Confirmation of Cage Effect and Prebiotic Production Potential of a -Mannanase, with SBM as Substrate Using Microscopy and Wet Chemistry

In vitro assays were carried out to investigate the solubilization of cell walls and generation of mannan oligosaccharides of a b-mannanase-containing commercial product on SBM. Using commercial dosages of the b-mannanase (500 g per ton of feed) cell wall degradation of mannan in SBM cell walls was visualized and an increase in reducing ends (0.12±0.02 mg/mL) and the generation of mannan oligosaccharides of degree of polymerization 2 and 4 (22.9±3.2 mg/L and 398.8±25.4 mg/L) were also measured using HPLC. Mannan, which is H-bonded to cellulose and xyloglucan, was solubilized using a single monocomponent enzyme, allowing for visualization of the disintegration of the entire SBM cell wall structure. This work is the first of its kind using strictly commercial dosage levels of enzyme for evaluating efficacy of the same microscopically. These data confirm the hypothesis that there most likely is a need for only a single relevant NSP enzyme targeting its specific substrate, independent of the concentration of the latter within the complex polysaccharide matrix in the plant cell wall to experience the beneficial effects of the enzyme both in vitro and in vivo. An analogy to compare our data would be destruction of the foundation (mannan) of a building or a bridge (soybean cell wall) which would inevitably lead to dismantling or demolition the entire building or bridge.


Introduction
Non-starch polysaccharide (NSP) degrading enzymes are added to help increase the metabolizable energy in fiber-containing monogastric diets. Many studies in the past have been conducted to improve the understanding of the mode of action of NSPases or fiber-degrading enzymes for use in animal feed. The three main modes of action postulated are (1) reduction in viscosity (2) destruction of cell walls by solubilization of specific polymers which make up the cell walls to release the starch/protein encapsulated within and (3) Generation of soluble oligosaccharides during cell wall destruction which have prebiotic effects (Bedford, 2018). It has been reported that the addition of NSP-degrading enzymes such as b-mannanases and xylanases improve animal growth performance by improving nutrient digestibility and increasing the levels of good gut bacteria (Mehri et al., 2010;Lan et al., 2017). However, the mechanisms by which NSP-degrading enzymes exert these effects are still under debate. A recent study showed that there was increased ileal expression of MUCIN 2 in pigs fed high-fiber diets with no changes in goblet cell number. Addition of NSP-degrading enzymes to the diet modulated the local immune profile of the ileum, increasing IL-1b expression and concentration and decreasing IL-4, IL-17A and IL-11 concentration (Ferrandis et al., 2018). It is also claimed NSPases can be considered as tools to train the microbiome to be better able to degrade fiber more effectively especially when they are accompanied initially with oligosaccharides. By breaking down the fiber they produce small amounts of oligosaccharides which have prebiotic effect, and they are postulated to play a role in signaling to the gut microbial population present to develop a higher fiber-degrading capacity (Bedford, 2018).
In this work using a b-mannanase, a NSPase product we have endeavored to study in vitro, where we clarify the two postulated modes of action of NSPases: namely cell wall degradation or decaging effect and generation of prebiotic oligosaccharides. One of the key anti-nutritional factors (ANF) in plant cell walls is b-mannan, an NSP fiber component. It is composed of a backbone of mannose and glucose units in β-1,4-linkages (Moreira & Filho, 2008), and may also be linked to galactose residues by α-1,6 linkage which increases its water solubility. The mannan structure in SBM is galactomannan having an average galactose to mannose ratio of 1:1.8 (Hsiao et al., 2006). Mannan is H-bonded to cellulose and xyloglucan and its interaction with cellulose resembles that of xyloglucan (Schröder et al., 2009).
One way of trying to understand the positive in vivo effects of NSPases, is to study the effects of the same in vitro. But cell wall breakdown theory with the current published in vitro data is also being challenged (Bedford, 2018). One of the analytical tools used is microscopy to visualize the enzyme's effect on feed substrates and understand cell wall breakdown theory as well as the composition and cell wall architecture of some raw materials (Pedersen et al., 2017). Commercial dosages of enzyme showed very little cell wall degradation (Tervila-Wilo et al., 1996), but cell wall degradation was clearly visualized when very high amounts of enzyme between 100-500X beyond commercial in-vivo dosages were used (Pedersen et al., 2017), which may not be economically relevant or be able to explain good in vivo effects (Le et al., 2013;Pedersen et al., 2017;Ravn et al., 2016). The objective of current study was to use commercially relevant dosages of the current b-mannanase product on SBM and prove that the hypothesis of cell wall degradation is true; to show the effective solubilization/breakdown of cell walls by an NSPase enzyme in vitro and production of oligosaccharides in order to understand positive in vivo results obtained in poultry, pigs and cattle (Ferreira et al., 2016;Kim et al., 2017;Park et al., 2019;Tewoldebrhan et al., 2017).

Chemicals
All chemicals used were from Sigma-Aldrich (USA), mannan oligosaccharides were purchased from Megazyme International, Ireland, antibodies were from Plant Probes, England and Thermo-Fisher Scientific.

Enzyme Product
CTCzyme obtained from CTCBio Inc. S. Korea, is a commercial monocomponent β-mannanase product produced by B. subtilis with a declaration of 800,000 U/kg mannanase. One enzyme unit is defined as generation of 1μmole of reducing sugar per min at pH6.0 and 50 °C using locust bean gum as substrate.

Enzyme Treatment of SBM Before Microscopy
Samples of SBM were incubated individually at pH 5 either without enzyme (control) or with the addition of either CTCzyme at commercial (1X) or 10X commercial dosages. In short, 1 g each of SBM samples was incubated with 0.1 M sodium acetate buffer pH 5 alone (control) or commercial β-mannanase product CTCzyme in the same buffer at 40 °C for 4 h with stirring at 500 rpm. After incubation, the samples were then centrifuged at 2500 × g for 10 min. The pellets obtained were washed once with MiliQ water, recentrifuged and dried overnight at 60 °C and used for microscopy analyses.

Embedding and Sectioning
Controls and enzyme treated SBM pellets were embedded in paraffin. Prior to the embedding procedure, a protocol according to Ravn et al. (2016) was followed whereby the control and enzyme treated SBM samples were first fixed in Karnovsky's fixative, washed progressively in 0.1 M cacodylate buffer pH 7.3 and demineralized water and dehydrated increasingly higher concentrations of ethanol (from 50-99%) before infiltration in melted paraffin at 60 °C using Histochoice clearing agent (Sigma-Aldrich). Approximately 7-10 μm thick sections of paraffin-embedded samples were sectioned on a Leica Reichert-Jung 2030 microtome.

Immunochemistry for Visualization of Samples Embedded in Paraffin and Staining With Calcofluor
The procedure was carried out as described in Pedersen et al. (2017). A 5% skimmed milk solution (from Sigma-Aldrich) in 1× PBS was used to block the paraffin sectioned SBM samples of control (Incubated with buffer alone) and b-mannanase-treated enzyme product for 1 h. Sections were then washed in PBS buffer followed by incubation for 1 h with 10-fold dilutions of the rat monoclonal antibody (LM21) diluted in the skimmed milk-PBS buffer solution. Samples were subsequently incubated for 1½ h with anti-rat IgG linked to an Alexa-555 fluorophore and washed in PBS buffer. The anti-fading agent Citiflour AF1(Agar Scientific, UK) was used to avoid fluorescence signal from bleaching. A negative control labelling was also carried out using only the secondary antibody. Slides were also stained for 2 minutes with a drop of Calcofluor stain as a counterstain to stain b-glucan linkages such as cellulose and xyloglucan in the presence and absence of LM21-the antibody to b-mannan.

Confocal Immunofluorescence Microscopy
A Confocal Laser Scanning Microscope (Olympus, Japan), was used to obtain confocal images of immunolabeled SBM samples. A 20x water-immersion objective was used for all images. An excitation laser line at 561 nm (green) and an emission spectrum from 590 to 630 nm designated red in the software was used to monitor the Alexa-555 fluorescein-signal from the immunolabelling. An excitation laser line at 488 nm (blue) and an emission spectrum from 500 to 540 nm designated green in the software was used to monitor the autofluorescence from protein globules. For the calcofluor staining, an excitation laser line of 405 nm and an emission spectrum from 430-480 nm, designated as blue in the software was used to monitor the fluorescence of the cell walls. Multicolor super resolution images in 3 different channels were simultaneously obtained.

Enzyme Reaction for Wet Chemistry Analyses
SBM was used at a concentration of 1%. The experiments were carried out in triplicate. In short, 3 g of SBM was mixed with 300 mL of 0.02 M phosphate buffer at pH 6.0 and 300 u/mL enzyme (commercial dosage) and incubated for a total of 180 mins at 50 °C with stirring. Samples were taken at several time intervals for reducing end assays and at the end of the reaction for analyses of oligosaccharides. After the incubation time, the reaction mixture was centrifuged, and supernatant were tested for reducing ends and were also run on an HPLC column (SUGAR SP0810, Shodex, Japan) for detection of MOS as described.

Reducing ends assay
DNS assay was carried out using SBM (1.0% w/v) as substrate. SBM was mixed with 20 mM sodium-phosphate buffer (pH 6.0) by stirring constantly at 50 °C. An aliquot of 300 U/ml enzyme was incubated with the SBM substrate at 50 °C up to 120 min. The reaction was stopped by the addition of DNS reagent and subsequent boiling for 5 min and reducing sugar was measured at 540 nm against the blank. The reducing sugars released were then determined against a standard curve obtained with mannose (Sigma-Aldrich) (Miller, 1959;Garriga et al., 2017).

HPLC
Mannan oligosaccharide concentrations were analyzed by high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) using an Agilent 1200 system (Agilent, CA) with SUGAR SP0810 analytical column (8 × 300 mm) at 80 °C. For analysis of oligosaccharides, deionized water was used as an eluent. The eluent flow was kept at 1.0 mL/min. Quantification was carried out using external standards: mannan oligosaccharides purchased from Megazyme, Ireland. Amounts were expressed as milligrams per liter (mg/L). Data were collected and analyzed with the program ChemStation software (Agilent, CA).

Statistical Analysis
All statiscal analyses of the sugar ends data were done in Excel (Microsoft Office, 2016) by T-test group comparison, one tailed test. Visualization of enzyme by microscopy was only observational and was done in triplicate.

Microscopy
Images were recorded with a confocal laser scanning microscope (CLSM) displaying the Alexa-555 fluorescein signal in red, protein autofluorescence signal is green and calcofluor signal as blue. Calcofluor is known to emit fluorescence when binding to b-glucan linkages such as cellulose (Herrera-Ubaldo & de Folter, 2018) and xyloglucan. Immunofluorescence labelling with monoclonal antibody LM21 b-mannan (Marcus et al., 2010) bound to the cell wall lining overlapping the blue color showing that b-mannan is very closely linked to the b-glucan linkages cellulose and xyloglucan the major NSP in SBM ( Figure 1A). After enzyme treatment, the monoclonal antibody signal was significantly reduced ( Figure 1B), or not detected ( Figure 1C), compared to samples incubated in acetate buffer alone ( Figure 1A). A notable loosening of cell walls can be seen on enzyme treatment making the protein more visible (Figures 1B and 1C). jas.ccsenet.

Reduci
An increas at 120 min

Oligom
Two speci each produ the degree times commercial dosage of an NSPase (Ravn et al., 2016) and 100 and 1000 times commercial dosage to measure fiber solubilization (Pedersen et al., 2017) and to visualize enzyme efficacy using microscopy. The use of such high amounts may most likely indicate that the concentration of relevant enzyme/s in the NSPase product are very low and very high dosages are needed to show in vitro efficacy or the enzymes are acting sub additively. The in vitro data in this paper shows that using commercial dosage of b-mannanase product CTCzyme was sufficient to solubilize mannan.

Microscop
An increase in reducing ends of sugars as well generation of mannose oligomers were measured in this study. The commercial prebiotic MOS-containing product Salmosan® S-βGM (produced from carob and guar bean mannan hydrolyses) protected epithelial barrier function in a Caco-2 cell model disrupted by Salmonella enteritidis and had the ability to agglutinate the same (Brufau et al., 2016). MOS increased Bacteroidetes proliferation (Teng & Kim, 2018); L. salivarius was shown to be most effective against Salmonella colonisation and L. crispatus is effective against both Salmonella and E. coli (Teng & Kim, 2018). Supplementing broiler diets with MOS resulted in a reduction in coccidiosis lesions caused by Eimeria species (Elmusharaf et al., 2007) due to improved immune function and increased their growth performance as well (Chand et al., 2016). Supplementation of 0.2 % dietary MOS to broilers significantly reduced E. coli profiles in the caecum and ileum of birds (Chacher et al., 2017). MOS having a degree of polymerization ≤ 5 are stated to have powerful prebiotic and butyrogenic effects (Tiwari et al., 2020). In vitro assays measuring MOS in this work showed the generation of MOS having DP of 2 and 4 after enzyme reaction on SBM, indicating the ability of the b-mannanase to produce oligomers having prebiotic potential.
The irrefutable conclusion of the efficacy of a feed enzyme is of course proof of improved performance in the animal. In vivo data showed that supplementing a corn-soy diet with CTCzyme in a broiler trial increased the apparent metabolizable energy (AME) and apparent metabolizable energy corrected for nitrogen (AMEn) of diets by 4.6 and 5.0% (Kong et al., 2011). In the same way, using suboptimal nutrient levels in a broiler trial Ferreira et al. (2016) showed an improved AMEn of 1.5% with the inclusion of CTCzyme. With supplementation of CTCzyme β-mannanase in a low-energy and low-protein diet, laying hens were able to maintain similar production performance when compared to a high-energy and high-protein diet during early and late egg production (Zheng et al., 2020). There was no inclusion of any low molecular weight fermentable oligosaccharides in the above mentioned in vivo work besides the enzyme as opposed to the work of Cordero et al. (2019) which included both enzyme and fermentable oligosaccharides (in the experiment: a xylanase and xylan oligosaccharides). This suggests the ability of the b-mannanase product to solubilize mannans creating bioactive potent oligosaccharides even early in the digestion process in the animal gut within its life span, thereby alleviating the need for addition of further fermentable oligosaccharides in the product. Further work to test the prebiotic effect of the generated mannose oligosaccharides in vitro as well as inclusion of low molecular weight fermentable oligosaccharides in vivo needs to be conducted.
Based on our in vitro data in this paper, and published in vivo data (Kong et al., 2011;Mussini et al., 2011, Zheng et al., 2020 we can confirm the two modes of action of CTCzyme as an NSPase: (1) Solubilization of mannan in the cells walls to loosen and destroy the compact cell wall architecture thereby making the protein more bioavailable, and (2) Release of mannose oligomers considered to have prebiotic functions for improving gut health and integrity.