 39.47c ± 1.84 38.28c ± 4.19

Means ± SD with different superscripts in the same row differ significantly at p < 0.05.

Domingo, Garcia-Ramos, and Sanchez-Cortes (2014) also reported that quercetin exhibited high instability in alkaline solution, and the OH group at position C3 is an important source of instability in flavonoids (e.g. quercetin) under alkaline conditions. The free hydroxyl group on the aromatic ring is responsible for the antioxidant properties (Flora, 2009) and the quercetin contains numerous completed double bonds and hydroxyl groups that can donate electrons through resonance to stabilize the free radicals (Machlin & Bendich, 1987). Therefore, increasing the antioxidant activity of quercetin in this study may be due to the participation of hydroxyl group by in vitro human digestion. In this study, we simulated mouth, stomach, small intestine, and large intestine digestion with enterobacteria of rutin, quercetin and chlorogenic acid, and found that its antioxidant activity and lipid digestibility were influenced by in vitro human digestion. These results indicate that an in vitro human

digestion system can be used to determine the digestibility and bioavailability of food materials.

4.

Conclusion

This study presented data on two in vitro human digestion procedures for the analysis of food digestibility and bioavailability. That includes for the first time full steps in in vitro human digestion system that simulates the mouth, stomach, small intestine, and large intestine with enterobacter bacteria. Previous in vitro human digestion studies utilized only a few enzymes or digestive juices before analysis of target materials. Including all steps in an in vitro model of human digestion is the most similar to in vivo digestion. In this study, lipid digestibility and antioxidant activity were analysed using this

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full in vitro human digestion system. The results indicated that this system is useful to animal or human studies as a preliminary and rapid screen for functional food digestibility and bioavailability.

Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2055131). This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Screening of starter cultures and development of utilization technology for Korean fermented sausage, Project No: PJ010860032015)” Rural Development Administration, Republic of Korea.

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