ENDOGLUCANASE-TREATED GLUCANS

Abstract

In one aspect, the present invention relates to an edible or thinkable composition that comprises alpha-1,6-mannans. In another aspect, the invention relates to an edible or drinkable composition that comprises endoglucanase-treated beta-1,3:1,6 glucans. The compositions are particularly suitable for use in a method of improving the immune and/or health status of a mammalian or non-mammalian subject. The present invention further relates to a method for decreasing the feed conversion ratio and/or increasing body weight in a non-human animal, comprising feeding the non-human animal with an edible or drinkable composition that comprises alpha-1,6-mannans and/or endoglucanase-treated beta-1,3:1,6 glucans. In a still further aspect, the invention relates to the use of an edible or drinkable composition that comprises alpha-1,6-mannans and/or endoglucanase-treated beta-1,3:1,6 glucans for improving the health or immune status of a mammalian or non-mammalian Finally, the invention relates to the use of an edible or drinkable composition that comprises alpha-1,6-mannans and/or endoglucanase-treated beta-1,3:1,6 glucans for decreasing the feed conversion ratio and/or increasing body weight in a non-human animal.

Claims

1. Edible or drinkable composition comprising endoglucanase-treated beta-1,3:1,6 glucans, wherein said composition comprises at least 3.0 g reducing sugars per 100 g dry matter of the composition, wherein the reducing sugars completely result from the decomposition of the glucans.

2. Edible or drinkable composition of claim 1, wherein said composition comprises at least 5.0 g reducing sugars per 100 g dry matter of the composition.

3. Edible or drinkable composition of claim 1, wherein said composition is a food or feed product.

4. Edible or drinkable composition according to claim 1, wherein said composition comprises glucose oligosaccharides in an amount of 1-300 mg/kg of the composition, preferably 10-200 mg/kg, and more preferably 20-100 mg/kg of the composition.

5. Edible or drinkable composition according to claim 1, wherein said beta-1,3:1,6 glucans are derived from yeast.

6. Edible or drinkable composition according to claim 5, wherein said beta-1,3:1,6 glucans are derived from yeast cell walls.

7. Edible or drinkable composition according to claim 1, wherein said composition comprise more than 60% (w/w) glucose oligosaccharides having a molecular weight (MW) of below 150 kDa, based on the total amount of glucans in the composition.

8. Edible or drinkable composition according to claim 1, wherein said yeast beta-1,3:1,6 glucans have been treated with an endo-1,3-beta-glucanase, an endo--1,3(4)-D-glucanase or another enzyme with endo-1,3-beta-D-glucanase activity.

9. Edible or drinkable composition according to claim 8, wherein said endo-1,3-beta-glucanase comprises or consists of the sequence set forth in SEQ ID NO:1.

10. Edible or drinkable composition comprising alpha-1,6-mannans, wherein said composition comprises a ratio of soluble mannan/total mannan of at least 0.1 and a ratio of soluble mannan/total dry matter of at least 0.05.

11. Edible or drinkable composition of claim 10, wherein said composition comprises a ratio of soluble mannan/total mannan of at least 0.65 and a ratio of soluble mannan/total dry matter of at least 0.1.

12. Edible or drinkable composition of claim 10, wherein said composition is a food or feed product.

13. Edible or drinkable composition according to claim 10, wherein said composition comprises alpha-1,6-mannans in an amount of 15-35% (w/w) of the composition.

14. Edible or drinkable composition according to claim 10, wherein said alpha-1,6-mannans are derived from yeast.

15. Edible or drinkable composition according to claim 14, wherein said alpha-1,6-mannans are derived from yeast cell walls.

16. A method of improving the health status of an animal or human, said method comprising feeding to the animal or human an edible or drinkable composition according to claim 1.

17. Method for decreasing the feed conversion ratio and/or increasing body weight in a non-human animal, comprising feeding the non-human animal with an edible or drinkable composition according to claim 1.

18. Method according to claim 17, wherein said non-human animal is selected from the group consisting of cow, pig, sheep, horses, cat, dogs, fish, shrimp and poultry.

19. A method for improving the health status of a human or animal, said method comprising feeding to the animal or human an edible or drinkable composition according to claim 10.

20. A method for decreasing the feed conversion ratio and/or increasing body weight in a non-human animal, said method comprising feeding the non-human animal with an edible or drinkable composition according to claim 10.

21. Method for producing an edible or drinkable composition comprising endoglucanase-treated beta-1,3:1,6 glucans, said method comprising: (a) providing a composition that comprises beta-1,3:1,6 glucans; (b) incubating said composition with an endoglucanase, such as an endo-1,3-beta-glucanase, an endo--1,3(4)-D-glucanase, or another enzyme with endo-1,3-beta-D-glucanase activity until the composition comprises at least 3.0 g reducing sugars per 100 g dry matter of the composition.

22. Method for producing an edible or drinkable composition comprising alpha-1,6-mannans, said method comprising: (a) providing a composition that comprises mannans; (b) adjusting the amount of mannans such that the composition comprises a soluble mannan/total mannan ratio of at least 0.1 and a soluble mannan/total dry matter ratio of at least 0.05.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0067] FIG. 1 shows the result of a size exclusion separation using the 3G-GlOS yeast cell wall product obtained in Example 1. It can be seen that the product contains a high amount of beta 1,3:1,6-linked oligosaccharides having 5-200 glucose units.

[0068] FIG. 2 shows the results of measuring the mortality of broiler fed with diets that were supplemented with 1G-YCW and 3G-GlOS. (1) Negative control, (2) positive control (Narasin), (3) 250 mg/kg 1G-YCW, (4) 1000 mg/kg 1G-YCW, (5) 62.5 mg/kg 3G-GlOS, (6) 250 mg/kg 3G-GlOS, (7) 1000 mg/kg 3 G-GlOS.

[0069] FIG. 3 shows the European Production Efficiency Factor (EPEF) of the different diets that were supplemented with 1G-YCW and 3G-GlOS. The factor was calculated using average daily gain, liveability and FCR.

[0070] FIG. 4 shows the results of measuring the total oocyst counts per gram of faecal material from broiler fed with diets that were supplemented with 3G-GlOS and BG.

[0071] FIG. 5 shows the results of measuring the amount of reducing sugars solubilized in the supernatant after endoglucanase treatment of yeast cell walls. The amount is expressed as g reducing sugar per litre.

[0072] The following examples illustrate various embodiments of the invention. It will be understood that the invention is not limited by the examples.

EXAMPLES

Example 1: Preparation of 1G and 3G Yeast Cell Walls

[0073] For the preparation of 1G yeast cell walls, cream yeast was inactivated for 2 hours at 90 C. 1.0 g/l of protease Protin PC-10 (Daiwa Kasei) was added, and the reaction was incubated for 10 hours at 40 C. The enzyme was inactivated for 40 seconds at 140 C., and the cell walls were separated from the intracellular matter by centrifugation. The cell walls (pellet) were washed 4 times with process water. The washed pellet contains the hydrolyzed yeast cell walls (=1G YCW).

[0074] For the preparation of 3G yeast cell walls, 1G YCW with a dry matter of 10-13% were heated to 50 C. and the pH was adjusted to 6.5 using NaOH. Subsequently 0.3% (in regard to the dry matter of yeast cell wall cream) Denazyme GEL-L1/R enzyme (laminarinase) was added and the mixture was incubated at 50 C. for 18 h. The pH was not adjusted during the reaction. After 18 h, the enzyme reaction was stopped by heat inactivation, and the resulting product was spray dried (=3G-GlOS).

[0075] The molecular size distribution of the molecules present in the 3G-GlOS product was analyzed by HPLC-Size Exclusion Chromatography. 3G-GlOS was dissolved in water to a concentration of 1% (w/v), filtered, and 20 l of this solution was analyzed on a PPS Proteema (100 , 5 m) column (Supplier: Polymer Standards Service) using purified water as the eluent at a flow rate of 1 ml/min. Compounds were detected by refractive index detection. The results of the size exclusion chromatography are shown in FIG. 1.

Example 2: Effect of 3G-GlOS on Broilers Exposed to a Clostridium perfringens Challenge

[0076] The objective of this experiment was to evaluate 1G YCW and 3G-GlOS on broiler chicken performance, intestinal health and microbiota under necrotic enteritis (NE)-challenged conditions.

[0077] The diets were wheat-soy based feeds for broiler chicks and formulated as shown in Table 1. The starter formulation was used during the first 3 weeks and the grower diet for the last 3 weeks of the trial. The starter diets were prepared as 2 mm and the grower diet as 4 mm pellets.

[0078] 616 healthy broilers of the breeding strain Ross 308 with an initial weight of between 40-45 g were grown for a period of 42 days at a stocking density of about 1.125 animals per m.sup.2. Conditions were 18 hours light at 20 lx followed by 6 hours darkness. The temperature was 32 C. at the beginning of the trial and was gradually decreased to 22 C.

[0079] As a positive control (PC), a commercial diet containing Monteban (ionophore) and Narasin (coccidiostat and antibiotic) was used. As a negative control, a diet without the addition of any YCW products was used. The diets to be tested were modified by the addition of the following yeast products: [0080] 250 mg/kg 1G-YWC, [0081] 1000 mg/kg 1G-YWC, [0082] 62.5 mg/kg 3G-GlOS, [0083] 250 mg/kg 3G-GlOS, [0084] 1000 mg/kg 3G-GlOS.

TABLE-US-00001 TABLE 1 Basic control diet. Starter % Grower % (d 0-21) (d 21-42) Wheat 58.79 64.36 Soybean meal 48 33.17 26.00 Sunflower oil 3.57 5.85 Monocalciumphosphate 1.34 1.07 Limestone 1.47 1.24 NaCl 0.42 0.38 Mineral premix* 0.20 0.20 Vitamin premix** 0.20 0.20 DL-Methionine 0.32 0.27 L-Lysine HCl 0.26 0.22 Threonine 0.16 0.11 Inert 0.10 0.10 Total 100 100 *Contents of the mineral premix: calcium 296.8 g/kg, iron 12.5 g/kg, copper 4 g/kg, manganese 25 g/kg, zinc 32.5 g/kg, iodine 0.225 g/kg, selenium 0.1 g/kg. **Contents of the vitamin premix calcium 331.3 g/kg, vitamin A 6000000 IU, vitamin D3 2250000 IU, vitamin E 30000, tocopherol 27270 mg/kg, vitamin K3 1505 mg/kg, vitamin B1 1257.3 mg/kg, vitamin B2 3000 mg/kg, vitamin B6 2009.7 mg/kg, vitamin B12 12.5 mg/kg, biotin 75 mg/kg, folic acid 504 mg/kg, niacin 20072 mg/kg, pantothenic acid 7506.8 mg/kg.

[0085] On day 14, the chicks were individually inoculated with 5000 oocysts of E. maxima (Biopharm, Czech Republic). The size of inoculum providing a clear but subclinical effect on performance was chosen. The oocysts were suspended in tap water one day before inoculation to improve the excystation efficiency. Oocysts were pipetted into the beak of each chick in 200 nl of suspension, which the chicks could easily swallow.

[0086] On day 18, each broiler chicken was orally inoculated with a fresh culture of Cl. perfringens (0.5-110.sup.9 live cells). The strain of Cl. perfringens was originally isolated from ileal contents of NE-affected broiler chicks. The strain has been repeatedly found to cause NE provided that the chickens are sensitized by E. maxima challenge.

[0087] The chicks were weighed on days 0, 14, 21 and 42. Correspondingly, feed intake per pen and the feed conversion ratio (FCR) were measured for the following periods: [0088] Days 0-14, early starter diet period and the period before challenges [0089] Days 14-21, late starter diet period and the period of E. maxima and Cl. perfringens inoculations [0090] Days 21-42, grower diet period and the recovery period after inoculations

[0091] On days 21 and 42, two birds from all pens were euthanised, abdominal cavity opened, blood samples taken by cardiac puncture and jejunum, ileum and paired caeca were removed. Jejunum was opened for scoring NE lesions using scale from 0 to 6 as described by Shojadoost et al. (Veterinary Research 2012, 43:74). Ileal and caecal digesta samples were collected during the trial and stored for possible later analysis. From three pens only one or no birds were euthanized on day 21 due to high mortality.

[0092] Results: Regarding mortality, the results are shown in Table 2. It can be seen that there was no effect of the diet on mortality prior to the NE challenge (days 0-14). Overall mortality was quite high and the C. perfringens infection was more severe than expected. It can be seen that the feeding of YCWs was effective in reducing mortality. The strongest reduction was clearly achieved with the 3G-GlOS. The results of the mortality measurement between days 0-42 is shown in FIG. 2. The figure shows (1) the negative control, (2) the positive control (Narasin), (3) a diet with 250 mg/kg 1G-YCW, (4) a diet with 1000 mg/kg 1G-YCW, (5) a diet with 62.5 mg/kg 3G-GlOS, (6) a diet with 250 mg/kg 3G-GlOS, and (7) a diet with 1000 mg/kg 3G-GlOS.

TABLE-US-00002 TABLE 2 Mortality of broilers fed with YCWs and exposed to a C. perfringens challenge on day 14 post-hatch and raised until day 42 post-hatch. PI = day post-inoculation with 5,000 oocysts of Eimeria maxima and 4 days later orally inoculated with a fresh culture of C. perfringens (0.5-1.0 10.sup.9 living cells). Mortality, % Dose, d 0-14 d 14-21 d 0-21 d 21-42 d 14-42 d 0-42 Trt Diet mg/kg 14 PI +7 PI +28 PI 1 NC 0 1.01 48.48.sup.a 49.50.sup.a 1.85 50.34.sup.a 51.35.sup.a 2 PC 0 0.00 2.02.sup.e 2.02.sup.e 3.86 5.88.sup.e 5.88.sup.d 3 1G- 250 0.00 31.31.sup.b 31.31.sup.b 1.85 33.16.sup.bc 33.16.sup.bc 4 YCW 1000 1G- 0.00 YCW 28.28.sup.bc 28.28.sup.bc 6.22 34.50.sup.b 34.50.sup.b 5 3G- 62.5 1.01 14.14.sup.d 15.15.sup.d 4.01 18.15.sup.de 19.16.sup.cd 6 GlOS 250 0.00 19.19.sup.cd 19.19.sup.bcd 4.48 23.67.sup.bcd 23.67.sup.bc 7 3G- 1000 GlOS 3G- 1.14 GlOS 22.73.sup.bcd 23.86.sup.bcd 0.00 22.73.sup.bcd 23.86.sup.bc SEM 0.89 4.28 4.49 2.51 4.92 5.12 Diet P value 0.5404 <0.0001 <0.0001 0.1131 <0.0001 <0.0001

[0093] Regarding body weight gain, the results are shown in Table 3. There was no effect of diet on initial body weight (BW, P=0.22) of 42 g. Feeding YCWs pre-challenge had a significant effect on BW gain, without influencing food intake (FI). Post-challenge, YCWs at the lower doses increased BW gain above that of the NC, but it wasn't until day 21-42 that BW gain was comparable to the PC in birds fed 3G-GlOS at the lowest doses. Overall, the supplementation of 3G-GlOS during a NE challenge decreased mortality compared to the NC.

TABLE-US-00003 TABLE 3 Body weight gain of broilers fed YCWs and exposed to a C. perfringens challenge on day 14 post-hatch and raised until day 42 post-hatch. PI = day post-inoculation with 5,000 oocysts of Eimeria maxima and 4 days later orally inoculated with a fresh culture of Clostridium perfringens (0.5-1.0 10.sup.9 living cells). Body weight gain, g Dose, d 0-14 d 14-21 d 0-21 d 21-42 d 14-42 d 0-42 Trt Diet mg/kg 14 PI +7 PI +28 PI 1 NC 0 514.7.sup.a 307.3.sup.d 822.0.sup.c 2249.1.sup.c 2556.4.sup.c 3071.1.sup.c 2 PC 0 507.8.sup.ab 593.2.sup.a 1101.1.sup.a 2568.9.sup.a 3162.1.sup.a 3669.9.sup.a 3 1G-YCW 250 505.1.sup.abc 366.3.sup.bc 871.4.sup.bc 2393.8.sup.bc 2760.2.sup.b 3265.2.sup.b 4 1G_YCW 1000 515.3.sup.a 344.2.sup.bcd 859.5.sup.bc 2356.9.sup.bc 2700.9.sup.bc 3217.4.sup.bc 5 3G-GlOS 62.5 481.7.sup.d 359.7.sup.bc 841.5.sup.c 2414.7.sup.abc 2774.4.sup.b 3256.2.sup.b 6 3G-GlOS 250 486.7.sup.cd 357.9.sup.bc 844.7.sup.bc 2433.0.sup.ab 2790.9.sup.b 3277.6.sup.b 7 3G-GlOS 1000 493.3.sup.bcd 336.2.sup.cd 829.5.sup.c 2329.6.sup.bc 2665.8.sup.bc 3159.1.sup.bc SEM 7.17 16.45 19.77 61.83 65.33 65.17 Diet P value 0.0070 <0.0001 <0.0001 0.0554 <0.0001 <0.0001

[0094] Regarding feed conversion ratio (FCR), it can be seen that mortality-corrected FCR was influenced by diet and day with a decrease in FCR in birds fed 3G-GlOS compared to the NC, with the lowest 3G-GlOS dosage resulting in the highest improvement in FCR, see Table 4.

TABLE-US-00004 TABLE 4 Feed conversion ratio of broilers fed YCWs and exposed to a Clostridium perfringens challenge on day 14 post-hatch and raised until day 42 post-hatch. PI = day post- inoculation with 5,000 oocysts of Eimeria maxima and 4 days later orally inoculated with a fresh culture of C. perfringens (0.5-1.0 10.sup.9 living cells). Mortality corrected feed conversion ratio, g:g Dose, d 0-14 d 14-21 d 0-21 d 21-42 d 14-42 d 0-42 Trt Diet mg/kg 14 PI +7 PI +28 PI 1 NC 0 1.141.sup.d 1.359.sup.ab 1.211.sup.abc 1.828.sup.a 1.780.sup.a 1.670.sup.a 2 PC 0 1.155.sup.d 0.989.sup.c 1.064.sup.d 1.713.sup.cd 1.577.sup.c 1.518.sup.d 3 1G- 250 1.162.sup.cd 1.251.sup.b 1.193.sup.bc 1.778.sup.abc 1.708.sup.ab 1.620.sup.abc 4 YCW 1000 1G- YCW 1.152.sup.d 1.270.sup.ab 1.189.sup.c 1.731.sup.bcd 1.671.sup.b 1.585.sup.bc 5 3G-GlOS 62.5 1.181.sup.bc 1.475.sup.a 1.297.sup.a 1.673.sup.d 1.689.sup.b 1.577.sup.cd 6 3G-GlOS 250 1.212.sup.a 1.381.sup.ab 1.277.sup.ab 1.751.sup.abcd 1.711.sup.ab 1.634.sup.abc 7 3G-GlOS 1000 1.207.sup.ab 1.436.sup.ab 1.288.sup.a 1.776.sup.abc 1.731.sup.ab 1.647.sup.ab SEM 0.009 0.077 0.030 0.033 0.032 0.023 Diet P value <0.0001 0.0027 <0.0001 0.0491 0.0029 0.0003

[0095] Due to the different effects of the YCWs on mortality, BW gain and FCR pre- and post-NE challenge, it is complicated to determine which parameter is most important and which YCW source performs best. Therefore, the European Production Efficiency Factor was calculated: EPEF=(average grams gain/day x % livability)/FCR10. The EPEF is generally used to compare results between different flocks and different regions using a standardized formula. The results are presented in FIG. 3. It can be seen that overall, the 3G-GlOS have a more positive effect than the 1G YCW or NC, and that the lowest dosage of 3G-GlOS gave the best results.

Example 3: Effect of 3G-GlOS and Beta-Glucans on Broilers Exposed to Coccidia Vaccine

[0096] The objective of this experiment was to evaluate the effect of 3G-GlOS and beta-glucan diets fed to animals that were exposed to a coccidia vaccine. For this purpose, 2000 healthy broilers of the breeding strain Cobb 500 with an initial weight of between 38-45 g were grown for a period of 42 days at a stocking density of about 0.93 animals per m.sup.2. Conditions were 20 hours light at 20 lx followed by 4 hours darkness. The temperature was 29.5 C. at the beginning of the trial and was gradually decreased to 22 C.

[0097] As a positive control (PC), a commercial diet containing Monteban (ionophore) and Narasin (coccidiostat and antibiotic) was used. As a negative control, a diet without the addition of any YCW products was used. The diets to be tested were based on the negative control that was modified by the addition of [0098] 125 mg/kg 3G-GlOS, [0099] 250 mg/kg 3G-GlOS, [0100] 125 mg/kg BG, [0101] 250 mg/kg BG

[0102] After 14 days, the animals were infected with Eimeria oocysts. Eimeria oocysts are ingested by the bird and invade the intestinal cells to undergo various stages of their lifecycle where they multiply, burst out, and re-invading the cells to cause damage to cellular integrity. The oocysts are eventually excreted in the faeces to be re-ingested by the bird. The Eimeria lifecycle can take 5-7 days, and an initial growth check is expected approximately 1 week post-vaccination and again at 21 days, as birds are exposed to a higher number of oocysts which activates a protective immune response. The overall effect is costly to growth and FCR and can lead to secondary infections such as Clostridum perfringens, the causative agent of necrotic enteritis.

Results:

[0103] It was found that supplementing broiler diets with 3G-GlOS at 125 mg/kg, FCR and EPEF comparable to birds fed a coccidiostat (data not shown). In addition 3G-GlOS at 125 mg/kg reduced total oocysts counts. The results are depicted in FIG. 4. 3G-GlOS treated broilers contained a lower total oocyst count than BG treated broilers. Moreover, the lowest 3G-GlOS dose, resulted in the strongest reduction in total oocyst counts.

TABLE-US-00005 TABLE 4 Eimeria oocysts per gram of faecal material from 21-day old birds exposed to 2x dose of live coccidia vaccine.sup.1 and fed 3G-GlOS or B-glucans from hatch. Dose, Acervulina/ Maxima/ Tenella/ Total/ Diet mg/kg g faeces g faeces g faeces g faeces Positive 0 258.5.sup.b 800.5.sup.c 133.4 1192.4.sup.c control.sup.2 Negative 0 9196.4.sup.a 1375.5.sup.bc 5160.9 15732.9.sup.ab control 3G-GlOS 125 5060.9.sup.ab 1150.6.sup.bc 962.4 7994.4.sup.bc 3G-GlOS 250 9938.4.sup.a 2192.8.sup.ab 5519.5 20076.9.sup.a B-glucan 125 6303.3.sup.a 2993.3.sup.a 2326.3 11622.5.sup.ab (BG) B-glucan 250 5035.9.sup.ab 1467.4.sup.bc 2959.8 10413.8.sup.b (BG) SEM 2078.5 486.0 1661.0 3216.7 Diet P-value 0.0628 0.0154 0.2103 0.0071 .sup.1Coccivac-B52 (Merck Animal Health) contains live occysts of Eimeria acervulina (duodenum), maxima (jejunum), mivati (duodenum) and tenella (ileum). .sup.2Basal diet supplemented with Robenz (Zoetis, US) coccidiostat at 33 mg/kg.

[0104] The results are also depicted in FIG. 4.

[0105] The above examples were also performed with pigs and piglets and similar results were achieved.

Example 4: Quantifications of Reducing Sugars in the 3 G-GlOS Product

[0106] The 3G-GlOS product was prepared as described in Example 1. Briefly, 1G YCW with a dry matter of 10-13% were heated to 50 C. and the pH was adjusted to 6.5 using NaOH. Subsequently 0.3% (in regard to the dry matter of yeast cell wall cream) Denazyme GEL-L1/R enzyme (laminarinase) was added and the mixture was incubated at 50 C. for 18 h. The pH was not adjusted during the reaction. During the reaction, samples were taken and analyzed in view of the amount of reducing sugars using a slightly amended version of the method described in Wood et al. (2012), Biomass and Bioenergy, vol. 44, pages 117-121. Briefly, the dinitrosalicylic acid (DNSA) reagent was prepared in the same way and in the same concentrations that was described by Wood et al. (2012). However, the reaction was performed in Eppendorf tubes (end volume 750 L), and not in 96-well plates as described by Wood. The sample and DNSA were mixed 1:2 (250 L sample+500 L DNSA reagent). The mixture was incubated at 99.5 C. for 5 min in a water bath and subsequently centrifuged at 5,000 rpm for 5 min. The supernatant was obtained and diluted 10 times before measuring absorbance at a wavelength of 530 nm. Reference samples were prepared by following all steps except heating. The absorbance of the reference sample was determined and subtracted from the absorbance of the reacted sample to eliminate unspecific turbidity.

[0107] The results are shown in FIG. 5. It can be seen that the 3G-GlOS product comprises up to 9 g/L reducing sugars in the supernatant. Drawn to the amount of cell wall material, this amounts to about 5.0-7.5 g reducing sugar/100 g cell wall material. This range was confirmed in further pilot trials.

Example 5

[0108] For separating the glucans and the mannans from the 3G-GlOS product, the 3G-GlOS product was essentially prepared as described in Example 1. However, instead of spray-drying, the product was subjected to ultrafiltration using the ultrafiltration unit DSS LabStack M38H according to the suppliers instructions. The ultrafiltration was carried out using a membrane area of 1.05 m.sup.2 (i.e. 0.15 m.sup.2 per plate), which corresponds to 7 membrane support plates and 14 flat sheet membranes with 10 kDa MWCO PES. In the middle section of the unit, a stop disc was placed in one of the membrane support plates in order to change the feed flow direction during the filtration. The filtration was conducted in batch mode at 50 C., overnight and stirring. De-ionized water was added to the feed tank when the retentate volume started to reduce during the filtration. The inlet pressure was kept at around 1-3 bar and the outlet at 2-3 bar, which are within the specification limits.

[0109] During the filtration, the oligosaccharide molecular weight (MW) distribution was evaluated by HPLC-SEC following the protocol mentioned before. As a result two fractions were obtained, a high MW fraction (retentate) and a low MW fraction (permeate). The retentate was sterilized at 140 C. for 40 seconds using an ultra-high temperature (UHT) unit and then spray-dried. The permeate was freeze-dried for 3 days and then dissolved with deionized water to a dry matter content of about 35% (w/w). Subsequently, it was also sterilized and spray-dried under the same conditions as the retentate. The dried forms of the Low molecular weight fraction (LMWF) and the High molecular weight fraction (HMWF) as well as the 3G-GlOS product were used to evaluate the effect on broiler performance and microbial populations from hatch to 42 days post-hatch during a NE challenge. For this purpose, 968 healthy broilers with an initial weight of between 38-45 g were grown for a period of 42 days at a stocking density of about 33 kg per m.sup.2. Conditions were 18 hours light followed by 6 hours darkness. The temperature was 32 C. at the beginning of the trial and was gradually decreased to 22 C. As a positive control (PC), a commercial diet containing Monteban (ionophore) was used. As a negative control, a diet without the addition of any YCW products was used. The diets to be tested were based on the negative control that was modified by the addition of [0110] 62.5 mg/kg 3G-GlOS, [0111] 125 mg/kg 3G-GlOS, [0112] 250 mg/kg 3G-GlOS, [0113] 14.38 mg/kg LMWF, [0114] 28.75 mg/kg LMWF, [0115] 57.50 mg/kg LMWF, [0116] 48.13 mg/kg HMWF, [0117] 96.25 mg/kg HMWF, [0118] 192.50 mg/kg HMWF.

[0119] The chicks were weighed on days 0, 14, 21 and 42. Correspondingly, feed intake per pen, body weight gain, and the feed conversion ratio (FCR) were measured

[0120] On days 21 and 42, two birds from all pens were euthanized, abdominal cavity opened, blood samples taken by cardiac puncture and jejunum, ileum and paired caeca were removed. Jejunum was opened for scoring NE lesions using scale from 0 to 6 as described by Shojadoost et al. (Veterinary Research 2012, 43:74). Ileal and caecal digesta samples were collected during the trial and stored for possible later analysis. From three pens only one or no birds were euthanized on day 21 due to high mortality.

[0121] By day 21, (7 days post E maxima challenge and 3 days post C. perfringens challenge), BW was as expected according to breed recommendations at 946 vs. 945 g respectively. The challenge was considerably more severe than expected, with 33% mortality in the NC treatment and this was significantly higher than birds fed the positive control.

[0122] The results for growth performance for the entire trial period between days 0-42 are shown in Table 5

TABLE-US-00006 TABLE 5 Growth performance from day 0-42. Feed intake, BW gain, FCR, Mortality, # Basal diet g g g:g % 1 Negative control 4260.sup.d 3254.sup.bc 1.309.sup.d 58.47.sup.a (NC) 2 Positive control.sup.1 5262.sup.a 3664.sup.a 1.437.sup.bc 10.86.sup.c 3 2G YCW, 62.5 3731.sup.e 2179.sup.d 1.676.sup.a 31.92.sup.a mg/kg 4 2G YCW, 125 4636.sup.bcd 3266.sup.bc 1.418.sup.bc 26.72.sup.b mg/kg 5 2G YCW, 250 4658.sup.bcd 3217.sup.bc 1.443.sup.bc 29.96.sup.a mg/kg 6 LMWF, 14.4 mg/kg 4349.sup.cd 3169.sup.bc 1.373.sup.cd 49.26.sup.a 7 LMWF, 28.8 mg/kg 4647.sup.bcd 3213.sup.bc 1.444.sup.bc 29.26.sup.a 8 LMWF, 57.5 mg/kg 4490.sup.bcd 3141.sup.c 1.431.sup.bc 34.08.sup.a 9 HMWF, 48.1 mg/kg 4795.sup.b 3316.sup.b 1.446.sup.bc 28.92.sup.b 10 HMWF, 96.3 mg/kg 4693.sup.bc 3160.sup.bc 1.488.sup.b 21.40.sup.b 11 HMWF, 193 mg/kg 4628.sup.bcd 3101.sup.c 1.492.sup.b 24.27.sup.b SEM 145.sup. 62 0.04 7.3 R.sup.2 .sup.0.47 0.81 0.43 0.29 RMSE 411.sup. 174 0.10 21 n 88 88 87 88 Diet P <0.0001 <0.0001 <0.0001 0.0021 .sup.1Contains Monteban. LMWF = low molecular weight fraction of the 3G-GlOS. HMWF = high molecular weight fraction of the 3G-GlOS.

[0123] The LMWF and HMWF were fed at concentrations that were equivalent to those found in the 3G-GlOS at each dose. Growth performance data are shown in Table 5. Feed conversion ratio's were not significantly better than the positive control. However, mortality clearly improved in birds treated with either 3G-GlOS or fractions of the 3G-GlOS, and the mortality improvements after treatment with HMWF were also statistically significant.