Effect of feeding acidified or fermented barley using Limosilactobacillus reuteri with or without supplemental phytase on diet nutrient digestibility in growing pigs

Abstract

Fermentation of cereal grains may degrade myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP₆) thereby increasing nutrient digestibility. Effects of chemical acidification or fermentation with Limosilactobacillus (L.) reuteri with or without phytase of high β-glucan hull-less barley grain on apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of nutrients and gross energy (GE), standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AAs), and standardized total tract digestibility (STTD) of P were assessed in growing pigs. Pigs were fed four mash barley-based diets balanced for water content: 1) unfermented barley (Control); 2) chemically acidified barley (ACD) with lactic acid and acidic acid (0.019 L/kg barley grain at a ratio of 4:1 [vol/vol]); 3) barley fermented with L. reuteri TMW 1.656 (Fermented without phytase); and 4) barley fermented with L. reuteri TMW 1.656 and phytase (Fermented with phytase; 500 FYT/kg barley grain). The acidification and fermentation treatments occurred for 24 h at 37 °C in a water bath. The four diets were fed to eight ileal-cannulated barrows (initial body weight [BW], 17.4 kg) for four 11-d periods in a double 4 × 4 Latin square. Barley grain InsP₆ content of Control, ACD, Fermented without phytase, or Fermented with phytase was 1.12%, 0.59%, 0.52% dry matter (DM), or not detectable, respectively. Diet ATTD of DM, CP, Ca, and GE, digestible energy (DE), predicted net energy (NE) value, and urinary excretion of P were greater (P < 0.05) for ACD than Control. Diet ATTD of DM, CP, Ca, GE, DE and predicted NE value, urinary excretion of P was greater (P < 0.05), and diet AID of Ca and ATTD and STTD of P tended to be greater (P < 0.10) for Fermented without phytase than Control. Diet ATTD of GE was lower (P < 0.05) and diet ATTD and STTD of P, AID and ATTD of Ca was greater (P < 0.05) for Fermented with phytase than Fermented without phytase. Acidification or fermentation with/without phytase did not affect diet SID of CP and AA. In conclusion, ACD or Fermented without phytase partially degraded InsP₆ in barley grain and increased diet ATTD of DM, CP, and GE, but not SID of CP and most AA in growing pigs. Fermentation with phytase entirely degraded InsP₆ in barley grain and maximized P and Ca digestibility, thereby reducing the need to provide inorganic dietary P to meet P requirements of growing pigs.

Introduction

In 2019, global barley grain production was 159 million tons (FAOSTAT, 2020). In Canada, 10.4 million tons barley was produced with 6.8 million tons used as animal feed (AAFC, 2020). In swine diets, barley grain provides starch as energy source and contains 0.4% total P (dry matter [DM] basis) with about 75% as myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate; InsP₆; Rodehutscord et al., 2016), and phytate, i.e., any salt of InsP₆ (Steward et al., 1988). Hydrolysis of InsP₆ in monogastric species is incomplete because small intestine mucosa lacks sufficient endogenous phytase secretion (Selle and Ravindran, 2008). Moreover, InsP₆ is an antinutritional factor, forming mineral complexes and thereby inhibiting absorption of cations and protein in pigs (Leenhardt et al., 2005; Woyengo and Nyachoti, 2013).

Processing techniques, such as sourdough fermentation, reduce pH and thereby promote InsP₆ hydrolysis by activating intrinsic phytase of barley (Leenhard et al., 2005) increasing InsP₆ solubility (Grynspan and Cheryan, 1983). Consequently, InsP₆ is degraded to lower inositol phosphate (InsP) forms prior to feeding that are more digestible by growing pigs (Blaabjerg et al., 2011). Previously, fermentation of wheat–barley-based diet with 750 FYT supplemental Aspergillus niger phytase per kg of feed increased InsP₆ degradation (Blaabjerg et al., 2011). However, effects of fermentation of barley grain using lactic acid bacteria with or without phytase supplementation on P digestibility have rarely been reported. Other methods, e.g., lactic acid acidification, altered complex carbohydrate fractions in barley grain, increased resistant starch and hemicellulose fractions, and may stimulate hindgut fermentation (HF) and mineral digestibility (Metzler-Zebeli, 2010, 2015). Fermentation of grains with the porcine foregut symbiont Limosilactobacillus (L.) reuteri additionally delivers high cell counts of probiotic bacteria to the swine gut (Walter, 2008; Yang et al., 2015).

The hypotheses of the present study were that chemical acidification or fermentation of high β-glucan barley grain using L. reuteri would break down complex of InsP₆ thereby increasing digestibility of nutrients and gross energy (GE) in growing pigs, and that supplementation of exogenous phytase during fermentation would further degrade InsP₆ thereby increasing digestibility of nutrients and GE compared with fermentation without phytase. The objectives were to compare: 1) apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of crude protein (CP), P, Ca, and GE, digestible energy (DE) and predicted net energy (NE) values; 2) standardized ileal digestibility (SID) of amino acids (AAs) and CP; and 3) standardized total tract digestibility (STTD) of P in growing pigs fed diets containing barley grain that was chemically acidified or fermented using L. reuteri with or without exogenous phytase.

Materials and Methods

Animal procedures were reviewed, and animal use was approved by the University of Alberta Animal Care and Use Committee for Livestock, and followed principles established by the Canadian Council on Animal Care (CCAC, 2009). The animal study was conducted at the Swine Research and Technology Centre, University of Alberta (Edmonton, AB, Canada).

Test ingredient and processing

A highly fermentable and high β-glucan hull-less barley grain cultivar (Fibar; Crop Development Centre [CDC], Saskatoon, SK, Canada) was used as test ingredient (Fouhse et al., 2017; Table 1). Barley grain was first ground using a hammer mill through a 2.36-mm opening screen. Ground barley grain was then mixed in diets in four different forms: 1) intact; 2) chemically acidified; 3) fermented with L. reuteri TMW1.656; and 4) fermented with L. reuteri TMW1.656 and phytase (500 FYT/kg barley grain; Ronozyme Hiphos GT, DSM Nutritional Products, Kaiseraugst, Switzerland).

Table 1.

Analyzed nutrient content and energy value of hull-less barley grain samples included in the experimental diets (DM basis)¹

Item, %	Hull-less barley
	Control	ACD	Fermented
			Without phytase	With phytase
GE, Mcal/kg	4.53	4.53	4.55	4.54
Starch	57.3	59.3	57.9	60.3
CP (N × 6.25)	15.0	14.8	15.2	15.2
Ether extract	0.84	1.70	2.04	2.19
TDF	11.4	10.6	10.2	10.2
Soluble fiber	0.16	0.15	0.09	0.10
Insoluble fiber	11.0	10.0	9.4	9.6
NDF	9.91	8.94	6.98	6.78
ADF	1.96	1.84	2.24	2.29
Crude fiber	1.44	1.53	1.59	1.60
Ash	2.00	2.00	2.07	2.05
Zn	0.004	0.004	0.004	0.004
Mn	0.002	0.002	0.002	0.001
Fe	0.01	0.01	0.01	0.01
Ca	0.04	0.04	0.04	0.04
Total P	0.44	0.47	0.48	0.48
InsP6	1.12	0.59	0.52	ND
InsP6–P	0.31	0.17	0.15	ND
Indispensable AA
Arg	0.63	0.63	0.59	0.57
His	0.29	0.29	0.30	0.31
Ile	0.52	0.51	0.54	0.53
Leu	0.96	0.94	0.95	0.94
Lys	0.50	0.49	0.52	0.51
Met	0.21	0.21	0.21	0.21
Phe	0.73	0.72	0.74	0.74
Thr	0.45	0.44	0.48	0.47
Trp	0.13	0.14	0.14	0.14
Val	0.70	0.69	0.73	0.73
Dispensable AA
Ala	0.54	0.53	0.61	0.61
Asp	0.77	0.77	0.82	0.81
Cys	0.32	0.32	0.33	0.33
Glu	3.50	3.41	3.45	3.44
Gly	0.54	0.53	0.56	0.55
Pro	1.54	1.50	1.56	1.57
Ser	0.53	0.51	0.53	0.52
Tyr	0.13	0.14	0.14	0.14
Chemically available Lys	0.48	0.48	0.50	0.49
Total AA	13.5	13.2	13.8	13.8

¹Control barley; ACD, chemically acidified barley; Fermented without phytase, fermented barley with L. reuteri; Fermented with phytase, fermented barley with L. reuteri and 500 FYT phytase/kg barley grain; InsP₆, myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate); ND, not detected (detection limit: InsP₆, 0.1 g/100 g).

For the intact barley, ground barley grain was merely mixed with an equal weight of tap water prior to feeding to pigs. The chemically acidified barley was prepared by the addition of water, lactic acid (80%; MilliporeSigma Canada Co., Oakville, ON, Canada), and glacial acetic acid (MilliporeSigma Canada Co.) in a ratio of 4:1 (vol/vol) (0.019 L/kg barley grain) according to an established protocol (Le et al., 2016). Feed-grade organic acids were used to simulate treatments with potential practical relevance. The chemically acidified barley was incubated for 24 h at 37 °C in a water bath. The chemically acidified barley was acidified to pH 4 (same pH as fermented barley) to stimulate intrinsic phytase activity (Leenhardt et al., 2005). The fermentation of barley grain was performed according to Yang et al. (2015). A sourdough isolate (L. reuteri TMW1.656) was grown on modified De Man, Rogosa, and Sharpe (MRS) agar (Meroth et al., 2003) and incubated anaerobically at 37 °C. In the laboratory, an initial seed sourdough stock was prepared by adding approximately 10⁸ colony-forming unit (CFU)/g L. reuteri to ground barley with, and tap water in a 1:1 ratio (wt/vol) and this mixture was incubated at 37 °C for 48 h. Before feeding to pigs, 10% seed sourdough stock was mixed with ground barley grain and water with 1:1 ratio (wt/vol) and incubated for 24 h at 37 °C in a water bath. Following incubation, 90% of the fermented barley was mixed with the other diet ingredients, the remaining 10% were used to inoculate the subsequent batch (back-slopping). After six fermentation cycles with 10% inoculum, another seed sourdough was prepared from the stock and used to inoculate the next six barley fermentation cycles. For the fermented barley and exogenous phytase treatment, 500 FYT/kg phytase was added to barley grain during the fermentation, a common level of phytase supplementation (Dersjant-Li et al., 2015). To avoid cross contamination, the daily back-slop was prepared within the fermented barley without phytase treatment.

For quality control, samples were taken after 24 h of fermentation to monitor pH, cell counts, and colony morphology. Fermentation of barley grain with or without phytase lowered pH to 4 after 24 h of incubation. Cell counts and cell morphology were measured by serial dilutions and surface plating on MRS agar plates (Yang et al., 2015). A uniform colony morphology indicates lack of contamination in sourdough using defined Lactobacilli strains (Lin and Gänzle, 2014).

Experimental diets and design

Pigs were fed four mash diets containing 50% of one of the four barley samples (Table 2): 1) intact (unfermented barley [Control]); 2) chemically acidified (ACD); 3) fermented (Fermented without phytase); and 4) fermented and supplemented with phytase (Fermented with phytase). Diets exceeded NRC (2012) requirements for most nutrients, except for Ca and P. Diets contained InsP₆ provided solely by barley grain and were deficient in Ca (30% and 26% of requirement for 11 to 25 kg body weight (BW) and 25 to 50 kg BW, respectively; NRC, 2012) and total P (39% and 34% requirement for 11 to 25 kg BW and 25 to 50 kg BW, respectively; NRC, 2012). Titanium dioxide was included as an indigestible marker. A 300-kg horizontal paddle mixer (model 3061; Marion Process Solutions, Marion, IA) was used to mix an ingredient blend without barley grain. Complete diets were then prepared in the barn every day mixing each treated barley grain samples at 1:1 with the ingredient blend. Diets for the entire day were mixed in the morning and split into two. The morning meal was fed immediately and the rest was stored at room temperature until the afternoon feeding. After daily mixing, diet samples were collected and stored at −20 °C. Upon completion of the trial, samples were pooled and subsamples were freeze-dried.

Table 2.

Ingredient composition of the experimental diets

Item, % as-fed	Hull-less barley1
	Control	ACD	Fermented
			Without phytase	With phytase
Barley2	50.0	—	—	—
Acidified barley3	—	50.0	—	—
Fermented barley4	—	—	50.0	50.0
Casein5	18.0	18.0	18.0	18.0
Cornstarch6	17.790	17.790	17.790	17.785
Lactose	8.00	8.00	8.00	8.00
Cellulose7	2.00	2.00	2.00	2.00
Canola oil	1.00	1.00	1.00	1.00
Limestone	0.70	0.70	0.70	0.70
Titanium dioxide	0.50	0.50	0.50	0.50
Salt	0.50	0.50	0.50	0.50
Vitamin premix8	0.50	0.50	0.50	0.50
Mineral premix9	0.50	0.50	0.50	0.50
KCl	0.20	0.20	0.20	0.20
DL-Met	0.10	0.10	0.10	0.10
L-Thr	0.06	0.06	0.06	0.06
MgO	0.05	0.05	0.05	0.05
Choline chloride, 60%	0.10	0.10	0.10	0.10
Phytase10	—	—	—	0.005

¹CDC Fiber, high-fermentable, high β-glucan, hull-less barley.

²Control barley.

³ACD, chemically acidified barley using lactic and glacial acidic acid (0.019 L/kg barley grain; ratio 4:1 [vol/vol]).

⁴Barley fermented with L. reuteri for 24 h at 37 °C.

⁵Sodium Caseinate 180 (NZMP, Auckland, New Zealand).

⁶Melojel (National Starch and Chemical Co., Bridgewater, NJ).

⁷Solka-floc (International Fiber Corp., North Tonawanda, NY).

⁸Provided the following per kilogram of diet: vitamin A, 7,500 IU; vitamin D, 750 IU; vitamin E, 50 IU; niacin, 37.5 mg; pantothenic acid, 15 mg; folacin, 2.5 mg; riboflavin, 5 mg; pyridoxine, 1.5 mg; thiamine, 2.5 mg; choline, 2,000 mg; vitamin K, 4 mg; biotin, 0.25 mg; and vitamin B₁₂, 0.02 mg.

⁹Provided the following per kilogram of diet: Zn, 125 mg as ZnSO₄; Cu, 50 mg as CuSO₄; Fe, 75 mg as FeSO₄; Mn, 25 mg as MnSO₄; I, 0.5 mg as Ca(IO₃)₂; and Se, 0.3 mg as Na₂SeO₃.

¹⁰500 FYT phytase/kg barley grain was added during the fermentation (Ronozyme HiPhos (GT); DSM Nutritional Products, Kaiseraugst, Switzerland).

Eight crossbred barrows (initial BW, 17.4 ± 1.6 kg; final BW, 51.9 ± 2.9 kg; average BW gain, 784 ± 47 g/d; Duroc × Large White/Landrace F1; Hypor, Regina, SK, Canada) were housed in individual metabolism pens (1.2 m wide, 1.5 m long, and 0.95 m high) with polyvinyl chloride walls with windows and plastic slatted flooring. Pens were equipped with a stainless-steel feeder attached to the front of the pen and a cup drinker beside the feeder assuring free access to water throughout the experiment. Room temperature was controlled at 22.9 ± 2.0 °C. Pigs were surgically fitted with a simple T-cannula at the distal ileum, approximately 5 cm prior to the ileocecal sphincter (Sauer et al., 1983; de Lange et al., 1989). After recovery, pigs were fed the four diets over four periods in a double 4 × 4 Latin square to achieve 8 observations per dietary treatment.

Daily feed allowance was adjusted to 3.0 times maintenance requirement for DE (3.0 × 110 kcal of DE/kg of BW^0.75; NRC, 2006) fed in two equal meals at approximately 0800 and 1500 h. Each 11-d experimental period consisted of a 7-d acclimation to the experimental diet, followed by a 2-d collection of feces and urine, and then a 2-d collection of ileal digesta. Feces were collected using plastic bags attached to a Velcro/leather ring glued to the skin around the anus (van Kleef et al., 1994). Total urine was collected via a tray underneath the entire pen that funneled into a bucket containing sulfuric acid (20 mL; 95.0% to 98.0% purity) covered with cheese cloth to prevent entry of solids. Digesta was collected between 0800 and 1600 h using plastic bags containing 15 mL of 5% formic acid attached to the opened cannula barrel with rubber band (Li et al., 1993). Feces, urine, and digesta samples were pooled for each pig within experimental period and stored at −20 °C. Upon completion of the trial, digesta, urine, and fecal samples were thawed, homogenized, subsampled, and freeze-dried.

Chemical analyses

Barley grain and lyophilized diets, feces, and digesta were ground using a 1-mm screen in a centrifugal mill (model ZM200; Retsch, Haan, Germany) and analyzed for moisture (method 930.15; AOAC, 2006), CP (method 990.03; N × 6.25; AOAC, 2006), GE using an adiabatic bomb calorimeter (model 5003; Ika-Werke, Staufen, Germany), and Ca (method 3051a for sample preparation; EPA, 2020) using an inductively coupled plasma-optical emission spectrometer (ICP-OES; Thermo Fisher Corp., Cambridge, United Kingdom). Barley grain, diets, feces, urine, and digesta were analyzed for P (method 965.17; AOAC, 2006). Barley grain and diets were analyzed for ash (method 942.05; AOAC, 2006), ether extract (method 920.39A; AOAC, 2006), starch (assay kit STA-20; Sigma, St. Louis, MO), acid detergent fiber (ADF) inclusive of residual ash (method 973.18; AOAC, 2006), neutral detergent fiber (NDF) assayed without heat stable amylase and expressed inclusive of residual ash (Holst, 1973), and InsP₆ (AOAC, 2006). Barley grain was analyzed for total dietary fiber (TDF), soluble and insoluble dietary fiber content (method 991.43; AOAC, 2006). The AA content in barley grain, diets, and digesta was analyzed by HPLC (method 982.30E; AOAC, 2006), and chemically available Lys was analyzed by spectrophotometry (method 975.44; AOAC, 2006). Diets, feces, and digesta were analyzed for titanium dioxide by spectrophotometry (Myers et al., 2004).

Calculations

Diet AID and ATTD of DM, CP, GE, Ca, and P were calculated using the index method (Adeola, 2001). To calculate AID and ATTD of Ca, the mean Ca content across diets was used. Apparent hindgut fermentation (AHF) was calculated as ATTD − AID. HF as percentage of ileal digesta was calculated as [(ATTD − AID)/(100 − AID)] × 100 (Woyengo et al., 2016). Diet NE value was calculated using equation 5 from Noblet et al. (1994) using determined diet DE value and analyzed content of ADF, starch, CP, and ether extract, as adopted by NRC (2012).

The AID of AA in diets was calculated using the index method (Adeola, 2001). The SID of CP and AA was calculated by correcting AID for basal ileal endogenous AA losses (Iend) using mean values (Jansman et al., 2002) and equation (Stein et al., 2007; equation 7): SID = [AID + (Iend/AA in diet)].

Excretion of P was calculated as follows: Excretion = P intake × [(100 – ATTD of P in diet)/100]. Retention of P was calculated according to Almeida and Stein (2010). The STTD of P was calculated by correcting ATTD of P for the reported basal endogenous P losses (EPL) of 190 mg/kg DM intake (NRC, 2012) according to equations 13–18 (NRC, 2012): STTD (%) = ATTD + [(basal EPL/P_diet) × 100].

Statistical analyses

Nutrient digestibility data were analyzed using the MIXED procedure of SAS (ver. 9.4; SAS Inst. Inc., Cary, NC). Normality and homogeneity of variance of the residuals for each variable were confirmed prior to ANOVA. Diet was the fixed effect, and square, period nested in square, and pig nested in square were random effects in the model.

Single-degree of freedom contrasts were used to test the effects of acid addition (Control vs. ACD), fermentation (Control vs. Fermented without phytase), and fermentation with phytase (Fermented without phytase vs. Fermented with phytase) on digestibility, mineral retention, and DE and predicted NE values (Littell et al., 2006). Data are presented as least squares means with SEM. To test the hypotheses, P < 0.05 was considered significant and 0.05 ≤ P < 0.10 was considered a trend.

Results

Pigs remained healthy and consumed their entire daily feed allowance throughout the trial.

Fermented with and without phytase and ACD barley grain samples did not alter CP and total P content, but reduced TDF and NDF content than Control (Table 1). InsP₆ content in barley grain was 53%, 46%, and 100% lower for ACD, Fermented without phytase or Fermented with phytase than Control. Across the four barley diets, CV for CP, NDF, ADF, P, and GE was 1.9%, 6.0%, 15.4%, 1.4%, and 0.2%, respectively (Table 3). Diet NDF content in Fermented with or without phytase was lower than Control.

Table 3.

Analyzed nutrient content and energy value of the experimental diets¹

Item, % DM	Hull-less barley
	Control	ACD	Fermented
			Without phytase	With phytase
GE, Mcal/kg	4.59	4.58	4.59	4.60
Starch	47.4	47.0	48.8	49.1
CP (N × 6.25)	26.6	25.6	26.0	26.6
NDF	5.21	5.09	4.68	4.62
ADF	2.05	2.76	2.79	2.21
Crude fiber	1.63	1.97	1.68	1.66
Ash	4.05	3.83	4.06	4.13
Zn	0.02	0.01	0.02	0.02
Cu	0.01	0.01	0.01	0.01
Mn	0.004	0.003	0.004	0.005
Fe	0.01	0.01	0.01	0.01
Ca	0.54	0.26	0.33	0.36
Total P	0.36	0.37	0.38	0.38
Ether extract	1.14	1.25	1.35	1.08
Indispensable AA
Arg	0.98	0.96	0.95	0.92
His	0.69	0.68	0.69	0.70
Ile	1.31	1.27	1.29	1.34
Leu	2.27	2.21	2.21	2.29
Lys	1.78	1.71	1.74	1.81
Met	0.68	0.67	0.69	0.70
Phe	1.34	1.32	1.33	1.36
Thr	1.10	1.04	1.08	1.09
Trp	0.33	0.32	0.32	0.33
Val	1.63	1.58	1.61	1.65
Dispensable AA
Ala	0.86	0.85	0.87	0.89
Asp	1.76	1.71	1.73	1.79
Cys	0.21	0.20	0.21	0.21
Glu	6.06	5.89	5.89	6.03
Gly	0.61	0.61	0.62	0.62
Pro	2.70	2.63	2.66	2.73
Ser	1.16	1.11	1.11	1.15
Tyr	0.94	0.97	1.01	0.86
Total AA	26.7	25.9	26.3	26.8

¹Control barley; ACD, chemically acidified barley; Fermented without phytase, fermented barley with L. reuteri; Fermented with phytase, fermented barley with L. reuteri and 500 FYT phytase/kg barley grain.

Diet AID and AHF of DM, CP, and GE was not affected by ACD or Fermented without phytase compared to Control, or Fermented with phytase compared to Fermented without phytase (Table 4). The HF of DM and GE was greater (P < 0.05) and HF of CP tended to be greater (P < 0.10) for ACD than Control. The HF of DM, CP, and GE was greater (P < 0.05) for Fermented without phytase than Control. Diet ATTD of DM, CP, GE, DE, and predicted NE value was greater (P < 0.05) for ACD and Fermented without phytase than Control. Fermented with phytase had lower (P < 0.05) diet ATTD of GE, and tended to have lower (P < 0.10) diet ATTD of DM than Fermented without phytase. The range between the greatest and lowest AID of GE was greater than for ATTD of GE; however, only ATTD of GE differed among treatments due to its 86% lower pooled SEM. Diet SID of AA was not affected by ACD and Fermented without phytase compared to Control, or Fermented with phytase compared to Fermented without phytase (Table 5).

Table 4.

AID, ATTD, AHF, and HF as percentage of ileal digesta of DM, CP, and GE and DE and predicted NE values of experimental diets (DM basis)^1,2

Item	Hull-less barley				Pooled SEM	P-value3
	Control	ACD	Fermented
			Without phytase	With phytase		Acid	Fermentation	Phytase
AID, %
DM	76.5	74.6	73.5	73.1	1.65	0.260	0.086	0.833
CP	85.3	85.7	84.5	84.2	1.32	0.774	0.550	0.786
GE	78.2	76.6	75.7	75.1	1.71	0.365	0.164	0.754
ATTD, %
DM	88.6	89.6	89.5	89.0	0.24	0.001	0.002	0.055
CP	89.0	90.9	91.6	90.9	0.70	0.016	0.002	0.360
GE	87.7	88.9	88.9	88.3	0.27	0.001	0.001	0.040
AHF4, %
DM	12.1	15.0	16.0	15.9	1.77	0.122	0.043	0.940
CP	3.68	5.17	7.07	6.77	1.65	0.377	0.057	0.861
GE	9.67	12.3	13.2	13.1	1.85	0.192	0.080	0.966
HF5, % of ileal digesta
DM	49.6	58.9	60.0	58.7	3.27	0.012	0.006	0.705
CP	18.5	35.8	44.4	40.1	8.47	0.059	0.008	0.617
GE	41.4	52.1	53.6	52.4	4.33	0.029	0.015	0.781
DE, Mcal/kg DM	4.02	4.07	4.08	4.06	0.013	0.003	0.001	0.246
NE6, Mcal/kg DM	2.80	2.84	2.85	2.84	0.009	0.001	<0.001	0.184

¹Control barley; ACD, chemically acidified barley; Fermented without phytase, fermented barley with L. reuteri; Fermented with phytase, fermented barley with L. reuteri and 500 FYT phytase/kg barley grain.

²Least squares means based on 7 or 8 observations per diet.

³Acid, acid addition (Control vs. ACD); Fermentation, Control vs. Fermented without phytase; Phytase, fermentation with phytase (Fermented without phytase vs. Fermented with phytase).

⁴AHF = ATTD − AID.

⁵HF = [(ATTD − AID)/(100 − AID)] × 100 (Woyengo et al., 2016).

⁶Diet NE values were calculated using equation 5 from Noblet et al. (1994).

Table 5.

SID of CP and AA of the experimental diets^1–3

Item	Hull-less barley				SEM	P-value4
	Control	ACD	Fermented
			Without phytase	With phytase		Acid	Fermentation	Phytase
CP	89.3	89.8	88.6	88.1	1.32	0.687	0.598	0.731
Indispensable AA
Arg	92.3	92.9	92.0	92.0	0.92	0.521	0.776	0.995
His	93.3	93.8	93.1	92.6	0.84	0.544	0.796	0.579
Ile	92.4	92.9	92.2	92.1	0.84	0.567	0.798	0.842
Leu	93.4	94.0	93.6	93.7	0.86	0.510	0.827	0.941
Lys	93.5	94.3	92.3	92.4	0.69	0.251	0.112	0.935
Met	95.8	96.4	96.0	96.0	0.41	0.172	0.687	0.855
Phe	92.7	93.4	93.2	93.3	1.04	0.555	0.663	0.925
Thr	88.2	87.9	86.7	86.2	1.44	0.809	0.312	0.692
Trp	93.7	94.9	94.5	94.0	1.49	0.425	0.588	0.715
Val	91.3	91.7	91.1	91.0	1.01	0.677	0.879	0.903
Dispensable AA
Ala	85.5	87.6	84.8	84.7	1.57	0.194	0.657	0.961
Asp	87.8	89.2	86.4	86.0	1.39	0.340	0.330	0.764
Cys	75.5	73.7	76.2	71.2	4.07	0.659	0.869	0.236
Glu	92.4	93.3	93.7	92.9	0.96	0.331	0.191	0.426
Gly	86.6	88.0	87.2	85.7	2.71	0.606	0.845	0.611
Pro	93.9	94.9	94.5	93.8	0.86	0.258	0.518	0.432
Ser	90.7	90.8	90.0	88.9	1.25	0.930	0.602	0.404
Tyr	94.9	95.3	94.7	94.0	0.93	0.626	0.858	0.428

¹Control barley; ACD, chemically acidified barley; Fermented without phytase, fermented barley with L. reuteri; Fermented with phytase, fermented barley with L. reuteri and 500 FYT phytase/kg barley grain.

²The SID of CP and AA was calculated by correcting the AID of CP and AA with values for basal endogenous losses (g/kg DM intake; Jansman et al., 2002): CP, 10.53; Arg, 0.40; His, 0.16; Ile, 0.29; Leu, 0.44; Lys, 0.36; Met, 0.10; Phe, 0.31; Thr, 0.51; Trp, 0.13; Val, 0.41; Ala, 0.45; Asp, 0.70; Cys, 0.17; Glu, 0.85; Gly, 1.01; Pro, 1.31; Ser, 0.51; Tyr, 0.30.

³Least squares means based on 8 pig observations per diet.

⁴Acid, acid addition (Control vs. ACD); Fermentation, Control vs. Fermented without phytase; Phytase, fermentation with phytase (Fermented without phytase vs. Fermented with phytase).

Diet AID of P was not affected by ACD and Fermented without or with phytase (Table 6). Diet ATTD and STTD of P tended to be greater (P < 0.10) for Fermented without phytase than Control. Diet ATTD and STTD of P was greater (P < 0.05) and fecal P excretion tended to be lower (P < 0.10) for Fermented without phytase than Fermented with phytase. Urinary excretion of P was greater (P < 0.05) for Fermented without phytase and ACD than Control. The retention:digestion ratio of P tended to be lower (P < 0.10) for ACD and Fermented without phytase than Control, and (P < 0.10) for Fermented with phytase than Fermented without phytase. Diet AID of Ca tended to be greater (P < 0.10) for Fermented without phytase than Control, and was greater (P < 0.05) for Fermented with phytase than Fermented without phytase. Diet ATTD of Ca was greater (P < 0.05) for ACD and Fermented without phytase than Control, and greater (P < 0.05) for Fermented with phytase than Fermented without phytase.

Table 6.

Retention of P, AID, ATTD of P, Ca, and InsP₆, and STTD of P of experimental diets^1–3

Item	Hull-less barley				SEM	P-value4
	Control	ACD	Fermented
			Without phytase	With phytase		Acid	Fermentation	Phytase
P
AID, %	68.7	70.4	69.7	79.0	4.99	0.740	0.839	0.081
ATTD, %	71.9	75.0	78.9	86.6	3.45	0.376	0.061	0.042
STTD, %	76.9	80.4	83.6	92.6	3.45	0.332	0.075	0.019
Intake, g/d	3.88	4.15	4.10	4.16	—	—	—	—
Fecal excretion, g/d	1.03	1.03	0.83	0.56	0.127	0.979	0.130	0.051
Urinary excretion, g/d	0.24	0.49	0.53	0.69	0.115	0.046	0.022	0.182
Retention, g/d	2.61	2.63	2.73	2.90	0.159	0.924	0.457	0.309
Retention:intake, %	66.1	64.7	67.7	70.4	4.34	0.751	0.717	0.543
Retention:digestion, %	91.7	86.3	86.3	81.4	2.72	0.065	0.064	0.097
Ca
AID, %	89.5	90.7	91.4	93.8	1.01	0.222	0.068	0.035
ATTD, %	89.0	93.2	92.5	95.3	1.22	0.004	0.011	0.037

¹Control barley; ACD, chemically acidified barley; Fermented without phytase, fermented barley with L. reuteri; Fermented with phytase, fermented barley with L. reuteri and 500 FYT phytase/kg barley grain.

²The STTD of P was calculated by correcting the ATTD of P with recommended basal endogenous losses (190 mg P/kg DM intake; NRC, 2012).

³Least squares means based on 7 to 8 pig observations per diet.

⁴Acid, acid addition (Control vs. ACD); Fermentation, Control vs. Fermented without phytase; Phytase, fermentation with phytase (Fermented without phytase vs. Fermented with phytase).

Discussion

Nutrient composition of barley grain

In the present study, hull-less barley grain contained 57% starch, similar to that reported previously (Jha et al., 2011; Fouhse et al., 2017). Total NSP in hull-less barley grain ranges 23% to 39% and is primarily composed of 7% to 12% arabinoxylans and 3% to 8% β-glucans (Aumiller et al., 2015). The hull-less barley grain in the present study contained high β-glucan (10.3%) and little amylose (0.20%; Fouhse et al., 2017). About 80% of β-glucan is distributed throughout the endosperm in high β-glucan hull-less barley grain cultivars (Zheng et al., 2000). In the present study, barley grain contained 0.44% P, similar to that reported previously (Rodehutscord et al., 2016). However, 71% of total P in barley grain was bound as InsP₆ or phytate (any salt of InsP₆) that is poorly digestible by nonruminants. Intrinsic phytase activity in barley grain can vary between 490 and 1,100 U/kg DM and differs among barley cultivars (Steiner et al., 2007; Rodehutscord et al., 2016). Although intrinsic phytase activity of barley grain was not analyzed in the present study, CDC Fibar barley grain of a different harvest year contained 1,120 U/kg DM intrinsic phytase activity. Thus, to increase digestibility of plant P in cereal-based diets, strategies to degrade InsP₆ to lower forms of InsP prior to feeding to pigs is warranted. Lower InsP forms (InsP₄ down to InsP₂) can be almost entirely digested by pigs (Rosenfelder-Kuon et al., 2020).

Cereal processing for human consumption such as sourdough fermentation may activate intrinsic proteases, amylases, pentosanases, and phytases that stimulate degradation of fiber and antinutritional factors thereby increasing nutrient availability (Leenhardt et al., 2005; Gänzle, 2014). In pigs, L. reuteri is a symbiont forming stable populations in the pars esophagus, and can be used for cereal fermentation (Walter, 2008; Su et al., 2012). During fermentation, L. reuteri metabolizes and degrades carbohydrates, proteins, and phenolic compounds by increased activity of intrinsic cereal enzymes so that end products may serve as substrates for further bacterial growth of L. reuteri in situ (Gänzle, 2014). In the present study, fermentation of barley grain decreased NDF content and thus hemicellulose. Fermentation or acidification may thus alter nutrient content. For example, fermentation with L. reuteri decreased CP, ADF, and NDF content of wheat grain (Le et al., 2016). The close structural relationship between dietary fiber and InsP₆ in the aleurone layer of cereal grain should be considered when fermenting barley grain to open up the fiber matrix and thereby increase access for enzymatic degradation and increase nutrient digestibility in pigs (Newkirk and Classen, 1998; Raboy, 2003; Blaabjerg et al., 2011; Fouhse et al., 2017).

In barley, InsP₆ and plant phytase are primarily located in the aleurone layer (Raboy, 2003). In the present study, fermentation with L. reuteri without phytase reduced InsP₆ content in barley grain by 54% compared with control resulting in a contribution of InsP₆–P to total P content of 31% instead of 71% in the control. Previously, decreasing pH moderately to 5.5 using sourdough fermentation or acidification using lactic acid reduced InsP₆ content in wheat flour by 70% (Leenhard et al., 2005). The decrease in InsP₆ in barley might not be as pronounced as in wheat because barley grain has lower intrinsic phytase activity (Egli et al., 2002; Leenhard et al. 2005). However, fermentation with L. reuteri and the addition of 500 FYT phytase/kg barley grain entirely degraded InsP₆ due to optimal conditions for exogenous and intrinsic phytase activity. For acidification, pH is important because plant phytase efficacy and solubility of phytate is greatest with pH below 4 (Grynspan and Cheryan, 1983). Acidification using 5% lactic acid lowered pH to 2.4 and reduced InsP₆–P in barley grain, by converting InsP₆ into lower InsP forms such as InsP₄ and InsP₅ (Metzler-Zebeli et al., 2014).

Nutrient and energy digestibility of diets

In contrast to the present study, fermentation of wheat grain using L. reuteri decreased diet ATTD of CP from day 15 to 21 after weaning in piglets (Le et al., 2016). Lack of changes in SID of CP and AA in the present study indicated that decreased pH and potentially related effects on N solubility and intrinsic proteases activity might be of minor importance for ACD and Fermented without phytase. Notably, the phytate:CP ratio is small in cereal grains compared with protein feedstuffs and P digestibility was substantial in the Control due to intrinsic phytase activity in barley. Thus, the conditions of the present study reduced effectiveness of the ACD and Fermented without phytase treatments if mediated through phytate–AA complexes. Incubation at pH 4 for ACD and Fermented without phytase pronouncedly degraded InsP₆ located in the aleurone layer of barley grain and changed fiber composition by increasing solubility of arabinoxylans, possibly opening up the barley fiber matrix and increasing diet ATTD of CP and GE in pigs (Nortey et al., 2008; Gänzle, 2014).

While dietary InsP₆ was not analyzed, actual InsP₆ content should be proportional to barley grain inclusion as barley was the major ingredient providing InsP₆ and lower InsP. The lower dietary InsP₆ content reduced antinutritional activity thereby increasing ATTD of energy and nutrients; however, the reason for unchanged AID of CP is unclear. Previously, AID and ATTD of CP and GE were lower for a high-InsP₆ than a low-InsP₆ diet in pigs (Liao et al., 2005).

In the present study, fermentation increased the AHF of DM in barley diets. The decrease in hemicellulose including soluble fiber might decrease diet viscosity and increase digesta passage rate thereby reducing retention time available for enzymatic digestion (Owusu-Asiedu et al., 2006). Consequently, more undigested nutrients reach the hindgut providing substrate for bacterial fermentation and increase ATTD of nutrients and energy for ACD, and increase AHF and ATTD of nutrients and energy for Fermented without phytase compared with Control. Moreover, fermentation and ACD increased HF, indicating that these treatments enhanced susceptibility of undigested residue to be fermented, similar to supplemental enzymes (Jha et al., 2015). However, the reason for decreased ATTD of GE for Fermented with phytase than Fermented without phytase cannot be explained and requires further investigation.

Mineral digestibility of diets

In the present study, the main dietary P sources were barley grain and casein with the former being the sole InsP-containing ingredient. The ACD, Fermented without phytase, or Fermented with phytase decreased InsP₆ in barley grain by 47%, 54%, and 100%, thus minimizing the antinutritional effect of InsP₆ to reduce mineral digestibility (Schlemmer et al., 2001). Exogenous microbial phytases are used extensively; however, their efficacy is affected by passage rate and pH within the gastrointestinal tract (Blaabjerg et al., 2011). The present results indicate that fermenting barley grain with exogenous phytase prior to feeding entirely degraded InsP₆ content and thus is promising to maximize the ATTD of P. Fungal phytase (A. niger 750 FYT/kg) addition to wheat- and barley-based mash diets without inorganic P increased ATTD of P by 23% to 66% in growing-finishing pigs (Poulsen et al., 2007). Moreover, fermentation of wheat–barley-based diets with 750 FYT exogenous A. niger phytase/kg feed at 20 °C for 17.5 h following pelleting at 90 °C and crumbling also entirely degraded InsP₆ prior to feeding (Blaabjerg et al., 2011). Degradation of InsP₆ into InsP₄ is the limiting step for plant P utilization (Rosenfelder-Kuon et al., 2020). Therefore, fermentation of barley grain with phytase is a promising strategy to decrease InsP₆ content as it likely triggers InsP₆ degradation to lower isomers. Recently, pigs fed corn–soybean meal-based diets with up to 3,000 FYT exogenous E. coli-derived 6-phytase/kg feed (Exp. 1) and corn–soybean meal or corn–soybean meal–rapeseed cake diets supplemented with 1,500 FYT exogenous E. coli-derived 6-phytase/kg feed (Exp. 2) had greater concentrations of InsP₄ in ileal digesta than those fed control diets (Rosenfelder-Kuon et al., 2020). The authors concluded that InsP₄ is the limiting isomer of InsP degradation for the used microbial phytase.

Fermentation of barley grain using L. reuteri for 24 h degraded InsP₆, thus tended to increased P digestibility of diets. In vitro studies indicated that one part of InsP₆ in cereals and oilseeds is readily degraded, whereas the residual part requires more time (Blaabjerg et al., 2010). The less degradable part of InsP₆ may consist of complexes formed with minerals and/or proteins or InsP₆ may be encapsulated within the cell wall matrix reducing accessibility for enzymes such as phytase (Newkirk and Classen, 1998; Blaabjerg et al., 2011). The increased digestibility of P did not increase P retention but increased urinary P excretion for ACD and Fermented without phytase. Likely, absorbed P from the Control diet was sufficient to meet P requirements. Previously, digestible P intake was regressed against daily urinary P excretion (Ekpe et al., 2002). This regression indicated that the 0.24 P g/d urinary excretion for pigs fed the Control diet was associated with those pigs receiving around 1.0 g/d digestible P above reaching minimal urinary P excretion of 0.05 P g/d. More available P in other treatments thus increased urinary excretion of P. Indeed, the 0.53 P g/d urinary excretion for pigs fed the Fermented without phytase diet indicated that fermentation increased digestible P intake by 0.57 g/d.

In the present study, the main dietary Ca source was limestone, a highly digestible Ca source for nonruminants (Zhang and Adeola, 2017). Although analyzed Ca varied among diets, diets were formulated with the same Ca inclusion level and calculated Ca:P ratio to avoid negative effects of Ca oversupply. The ACD increased ATTD of Ca, whereas Fermented without phytase increased AID and ATTD of Ca compared with Control and was further increased by phytase supplementation during fermentation possibly by increased cation solubility, InsP₆ hydrolysis (Siener et al., 2001), and related opening of the cereal cell wall matrix. The antinutritional effect of InsP₆ is caused by its negatively charged character therefore forming complexes with positively charged molecules and salts (Angel et al., 2002). The potential of InsP₆ to bind cations decreases at a lower pH reducing the risk of complexing with cations such as Ca (Angel et al., 2002).

In conclusion, feeding ACD or Fermented without phytase barley grain diets to growing pigs partially degraded InsP₆ and increased diet ATTD of DM, CP, and GE, but did not affect SID of CP and most AA. Fermentation with phytase entirely degraded InsP₆ and maximized both P and Ca digestibility. Therefore, combining fermentation with supplemental phytase entirely degraded InsP₆ and thereby enhanced P and Ca digestibility thereby reducing the need to provide inorganic dietary P to meet P requirements of growing pigs.

Glossary

Abbreviations

AA

amino acid

ADF

acid detergent fiber

AHF

apparent hindgut fermentation

AID

apparent ileal digestibility

ATTD

apparent total tract digestibility

BW

body weight

CFU

colony-forming unit

CP

crude protein

DE

digestible energy

DM

dry matter

GE

gross energy

HF

hindgut fermentation

Iend

basal ileal endogenous amino acid losses

InsP

inositol phosphate

InsP₆

myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate)

MRS

De Man, Rogosa, and Sharpe

NDF

neutral detergent fiber

NE

net energy

SID

standardized ileal digestibility

STTD

standardized total tract digestibility

TDF

total dietary fiber

Conflict of interest statement

All authors declare no real or perceived conflicts of interest.