Effect Of Inulin Suplementation Of Milk To Prepare Fermented Biomilks

Transcription

1 Effect Of Inulin Suplementtion Of Milk To Prepre Fermented Biomilks Ricrdo Pinheiro de Souz OLIVEIRA 1, Ptrizi PEREGO 2, Attilio CONVERTI 2, Mricê Nogueir de OLIVEIRA 1 1 Deprtmento de Tecnologi Bioquímico-Frmcêutic, Fculdde de Ciêncis Frmcêutics, Universidde de São Pulo. Av. Prof. Lineu Prestes, São Pulo, SP Brsil. 2 Diprtimento di Ingegneri Chimic e di Processo, Università degli Studi di Genov, Vi Oper Pi 15, I Genov, Itly Actully, functionl fermented milks hve to exhibit multiple benefits for the helth, ssocited to good orgnoleptic chrcteristics. These re strengthened by the ddition of probiotics s well s certin soluble fibers, mong which inulin. The im of this work ws the evlution of the effect of inulin supplementtion of milk bsis on the production of probiotic fiber-enriched fermented milks. To this purpose, we investigted the kinetics of cidifiction of inulin supplementtion of milk (0, 1, 2 nd 4 g/100 g) throughout the fermenttion, the probiotic survivl, the post-cidifiction nd the firmness of fermented fiber-enriched milks, stored t 4 C either for 24 h or fter 7 dys since their preprtion. This work shows tht the biomilk preprtion is influenced either by the supplemented mount of inulin or by the co-culture composition. According to the selected co-culture, inulin ddition to the milk influenced the kinetic prmeters of cidifiction, the concentrtion of probiotics, the post-cidifiction nd the firmness of fermented fiber-enriched milk. 1. Introduction Nowdys, consumers re demnding for foods with incresingly properties, such s plesnt flvor, low-clorie vlue or low ft content, nd benefic helth effects. Within this context, food industry hs been trying to offer products with improve flvor nd ppernce. In ddition, functionl diry products offer requirements, benefits to helth tht re strengthened by the ddition of probiotics s well s by certin types of soluble fibers known s prebiotic. Prebiotics re non-digestible food components tht beneficilly ct on the host becuse they selectively stimulte either the prolifertion or the ctivity of bcteril popultions tht re desirble in the colon. Moreover, prebiotics my inhibit pthogen multipliction, ensuring dditionl benefits to host s helth. Such components mostly

2 ct on the lrge intestine, even though they cn lso impct the microorgnisms inside the smll one (Roberfroid 2000; Mttil-Sndholm et l. 2002). Lctic-cid bcteri (LAB) isolted from humns nd nimls gstrointestinl trct is known s probiotics. These orgnisms, when used in lrge mounts in the preprtion of foods nd diry products, should survive the pssge through the upper digestive trct. Besides, probiotic microorgnisms should be ble to dhere to intestinl cells, providing beneficil effects in the intestinl trct (Gillilnd 1989). When such orgnisms re lrgely consumed, they help in the intestinl blnce (Ferreir 2003). In order to produce the desired benefits, probiotics bcteri should be present in the products in vible counts during their whole shelf-life. To this purpose, we investigted throughout the fermenttion by Streptococcus thermophilus in co-culture with Lctobcillus delbrueckii subsp. bulgricus, Lctobcillus cidophilus, Lctobcillus rhmnosus nd Bifidobcterium nimlis subsp. Lctis the kinetics of cidifiction of inulin supplementtion of milk (1, 2 nd 4 g. 100 g -1 ), the probiotic survivl, the post-cidifiction nd the firmness of fermented fiber-enriched milks, stored t 4 C either for 24 h or fter 7 dys since their preprtion. 2. Mteril nd Methods 2.1 Experimentl Procedure Five strins of pure commercil strter freeze-dried cultures (Dnisco, Sssenge, Frnce) were used: Streptococcus thermophilus TA040 (St) nd Lctobcillus delbrueckii subsp. bulgricus LB340 (Lb) (yogurt microorgnisms) nd Lctobcillus cidophilus LAC4 (L), Lctobcillus rhmnosus LBA (Lr), nd Bifidobcterium nimlis subsp. lctis BL 04 (Bl) (probiotics microorgnisms). Precultures were prepred in which bcteri verge counts in the different pre-cultures rnged from 6.1 to 6.5 LogCFU.mL -1. Milk prepred dding 13 g of skim powder milk (Molico, Nestlé, Arçtub, Brzil) in 100 g of distilled wter (M) ws supplemented with inulin (Beneo TM, ORAFTI Active Food Ingredients, Oreye, Belgium), specificlly 1 (SM1), 2 (SM2) nd 4 (SM4) g.100g -1, nd them thermlly treted t 90 C for 5 min in wter bth (550 THE, Fistom, São Pulo, Brzil). The het treted milk ws collected into 1.0 L sterile flsks, cooled in ice bth, distributed into 250 ml sterile Shott flsks inside lminr flow chmber, nd stored t 4 C for 24 h, before using. After inocultion, flsk smples were trnsferred on wter bth equipment ssembled to CINAC (Cynetique d cidifiction, Ysebert, Frépillon, Frnce) system nd, btch fermenttions were performed t 42 o C up to ph 4.5 corresponding to the finl fermenttion time. Fermenttions were crried out in duplicte nd monitored using the CINAC system which llows to continuously mesure nd record of ph s well s to evlute cidifiction rtes throughout the run (Spinnler nd Corrieu, 1989). From the collected dt, the cidifiction rtes (dph/dt) were clculted s the time vrition of ph nd expressed s 10-3 ph units. min -1 (V mx ). At the end of the incubtion period, the following kinetic prmeters were lso clculted: (i) t mx (h): time in which the

3 mximum (V mx ) cidifiction rtes were reched nd (ii) t ph4.5 (h): time to rech ph 4.5. Not supplemented milk (M) ws used s control. 2.2 Milk Chemicl Composition nd Firmness Anlyses Physico-chemicl nlyses (ft, gross protein, lctose nd totl solids) were crried out in inulin-supplemented milk ccording to Schmidt-Hebbel (1956), A.O.A.C (1995) nd INSTITUTO ADOLFO LUTZ (1985) methods, in triplicte. The results were expressed in g.100 g -1. Fermented milk post-cidifiction ws determined by ph mesurement using phmeter model Q-400M1 (Quimis, São Pulo, Brzil). Acidity, expressed in percentge of lctic cid, ws evluted by titrtion using 0.11N NOH solution ccording to AOAC (1995) methodology No Fermented milk firmness ws mesured t 8ºC using TA-XT2 Texture Anlyzer (Stble Micro Systems Ltd., Godlming, United Kingdom). The opertion conditions were: probe type crylic P/25P (2.5 cm dimeter), 10 mm penetrtion, nd 10 mm s -1 speed). Firmness ws expressed s the mximum penetrtion, in N. 2.3 Counts of Probiotic Bcteri Bcteril enumertions were crried out either fter 24h or fter seven dys since storge of fermented milks t 4ºC. One ml of smple ws diluted with 9 ml of 1% sterile peptonted wter. Afterwrds, seril dilutions were done nd bcteri were counted pplying the in-depth pltelet technique. All medi were obtined from Merck (Drmstdt, Germny). St colonies were enumerted in M17 gr by erobic incubtion t 37 ºC for 48 h. Lb enumertion ws crried out in MRS Agr medium with ph djustment t 5.4 by cetic cid ddition, nd erobic incubtion t 37 ºC for 48 h. L nd Lr were enumerted in the sme medium with ph djustment t 5.4 by cetic cid ddition, nd nerobic incubtion t 37 ºC for 72 h. Bl ws enumerted s L nd Lr without ny ph djustment (IDF 1996, 1997, 2003). 2.4 Sttisticl Anlyses Results were submitted to nlyses of vrince (ANOVA) using the Sttistic Softwre 6.0. Men vlues were compred using the Tukey test t P< Results nd Discussion 3.1 Milk Chemicl composition Ft, protein nd solid contents rnged respectively from 0.03 to 0.06 g. 100 g -1, 4.00 to 4.10 g. 100 g -1, nd to g. 100 g -1. No sttisticlly significnt difference ws observed in ft nd protein contents wheres those of totl solids vlues were

4 sttisticlly different. Lctose content rnged from 4.43 to 4.63 g. 100 g -1, with significnt sttisticl differences when 4 g. 100 g -1 of inulin ws dded. 3.2 Kinetics of Acidifiction The fermenttion times (t ph4.5 ) rnged between 5.2 to 11.2 h. There ws sttisticlly significnt vrition in this prmeter, which ws shorter in milks tht received inulin s supplement mostly t 4 g.100 g -1 concentrtions, wheres St-Lr cultures ws strongly ffected t such inulin level. However, for St-Lb (SM1), St-Lr (SM1 nd SM2), nd St- Bl (SM1) co-cultures, the fermenttion times were close to those obtined t the other concentrtions (Figure 1) d d d bc 9 b tph4.5 (h) Inulin Content (g.100g -1 ) Fig. 1. Effect of inulin ddition to milk on the durtion of fermenttion (tph 4.5 ) by Lctobcillus bulgricus, Lctobcillus cidophilus, Lctobcillus rhmnosus, nd Bifidobcterium lctis in co-culture with Streptococcus thermophilus. (ο) St-L;( ) St- Lb; ( ) St-Lr; ( ) St-Bl. Inulin influenced significntly the cidifiction kinetics of the milk for probiotics cultures. Mximum cidifiction rtes (V mx ) incresed with inulin contents. Milk supplementtion with 4 g.100 g -1 of inulin reduced the fermenttion time of S. thermophilus nd L. rhmnosus co-culture. 3.3 Post-Acidifiction nd Acidity ph vlues rnged from 4.32 (St-L/M) to 4.48 (St-Lr/M) fter one dy of fermenttion (D1), nd from 4.15 (St-L/SM4) nd 4.37 (St-L/M) fter seven dys (D7). On

5 verge, there ws 0.14 uph decrese fter seven-dy storge t 4 C. Using the St-Lb nd St-L co-cultures, the post-cidifiction ws stronger when using 4 g.100 g -1 of inulin, while there ws higher post-cidifiction of milk supplemented with 1 g.100 g -1 of inulin using St-Lr nd St-Bl co-cultures. After one dy of fermenttion, the cidity rnged from 0.81 (St-Bl/M) to 1.03 g.100g -1 of lctic cid (St-L/SM2). Using the St-Lb co-culture the mount of lctic incresed with inulin concentrtion in the formultion, but only the highest inulin concentrtion (4 g.100 g -1 ) exhibited sttisticlly significnt difference. Using the St-L co-culture the ddition of 2 g.100 g -1 of inulin yielded the highest production of lctic cid compred to ll tested co-cultures. Concerning St-Lr co-culture the highest production of lctic cid (0.94 g.100 g - 1) ws obtined using 1 g.100 g -1 of inulin. The lowest vlues of lctic cid were found using the St-Bl co-culture, nd they rnged from 0.81 g.100 g - 1 of lctic cid in Ml to 0.86 g.100 g - 1 in SM1, with sttisticlly significnt difference between them. After seven dys of fermenttion (D7), lctic cid production ws lwys higher thn fter one dy (D1), rnging from 0.90 g.100 g -1 (St-Lr/M) to 1.20 g.100 g -1 of lctic cid (St-L/SM2). Tken s n verge, the increse in lctic cid production ws 0.13 g.100 g -1. The ddition of inulin supported higher post-cidifiction for ll investigted co-cultures. 3.4 Probiotic Bcteri Counts The counts for S. thermophilus fter 1 dy of fermenttion rnged from 8.67 (St-Bl) to 9.45 Log CFU.mL -1 (St-Lr) in SM1, without no sttisticlly significnt difference between the cultures. After 7 dys of fermenttion, counts rnged from 8.18 (St-Bl) to 9.42 Log CFU.mL -1 (St-Lr) in the sme medium, with sttisticlly significnt differences. In verge, the ddition of 4g100 g -1 of inulin resulted in 0.40 log CFU.mL -1 decrese for L nd 0.69 Log CFU.mL -1 for Lr. After one dy of fermenttion, probiotic counts rnged from 7.37 (St-Lr/ M) to 9.13 Log CFU.mL -1 (St-Bl/SM4). There ws sttisticlly higher growth of St-Lb in milks supplemented with inulin. Compred to the control the growth of L in milk supplemented with inulin ws slightly higher, with sttisticlly significnt difference. However, no pprecible vrition between counts of Lr in milk supplemented or not with 1 g.100 g -1 of inulin ws observed, wheres it becme pprecible when 2 nd 4 g.100 g -1 of inulin ws dded. Indeed, inulin showed sttisticlly significnt in vitro bifidogenic effect, with higher counts in milks supplemented with inulin. After 7 dys of fermenttion he vibility of L. cidophilus nd B. lctis, probiotic bcteri, decresed by ~0.04 nd ~0.15 Log CFU.mL -1, respectively. However, counts remined stble for L. rhmnosus nd L. bulgricus cultures. Milk supplemented with inulin hd significnt influence on the survivl of these probiotic bcteri. Probiotic counts were higher thn 7.37 Log CFU.mL -1. The inulin showed n in vitro bifidogenic effect, stimulting the growth of B. lctis; this effect ws more pronounced using B. lctis thn the other probiotic cultures. Counts remined lmost stble fter seven dys of storge t 4 C.

6 3.5 Firmness After one dy of fermenttion (D1), firmness rnged from 0.28 (St-Lb/M) to 0.47 N (St-Lr/SM4). At seven dys (D7) it incresed, on verge, when compred to D1 by ~0.06 N, vrying from 0.33 (St-Lb/M) to 0.54 N (St-Lr/SM4). Such results demonstrte tht the supplementtion of milk with inulin resulted in stronger firmness for ll products nd gree with those reported by MARTIN et l. (1999) nd OLIVEIRA et l. (2001). Acknowledgement The uthors thnk the FAPESP (Reserch Foundtion of the Stte of São Pulo), DANISCO nd ORAFTI Active Food Ingredients for furnishing the cultures nd the inulin, respectively. 4. References AOAC. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS. Officil Methods of Anlysis 16.ed. Wshington, p. FERREIRA, C L L (2003). Grupo de bctéris látics: crcterizção e plicção tecnológic de bctéris probiótics. In Prebióticos e Probióticos: Atulizção e Prospecção, pp Ferreir, C.L.L. ed., Viços - MG: UFV. GILLILAND, S E (1989). Acidophilus milk products: review of potentil benefits to consumers. Journl of Diry Science IDF (1996) Preprtion of Smples nd Dilutions for Microbiologicl Exmintion, Stndrd No. 122C, Interntionl Diry Federtion, Brussels, Belgium. IDF (1997) Diry Strter Cultures of Lctic Acid Bcteri (LAB) Stndrd of Identity, Stndrd No. 149A, Interntionl Diry Federtion, Brussels, Belgium. IDF (2003) Yoghurt / Enumertion of Chrcteristic Microorgnisms Colony Count Technique t 37 C, Stndrd No. 117, Interntionl Diry Federtion, Brussels, Belgium. INSTITUTO ADOLFO LUTZ. (1985) Norms Anlítics do Instituto Adolfo Lutz: Métodos químicos e físicos pr nálise de limentos. 3.ed. São Pulo: Imprens Oficil do Estdo de São Pulo, v.1, 533p. MARTIN, N C, SKOKANOVA, J, LATRILLE, E, BÉAL, C nd CORRIEU, G (1999) Influence of fermenttion nd storge conditions on the sensory properties of plin low ft stirred yoghurts. Journl of Sensory Studies MATTILA-SANDHOLM, T, MYLLÄRINEN, P, CRITTENDEN, R, MOGENSEN, G, FONDÉN, R nd SAARELA, M (2002). Technologicl chllenges for future probiotic foods. Interntionl Diry Journl

7 OLIVEIRA, M N, SODINI, I, REMEUF, F nd CORRIEU, G (2001). Effect of milk supplementtion nd culture composition on cidifiction, texturl properties nd microbiologicl stbility of fermented milks contining probiotic bcteri. Interntionl Diry Journl ROBERFROID, MB (2000) Defining functionl foods. In: Gibson,G.R.; Willins, C.M. Functionl foods-concept to product. Boc Rton: CRC, Chpter.1, p SCHMIDT-HEBBEL, H (1956) Quimic y tecnologi de los limentos. Sntigo: Editoril Slesin, 313p. SPINNLER, H E nd CORRIEU, G (1989). Automtic method to quntify strter ctivity bsed on ph mesurement. Journl of Diry Reserch