In-vitro digestion models: a critical review for human and fish and a protocol for in-vitro digestion in fish

Figures & data

Figure 1. Current review articles on in-vitro gastrointestinal digestion of humans, ruminants, and aquatic species for several different topics. Proximity of the references to different topics indicates the salient feature of the review articles encompassing multiple topics. Numbered references are listed in Supplementary References

Table 1. Dynamic in-vitro gastrointestinal model

Code*		Name	Reference
1	Mono-compartmental	Dynamic Gastric Model (DGM)	[153]
2		Human Gastric Simulator (HGS) v1.0 and v.2.0	[51,154]
3		Gastric Simulation Model (GSM)	[155]
4		Gastric Digestion Simulator (GDS)	[156]
5		In-vitro Mechanical Gastric System (IMGS)	[157]
6		Dynamic In-vitro Human Stomach (DIVHS)	[158,159]
7		Artificial Colon (ARCOL)	[160]
8		Dynamic Colon Model (DCM)	[161]
9	Multi-compartmental	Dynamic gastrointestinal digester (DIDGI®)	[59]
10		TNO gastrointestinal model (TIM-1, TIM-2, tiny-TIM)	[60,162]
11		Simulator of the Human Intestinal Microbial Ecosystem (SHIME)	[163]
12		Simulator of the Gastrointestinal Tract (SIMGI)	[164]
13		Engineered Stomach And Small Intestine Model (ESIN)	[165]
14		SimuGIT	[39]
15		Membrane bioreactor with dialysis cell	[102,103]
*The references in parentheses throughout features of dynamic digestion models indicate the code number in this table

Figure 2. Block flow diagram of the human gastrointestinal digestion process modeled as various unit operations adapted from Bornhorst et al. [50], with the simulation scope of several current developed dynamic in-vitro gastrointestinal models

Table 2. Recent In-vitro digestibility assay performed on fish

pH	E:S ratio (U/mg protein)	T°C	Time (min)	Ingredient	Measured parameter	Species	Note	Ref
2 & 8	-	25	60 & 60	24 protein ingredients	DH	Onchorychus mykiss, cobia, tilapia	Two stage pH stat. Compare caged and farm tilapia	[136]
2 & 8	12.5 & 16.7	37	60 & 180	Fish and soybean meal	Peptide fraction, FAA Residual protein	Pacific bluefin tuna	Two stage digestion, with commercial pepsin and fish crude intestinal extract	[146]
2 & 8	12.5 & 16.7	37	60 & 180	Fish and poultry meal	Peptide fraction, FAA Residual protein	Pacific bluefin tuna	Two stage digestion, with commercial pepsin and fish crude intestinal extract	[147]
8		37	60	Soybean meal on different composition with fish meal	DH, pHdrop, zymography, APD	Mystus nemurus	One stage intestinal digestion with 4 enzyme system: fish crude extract and 3 mammalian enzymes mixture	[84]
3 & 8	24.125 & 2.875	37	15 & 45	13 protein ingredients	DH FAA Zymography	Lutjanus guttatus	One and Two stage pH stat. Comparison between two fish developmental stage	[166]
3.5 & 8	6.25	37	15 & 45	Soybean concentrate on 7 different level	DH, Nutrient retention (N, P), Invivo	Totaba magnodali		[83]
8	nd	25	60 min	Nannochloropsis granulata	DH	Oncorhynchus mykiss Litopenaeus vannamei	Evaluate different treatment by supercritical CO2 extraction	[167]
3.5–6.5 & 6.5–8.5	125–500 & 50–200	nd.	240–480 & 480–640	Comerical feed	FAA Reducing sugar	Sparus aurata	Two stage membrane reactor, with fish crude extract	[23]
3.5 & nd.	nd.	37	15	11 protein ingredients	DH, FAA APD	Centropomus undecimalis	Two stage pH stat	[87]
8	-	25	60	10 protein ingredients	DH, invivo	Acipenser baeri	One step intestine stage,	[168]
3.4&7.8	256 & 1460	28	90 acid	Poultry by product on several different grade	Pepsin assay FAA APD	Lates calcarifer	Two stage membrane reactor, with commercial pepsin and fish crude intestinal extract	[169]
9	2.5	25	90	4 microalgae	SDS page, FAA	Senegalese sole	One stage intestinal digestion	[170]
2 & 7	nd	37	120 & 120	Three commercial diet	In vivo assessment Residual protein Minerals (P, Fe, Zn)	P.mesopotamicus	Two step digestion with final product dialysis separation. Added bile extract	[16]
3.5 & 8	-	-	15&45	16 protein ingredients	DH, FAA	Amphilophus trimaculatus	Intestinal extract	[90]
2.1 & 8	-	-	60 &240	Two mixed diet	Minerals	Onchorychus mykiss	Intestinal extract Compared with and without inorganic supplementation	[171]
nd	13.02 &11.27	37	nd	Soybean β-conglycinin	SDS page, FAA, qPCR,	Ctenopharyngodon idella	Stomach and pyloric extract	[143]
8	nd.	37	10	Fish, soybean, and squid meal	pH drop	Mystus nemurus	One stage intestinal digestion with 4 enzyme system: fish crude extract and 3 mammalian enzymes mixture	[85]
The notation ‘&’ signifies pH, ES ratio, and duration of gastric and intestinal phase. Data without notation ‘&’ indicate only single intestinal phase. nd. = not determined FAA for free amino acids; DH for pH-stat degree of hydrolysis; APD for in-vivo based apparent protein digestibility.

Table 3. In-vitro digestibility assay on different species of fish. Coded references are listed in Supplementary references

Species	Reference	Count
Trout	5 6 9 16 21 22 23 24 30 32 33 38 40 41 47 55	16
Larvae	8 11 12 14 28	5
Bream	26 31 34 36 48	5
Salmo salar	10 19 25 20 39	5
Tuna	7 35 42 43	4
Tilapia	3 18 37 41	4
Carp	1 2 32 56	4
Catfish	44 57	2
Flatfish	4 13	2
Other cichilid	54	1
Pacu	53	1
Barramundi	51	1
Siberian Sturgeon	50	1
Snook	49	1
Totoaba	46	1
Snapper	45	1
Cobia	41	1
Meagre	37	1
European bass	37	1
Cod	27	1

Table 4. In-vitro digestibility assay on different protein ingredient. Coded references are listed in Supplementary references

Feed	Reference	Count
Fish meal	2 5 6 9 10 11 13 16 18 19 20 23 25 27 28 30 33 34 35 36 38 41 42 43 45 50 54 57	28
Soybean meal	2 5 9 13 16 18 19 27 28 32 34 36 38 41 42 44 45 46 49 50 54 56 57	23
Mixed diet	7 11 12 14 15 17 20 26 29 31 37 39 40 48 53 55	16
Casein	2 9 11 13 16 24 28 34 36 45 54	11
Poultry (byproduct) meal	9 21 27 41 43 45 49 50 51 54	10
Corn gluten	5 9 13 27 34 41 45 49 50 54	10
Wheat gluten	9 16 27 28 41 45 49 50 54	9
Meat meal	16 18 19 34 45 50 54	7
Squid meal	11 13 28 45 54 57	6
Rapeseed meal	27 41 45 49 50	5
Other seeds*	2 18 19 27 41	5
High-starch meal*	5 9 18 41 54	5
Hydrolyzed feather meal	9 27 41 50 54	5
Blood meal	34 41 50 54	4
Krill meal	13 27 28 45	4
Algae	1 47 52	3
Single-cell protein	1 22 50	3
Shrimp meal	18 27 54	3
Peameal	13 27 32	3
Zooplankton	8 28	2
Haemogoblin	45 54	2
Dried whey	11 49	2
Mussel and crab meal	13 27	2
Lupin meal	27 34	2

Figure 3. Employed operating condition throughout 16 reviewed studies of in-vitro digestion for Onchorychus mykiss.

Figure 4. (a) Soybean (b) fish meal in-vitro protein digestibility using different analytical method

Table

Acid (pH ≤ 5):	$\mathit{DH}=100\mathit{x}\frac{\mathit{V}\times \mathit{N}}{\mathit{m}\times {\mathit{h}}_{\mathit{tot}}}\times \frac{1}{1-{\mathit{\alpha }}_{\mathit{COOH}}}$	${\alpha }_{\mathit{COOH}}=\frac{{10}^{pH-p{K}_a^{COOH}}}{1+{10}^{pH-p{K}_a^{COOH}}}$	(1)
Neutral/Basic (pH ≥ 7) :	$\mathit{DH}=100\mathit{x}\frac{\mathit{V}\times \mathit{N}}{\mathit{m}\times {\mathit{h}}_{\mathit{tot}}}\times \frac{1}{{\alpha }_{N{H}_2}}$	${\alpha }_{N{H}_2}=\frac{{10}^{pH-p{K}_a^{N{H}_2}}}{1+{10}^{pH-p{K}_a^{N{H}_2}}}$	(2)

Table 5. Correlation of pH-stat method with in-vivo protein digestibility

Species	Correlation Equation	R^2	Notes	Reference
Rainbow trout	y = 1.98 x + 39.4	0.64	Mammalian enzymes	[98]
	y = 1.67 x + 41.2	0.82	Trout enzymes
Gadoids	y = 1.30x + 70.57	0.34	Recalculated from the provided data	[119]
Catfish	y = 1.33x + 29.28	0.930	Fish enzymes	[84]
	y = 1.05x + 56.21	0.853	Single enzyme
	y = 1.07x + 33.56	0.895	Three enzyme mixture
	y = 1.54x + −17.66	0.924	Four 4 enzyme mixture
Totaba magnodali	y = 12.59x – 1.8018	0.792	Recalculated from the data	[83]
Siberan Sturgeon	y = 10.62x + 32.08	0.879	Animal based meal	[168]
	y = 6.627x + 43.531	0.967	Plant based meal
	y = 1.30x + 70.57	0.34	Overall
Snook, Centropomus undecimalis	Y = 1.8968 x + 88.52	0.73	Fish digestive enzyme for 11 ingredient	[87]

Figure 5. Suggested protocol for in-vitro digestion study for fish. *the total volume and amount of digestive fluid added could be scaled with the amount of sample. **the digestive fluid contains the amount of enzymes determined during the preliminary study

Vardakou M, Mercuri A, Barker SA, et al. Achieving antral grinding forces in biorelevant in vitro models: comparing the USP dissolution apparatus II and the dynamic gastric model with human in vivo data. AAPS PharmSciTech. 2011;12(2):620–626.

 PubMed Web of Science ®Google Scholar

Mennah-Govela YA, Bornhorst GM. Breakdown mechanisms of whey protein gels during dynamic in vitro gastric digestion. Food Funct. 2021;12(5):2112–2125.

 PubMed Web of Science ®Google Scholar

Kong F, Singh RP. A human gastric simulator (HGS) to study food digestion in human stomach. J Food Sci. 2010;75(9):E627–35.

 PubMed Web of Science ®Google Scholar

Li Y, Fortner L, Kong F. Development of a Gastric Simulation Model (GSM) incorporating gastric geometry and peristalsis for food digestion study. Food Res Int. 2019;125. https://doi.org/10.1016/j.foodres.2019.108598

 Web of Science ®Google Scholar

Kozu H, Nakata Y, Nakajima M, et al. Development of a Human Gastric Digestion Simulator Equipped with Peristalsis Function for the Direct Observation and Analysis of the Food Digestion Process. Food Sci Technol Res. 2014;20:225–233.

 Web of Science ®Google Scholar

Barros L, Retamal C, Torres H, et al. Development of an in vitro mechanical gastric system (IMGS) with realistic peristalsis to assess lipid digestibility. Food Res Int. 2016;90:216–225.

 PubMed Web of Science ®Google Scholar

Wang J, Wu P, Liu M, et al. An advanced near real dynamic in vitro human stomach system to study gastric digestion and emptying of beef stew and cooked rice. Food Funct. 2019;10:2914–2925.

 PubMed Web of Science ®Google Scholar

Chen L, Wu X, Chen XD. Comparison between the digestive behaviors of a new in vitro rat soft stomach model with that of the in vivo experimentation on living rats – motility and morphological influences. J Food Eng. 2013;117:183–192.

 Web of Science ®Google Scholar

Gerard-Champod M, Blanquet-Diot S, Cardot JM, et al. Development and validation of a continuous in vitro system reproducing some biotic and abiotic factors of the veal calf intestine. Appl Environ Microbiol. 2010;76:5592–5600.

 PubMed Web of Science ®Google Scholar

Stamatopoulos K, Batchelor HK, Simmons MJH. Dissolution profile of theophylline modified release tablets, using a biorelevant Dynamic Colon Model (DCM). Eur J Pharm Biopharm. 2016;108:9–17.

 PubMed Web of Science ®Google Scholar

Ménard O, Cattenoz T, Guillemin H, et al. Validation of a new in vitro dynamic system to simulate infant digestion. Food Chem. 2014;145:1039–1045.

 PubMed Web of Science ®Google Scholar

Minekus M, Marteau P, Havenaar R, et al. A multicompartmental dynamic computer-controlled model simulating the stomach and small intestine. ATLA Altern Lab Anim. Altern Lab Anim. 1995;23(2):197–209.

 Web of Science ®Google Scholar

Minekus M, Smeets-Peeters M, Bernalier A, et al. A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products,”. Appl Microbiol Biotechnol. 1999;53:108–114.

 PubMed Web of Science ®Google Scholar

Molly K, Vande Woestyne M, Verstraete W. Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem. Appl Microbiol Biotechnol. 1993;39:254–258.

 PubMed Web of Science ®Google Scholar

Barroso E, Cueva C, Peláez C, et al. Development of human colonic microbiota in the computer-controlled dynamic SIMulator of the GastroIntestinal tract SIMGI. LWT - Food Sc Technol. 2015;61:283–289.

 Web of Science ®Google Scholar

Guerra A, Denis S, Le Goff O, et al. Development and validation of a new dynamic computer-controlled model of the human stomach and small intestine. Biotechnol Bioeng. 2016;113:1325–1335.

 PubMed Web of Science ®Google Scholar

Rivas-Montoya E, Miguel Ochando-Pulido J, Manuel López-Romero J, et al. Application of a novel gastrointestinal tract simulator system based on a membrane bioreactor (SimuGIT) to study the stomach tolerance and effective delivery enhancement of nanoencapsulated macelignan. Chem Eng Sci. 2016;140:104–113.

 Web of Science ®Google Scholar

Savoie L, Gauthier SF. Dialysis Cell for the In Vitro Measurement of Protein Digestibility. J Food Sci. 1986;51(2):494–498.

 Web of Science ®Google Scholar

Gauthier SF, Vachon C, Jones JD, et al. Assessment of Protein Digestibility by In Vitro Enzymatic Hydrolysis with Simultaneous Dialysis. Journal Nutr. 1982;112(9):1718–1725.

 PubMed Web of Science ®Google Scholar

Bornhorst GM, Gouseti O, Wickham MSJ, et al. Engineering Digestion: multiscale Processes of Food Digestion. J Food Sci. 2016;81(3):R534–43.

 PubMed Web of Science ®Google Scholar

Yasumaru F, Lemos D. Species specific in vitro protein digestion (pH-stat) for fish: method development and application for juvenile rainbow trout (Oncorhynchus mykiss), cobia (Rachycentron canadum), and Nile tilapia (Oreochromis niloticus). Aquaculture. 2014;426-427:74–84.

 Web of Science ®Google Scholar

Román-Gavilanes AI, Martínez-Montaño E, Viana MT. Comparative Characterization of Enzymatic Digestion from Fish and Soybean Meal from Simulated Digestive Process of Pacific Bluefin Tuna,Thunnus orientalis. J World Aquacult Soc. 2015;46(4):409–420.

 Web of Science ®Google Scholar

Castillo-Lopez E, Espinoza-Villegas RE, Viana MT. In vitro digestion comparison from fish and poultry by-product meals from simulated digestive process at different times of the Pacific Bluefin tuna, Thunnus orientalis. Aquaculture. 2016;458:187–194.

 Web of Science ®Google Scholar

Rahmah S, Aliyu-Paiko M, Hashim R. In vivoandin vitroprotein digestibility in juvenile bagrid catfishMystus nemurus(Cuvier and Valenciennes 1840) fed soybean meal-based diets. Aquacult Res. 2016;47(5):1392–1401.

 Google Scholar

Peña E, Hernández C, Ibarra-Castro L, et al. In vitro protein digestibility of different grow-out stages of spotted rose snapper (Lutjanus guttatus, Steindachner, 1869). Aquaculture Nutr. 2017;23:1204–1215.

 Web of Science ®Google Scholar

Trejo-Escamilla I, Galaviz MA, Flores-Ibarra M, et al. Replacement of fishmeal by soya protein concentrate in the diets ofTotoaba macdonaldi(Gilbert, 1890) juveniles: effect on the growth performance,in vitrodigestibility, digestive enzymes and the haematological and biochemistry parameters. Aquacult Res. 2017;48(8):4038–4057.

 Web of Science ®Google Scholar

Tibbetts SM, Yasumaru F, Lemos D. In vitro prediction of digestible protein content of marine microalgae (Nannochloropsis granulata) meals for Pacific white shrimp (Litopenaeus vannamei) and rainbow trout (Oncorhynchus mykiss). Algal Res. 2017;21:76–80.

 Google Scholar

Gilannejad N, Martinez-Rodriguez G, Yufera M, et al. Modelling digestive hydrolysis of nutrients in fish using factorial designs and desirability function. PLoS One. 2018;13(11):e0206556.

 PubMed Web of Science ®Google Scholar

Lemus I, Maldonado C, Cuzon G, et al. In VitroandIn VivoFeedstuff Digestibility for Snook,Centropomus undecimalis, Juveniles. J World Aquacult Soc. 2018;49(1):205–215.

 Web of Science ®Google Scholar

Mirzakhani MK, Abedian Kenari A, Motamedzadegan A. Prediction of apparent protein digestibility by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for Siberian sturgeon (Acipenser baeri, Brandt 1869). Aquaculture. 2018;496:73–78.

 Web of Science ®Google Scholar

Lewis MJ, Francis DS, Blyth D, et al. A comparison of in-vivo and in-vitro methods for assessing the digestibility of poultry by-product meals using barramundi (Lates calcarifer); impacts of cooking temperature and raw material freshness. Aquaculture. 2019;498:187–200.

 Web of Science ®Google Scholar

Vizcaíno AJ, Sáez MI, Martínez TF, et al. Differential hydrolysis of proteins of four microalgae by the digestive enzymes of gilthead sea bream and Senegalese sole. Algal Res. 2019;37:145–153.

 Google Scholar

Cian RE, Bacchetta C, Cazenave J, et al. In vitro assays predicts mineral retention and apparent protein digestibility of different fish feed measured using a juvenile P. mesopotamicusmodel. Aquaculture Research. 2018;49(6):2267–2277.

 Web of Science ®Google Scholar

Toledo‐Solís FJ, Martínez‐García R, Díaz M, et al. Potential bioavailability of protein and lipids in feed ingredients for the three-spot cichlidAmphilophus trimaculatus: an in vitro assessment. Aquacult Res. 2020;51(7):2913–2925.

 Web of Science ®Google Scholar

Silva MS, Prabhu PAJ, Ornsrud R, et al. In vitro digestion method to evaluate solubility of dietary zinc, selenium and manganese in salmonid diets. J Trace Elem Med Biol. 2020;57:126418.

 PubMed Web of Science ®Google Scholar

Duan X-D, Feng L, Jiang W-D, et al. The dynamic process of dietary soybean β-conglycinin in digestion, absorption, and metabolism among different intestinal segments in grass carp (Ctenopharyngodon idella). Fish Physiology and Biochemistry. 2020;46(4):1361–1374.

 PubMed Web of Science ®Google Scholar

Rahmah S, Hashim R, El‐Sayed A-FM. Digestive proteases and in vitro protein digestibility in bagrid catfishMystus nemurus(Cuvier and Valenciennes 1840). Aquacult Res. 2020;51(11):4613–4622.

 Web of Science ®Google Scholar

Dimes LE, Haard NF. Estimation of protein digestibility - I. Development of an in vitro method for estimating protein digestibility in salmonids (Salmon gairdneri). Comp Biochem Physiol. 1994;108:249–362.

 Google Scholar

Tibbetts SM, Verreth JAJ, Lall SP. In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 2. In vitro protein digestibility of common and alternative feed ingredients. Aquaculture. 2011;319(3–4):407–416.

 Web of Science ®Google Scholar