Proceedings of the 7th Intertuitumal Working Conference on Stored-product Protection - Volume 1

Storage of compound feeds for animals: shelf-life and real-time detection of fungal development

Joseph Le Bars! and Pierrette Le Bars

Abstract

Storage of compound feeds for arnmals is quite different fromgrain one. Indeed, they are a mixture of numerous crushedraw matenals, mcluding by-products from different regionsor countnes. Moreover they are often stored in hazardousconditions and silos are usually reloaded before bemgemptied. All these conditions lead to a high susceptibility tofungal alteration. So m order to study the quantitativeeffects of the mam parameters on shelf -hie, a complexexperimental system was elaborated: kmetics of naturalfungal evolution m commercial compound feeds wasreproduced m 96 mini-silos, equipped with sensors (mtcmaltemperature, Aw CCh), lmked to an automatic acquisitioncomputer allowmg Real-TIme Detection (RTD) of the firststep of fungal development (European Contract PL 94 -1325, 1994 - 1998). Among several physico-chermcalcriteria for RTD in such mert feed, the earliest detectionwas obtamed with CCh increase, but the most suitablecriteria for such a multi-silos device, workmg m factorialplans, was the production of calones, automatically detectedby temperature increase Determmation of the mainparameters and quantification of therr effects on the shelf-life was conducive to useful diagrams for mdustnals andbreeders, improving prevision and adequacy of a treatment.Thus, safe storage penod decreases approximately two-foldfor every increase of + 5·C temperature or + 0.5% watercontent ThIS period is doubled If the mixed feed ISpelletized. Currently tlus methodology IS applied toevaluation of mould inhibitors m compound feeds and ISchecked on a pilot scale.

Introduction

Fungal development m foods and feeds leads to manydetnmental effects: - alteratlons of aspect and technologIcalproperties; - modifIcatlOn of nutrItlve value and/oroccurrence of refuse factors; - development of mycotlc and/or allergIC agents; - mycotoxm contaminatlon. In animal

1 Laboratory Pharmacology & TOXIcology, INRA, BP3, 31931Toulouse Cedex 9, France

feed industry, conditions are much different from gramstorage and are more propitious to such deletenous effects.smce raw matenals and by-products used are crushed andmay be from numerous regions and countnes that may leadto a high diverst ty of fungal species; the final compoundfeeds are often stored m precanous conditions on farmwhere silos are usually reloaded before being emptied,Investigations on prevision and prevention (European

Contract AIR - 3, 94 - 1325) led to a Real-TIme Detectiondevice (R T. D ) of fungal development in compound feeds,on both laboratory and pilot-scale that was conducted byquantitatively studying relations between the mamparameters and feed shelf- life

Materials and methods

The device (figure 1) principally compnses mnu-silos,incubators, sensors and mformatory system. The fungalpopulation (from xerophihc species to more hygrophihcones) in commercial compounds feeds m typical mnu-silos (6groups of 16, msulated cylmders WIth holes for sensors andsamplmg, 30 em m diameter and height) was contmuouslyexammed in order to reproduce the natural kmetics of fungalpopulation, in a rmmmal volume. After slightlyhomogeneous hydration (water contents varied from 12.8 to14.5 %, CV % 0.07 - 0.3), feed lots (purchased fromfactones ) are distributed in SIlos WhICh can be placedsimultaneously in four air-conditioned situanons (20 to35·C ). Each silo IS equipped WIth special home-madethermal sensors (Pt 100, o.orc i 16 thermo-hygrometercapacitive sensors (Rotronic 11021) and Gaspace 2 (SystechInstruments) were used for periodical AW and CCh - Chmeasurements. All these sensors were venfied andcalibrated two times a year. They were connected to a 128channels data collection system (16 bits). A data acquisitionprogram, elaborated from Labview on a PC, allowedvisuahsation of 'Real-TIme' analysis of the 128 lmes (bothtreatments and controls at each step of the entlre devIce).Data were automatically pnnted and stored on a hard drIve.All the system was secure over months with an automatlcpower management and was placed in an air-condItionedroom (72 m3, 20·C, 65 % RH).

PhYSIco-chemical parameters studIed for Real-TImeDetection were amounts of calories (temperature mcrease),

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Proceedings of the 7 th International Working Conference on Stored-product Protection - Volume 1

CQ and H20 (Aw increase) produced. Shelf life or lag timewas defined quantitatively as the Delay (D) in days before

an increase of 2°C in temperature in the followingexperiments.

000000000000-, __4 Incubators

Mini-silos

n=96

rn •Air conditioned room(200 C , RH 65 %)

Fig.l. General scheme of the Real-Time Detection device.

Results

Concerning physico-chemical criteria for Real-TimeDetection of fungal development, the increase in CQ wasthe first parameter significant to be detected. Rise intemperature ( + 2"C) will be 1.5 - 3 days (depending onhydrothermic conditions) later and 5 - 7 days later for Awincrease. The first two criteria were more precocious than asignificative increase in fungal numbers estimated bymycological analysis. The most suitable criterion for such amini-silos device, working in factorial plans, was theproduction of calories, so, for further experiments, theshelf-life was determined by the delay in days before a + 2"Cby self-heating (Figure 2).Variation inside one mini-silo was less than 0.5 % « 3

hours over 1 - 1.5 months) and inter-silos variation for asame treatment was less than 1.25 % « 0.5 days).

-

Numerous assays in factorial plans allowed to modelpractical relations between main parameters and the delaybefore fungal development. When Aw is equal to or over0.70 (as actual situation occurring in certain parts of silos),an increase in fungi numbers by a factor of 102 equals abouttwo fold the Delay, which is related to initial xerophilepopulation in commercial compound feeds, but has little todo with temperature (20, 25, 30, 35"C), water content(< 14.5 %) and feed form (powder, pellets).The Delay (D) sharply decreased with increasing water

content (we %):LogD = - a . wc % + b (R > 0.98) (figure 3).

According to the following relation, the higher was theconstant storage temperature (t), the shorter would be thedelay, regardless the water content « 14.5%) and feedform (figure 3) :

I/D= at + b (R>0.98).

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Proceedmos of the 7th International Workmg Conference on Stored-product Protectum - Volume 1

dtoc10

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7

6 .,. + .

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Fig.2. Example of results obtamed With the R T. D device: effect of water content (w c %) on shelf-life of a compound feedstored at 25'C .

log +-.....,L..l ...........---4-_~~-4--_ol----t--4_--'---Io-....4- __ -l--...........~--f-ys1.8

6013 ~

1.6

1.5

1.4

1.3

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0.8

0.7

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Fig.3. Diagram on the effect of hydrothermal status of compound feed on the Delay before fungal rrutiation

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Proceedings of the 7th International Working Conference on Stored-product Protectwn - Yolume 1

Whatever the other parameters except the imtial fungalcontammation may be, pelleuzation generally doubles the Dm companson with the initial powder feed. Extension of thedelay by pelletization was limited to + 20% when xerophihcfungal population was low m rruxed powder (about 103 cfu/g) and up to + 100 - 150% when the number of xerophihcexceeded 104/ g.Feed composition practically did not change the delay.

The delay IS slightly mcreased for feeds with high cellulosecontent (1. e. compound feed for rabbits) and decreased Withhigh lipid content, which are m agreement WIthAw effecton fungal germmation.

Discussion

It ISbest to detect fungal deterioration of raw matenals andfeed at an early stage, when prevention measures are stillfeasible and economical. When heatmg and cakmg becomeconspICUOUS,irreversible quality loss has occurred (Le Bars,1988)The aIDlIS to reflect a current metabohc fungal activity to

warn m real-time the supervisor and then to take urgentmeasures (drying or treatment with fungistatics ). Suchdynamic cnteria have to possess the followmg generalcharacteristics.To be sufficiently general m order to SIgnal a fungal

development whatever is the fungus concerned.To be sufficiently sensitive and measurable at the earhest

stage of fungal developmentTo be practicable for automatic supervision m mdustnal

silos in animal feed mdustry and on farms.Volatile compounds are produced by fungi in stored

commodities, and many can be distinguished qualitativelyfrom those produced by bactena or by the dormant seeds.Several mvesugators have monitored the productIOn ofvolatiles m gram storage and have suggested that theirappearance could be used to momtor early quahty loss(Richard-Molard et al , 1976; Kammski and WasowIcz,1991 ). QuahtatIve and quantitative dIfferences m thereported productIon of volatIles metabohtes appear todepend, in decreasmg order, on fungal speCIes, substratenature, physiologIcal state of fungI and enVIronmentalparameters.So, such specific bIOchemIcal mdicators are not yet

transposable to the numerous SItuatIonsand compounds (rawmaterials, end products) mammal feed mdustry.The present study on Real-TIme DetectIon of fungal

development was based on the general bIochemIcal reactionof aerobe orgamsms leadmg to COz, H20 and calonesproductIon (Srour, 1982)UtIhsation of these three end-products as cntena may be

theoretIcally envIsaged m the partIcular SItuation of ammalfeed mdustry and storage of compound feeds for the

following reasons:- Most commodities are biologically inert, 1 e. they arenot m a latent life state as cereals WIth a minimalbreathmg.They have too low a water activity to allow bactenalgrowth, and so there IS no mterference with bacterialmetabohtes.Storage penod IS usually too short for msects toprohferate. However, If It is the case, their undesirabledevelopment favors fungal rmtiation and can be detected.

- They are stored m normal air, not in confmed or mertatmosphere sometimes used for long term storage ofgrams, or anaerobically for SIlages.Effectiveness of this methodology and device was

confirmed by three years of quahtative and quantitativeexpenments on natural evolution of mycoflora m commercialcompound feeds m mmi-silos The mycoflora species rangedfrom xerophiles (Aspergtllus glaucus gr., Wallemwichthyophaga, etc.) to more hygrophihc ones (otherAspergillus and/or Pemallcum species) depending on theirutial hydrothermic status (Pelhate , 1979). In mini-silo,conditions are homogeneous, whereas m a great silo,conditions are heterogeneous. Such heterogeneity m actualsituations (1. e. temperature differences at the opposite SIdesof silos) IS the mam cause of the start of fungal developmentin compound feeds.Obtamed diagrams on the relations between the main

parameters and shelf-hfe are to be apphed to the observedcritical pomts m order to aVOIdfungal germmation andsubsequent exponential invasion of the substrate by mycelialnetworkThese relations may be summanzed as follows: safe

storage penod decreases approximately two-fold for everymcrease of 50C or 0.5 % water content, and is doubled Ifcompound feed ISpelletized.Based fIrst on the parameter - effect relationshIps, and

second on the phySIco-chemIcal condItions occurring atcntIcal pomts III a partIcular sIlo during a partIcular season,It ISpoSSIbleto estImate a preVISIonon safe storage penod ofa feed lot, or to settle means of prevention (including feedpreserva tIves If necessary)Currently thISmethodology ISapphed to evaluate effIcacy

of fungistatIc treatments on compound feeds for ammals andto establIsh dose-effect relatIonships for molecules and theIraSSOCIationsMoreover, checkmg and adjustment of thISReal-TIme DetectIon methodology and of the obtaineddIagrams on shelf lIfe of compound feeds are in progress on apilot scale m order to actually Improve prevention ofdeletenous effects on ammals resultmg from fungalalteratIons

References

KaminskI, E. and WasoWIcs,E. 1991. The usage of volatile

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Proceedmqs of the 7th International Work'tng Conference on Stored-product Protection - Volume 1

compounds produced by moulds as indicators of gramdeterioration. In Chelkowski, ]. ed. 'Cereal grain,mycotoxins, fungi and quality m drying and storage'.Elsevier N. Y , 229 - 258.Le Bars, ]. 1988. Toxigenesis as a function of the ecologicalconditions of the grain/microorganism system. In:Mutton, ]. 1. ed., Preservation and storage of grains,seeds and their by-products. Lavoisier pub. New York,Paris, 347 - 366.Pelhate, J. 1979. Mycoflore des semences et qualitegerminative. Bulletin Societe Mycologic de France, 95,

335 - 353.Richard-Molard, D., Cahagnier, B., Poisson, ].,Drapron, R. , Desserme, C. 1976. Comparative evolutionof volatile constituants and microflora in maize storedunder different conditions of temperature and humidity.Annales Technologie Agricole, 25, 29 - 44.Srour, S. 1982. Proprietes thermiques des grains,production de chaleur et de ~. In : Multon, J. 1. ed.,Conservation et stockage des grains et graines. Lavoisierpub., Paris, 217-230.

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