Screening of two probiotic products for use in fermented liquid feed

Screening of two probiotic products for use in

fermented liquid feed ☆

J.A.M.Missotten a,?,J.Michiels a ,J.Goris b ,L.Herman b ,M.Heyndrickx b ,

S.De Smet a ,N.A.Dierick a

a

Laboratory for Animal Nutrition and Animal Product Quality,Department of Animal Production,

Ghent University,Proefhoevestraat 10,B-9090Melle,Belgium

b

Institute for Agricultural and Fisheries Research,Technology and Food Unit,Brusselsesteenweg 370,B-9090Melle,Belgium

Abstract

In this trial two commercial probiotic products (Bactocell?and Adjulact?Pro)were investigated in vitro for their use as microbial inoculum for the production of fermented liquid feed (FLF)for pigs.Bactocell?was applied at a dose of 9and 10log 10CFU/kg and Adjulact?Pro at a level of 9log 10CFU/kg.The FLF (control and treatments)was prepared with a water to feed ratio of 4:1and run in batch for 72h at 30°C.The microbial population was followed with plate countings and the lactic acid,acetic acid and ethanol concentration was determined at different time points in the FLF.After 24h,significant differences (P b 0.05)were found between the control and the Adjulact?Pro FLF for pH (4.7vs 4.3),lactic acid (57.9vs 91.5mmol/L),acetic acid (23.1vs 6.8mmol/L),ethanol (24.5vs 1.1mmol/L),coliforms (7.2vs 4.3log 10CFU/mL)and E.coli (6.2vs 4.4log 10CFU/mL).Bactocell?addition did not alter the fermentation characteristics compared to the control FLF.After 72h no significant differences between treatments were noted,except for the yeast count which was higher in the FLF inoculated with Adjulact?Pro.

?2007Elsevier B.V .All rights reserved.

Keywords:Probiotics;Fermentation;Fermented liquid feed;Lactic acid bacteria;Lactobacillus spp.;Pig feed

1.Introduction

From January 2006onwards,the use of antibiotics as growth promoters in pig feed is prohibited in the EU (EC 1831/2003).Feeding fermented liquid feed

(FLF)to pigs has been shown to improve pig performance and to reduce the numbers of enteric pathogens (Jensen and Mikkelsen,1998)and could therefore be an alternative for feed https://www.360docs.net/doc/685958626.html,ually FLF is prepared by spontaneous fermentation or by use of lactic acid bacteria (LAB)culture as microbial inoculum.For effective pathogen suppression,a low final pH (high lactic acid concentration)and a rapid initiation of lactic acid production are equally important.The objective of this study was to evaluate the potential of two commercially available probiotic products as microbial inoculum for the production of FLF for

pigs.

Livestock Science 108(2007)232–

235

https://www.360docs.net/doc/685958626.html,/locate/livsci

☆

This paper is part of the special issue entitled “Digestive Physiology in Pigs ”guest edited by JoséAdalberto Fernández,Mette Skou Hedemann,Bent Borg,Jensen,Henry J?rgensen,Knud Erik Bach Knudsen and Helle Nygaard L?rke.

?Corresponding author.Tel.:+3292649014;fax:+3292649099.E-mail addresses:Joris.Missotten@UGent.be ,lanupro@UGent.be (J.A.M.Missotten).

URL:https://www.360docs.net/doc/685958626.html,nupro.UGent.be (J.A.M.Missotten).1871-1413/$-see front matter ?2007Elsevier B.V .All rights reserved.doi:10.1016/j.livsci.2007.01.078

2.Materials and methods

2.1.Probiotic products

Probiotic products from Lallemand S.A.(Toulouse, France)were used in vitro for producing FLF.The products tested were Bactocell?(Pediococcus acidilactici CNCM MA18/5M)and Adjulact?Pro(Streptococcus infantarius CNCM I-841and Lactobacillus plantarum CNCM I-840).Bactocell?was applied at the level of 9log10CFU/kg,as recommended for use in pigs,and at a 10times higher level,as recommended for the use in chicken(Council Directive70/524/EEC).Adjulact?Pro was added at its recommended level of9log10CFU/kg for calves(Council Directive70/524/EEC).

2.2.Batch fermentations

The batch fermentations were performed in triplicate in a warm water bath(30°C)for72h with6flasks of 500mL.The FLF was prepared with a water to feed ratio of4:1(400mL water:100g feed)and was stirred continuously.A complete weaner feed was used(corn/ soybean flour(67%/27.5%respectively);no Cu or Zn beyond requirements).Parameters recorded at0,1,2,4,8, 24,48and72h were:microbial population(1g FLF); lactate and volatile fatty acids(VFA)production(10g FLF);pH and ethanol production(10g FLF).pH was measured with a SenTix41electrode(WTW Wissenschaf-tlich,Welheim,Germany)that was calibrated every day.

2.3.Microbial population

Plate counting was applied according to the tech-nique of Van der Heyde and Henderickx(1963).Plates of MRS(de man,Rogosa,Sharpe agar;total LAB population)were incubated with90%N2and10%CO2 for48h at37°C.Plates of EMB(Eosin Methylene Blue agar;coliforms)and TBX(Tryptone Bile X-Glucuro-nide;E.coli)were incubated for24h at37°C.For yeasts RBCA(Rose-Bengal Chlorampenicol agar)was incubated for4d at room temperature.Bifidobacteria were plated on TPY(Tryptone Peptone Yeast extract agar;Rada and Petr,2002)and incubated with90%N2 and10%CO2for3d at37°C.All media,except TPY (Scharlau Chemie,Barcelona,Spain),were from Oxoid (Basingstoke,England).

2.4.Gas chromatography analysis

The concentrations of lactate and VFA were deter-mined according to a slight modification of the gas chromatography(GC)method described by Jensen et al.(1995).Ethanol concentration was determined with an in house GC-method.In short,a sample of 10mL FLF was acidified with2%12N H2SO4and centrifuged for10min at2000g.The supernatant was filtered(Rotilabo?,Type15A)and1μL was injected in the GC.The column was a packed column with Chromosorb60/80(RSL,Eke,Belgium)and Tween 80.The temperature program was5min at80°C, followed by an increase of15°C/min to a final temperature of140°C,which was kept for4min.The injector and detector temperatures were160°C and 150°C,respectively.

2.5.Statistical analyses

Bacterial counts were log transformed to fit a normal distribution prior to statistical analysis. The statistical program used was S-PLUS 6.1for Windows.

Table1

pH,ethanol,lactic acid and acetic acid concentration(mmol/L)at different times during batch fermentations of untreated liquid piglet feed(control),and piglet feed inoculated with Bactocell?or Adjulact?Pro

Control Bactocell?10×

Bactocell?

Adjulact?Pro

pH

0h 5.8 5.8 5.8 5.8

8h 6.0a 5.9a,b 5.7b 5.2c

24h 4.7a 4.7a 4.6a 4.3b

48h 4.3a 4.2a 4.2a 4.0b

72h 4.1 4.0 4.0 4.0

Lactic acid

0h n.d.?n.d.n.d.n.d.

8h n.d.n.d. 4.8±0.8a18.0±4.4b

24h57.9±6.5a57.0±1.3a66.5±4.9a91.5±11.6b 48h103.7±4.7a120.7±12.0a,b115.9±3.4a,b134.9±13.1b 72h126.7±10.3141.7±11.1151.1±19.6150.1±4.1 Acetic acid

0h 3.3±0.5 3.0±0.5 2.9±0.3 3.0±0.3

8h 3.6±0.5 3.3±0.2 3.4±0.4 3.3±0.4

24h23.1±1.5a,b23.5±1.2a20.2±1.5b 6.8±0.7c

48h30.3±1.8a34.1±3.7a32.7±5.1a15.4±5.8b

72h43.3±4.248.6±4.347.3±7.230.6±17.0 Ethanol

0h0.2±0.40.5±0.20.2±0.20.6±0.2

8h0.6±0.50.4±0.10.6±0.30.9±0.2

24h24.5±7.6a16.5±2.7a9.6±8.5a,b 1.1±0.2b

48h37.5±7.1a26.3±4.7a,b15.7±14.7a,b4.9±6.7b

72h38.2±5.624.2±4.117.0±15.717.6±14.9 Means±standard deviation.

a,b,c:values in the same row with different superscript differ significantly at P b0.05(Tukey).

?n.d.:not detected.

233

J.A.M.Missotten et al./Livestock Science108(2007)232–235

3.Results

A significantly faster pH drop was noticed during the first24h in FLF inoculated with Adjulact?Pro compared to the other treatments,also reflected in significantly higher lactic acid levels(Table1). Adjulact?Pro addition also resulted in significantly lower acetic acid and ethanol levels compared to the control FLF after24and48h(Table1).Bactocell?added at9or10log10CFU/kg did not influence the fermentation pattern much(Table1).The bifidobac-terial counts showed a trend(not significant)to be higher in the Adjulact?Pro FLF(Table2).The yeast counts were significantly higher at48and72h in the Adjulact?Pro FLF compared to the control and Bactocell?FLF,whereas the coliform and E.coli counts were significantly lower at24and48h (Table2).4.Discussion

Van Winsen et al.(2001)stated that FLF should have the following characteristics:pH b4.5;LAB N9log10 CFU/mL;lactic acid concentration N150mmol/L;acetic acid concentration b40mmol/L and ethanol concentra-tion b0.8mmol/L.Adjulact?Pro was successful in producing FLF with the desirable characteristics (Tables1and2),except for the count of LAB which was slightly lower than9log10CFU/mL and the ethanol concentration which was higher than0.8mmol/L (Table1).This is probably due to the batch type of fermentation process that was used in this study. Furthermore,a L.plantarum strain isolated from the ileum of a pig,showed a fermentation pattern similar to Adjulact?Pro(results not shown).So it is possible that the L.plantarum strain present in Adjulact?Pro was responsible for its beneficial fermentation characteristics.

The use of Bactocell?as microbial inoculum did not result in a product of beneficial properties.In fact,the Bactocell?supplementation followed the control fermentation.Furthermore,addition of Bac-tocell?at a10×higher level also did not alter the fermentation pattern.Geary et al.(1999)supplemen-ted FLF with Bactocell?to produce FLF with a pH of±4–4.5,but P.acidilactici did not become the dominant micro-organism in the FLF.Thus it is possible that the indigenous flora present on the feed dominated the fermentations in the Bactocell?and control fermentations.

Recently,Niven et al.(2006)performed experi-ments with strains from Lallemand.They used a fermentation temperature of35°C and found that the strains L.plantarum CNCM MA18/5U and P. acidilactici CNCM MA18/5M(present in Bacto-cell?)produced FLF with desirable characteristics. In our results,Bactocell?did not give an acceptable fermentation pattern during the first 48h,possibly because we used the commercial product and not the pure strain.Also the feed composition and the fermentation temperature were different(30°C vs35°C).

5.Conclusion

Adjulact?Pro was found to produce FLF with desirable characteristics in batch fermentations, probably due to the L.plantarum strain present in the product.Bactocell?did not offer any benefit and resulted in a similar fermentation pattern as the con-trol FLF.

Table2

Microbial enumerations(log10CFU/mL)at different times during the batch fermentations of untreated liquid piglet feed(control),and piglet feed inoculated with Bactocell?or Adjulact?Pro

Control Bactocell?10×

Bactocell?Adjulact?Pro

#lactic acid

bacteria

0h 2.3±0.4a 5.1±0.6b 6.1±0.5b 5.0±0.1b

24h9.1±0.39.1±0.29.1±0.18.8±0.8

48h9.0±0.29.3±0.29.2±0.18.4±0.9

72h9.2±0.19.4±0.49.4±0.18.9±0.7

#bifidobacteria

0h2?2? 2.2±0.32?

24h 3.9±0.1 4.5±0.5 4.7±0.4 4.1±0.1

48h 4.3±0.5 4.7±0.1 5.4±0.3 5.6±1.3

72h 4.5±0.2 4.7±0.4 4.8±0.5 6.3±1.7

#coliforms

0h 3.5±0.8 3.2±0.7 3.1±0.8 3.8±0.4

24h7.2±0.7a 6.7±0.3a 6.4±0.6a 4.3±0.4b

48h 6.3±1.1a 6.2±1.1a 6.4±1.2a 4.0±0.2b

72h 4.0±1.5 4.5±0.8 3.1±0.5 4.2±0.4

#E.coli

0h2?2?2?2?

24h 6.2±0.7a 5.9±0.3a 6.0±0.7a 4.4±0.1b

48h 6.1±0.6a 6.2±0.5a 5.6±0.6a2?,b

72h 3.4±2.0 4.1±1.9 2.5±0.8 2.4±0.8

#yeasts

0h 2.6±0.3a 2.8±0.2a,b 3.0±0.1a,b 3.1±0.1b

24h 5.2±0.3 5.5±0.2 5.5±0.5 5.8±0.9

48h 5.1±0.1a 5.3±0.3a 5.5±0.6a 6.7±0.3b

72h 5.8±0.2a 5.7±0.5a 5.3±0.4a7.2±0.8b

Means±standard deviation.

a,b:values in the same row with different superscript differ significantly

at P b0.05(Tukey).

2?:counts were lower than detection limit(2log10CFU/mL).

234J.A.M.Missotten et al./Livestock Science108(2007)232–235

Acknowledgements

This work was financially supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders(IWT),Brussels,Belgium. Lallemand S.A.(Toulouse,France)is thanked for providing the probiotic products.

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