Effect of lactic acid fermentation of wheat and barley whole meal flour on carbohydrate composition and digestibility in mink (Mustela vison)

Animal Feed Science and Technology 90 (2001) 199±212

Effect of lactic acid fermentation of wheat and barley whole meal ¯our on carbohydrate composition and digestibility in mink (Mustela vison) G. Skredea,*, S. Sahlstrùma, A. Skredeb, A. Holcka, E. Slindec a MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 AÊs, Norway Department of Animal Science, Agricultural University of Norway, P.O. Box 5025, N-1432 AÊs, Norway c Department of Aquaculture, Institute of Marine Research, P.O. Box 1870, Nordnes, N-5024 Bergen, Norway b

Received 1 December 1999; received in revised form 5 June 2000; accepted 17 December 2000

Abstract Wheat and barley whole meal ¯ours (WMFs) were subjected to treatment by fermentation, autoclaving, and fermentation followed by autoclaving. The WMFs were analysed for chemical composition, formulated into wet diets (282 g kg 1) and fed to adult mink (Mustela vison) for determination of total tract digestibility of total starch, total carbohydrate, crude protein and fat. Fermentation of WMF/water mixtures inoculated with a Lactobacillus sp. (strain AD2) was performed at 308C for 16 h. Autoclaving was carried out for 60 min at 1208C. Fermentation increased colony-forming units (CFUs) to about 108 g 1 and lowered pH to 3.7±3.8 in both WMFs. All carbohydrate parameters were affected by type of cereal, and were, except for total starch, affected by treatment. Levels of total dietary ®bre and b-glucans decreased by fermentation in both WMFs. The decrease in total b-glucans from 33.5 to 18.4 g kg 1 in barley WMF, was mainly restricted to the soluble fraction. Glucose levels in barley WMF increased simultaneously from 0.6 to 12.3 g kg 1. The main effects of autoclaving were increased levels of total dietary ®bre, maltose, and increased hydration capacity. With fermentation prior to autoclaving, increases in levels of the ®bre fractions and maltose were prevented while hydration capacity prevailed as an effect of autoclaving. Compared with fermentation alone, the combined treatment increased damaged starch levels and hydration capacity. Digestibilities of total carbohydrate, crude protein and fat were signi®cantly higher for wheat than for barley. Fermentation had no effect on digestibility of total starch or total carbohydrate of wheat, but increased digestibility of total starch of barley signi®cantly from 0.742 to 0.880, and of total carbohydrate from 0.457 to 0.616. Autoclaving had no signi®cant effect on digestibility of total starch and total carbohydrate of wheat. Digestibility of *

Corresponding author. Tel.: ‡47-649-701-00; fax: ‡47-649-703-33. E-mail address: [email protected] (G. Skrede). 0377-8401/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 2 2 2 - X

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total starch and total carbohydrate in barley increased signi®cantly after autoclaving. Total starch and total carbohydrate digestibility of both wheat and barley were signi®cantly enhanced by combined fermentation and autoclaving compared with fermentation alone. Compared with autoclaving alone, combined fermentation and autoclaving promoted no signi®cant improvement of total starch and total carbohydrate digestibility in wheat, whereas total carbohydrate digestibility in barley increased from 0.605 to 0.672. Fat digestibility was slightly improved by both fermentation and autoclaving. Autoclaving of cereals reduced signi®cantly the faecal dry matter contents of mink. This effect could be counteracted by preceding fermentation. In conclusion, lactic acid fermentation of wheat and especially barley provided chemical changes of bene®t for carbohydrate digestion in the mink. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Lactic acid fermentation; Barley; Wheat; Whole meal ¯our; Starch; b-glucans; Digestibility; Mink

1. Introduction Lactic acid fermentation of wheat and rye has a long tradition in the processing of sourdough bread (Saunders et al., 1972; Barber et al., 1991; MartõÂnez-Anaya et al., 1993). During lactic acid fermentation of cereal ¯our, carbohydrate composition is modi®ed as a result of microbial metabolism and to some extent due to activation of inherent enzymes in the cereal (Barber et al., 1991; Marklinder, 1996). The ability of lactic acid bacteria to metabolise sugars varies with the bacterial strain, and this characteristic is used for classi®cation of the bacteria (Spicher and SchroÈder, 1978). In wheat ¯our, lactic acid fermentation yields increased levels of glucose, fructose, maltose and reduced levels of sucrose and raf®nose (Barber et al., 1991; Rouzaud and MartõÂnez-Anaya, 1993). Oligosaccharide contents (chain length 3±5) have been shown to increase when Lactobacillus plantarum was used as a bacterial starter (Rouzaud and MartõÂnez-Anaya, 1993). In barley, which is rich in mixed-linked …1 ! 3†…1 ! 4†-b-glucans (Bach Knudsen, 1997), lactic acid fermentation can reduce the levels of total b-glucans by up to 31% depending upon ¯our and strain of Lactobacillus (Marklinder and Johansson, 1995). Also solubility of b-glucans decreases during fermentation (Marklinder, 1996). Lactic acid fermentation of cereals may be an interesting approach to increase utilisation of starch, non-starch polysaccharides (NSPs) and possibly other nutrients in carnivorous animals. As a carnivore with a short digestive tract, limited amylase activity, rapid gastrointestinal passage, and only minor microbial activity in the hindgut, the mink (Mustela vison) has a lower capacity to digest native starch than most mammalian species (Glem-Hansen et al., 1977; Bùrsting et al., 1995; Ahlstrùm and Skrede, 1998). Depending on its source, starch gelatinisation achieved by cooking, extrusion, or other methods involving heat treatment improves digestibility of starch in mink (Bùrsting et al., 1995). Non-ruminant animals lack gastrointestinal b-glucanase secretion and are therefore incapable of digesting b-glucans, unless the enzyme is supplied by the feed (Cheeke, 1999). In poultry, b-glucans reduce energy digestibility, and cause viscous digesta and reduced feed intake, thus affecting general performance negatively (Hesselman and Ê man, 1986; Almirall et al., 1995; Svihus et al., 1995). From this, it has been postulated A that lactic acid fermentation of the cereal component of the feed may be bene®cial for digestion and performance in the mink.

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The present investigation was undertaken to study effects of controlled lactic acid fermentation of whole meal ¯ours (WMFs) of wheat and barley on carbohydrate composition as well as nutrient digestibility in mink. The lactic acid fermentation of cereals was investigated as the sole treatment method and in combination with heat treatment by autoclaving, prior to use in mixed wet feed. 2. Materials and methods 2.1. Cereals, fermentation procedure and cereal treatments Commercial WMFs of wheat and barley, ground through a 3.5 mm screen, were obtained from Statkorn AS, Oslo, Norway. The falling number of the whole wheat ¯our was 338 s. Falling number is the time (s) a standardised weight needs to pass through a boiled ¯our/water solution. The time is a function of the viscosity of the solution and thereby a measure of structural properties of the starch. Falling number is used as an indication of sprouting damage in wheat. According to Norwegian standards, a falling number higher than 200 s is required to allow use of WMF in bread baking. The WMFs were fermented using a Lactobacillus strain (AD2) from a spontaneously fermented sourdough from Norwegian rye, isolated on deMan, Rogosa, Sharpe (MRS) agar (Oxoid, Basingstoke, England). The standard fermentation procedure included mixing of the WMFs with water, inoculating with 1  106 bacteria g 1 and incubating at 308C for 16 h. For wheat, 1 kg water kg 1 WMF was used and for barley 1.2 kg water kg 1 WMF. Diets were formulated with the wheat or barley WMF as the sole source of carbohydrate (Table 1). The experiment included four treatments for each of the cereals: untreated, fermented, autoclaved, and fermented followed by autoclaving. To the nonfermented WMFs, water was added prior to autoclaving. The WMF/water mixture nearly ®lled 5-l glass beakers and the conditions for the autoclaving (Autoclave Emmer, Mùglestue, Oslo, Norway) were 1208C for 60 min, followed by 20 min holding in the autoclave without further heating. The ®nal core temperature of the mixtures was 788C. The cereal batches were stored at 48C until the next day when they were processed into

Table 1 Composition of experimental diets given to mink Formulation

g kg

Untreated or fermented wheat or barley WMF Fish meala Raw ®sh ®lleting scrapb Soybean oil Vitamin mixturec Water (to suitable consistency)

282 137 505 74 2

a

Norseamink, Norsildmel, Bergen, Norway. From cod (Gadus morrhua), containing DM of 215 g kg 1. c Composition as shown by Skrede (1979). b

1

as fed

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wet diets. Cereal samples for bacterial counts were analysed immediately after fermentation and autoclaving. For the chemical analyses, duplicate samples from each batch were frozen ( 208C) and analysed once within 1 month. 2.2. Mink experiments Experimental animals were 10-month-old male mink of the standard genotype, with an average initial weight of 2146 g …S:D: ˆ 85†. The animals were kept in individual metabolism cages equipped for controlled feeding and quantitative collection of faeces (Jùrgensen and Glem-Hansen, 1973). The animals were allotted randomly into eight groups of four animals each. Wet feed rations providing approximately 900 kJ metabolisable energy (ME) per animal per day were used. The daily rations were kept at 228C until 24 h before feeding when they were placed in a refrigerator for thawing. Each diet was fed individually to four animals throughout a 3-day preliminary period followed by a 4-day faecal collection period. Feeding and quantitative collection of faeces were carried out once a day. Water was available ad libitum. The animals were cared for according to guidelines given by the Norwegian Animal Research Authority. Faeces were stored deep-frozen in plastic boxes with tightly ®tting lids between collections. Immediately after termination of the experiment, pooled faeces from each animal were freeze-dried, ground and sieved for removal of hair pending analysis. Apparent total tract digestibility coef®cients were determined as ……a b†=a†, where a is the nutrient intake and b the amount of nutrient in faeces. 2.3. Analyses Dry matter (DM), ash, crude protein …N  6:25† and fat contents were determined by the AOAC (1980) methods. Total carbohydrate were calculated from the contents of DM, crude protein, ash and fat as: total carbohydrate ˆ DM …N  6:25† ash fat. Total and insoluble dietary ®bre were analysed according to the method given by AOAC (1990). Total starch, damaged starch, total and insoluble mixed-linked …1 ! 3†…1 ! 4†-b-glucans and raf®nose-series oligosaccharides (RSOs) were all determined enzymatically applying commercial kits (Megazyme PTY, Wicklow, Ireland). Total starch determination was performed without previous ethanol extraction and thus included mono-, di-, and oligosaccharides. Further DMSO treatment of samples was omitted, thus excluding any resistant starch from the total starch content. The fungal aamylase used for determination of damaged starch, solubilises starch available in damaged starch granules while undamaged granules are left intact. In the present study, damaged starch is used as an indicator of starch gelatinisation. RSO was calculated as raf®nose with molecular weight 504. Soluble fractions of dietary ®bre and b-glucans were calculated as the difference between the total and insoluble fractions of the two. Maltose, sucrose, glucose and fructose were determined by HPLC (Sharma et al., 1988) after extraction of samples with 80% ethanol at 808C, evaporation of the alcohol and resolving of the residue in water. The column (Carbohydrate Pb, 250  7:8 mm ID, Chrompack International BV, the Netherlands) was eluted with water (0.4 ml min 1) at 808C. Sugars were detected by a Gilson 132 RI-detector (Gilson, Middleton, WI) and

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identi®ed and quanti®ed by external standards. Lactic acid was determined by HPLC after hot water (608C) extraction and puri®cation of the extracts by solid phase extraction (Sep-Pak C18, Waters Assoc., Milford, MA). The column (HPX-87H, 300  7:8 mm ID, Bio-Rad Laboratories, Richmond, CA) was eluted with 0.005 N H2SO4 (0.6 ml min 1) at 458C (éyaas et al., 1995). External lactic acid standard was used for quanti®cation. Hydration capacity of untreated and treated WMF was determined as weight of sediment per weight of dry sample (g g 1 DM) after hydration and centrifugation (AACC, 1983). Colony-forming units (CFU g 1) were obtained by spreading on plate agar and incubation at 258C for 3 days (NMKL, 1994). The bacteria were characterised according to standard microbial methods. Carbohydrate metabolism of the bacterial strain was determined using an API 50 CHL test kit (Biomerieux SA, Lyon, France). In short, the bacteria were incubated anaerobically at 308C. After 24 and 48 h, the ability of the bacteria to utilise the 49 different carbohydrates in the test was reported as positive, partly positive or negative. Gas production during the incubation period was also recorded. Bacterial growth on agar plates containing 0.1 g 100 ml 1 of barley mixed-linked …1 ! 3†…1 ! 4†-b-glucans (Sigma, St. Louis, MO) as the only source of carbohydrates was evaluated according to Jonsson and Hemmingsson (1991). In this test, breakdown of b-glucans in the agar is veri®ed by ¯ooding the bacterial colonies with 1 mg ml 1 Congo red in water and observing clearing zones. 2.4. Statistics Two-way ANOVA was performed on chemical data, digestibility coef®cients and faecal DM using the general linear model procedure of the Statistical Analysis System (Statistical Analysis System, 1998) with cereal (2) and treatment (4) as the main effects. Differences between pairs were evaluated by Boniferroni test with four planned comparisons (fermented vs untreated, autoclaved vs untreated, fermented and autoclaved vs autoclaved, fermented and autoclaved vs fermented). For parameters with no signi®cant cereal  treatment interaction, the Boniferroni test was performed within the two-way ANOVA. For parameters with signi®cant interaction between effects, one-way ANOVAs and Boniferroni tests were performed for each cereal individually. Root of the mean square error (RMSE) for each parameter was calculated as the square root of the residual mean squares of the two-way ANOVA. The signi®cance level was 95% …p  0:05†. 3. Results 3.1. Characteristics of bacteria and fermentation process The bacterial strain (AD2) used in the present study was selected from a spontaneously fermenting rye sourdough due to its rapid pH-reducing ability during fermentation. The bacterium was isolated on MRS agar suitable for the growth of lactobacilli and tentatively identi®ed as a Lactobacillus sp. due to the typical criteria: rod-shaped, gram positive, catalase negative, and gas producing (heterofermentative). Among the 49 different

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carbohydrates tested in the API-test, the bacteria showed positive reaction for 22, partly positive reactions for ®ve and negative reactions for 20 while only gas and no acids were produced for two carbohydrates. All carbohydrates giving positive reactions also promoted gas production. The metabolisable carbohydrates relevant to the fermented wheat and barley WMFs were glucose, fructose, sucrose, maltose, cellobiose and raf®nose. The AD2 bacterium failed to metabolise starch. The AD2 formed colonies when spread on agar containing barley mixed-linked …1 ! 3†…1 ! 4†-b-glucans (in the following called b-glucans) as the sole source of carbohydrates. The growth was slower than when the agar contained glucose. Flooding the plates with Congo red resulted in colourless zones surrounding each bacterial colony, while the remaining agar developed a red colour typical of the association of Congo red and b-glucans. This indicated that the b-glucans were metabolised by the bacteria. The untreated wheat and barley WMFs initially contained a mixed ¯ora consisting of lactic acid bacteria, mould and yeast, and about 4 g lactic acid kg 1 (Table 2). After inoculation and incubation with AD2, viable counts of lactobacilli increased to about 108 g 1 of the WMF/water mixtures and lactic acid levels increased to 18±19 g kg 1 DM for both cereals. The pH was reduced from nearly neutral to 3.7±3.8 during fermentation. Autoclaving inactivated the initial micro¯ora of the WMFs and had no effect on the levels of lactic acid. There appeared to occur an initial bacterial activity during autoclaving of fermented WMFs, as the lactic acid levels after the combined treatment were higher than those obtained by fermentation alone. 3.2. Effects of cereal and treatment on carbohydrate composition Carbohydrate composition of differently treated wheat and barley WMFs is presented in Table 2. The two-way ANOVA revealed signi®cant effects of cereal for all carbohydrate parameters, with barley WMF having higher levels of all carbohydrates analysed, except total starch, maltose and fructose where wheat WMF levels were higher. Signi®cant effects of treatment were found for all carbohydrate parameters except total starch. There were signi®cant interactions …cereal  treatment† for all carbohydrate parameters. Thus, signi®cant differences between treatments were studied within each cereal by one-way ANOVA and four-comparison Boniferroni test. 3.3. Carbohydrate composition after fermentation Fermentation of wheat WMF signi®cantly decreased levels of total dietary ®bre and b-glucans (Table 2). Among the low-molecular sugars, maltose, sucrose and RSO nearly disappeared, while glucose and fructose increased signi®cantly during fermentation. In barley WMF, levels of total dietary ®bre and total b-glucans as well as of sucrose and fructose decreased signi®cantly during fermentation, while there was a substantial increase in glucose from 0.6 to 12.3 g kg 1 DM. The decrease in total b-glucans from 33.5 to 18.4 g kg 1 DM during fermentation of barley WMF would correspond to the formation of 17 g glucose kg 1 DM. The fermentation primarily in¯uenced the soluble fraction of the b-glucans, only 38% of the initial soluble b-glucans remained in the barley WMF after fermentation. Fermentation had no effect on hydration capacity of any of the

Table 2 Effect of fermentation, autoclaving and fermentation with subsequent autoclaving on DM, pH, lactic acid bacteria (CFU), lactic acid, dietary ®bre, …1 ! 3†…1 ! 4†-bglucans, total and damaged starch, maltose, sucrose, glucose, fructose, RSO and hydration capacity in wheat and barley WMFs

Dietary ®bre (g kg Total Insoluble Soluble

1

DM)

…1 ! 3†…1 ! 4†-b-glucans (g kg Total Insoluble Soluble Total starch (g kg 1 DM) Damaged starch (g kg 1 DM) Maltose (g kg 1 DM) Sucrose (g kg 1 DM) Glucose (g kg 1 DM) Fructose (g kg 1 DM) RSOe (g kg 1 DM) Hydration capacity (g g 1 DM) a

Barley WMF

Untreated

Fermented Autoclaved Fermented/ autoclaved

Untreated

Fermented Autoclaved Fermented/ autoclaved

867 8  102 d 4.1 6.6

442 1  108 18.8 3.7

423 20 3.9 6.7

466 <10 24.6 4.0

868 4  104 d 4.5 6.0

428 8  107 18.1 3.8

410 <10 3.8 6.4

417 <10 27.3 3.9

129 120 10

118 109 9

162 146 16

135 125 11

215 169 46

184 165 19

220 175 45

1 DM) 5.9 3.5 2.4 679 12 0.3 5.5 0.5 1.4 7.0 0.86

3.7 2.4 1.3 675 7 0.0 0.9 3.4 2.0 0.8 0.67

6.1 4.6 1.5 639 212 50.1 5.2 3.8 2.4 5.6 2.72

3.6 2.6 1.0 662 119 0.0 1.0 2.7 2.6 0.7 2.54

33.5 10.9 22.7 546 9 0.2 6.2 0.6 1.4 5.6 1.18

18.4 9.6 8.7 561 22 0.2 1.2 12.3 0.7 4.8 1.16

36.1 18.7 17.4 571 214 30.6 4.7 3.9 2.7 5.2 2.74

Root of mean square error (two-way ANOVA). Not analysed. c CFU g 1 WMF/water mixture. d Mixed ¯ora with mould and yeast. e RSOs calculated as raf®nose (MW 504). b

Significance Cereal

Treatment

Interaction

nab na 0.4 na

na na 0.023 na

na na <0.001 na

na na 0.002 na

193 169 24

4 3 4

<0.001 <0.001 <0.001

<0.001 <0.001 <0.001

0.006 0.002 <0.001

19.4 9.2 10.3 542 223 0.2 1.3 11.5 1.2 4.4 2.55

0.4 0.5 0.5 8 na 0.4 0.1 0.4 0.1 0.3 0.16

<0.001 <0.001 <0.001 <0.001 na <0.001 0.007 <0.001 <0.001 <0.001 <0.001

<0.001 <0.001 <0.001 0.072 na <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

<0.001 <0.001 <0.001 0.002 na <0.001 0.001 <0.001 <0.001 <0.001 <0.001

G. Skrede et al. / Animal Feed Science and Technology 90 (2001) 199±212

DM (g kg 1) Lactic acid bacteria (CFU)c Lactic acid (g kg 1 DM) pH

RMSEa

Wheat WMF

205

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cereals. There appeared to be a slight increase in damaged starch during fermentation of barley WMF. 3.4. Carbohydrate composition after autoclaving Autoclaving of untreated wheat WMF caused a signi®cant increase in the detectable level of total dietary ®bre and signi®cantly increased insolubility of both dietary ®bre and b-glucans (Table 2). Simultaneously there was a tendency towards reduced level of total starch in the autoclaved wheat WMF. In barley WMF, a higher level of total b-glucans was detected after autoclaving. Autoclaving of barley WMF also signi®cantly decreased b-glucan solubility compared with the untreated WMF. Although there were no signi®cant effects of autoclaving on total starch for any of the cereals, the maltose levels increased extensively during autoclaving from 0.3 to 50.1 g kg 1 DM in wheat WMF and from 0.2 to 30.6 g kg 1 DM in barley WMF. Slight but signi®cant increases in glucose and fructose content also occurred during autoclaving. RSO decreased signi®cantly in wheat but not in barley WMF during autoclaving. The gelatinisation of the starch during autoclaving caused substantial increases in both damaged starch and hydration capacity of both wheat and barley WMFs. 3.5. Carbohydrate composition after combined fermentation and autoclaving Fermentation prior to autoclaving partly prevented increases in total dietary ®bre and b-glucans, as well as increasing ®bre insolubility otherwise seen by autoclaving alone (Table 2). The most pronounced effect of the preceding fermentation on low-molecular carbohydrates was the prevention of maltose formation. Level of damaged starch and hydration capacity after combined fermentation and autoclaving appeared to be primarily a function of autoclaving. Compared with fermentation alone, the main signi®cant effects on carbohydrate composition after combined fermentation and autoclaving were higher levels of total and insoluble dietary ®bre in the wheat WMF, and substantially higher levels of damaged starch and hydration capacities of both cereals. 3.6. Digestibility and faecal characteristics in mink The experiments were carried out without problems concerning animal health or appetite, and there were only minor feed refusals. Total tract digestibility in mink is shown in Table 3. There was no signi®cant difference between wheat and barley in total starch digestibility, but signi®cant effects of treatment and cereal  treatment interaction. Fermentation had no signi®cant effect on digestibility of total starch in wheat, but improved total starch digestibility of barley signi®cantly from 0.742 to 0.880. Similarly, autoclaving alone failed to affect total starch digestibility of wheat signi®cantly but increased total starch digestibility of barley. Total starch digestibilities of fermented and subsequently autoclaved wheat and barley were signi®cantly higher than corresponding ®gures for fermented wheat and barley, respectively. The combined treatment of wheat and barley WMFs by fermentation and autoclaving did not promote signi®cantly higher total starch digestibility than autoclaving as the sole treatment method.

Wheat WMF

RMSEa

Barley WMF

Untreated Fermented Autoclaved Fermented/ Untreated Fermented Autoclaved Fermented/ autoclaved autoclaved Digestibility Total starch Total carbohydrate Crude protein Fat Faecal DM (g 100 g 1) a

0.760 0.552 0.874 0.940 36.2

0.702 0.537 0.863 0.948 39.1

0.940 0.698 0.857 0.950 32.5

Root of mean square error (two-way ANOVA).

0.968 0.765 0.864 0.953 36.5

0.742 0.457 0.843 0.932 37.5

0.880 0.616 0.836 0.946 37.8

0.925 0.605 0.835 0.943 29.5

0.945 0.672 0.820 0.943 37.4

0.071 0.057 0.013 0.004 2.3

Significance Cereal

Treatment Interaction

0.236 0.020 <0.001 <0.001

<0.001 <0.005 0.110 <0.001

0.021 0.012 0.391 0.334

0.515

<0.001

0.230

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Table 3 Effect of fermentation, autoclaving and fermentation with subsequent autoclaving on apparent digestibility and faecal DM in mink fed diets with wheat or barley WMF

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Digestibility of total carbohydrate was signi®cantly higher for wheat than for barley. Separate treatment by fermentation or autoclaving had no signi®cant effect on total carbohydrate digestibility of wheat, but both methods increased the total carbohydrate digestibility of barley. Total carbohydrate digestibility of both wheat and barley was enhanced by combined fermentation and autoclaving compared with fermentation alone. In wheat, there was no signi®cant increase in the total carbohydrate digestibility after the combined fermentation and autoclaving by comparison with autoclaving. In barley, the combined treatment increased total carbohydrate digestibility signi®cantly from 0.605 to 0.672 obtained after autoclaving alone. Crude protein digestibility was signi®cantly higher with wheat diets than with barley diets. There were no signi®cant effects of treatment or cereal  treatment interaction on crude protein digestibility. There were slight but signi®cant effects of cereal and treatment on fat digestibility, but no cereal  treatment interaction. Diets containing barley revealed lower fat digestibility than the wheat diets. Both fermentation and autoclaving increased fat digestibility compared with untreated cereal. Combined fermentation and autoclaving had no additional effect on fat digestibility compared with fermentation or autoclaving alone. There were no effects of cereal and no cereal  treatment interactions on contents of DM in mink faeces (Table 3). While treatment by fermentation had no effect, autoclaving caused a signi®cant reduction in faecal DM content. Preceding fermentation counteracted this effect. 4. Discussion The bacterial strain used for fermentation was isolated from a culture in a spontaneously fermented Norwegian rye sourdough, under conditions similar to those reported by LoÈnner et al. (1986) as suitable for growth of a micro¯ora dominated by Lactobacillus spp. By comparing with the metabolising sugar pro®les of bacteria isolated from sourdoughs, the bacterial strain appeared different from the Lactobacilli described by Spicher and SchroÈder (1978). Gas-producing lactic acid bacteria as the AD2 strain of the present study metabolise hexoses with the production of equimolar amounts of lactic acid, acetic acid or ethanol and carbon dioxide (LoÈnner, 1988). The levels of lactic acid in the fermented cereals thereby re¯ect the amount of carbon dioxide produced during fermentation. The gas escapes from the fermentation mixture and causes loss in cereal DM. In the present study, the maximum lactic acid level of 27.3 g kg 1 DM for the fermented and autoclaved barley WMF would correspond to a carbon dioxide production, and thus a maximum loss in DM, of 13.3 g kg 1 DM. Losses in cereal DM due to carbon dioxide production during fermentation is therefore considered to be of minor importance when evaluating the effects of lactic acid fermentation on feed value. Sugar levels decrease during lactic acid fermentation unless generated at least as rapid as metabolised (MartõÂnez-Anaya et al., 1993). From the API-test it was concluded that the Lactobacillus strain used for fermentation in the present study metabolised mono- and disaccharides occurring in the wheat and barley WMFs. As a consequence, sucrose and fructose decreased during fermentation. Glucose, however, increased during fermentation

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especially in the barley WMF and appeared to be liberated from other carbohydrates present. Rouzaud and MartõÂnez-Anaya (1993) have previously reported increased levels of glucose during lactic acid fermentation of wheat ¯our. Starch may be excluded as the source of glucose in the present study, as the AD2 bacteria were unable to metabolise starch. Most likely, the increased glucose content of barley WMF originated from b-glucans, as the AD2 bacteria hydrolysed the …1 ! 4†-b-glucosidic binding of cellobiose, the same type of binding as in the barley mixed-linked …1 ! 3†…1 ! 4†-b-glucans. The AD2 bacteria also grew on agar with mixed-linked …1 ! 3†…1 ! 4†-b-glucans as the sole carbohydrate. Lactobacilli capable of degrading mixed-linked …1 ! 3†…1 ! 4†-b-glucans have previously been isolated from pigs (Jonsson and Hemmingsson, 1991) and used in barley sourdoughs (Marklinder et al., 1996). However, endogenous b-glucanase activity in barley ¯our may contribute to the reduction in b-glucan content during fermentation (Marklinder, 1996). The present ®nding that only soluble b-glucans were subjected to degradation during fermentation agrees with earlier observations by Marklinder (1996). This may be of nutritional importance since the ability of b-glucans to bind water and form viscous solutions is associated with the soluble fraction of the polymer Ê man et al., 1990). (A Autoclaving caused starch gelatinisation, shown by increased levels of damaged starch and hydration capacity in both wheat and barley. In wheat WMF, autoclaving alone tended to decrease levels of total starch. Simultaneously there was a comparable increase in the levels of total dietary ®bre, indicating that autoclaving may have caused formation of resistant starch in the wheat WMF. Berry (1986) reported progressively increased yields of resistant wheat starch by autoclaving with water levels increasing from 150 to 800 g kg 1. Thus, the water content (577 g kg 1 DM) during autoclaving in the present study was within the range known to allow formation of resistant starch in wheat. With both cereals, autoclaving caused extensive starch degradation. This was not re¯ected in the levels of total starch, but was veri®ed by an increase in maltose. In the present study, total starch included all oligomers with …1 ! 4†-a-glucosidic bindings as well as glucose. Thus, endogenous b-amylase in the WMF must have been active during autoclaving. The fermentation process apparently inactivated the b-amylase, as no maltose formation occurred when the WMFs were fermented prior to autoclaving. We have no explanation as to the increased level of b-glucans in the barley WMF after autoclaving, except that autoclaving may have increased the availability of the b-glucans to the analytical enzymes. Total tract digestibility in mink was measured in the present study. In mink there are only minor effects of post-ileal fermentation on digestibility (Skrede, 1979). Thus, enzymatic digestion and disappearance in the small intestine mostly determine total tract digestibility in this species. Different particle size of wheat and barley is known to in¯uence carbohydrate digestibility in mink (Glem-Hansen and Jùrgensen, 1977). Fine grinding is generally recommended for practical diets due to increased digestibility and improved feed consistency. In the present study, wheat and barley were coarsely ground to pass a 3.5 mm screen to avoid masking of the effects of fermentation and autoclaving. Bùrsting et al. (1995), working with ®nely ground (0.5 mm screen) raw and boiled whole wheat kernels fed to mink, found average values of total crude carbohydrate digestibilities of 0.645 and

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0.737, respectively. The latter values are somewhat higher than those obtained for raw and autoclaved wheat in the present study. The starch digestibility of raw wheat kernels was 0.844 (Bùrsting et al., 1995), while digestibility of starch in raw wheat was 0.760 in the present study. It is interesting to note, however, that combined fermentation and autoclaving in the present study gave higher total carbohydrate digestibility than boiled wheat kernels in the study of Bùrsting et al. (1995), in spite of the great difference in particle size. With boiled wheat kernels the starch digestibility was 0.907 (Bùrsting et al., 1995), whereas the present study showed starch digestibilities of 0.940 for autoclaved wheat and 0.968 after combined fermentation and autoclaving. However, comparison of results from the present study with digestibility ®gures from other studies applying smaller particle size, should be done with great caution. In the present study, mink were fed diets with all the carbohydrates originating from the investigated cereals. Digestibility coef®cients for total starch and total carbohydrate would thus be representative of the effects of cereal type and treatment. However, it should be noted that total carbohydrate were estimated by difference, thus being in¯uenced by analytical errors for DM, crude protein, fat and ash. Total starch was determined enzymatically without alcohol extraction, thus including glucose, maltose and oligosaccharides in diets as well as in faeces. Using this approach partly degraded starch in faeces is recovered as undigested. Crude protein and fat in the diets used originated mostly from other ingredients than the investigated cereals. Hence, differences in the protein and fat digestibilities may be indicating associative effects of different cereals in the diet, rather than direct effects. Since there were no differences in protein digestibility and only minor differences in fat digestibility among mink given diets with differently treated wheat or barley, it is unlikely that there were substantial associative effects of fermentation on protein and fat digestibility. However, barley b-glucans reduce fat digestibility in other species (Bergh et al., 1999), and the slightly improved fat digestibility in mink fed fermented cereal may be caused by a reduction in soluble b-glucans. Thus, soluble dietary ®bre may affect fat digestibility by reduced emulsi®cation of dietary fat (Pasquier et al., 1996), and by inhibited formation of lipid micelles (Cheeke, 1999). However, in the present study autoclaving improved fat digestibility to about the same extent as fermentation. Fermentation of wheat and barley may have major nutritional effects by eliminating or reducing the negative impact of NSP in both cereals. This is not achieved by autoclaving. On the other hand, heat treatment of cereals has bene®cial effects on carbohydrate digestibility in mink due to gelatinisation of the starch (Bùrsting et al., 1995). Accordingly, combined fermentation and heat treatment may serve a dual purpose by removal of antinutritive components, and thereby increasing carbohydrate digestibility and reducing the water-binding action of soluble indigestible carbohydrates, and in addition improval of starch digestibility by gelatinisation. This could result in improved energy utilisation and performance, especially in species with limited a-amylase secretion and hindgut fermentation, and restricted ability to digest native starches, like the mink (Bùrsting et al., 1995) and other carnivores. In the present study, fermentation of barley without subsequent gelatinisation by autoclaving enhanced total carbohydrate and starch digestibility in mink, whereas identical treatment of wheat had no such effect. Since the lactic acid bacteria used were

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found to metabolise soluble sugars and b-glucans rather than starch, improved barley starch digestibility caused by fermentation could be an indirect effect related to breakdown of components capable of encapsulating starch or creating a viscous environment in the gastrointestinal tract (Bergh et al., 1999). This could also explain why the effect of combined fermentation and autoclaving of barley on total carbohydrate digestibility in mink exceeded the effects of fermentation and autoclaving separately. Barley as a component of extruded diets has been shown to decrease faecal DM percentages in dogs (GroÈner and Pfeffer, 1997). This could be due to b-glucans, since Gohl and Gohl (1977) showed that b-glucans increased weight of faeces in rats. In the present study, autoclaving of cereals without previous fermentation reduced faecal DM percentages in mink, possibly due to inactivation of endogenous enzymes in the cereal. Thus, fermentation prior to autoclaving prevented the reduction in faecal DM caused by autoclaving, possibly due to partial degradation of b-glucans and other ®bre components. Although the signi®cance of the latter observations remains to be veri®ed, it may be of importance to reduce risk of dehydration in lactating mink females. References AACC, 1983. Hydration Capacity of Pregelatinized Cereal Products. Method 56-20. Approved Methods of the American Association of Cereal Chemists, 8th Edition, St. Paul, MN. Ahlstrùm, é., Skrede, A., 1998. Comparative nutrient digestibility in dogs, blue foxes, mink and rats. J. Nutr. 128, 2676S±2677S. Almirall, M., Francesch, M., Perez-Vendrell, A.M., Brufau, J., Esteve-Garcia, E., 1995. The differences in intestinal viscosity produced by barley and b-glucanase alter digesta enzyme activities and ileal nutrient digestibilities more in broiler chicks than in cocks. J. Nutr. 125, 947±955. Ê man, P., Pettersson, D., Graham, H., 1990. Chemical and nutritional evaluation of high-moisture barley and A high-moisture barley treated with Lactobacilli or Lactobacilli and yeast. Anim. Feed Sci. Technol. 29, 223±235. AOAC, 1980. Of®cial Methods of Analysis, 13th Edition. Association of Of®cial Analytical Chemists, Washington, DC, pp. 125±132. AOAC, 1990. Of®cial Methods of Analysis, 15th Edition. Association of Of®cial Analytical Chemists, Arlington, VI, pp. 1105±1106. Bach Knudsen, K.E., 1997. Carbohydrate and lignin content of plant materials used in animal feeding. Anim. Feed Sci. Technol. 67, 319±338. Barber, S., BaÂguena, R., de Barber, C.B., MartõÂnez-Anaya, M.A., 1991. Evolution of biochemical and rheological characteristics and breadmaking quality during a multistage wheat sour dough process. Z. Lebensm. Unters. Forsch. 192, 46±52. Ê man, P., 1999. Nutritional in¯uence of broiler chicken diets based on covered Bergh, M.O., Razdan, A., A normal, waxy and high amylose barleys with or without enzyme supplementation. Anim. Feed Sci. Technol. 78, 215±226. Berry, C.S., 1986. Resistant starch: formation and measurement of starch that survives exhaustive digestion with amylolytic enzymes during the determination of dietary ®bre. J. Cereal Sci. 4, 301±314. Bùrsting, C., Bach Knudsen, K.E., Steenfeldt, S., Mejborn, H., Eggum, B.O., 1995. The nutritive value of decorticated mill fractions of wheat. 3. Digestibility experiments with boiled and enzyme treated fractions fed to mink. Anim. Feed Sci. Technol. 53, 317±336. Cheeke, P.R., 1999. Applied Animal Nutrition. Feed and Feeding, 2nd Edition. Prentice-Hall, Englewood Cliffs, NJ, 523 pp. Glem-Hansen, N., Jùrgensen, G., 1977. Fordùjelighed og kemisk sammensñtning af fodermidler. Dansk Pelsdyravl 40, 289±291.

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Glem-Hansen, N., Christensen, K.D., Jùrgensen, G., 1977. Content of different carbohydrate fractions related to the digestibility of carbohydrates in diets for mink. Scientifur 1 (4), 28±33. Gohl, B., Gohl, I., 1977. The effect of viscous substances on the transit time of barley digesta in rats. J. Sci. Food Agric. 28, 911±915. GroÈner, T., Pfeffer, E., 1997. Digestibility of organic matter and digestible energy in single ingredients of extruded dog feeds and their effects on faecal dry matter concentration and consistency. J. Anim. Physiol. Anim. Nutr. 77, 214±220. Ê man, P., 1986. The effect of b-glucanase on the utilization of starch and nitrogen by broiler Hesselman, K., A chickens fed on barley of low- and high-viscosity. Anim. Feed Sci. Technol. 15, 83±93. Jonsson, E., Hemmingsson, S., 1991. Establishment in the piglet gut of lactobacilli capable of degrading mixedlinked b-glucans. J. Appl. Bact. 70, 512±516. Jùrgensen, G., Glem-Hansen, N., 1973. A cage designed for metabolism and nitrogen balance trials with mink. Acta Agric. Scand. 23, 3±4. LoÈnner, C., 1988. Starter cultures for rye sour doughs. Characteristic and functions of lactic acid bacteria. Ph.D. Thesis. Lund University, Lund, Sweden, pp. 14±17. LoÈnner, C., Welander, T., Molin, N., DostaÂlek, M., 1986. The micro¯ora in a sour dough started spontaneously on typical Swedish rye meal. Food Microbiol. 3, 3±12. Marklinder, I., 1996. Lactic acid-fermented oats and barley for human dietary use with special reference to Lactobacillus spp. and to nutritional and sensory properties. Ph.D. Thesis. University of Uppsala, Uppsala, Sweden, pp. 38±42. ISBN 91-554-3687-0. Marklinder, I., Johansson, L., 1995. Sour dough fermentation of barley ¯ours with varied content of mixedlinked …1 ! 3†, …1 ! 4†-b-D-glucans. Food Microbiol. 12, 363±371. Ê ., Johansson, L., 1996. In¯uences of lactic acid bacteria on technological, nutritional, Marklinder, I., Haglund, A and sensory properties of barley sour dough bread. Food Qual. Preference 7, 285±292. MartõÂnez-Anaya, M.A., Pitarch, B., de Barber, C.B., 1993. Biochemical characteristics and breadmaking performance of freeze-dried wheat sour dough starters. Z. Lebensm. Unters. Forsch. 196, 360±365. NMKL, 1994. Aerobic Microorganisms (Plate Count). Determination by the Plate Count Method at 308C in Milk, Cream and Icecream. Method No. 27, 3rd Edition. Nordic Committee on Food Analysis, Espoo, Finland, 4 pp. éyaas, J., Storrù, H., Lewin, D.W., 1995. The effective diffusion coef®cient and the distribution constant for small molecules in calcium alginate gel beads. Biotechnol. Bioeng. 47, 422±500. Pasquier, B., Armand, M., Castelain, C., Guillon, F., Borel, P., Lafont, H., Lairon, D., 1996. Emulsi®cation and lipolysis of triacylglycerols are altered by viscous soluble dietary ®bres in acidic gastric medium in vitro. Biochem. J. 314, 269±275. Rouzaud, O., MartõÂnez-Anaya, M.A., 1993. Effect of processing conditions on oligosaccharide pro®le of wheat sourdoughs. Z. Lebensm. Unters. Forsch. 197, 434±439. Saunders, R.M., Ng, H., Kline, L., 1972. The sugars of ¯our and their involvement in the San Francisco sour dough French bread process. AACC 49, 86±91. Sharma, D.D., D'Souza, V.F., McConnell, M.B., Mazza, G., 1988. Identi®cation and quanti®cation of sugars in winter-hardy apples by high performance liquid chromatography. Can. Inst. Food Sci. Technol. J. 21, 435±437. Skrede, A., 1979. Utilization of ®sh and animal byproducts in mink nutrition. IV. Fecal excretion and digestibility of nitrogen and amino acids by mink fed cod (Gadus morrhua) ®llet or meat-and-bone meal. Acta Agric. Scand. 29, 241±257. Spicher, G., SchroÈder, R., 1978. Die Mikro¯ora des Sauerteiges. IV. Mitteiling: Untersuchungen uÈber die Art der ``Reinzuchtsauern'' anzutreffenden staÈbchenfoÈrmigen MilchsaÈurebakterien (genus Lactobacillus beijerinck). Z. Lebensm. Unters. Forsch. 167, 342±354. Statistical Analysis System, 1998. Statistix for Windows 2.0. Analytical Software, Tallahassee, FL. Svihus, B., Selmer-Olsen, I., BraÊthen, E., 1995. Effect of different preservation methods for high moisture barley on feeding value for broiler chickens. Acta Agric. Scand. 45, 252±259.