WO1995007090A1

WO1995007090A1 - Probiotics

Info

Publication number: WO1995007090A1
Application number: PCT/GB1994/001849
Authority: WO
Inventors: Derek George Waterworth; Keith Hazell; Geoffrey White
Original assignee: Zeneca Limited
Priority date: 1993-09-06
Filing date: 1994-08-24
Publication date: 1995-03-16
Also published as: GB9318439D0; AU7465794A

Abstract

Probiotics for pigs contain Lactobacillus delbrueckii; they are preferably fed during the suckling and post suckling stages.

Description

PROBIOTICS

THIS INVENTION relates to probiotics. In animal husbandry it is commonly desired that newly born animals should gain weight rapidly, usually with a view to early slaughter.

It is proposed in European Patent 203,586 to administer a strain of the species Lactobacillus fermentum to animals such as pigs, cows, sheep, goats and horses in order to prevent gastrointestinal disease. The use of a strain of Lactobacillus lactis is proposed in US Patent 3,953,609 and the use of a mixture of Lactobacillus acidophilus and non pathogenic E.coli is disclosed in US Patent 3,320,130. Other strains of Lactobacillus are disclosed for treating infectious disease in US Patent 4,314,995. The above and other references are discussed in European Patent 203,586. The use in humans of certain strains of Lactobacillus acidophilus is disclosed in European Patent 199,535, and the use of species of Lactobacillus and other microorganisms is disclosed in the treatment of animals in European Patent 271,364. It is proposed to administer probiotics to animals prior to weaning in patent application WO 93/00012.

We have found that the use of Lactobaci1lus delbrueckii has particularly beneficial characteristics in such applications, especially as regards piglets and particularly when used together with Lactobacillus acidophilus and preferably also with Lactobacillus fermentum and that the piglet is preferably fed with compositions comprising such microorganisms both during the suckling and in the post suckling stage. By "post suckling" is meant the stage by which the piglet is able to obtain most of its nutritional intake from sources other than milk, and preferably a stage by which it no longer consumes mother's milk. The invention involves a process of increasing the growth rate of pigs which comprises conditioning their digestive tracts by administrating to them, preferably both in the suckling and in a post suckling stage a biologically pure culture of Lactobacillus delbrueckii in an orally and digestively acceptable composition optionally formed by admixture of such a culture with at least one other substance. The culture may comprise a nutritionally acceptable carrier which may be water, a food for example milk or a milk product or starch and/or other materials for example adjuvants, lubricants or carriers .

Other substances with which it is preferably admixed include biologically pure cultures of Lactobaci1lus acidophilus and preferably also Lactobacillus fermentum. By "digestively acceptable" is meant capable of oral administration without detriment to the digestive system.

The invention also comprises a digestively acceptable composition for conditioning the digestive system of pigs formed by admixture of a biologically pure culture of Lactobacillus delbrueckii with a biologically pure culture of Lactobacillus acidophilus and Lac obaci1lus fermentum, preferably together with milk, a milk product and/or starch. The composition may be allowed to ferment before use if desired. The composition conditions the intestines.

The invention also comprises a process of making a composition as aforesaid by admixture of the said ingredients and optionally subsequently fermentating the composition.

EXAMPLES A probiotic preparation which consists of 3 selected lactobacilli, deposited at the National Collections of Industrial and Marine Bacteria Limited, 23 Machar Drive, Aberdeen AB2 1RY Scotland UK on 2 April 1990 namely Lactobacillus fermentum (L94) NCIMB 30016, Lactobacillus delbrueckii (L99) NCIMB 30017 and Lactobacillus acidophilus (L83) NCIMB 30015 which are produced separately by fermentation and freeze drying was produced by mixing the lactobacilli in the correct proportion to provide viable cells of the specified colony forming units per gram (cfu/g) of product. The deposits are subject to the terms of the Budapest Treaty Fermentation

The lactobacilli were produced separately by growing each of them in a 30L batch fermenter by the following procedure.

1 An inoculant freeze dried culture was grown up on 200-400ml of MRS broth at 37°C to provide a rich culture overnight.

2 The 30L fermenter was charged with the following and sterilised at 121°C for 30 min: yeast autolysate 30g/L

MgS0₄.7 H₂0 0.21g/L

MnS0₄.4H₂0 O.Q5g/L

"Tween" 80 Surfactant 1.0 ml/L "Silcolapse" 5000 foam suppressant 2.0 ml/L

H₃P0₄ (17 Molar) • 1.0 ml/L Sterilised Glucose to give 50-100g/L

The temperature was adjusted to 30°C and the pH to 6.0. The fermenter was run aerobically for L94 and L83 and anaerobically for L99 and harvesting the lactobacilli when growth was complete. Harvesting consisted of centrifuging the cells and freeze drying the cells. ADMINISTRATION OF THE PROBIOTIC LACTOBACILLI TO PIGLETS Product administered in the suckling period The product was administered to the piglets as an oral dose as soon as possible after birth and periodically during the suckling period, e.g. 0 and 14 days and on the day of weaning. The drench took the form of a mixture of the 3 lactobacilli as a freeze dried powder mixed with water, or with sufficient phosphate buffer saline solution (PBS) in quantities to administer to at least one litter.

The dose aimed for was 3 x 10⁹ cfu of each lactobacillus per piglet/dose. The control piglets were dosed with the corresponding liquid, ie water or PBS.

Product administered in the post weaned period

The mixture of freeze dried Lactobacilli were mixed with skimmed milk powder (SMP) in a ratio to supply the necessary quantities in the feed. The calculated quantity administered in the feed was approximately 1 x 10^s cfu per pig per day of each lactobacillus over the 4 week post weaned period.

The probiotic mixture was administered as a top dressing to the feed pellets, put into the feed hopper or mixed directly into the weaner feed. EXAMPLES OF THE EFFECT OF ADMINISTERING THE 3

LACTOBACILLI TO PIGLETS

EXAMPLE 1

Design Thirty two Landrace/Large White sows were paired according to genotype, parity and service data and reared and fed according to the standard procedure of the unit during the pregnancy period. At farrowing the sows were randomly assigned to farrowing accommodation so that litters destined to become control or test litters were assigned to accommodation so that probiotic lactobacilli could not be inadvertently transferred from test to control piglets through piglet contact or via the pigman.

Treatments - Lactation Period The treated pigs were dosed with 3 ml (10⁹ of each

Lactobacillus species/dose) suspension of the probiotic lactobacilli and the control pigs were dosed with water.

Post Weaning Period

The probiotic powder was sprinkled onto the feed daily and the corresponding SMP carrier was sprinkled onto the feed of the control pigs.

Experimental

During the first few days piglets were fostered between litters before dosing or within treatment groups after dosing.

Normal husbandry practices were adopted throughout the experiment. Creep feed was not provided. Litters were checked daily for signs of ill health. The pigs were weaned at 25 days of age. At weaning the pigs were weighed individually and approximately half from each treatment allocated randomly, within treatment, to rearing pens at 8-10 pigs/pen. Water and feed were provided ad libitum.

Results

Suckling Period: There was no difference between treatments for: the number of pigs born or weaned per litter; the health status of the piglets.

There was a 7.5% increase in daily liveweight gain of the probiotic treated piglets. Post Weaned Period

There was a 17% improvement (P<0.05) in liveweight gain and feed intake by the pigs given the probiotic.

(Table 1) .

When covariate corrections for group size were taken into account there was a 25% improvement in these parameters.

Table 1 - Growth Performance in the Post Weaned Period

Sucking Period Control Probiotic

Litter size at birth 11.6 11.3

Litter size at weaning 10.3 9.3 Piglet weight at birth (Kg) 1.56 1.49

Piglet weight at weaning (Kg) 6.87 7.15

Piglet liveweight gain (g/day) 212 228

Weaned Period

Group Size 10.9 8.7 Age at weaning (days) 23.0 22.4

Initial weight (Kg) 6.53 6.45

Pig liveweight gain (g/day) 313 365

Feed Intake (g/day) 470 553

Covariate Correction for Group Si Lzzee Pig liveweight gain (g/day 301 377

Feed intake (g/day) 465 558 CONCLUSION

There was an increase in liveweight gain in the suckling period when there was no background disease in evidence. There was a significant liveweight gain and feed intake increase in the post weaned period when the probiotic was administered in both the suckling and the post weaned period.

EXAMPLE 2 Design Forty-eight hybrid sows mated to conventional sires were randomly assigned pre-farrowing to control or treatment. The treatment litters were orally dosed with 3ml of 2 of the lactobacilli L. fermentum and L. delbrueckii. The control litters were dosed with distilled water.

Litter numbers were approximated to 10/litter by cross-fostering. Treatments : Lactation Period

The treated piglets were dosed with 3ml of a suspension of the 2 lactobacilli in quarter strength Ringers solution. Control pigs were dosed with 3ml quarter strength Ringers solution. Each 3ml dose provided 1 x 10⁹ of each Lactobacillus species. Post Weaning After weaning the pigs were penned in groups of approximately 20 according to the treatments in the suckling period. No probiotic treatment was given in this period. All the pigs were fed a standard commercial pelleted rearing diet. Experimental

Sows and pigs were subjected to normal husbandry procedures. Creep feed was not given during the suckling period.

The piglets were orally dosed on days 1, 14, and the day of weaning. At weaning the pigs were transferred to commercial weaner pens holding approx 20 pigs/pen.

Individual pig weights were measured at weaning and at 4 weeks post weaning. The feeding system in the post weaning period did not allow for accurate measurement of feed intake. Two batches of pigs were weaned and put through the post weaning unit. Results In the Suckling Period there were no health problems in either of the control or treatment pigs; there was no effect of the probiotic on the number weaned or on the growth rate to weaning. In the Post Weaned Period (Table 2):

Two batches of piglets, consisting in total of 400 pigs from both suckled probiotic and suckled control pigs were fed a standard pig rearer diet in the post weaned period.

The pigs which were probiotic dosed in the suckling period gained 3.5% more liveweight in the post weaned period than the control pigs. Table 2 - Growth Performance in the Post Weaned Period

Control Probiotic Pigs initial weight (Kg) 6.51 6.44

Pigs final weight (Kg) 19.40 19.59

Mean liveweight gain (g/day) 345 357 There were no differences between treatments in terms of health status of the pigs. EXAMPLE 3

This experiment was designed as an E.coli challenge experiment on very early weaned piglets rather than as a growth experiment. The challenging E.coli failed to induce a pathogenic response. However, the pigs were weighed at relevant times and the effect of the probiotic on growth performance was assessed.

Design

The objective of the original design was to administer the probiotic daily to piglets from birth, challenge with E.coli K88 once on a day prior to weaning and assess the protective effect of the probiotic against scours caused by the E.coli. Experimental Piglets from paired sows were earmarked for treatment or control probiotic dosing from birth and every other day thereafter. The treatment dose consisted of the three lactobacilli L94, L99, L83 delivered in 2ml to supply 1-10⁹ of each lactobacilli. Additional doses were given on the day before and after weaning on day 14. (The E.coli was given on day 7) .

The pigs were weaned into individual cages. Health status was checked daily and they were also weighed daily. Results

None of the pigs scoured. One of the pigs on the probiotic treatment died 8 days post weaning. This pig had performed badly from the start and was omitted from the growth performance calculations. In the suckling period there was no effect of the probiotic on growth. In the post weaned period the daily liveweight gain was increased by 5%, feed intake was reduced by 3%, and feed conversion improved by 7% (Table 2) .

Table 3 - Growth Performance in the Post Weaned Period Control Probiotic

Pigs initial weight (Kg) 4.53 4.55

Final weight (Kg) 8.45 8.82

Food intake (g/day) 265 256

Weight gain (g/day) 210 221 Feed conversion ratio 1.33 1.16

Conclusion

The growth data indicated that even in a situation where there is no health problem a growth performance improvement is observed.

Claims

1 A process of growing pigs in which their growth rate is increased which comprises conditioning their digestive tracts by administering to them a digestively acceptable composition obtained from a biologically pure culture of Lactobacillus delbrueckii.

2 A process as claimed in Claim 1 in which the composition comprises a nutritionally acceptable carrier.

3 A process as claimed in Claim 2 in which the nutritionally acceptable carrier comprises water.

4 A process as claimed in any preceding claim in which the composition comprises a pH buffer.

5 A process as claimed in Claim 1 or 2 in which the composition is derived from biologically pure cultures of Lactobacillus delbrueckii and Lactobacillus acidophilus.

6 A process as claimed in any preceding claim in which the composition is administered to piglets in their suckling and post suckling stages.

7 A process as claimed in any preceding claim in which the composition is derived from biologically pure cultures of Lactobacillus delbrueukii, Lactobacillus acidophilus and Lactobacillus fermentum.

8 A digestively acceptable probiotic composition formed by mixing biologically pure cultures of Lactobacillus delbrueckii, Lactobacillus acidophilus and Lactobacillus fermentum.

9 A composition according to Claim 8 which comprises milk, a milk product and/or starch. A composition according to Claim 8 or 9 which comprises a buffer. A composition according to Claims 8, 9 or 10 which has fermented. A process for preparing a probiotic which comprises mixing biologically pure cultures of Lactobacillus delbrueckii, Lactobacillus acidophilus and Lactobacillus fermentum to form a digestively acceptable composition.