Inhibition of the growth of yeasts in fermented salads

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  • International Journal of Food Ali('n)hiolog)" 17 (It)t)3) 311-320 311 , It193 Elsc~ricr Science Publb, hcrs B.V. All right~, rc.,,cncd OIl)N-Iol)5./t,)3/S116.|10

    FOOD 00547

    Inhibition of the growth of yeasts in fermented salads

    M.H. Bonestroo, J.C. de Wit, B.J.M. Kusters and F.M. Rombouts I)cpartmc~t of l:ood S('i(w('('..-Igri('uhural Unit cr.',ity. II'(tg('ni~gcn Th(" ;~)'ctherhmd.~

    (Received 1) i)cccn)bcr Itltil ' rc~b, ion rccci,.cd 17 June 1~1~,)2, accepted 3 Augu.~! It}g2)

    5:|l;.ids (,:OlllpOsL.'d of ~cgchlhlc~, :.lntl/or mual ill an oil-ill-v.;ller cnzttlr..ioll x~,crt" prepared by l'~rmunt:_|liOl) for 7 h at 42'(" or 45"(" ~ith MrailP, of /.,l('l,)t,,t(illtt~ H~p. Their ,,tabilit.x Io~ards spoilage yea~,ts t~'as ~,tudicd I.l.~illg .~a('l'hllrottly('('.~ ('('r('tl.shl('. ,~d('('h0)r,)ttw,++'s ,'+ttrlol), ~md T, ruhtvnJra &'lhn~t'('kii, isohttcd from salads, as ~ell ;is Pihh~ Ilh'll)lhrallat'til('iclP, alltl Z}'~oslll'(hlll'Otllt'(t'~ I)ailii. Salads fermented x~'ith good lactic shlrlcrs usually Ilad pll valuc~, of _< 4.2 and lactic acid conccntration,~ of ().2N Io 0.43~i (v./v,). Iligh nl.lnll')crs t)f spoilage yca~,l~, (and prod).lClion of htrgc ~.Ohlll'lc~ o[ (.'(.),) ~crc not attained ill these salad.~, provided the initial t'ollClllldtion of ,~poil:lt~c ~C;.I~I>, ~.~,a~ ,qlllicicntl h)v,- { _< IIX} ( 'FU/g) . Inhibition of ~poilagc ycast~ ill I:tclic fcrnlenlcd .~alittb, it, probabl.~ title to h.tctic acid, the Io~. t, storage h:mpcraturc and Ihc Iov, rc~,idual oxygen con~:nlr:lliOl).

    Key word,< Saladr,: FerlllCnlalion: l.~|clil: acid baclcria:'lol)d(tl/It~: ~l'ci.|sl~,; .~a(tharonlyt'e.~; Tortdav)ora: (;ro~lh inhibition


    Salads, composed of vegetables and/or me~t in an oil-in-water cmui,,ion with a h)w pH ( < 4.8) due to the addition of acids, are popular in Western countries. "lhe shelf life of these salads is influenced mainly by low pH, storage temperature and the use of chemical preservatives, i.e. sorbic and benzoic acids. Higher demands on quality of raw materials, better preparation methods and especially the use of preservatives, have led to a shelf life of these salads of 6-8 weeks, if stored below 7C. However, consumer trends arc towards salads without chemical preservatives and with a less sour taste. The omission of preservatives limits the shelf life of salads to 2-3 weeks, if stored below 7C (Gromzik, 19t) l ). Micro-organisms causing spoilage arc lactic acid bacteria, yeasts, moulds and bacilli (Smittle, 1977: Kirsop and Brocklchurst, 1982: Brocklehurst et al., It)83: Lund et al., 1984). According to literature data. the most important spoilage yeasts arc Saccharomyces eriguus and Saccharomyces dairensix. Pichia membramt~]'(wienx. Debat3"omyces kloeckeri.

    ('orrc,v~mdeswe addrc.~,~: M.]I. l]onc~troo. Dcpartmcn! of Food Science, Wagcningcn Agricultural University. Bomcn~'eg 2. ()703 l id Wageni,lgcn. The Netherlands. Fax: + 31 $370 N4S93.

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    Geotrichum cam~Mum, some CamlMa spp. and Zygosaccharomyces bailii are al~ found in salads (Smittle, 1977: Baumgart, 1977: Smittle and Flowers, 1982; Brock- lehurst et al., 1983; Brocklehurst and Lund, 1984).

    It is generally accepted that the antimicrobial activity of weak acids such as acetic acid and lactic acid is due to the undissociated acid molecules. An increase in concentration of these molecules, independent from a decrease in pH levels, will improve the microbial stability of salads. This can be achieved by using buffer systems of weak organic acids and their salts (Debevere, 1987). it has been suggested that an additional advantage would be that taste problems are dimin- ished. The shelf life of salads could also be extended by technological solutions, such as the production of salads under strict hygienic conditions, in closed systems with rigorous exclusion of oxygen and the use of modified atmospheres. These methods require, however, large capital investments. Moreover. the growth of yeasts having a fermentative ability and especially lactic acid bacteria will not be prevented (Buick and Damoglou, 1989: Farber, 1991).

    Lactic acid fermentation of foods generally is a low-cost method of food preservation in which spoilage and pathogenic organisms are inhibited, mostly by acid production and pH reduction but also by lowering the oxidation-reduction potential, competition for essential nutrients and possibly by the production of inhibitory compounds: antibiotics and hydrogen peroxide. In a previous paper (Boncstroo et al., 1992) lactic acid fermentation was proposed as a novel way of preparation of salads, if sufficiently attractive, a certain interest may be expected from the market fi~r these fermented salads, because of their mildly sour taste, their possible health effects and especially because the use of preservatives can be abandoned.

    The objective of this study, as part of an investigation on lactic acid fermented salads, was to investigate the possible inhibitory effect of fermentation on the growth of yeasts. Therefi)re, yeasts, isolated amongst others from spoiled, industri- ally produced salads, were deliberately added to salads, which were then fer- mented. Changes in microbiological condition and chemical composition were monitored during storage at 7C.

    Materials and methods

    Organisnls and culture comlitions Lactobacillus platHariln~ and Lactobacillus acidophihs wcrc obtained from our

    laboratory collection (Boncstroo ct al., 1992). Prior to use the strains were cultivated for 24 h at 30C or 42C in a broth with the same composition as MRS broth (Merck, Darmstadt, FRG), but containing sucrose (Merck) as carbohydrate, in,~tcaa~ ef glucose, followed by subsequent cultivation in the same broth for 16 h at 30C or 42C. Pichia tnembranaefaeciens CBS 107 and Zygosaccharomyces bailii QST2877 were kindly provided by Dr W.J. Middelhoven (Department of Microbi- ology, Wageningen Agricultural University, The Netherlands) and Mr P. Breeuwer (Department of Food Science, Wageningen Agricultural University, The Nether-

  • 313

    lands), respectively. Other yeast strains were isolated, using Oxytetracycline Glu- cose Yeast Agar (OGYA, CM545, Oxoid Ltd., Basingstoke, UK) incubated at 25C, from fresh salads, sell-by date salads and spoiled salads with bulging packings, all produced without preservatives. Strains were purified and after morphological screening, preliminary characterized with the API ATB 32 C identification kit (AP! System S.A., France). Predominant yeast species were identified by Dr M. Th. Smith (CBS Yeast Division, Identification Service. Delft, The Nethcrla~|ds). Yeast cultures were maintained at 4C on Malt Extract Agar (CM59, Oxoid). Prior to usc the yeast cultures were cultivated twice, at 25C for 48 h. consecutively, in Malt Extract Broth (CM57, Ox,,~id).

    Prt'l~ttrtltion and storage of scd, ds Four types of salads wcrc prcparcd, onc containing 65c; (w/w) pcclcd, sliced (3

    mm) and ctR~ked (5-10 rain, t~)-I00C) potato tubers (cultivar Bintje} and 35c~ {w/w) dressing; the second containing 23~/" (w/w) shredded (10 x 6 3 ram) leek, 23~ {w/w) shredded ( 15 ill 3 ram) cabbage, 10~; (w/w} cooked ham and 44~ dressing: the third containing 5{}c; (w/w) gratcd {30 3 3 ram) and blanched ( I min, I(X}C} carrots and 5(); (w/w) dressing: the fi~urth containing 50c/,; (w/w) shredded ( 15 10 3 ram) cabbage and 5(}c,~ dressing. Thc dressing was com- posed of wlter (35.0-52.2~ (w/w)), soybean oil (35-5t}r~; {w/w)), sucrose (6; (w/w)), salt (1-1.8~ {w/w)), cgg yolk {3-3.5r,~ (w/w)) and thickening agents (2.8r~, (w/w)). Occasionally small amounts of seasonings were added to the salads. The salads were inoculated with 10"-!0 v cells of lactic acid bacteria per g and filled in glass jars (liHI g salad, hcadspacc approx. 50 ml) air-tightly sealed with metal Vapor-Vacuum"" Twist-Off" caps (Whitc Cap international, USA). Thc jars were incubated in a watcrbath for 7 h at 42 or 45C, and then chilled in ice watt! to a temperature of 7C and stored at this temperature. Uninoculatcd, acidified (0.4~ (w/w) lactic acid) salads were used as control.

    Growth at dif]'t'rent tonperclturt',~ Yeast strains, grown twice at 25C fi~r 48 h, consecutively, were inoculated into

    Malt Extract Broth (CM57, Oxoid) and cultivated at different temperatures. Growth was monitored either by pipetting aliquots in micro-titer plates and reading optical densities at 620 nm or by enumerating the yeasts on Oxytetracy- cline Glucose Yeast Agar (CM545, Oxoid).

    Et'ah~ation of the quafiO" oj'li'rmer:ted ,scd, d,~ Me, surement oJ'pH. The pH of the salads was detcrmiJlcd with a pH-eiectrode

    (NOl, Schott Ger[itc GmbH, Hofheim a. Ts., FRG) coupled to a combined pH/mV-metcr (pH522, Wisscnschaftlich-Technische Werkst~itten GmbH, Weil- heim, FRG).

    Microbiological qualiO'. The vegetable salads were judged on the basis of microbiological analyses directly after fermentation and up to three weeks of storage at 7C. Mesophilic aerobic spoilage bacteria were enumerated using Gelysate Agar (GEL): 5 g Bacto-peptone (Difco, Detroit, Michigan, USA), 5 g

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    sodium chloride (Merck) and 14 g agar (LI3. Oxoid) per !, pH 7.6 _+ 0.1, incubated at 30C for 3 days. Enterobacteriaceae were counted on Violet Red Bile Glucose Agar (VRBG: CM485, Oxoid) with a top layer of the same medium and incubated fi~r 24 h at 30C. Lactic acid bacteria were selectively enumerated on MRS medium (Merck) with 12 g of agar (L13, Oxoid) and 2 g of Delvocid (Gist Brocades. Delft, The Netherlands) per I. incubated under anaerobic conditions (Anaerocult system, Merck) at 30C for 3 days. Yeasts and moulds were counted on Oxytetracyclinc Glucose Yeast Agar (CM545. Oxoid), incubated at 25C fi)r 5 days, as recommended by Banks and Board (1987).

    Determbtation of sugars, etha,ol a,d orgallic acMs. Sugars. ethanol and organic acids in fermented salads were determined by means of HPLC as described earlier (Bonestr,ao ct al.. 1992).

    Results and Discussions

    In most fermentation processes a rapid pH decrease, in addition to a low final pH, is necessary to achieve a microbiologically stable product. As can bc seen in Table I a low pH ( < 3.9) was reached in the salads after 7 h of fermentation at 42C. Although the fermented salads contained raw ingredients, i.e. cabbage, no growth of yeasts occurred. During storage at 7C the pH remained low and the fermented sahtds were microbiologically stable fl~r at least 3 weeks. The fermented salads contained 11.35 ___ ().113C/ (w/w) lactic acid alter 7 h of fermentation at 42C, increasing to 0.46 ll.I)9/~ (w/w) ;.liter subsequent storage at 7C for 3 weeks. Little or no acetic acid could be found in the fermented salads, whilst ethanol

    TABI,[" I

    Microbioh)gical condition and pl I of fermented salads composed of cabbage and dressing

    Starter pit ('tiLl d after 7 h I, (log N /g) ( 'FU alter storage c (log N /g)

    no. " Inili~.l Al'lcr After ENT " Y[-A I,AB MAB ENT YEA LAB MAll 7 h i, storage "

    9 5.117 3.~11 3.ll4 < I.I)l) < 1.1111 9.12 < 3.711 < I.|1t1 < 1.1)1) S.~)9 ,,~ I.|H) 21) 5.112 3.82 3.57 < I.Ill) < I.()l) 8.71 < 3.711 < I.II(| < I.qll) 8.42 2.114 21 .1.1.)3 3.87 3.62 < I.IIt) < I.(lll 8.83 < 3.711 < I.(ll) < I.|HI 8.5f~ < 1.1111 23 5.(17 3.811 3.(14 < I.(li) < I.I)0 8.8~ < 3.71) < I.(lll < I.(ltl 8.69 < I.(ll| 24 5.13 3.84 3.78 < 1.1)1) < 1.11t) 8.91 < 3.711 < I.II11 < I.Illl 8.27 < I.II{)

    Resttlb~ ;are the nle;.ins of deterfflin;.itions of dttplicilte s;.tmples. ~ Ntmlber,', refer to'lo~at'ilhl.~ I~ho,ar, t , strain numbel:., tlcscribcd ill Bol|Mroo cl it(. (Iqq2L h After 7 h of fermentation at 42C. Alter 7 II of fermentalion at 42C iliad subsequent storage ill 7C for 21 days. d CFU, Colony filt'lning units. c ENT. ]',',terobacteritlcette" YEA, yeasts" LAFL lactic acid bacteria: MAB. mes~,philic aerobic spoilage


  • 315

    production was absent and minor amounts of sugars, generally less than ().Sr~ (w/w). were used. by the lactic acid bacteria (results not shown).

    ,~'a('('haronlyce.~ t:~'igtltt.s, Sat't'hto'omyt't'.s cercrisiac, "l~icho.woron beigelii and Tortt- laspora delbnwckii were isolated from fresh salads, sell-by date salads and spoiled salads with bulging packings, all industrially produced without prese~'alivcs. The strains failed to grow in Malt Extract Broth at 3-3.5C, but they showed g~d growth at 4-4.5C (data not shown). They showed good growth at higher tempera- lures, except for Saccharomyces (:riguu.~" and Toruh~xlu}ra dclhrueckii, of which no growth was observed at 3(1C. None of these strains showed growth at 42C. At a temperature of 7",_ ", all strains reached maximum cell numbers within 7-14 days (results not shown). Most strains possess a fermentative mcta~flism and arc thus able to grow in the absence of oxygen, except Trit'ho, vporotl I~cigelii, which possesses an oxidative metabolism.

    Trichosporon hdgelii, isolated from fresh salads, failed to grow in Malt Extract Broth at ptt 4.5 and ptt 4.0 at 7C in the presence of I).5C; acetic acid. The other strains mentioned ab~wc showed moderate to good growth under these conditkms. With the exception of .~ilt't'htlronlyt't's ccreri.shw, which could tolerate IC~ acetic acid at pH 4.5, but not at pH 4.(I, they tolerated I.O(:~; acetic acid at both pH levels. It wits clear that acetic acid is more inhibitory to the yeasts than lactic acid. Addition of lactic acid in concentrations of 0.5~ and l.l) to t}.5c~ , acetic acid did not change the growth patterns of th~ 4 yeaq s!rains (data not included).

    Various salads, deliberately inoculated with a mixture of tile above mentioned yeasts (approx. 10[} cells/g salad), fermented with l, at'tobat'ilhls spp. tbr 7 h at 42C and stored at 7C, remained microbiologieally stable ibr at least 3 weeks, as filr its yeast growth is concerned. Growth of yeasts was obviously suppressed. However, with higher inoculation levels ( > 10t]O ccl ls/g salad), growth of yeasts within 2 weeks generally could not bc prevented (data not included).

    Other experiments, in which carrot salads were inoculated with single strains of the above yeasts ( >_ 250 cells/g salad) and the yeasts Pichhl memhranaefaciens and Zygosacchatwnlyces hailii, fermented fi,r 7 h at 42C with different Lactohacillus plantanon strains and subsequently stored at 7C, gave comparable results (Table !!). Strains of Tonlht.~pora delhnwckii, Pit'hhl membnmaclbcit'ns and especially Sat'charomyce.s t'elrri,shle showed abundant growth in the fermented salads after ll} days of storage at 7C, while growth of other strains was more or less inhibited. After 20 days storage at 7C, most salads were spoiled by yeasts, except fi}r the salads inoculated with Stlt'charoltl)'t't'x t:~gulls 2 and Zygostlt'charonlyces btlilii, and the salad inoculated with "l)'it'ho,~poron be(gelii fermented with Lactobacilhts pltm- tarum no. 20. The fermented salads contained 0.28 (I.(}2q; (w/w) lactic acid after l0 days storage at 7C, increasing to 0.35 +_ O.07f~ (w/w) after 20 days. Little or no acetic acid...


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