View of Biological quality of fermented fish offal and chicken by-products

Vol.4: 27-33.

Biological quality of fermented fish offa and chicken by-products

T.Mikael Lassén

Division of^{AnimalNutrition,Department}ofAnimal Science and AnimalHealth,Royal Veterinary andAgricultural University, Bulowsvej 13,DK-1870Frederiksberg C„Denmark

Thebiological quality of fermented animal by-products prepared from fish and chicken offal was evaluated. Aquality index (QI) based on analyses ^ofa few importantfree amino acids and their corresponding biogenic amineswasgiven. Agroupseparation method based onionexchange chro- matography wasfound suitable for isolating and purifying aminesand amino acids in biological samples. Qualitywasevaluated insamplesfermented with different startercultures,inoculation siz- esand substrate levels.^Sloworincompletefermentation led to the accumulation ofbiogenic amines, especially tyramine,and resulted inalowQI.^Fast initialand continuous stable fermentation for four weeks resulted inlow concentrations ofbiogenic amines andhigh concentrations of free amino acids andconsequently ^ahighQI.The alanine concentration wasconsidered to beauseful tool for estimat- ing proteolysis,and QI^{related tochanges in}alanineconcentration,lactic acid production and redox potential wereconsidered togivethe most useful estimation of thebiological qualityof fermented fish offal andpoultry waste.

Key^words',biological silage,animal offal,biogenic amines,aminoacids,HPLC

ntroduction

Biogenic amines comprise alarge and heteroge- neousgroup of naturalproducts of special inter- estowingtotheir physiological effects. Theyare produced in enzyme catalysed reactions with amino acidsasdirect precursors, someof which are commontoall living cells. Amino acids are transformed into biogenic amines in sequences of reactions with severalintermediates, some of which may be specific for biochemical reactions

inmany^organisms,organs, tissuesorcells (Egg- um et al.

1988

a). Amino acid decarboxylases (ADC) from microorganisms areimportant fac- torsfor production of biogenic amines ^(Bprre- sen et al. 1988, Eggum etal. 1988^a).Adverse effects on animal growth and health may be a result of excessively high dietary concentrations of both psychoactive and vasoactive biogenic amines (Eggum etal. 1988^a).

The presence ofbiogenic amines ⁱⁿanimal by-products suchasfish and slaughter offal may have toxicological implications. Scombroid fish

©Agricultural ^ScienceinFinland ManuscriptreceivedFebruary 1994

poisoning of humans occurs because of inges- tion of pelagic ^fish, e.g. tunaand ^mackerel, which contain unusually high levels ofhistamine (Taylor 1986, 1988).Animal feeds often include whole fish, fish offalor fish meal, and large amounts of fishery products in feed have been implicated in outbreaks ofillness,e.g.,diarrhoea, and poor growth performance among animals.

High levels of histamine or other biogenic amines have been proposed ascausativeagents (Skadborg 1985,Eggum et al. 1988^{b, Klausen}

1988).

Biogenic amines or their products are con- sidered of interest in relation tothe quality of foodstuffs of animal origin and fur-animal feed.

Informationon types,combinations andconcen- trations of the actually harmful compounds caus- ing the problems observed in fermented offal of animal origin (Lassén etal. 1990,Urlings ¹⁹⁹²⁾ isscanty. Some of the problems associated with poorquality mink feed and the role of protein decomposition products in feed deterioration have been studied by Eggum etal. (1987). The results indicate that several problems still need tobe solved, and that reproducible, simple and efficient methods for feed quality control have

to be developed and evaluated. This study fo- cusedonimproving the analytical control of fer- mented silagetobe usedasanimal feed by High Performance Liquid Chromatography ^(HPLC) determination of free amino acids and biogenic amines,and the relationship between them.

Material and methods

Biogenic amines and free amino acidswere ana- lysed atthe Chemistry Department, Royal Vet- erinary and Agricultural University, Denmark.

To obtain fish silage of different qualities (silos 1to 3), herring offal was fermented with 10⁷ colony forming units (cfu) Lactobacillus plantarum (L.pl) g and 0,2, and 5% (w/w)dex- trose at25°C(Lassén 1993^a).The effects of dif- ferent starter cultures on the biological quality

of differentraw materials were studied by fer- menting herring offal(silos 4to7) and poultry waste(silos 8-11) with four different_startercul- tures(L.p, L.p :Pediococcus pentosaceus (P.p), L.p -.Pediococcus acidilactici (P.a), and Pelzyme®), and 5 ^% (w/w) dextrose at 25°C (Lassén 1993^b). Analyses for amino acids and biogenic amines _were made after 0, 1,2, and 7 days'storage (silos 1-3), and in samples taken weekly during the4 weeks' storage period (si- los 4-11). Samples were frozen immediately, freeze dried, minced and homogenized. They wereanalysed for free amino acids and biogen- ic amines using modifications of methods de- scribed by Bjergetal. (1984). Samples composed of200 mg of fermented material and 200 pi of internal standard solution (2 pmol 3,4-Dimetoxy Phenylmetylamin and 3 pmol Norvaline/g)_were extracted three times in 5 ml of 70% boiling methanol. The raw ^extract was air driedover- night, dissolved in2 ml ofwaterand separated into basic amino acids and biogenic aminesus- ing CM-Sephadex 25® (Pharmacia, Sweden)ion exchange column(A)and into neutral and acid- ic amino acids using Dowex50w x 8 200 mesh®

(Pharmacia, Sweden) ion exchange column(B).

The A column was eluated with 4 M acetic acid:methanol solution(1:1) and the B column with2 M Pyridine solution. Both eluates were air dried overnight and dissolved in 500 pi of waterand purifiedover Imm Bondapac ClB®

(Pharmacia, Sweden). The Bondapac column waseluated with 1.5 ml of^waterand the eluates were air dried overnight and resolved in 200 pi ofwater. 10 pi of solution was used for quanti- tative determination by HPLC. The HPLC meth- od usedwaspre-column derivation with OPA (25 mgo-PhatalaldehydeOPA, 2 mlmethanole,250 pi potassiumborate,and 25 pi mercapto propi- onicacid) (B-eluates) and NAP ^(16.5 mg p- Phatalaldehyde, 1.32 ml methanole, 165 pi po- tassiumborate, and29.73 mg N-acetyl-D-peni- cillum,NAP) (A-eluates). A-eluateswereeluat- ed for60 minutes with 25 mM phosphatebuffer (A) and 90% acetonitrile (C) using the follow- ing gradient: ⁰min 90%(A):10%(B);45 65:35;

50^40:60; 60^90:10;B-eluates_were eluated for

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60 minutes with 25 ^mMphosphatebuffer(A)and 50% acetonitrile:phosphatebuffer ^(B) with the following gradient: 0 min 100%(A):0% (B);20 80:20;40 65:35; 50 40:60; 60 100:0. The HPLC columnwas aSuperPac spherisorbODS2® ^3p.m x 125 mm (Pharmacia, Sweden) and the column temperature was 30°C,flowrate was 1ml/min, and the absorbancewasmeasuredat340nm.The raw data _were processed by computerwith _an

HPLC manager (Pharmacia, Sweden).

Feed quality was presented by aquality in- dex (QI)expressed by the following formula;

(Lysine ⁺Arginine⁺Tyrosine) Ql⁼

(Ornithine⁺Cadaverine⁺Putrescine⁺Tyramine) Samples from the 11 differenttreatmentsde- scribed abovewereanalysed for^contentsof free amino acids and biogenic amines,and the qual- ity of the silage _was evaluated according to

QI.

To estimate the degree of proteolysis, the alanine content wasrecorded in additiontoQI.

Results

The study showed that herring offal (silo 1) could be stored anaerobically for two days at 25°C

without affecting quality as measured by QI;

however,it putrefied afterone week ofstorage.

Offal in silo 2 also putrefied after one week of storage, but fermentation with5% dextrose (silo 3)resulted in stable silage withahigh

QI

^(0.75)

afterone week ofstorage (Fig. 1).The alanine content increased from _an initial concentration of 18.7 pmol/gto 212.2 pmol/g afterone week in silo 2, and from 16.5 pmol/g to 148.4 pmol/g in silo 3. If no dextrose was added (silo 1), alanine decreased from_an initial concentration of24 pmol/g to 7.9 pmol/g aftertwo days and then increased once more ^to 14.0 pmol/g after one week (Table 1).

In herring offal (silos 4-7)

QI

increased dur- ing the first week ofstorageand then decreased

Table 1.^Changesinalanine content (pmol/g)duringstorage forsilageof different organoleptic qualities (silos 1-3) and forherringoffal (silos 4-7) andpoultrywaste (silos 8-11)fermented with different starter cultures.

StoragetimeDays Storagetime Weeks

S* 0 1 2 7 S* 0 I 2 3 4

1 24.0 13.7 7.9 14.0

2 18.7 43.3 58.6 212.2

3 16.5 26.6 50.6 148.5

4 14.5142.2 145,0 218.8214.3

7.856.5 89.2103.3 120.5

11.355.6 97.1123.3 149.6

25.059.8 80.8184.1 104.1

20.197.4 120.1144.7 183.8

44.098.9 112.5134.6 144.0

34.485.94 113.0135.7 156.4

35.098.2 104.3139.2 203.1

5 6 7 8 9 10 II

*S⁼silo number

Fig. 1. Qualityindex (QI) for herringoffal (silos 1-3) fermented with 10⁷ colony forming units/gLactobacillus plantarum and0,2,and5%dextrose at 25°C.

and stabilizedatabout0.75 for good quality si- lage and 0.25 for poor quality silage (Figure 2.).

In this type ofraw material alanine increased during the first three weeks from _aninitial_con- centration of 14.5 pmol/gto218.8 pmol/g (L.p), and from 25.0 pmol/g to 184.1 pmol/g (Pelzyme®), and then decreasedto214.3 pmol/

g, and 104.1 pmol/g, respectively. For L.p:P.p and L.p:P.a alanine increased throughout the period from initial concentrations of 7.8 pmol/g and 11.3 pmol/g to 120.5 pmol/g and 149.6 pmol/g, respectively (Table 1), but increasing concentrations of biogenic amines, especially tyramine, resulted in a low QI. The quality of the fermented silagewas acceptable for all four typesof silage, Pelzyme® yielding the mostac- ceptable odour and texture.

There was nomajor difference in

QI

^between

poultry wastes (silo 8-11), except slightly in- creased

QI

for silage fermented withL.p:P.p and L.p:P.a after^twoweeks ofstorage,but after four weeks of storage the final QI was almost the same (Figure 3). Alanine _content(Table 1) in- creased throughout the period of storage from aninitial concentration of20.1 pmol/gto 183.8 pmol/g (L.p), 44.0 pmol/g ^to 134.6 pmol/g (L.p:P.p), 34.4 pmol/gto 156.4pmol/g (L.p:P.a), and35 pmol/g to203.1 pmol/g (Pelzyme®). The quality of the fermented silage was acceptable for all silages witha primarilysourodour after four weeks ofstorage. The offal liquefied after

one week of fermentation, and separated into oily, aqueous and solid fractions due to hydrol- ysis of the material; herring offal_was the most liquefied.

Discussion

QI is a new way ofpresenting analytical infor- mation obtained from analyses of the amino acids released from protein and accumulated bio- genic amines. The amino acids lysine, arginine, and tyrosine wereused in the index for the esti- mation of quality, because they arereadily re- leased from protein and metabolisedtothe cor- responding biogenic amines (cadaverine, orni- thine and putrescine, and tyramine). Histidine and histamine were not chosen, because in- creased concentrations of histamine in ferment- ed products (herring offal and chicken offal) wereonly detected when the productwasalready organoleptically unacceptable. The alaninecon- tentwaschosenas atool tocompare theamounts of amino acids released from protein over time, because alanine is readily released but hardly metabolisedatall during storage. Therefore,the reliability of

QI

improves with simultaneous_re- cording of alaninecontent in the product.

The finding ofa low QI in spoiled fish offal Fig. 2. Quality index (QI) forherringoffal (silos 4-7)

fermented with different lactic acid bacteria cultures ⁽ 10*

colony forming units/g, 5%dextrose at 25°C).

Fig. 3. Qualityindex (QI) for chicken offal (silos 8-11) fermented with different lactic acid bacteria cultures (1O*col-

ony forming units/g, 5% dextrose and 12% extruded wheatmeal:feathers (2:1) at 25°C).

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(stored at25°C forone week, silo 1)was as ex- pected, due to the high concentrations of bio- genic amines. Spoiled herring offal was, how- ever, characterized by anincrease in

QI

^during

the first two days of storage, primarily due to low concentrations of biogenic amines. This is probably _{an outcome} of less hydrolase activity and slower release of amino acids from protein in unfermented herring offal, which was con- firmed by lower concentrations of free alanine.

Afterone week at 25°C almost all the released amino acids had decomposedtobiogenic amines (cadaverine and tyramine) andornithine, where- asthemorestable silage (silo 3) hadahigher QI due tolower concentrations of biogenic amines.

High concentrations of biogenic amines were also found in poor quality silage (silo 2) after oneweek ofstorage(low QI). When herring of- falwasfermented with different lactic acid bac- teria (LAB) (silos 4-7), a difference in QI de- velopment between the cultures was observed, the silage culture Pelzyme® generally having a higher QI than the others. For chicken offal fer-

mented under similar conditions (silos8-11)no major difference between the LAB culturestest- ed_wasobserved. Fermented silagewasgeneral- ly characterized by_anincreased alanine_concen- trationover time, which describes the hydroly- sation and proteolytic activity in the silage.

Using QI notonly provides a simple way of expressing feed quality, but also a way ofcom- paring analyses based on wetweight (ww)and drymatter.Data fromastudy by Klausen (1988), recalculated to QI, give the following informa- tion. At the beginning of thestorage,herring vis- cera had a very high QI (10°C:10.2 and

20°C;6.9),due^tothe high contentof free amino acids and low concentrations of biogenic amines.

After 24 h

QI

^{decreased to} 1.15 and 0.98, re- spectively, and finally decreased to 0.29 and 0.20, at which point the herring viscera were considered putrefied and spoiled.

Both in the present study and in that of Klausen(1988), low QI could be explained by a high content of biogenic amines (tyramine and cadaverine) and low concentrations of free ami-

noacids in the herring viscera. An initial increase

in

QI

^canbe attributetdtorelease of amino acids from protein with littleor no transformation into biogenic amines. This was often observed dur- ing the first week ofstorageafter successful in- itial fermentation (silos 4,6, and 7). However, after_oneweek of_storage,QIdecreased,and high concentrations of biogenic amines and ornithine wereobserved after four weeks ofstorage,even if the pH and redox potential in the silagewere still low enough (Lassén

1993

^{b) to inhibit en-}

zymeactivity. The lactic acid producing bacte- ria itself might be responsible for ADC activity atlow pH, becausemostof the spoilage bacteria areinhibitedatpH^<4.5orlack cellular activity (Lindgren 1985). The biochemistry of fish vis- cerais obviously quite different from that of the fillet(Bprresen etal. 1988),but studies_on the biochemistry offish visceraare scanty.Stede and Stockemer(1981)found for whole herring stored at6°C that the viscera contained 1.00 (imol his- tamine/g ww after 2 daysat6°C. Corresponding values for herring fillet were 0.41 pmol hista- mine/g ww. Klausen(1988) studied thecontent of free amino acids and biogenic amines in her- ring fillet and herring viscera storedatO°C for

10 days, and found the contents of tyramine, cadaverine and agmatine in the viscera to be approximately 1000, 200 and 125times,respec- tively, thecontentsfound in the fillet.^Bprresen etal. (1988) found the concentration ofmost biogenic amines in freshly caught herring tobe lower than 15 nmol/g ww in thefillet, and 1.5 pmol/g ww in theviscera, whichwas supported by the results obtained here.

The biochemistry of fermented poultry offal is largely unknown,but Urlings (1992) studied the concentrations of biogenic amines in poul- tryoffal fermented with10% beet pulp, 2% dex- troseand Lactobacillus plantarum and storedat

15°C for21 days, and found lowerconcentra- tions than those reported by Eggum etal. (1987,

1988

^b).

Eggum etal. (1987) found very low concen- trations of biogenic amines in offal from freshly slaughted poultry, but cadaverine and tyramine increased heavily after24 h ^at 20°C. This find- ingwassupported by thepresentstudy, in which

cadaverine and tyramine increased during the first week of fermentation from almost undetec- tableconcentrations, (0.6, and 0.3 (tmol/g DM, respectively)to 12.7 pmol/gDM,and 13.3 (imol/

gDM, respectively.

There are various possible mechanisms for explaining the accumulation of free amino acids and biogenic amines in fermented silage (Egg- umetal. 1988

a,

^Klausen 1988).Undoubtedly the pattern observed in the present study resulted from the combined effect of autolytic and mi- crobial enzyme activities in which amino acids werereleased from protein. Biogenic amines were formed from free amino acids by autolytic decarboxylationordecarboxylation by microbi- al enzymes and removed by oxidative deamina- tion or other unidentified pathways. The de- creased alanine concentration in putrefied silage might be explained by the Sticklandreaction, in which alanines _react with glycine and H₂O to form acetic acid, ammonia and carbon dioxide (Schlegel 1986).

From thepresentand earlier studies(Lassén etal. 1990, Urlings ^1992),it canbe concluded

that accumulation ofbiogenic amines in ferment- ed animal by-products is aproblem due to the decarboxylase activity caused by the LAB cul- turesadded.

QI

^{might bean}informative way of presenting and comparing dataonbiological feed quality in fermented animal by-products, and its informative value increases if combined with concentration of free alanine andparameters

suchas lactic acid production, pH and changes in redoxpotential. Changes in concentrations of both biogenic amines and free amino acids dur- ing storage ought, however, tobe presented in pmol active molecules/g material regardless of whether the analyses are performed on dry or wet material.

Acknowledgements. Financial support for this study was provided bytheAcademyof Finland. Sinceregratitudeis due to Mrs Birthe lessen of Chr. Hansen’sLab,A/S,Hors- holm,Denmark forproviding the startercultures,to Mr PeterMöllerof theChemistry Department, forhelpfuland skilful assistance with HPLC analyses, and to Associate Professors Niels EnggaardHansen,Anne-Helene Tauson and HilmerSorensen for valuable suggestionsand ^com- ments regardingthemanuscript.

References

Bjerg, 8., Olsen, 0., Rasmussen, K. W. ^&Sorensen, H. 1984. Newprinciplesof ion exchange techniques suit- ableto sample preparation and group separation of nat- ural products prior to liquid chromatography. Journal of Liquid Chromatography7: 691-707.

Borresen,T.,Klausen,N.K, Larsen, L. M.^&Sorensen, H. 1988. Aminosyredekarboxylaser; Egenskaber ogre- lation til biogene aminer og kvalitet af pelsdyrfoder. Faglig Årsberetning1987.^DanskPelsdyravlerforening.p. 174- 184.

Eggum, B. 0,, Hansen, N.E., Henriksen, P. ^& So- rensen, H. 1987.Biogeneamineripelsdyrfoder. Faglig Årsberetning1986.Dansk Pelsdyravlerforening. p.233- 245.

-,Hansen, N.E., Henriksen, P.^&Sorensen, H.1988b.

Biogeneaminerirelation til kvalitet af pelsdyrfoder. Faglig Årsberetning 1987.Dansk Pelsdyravlerforening. p. 185- 207.

-,Hansen,N. E.^&Sorensen,H. 1988a. Amino acid pre-

cursors^{ofbiogenic amines.} In: Friedman, M. (ed.). Ab- sorptionand utilization of amino acids. CRCPress,Boca Raton, Florida.42^p.

Klausen, N. K. 1988.Decarboxylationof tyrosinein re-

lation to metabolism of amino acids and biogenic amines in fish during storage: Enzymes, Kinetics, _{and impor-} tance. Ph.D Thesis, Chemistry Department, RoyalVet- erinary^andAgricultural University,Denmark. ^{185 p.}

Lassén, T. M. 1995a, Evaluation of conditions for fer- mentation of fish offal. Agricultural Science inFinland 4:

11-17.

-1995b. Lactic acid fermentation of fish offal and chick- enby-productwith different starter cultures. Agricultural Science in Finland 4: 19-26.

Hildén,A.,Hildén, B.H.^&Laitinen, M.J.1990.Prak- tisk tillämpning av erfarenheter från försök med biolo- giskkonserverad ensilageifoder till mink ochräv.NJF- Utredning/RapportNr.^{60. 20 p.}

Lindgren, S.E. 1985.Användningavmjölksyrajäsande bakterier för konservering av animaliska råvaror. NJF- Seminarium Nr. 85.Aalborg,Denmark. 6p.

Schlegel,H. G. 1986. General Microbiology. Sixth Edi- tion Cambridge University Press. Cambridge, UK. p. 293- 302.

Skadborg,J. 1985. Sundhedsskadeligestaffer dannet ved mikrobiologisk, enzymatisk og oxidativ aktiviteti^foder

till pelsdyr. HovedopgaveiPelsdyrproduktion, Royal^Vet-

Vol. 4: 27-33.

erinaryand Agricultural University, Denmark. 64p.

Stede,M.^&Stockemer, J. 1981.Bildungvon Histamin intrischen Heringen und^Makrelen.Fleischwirtschaft61:

1746-1749.

Taylor,S. L. 1986. Histamine Food Poisoning: Toxicolo- gy and Clinical Aspects. CRC Critical Reviews inToxi- cology17: 91-128.

- 1988.Marine toxins of microbial origin. Food Technol- ogy42: 94-98.

Urlings,H. A. P. 1992.Fermentation of animal by-prod- ucts. Microbiological aspects of processing, epidemiolo- gy and animal nutrition. Diss. Utrecht University, Faculty of Veterinary Medicine, Departmentof the Science of Food of Animal Origin,Utrecht,The Netherlands. 135p.

SELOSTUS

Fermentoitujen kala- ja kanajätteiden biologinen laatu

T. Mikael Lassén

Royal Veterinary andAgricultural University,Tanska

Tutkimuksessa selvitettiin fermentoitujenkala- ja kanajätteiden biologista laatua. Tutkituille jätteille määritettiinlaatuindeksi,jonkamäärittämiseksiana- lysoitiintärkeät vapaataminohapot janiitä vastaavat biogeeniset aminit. Näytteet analysoitiin ryhmäerot- telu-menetelmällä,jokaperustuu ioninvaihtokroma- tografiaan. Biologinen laatututkittiinnäytteistä, jot- ka olivat eribakteeriviljelmistä, erikokoisista siirros-

tuksistajaerilaisilta kasvualustoilta.

Hidas tai epätäydellinen fermentaatio aiheutti biogeenisten aminien, erityisesti tyramiinin, määrän

kasvun ja lopputuloksena oli alhainen laatuindeksi.

Nopean alkufermentaationja jatkuvan vakaanneljän viikon fermentaation seurauksenaoli alhainen bio- geenisten aminienpitoisuus,korkea vapaidenamino- happojen pitoisuus jakorkea laatuindeksi.

Alaniinipitoisuuden todettiin olevankäytännölli- nen proteolyysin arviointiväline. Lisäksi muutokset alaniinipitoisuudessa vaikuttivatlaatuindeksiin.^Mai- tohappopitoisuus ja redox-potentiaali soveltuivat kaikkeinparhaiten fermentoitujenkala-ja kanajättei- denbiologisen laadun arviointiin.