Heating and Soaking Influence in Vitro Hindgut Fermentation of

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Heating and soaking influence the in vitro hindgut fermentation in pigs of tropical legume grains Julieta Torres, Luz Munoz, Michael Peters, and Carlos Montoya J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04751 • Publication Date (Web): 09 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Journal of Agricultural and Food Chemistry

1 Heating and soaking influence the in vitro hindgut fermentation in pigs of tropical legume grains

Julieta Torresa, Luz S. Muñoza, Michael Petersb, Carlos A. Montoyaa,c*

a

Universidad Nacional de Colombia, Dept. de Produccion Animal, Carrera 32 Chapinero,

Palmira, Colombia. b

Centro Internacional de Agricultura Tropical, AA 6713, Cali, Colombia.

c

Massey Institute of Food Science and Technology; Riddet Institute, Massey University.

Private Bag 11, 222 Palmerston North 4442, New Zealand.

* Corresponding author: Carlos A. Montoya Tel: +64 (06) 3505799 ext 84264, fax 64 (06) 3505655 Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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The effect of different thermal (raw versus autoclaving or boiling for 5 and 20 min) and

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soaking (with or without) treatments on the in vitro hindgut fermentation in pigs of undigested

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residue collected after in vitro foregut digestion of tropical legumes’ grains (Canavalia

5

brasiliensis; Lablab purpureus; pink, red and white Vigna unguiculata) were investigated.

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The undigested residue was fermented with a pig faecal inoculum to determine fermentability,

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gas and short-chain fatty acid (SCFA) productions. Soaked raw legumes increased the

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production of SCFAs (e.g. butyric acid) and fermentability, while autoclaving reduced them.

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The production of butyric acid and energy derived from SCFAs differed between legumes,

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with canavalia and lablab having the lowest and highest values, respectively. SCFAs and

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energy productions were highly related to the predicted nutrients entering the hindgut. In

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conclusion, different heating and soaking treatments can be applied to legumes to modulate

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the production of target SCFAs.

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Keywords. Tropical legume; in vitro hindgut fermentation; short-chain fatty acid production;

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heating; soaking


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INTRODUCTION Grains of raw legumes are well known for their low in vivo and in vitro foregut digestion,

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which is partially explained by high resistance of starch and protein content 1-3. This has

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encouraged research to investigate the effect of treatments such as heating to render starch

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and protein more susceptible to hydrolysis 1-2, 4-6. Some of these studies have been

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successfully able to reduce the amount of resistant starch and protein. For example, the

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amount of resistant starch of raw green peas (32%) decreased after soaking in water for 16 h

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and autoclaving for 10 min (9%) 6. However, depending on the treatment and legume used

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this reduction has been variable. For example, soaking and boiling reduced to different degree

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the resistant starch content of green (-73%) and yellow (-39%) peas 6. Previous in vitro

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studies determining the effect of heating and soaking treatments have mainly been focused on

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the foregut digestion of protein and starch 1-2, 4-6 despite that a considerable amount of

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undigested protein and starch and non-starch polysaccharides enter the hindgut. These

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undigested nutrients can be fermented by the hindgut microbial population producing

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different metabolites with either beneficial [e.g. short-chain fatty acids (SCFAs)] or

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detrimental (e.g. hydrogen sulphide) effects on the host health as have been extensively

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reviewed 7-9. For example, SCFAs have beneficial effect on local (e.g. prevention of colonic

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diseases, intestinal tissue proliferation, enhanced absorption of minerals and water) and

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systemic (e.g. reduction of blood cholesterol) 10-13 health, and can contribute up to 10% of the

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energy requirement in humans 14 and 13% in pigs as reviewed elsewhere 15. Therefore,

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understanding the fermentation of the material entering the hindgut is a very important

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undertaken to determine both the complete effect of processing (heating and/or soaking) on

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the overall nutritional value of the processed legumes and its potential effect on health.

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Surprisingly, there are only few studies 16-19 that have evaluated the effect of the treatments on

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the hindgut fermentation of the undigested material of legumes entering the hindgut. For



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example, in an in vivo study the caecal concentration of propionic acid was 27% lower for rats

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fed soaked (12 h in water) autoclaved for 20 min common beans when compared to their

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counterparts fed soaked (20 min) boiled for 70 min common beans 16. It is important to

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consider that SCFA concentrations in digesta represent only the unabsorbed SCFAs and that

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total production of SCFAs may be quite different 20. Few in vitro studies have compared

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legumes, but without considering the treatment effect 21-22. Recently however, an in vitro

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study compared the effect of heating different protein sources (insects, meats and traditional

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legume grains) on several in vitro hindgut fermentation parameters. In general, the effect of

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heating on the parameters varied between protein sources. For example, cooking chicken

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breast in the oven did not affect the production of total SCFAs, while it increased the

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production of total SCFAs for house crickets when compared to their own raw counterparts 18.

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Overall, there is a lack of information concerning the effect of processing (heating and

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soaking) legume grains on the hindgut fermentation.

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The aim of this study was to explore the effect of different combinations of heating (raw,

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autoclaving and boiling for 5 and 20 min) and soaking (unsoaked or soaked) treatments on

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several parameters of in vitro hindgut fermentation of the undigested material of tropical

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legume grains in pigs. The undigested material was collected after sequential in vitro pepsin-

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pancreatin (120-240 min) digestion. The soaking and heating treatments applied to the legume

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grains in this study were selected as previous studies have shown that they influence the

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degree of digestion of protein 4 and starch (Torres et al., unpublished). Tropical legumes were

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also selected, as legume grain models, due that little information about their complete

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nutritional value is available and they are alternative sources of protein and energy in the

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tropic 3.

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MATERIAL AND METHODS


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Material and treatment. Tropical legume grains [canavalia (Canavalia brasiliensis, CB),

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lablab (Lablab purpureus, LP) and 3 varieties of cowpea (Vigna unguiculata; pink PVU, red

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RVU and white WVU colour hulls)] were simultaneously grown at the International Centre of

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Tropical Agriculture (Cali, Colombia) and produced exclusively for this study.

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Unsoaked or soaked (overnight in distilled water at room temperature 1:3, w:v) raw,

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boiled (96 °C for 5 min, B5) and autoclaved (121 °C for 5 min, A5) legumes were in vitro

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digested with pepsin (120 min) and pancreatin (360 min) as described elsewhere 4. To

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determine the effect of extended heating time, soaked legumes were boiled and autoclaved for

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20 min (B20 and A20) prior to being in vitro digested. After in vitro digestion, the undigested

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residue was recovered using a Nylon cloth (42 µm), washed and dried before in vitro

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fermentation 3. Due to the small amount of residue recovered after each hydrolysis, the same

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hydrolysis procedure was repeated six times, and the residues were combined to ensure that

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there was sufficient residue material for chemical composition and in vitro fermentation. This

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was repeated (n=4) to create the replicates used for the statistical analysis.

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In vitro hindgut fermentation. The in vitro hindgut fermentation of the undigested legume

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residues after in vitro digestion was performed using the gas production technique described

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previously for pigs 3, 23. Briefly, a buffer inoculum was prepared mixing the fresh faeces of

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three pigs (136 g faeces in 1 L of buffer solution), which were fed a commercial diet free of

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antibiotics, to a buffer solution composed of salts and minerals ratio. The gas-test was

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performed by transferring 30 mL of inoculum into 100 mL-glass syringes containing 200 mg

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of residue for each pre-caecal hydrolysate replicate (n=4). Additionally, three syringes

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containing only the inoculum were used as blanks. Syringes were incubated at 39 °C and

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volumes of gas production were recorded over 72 h of incubation 23. At 72 h, the content of

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every syringe was centrifuged (10,000 g, 20 min, 4 °C). An aliquot of supernatant (0.8 mL)



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was taken and mixed with 250 g/L metaphosphoric acid (0.16 mL) for SCFAs analysis. The

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remaining supernatant was discarded and the pellet was frozen and freeze-dried for dry matter

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(DM) determination.

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Short-chain fatty acids analysis. Concentration of SCFAs in fermentation supernatants

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were determined as described elsewhere 3 using HPLC (CL-10A, Shimadzu) equipped with

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an organic acid analysis column (300 mm x 7.8 mm id; Aminex HPX-87H, Bio-Rad

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laboratories, Hercules, CA, USA) which was maintained at 60 °C. A mobile phase consisting

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of 6 mM H2SO4 was pumped at 0.9 mL/min isocratically and the absorbance at 210 nm was

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recorded (shimadzu SPD 10AV UV-VIS Detector, Shimadzu).

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Chemical analysis. The undigested residues were analysed for DM (method 930.15 24) and

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total starch using concentrated alkali as chaotropic agent 25. A pooled sample of undigested

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residue from the four replicates of each treatment was used to determine the ash content

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(method 942.05 24). The residues after fermentation were analysed for DM as described

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above.

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Calculations. The predicted amount of undigested DM entering the hindgut was calculated

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considering the in vitro digestion of DM, while the amount of undigested crude protein and

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starch entering the hindgut was calculated considering the in vitro degree of hydrolysis of

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protein 4 and starch (Torres, Munoz, Peters, & Montoya, unpublished) as follows:

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DigestionDM (%) = (BeforeDM – AfterDM) / BeforeDM x 100

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UndigestedDM (mg/g DM) = [1000 – (1000 x DigestionDM) / 100]

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Undigestedcrude protein or starch (mg/g DM) = [1000 – (1000 x DigestionProtein or starch) / 100]


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Gas accumulation curves recorded over 72 h were modelled using the mathematical model proposed by France et al. 26: G = 0,

if 0 < t < L

= Gf (1 – exp [-(b(t – L) + c (√t – √L))]), if t ≥ L

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where G (mL/g DM incubated) represents the gas accumulation over time, Gf (mL/g DM

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incubated) the maximum gas volume for t = ∞ and L (h) the lag time before the fermentation

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starts. The constants b (h-1) and c (h-1/2) determine the fractional rate of degradation of the

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substrate µ (h-1), which is postulated to vary with time as follows:

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µ = b + (c / 2√t), if t ≥ L

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The kinetics parameters (Gf, µt=T/2 and T/2) were compared in the statistical analysis, T/2 is

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the time to half asymptote when G= Gf /2. At this time, the rate of gas production is in a linear

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phase, near its maximum.

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The in vitro hindgut fermentability of DM was calculated as follows: Hindgut fermentabilityDM (%) = (BeforeDM – [AfterDM – BlankDM]) / BeforeDM * 100

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Some of the parameters of fermentation were recalculated per weight (gram) of initial

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legume grains. The predicted fermented and digested DM content as well as the production of

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SCFAs and total gas per gram of legumes were calculated as follows:

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FermentedDM (mg/g DM initial legume) = UndigestedDM x Hindgut fermentabilityDM /

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100

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DigestedDM (mg/g DM initial legume) = (1000 x DigestionDM / 100) + FermentedDM

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SCFA (mg/g DM initial legume) = UndigestedDM x SCFA concentration (mg/g

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undigested incubated material) / 1000



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Total gas (mL/g DM initial legume) = [1000 – (1000 x DigestionDM) / 100] x gas

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production (mL/g undigested incubated material) / 1000

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Statistical analysis. The statistical analyses were performed using the Mixed Model

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procedure of SAS (SAS/STAT Version 9.4, SAS Institute Inc., Cary, NC, USA). To examine

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the effect of legume (CB, LP, PVU, RVU and WVU), heating (raw, E5 and A5), soaking

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(with and without) and all their interactions on the variables of fermentation, a complete

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randomised block factorial treatment arrangement (5 x 3 x 2) was firstly performed, using the

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inoculum as a random effect. For all the tested variables, there was no significant effect of the

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inoculum, therefore, the random effect was removed from the final model. To examine the

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effect of extended cooking time (i.e. soaked B5 vs. soaked B20 and soaked A5 vs. soaked

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A20) on the studied variables, including the undigested residue (e.g. undigested starch), a

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completely randomised design was conducted to compare all the treatments (i.e. raw, B5,

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B20, A5, A20 for soaked and raw, B5 and A5 for unsoaked legumes).

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The model diagnostics (e.g. normal distribution, equal variance across treatments) of each

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variable were tested using the ODS Graphics options of SAS prior comparing the fitted

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means. When the F-value of the analysis of variance was significant (P < 0.05), the fitted

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means were compared using the adjusted Tukey tests. When a triple interaction was

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significant, heating and soaking treatments combinations were compared to determine their

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effect within each legume. In contrast, when a double interaction or main factor were

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significant, all treatments were compared.

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Regression analyses were carried to determine the relationship between the production of

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SCFAs and the energy derived from SCFAs with the predicted amount of nutrients entering

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the hindgut using the PROC REG of SAS. The regression analysis did not include intercept as

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the mean value was expected to be cero, after correcting for the blank, if there are no nutrients


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(i.e. substrate) in the fermentation medium. The nutrients considered in the regression were

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undigested protein, undigested starch and non-defined material (OM – undigested protein –

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undigested starch). The non-defined fraction was assumed to be mainly composed of

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hemicellulose, cellulose and lignin as the amount of lipids is relative low in the legumes (15-

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55 mg/g DM legume; 3), and expected to be highly digested before entering the hindgut and

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the fermentation of lipids and SCFA production, if any, is very low.

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RESULTS

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Undigested residue collected after in vitro pepsin-pancreatin digestion. The undigested

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residue collected after digesting in vitro one gram of legumes’ DM with pepsin-pancreatin

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(120-240 min) varied from 613 mg for B5 unsoaked LP to 313 mg for A20 soaked WVU

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(Table 1). Similarly, high differences were observed within each legume for DM, resistant

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starch and resistant protein (e.g., for PVU, there was a difference of 228 mg DM between the

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most and the least resistant treatment) (P < 0.001). CB and LP had more undigested DM and

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protein (508 and 114 mg, on average across all treatments for CB and LP, respectively) than

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cowpeas (400 and 90 mg, on average across treatments and all cowpeas, respectively). In

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contrast, the amount of undigested starch was similar among all legumes (215 mg for CB and

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LP and 224 mg for all cowpeas). These differences explain the variability in the composition

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of the undigested residue. For example, the composition of starch varied from 35 to 54% for

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undigested CB. The composition also varied between legumes. For example, the average

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composition of starch for all undigested LP was 42%, while it was 61% for all undigested

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WVU.

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Hindgut fermentation of undigested residue. The variables of hindgut fermentation

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reported in this section were estimated considering the predicted amount of undigested



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material entering the hindgut after digesting one gram of initial legume (Table 1). Extending

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the boiling and autoclaving time from 5 to 20 min did not affect any of the response variable

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tested (data not shown).

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There was a significant interaction between legume, heating and soaking on the predicted

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fermented DM and gas production (P < 0.001) (Table 2 and Figure 1). Soaked raw CB and

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PVU, and unsoaked B5 LP had higher fermented DM content and gas production than their

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unsoaked raw and soaked A5 counterparts (P < 0.05). In addition, soaked raw PVU and

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unsoaked B5 WVU had higher DM fermented content and gas production than its soaked B5

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(except gas production for WVU) and unsoaked A5 counterparts. Other specific differences

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were observed within each legume for both DM fermented content and gas production. For

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example, soaked A5 CB had lower gas production when compared to the other treatments (P

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< 0.05), while the difference between soaked A5 and other treatments varied for LP

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(unsoaked B5 and A5 and soaked raw and B5), PVU (soaked raw and unsoaked B5), RVU

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(unsoaked raw and A5 and soaked raw) and WVU (unsoaked B5) (P < 0.05).

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There was a significant interaction of heating and soaking on the production of acetic,

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propionic and butyric acids and on the predicted energy derived from SCFAs (P < 0.001). The

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production of these SCFAs and energy was higher for the soaked raw and unsoaked B5

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legumes when compared to the unsoaked raw legumes (P < 0.05). In addition, for butyric

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acid, these treatments were also higher than soaked B5 and A5 and unsoaked A5 (P < 0.05). A

210

significant interaction between legume and heating was observed for the production of acetic

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and propionic acids (P < 0.05). B5 LP had higher production of acetic and propionic acids

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than B5 PVU, and A5 LP had highest production of acetic acid than A5 RVU and CB.

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Unsoaked LP had higher acetic acid production than the other unsoaked legumes (P < 0.05 for

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the interaction legume and soaking). There was an effect of the legume on the digested DM,

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butyric acid production and energy production (P < 0.05). The digested DM content was, on


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average, 5% higher for the cowpeas when compared to the CB and LP (P < 0.05). The

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production of butyric acid and energy content was higher for LP when compared to CB (P
131 g/kg DM) compared to cowpeas (

Heating and Soaking Influence in Vitro Hindgut Fermentation of

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