Effect of a Kestose and Nystose Preparation on Growth Performance and Gastrointestinal Tract Function of Turkeys Z. Zdun´czyk,* J. Jus´kiewicz,*1 J. Stan´czuk,* J. Jankowski,† and B. Kro´l‡ *Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, 10-747 Olsztyn; †Department of Poultry Science, University of Warmia and Mazury in Olsztyn, 10-718, Poland; and ‡Institute of Chemical Technology, Technical University of Lo´dz´, 90-924, Poland ABSTRACT The aim of the present study was to determine the effects of dietary administration of a fructooligosaccharide preparation rich in kestose and nestose on growth performance and gastrointestinal parameters in young turkeys. The kestose and nestose preparation was obtained through bioconversion of sucrose using fungi fructosyl transferase and contained in DM 39.9% of kestose, 17.6% of nystose, as well as 26.5% of glucose and 14.7% of sucrose. Three dietary levels of the sum of kestose and nystose (0.3, 0.6, and 1.2%) were fed to growing turkeys for 8 wk. When compared with the control treatment, addition of the kestose and nestose preparation had no effect on feed intake, feed conversion, and BW. The kestose and nestose-supplemented diet, especially the medium level of kestose and nystose, influenced mi-

crobial metabolism, especially in the ceca. Compared with the control group, the medium level of kestose and nestose decreased relative weight of gizzard (from 18.67 to 16.51 g/kg of BW) and weight of small intestine tissue (from 23.3 to 19.6 g/kg of BW) and increased weight of ceca digesta (from 3.51 to 4.77 g/kg of BW) as well as activities of microbial β-glucosidase (an increase from 0.22 to 0.38 U/g) and α-galactosidase (an increase from 0.90 to 1.61 U/g), pH of digesta (a decrease from 6.13 to 5.79), concentration of NH3 (an increase from 0.60 to 0.98 mg/g), and concentration of total short-chain fatty acids (an increase from 81.1 to 107.7 ␮mol/g) in the cecal digesta. A high content of kestose and nestose in the diet caused a decrease in ileal and cecal pH (to 5.42 and 5.49, respectively).

Key words: kestose, nystose, fructooligosaccharide, gastrointestinal tract, metabolism 2007 Poultry Science 86:1133–1139

INTRODUCTION Earlier research has shown that fructooligosaccharides (FOS) are not hydrolyzed by salivary and pancreatic amylases and that few or none are hydrolyzed by intestinal brush border enzymes (Oku et al., 1984). Consequently, they reach the large intestine, where they act as specific growth substrates for bacteria (Howard et al., 1995) and are thought to be responsible for an increase in the density of beneficial bacteria in the gastrointestinal tract (Williams et al., 1994). As a result of these properties of FOS, diets with their content can reduce susceptibility to Salmonella colonization in the gastrointestinal tract (Bailey et al., 1991; Fukata et al., 1999) and can improve health and performance of poultry (Monsan and Paul, 1995; Patterson and Burkholder, 2003). However, the results of previous investigations have been inconclusive in establishing optimal doses of FOS for different species and feeding programs in poultry. Some reports have indicated

©2007 Poultry Science Association Inc. Received November 10, 2006. Accepted February 4, 2007. 1 Corresponding author: [email protected]

that in a diet for broiler chickens the content of FOS in diets ranged from 0.2 to 2% (Du¨rst, 1996; Farnworth et al., 1996; Patterson et al., 1997). In the case of turkeys, Jus´kiewicz et al. (2002a) found that a low dose of FOS (0.4%) had no significant effect on BW of poults and negligibly changed the short-chain fatty acid (SCFA) concentration in the cecal digesta. Jus´kiewicz et al. (2006) also found that increasing FOS content to 2% of a diet caused improvement of fermentation processes in ceca, without any influence on growth performance of turkeys. The FOS occur in many plants as homogenous oligosaccharides built exclusively of fructose and as heterogeneous oligosaccharides formed by the successive binding of fructose units (up to 8 residues) to the fructose moiety of sucrose (Niness, 1999). For food purposes, FOS preparations are usually produced through partial depolymerization of inulin extracted from chicory root. Another way to obtain a FOS preparation is sucrose conversion mediated by transfructosylating enzymes, especially by fructosyl transferase (Iiang, 2002). The process of transfructosylation of sucrose has been shown to cause the synthesis of FOS with a low degree of polymerization (Byung and Yun, 1998). The use of fructosyl transferase affords an opportunity to obtain a product containing about 60% of

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FOS, mainly kestose, nystose, and fructosyl nystose forms. The rest consists of unprocessed sucrose and glucose residues, which suppress the transfructosylation process. Such a product could be less expensive than pure FOS, whose production requires additional enzyme, β-Dfructofuranosidase (Kurakake et al., 1996), or purification using low-pressure chromatography (Bornet et al. 2002). Moreover, several studies with chicory inulin and shortchain FOS (Xu et al., 2003; Zdun´czyk et al., 2005) have indicated that the kestose and nystose preparation can be well utilized in the gastrointestinal tract of turkeys, which is characterized by the short relative length of the midgut and hindgut. The aim of the present study was to determine the effects of dietary administration of a short-chain FOS preparation, containing kestose and nystose, on growth performance and gastrointestinal functioning in young turkeys.

MATERIALs AND METHODS Preparation of FOS The kestose and nystose preparation was produced at Polfarmex SA (Kutno, Poland) through bioconversion of sucrose with the use of fructosyl transferase from Aspergillus niger (Kro´l and Klewicki, 2004). The final preparation contained 58.8% of FOS (39.9% of kestose and 17.6% of nystose and 1.3% of fructosyl nystose) and 41.2% monoand disaccharides (14.7% of sucrose and 26.5% of glucose). The kestose and nystose preparation was applied in experimental diets for turkeys in the first 8 wk of feeding.

Diets, Bird Management, and Sample Collection The 8-wk experiment was conducted on 320 three-dayold BUT-9 (Hatchery Grelavi Co., Ketrzyn, Poland) male turkey poults, randomly assigned to 4 dietary treatments, each consisting of 4 pens of 20 birds per pen. The birds were vaccinated at 1 d of age using the Aviffa-RTI (Rhone Merieux, Lyon, France), a vaccine against turkey rhinotracheitis (an aerosol-spraying method). The poults were raised in floor pens and given 16L:8D per day. Room temperature was maintained at 32°C for the first 5 d and gradually reduced according to normal management practices until a temperature of 22°C was reached. Birds were given free access to mash diets formulated to meet nutrient requirements of turkeys (NRC, 1994). The 4 experimental diets contained 0, 0.5, 1.0, or 2% FOS preparation, added at the expense of wheat (Table 1). The content of pure kestose and nystose was estimated at 0, 0.3, 0.6, and 1.2%, respectively. All procedures were approved by the University of Warmia and Mazury Animal Care and Use Committee. At the end of each 4-wk period, birds were weighed, and feed consumption was recorded. Weight gain and feed efficiency at each 4-wk period were calculated. For performance indices, each pen was considered an experimental

Table 1. Composition (%) and calculated analysis of basal diet1 Feeding period, wk Component Wheat Corn Soybean meal Fish meal Chicken fat NaCl Limestone Monocalcium phosphate DL-Met 99 L-Lys 99 MonohydroCl L-Thr Mineral and vitamin premix2 Calculated composition ME, kcal/kg CP, % Crude fiber, % Crude fat, % Lys, % Met + Cys, % Ca, % Available P, % Na, % Cl, %

1 to 4

5 to 8

23.75 20.00 44.00 4.00 2.50 0.18 1.00 2.90 0.25 0.42 — 1.00

27.57 20.00 39.00 3.00 4.50 0.23 0.90 2.95 0.35 0.40 0.10 1.00

2,797 27.95 3.40 4.57 1.80 1.10 1.29 0.69 0.15 0.24

2,874 25.43 3.28 5.51 1.61 1.13 1.23 0.69 0.15 0.25

1 Four experimental diets contained 0, 0.5, 1.0, or 2.0% fructooligosaccharide preparation, added at the expense of wheat. 2 For 1 to 4 and 5 to 8 wk of feeding, the vitamin and mineral premix supplied the following per kilogram of diet: vitamin A, 15,000 and 13,000 IU, and vitamin E, 40 and 35 mg, respectively. For 1 to 8 wk of feeding, the vitamin and mineral premix supplied the following per kilogram of diet: Se, 0.3 mg; Mn, 150 mg; Zn, 90 mg; Fe, 60 mg; Cu, 15 mg; I, 1 mg; diclazuril, 1 mg; vitamin D3, 4,500 IU; vitamin K3, 2.5 mg; thiamin, 3.5 mg; riboflavin, 10 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; folic acid, 2 mg; biotin, 0.36 mg; niacin, 75 mg; pantothenic acid, 21 mg; and choline, 600 mg.

unit. The experiment lasted 8 wk, after which time the birds were weighed, and 8 turkeys (2 birds from each replicate) representing an average BW of each group were killed by cervical dislocation according to the recommendations for euthanasia of experimental animals (Close et al., 1997). Segments of the digestive tract (crop, gizzard, small intestine, ceca, and colon) were removed and weighed.

Measurement of Gastrointestinal Tract Indices As soon as possible after euthanasia (ca. 30 min), ileal and cecal pH was measured using a microelectrode and pH-ion meter (model 301, Hanna Instruments, Vila do Conde, Portugal). The crop and gizzard tissues were cleaned and weighed. Samples of ileal (from the central ileum) and cecal contents were immediately transferred to microfuge tubes, which were stored at −70°C. The cecal wall was flushed clean with ice-cold saline, blotted on filter paper, and weighed (cecal wall weight). The same procedure was applied to the small intestine and colon. Dry matter of intestinal and cecal digesta was determined at 105°C. In fresh cecal digesta samples, NH3 was extracted and trapped in a solution of boric acid in Conway

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KESTOSE AND NYSTOSE PREPARATION Table 2. Feed intake, feed conversion ratio (FCR), and BW of turkeys fed diets without or with different content of kestose and nystose preparation

Item Feed intake, kg 1 to 4 wk 5 to 8 wk BW, kg 4 wk 8 wk FCR, kg/kg 1 to 4 wk 5 to 8 wk 1 to 8 wk

Control

0.3% kestose and nystose

0.6% kestose and nystose

1.2% kestose and nystose

SEM

1.33 5.24

1.34 5.13

1.38 5.26

1.34 5.37

0.01 0.04

0.88 3.84

0.88 3.84

0.91 3.81

0.91 3.89

0.01 0.02

1.52 1.77 1.71

1.53 1.75 1.70

1.52 1.76 1.71

1.47 1.79 1.71

0.01 0.01 0.01

dishes and was determined by direct titration with H2SO4 (Hofirek and Haas, 2001).

Data Analysis

Activity of Microbial Enzymes and SCFA Analysis

The results of the experiment were analyzed using the one-way ANOVA test, and significant differences between groups were determined by Duncan’s multiple range test. Differences were considered significant at P ≤ 0.05.

The glycolytic activity in the cecal digesta was measured by the rate of p- or o-nitrophenol release from their nitrophenylglucosides according to the modified method of Djouzi and Andrieux described by Jus´kiewicz et al. (2002b). The following substrates were used: p-nitrophenyl-α-D-glucopyranoside (for α-glucosidase) and p-nitrophenyl-β-D-glucopyranoside (for β-glucosidase), p-nitrophenyl-α-D-galactopyranoside (α-galactosidase), o(β-galactosidase), nitrophenyl-β-D-galactopyranoside and p-nitrophenyl-β-D-glucuronide (for β-glucuronidase). The reaction mixture contained 0.3 mL of substrate solution (5 mM) and 0.2 mL of a 1:10 (vol/vol) dilution of the cecal sample in 100 mM phosphate buffer (pH 7.0) after centrifugation at 10,000 × g for 15 min. Incubation was carried out at 37°C, and p-nitrophenol was quantified at 400 nm and at 420 nm (o-nitrophenol concentration) after the addition of 2.5 mL of 0.25 M cold Na2CO3. The enzymatic activity (α- and β-glucosidase, α- and β-galactosidase, and β-glucuronidase) was expressed as micromoles of product formed per minute (IU) per gram of digesta. The protein content in the supernatant was determined by Lowry’s method (Lowry et al., 1951) using BSA as the standard. Cecal digesta samples were subjected to SCFA analysis using gas chromatography (Shimadzu GC-14A, Shimadzu Co., Kyoto, Japan). The samples (0.2 g) were mixed with 0.2 mL of formic acid, diluted with deionized water, and centrifuged at 10,000 × g for 5 min. Supernatant was loaded onto the chromatography glass column (2.5 m × 2.6 mm) packed with 10% SP-1200 and 1% H3PO4 on 80/ 100 Chromosorb W AW (Supelco Co., Bellefonte, PA). The chromatograph was coupled to a flame ionization detector, and we used a column temperature of 110°C, detector temperature of 180°C, and injector temperature of 195°C. Cecal SCFA pool size was calculated as the sum of SCFA concentration in digesta and cecal digesta mass.

RESULTS AND DISCUSSION Growth Performance Diet intake, feed efficiency, and BW of turkeys were similar for all treatments (Table 2). Addition of 0.3 to 1.2% of kestose and nystose to turkey diets had no effect on performance parameters. Similar results have been obtained in other experiments by supplementation of a broiler diet with 2% kestose (Patterson et al., 1997) and a turkey diet with 0.5 to 2% of chicory FOS (Jus´kiewicz et al., 2006). Opposite results (i.e., an increase in BW of broilers) have been reported after supplementation of a broiler diet with 0.4% FOS (Xu et al., 2003). Addition of 1% sucrose thermal oligosaccharide caramel (mainly kestose) has been shown to increase BW gain of broilers compared with the control group (Orban et al., 1997). In that experiment, the increased content of sucrose thermal oligosaccharide caramel to 3% of a diet did not cause an additional improvement in diet intake, feed conversion, and BW of broilers.

Gastrointestinal Tract Indices Parameters of the upper part of the gastrointestinal tract of turkeys fed diets without or with different content of FOS are shown in Table 3. The relative weight of crop was similar among all treatments, whereas the medium as well as high dose of the kestose and nystose preparation was characterized by a lower relative gizzard weight compared with the control treatment. The relative mass of ileal tissue was significantly higher for the control group, and the weight of ileal digesta was the highest when 1.2% of kestose and nystose was added to a diet (P < 0.05 vs. the control and 0.6% kestose and nystose

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Table 3. The effect of fructooligosaccharide addition on intestinal segment weights and ileal pH values in turkeys after 8 wk of growth

Control

0.3% kestose and nystose

0.6% kestose and nystose

1.2% kestose and nystose

SEM

Crop, g/kg of BW Gizzard, g/kg of BW

4.42 18.67a

4.37 17.71ab

4.00 16.51b

4.54 16.51b

0.10 0.35

Small intestine Tissue, g/kg of BW Ileal digesta, g/kg of BW DM of ileal digesta, % pH of ileal digesta

23.3a 22.4b 15.2 6.04a

20.2b 25.9ab 14.4 5.73ab

19.6b 22.2b 15.0 5.56ab

18.99b 28.0a 16.0 5.42b

0.44 0.93 0.29 0.09

Item

Means within rows with no common superscripts differ significantly (P < 0.05).

a,b

groups). Dry matter concentration of ileal digesta was similar in all dietary treatments, but pH of digesta was the highest in the control group and the lowest in the group fed the highest level of kestose and nystose. Overall, a gradual decrease in the pH value was observed; the higher the amount of kestose and nystose in a diet, the more ileal digesta was acidified. It has been reported that the microflora of the small intestine may be responsible for initial utilization of short-chain FOS, thus creating more favorable intestinal environment (i.e., low pH) for growth (Xu et al., 2003). Similar results have been obtained in an experiment in which turkeys were fed a diet containing chicory short-chain FOS (Zdun´czyk et al., 2005; Jus´kiewicz et al., 2006). Application of short-chain oligofructose has been shown to more effectively decrease ileal pH than long-chain inulin in turkeys (Jus´kiewicz et al., 2004; Zdun´czyk et al., 2005). In another study by Farnsworth et al. (1996), supplementation of a broiler diet with 2% FOS had no effect on the pH in the upper parts of the gastrointestinal tract. In the present study, supplementation of a diet with the kestose and nystose preparation changed some cecal parameters (Table 4). The highest weight of cecal tissue was observed in the group fed 0.3% kestose and nystose preparation, and it is not clear why the lowest value was observed for the 0.6% kestose and nystose treatment.

Compared with the control group, higher weights of cecal digesta were observed in the groups fed the diets with low and medium levels of kestose and nystose. Dry matter of cecal digesta was similar in all dietary treatments, but total amount of DM in ceca (calculated to kg of BW) was significantly higher in the group fed the diets with low and medium levels of kestose and nystose. Compared with the control group, lower pH of digesta and, simultaneously, the highest concentration of NH3, was observed in the group fed a diet with a high content of kestose and nystose. When compared with the control treatment, the low and medium levels of the kestose and nystose preparation also caused a significant increase in the concentration of NH3 in the cecal digesta. In all these kestose and nystose groups, numerically higher content of protein in cecal digesta was observed. The relative weight of colon tissue was similar among groups; however, the amount of colonic digesta gave ambiguous response to the experimental factor (e.g., the 1.2% kestose and nystose group was characterized by 2.5 times lower colonic digesta content than that observed with the medium dose of FOS preparation. Results of numerous in vivo experiments indicate that dietary FOS increase large bowel mass and decrease pH of cecal digesta (Campbell et al., 1997; Kleessen et al., 2001). Such responses have been observed for broilers fed

Table 4. The effect of fructooligosaccharide addition on cecal and colonic weights and on indicators of cecal metabolism in turkeys after 8 wk of growth

Item

Control

0.3% kestose and nystose

Ceca Tissue, g/kg Digesta, g/kg of BW DM of digesta, % DM, g/kg of BW pH of digesta NH3, mg/g NH3, mg/kg of BW Protein, mg/g Protein, mg/kg of BW

5.45ab 3.51b 16.6 0.59b 6.13a 0.60b 2.54b 0.39 1.64

5.75a 5.00a 17.0 0.85a 5.89ab 0.89a 4.34a 0.44 2.09

4.77b 4.77a 17.9 0.85a 5.79ab 0.98a 4.18a 0.47 2.05

5.20ab 3.68b 16.2 0.60ab 5.49b 1.06a 4.18ab 0.44 1.99

0.14 0.24 0.47 0.04 0.08 0.05 0.29 0.01 0.12

2.88 1.66ab

2.81 1.61ab

2.64 2.24a

2.76 0.95b

0.09 0.20

Colon Tisssue, g/kg of BW Digesta, g/kg of BW

0.6% kestose and nystose

1.2% kestose and nystose

SEM

Means within rows with no common superscripts differ significantly (P < 0.05).

a,b

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KESTOSE AND NYSTOSE PREPARATION Table 5. Bacterial enzyme activity (U/g of digesta) in the ceca of turkeys

Enzyme α-Glucosidase β-Glucosidase α-Galactosidase β-Galactosidase β-Glucuronidase

Control

0.3% kestose and nystose

0.6% kestose and nystose

1.2% kestose and nystose

SEM

0.81 0.22ab 0.90b 1.66 0.51

1.20 0.24ab 1.30ab 2.00 0.49

1.14 0.38a 1.61a 1.73 0.34

0.74 0.19b 0.85ab 1.80 0.48

0.11 0.03 0.12 0.25 0.07

Means within rows with no common superscripts differ significantly (P < 0.05).

a,b

diets containing chicory FOS (Farnworth et al., 1996) and sucrose thermal oligosaccharide caramel (Orban et al., 1997). Application of 2% chicory FOS in a broiler diet has been shown to reduce the cecal pH by 0.4 units (Farnworth et al., 1996). Several reports have indicated that fermentation of FOS in ceca causes a lower level of NH3 in the digesta (Terada et al., 1994). On the other hand, the proliferation of bacteria, which is a prerequisite for SCFA production, is the fixing of N as bacterial protein. The main source of N is NH3 derived from urea (Topping, 1996). That process leads not only to lowering blood urea concentration but also to a temporary raising of intracecal NH3 concentration and consequently to an increase in NH3 pool observed in birds fed diets with kestose and nystose in our study (Jus´kiewicz et al., 2004, 2006). In our experiment, enhanced amount of cecal digesta was determined in groups fed the diet with the low and medium levels of kestose and nystose. Compared with the control group, the pH of cecal digesta was numerically lower (by 0.24 to 0.34 units) and the concentration of NH3 significantly higher in those groups. A high dose of kestose and nystose significantly decreased the pH of digesta, but the amount of the content was similar as in the control group. It may result from a faster cecal and colonic transit time when the birds were treated with the highest dose of the kestose and nystose preparation.

Activity of Microbial Enzymes and SCFA Concentration Bacterial enzyme activity in cecal digesta of turkeys is shown in Table 5. In all dietary treatments, a similar activity of α-glucosidase, β-galactosidase, and β-glucuronidase was determined. The highest activity of bacterial β-glucosidase was observed in the 0.6% kestose and nystose group and the lowest in the 1.2% kestose and nystose treatment. Compared with the control group, a higher activity of α-galactosidase was observed for the diet containing a medium level of FOS. In the latter group, a significantly higher concentration of total SCFA, propionate and valerate, as well as numerically higher concentrations of acetate and butyrate, were determined (Table 6). The increase in the concentration of SCFA was less distinct in the groups fed diets containing low and high doses of kestose and nystose. The highest SCFA pool was observed in birds fed the low and medium doses of FOS preparation; however, the results did not differ statistically among treatments. An analysis of SCFA profile pointed to beneficial changes in the composition of individual major acids following kestose and nystose addition [i.e., a lower proportion of acetic acid (the 0.6% kestose and nystose group) and higher proportion of propionic and butyric acids (the 0.6 and 1.2% treatments, respectively)]. It has been reported that propionate may alter

Table 6. Concentration (␮mol/g of fresh digesta), total content (␮mol/kg of BW), and profile (%) of short-chain fatty acids (SCFA) in the ceca of turkeys

Item SCFA concentration, ␮mol/g Total SCFA Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate SCFA pool, ␮mol/kg of BW SCFA profile, % Acetate Propionate Butyrate

0.3% kestose and nystose

0.6% kestose and nystose

1.2% kestose and nystose

SEM

284.8

84.2b 53.8 7.37b 0.89 18.81 1.06 2.26b 421.1

107.7a 61.1 19.63a 1.05 21.61 1.07 3.30a 421.6

93.0ab 58.4 5.46b 1.12 24.67 1.49 1.95b 342.0

3.26 1.85 1.23 0.07 1.06 0.10 0.17 24.55

67 a 5c 23 ab

64 a 9b 22 ab

63 a 6 bc 26 a

1.03 1.08 0.84

Control 81.1b 53.8 4.28b 1.13 19.19 1.07 1.63b

55 b 18 a 20 b

Means within rows with no common superscripts differ significantly (P < 0.05).

a–c

´ CZYK ET AL. ZDUN

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the cholesterol pool or hepatic cholesterogenesis (Wright et al., 1990). Butyrate, in addition to its trophic effect on the mucosa, is an important energy source for the cecal and colonic epithelium, and it regulates cell growth (Lupton, 2004). It is accepted that limited supplementation of diets with oligosaccharides is sufficient to stimulate enzyme production by intestinal microflora, especially glycolytic ones (Okumara et al., 1994; Monsan and Paul, 1995). Products of bacterial digestion of oligosaccharides are SCFA, mainly acetate, propionate, valeriate, and butyrate. Supplementation of a diet has been shown to usually cause a lower content of acetate and a higher content of propionate and butyrate in the sum of SCFA (Levrat et al., 1991; Kleessen et al., 2001). In conclusion, different amounts of kestose and nystose (0.3, 0.6, and 1.2% of a diet) did not influence the productivity nor the performance of turkeys after feeding for 8 wk. We found that the microbial metabolism was affected by dietary short-chain FOS to some extent. The increase in dietary kestose and nystose addition was followed by a reduction in the ileal and cecal pH of digesta. The kestose and nystose-rich preparation increased, especially at the 0.6% dose, the concentration of total SCFA in the ceca and the proportion of propionic acid at the expense of acetate.

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